Engineered chimeric fusion protein compositions and methods of use thereof

Abstract

The present disclosure provides compositions and methods for making and using engineered cells, such as engineered myeloid cells expressing chimeric fusion proteins having binding domains capable of binding to surface molecules on target cells, such as diseased cells.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 511,274, filed June 30, 2023; and U.S. Provisional Application No. 63 / 579,422, filed August 29, 2023; each of the aforementioned U.S. Provisional Applications is incorporated herein by reference in its entirety. Background Technology

[0003] Cellular immunotherapy is a promising new technology for combating difficult-to-treat diseases such as cancer, as well as certain diseases with persistent infections and resistance to other forms of treatment. The discovery of CAR-T cells and their potential use in immunotherapy has been a major breakthrough. CAR-T cells are T lymphocytes that express chimeric antigen receptors, which help target specific diseased cells, such as cancer cells, and can induce cytotoxic responses or immunosuppression and / or tolerance aimed at killing target cancer cells, depending on the intracellular domains employed and the co-expressed immunosuppressive cytokines. Although CAR-T cells remain a promising tool for cancer therapy, several limitations along the way have slowed progress and diminished their prospects in clinical trials.

[0004] Understanding the limitations of CAR-T cells is crucial for leveraging this technology and continuing to innovate towards better immunotherapy models. Specifically, CAR-T cells appear to face a major challenge in T-cell malignancies. CAR-T cells and malignant T cells share surface antigens in most T-cell lymphomas (TCLs), thus CAR-T cells are affected by cytotoxicity in the same way as cancer cells. In some cases, CAR-T products can be contaminated by malignant T cells. Additionally, T-cell hypoplasia is a potential problem due to the prolonged persistence of CAR-T cells. Other limitations include the poor ability of CAR-T cells to penetrate into solid tumors and the potent tumor microenvironment that downregulates their anti-tumor potential. CAR-T cell function is also negatively affected by the immunosuppressive tumor microenvironment (TME), which leads to the inactivation and exhaustion of endogenous T cells.

[0005] Myeloid cells, including macrophages, are cells derived from the myeloid lineage and belong to the innate immune system. They originate from bone marrow stem cells, which flow into the bloodstream and can migrate to tissues. Some of their key functions include phagocytosis, activation of T cell responses, and clearance of cellular debris and extracellular matrix. They also play important roles in maintaining homeostasis and in initiating and resolving inflammation. Furthermore, myeloid cells can differentiate into many downstream cell types, including macrophages, which can exhibit different responses ranging from pro-inflammatory to anti-inflammatory depending on the type of stimulus they receive from the surrounding microenvironment. In addition, tissue macrophages have been shown to play a broad role in regulating and activating other immune cell types, including CD8+ and CD4+ T effector cells, NK cells, and regulatory T cells. Macrophages have been shown to be major immune infiltrators in malignancies and have been shown to have broad immunosuppressive effects on effector immune infiltration and function. Summary of the Invention

[0006] The diverse functions of myeloid cells make them ideal candidates for cell therapy, and they can be engineered to have a variety of therapeutic effects. This disclosure relates to immunotherapy using myeloid cells of the immune system (e.g., CD14+ cells), particularly phagocytes. Myeloid cells can be used to consider many therapeutic indications. For example, myeloid cell immunotherapy may be extremely important in treating cancer, autoimmune diseases, fibrotic diseases, and infections. This disclosure relates to immunotherapy using myeloid cells, including phagocytes of the immune system, particularly monocytes. The object of the invention disclosed herein is to utilize one or more of these functions of myeloid cells for therapeutic purposes. For example, the object of the invention disclosed herein is to utilize the phagocytic activity of myeloid cells (including engineered myeloid cells) for therapeutic purposes. For example, the object of the invention disclosed herein is to utilize the ability of myeloid cells (including engineered myeloid cells) to promote T cell activation. For example, the object of the invention disclosed herein is to utilize the ability of myeloid cells (including engineered myeloid cells) to promote the secretion of tumoricidal molecules. For example, the object of the invention disclosed herein is to utilize the ability of myeloid cells (including engineered myeloid cells) to promote the recruitment and transport of immune cells and molecules. In one aspect, this disclosure provides novel and useful chimeric constructs that, when expressed in myeloid cells, can drive targeted attack and phagocytosis of molecules, molecular assemblies, objects, or cells containing targets (e.g., target antigens on their surface). One of the many aspects of this disclosure is (i) enhancing the phagocytic capacity of myeloid cells (e.g., engineered myeloid cells expressing novel and improved chimeric constructs); helping to initiate coordinated and sustained immune responses against targets (e.g., target antigens). This disclosure provides innovative methods and compositions that can successfully transfect or transduce myeloid cells, or otherwise induce genetic modifications in myeloid cells, with the aim of further enhancing functional aspects of myeloid cells without impairing their differentiation capacity, maturation potential, and / or plasticity. The resulting cells may be referred to as therapeutically effective engineered myeloid cells or effector myeloid cells. One strategy for improvement described herein is to induce an inflammatory phenotype in myeloid cells to develop effector myeloid cells. One strategy is to generate effector myeloid cells that produce an inflammatory phenotype when bound to a target.

[0007] This document provides a composition comprising a recombinant polynucleotide, wherein the recombinant polynucleotide contains a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (i) an extracellular domain comprising a TROP2 antigen-binding domain, (ii) a transmembrane domain operatively connected to the extracellular domain; and (iii) optionally an intracellular domain; wherein the transmembrane domain binds to the transmembrane domain of an endogenous FcR receptor when the mRNA is expressed in the cell; wherein the CFP encoded by the mRNA is expressed on the cell surface when the transmembrane domain encoded by the mRNA binds to the endogenous FcR receptor.

[0008] In some embodiments, the antigen-binding domain has HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the antigen-binding domains listed in Table 1A.

[0009] In some embodiments, the antigen-binding domain has a VH that has at least 80% sequence identity with the VH of the antigen-binding domain in Table 1A, and a VL that has at least 80% sequence identity with the VL of the antigen-binding domain in Table 1A.

[0010] In some embodiments, CFP further comprises a signal peptide.

[0011] In some embodiments, the signal peptide has a signal peptide sequence according to Table 1B.

[0012] In some embodiments, the extracellular domain comprises a sequence having at least 80% sequence identity with the sequence in Table 1C.

[0013] In some embodiments, the transmembrane domain comprises a sequence that has at least 80% sequence identity with the sequences in Table 1D.

[0014] In some embodiments, each of the at least two intracellular signal transduction domains has a sequence that has at least 80% sequence identity with the sequences in Table 2.

[0015] In some embodiments, the intracellular domain has a sequence that has at least 80% sequence identity with the sequences in Table 3.

[0016] In some embodiments, the CFP has a sequence that has at least 80% sequence identity with the sequences in Table 4.

[0017] This article also provides a composition comprising a recombinant polynucleotide, wherein the recombinant polynucleotide contains a sequence encoding a chimeric fusion protein (CFP), wherein the CFP has a sequence having at least 95% sequence identity with the sequences in Table 4.

[0018] In some embodiments, the CFP has a sequence that has at least 98%, 99%, or 100% sequence identity with the sequences in Table 4.

[0019] In some embodiments, the intracellular domain includes at least one additional intracellular signal transduction domain.

[0020] In some embodiments, the antigen-binding domain comprises an antibody or a fragment thereof.

[0021] In some embodiments, the antigen-binding domain includes scFv.

[0022] In some embodiments, the antigen-binding domain includes an extracellular domain, which includes a hinged domain connecting the antigen-binding domain and the transmembrane domain.

[0023] In some embodiments, the recombinant polynucleotide is mRNA.

[0024] In some embodiments, the recombinant polynucleotide is associated with one or more lipids.

[0025] In some embodiments, the recombinant polynucleotide is encapsulated in liposomes.

[0026] In some embodiments, liposomes are lipid nanoparticles.

[0027] This document provides a pharmaceutical product comprising a recombinant mRNA encoding a chimeric antigen receptor comprising: an anti-TROP2 binding scFv extracellular domain; and a CD89 transmembrane domain; wherein the pharmaceutical product is formulated in an aqueous formulation for systemic delivery. In some embodiments, the pharmaceutical composition comprises an scFv comprising a heavy chain and a light chain, the heavy chain comprising a CDR3 having the sequence GGFGSSYWYFDV, and the light chain comprising a CDR3 having the sequence QQHYITPLT. In some embodiments, the heavy chain further comprises a CDR1 sequence of NYGMN and a CDR2 sequence of WINTYTGEPTYTDDFKG; and the light chain further comprises a CDR1 sequence of KASQDVSIAVA and a CDR2 sequence of SASYRYT. The above pharmaceutical composition comprises a CD89 transmembrane domain having the sequence LIRMAVAGLVLVALLAILV.

[0028] In some embodiments, the recombinant polynucleotide comprises a vector.

[0029] This document also provides a composition comprising a nanoparticle delivery medium and a recombinant polynucleotide as described herein, wherein the recombinant polynucleotide is associated with or within the nanoparticle delivery medium.

[0030] In some embodiments, the transmembrane domain is a transmembrane domain derived from a protein that dimerizes with the endogenous FcRγ receptor in myeloid cells.

[0031] In some embodiments, the transmembrane domain includes a transmembrane domain derived from CD16a, CD64, CD68, or CD89.

[0032] In some embodiments, the recombinant polynucleotide is associated with or within a nanoparticle delivery medium, wherein the nanoparticle delivery medium comprises lipid nanoparticles or polymer nanoparticles.

[0033] In some embodiments, the recombinant polynucleotide is mRNA, and the mRNA is encapsulated by nanoparticles.

[0034] This document also provides a composition comprising cells containing the recombinant polynucleotides described herein.

[0035] In some embodiments, the cells are immune cells.

[0036] In some embodiments, the cells are myeloid cells, lymphoid cells, precursor cells, stem cells, or induced pluripotent cells.

[0037] In some embodiments, the cells are CD14+CD16- cells.

[0038] This document also provides a pharmaceutical composition comprising the above-described composition, and a pharmaceutically acceptable excipient.

[0039] This article also provides a method for treating cancer in a subject, the method comprising administering a pharmaceutical composition described herein to the subject.

[0040] On one hand, this article provides a pharmaceutical composition comprising a polynucleotide encoding a chimeric antigen receptor comprising: an anti-TROP2 binding scFv extracellular domain; and a CD89 transmembrane domain, the polynucleotide being formulated in an aqueous formulation for systemic delivery at a therapeutically effective amount and time interval suitable for treating a cancer in a subject. In some embodiments, the subject is a human subject. In some embodiments, the polynucleotide is engineered RNA. In some embodiments, the polynucleotide is engineered mRNA. In some embodiments, the anti-TROP2 binding scFv comprises a heavy chain and a light chain, wherein the heavy chain comprises a CDR3 having the sequence GGFGSSYWYFDV, and the light chain comprises a CDR3 having the sequence QQHYITPLT. In some embodiments, the heavy chain further comprises a CDR1 sequence of NYGMN and a CDR2 sequence of WINTYTGEPTYTDDFKG; and the light chain further comprises a CDR1 sequence of KASQDVSIAVA and a CDR2 sequence of SASYRYT.

[0041] In some embodiments, the CD89 transmembrane domain comprises the sequence LIRMAVAGLVLVALLAILV.

[0042] In some embodiments, the pharmaceutical composition further comprises a lipid nanoparticle delivery medium. In some embodiments, the lipid nanoparticle delivery medium comprises cationic lipids, non-cationic lipids, neutral lipids, PEGylated lipids, or combinations thereof. In some embodiments, the subject is a human subject. In some embodiments, the cancer is epithelial cancer. In some embodiments, the subject has epithelial cancer. In some embodiments, the cancer is selected from urothelial carcinoma, cervical cancer, ovarian epithelial cancer, triple-negative breast cancer, HR+ / HER2- breast cancer, pancreatic ductal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, non-small cell lung cancer, and colorectal cancer.

[0043] In some embodiments, the cancer is metastatic.

[0044] In some embodiments, the pharmaceutical composition is formulated for systemic delivery.

[0045] In some embodiments, the pharmaceutical composition is formulated for intravenous delivery.

[0046] In some embodiments, the pharmaceutical composition is formulated for intravenous injection or infusion.

[0047] In some embodiments, the therapeutically effective dose comprises 1-5000 micrograms per microliter of the engineered RNA.

[0048] In some embodiments, the pharmaceutical composition comprises, per dose of the pharmaceutical composition, the following amounts of the engineered RNA: about 0.0005 mg / kg to about 0.001 mg / kg, about 0.001 mg / kg to about 0.005 mg / kg, about 0.005 mg / kg to about 0.01 mg / kg, about 0.01 mg / kg to about 0.05 mg / kg, about 0.05 mg / kg to about 0.1 mg / kg, or about 0.1 mg / kg to about 0.5 mg / kg.

[0049] In some embodiments, the pharmaceutical composition comprises, per dose of the pharmaceutical composition, the following amounts of engineered RNA per kg of the subject's body weight (mg / kg): about 0.001 mg to about 0.0015 mg, about 0.0015 mg to about 0.002 mg, about 0.002 mg to about 0.0025 mg, about 0.0025 mg to about 0.003 mg, about 0.003 mg to about 0.0035 mg, about 0.0035 mg to about 0.004 mg, about 0.004 mg to about 0.0045 mg, about 0.0045 mg to about 0.005 mg, about 0.005 mg to about 0.0055 mg, about 0.0055 mg to about 0.006 mg, about 0.006 mg to about 0.0065 mg, about 0.0065 mg to about 0.007 mg, about 0.007 mg to about 0.0075 mg, ... mg to about 0.008 mg, about 0.008 mg to about 0.0085 mg, about 0.0085 mg to about 0.009 mg, about 0.009 mg to about 0.0095 mg, or about 0.0095 mg to about 0.01 mg.

[0050] In some embodiments, the pharmaceutical composition comprises, per dose of the pharmaceutical composition, the following amounts of the engineered RNA: about 0.01 mg / kg to about 0.015 mg / kg, about 0.015 mg / kg to about 0.02 mg / kg, about 0.02 mg / kg to about 0.025 mg / kg, about 0.025 mg / kg to about 0.03 mg / kg, about 0.03 mg / kg to about 0.035 mg / kg, about 0.035 mg / kg to about 0.04 mg / kg, about 0.04 mg / kg to about 0.045 mg / kg, about 0.045 mg / kg to about 0.05 mg / kg, about 0.05 mg / kg to about 0.055 mg / kg, about 0.055 mg / kg to about 0.06 mg / kg, about 0.06 mg / kg to about 0.065 mg / kg, about 0.065 mg / kg to about 0.07 mg / kg, about 0.07 mg / kg. mg / kg to about 0.075 mg / kg, about 0.075 mg / kg to about 0.08 mg / kg, about 0.08 mg / kg to about 0.085 mg / kg, about 0.085 mg / kg to about 0.09 mg / kg, about 0.09 mg / kg to about 0.095 mg / kg, or about 0.095 mg / kg to about 0.1 mg / kg.

[0051] In some embodiments, the pharmaceutical composition comprises, per dose of the pharmaceutical composition, the following amounts of the engineered RNA: about 0.001 mg / kg, about 0.0015 mg / kg, about 0.002 mg / kg, about 0.0025 mg / kg, about 0.003 mg / kg, about 0.0035 mg / kg, about 0.004 mg / kg, about 0.0045 mg / kg, about 0.005 mg / kg, about 0.0055 mg / kg, about 0.006 mg / kg, about 0.0065 mg / kg, about 0.007 mg / kg, about 0.0075 mg / kg, about 0.008 mg / kg, about 0.0085 mg / kg, about 0.009 mg / kg, about 0.0095 mg / kg, or about 0.01 mg / kg.

[0052] In some embodiments, the pharmaceutical composition comprises the engineered RNA in the following doses per dose of the pharmaceutical composition: about 0.01 mg / kg, about 0.015 mg / kg, about 0.02 mg / kg, about 0.025 mg / kg, about 0.03 mg / kg, about 0.035 mg / kg, about 0.04 mg / kg, about 0.045 mg / kg, about 0.05 mg / kg, about 0.055 mg / kg, about 0.06 mg / kg, about 0.065 mg / kg, about 0.07 mg / kg, about 0.075 mg / kg, about 0.08 mg / kg, about 0.085 mg / kg, about 0.09 mg / kg, about 0.095 mg / kg, or about 0.1 mg / kg.

[0053] In some embodiments, the pharmaceutical composition comprises, per dose of the pharmaceutical composition, the following amounts of the engineered RNA: at least about 0.001 mg / kg, at least about 0.0015 mg / kg, at least about 0.002 mg / kg, at least about 0.0025 mg / kg, at least about 0.003 mg / kg, at least about 0.0035 mg / kg, at least about 0.004 mg / kg, at least about 0.0045 mg / kg, at least about 0.005 mg / kg, at least about 0.0055 mg / kg, at least about 0.006 mg / kg, at least about 0.0065 mg / kg, at least about 0.007 mg / kg, at least about 0.0075 mg / kg, at least about 0.008 mg / kg, at least about 0.0085 mg / kg, at least about 0.009 mg / kg, at least about 0.0095 mg / kg, or at least about 0.01 mg / kg.

[0054] In some embodiments, the pharmaceutical composition comprises, per dose of the pharmaceutical composition, the following amounts of the engineered RNA: at least about 0.01 mg / kg, at least about 0.015 mg / kg, at least about 0.02 mg / kg, at least about 0.025 mg / kg, at least about 0.03 mg / kg, at least about 0.035 mg / kg, at least about 0.04 mg / kg, at least about 0.045 mg / kg, at least about 0.05 mg / kg, at least about 0.055 mg / kg, at least about 0.06 mg / kg, at least about 0.065 mg / kg, at least about 0.07 mg / kg, at least about 0.075 mg / kg, at least about 0.08 mg / kg, at least about 0.085 mg / kg, at least about 0.09 mg / kg, at least about 0.095 mg / kg, or at least about 0.1 mg / kg, or at least about 0.2 mg / kg, or at least about 0.3 mg / kg, or at least about 0.4 mg / kg. mg / kg, or at least about 0.5 mg / kg, or at least about 0.6 mg / kg, or at least about 0.7 mg / kg, or at least about 0.8 mg / kg, or at least about 0.9 mg / kg, or at least about 1 mg / kg.

[0055] In some embodiments, when stored in a container, the concentration of the engineered RNA in the pharmaceutical composition is between about 0.5 mg / mL and about 1.5 mg / mL, or between about 0.7 mg / mL and about 1.3 mg / mL.

[0056] In some embodiments, the container is a single-use or reusable vial.

[0057] In some embodiments, the total volume of the aqueous formulation is about 1.0 mL to about 3.0 mL, about 1.5 mL to about 2.5 mL, or about 2.0 mL.

[0058] In some embodiments, the dosing interval is 14 days for a total of 3 doses, followed by an interval of 28 days for a total of 3 doses.

[0059] On the one hand, this article provides a method for treating cancer in a subject, the method comprising: administering to the subject the pharmaceutical composition of any of the above embodiments.

[0060] In some embodiments, the subject is a subject who is over 18 years of age.

[0061] In some embodiments, the subject exhibits progressive disease at baseline, or exhibits refractory or recurrent disease in response to standard care.

[0062] In some embodiments, the subject is neither pregnant nor a sperm donor.

[0063] In some embodiments, the subject did not have CNS metastases or carcinomatous meningitis.

[0064] In some embodiments, the pharmaceutical composition or treatment method described herein includes reducing the tumor by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or at least 10% after treatment.

[0065] In some embodiments, the pharmaceutical composition or treatment method described herein includes the relief of at least one of the symptoms associated with the cancer.

[0066] In some embodiments, the pharmaceutical composition comprises engineered RNA encapsulated in lipid nanoparticles, wherein the engineered RNA contains a sequence encoding a chimeric antigen receptor (CAR), the CAR having: an extracellular antigen-binding domain comprising an scFv that binds to TROP2, the scFv having a heavy chain comprising a CDR1 sequence of NYGMN, a CDR2 sequence of WINTYTGEPTYTDDFKG, and a CDR3 sequence of GGFGSSYWYFDV; and the light chain further comprising a CDR1 sequence of KASQDVSIAVA. The drug composition contains the CDR2 sequence of SASYRYT and the CDR3 sequence of QQHYITPLT; a transmembrane domain and an intracellular domain derived from CD89, the transmembrane domain and the intracellular domain having the sequence DSIHQDYTTQNLIRMAVAGLVLVALLAILVENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK, wherein when administered at a dose of at least twice weekly for about 18 weeks, the drug composition has no dose-limiting toxicity (DLT) in human subjects of need at a dose of at least up to 0.03 mg / kg.

[0067] In some embodiments, the extracellular antigen-binding domain comprises the sequence QVQLQQSGSELKKPGASVKVSCKASGYTFT. NYGMN WVKQAPGQGLKWMG WINTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSSGGGGGSGGGGSGGGGSDIQLTQSPSSSLSASVGDRVSITC KASQDVSIA VAWYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLTF GAGTKVEIKR (SEQ ID NO: 3) has at least 90% identical sequences.

[0068] In some embodiments, the engineered CAR contains at least 95% identical sequences to the sequence of SEQ ID NO: 3.

[0069] In some embodiments, the engineered CAR contains at least 95% identical sequences to the sequence of SEQ ID NO: 3.

[0070] In some embodiments, the engineered CAR contains at least 99% identical sequences to the sequence of SEQ ID NO: 3.

[0071] In some embodiments, the engineered CAR contains at least 90% identical sequences to either of the sequences of SEQ ID NO: 26 and SEQ ID NO: 41.

[0072] In some embodiments, the engineered CAR contains at least 90% identical sequences to either of the sequences of SEQ ID NO: 27 and SEQ ID NO: 34.

[0073] In some embodiments, the engineered CAR contains at least 90% identical sequences to either of the sequences of SEQ ID NO: 28 and SEQ ID NO: 35.

[0074] In some embodiments, the engineered CAR contains at least 90% identical sequences to either of the sequences of SEQ ID NO: 29 and SEQ ID NO: 36.

[0075] In some embodiments, the engineered CAR contains at least 90% identical sequences to either of the sequences of SEQ ID NO: 30 and SEQ ID NO: 37.

[0076] In some embodiments, the engineered CAR contains at least 90% identical sequences to either of the sequences of SEQ ID NO: 31 and SEQ ID NO: 38.

[0077] In some embodiments, the engineered CAR contains at least 90% identical sequences to either of the sequences of SEQ ID NO: 32 and SEQ ID NO: 39.

[0078] In some embodiments, the engineered CAR contains at least 90% identical sequences to either of the sequences of SEQ ID NO: 33 and SEQ ID NO: 40.

[0079] In some embodiments, the engineered CAR contains at least 95% identical sequences to any of the sequences in SEQ ID NO: 26-41.

[0080] In some embodiments, the treatment method further comprises administering one or more therapeutic agents to the subject in combination with or in addition to administering the pharmaceutical composition.

[0081] In some embodiments, the administration of the one or more therapeutic agents comprises administering the therapeutic agent prior to administering the pharmaceutical composition.

[0082] In some embodiments, the administration of the one or more therapeutic agents includes administering the therapeutic agent simultaneously with the administration of the pharmaceutical composition.

[0083] In some embodiments, the administration of the one or more therapeutic agents comprises administering the therapeutic agent after administering the pharmaceutical composition.

[0084] In some embodiments, the method includes administering the drug composition to the subject for at least one cycle. In some embodiments, the method includes administering the drug composition to the subject for at least two, three, or four cycles. In some embodiments, during a first cycle, the drug composition is administered to the subject about once a week, about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, or about once every nine weeks. In some embodiments, the method includes administering the drug composition to the subject once a week or once every two weeks during the first cycle.

[0085] In some embodiments, the method includes administering the pharmaceutical composition to the subject at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least eleven times, or at least twelve times for each cycle.

[0086] In some embodiments, the method includes administering the pharmaceutical composition to the subject about once to about three times, about three times to about six times, about six times to about nine times, or about nine times to about twelve times for each cycle.

[0087] In some embodiments, the method includes administering the pharmaceutical composition to the subject up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times for each cycle.

[0088] In some embodiments, the method includes administering the drug composition to the subject between 1 and 12 times for each cycle.

[0089] In some embodiments, the method includes administering the drug composition to the subject between about 1 and about 3 times, about 3 and about 6 times, about 6 and about 9 times, or about 9 and about 12 times for each cycle. In some embodiments, the method includes administering the drug composition to the subject 3 times in a first cycle. In some embodiments, a second cycle follows the first cycle.

[0090] In some embodiments, the method further includes administering the pharmaceutical composition to the subject during the second cycle. In some embodiments, during the second cycle, the pharmaceutical composition is administered to the subject about once a week, about once every two weeks, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, or about once every nine weeks. In some embodiments, the method includes administering the pharmaceutical composition to the subject every four weeks during the second cycle. In some embodiments, the method includes administering the pharmaceutical composition to the subject at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least eleven times, or at least twelve times during the second cycle. In some embodiments, the method includes administering the pharmaceutical composition to the subject about once to about three times, about three times to about six times, about six times to about nine times, or about nine times to about twelve times during the second cycle. In some embodiments, the method includes administering the drug composition to the subject up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times during the second cycle. In some embodiments, the method includes administering the drug composition to the subject between 1 and 12 times during the second cycle. In some embodiments, the method includes administering the drug composition to the subject between about 1 and about 3 times, about 3 and about 6 times, about 6 and about 9 times, or about 9 and about 12 times during the second cycle. In some embodiments, the method includes administering the drug composition to the subject 3 times during the second cycle. In some embodiments, the method includes administering the drug composition, wherein the therapeutically effective dose comprises an amount of 0.01 mg / kg to 1 mg / kg at intervals of once every two weeks to once a week over a period of about 18 to about 50 weeks.

[0091] On one hand, this document provides a pharmaceutical composition comprising an engineered mRNA that includes a sequence encoding the sequence of SEQ ID NO: 41, and wherein the pharmaceutical composition comprises a dose of the engineered mRNA of about 0.005 mg / kg to about 0.15 mg / kg, wherein the dose is based on a subject's weight. In some embodiments, the pharmaceutical composition comprises a dose of the engineered mRNA of about 0.005 mg / kg, 0.015 mg / kg, 0.03 mg / kg, 0.06 mg / kg, 0.10 mg / kg, or 0.15 mg / kg. In some embodiments, the engineered mRNA is encapsulated in lipid nanoparticles.

[0092] On one hand, this article provides a method for treating cancer in a subject in need, the method comprising administering a pharmaceutical composition comprising an engineered mRNA at a dose of about 0.005 mg / kg to about 0.15 mg / kg, the engineered mRNA comprising a sequence encoding the sequence of SEQ ID NO: 41, wherein the dose is based on a measurement of the subject's weight.

[0093] In one embodiment, this document provides a method for treating cancer in a subject of need, the method comprising administering a pharmaceutical composition comprising an engineered mRNA at a dose of about 0.005 mg / kg to about 0.15 mg / kg, the engineered mRNA comprising a sequence encoding a sequence having at least 95% identity with the sequence of SEQ ID NO: 41, wherein the dose is based on a measurement of the subject's weight. In some embodiments, the engineered mRNA comprises a sequence encoding a sequence having at least 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 41, or a sequence without a signal peptide sequence. In some embodiments, the scFv VH and VL are derived from humanized murine antibodies. In some embodiments, the scFv is derived from a fully human antibody.

[0094] On the one hand, this article provides a method for treating cancer in a subject of need, the method comprising administering a pharmaceutical composition comprising an engineered mRNA at a dose of about 0.005 mg / kg to about 0.15 mg / kg, the engineered mRNA comprising a sequence encoding the sequence of SEQ ID NO: 26, wherein the dose is based on a measurement of the subject's weight; or a sequence having at least 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 26.

[0095] In some embodiments, the engineered mRNA is encapsulated in lipid nanoparticles. The lipid nanoparticles may contain a variety of lipids, which may generally be referred to herein as lipid 1 or lipid 2, etc., wherein at least one lipid is a polar lipid and another lipid is at least a nonpolar lipid.

[0096] In some embodiments, the dose of the engineered mRNA is about 0.005 mg / kg, 0.015 mg / kg, 0.03 mg / kg, 0.06 mg / kg, 0.10 mg / kg, or 0.15 mg / kg.

[0097] In some embodiments, the pharmaceutical composition is administered to the subject once every 7 days, once every 14 days, or once every 28 days.

[0098] In some embodiments, the pharmaceutical composition is administered to the subject for at least four cycles. In some embodiments, the pharmaceutical composition is administered at a dose of about 0.05 mg / kg, 0.15 mg / kg, or 0.03 mg / kg, wherein the pharmaceutical composition is administered every 14 days in cycle 1 and every 28 days in cycles 2-4. In some embodiments, the pharmaceutical composition is administered at a dose of about 0.03 mg / kg, wherein the pharmaceutical composition is administered every 7 days in cycle 1 and every 28 days in cycles 2-4.

[0099] In some embodiments, the pharmaceutical composition is administered at a dose of about 0.06 mg / kg, 0.10 mg / kg, or 0.15 mg / kg, and the pharmaceutical composition is administered every 14 days.

[0100] In some embodiments, the period includes at least 28 days, 42 days, or 56 days.

[0101] By incorporating via reference

[0102] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same degree, as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference. Attached Figure Description

[0103] The novel features of the invention are set forth in the appended claims. A better understanding of these features and advantages of the invention will be obtained by referring to the following detailed description of illustrative embodiments, which utilize the principles of the invention.

[0104] Figure 1An exemplary schematic view of a chimeric antigen receptor (CAR) peptide construct for in vivo delivery is depicted. The CAR includes a cancer cell-specific extracellular antigen-binding domain comprising an scFv that has antigen-binding specificity to a target cancer antigen (e.g., TROP2 antigen). The extracellular antigen-binding domain (e.g., containing the scFv) is operatively linked to a CD89 transmembrane domain (TM) and a CD89 cytoplasmic domain (Cyto). When the antigen-binding domain is a TROP2-binding domain, the chimeric protein illustrated in the figure is referred to as an anti-TROP2-CD89 CAR (interchangeably represented as an anti-TROP2 chimeric fusion protein (CFP)). The figure shows a cross-section of a cell membrane expressing the CAR peptide, wherein the CAR transmembrane domain is associated with an endogenous FcRγ chain that stabilizes its expression in the cell.

[0105] Figure 2 The inhibition of tumor growth in mice with gp75+ tumors following intravenous administration of a lipid nanoparticle (LNP) composition containing mRNA (mRNA-LNP) was described, wherein the mRNA encodes an anti-GP75-CD89 CAR. The anti-GP75-CD89 CAR is similar to [previous model] except that the extracellular anti-TROP2 domain is replaced by an anti-GP75 domain. Figure 1 The anti-TROP2-CD89 CAR is represented graphically. Both 0.5 mg / kg and 2 mg / kg showed tumor regression. The administration schedule of the LNP formulation containing mRNA expressing anti-GP75-CD89 CAR (anti-GP75-CD89 CAR-LNP) is indicated by arrows below the X-axis.

[0106] Figure 3 Depicting in Figure 1 The expression of anti-GP75CFP in tumors was observed after intravenous administration of the mRNA-LNP composition of the construct described herein.

[0107] Figures 4A-4C T cell activation in a mouse model of GP75 tumors was depicted. These mice had GP75+ tumors. Mice were administered an mRNA-LNP modulator in which the mRNA encodes an anti-GP75 CAR having an extracellular anti-GP75 domain operatively linked to both the CD89 transmembrane domain (TM) and the CD89 cytoplasmic domain (Cyto). Figure 4A (Left panel) shows a dot plot depicting activation and depletion (TIM3 / PD-1) of T cells isolated from mice after treatment, demonstrating an increase in cells expressing CAR from mice treated with the CAR construct, compared to empty LNPs, by flow cytometry, of triple positivity for the markers CD8+TIM3+ and PD1+. Figure 4A (Right) shows a bar chart of the data shown in the dot plot on the left. Figure 4B The diagram shows a dot plot (left) and a bar plot (right) depicting proliferating T cells (CD8+ T cells that are also positive for the Ki-67 proliferation marker) indicating immune activation following administration of the CAR construct. Figure 4C The diagram shows a dot plot (left) and a bar plot (right), which depict a greater number of cells with cytolytic activity, as indicated by higher positive staining for the marker granzyme B by flow cytometry.

[0108] Figures 5A-5E The expression of anti-TROP2 CFP in myeloid cells derived from isolated whole blood was depicted in a TROP2+ HCC-1954 subcutaneous xenograft model in NCG mice following intravenous infusion of a test composition (test composition) containing mRNA encoding anti-TROP2 CFP. Figure 5A Total Ly6C+ cells were depicted; Figure 5B Ly6C+ CD11b+ cells were depicted; Figure 5C Ly6C+ CD11c+ cells were depicted; and Figure 5D Ly6G+ cells were depicted. Figure 5E This is an experiment illustrating CAR expression in myeloid cell types. Parent = total cells isolated with the markers shown in the figure (e.g., CD11b+Ly6C+).

[0109] Figure 6 The mean tumor volume after administration of the mRNA-LNP composition in the TROP2+ HCC-1954 subcutaneous xenograft model of NSG mice was depicted.

[0110] Figure 7 Experiments demonstrating the antitumor activity of anti-TROP2 CAR mRNA-LNP in inhibiting tumor growth are shown.

[0111] Figure 8 The frequency of anti-TROP2 CFP in mononuclear cells following intravenous infusion of the composition in cynomolgus monkeys was depicted.

[0112] Figure 9 Tumor killing (left) and cytokine production in PBMCs transfected with the anti-TROP2-CD89 construct are depicted.

[0113] Figure 10A A schematic diagram depicts the design of a first-in-human clinical trial for an anti-TROP2-CD89-CAR LNP formulation for the treatment of epithelial cancer.

[0114] Figure 10BAn updated schematic diagram depicts the design of a first-in-human clinical trial for an anti-TROP2-CD89-CAR LNP formulation for the treatment of epithelial cancer.

[0115] Figure 11 A flowchart of the FIH experiment designed using BOIN is shown. Detailed Implementation

[0116] T-cell therapy has revolutionized cancer treatment for many patients. However, sustained clinical benefit has not yet been achieved for most patients with advanced solid tumors. Unlike T cells, myeloid cells tend to accumulate in tumors, sometimes comprising up to 50% of tumor mass. Myeloid cells can be specifically engineered to become highly effective anti-tumor cells, known as Activated, Targeted, Attack, and Kill (ATAK) cells, which specifically target, engulf, and lyse tumor cells and coordinate in vivo immune activation against them.

[0117] All terms are intended to be understood in the manner that is commonly understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0118] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0119] While various features of this disclosure may be described in the context of a single embodiment, these features may also be provided individually or in any suitable combination. Conversely, although this disclosure may be described herein in the context of a single embodiment for clarity, it may also be implemented in a single embodiment.

[0120] References to “some embodiments,” “one embodiment,” “an embodiment,” or “other embodiments” in the specification mean that the features, structures, or characteristics described in connection with the embodiments are included in at least some embodiments, but not necessarily all embodiments of this disclosure.

[0121] As used in this specification and claims, the terms “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of inclusion, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional elements or method steps not listed. It is to be considered that any embodiments discussed in this specification can be implemented with respect to any method or composition of this disclosure, and vice versa. Furthermore, the compositions of this disclosure can be used to implement the methods of this disclosure.

[0122] When referring to measurable values ​​such as parameters, quantities, or durations, the terms “about” or “approximately” as used herein mean to encompass + / -30% or less, + / -20% or less, + / -10% or less, + / -5% or less, or + / -1% or less of the specified value and variations from the specified value, provided that such variations are suitable for implementation in this disclosure. It should be understood that the values ​​referred to by the modifier “about” or “approximately” are themselves specifically disclosed.

[0123] "Pharmaceutical" can refer to any cell, small molecule chemical compound, antibody or fragment thereof, nucleic acid molecule or polypeptide.

[0124] "Change" or "transformation" can refer to an increase or decrease. For example, a change can be an increase or decrease of 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or 40%, 50%, 60%, or even up to 70%, 75%, 80%, 90%, or 100%. For example, a change can be an increase or decrease of 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, or even up to 70, 75, 80, 90, or 100 times.

[0125] As used herein, “antigen-presenting cells” or “APCs” include specialized antigen-presenting cells (e.g., B lymphocytes, macrophages, monocytes, dendritic cells, Langerhans cells) as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes, thymic epithelial cells, thyroid epithelial cells, glial cells (brain), pancreatic β cells, and vascular endothelial cells). APCs can express major histocompatibility complex (MHC) molecules and can display antigens complexed with MHC on their surface, which can be recognized by T cells and trigger T cell activation and immune responses. Specialized antigen-presenting cells, particularly dendritic cells, play a key role in stimulating primary T cells. Non-specialized antigen-presenting cells, such as fibroblasts, may also contribute to this process. APCs can also cross-present peptide antigens by processing exogenous antigens and presenting the processed antigens on class I MHC molecules. Antigens that produce proteins that associate with and are recognized by class I MHC molecules are typically proteins produced within cells, and these antigens are processed and associated with class I MHC molecules.

[0126] "Biological sample" can refer to any tissue, cell, fluid or other material derived from a living organism.

[0127] In some embodiments, a drug, such as a pharmaceutical product, therapeutic substance, or drug product, may be referred to by its pharmaceutical substance (DS), which is the component within the drug that causes the pharmaceutical effect; it may also be understood as the active pharmaceutical ingredient (API) of a pharmaceutical composition, for example, an engineered mRNA encoding a chimeric antigen receptor (CAR) is an API of a pharmaceutical composition, which further comprises LNPs and / or solutions (excipients), etc. The final composition administered may be referred to as a pharmaceutical product (DP).

[0128] The term "epitope" can refer to any protein determinant, such as sequence or structure or amino acid residues, capable of binding to an antibody or its binding fragment, a T-cell receptor, and / or antibody-like molecules. Epitope determinants typically consist of chemically active surface groups of molecules such as amino acids or sugar side chains, and usually possess specific three-dimensional structural characteristics and specific charge properties. "T-cell epitope" can refer to a peptide or peptide-MHC complex recognized by a T-cell receptor.

[0129] Engineered cells, such as engineered myeloid cells, can refer to cells that have at least one exogenous nucleic acid sequence in their cells, even if expressed transiently. Expression of exogenous nucleic acids can be performed by various methods described elsewhere and encompassing methods known in the art. This disclosure relates to the preparation and use of engineered cells, such as engineered myeloid cells, like engineered phagocytes. This disclosure particularly relates to an engineered cell containing exogenous nucleic acid encoding, for example, a chimeric antigen receptor (CAR) (which may be interchangeably referred to herein as a chimeric fusion protein (CFP)).

[0130] Engineered polynucleotides are polynucleotides that do not naturally exist in nature. They are generated using molecular biology techniques, such as cutting and joining segments of polynucleotides (e.g., DNA) from the same or different genes or nucleic acid fragments, where the segments originally existed in an orientation or organization different from that present in the engineered polynucleotide. Engineered polynucleotides can be plasmids, recombinant DNA, synthetic DNA, synthetic RNA, in vitro transcribed RNA, circularized RNA, etc.

[0131] The term "immune response" includes, but is not limited to, T-cell-mediated, NK-cell-mediated, and / or B-cell-mediated immune responses. These responses may be influenced by T-cell co-stimulation and NK-cell co-stimulation. Exemplary immune responses include T-cell responses, such as cytokine production and cytotoxicity. Additionally, immune responses include those indirectly influenced by NK-cell activation, B-cell activation, and / or T-cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells, such as macrophages. Immune responses include adaptive immune responses. The adaptive immune system can respond to foreign molecular structures, such as antigens invading an organism. Unlike the innate immune system, the adaptive immune system is highly specific to pathogens. Adaptive immunity can also provide durable protection. Adaptive immune responses include humoral immune responses and cell-mediated immune responses. In humoral immune responses, antibodies secreted into the body fluids by B cells bind to pathogen-specific antigens, leading to the elimination of pathogens through various mechanisms, such as complement-mediated lysis. In cell-mediated immune responses, T cells capable of destroying other cells are activated. For example, if disease-related proteins are present in cells, they can be fragmented into peptides by proteolysis within the cell. Specific cellular proteins can then attach themselves to antigens or peptides formed in this way and transport them to the cell surface, where they can be presented to molecular defense mechanisms such as T cells. Cytotoxic T cells can recognize these antigens and kill cells containing them.

[0132] "Ligand" can refer to a molecule capable of binding to or forming a complex with another molecule, such as a receptor. Ligands can include, but are not limited to, proteins, glycoproteins, carbohydrates, lipoproteins, hormones, fatty acids, phospholipids, or any component that binds to a receptor. In some embodiments, the receptor has a specific ligand. In some embodiments, the receptor can co-bind with ligands, in which case it can bind to several ligands that share at least similarity in structural conformation, charge distribution, or any other physicochemical properties. Ligands can be biomolecules. Ligands can be biological materials. For example, a ligand can be a negatively charged particle, which is a ligand for the scavenger receptor MARCO. For example, a ligand can be TiO2, which is a ligand for the scavenger receptor SRA1. In the context of CFPs described herein, an extracellular binding domain can bind to a ligand, which is also designated as a target of the binding domain. In some embodiments, the target is an antigen expressed on diseased cells (such as cancer cells), and the diseased cell is the target cell in this case in the sense that the target antigen bound by the extracellular antigen-binding domain of the CFP expressed on its cell surface. In this disclosure, the terms anti-(target) binding domain, anti-(target) binding extracellular domain, or anti-(target) CFP are generally used interchangeably with terms such as (target) binding domain, (target) binding extracellular domain, or (target) CFP. For example, HER2 expressed on cancer cells is the antigen (ligand) bound by the anti-HER2 binding extracellular domain of CFP; or alternatively, the antigen (ligand) bound by the HER2 binding extracellular domain of CFP may be referred to as the antigen (ligand) bound by the HER2 binding extracellular domain of CFP.

[0133] The terms "major histocompatibility complex (MHC)," "MHC molecule," or "MHC protein" can refer to proteins capable of binding to and presenting antigenic peptides to T lymphocytes. Such antigenic peptides can represent T cell epitopes. Human MHC is also known as the HLA complex. Therefore, the terms "human leukocyte antigen (HLA)," "HLA molecule," or "HLA protein" are used interchangeably with the terms "major histocompatibility complex (MHC)," "MHC molecule," and "MHC protein." HLA proteins can be classified into HLA class I or HLA class II. The proteins in both HLA classes are structurally very similar; however, they have very different functions. Class I HLA proteins are present on the surface of almost all cells in the body, including most tumor cells. Class I HLA proteins carry antigens, which are usually derived from endogenous proteins or pathogens present inside the cell, and are then presented to primary or cytotoxic T lymphocytes (CTLs). HLA class II proteins are present on antigen-presenting cells (APCs), including but not limited to dendritic cells, B cells, and macrophages. It primarily presents external antigen sources, such as peptides processed outside the cell, to helper T cells.

[0134] In the HLA class II system, phagocytes such as macrophages and immature dendritic cells can absorb proteins into phagosomes through phagocytosis. However, B cells exhibit a more general endocytosis into endosomes, which fuse with lysosomes, where acidic enzymes cleave the ingested proteins into many different peptides. Autophagy is another source of HLA class II peptides. The most studied subclass II HLA genes are: HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1.

[0135] HLA class II peptides presented to CD4+ helper T cells can generate an immune response to foreign antigens. Once activated, CD4+ T cells can promote B cell differentiation and antibody production, as well as CD8+ T cell (CTL) responses. CD4+ T cells can also secrete cytokines and chemokines, which activate and induce the differentiation of other immune cells. HLA class II molecules are typically heterodimers of α and β chains that interact to form a peptide-binding groove that is more open than that of class I peptides.

[0136] HLA alleles are typically expressed in a codominant manner. For example, each individual carries two alleles of each of the three class I genes (HLA-A, HLA-B, and HLA-C), and can therefore express six different types of class II HLA. Within the class II HLA loci, each individual inherits a pair of HLA-DP genes (DPA1 and DPB1, encoding the α and β chains), HLA-DQ (DQA1 and DQB1, targeting the α and β chains), one gene HLA-DRα (DRA1), and one or more genes HLA-DRβ (DRB1 and DRB3, -4, or -5). For example, HLA-DRB1 has over 400 known alleles. This means that a heterozygous individual can inherit six or eight functional class II HLA alleles: three or more from each parent. Therefore, HLA genes are highly polymorphic; many different alleles exist among different individuals within a population. Genes encoding HLA proteins have many possible variations, allowing each person's immune system to respond to a variety of foreign invaders. Some HLA genes have hundreds of identified versions (alleles), each assigned a specific number. In some embodiments, class I HLA alleles are HLA-A*02:01, HLA-B*14:02, HLA-A*23:01, and HLA-E*01:01 (non-classical). In some embodiments, class II HLA alleles are HLA-DRB*01:01, HLA-DRB*01:02, HLA-DRB*11:01, HLA-DRB*15:01, and HLA-DRB*07:01.

[0137] "Myeloid cells" can be broadly defined to refer to cells of the myeloid lineage of the hematopoietic system, excluding, for example, lymphocyte lineages. Myeloid cells include, for example, cells of the granulocyte and monocyte lineages. Myeloid cells are the main cellular compartment of the immune system, comprising monocytes, dendritic cells, tissue macrophages, and granulocytes. In recent years, models of myeloid cell cytogenesis, activation, differentiation, and tissue-specific functions have been re-examined with surprising results. However, their enormous plasticity and heterogeneity during both homeostasis and disease remain not fully understood. Despite the many functions of myeloid cells, including phagocytosis and their ability to activate T cells, the therapeutic uses of these functions remain difficult to understand. Therefore, it is necessary to explore new avenues for developing improved therapeutics using other cell types, including but not limited to T-cell malignancies.

[0138] Myeloid cells are common progenitor cells derived from hematopoietic stem cells in the bone marrow. Commitment to the myeloid cell lineage can be controlled by the activation of various transcription factors, and therefore myeloid cells can be characterized as cells with a certain level of plasticity, which can be described as the ability to further differentiate into terminal cell types based on extracellular and intracellular stimuli. Myeloid cells can be rapidly recruited to local tissues via various chemokine receptors on their surface. Myeloid cells respond to a variety of cytokines and chemokines.

[0139] For example, myeloid cells can be cells derived from bone marrow stem cells 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, myeloid cells are progenitor cells. In some embodiments, myeloid cells can be cells possessing common myeloid progenitor cells or granulocyte progenitor cells, myeloblasts or monocyte-dendritic cell progenitor cells, or combinations thereof. Myeloid cells can include granulocytes or monocytes or their precursor cells. Myeloid cells can include immature granulocytes, immature monocytes, immature macrophages, immature neutrophils, and immature dendritic cells. Myeloid cells can include monocytes or pre-monocytes or monocyte precursors. In some cases, as used herein, myeloid cells can refer to monocytes with M0, M1, or M2 phenotypes. Myeloid cells can include dendritic cells (DCs), mature DCs, monocyte-derived DCs, plasmacytoid DCs, pre-dendritic cells, or precursors of DCs. Myeloid cells can also include neutrophils, which can be mature neutrophils, neutrophil precursors, or polymorphonuclear cells (PMNs). Myeloid cells can also include macrophages, monocyte-derived macrophages, tissue macrophages, and macrophages with M0, M1, or M2 phenotypes. Monocytes or macrophages exhibit polarization. As used herein, “polarization” can refer to the process by which macrophages exhibit different functional phenotypes in response to specific microenvironmental stimuli and signals, often referred to as physiological states. In some cases, macrophages can transition from one polarization state to another. For example, macrophages can be polarized into classically activated (M1) and alternatively activated (M2) macrophages. M2 macrophages are further subdivided into M2a, M2b, M2c, and M2d subclasses. These macrophages differ in their cell surface markers, secreted cytokines, and biological functions. M1 macrophages are typically characterized by the expression of TLR-2, TLR-4, CD80, CD86, iNOS, and MHC-II on their cell surface. These cells release various cytokines and chemokines, such as TNF-α, IL-1α, IL-1β, IL-6, IL-12, CXCL9, and CXCL10, and generally exhibit activation of transcription factors that regulate the expression of M1 genes, such as NF-κB, STAT1, STAT5, IRF3, and IRF5. NF-κB and STAT1 are believed to be two major pathways involved in M1 macrophage polarization. The M1 phenotype is associated with macrophages' antimicrobial and antitumor functions, exhibiting high phagocytic and inflammatory activity. On the other hand, tumor-associated macrophages typically become more M2 polarized under immunosuppressive conditions. Myeloid macrophages can include tumor-infiltrating monocytes (TIMs).Myeloid cells can include tumor-associated monocytes (TAMs). Myeloid cells can include myeloid-derived suppressor cells (MDSCs). Myeloid cells can include tissue-resident macrophages. Myeloid cells can include tumor-associated dendritic cells (TADCs). Therefore, myeloid cells can express one or more cell surface markers, such as CD11b, CD14, CD15, CD16, CD38, CCR5, CD66, Lox-1, CD11c, CD64, CD68, CD163, CCR2, CCR5, HLA-DR, CD1c, CD83, CD141, CD209, MHC-II, CD123, CD303, CD304, SIGLEC family proteins, and CLEC family proteins. In some cases, myeloid cells may be characterized by high or low expression of one or more cell surface markers, such as CD11b, CD14, CD15, CD16, CD66, Lox-1, CD11c, CD64, CD68, CD163, CCR2, CCR5, HLA-DR, CD1c, CD83, CD141, CD209, MHC-II, CD123, CD303, CD304, or combinations thereof. In one embodiment, it is desirable to activate M1 polarization in macrophages using the methods described herein.

[0140] "Phagocytosis" and "phagocytosis" are used interchangeably and can refer to the process by which cells engulf particles such as cancer cells or infected cells. This process can create internal compartments (phagosomes) containing particles. This process can be used to take up particles from the body and / or remove particles such as cancer cells or infected cells. Phagocytic receptors can be involved in the process of phagocytosis. The process of phagocytosis can be closely linked to immune responses and antigen presentation. The processing of exogenous antigens follows their uptake into specialized antigen-presenting cells through some type of endocytosis. Phagocytosis can also promote antigen presentation. For example, antigens or pathogens (including cancer antigens) from phagocytosed cells can be processed and presented on the cell surface of APCs.

[0141] A "peptide" can refer to a molecule containing amino acids linked together by peptide bonds, such as glycoproteins, lipoproteins, cellular proteins, or membrane proteins. A peptide can contain one or more subunits of a protein. A peptide can be encoded by a recombinant nucleic acid. In some embodiments, a peptide can contain more than one peptide sequence in a single amino acid chain, which can be separated by spacers, linkers, or peptide cleavage sequences. A peptide can be a fused peptide. A peptide can contain one or more domains, modules, or portions.

[0142] In some embodiments, a drug under investigation can be evaluated through pharmacokinetic (PK) and pharmacodynamic (PD) studies (pharmacokinetics). Such studies typically include investigating how a drug enters, is processed, and leaves the metabolic system, including but not limited to its absorption by different tissues, its accumulation in tissues or organs, and its excretion, the rate at which it passes through tissues or organs or biological systems.

[0143] A "receptor" can refer to a chemical structure composed of a polypeptide that transduces signals, such as polypeptides that transduce extracellular signals into cells. Receptors can be used to transmit information in cells, cell formation, or organisms. A receptor contains at least one receptor unit and may contain two or more receptor units, each of which contains a protein molecule, such as a glycoprotein molecule. Receptors may contain structures that bind to ligands and can form complexes with ligands. Signal transduction information can be transmitted through conformational changes in the receptor following binding to a ligand on the cell surface.

[0144] The term "antibody" can refer to a class of proteins commonly known as immunoglobulins, including but not limited to IgG1, IgG2, IgG3 and IgG4, IgA (including IgA1 and IgA2), IgD, IgE, IgM, and IgY. The term "antibody" includes, but is not limited to, full-length antibodies, single-chain antibodies, single-domain antibodies (sdAbs), and their antigen-binding fragments. Antigen-binding antibody fragments include, but are not limited to, Fab, Fab', and F(ab')2, Fd (composed of V...). H and C H 1. Composition), single-chain variable fragment (scFv), single-chain antibody, disulfide-linked variable fragment (dsFv), and V L and / or V H Fragments of structural domains. Antibodies can be derived from any animal source. Antigen-binding antibody fragments, including single-chain antibodies, may contain variable regions, alone or in combination with one or more of the hinge region, CH1 domain, CH2 domain, and CH3 domain. Any combination of variable regions and hinge regions, CH1, CH2, and CH3 domains is also included. Antibodies can be, for example, monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies that specifically bind to HLA-associated peptides or HLA-peptide complexes.

[0145] The term "recombinant nucleic acid" refers to nucleic acids prepared, expressed, produced, or isolated through recombination. Recombinant nucleic acids may contain nucleotide sequences that are not naturally occurring. Recombinant nucleic acids can be synthesized in a laboratory. Recombinant nucleic acids can be prepared using recombinant DNA techniques, such as enzymatic modifications of DNA (e.g., enzymatic restriction digestion, ligation, and DNA cloning). Recombinant nucleic acids can be DNA, RNA, analogs thereof, or combinations thereof. Recombinant DNA can be transcribed in vitro or in vitro to produce messenger RNA (mRNA). Recombinant mRNA can be isolated, purified, and used for transfection of cells. Recombinant nucleic acids can encode proteins or polypeptides. Throughout this specification, nucleic acid sequences are described that may comprise deoxyribonucleotides (DNA), ribonucleotides (RNA), or, in some embodiments, modified deoxyribonucleotides or modified ribonucleotides. For example, modified nucleotides may be 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 7-methylguanosine, pseudouridine, dihydrouridine, etc. Those skilled in the art can readily determine RNA sequences, such as mRNA sequences, from a given polynucleotide sequence. The sequence can be codon-optimized.

[0146] The process of introducing or incorporating nucleic acids into cells can be achieved through transformation, transfection, or transduction. Transformation is the process by which bacterial cells take up foreign nucleic acids. This process is suitable for the propagation of plasmid DNA, protein production, and other applications. Transformation introduces recombinant plasmid DNA into competent bacterial cells that have taken up extracellular DNA from the environment. Some bacterial species are naturally competent under certain environmental conditions, but competence is artificially induced in a laboratory setting. Transfection is the introduction of small molecules such as DNA, RNA, or antibodies into eukaryotic cells. Transfection can also refer to the introduction of bacteriophages into bacterial cells. 'Transduction' is primarily used to describe the introduction of recombinant viral vector particles into target cells, while 'infection' can refer to the natural infection of humans or animals with wild-type viruses.

[0147] The term "vector" can refer to a nucleic acid molecule capable of autonomous replication in a host cell and allowing for the cloning of nucleic acid molecules. As known to those skilled in the art, vectors include, but are not limited to, plasmids, granules, bacteriophages, viral vectors, phage vectors, yeast vectors, mammalian vectors, etc. For example, a vector for the transformation of exogenous genes can be a plasmid. In some embodiments, a vector contains a nucleic acid sequence containing an origin of replication and other elements necessary for replication and / or maintenance of the nucleic acid sequence in a host cell. In some embodiments, the vectors or plasmids provided herein are expression vectors. Expression vectors are capable of directing the expression of genes and / or nucleic acid sequences operatively linked thereto. In some embodiments, expression vectors or plasmids are in the form of circular double-stranded DNA molecules. Vectors or plasmids may or may not be integrated into the genome of a host cell. In some embodiments, the nucleic acid sequence of the plasmid is not integrated into the genome or chromosome of the host cell after introduction. For example, a plasmid may contain elements for transient or stable expression of a nucleic acid sequence in a host cell, such as genes or open reading frames contained in the plasmid. In some embodiments, the vector is a transient expression vector. In some embodiments, the vector is a stable expression vector that replicates autonomously in a host cell. In some embodiments, the nucleic acid sequence of the plasmid is integrated into the host cell's genome or chromosome upon introduction into the host cell. Expression vectors that can be used in the methods disclosed herein include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, or viral vectors. The vector can be a DNA or RNA vector. In some embodiments, the vectors provided herein are RNA vectors, such as retroviral or lentiviral vectors, that are capable of integrating into the host cell's genome upon introduction into the host cell (e.g., by reverse transcription). Other forms of expression vectors with equivalent functionality known to those skilled in the art may also be used, such as self-replicating extrachromosomal vectors or vectors capable of integrating into the host genome. Exemplary vectors are those capable of autonomously replicating and / or expressing nucleic acids linked thereto.

[0148] In some embodiments, nucleic acids can be delivered to a living system in the form of nanoparticles. The nucleic acid sequences disclosed herein can be delivered in vivo via suitable nanoparticles (e.g., liposomes, lipid nanoparticles, or polymer nanoparticles). Lipid nanoparticles may comprise polar lipids. In some embodiments, lipid nanoparticles comprise cationic lipids. In some embodiments, lipid nanoparticles comprise cationic and non-cationic lipids. In some embodiments, lipid nanoparticles comprise neutral lipids. In some embodiments, lipid nanoparticles comprise PEGylated lipids.

[0149] Alternatively, in some embodiments, nucleic acids can be electroporated in vitro within living cells to prepare cell therapies, wherein the cells are myeloid cells.

[0150] In some embodiments, the term "spacer" or "linker" as used herein with reference to fusion proteins may refer to a peptide sequence that connects two other peptide sequences of the fusion protein. In some embodiments, the linker or spacer has no specific biological activity other than connecting or preserving some minimum and / or maximum distance or imparting some other spatial relationship between two or more peptide sequences of the fusion protein. In some embodiments, the constituent amino acids of the spacer may be selected to influence some properties of the fusion protein molecule, such as folding, flexibility, net charge, or hydrophobicity. Suitable linkers for embodiments of this disclosure are well known to those skilled in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. In some embodiments, the linker is used to separate two or more polypeptides, such as two antigenic peptides, by a distance sufficient to ensure that each antigenic peptide folds correctly. Exemplary peptide linker sequences employ a flexible extended conformation and do not exhibit a tendency to form ordered secondary structures. The amino acids in the flexible linker protein region may include Gly, Asn, and Ser, or any arrangement of amino acid sequences containing Gly, Asn, and Ser. Other near-neutral amino acids such as Thr and Ala may also be used in the linker sequence.

[0151] In some aspects, this disclosure provides a method and composition for treating a disease or condition, wherein treatment and its grammatical variations can refer to the administration of a composition (e.g., a pharmaceutical composition) for alleviating, preventing, or improving a condition and / or symptom associated therewith (e.g., tumor formation or tumor, infectious agent, or autoimmune disease). "Treatment" can mean administering a therapy to a subject after the onset or suspected onset of a disease (e.g., cancer, or infection with an infectious agent or autoimmune disease). "Treatment" can include the concept of "remission," which can refer to reducing the frequency or severity of the occurrence or recurrence of at least one symptom or adverse reaction associated with the disease and / or side effects related to the therapy. Treatment can also encompass the concept of "management," which can refer to reducing the severity of a patient's disease or condition, such as prolonging the lifespan of a patient with said disease or increasing their survival rate, or delaying its recurrence, such as prolonging progression-free survival of a patient with a disease such as cancer. It should be understood that, although not excluded, treating a condition or symptom does not require the complete elimination of the associated condition, symptom, or symptom. In some embodiments, the terms “prevent,” “preventing,” “prevention,” and their grammatical equivalents as used herein can refer to avoiding or delaying the onset of such symptoms in a subject who has not yet developed symptoms associated with a disease or symptom at the start of administration of a drug or compound. In some embodiments, treating a subject or patient as described herein comprises administering a therapeutic composition, such as a drug, metabolite, preventative component, nucleic acid, peptide, or protein encoding or otherwise forming a drug, metabolite, or preventative component. In some embodiments, treatment comprises administering cells or cell populations to a subject in need. In some embodiments, treatment comprises administering one or more of the engineered cells described herein, such as one or more engineered myeloid cells, such as phagocytes, to a subject. Treatment comprises treating a disease or symptom or syndrome, which may be a pathological disease, symptom, or syndrome, or an underlying disease, symptom, or syndrome. In some embodiments, treatment may comprise administering a therapeutic agent, such as a vaccine. In some embodiments, engineered phagocytes are administered to a patient or subject. In some embodiments, cells administered to a human subject result in reduced immunogenicity. For example, engineered phagocytes may not cause or may reduce graft-versus-host disease (GVHD) or fratricide effects. In some embodiments, the engineered cells administered to a human subject are immunocompatible with the subject (i.e., having a matching HLA subtype naturally expressed in the subject). The subject-specific HLA alleles or HLA genotype of the subject can be determined by any method known in the art.In an exemplary embodiment, the method includes determining a polymorphic gene type, which may include generating alignments of reads extracted from a sequencing dataset with a gene reference set containing allelic variants of the polymorphic gene; determining a first post-probability or post-probability derived score for each allelic variant in the alignment; identifying an allelic variant with the highest first post-probability or post-probability derived score as a first allelic variant; identifying one or more overlapping reads aligned with the first allelic variant and one or more other allelic variants; determining a second post-probability or post-probability derived score for the one or more other allelic variants using a weighting factor; identifying a second allelic variant by selecting an allelic variant with the highest second post-probability or post-probability derived score; the first allelic variant and the second allelic variant defining the gene type of the polymorphic gene; and providing outputs for the first allelic variant and the second allelic variant.

[0152] In some embodiments, a “fragment” may refer to a portion of a protein or nucleic acid. In some embodiments, the fragment retains at least 50%, 75%, or 80%, or 90%, 95%, or even 99% of the biological activity of a reference protein or nucleic acid.

[0153] The terms “isolated,” “purified,” “biologically pure,” and their grammatical equivalents generally refer to materials found to varying degrees in their natural state and typically accompanied by components. “Isolated” indicates the degree of separation from the original source or surrounding environment. “Purified” indicates a degree of separation beyond isolation. A “purified” or “biologically pure” protein is sufficiently free of other material such that any impurities do not materially affect the protein’s biological properties or cause other adverse consequences. That is, the nucleic acid or peptide of this disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” can indicate that the nucleic acid or protein produces essentially one band in the electrophoresis gel. For proteins that can be modified, such as phosphorylated or glycosylated, different modifications can produce different isolated proteins, which can be purified individually.

[0154] In some embodiments, "tumor formation" or "cancer" can refer to any disease caused or resulting from an inappropriately high level of cell division, an inappropriately low level of cell apoptosis, or both. Glioblastoma is a non-limiting example of tumor formation or cancer. The terms "cancer," "tumor," or "hyperproliferative disorder" refer to the presence of cells with typical characteristics of cancerous cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and certain characteristic morphological features. Cancer cells typically take the form of tumors, but such cells can exist alone in an animal or can be non-tumorigenic cancer cells such as leukemia cells.

[0155] In some embodiments, "vaccine" can be understood to mean a composition for generating immunity for the prevention and / or treatment of a disease (e.g., tumor formation / tumor / infectious agent / autoimmune disease). Thus, a vaccine, as used herein, is a medicine comprising recombinant nucleic acids or cells comprising and expressing recombinant nucleic acids, and is intended for use in humans or animals to generate specific defensive and protective substances through vaccination. A "vaccine composition" may include pharmaceutically acceptable excipients, carriers, or diluents. Aspects of this disclosure relate to the use of techniques in the preparation of phagocyte-based vaccines.

[0156] In some embodiments, "pharmaceuticalally acceptable" may mean an animal approved or recognized by a federal or state regulatory agency, or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in humans. In some embodiments, "pharmaceuticalally acceptable excipient, carrier, or diluent" may mean an excipient, carrier, or diluent that can be administered to a subject together with the pharmaceutical agent and that does not destroy its pharmacological activity and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the pharmaceutical agent.

[0157] Nucleic acid molecules that can be used in the methods of this disclosure include, but are not limited to, any nucleic acid molecule having activity or encoding a polypeptide. Polynucleotides having substantially the same sequence identity as the endogenous sequence are generally capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. In some embodiments, “hybridization” can refer to when nucleic acid molecules pair up under various stringent conditions to form a double-stranded molecule between complementary polynucleotide sequences or portions thereof. (See, for example, Wahl, GM and SL Berger (1987) *Methods Enzymol.* 152:399; Kimmel, AR (1987) *Methods Enzymol.* 152:507). For example, stringent salt concentrations can typically 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-tightness hybridization can be achieved in the absence of organic solvents (e.g., formamide), while high-tightness hybridization can be achieved in the presence of at least about 35% or at least about 50% formamide. Tight temperature conditions typically include temperatures of at least about 30°C, at least about 37°C, or at least about 42°C. Various other parameters, such as hybridization time, detergent concentration (e.g., sodium dodecyl sulfate (SDS)), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Different levels of tightness can be achieved by combining these different conditions as needed. In an exemplary embodiment, hybridization can be performed at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another exemplary embodiment, hybridization can be performed at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 µg / ml denatured salmon sperm DNA (ssDNA). In another exemplary embodiment, hybridization can be performed at 42°C with 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 µg / ml ssDNA. Useful variations of these conditions will be apparent to those skilled in the art. For most applications, the washing step following hybridization may also vary in terms of stringency. Wash stringency conditions can be defined by salt concentration and temperature. As described above, wash stringency can be increased by decreasing the salt concentration or by increasing the temperature. For example, the stringent salt concentration used for the washing step 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. The stringent temperature conditions used for the washing step can include temperatures of at least about 25°C, at least about 42°C, or at least about 68°C.In an exemplary embodiment, the washing step may be performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In other exemplary embodiments, the washing step may be performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In yet another exemplary embodiment, the washing step may be performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations of these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in the following literature: Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proceedings of the National Academy of Sciences 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).

[0158] In some embodiments, "substantially identical" can refer to a polypeptide or nucleic acid molecule that exhibits at least 50% identity with a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Such sequences may be at least 60%, 80%, or 85%, 90%, 95%, 96%, 97%, 98%, or even 99% or more identical at the amino acid level or nucleic acid level to the sequence used for comparison. Sequence identity is typically measured using sequence analysis software, such as the sequence analysis packages BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs from the Genetic Computing Group at the University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705. Such software matches identical or similar sequences by specifying the degree of homology for various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions from the following group: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In exemplary methods for determining the degree of identity, a BLAST procedure can be used, where a probability score between e-3 and em indicates closely related sequences. In some embodiments, a “reference” is a comparison standard. It should be understood that the numbering of a particular position or residue in the corresponding sequence depends on the specific protein and numbering scheme used. For example, numbering may differ in mature proteins and precursors of the mature protein itself, and sequence differences from species may affect numbering. Those skilled in the art will be able to identify any homologous protein and corresponding residues in the corresponding encoding nucleic acid using methods well-known in the art, such as sequence alignment with a reference sequence and determination of homologous residues.

[0159] The terms “subject” or “patient” can refer to an organism such as an animal (e.g., a human) used for therapeutic, observational, or experimental purposes. By way of example only, subjects include, but are not limited to, mammals, including, but not limited to, non-human primates, rats, cattle, horses, dogs, sheep, or cats, and other human or non-human mammals.

[0160] In some embodiments, a therapeutic effect may be noted or anticipated, which may refer to the degree of relief of one or more symptoms associated with a condition (e.g., tumor formation, infection with an infectious agent, or an autoimmune disease), or in some way a measurable effect related to disease-related pathology, such as viral titers in biological samples from subjects treated with antiviral therapeutic agents. In one embodiment, a therapeutic effect may indicate a reduction in symptoms of the disease after administration of the therapy to a subject, such as a 5%, 10%, 20%, 30%, or other reduction in tumor mass after administration of the therapeutic composition. In another embodiment, a therapeutic effect may involve partial or complete relief of one or more symptoms or improvement of the disease. A therapeutically effective amount, as used herein, may refer to an amount of an agent, such as a pharmaceutical composition, that is effective at a time following administration, i.e., upon single or repeated (e.g., dose) administration, showing an indication of reduction or improvement of symptoms or parameters associated with the disease to which the therapeutic agent is administered. For example, a therapeutically effective amount may be an amount associated with prolonged survival in patients with the condition. In some embodiments, a therapeutically effective amount that reduces one or more signs or symptoms of the condition, prevents or delays the onset of the condition, etc., exceeds an amount expected in the absence of such treatment. Therapeutic effective doses are typically designed to quantify the amount required to achieve a therapeutic effect. Such therapeutic effects can be closely related to dosage (the amount of the therapeutic agent), schedule, or treatment regimen, and can vary from one subject to another. A schedule or treatment regimen can be understood as the frequency of administration of the therapeutic agent within a given time interval, such as once daily, once weekly, once every two weeks, etc., combined with the total duration of treatment administration. Thus, an example of a treatment regimen might involve administering a therapeutic agent (e.g., a pharmaceutical composition) at a dosage (e.g., an amount) at seven-day intervals for a duration of six months (e.g., a total period), during which or after which a therapeutic effect can be noticed or anticipated. For example, the therapeutic effect of epithelial cancer could be a reduction in cancerous lesions on the epithelial tissue. A skilled physician or veterinarian can easily determine and prescribe the required “therapeutic effective dose” (e.g., ED50) of the pharmaceutical composition.

[0161] This article provides engineered myeloid cells (including, but not limited to, neutrophils, monocytes, myeloid dendritic cells (mDCs), mast cells, and macrophages) designed to specifically bind to target antigens. Target antigens may be expressed only on target cells, such as infected cells, damaged cells, malignant cells, leukemia cells, or tumor cells. Therefore, these engineered myeloid cells can directly (e.g., through phagocytosis) and / or indirectly (e.g., through T cell activation) attack and kill target cells. In some embodiments, the target cells are cancer cells.

[0162] While cancer is one exemplary embodiment described in detail in this disclosure, the methods and techniques described herein are conceivable for targeting infected or otherwise diseased cells in vivo. Similarly, therapeutic compositions and vaccine compositions using engineered cells are described herein.

[0163] Myeloid effector cells can be generated from isolated myeloid cells derived from human biological samples and modified in vitro to prepare therapeutically desired cells using methods that engineer such cells, ensuring that the modifications do not alter the plasticity of these cells. Monocyte lineage cells are phagocytic and highly efficient antigen-presenting cells. In one aspect, this invention stems from the important discovery that engineered myeloid cells can be a highly effective therapeutic approach for treating many diseases, including cancer. Myeloid cells can be engineered to express chimeric antigen receptors that enhance the immune function of myeloid cells, where the cells are highly phagocytic and capable of attacking and killing diseased or infected cells in the body. The chimeric antigen receptor is a recombinant construct designed and specifically modified as described herein (a) highly targeted, specifically directed to bind to a target antigen, having an extracellular antigen-binding domain, and (b) an intracellular domain highly specialized to activate myeloid cells to achieve an activated phagocytic phenotype. For example, highly specialized intracellular domains are designed to generate chimeric receptors that, upon activation by binding to a target via the receptor's extracellular region, can generate intracellular signaling cues that activate the intracellular interferon signaling cascade, as well as transcription factors, specifically IRFs (internal radiofrequency receptors) that guide the activation of IFN regulators. Additionally, the methods and compositions described herein are also used in gene therapy, in which a recombinant nucleic acid encoding a chimeric antigen receptor is administered locally or systemically to a subject in need, resulting in specific expression of the recombinant nucleic acid in myeloid cells in vivo, thereby generating activated myeloid cells with therapeutic capacity. In some embodiments, the nucleic acid is mRNA. In some embodiments, the mRNA is delivered in an LNP.

[0164] Bacteriophages are typically designated as natural sentinels of the immune system, forming the first line of defense in the body. They engulf pathogens, pathogen-infected cells, foreign bodies, or cancer cells and remove them from the body. Most potential pathogens are rapidly neutralized by this system before they can cause, for example, an obvious infection or disease. This can involve receptor-mediated uptake via clathrin-coated pit systems, pinocytosis, and especially macropinocytosis resulting from membrane folding and phagocytosis. Therefore, phagocytes can be activated by a variety of non-self (and self) components and exhibit a degree of plasticity in recognizing their “targets.” Most phagocytes express scavenger receptors on their surface, which are pattern recognition molecules and can bind to a wide range of foreign particles, as well as dead cells, debris, and unwanted particles in the body. On the other hand, recombinant nucleic acids encoding chimeric antigen receptors (CARs) can be expressed in cells. CARs can be engineered to attack specific tumor cells, and myeloid effector cells expressing CARs can be activated to phagocytose and kill tumor cells. CARs can be engineered to produce phagocytic receptors that are specifically activated in response to target binding, and the phagocytic potential of macrophages is enhanced by specifically engineered intracellular domains of the receptors. The myeloid CAR platform described herein is engineered such that no tetanic signaling is detected in myeloid cells at any time before administration in vivo or prior to binding of the CAR to its target. This is typically tested in vitro. Simultaneously, CAR-expressing myeloid cells can further differentiate into M0, M1, or M2 phenotypes in the presence of appropriate stimulation, and retain cellular plasticity at least upon administration. Furthermore, CAR-expressing myeloid effector cells can migrate to lymph nodes and cross-present antigens to primary T cells within the lymph nodes, thereby activating adaptive responses.

[0165] In some embodiments, compositions and methods for generating myeloid cells isolated from biological samples and engineered in vitro to express recombinant proteins, and formulated into pharmaceutical compositions such that the myeloid cells of the compositions are highly efficient “effective” myeloid cells that induce immune activation in vivo. In some embodiments, the myeloid cells of the compositions are referred to as 'ATAK' myeloid cells, wherein the cells are myeloid-efficient in attacking and destroying target cells. The ATAK myeloid cells disclosed herein are engineered myeloid cells that express recombinant proteins, such as chimeric receptors, such as chimeric antigen receptors, which contain at least one intracellular signaling domain derived from interferon-inducible proteins in immune cells. In some embodiments, the methods and compositions described herein relate to causing engineered myeloid cells to exhibit an effector phenotype. In some embodiments, the engineered myeloid cells, such as monocytes, are M0 or M1 phenotype monocytes, and activation of the chimeric antigen receptor expressed in the myeloid cells causes the cells to exhibit an M1 phenotype. When engineered to target tumor cells, the M1 phenotype exhibited by the engineered cells makes the cells highly tumor-killing.

[0166] This article provides compositions and methods for treating diseases or conditions such as cancer. The compositions and methods provided herein utilize human myeloid cells, including but not limited to neutrophils, monocytes, myeloid dendritic cells (mDCs), mast cells, and macrophages, to target diseased cells such as cancer cells. The compositions and methods provided herein can be used to eliminate diseased cells such as cancer cells and / or diseased tissues through a variety of mechanisms, including T cell activation and recruitment, effector immune cell activation (e.g., CD8 T cell and NK cell activation), antigen cross-presentation, enhanced inflammatory responses, regulatory T cell reduction, and phagocytosis. For example, myeloid cells can be used to maintain an immune response against cancer cells.

[0167] The compositions previously described by the applicant comprise recombinant nucleic acids encoding chimeric fusion proteins (CFPs), such as phagocytic receptor (PR) fusion proteins (PFPs), scavenger receptor (SR) fusion proteins (SFPs), integrin receptor (IR) fusion proteins (IFPs), or caspase recruitment receptors (caspase-CAR) fusion proteins. The CFP encoded by the recombinant nucleic acid may include an extracellular domain (ECD) containing an antigen-binding domain that binds to an antigen on a target cell. The extracellular domain may be fused with a hinge domain or extracellular domain derived from a receptor such as CD2, CD8, CD28, CD68, a phagocytic receptor, a scavenger receptor, or an integrin receptor. The CFP encoded by the recombinant nucleic acid may further include 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, the CFP encoded by the recombinant nucleic acid further includes an intracellular domain comprising an intracellular signaling domain, such as an intracellular signaling domain derived from a phagocytic receptor, scavenger receptor, or integrin receptor. For example, the intracellular domain may comprise one or more intracellular signaling domains derived from a phagocytic receptor, scavenger receptor, or integrin receptor. For example, the intracellular domain may comprise one or more intracellular signaling domains that promote phagocytic activity, inflammatory responses, 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, the CFP encoded by the recombinant nucleic acid further includes an intracellular domain comprising a recruitment domain. For example, intracellular domains may contain one or more PI3K recruitment domains, caspase recruitment domains, or caspase activation and recruitment domains (CARDs).

[0168] This document provides modified immunogenic CAR compositions, such as recombinant nucleic acids encoding chimeric fusion proteins (CFPs, interchangeably referred to as chimeric antigen receptor CARs), which contain intracellular domains that activate interferon responses in cells expressing the CAR. This document provides immunogenic CFPs containing at least one intracellular domain containing the pLxIS motif. The recombinant nucleic acid can be DNA or RNA. The recombinant nucleic acid encoding the CAR can be contained in a vector. When expressed in cells, the recombinant CAR activates type I interferon production in the cells. Such cells are mammalian cells capable of producing type I interferon responses. These cells are immune cells, such as lymphocytes or myeloid cells.

[0169] In some embodiments, the recombinant nucleic acid encoding the chimeric receptor contains a specific sequence encoding a pro-inflammatory intracellular domain of the chimeric receptor. In some embodiments, the chimeric receptor protein described herein includes an intracellular domain capable of activating an interferon response gene or signaling cascade to induce type I interferon production in cells expressing the chimeric antigen receptor upon binding to its target at an extracellular domain. In some embodiments, the chimeric receptor protein described herein includes a domain derived from an innate immune pathway adaptor protein, such as mitochondrial antiviral signaling protein (MAVS), an interferon gene stimulator (STING), an IFN-inducing adaptor containing a Toll / IL-1R domain (TRIF), and a TLR adaptor (TASL) interacting with the endolysosomal SLC15A4 protein, or a portion thereof. In some embodiments, domains or fragments of innate immune pathway adaptor proteins, such as MAVS, STING, TRIF, or TASL proteins, can be incorporated into intracellular domains of CFP or CAR as described herein using recombinant DNA technology. These domains or fragments contain the pLxIS motif (where p represents a hydrophilic residue, x represents any residue, and S represents a phosphorylation site), which is phosphorylated by TBK1 or IKKε and mediates the recruitment of IRF-3 to the signal transduction complex.

[0170] In some embodiments, the chimeric receptor protein described herein includes an intracellular domain capable of activating nuclear factor κB response genes or signaling cascades to induce an NF-κB response in cells expressing the chimeric antigen receptor when it binds to its target at an extracellular domain.

[0171] Effector myeloid cells and interferon activation

[0172] Type I and type II interferons (IFNs) play important roles in regulating immune responses during infection and cancer. Type I is represented by multiple subtypes, including many members of the IFNα family, IFNβ, IFNδ, IFNε, IFNκ, IFNτ, and IFNω, all of which utilize the same cell surface receptor IFNαR, which contains heterodimers of the IFNαR1 and IFNαR2 proteins. Type II IFNs are represented by IFNγ. These two IFN types bind to different cell surface receptors expressed by virtually all cells to trigger signal transduction events and elicit different cellular responses. Myeloid cells are key targets of interferons, particularly during the early immune response to intracellular bacterial infection. Activated natural killer (NK) and T cells are the source of IFNγ production. During the early stages of infection, the production of the cytokines interleukin (IL)-12 and IL-18 drives these lymphocyte populations to produce antigen-nonspecific IFNγ. Antigen-specific CD4++ and CD8 +T cells can also produce IFNγ in response to these pathogens. There are numerous individual type I IFNs, including approximately 20 IFNα proteins and a single IFNβ. Each of these type I IFNs signals the host cell by binding to a conserved cell surface type I IFN receptor, IFNαR. The binding of cell surface IFNαR induces the expression of many antiviral immunostimulatory gene (ISG) products and thus protects the host from certain viral infections (Sadler AJ, Interferon-inducible antiviral effectors. (Review) Nature Review Immunol. July 2008; 8(7):559-68). However, responses to type I IFN are also significantly associated with increased susceptibility to many intracellular bacterial infections (Rayamajhi M. et al., Antagonistic crosstalk between type I and II interferons and increased host susceptibility to bacterial infections. Virulence, Sep-October 2010; 1(5):418-22), including Listeria monocytogenes, Mycobacterium tuberculosis, and Fransicella tularensis. IFNγ is secreted as a homodimer and acts on host cells by linking to cell surface receptors. Each IFNγ receptor is a heterodimer containing two type I monomeric membrane subunits, IFNγR1 and IFNγR2. The binding of IFNγ homodimer to cells leads to the aggregation of two receptor complexes, resulting in two IFNγR1 subunits and two IFNγR2 subunits, as well as additional signal transduction components.Although signal transduction requires two subunits, the actual binding site for IFNγ is located on IFNγR1 (Kearney S. et al., Different effects of type I and II interferons on myeloid cells and resistance to intracellular bacterial infections. *Immunol Res.*, March 2013; 55(0): 187-200). When IFNγ interacts with the IFNγR1 subunit, it induces a conformational change that allows the IFNγR1 and IFNγR2 subunits to associate more tightly. These rearrangements in the receptor induce autophosphorylation and cross-phosphorylation of Janus-associated kinases (JAK), which are constitutively related to the receptor. IFNγR1 contains the JAK1 binding motif, and IFNγR2 contains the JAK2 binding motif. Phosphorylation of JAK proteins stimulates their catalytic activity, and then they cause tyrosine residues (Y) at the C-terminus of IFNγR1. 440 Phosphorylation. This phosphorylated tyrosine residue provides a docking site for the SH2 domain on the signal transducer and activator of transcription-1 (STAT-1) protein. Because each receptor complex contains two IFNγR1 subunits, both STAT-1 proteins are able to bind to the receptor. JAK1 and JAK2 maintain receptor association and enable tyrosine residue 701 (Y 701 Each recruited STAT-1 protein is phosphorylated at the receptor. This phosphorylation allows the release of STAT-1 monomers from the receptor, as well as the formation of their homodimers. The STAT-1 homodimer translocates to the nucleus and binds to the gamma-activating sequence (GAS) in the promoter DNA of the IFN-stimulated gene (ISG), leading to increased transcription. Type I IFN signals via the typical JAK / STAT pathway, similar to the pathway activated by IFNγ. Ligand binding to IFNαR triggers dimerization of both receptor subunits and transphosphorylation of their associated TYK2 and JAK1 kinases. The kinases phosphorylate residues in the cytoplasmic tails of IFNαR1 and IFNαR2 to recruit STAT1 and STAT2 proteins via their SH2 domains. The docking of these STAT proteins with the receptor subunits allows them to be activated at the γ-axis of STAT-1. 701 Y at STAT-2 690Phosphorylation of activated JAK proteins occurs at the site. Phosphorylation of STAT monomers releases them from their docking sites, allowing them to dimerize and combine with IRF9 in homodimer or heterodimer form to produce the transcription factor ISG factor 3 (ISGF3). ISGF3 translocates to the nucleus to identify ISGs and induce their transcription. ISGs induced by type I IFN signaling typically contain an interferon-stimulated response element (ISRE) or a gamma-activated sequence (GAS) element within their promoter, although there is a clear preference for genes containing ISREs. Some instances of ISG transcribed from type I IFN are genes containing ISRE, such as ISG15, IP-10, IRF-7, and PKR

[66] , and genes containing GAS, such as IRF-1, IRF-2, IRF-8, and IRF-9 (Kearney S. et al., Differential effects of type I and II interferons on myeloid cells and resistance to intracellular bacterial infections. Immunol Res. March 2013; 55(0): 187-200).

[0173] TROP2-binding chimeric receptor for phagocytic cell activation

[0174] Extracellular antigen-binding domain

[0175] Table 1A shows exemplary sequences of chimeric fusion protein domains and / or fragments thereof, which are intended to be non-limiting in this disclosure. Underlined lines indicate the CDR sequences of the corresponding heavy and light chains in the order of CDR1, CDR2, and CDR3 according to the Kabat numbering system.

[0176] Table 1A. Exemplary chimeric fusion protein antigen-binding domains

[0177]

[0178]

[0179] Transmembrane domain

[0180] In some embodiments, the transmembrane domain and the antigen-binding domain are operatively connected via a connector. In some embodiments, the transmembrane domain and the antigen-binding domain are operatively connected via a connector such as a hinge region of CD8α, IgG1, or IgG4.

[0181] In some embodiments, the extracellular domain comprises a polymerized scaffold.

[0182] In some embodiments, the transmembrane domain includes a CD8 transmembrane domain. In some embodiments, the transmembrane domain includes a CD28 transmembrane domain. In some embodiments, the transmembrane domain includes a CD68 transmembrane domain. In some embodiments, the transmembrane domain includes a CD2 transmembrane domain. In some embodiments, the transmembrane domain includes an FcR transmembrane domain. In some embodiments, the transmembrane domain includes an FcRγ transmembrane domain. In some embodiments, the transmembrane domain includes an FcRα transmembrane domain. In some embodiments, the transmembrane domain includes an FcRβ transmembrane domain. In some embodiments, the transmembrane domain includes an FcRε transmembrane domain. In some embodiments, the transmembrane domain includes a transmembrane domain from a synaptic fusion protein such as synaptic fusion protein 3, synaptic fusion protein 4, or synaptic fusion protein 5. In some embodiments, when CFP is expressed in cells, the transmembrane domain oligomerizes with the transmembrane domain of the endogenous receptor. In some embodiments, when CFP is expressed in cells, the transmembrane domain oligomerizes with the transmembrane domain of the exogenous receptor. In some embodiments, when CFP is expressed in cells, the transmembrane domain dimers with the transmembrane domain of the endogenous receptor. In some embodiments, when CFP is expressed in cells, the transmembrane domain dimers with the transmembrane domain of the exogenous receptor. In some embodiments, the transmembrane domain originates from a protein from which the intracellular signaling domain originates. In some embodiments, the transmembrane domain originates from a protein from which the extracellular domain originates. In some embodiments, the transmembrane domain comprises the transmembrane domain of a phagocytic receptor. In some embodiments, the transmembrane domain and the extracellular domain originate from the same protein. In some embodiments, the transmembrane domain originates from the same protein as the intracellular signaling domain. In some embodiments, the recombinant nucleic acid encodes the DAP12 recruitment domain. In some embodiments, the transmembrane domain comprises a transmembrane domain oligomerized with DAP12.

[0183] In some embodiments, the length of the transmembrane domain is at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids. In some embodiments, the length of the transmembrane domain is at most 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids.

[0184] Intracellular domains

[0185] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocytic receptor. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocytic receptor other than Megf10, MerTk, FcRα, or Bai1. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocytic receptor selected from the group consisting of TNFR1, MDA5, CD40, lectins, and dectin. 1. CD206, scavenger receptor A1 (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, Fcα receptor I, CR1, CD35, CD3ζ, CR3, CR4, Tim-1, Tim-4, and CD169. In some embodiments, the intracellular signal transduction domain includes 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 inhibitory domain. In some embodiments, the intracellular domain comprises a Rac inhibitory domain, a Cdc42 inhibitory domain, or a GTPase inhibitory domain. In some embodiments, the Rac inhibitory domain, Cdc42 inhibitory domain, or GTPase inhibitory domain inhibits Rac, Cdc42, or GTPase at the phagocytic cuvette of a PFP-expressing cell. In some embodiments, the intracellular domain comprises an F-actin unassembly activation domain, an ARHGAP12 activation domain, an ARHGAP25 activation domain, or an SH3BP1 activation domain. In some embodiments, the intracellular domain comprises a phosphatase inhibitory domain. In some embodiments, the intracellular domain comprises an ARP2 / 3 inhibitory domain. In some embodiments, the intracellular domain comprises at least one ITAM domain. In some embodiments, the intracellular domain comprises at least one, two, three, four, five, six, seven, eight, nine, ten, or more ITAM domains.In some embodiments, the intracellular domain comprises at least one ITAM domain selected from the following: CD3ζ, CD3ε, CD3γ, CD3δ, Fcε receptor 1 chain, Fcε receptor 2 chain, Fcγ receptor 1 chain, Fcγ receptor 2a chain, Fcγ receptor 2b1 chain, Fcγ receptor 2b2 chain, Fcγ receptor 3a chain, Fcγ receptor 3b chain, Fcβ receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, a functional fragment thereof, and an amino acid sequence having at least one, but no more than 20, modified thereon. 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 comprises an F-actin depolymerization activation domain. In some embodiments, the intracellular domain lacks enzymatic activity.

[0186] In some embodiments, the intracellular domain does not include a domain derived from the CD3ζ intracellular domain. In some embodiments, the intracellular domain does not include a domain derived from the MerTK intracellular domain. In some embodiments, the intracellular domain does not include a domain derived from the TLR4 intracellular domain. In some embodiments, the intracellular domain includes a CD47 repressive domain. In some embodiments, the intracellular signaling domain includes a domain that activates integrins, such as the intracellular region of PSGL-1.

[0187] In some embodiments, the intracellular signaling domain includes a domain that activates the Rap1 GTPase, such as domains derived from EPAC and C3G. In some embodiments, the intracellular signaling domain is derived from paxillin. In some embodiments, the intracellular signaling domain activates focal adhesion kinase. In some embodiments, the intracellular signaling domain is derived from a single phagocytic receptor. In some embodiments, the intracellular signaling domain is derived from a single scavenger receptor. In some embodiments, the intracellular domain includes a phagocytosis-enhancing domain.

[0188] In some embodiments, the intracellular domain comprises a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain comprises a kinase activation domain or a kinase binding domain. In some embodiments, the pro-inflammatory signaling domain comprises an IL-1 signaling cascade activation domain. In some embodiments, the pro-inflammatory signaling domain comprises an intracellular signaling domain derived from: TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, IFN-receptor, STING, NLRP family members, NLRP1-14, NOD1, NOD2, Pyrin, AIM2, NLRC4, FCGR3A, FCERIG, CD40, Tank1-binding kinase (TBK), caspase domain, caspaseogen-binding domain, or any combination thereof.

[0189] In some embodiments, the intracellular domain includes a signal transduction domain, such as an intracellular signal transduction domain derived from a TLR protein. In some embodiments, the intracellular domain may include an intracellular signal transduction domain of an endosomal TLR (e.g., TLR3, TLR7, TLR8, or TLR9). In some embodiments, the intracellular signal transduction domain may be derived from a TLR3 protein. In some embodiments, the intracellular signal transduction domain may be derived from a TLR7, 8, or 9 protein. In some embodiments, the intracellular domain may include intracellular signal transduction domains of cell surface TLRs 1, 2, 4, 5, 6, and 10.

[0190] In some embodiments, an intracellular signaling domain specifically pairs with another intracellular domain or transmembrane domain to maximize the efficiency and phagocytic potential of myeloid cells expressing the construct. For example, in some embodiments, a TM domain containing CD64 TM or a portion thereof may specifically pair with an intracellular signaling domain containing an innate immune adaptor protein ICD or a PI3 kinase recruitment domain, or both. In some embodiments, the combination of domains of the chimeric receptor intracellular domain and / or transmembrane domain involves maximizing the phagocytic index of cells expressing the construct (e.g., myeloid cells). In some embodiments, the combination of domains of the chimeric receptor intracellular domain and / or transmembrane domain involves maximizing the inflammatory potential of cells expressing the construct, enabling cells to lyse target cells and activate immune response pathways to generate a long-term immune response. In some embodiments, the combination of domains of the chimeric receptor intracellular domain and / or transmembrane domain involves minimizing or eliminating any tetanic signaling by cells expressing the chimeric protein. In some embodiments, the combination of domains of the chimeric receptor intracellular domain and / or transmembrane domain involves maximizing the specificity of the immune response.

[0191] In some embodiments, the CFP does not contain a full-length intracellular signal transduction domain. In some embodiments, the length of the intracellular domain is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids. In some embodiments, the length of the intracellular domain is at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids.

[0192] In some embodiments, the recombinant nucleic acid encodes an extracellular domain, a transmembrane domain, and / or an intracellular domain of the FcRα chain. In some embodiments, the recombinant nucleic acid encodes an extracellular domain, a transmembrane domain, and / or an intracellular domain of the FcRβ chain. In some embodiments, when expressed in cells, the FcRα or FcRβ chain forms a complex with FcRγ. In some embodiments, when expressed in cells, the FcRα or FcRβ chain forms a complex with endogenous FcRγ. In some embodiments, the FcRα or FcRβ chain does not incorporate into the cell membrane of cells that do not express FcRγ. In some embodiments, the CFP does not contain an intracellular signaling domain of the FcRα chain. In some embodiments, the CFP does not contain an intracellular signaling domain of the FcRβ chain. In some embodiments, the recombinant nucleic acid encodes an extracellular domain, a transmembrane domain, and / or an intracellular domain of the TREM. In some embodiments, TREM is TREM1, TREM2, or TREM3.

[0193] In some embodiments, the recombinant nucleic acid comprises a sequence encoding a pro-inflammatory polypeptide. In some embodiments, the composition further comprises a pro-inflammatory nucleotide or a nucleotide in the recombinant nucleic acid, such as ATP, ADP, UTP, UDP, and / or UDP-glucose.

[0194] Intracellular interferon response domain

[0195] Most TLRs activate an adaptor protein called MyD88, which in turn activates the transcription factor protein NF-κB, driving the expression of pro-inflammatory genes as part of the immune response. Subgroups of TLRs (TLR3 and TLR4) can bind to the protein TRIF, which acts as a scaffold, allowing the kinase to add a phosphate ester group to the transcription factor IRF3. This phosphorylation activates IRF3, a member of a family of transcription factors called interferon regulatory factors (IRFs), which activate a wide range of gene expression programs. A hallmark of these programs is the production of type I interferon molecules. Interferons are potent drivers of a branch of the immune system known as the adaptive immune response, and therefore their presence carries the risk of autoimmunity. To prevent such attacks by the host's own immune system, the interferon response must be tightly regulated. As a safeguard, the specific amino acid residue sequence pLxIS motif in TRIF must be phosphorylated before IRF3 can be activated. This control mechanism provides a 'permission step' that is not only specific to TRIF as an adaptor protein for TLR signaling, but also a general marker of sensing pathways that bind to IRF3 or the associated protein IRF7 to drive interferon expression. With one exception, each identified innate sensing pathway that links nucleic acid recognition to type I interferon production has previously been shown to signal via one of three adaptor proteins known to date to contain the pLxIS motif: TRIF, MAVS, and STING. Thus, adaptor proteins containing the pLxIS motif specifically hardwire nucleic acid recognition to antiviral defense. In some embodiments, the intracellular signaling domain of CFP contains an ICD of an innate immune response protein.In some embodiments, innate immune response proteins are selected from intracellular signal transduction domains derived from TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, IFN receptor, STING, MAVS, TRIF, TASL, NLRP1, NLRP2, NLRP3, NLRP4, NLRP5, NLRP6, NLRP7, NLRP89, NLRP9, NLRP10, NLRP11, NLRP12, NLRP13, NLRP1-14, NOD1, NOD2, Pyrin, AIM2, NLRC4, FCGR3A, FCERIG, CD40, Tank1-binding kinase (TBK), TNFR1, chemokines, and MHC. Type II transactivator (CIITA), IPAF, BIRC1, RIG-I-like receptor (RLR) protein, macrophage galactose lectin (MGL), DC-SIGN (CLEC4L), islet protein (Langerin, CLEC4K), myeloid DAP12 lectin (MDL)-1 (CLEC5A), DC-associated C-type lectin 1 (Dectin1) subfamily protein, dectin 1 / CLEC7A, DNGR1 / CLEC9A, myeloid C-type lectin-like receptor (MICL) (CLEC12A), CLEC2 (CLEC1B), CLEC12B, DC immune receptor (DCIR) subfamily protein, DCIR / CLEC4A, Dectin2 / CLEC6A, blood DC antigen 2 (BDCA2) (CLEC4C), and mincle (macrophage-inducible C-type lectin) (CLEC4E). In some embodiments, the CFP includes at least one intracellular signaling domain, the at least one intracellular signaling domain including the amino acid sequence motif pLxIS.

[0196] In some embodiments, the composition further comprises a pro-inflammatory peptide. In some embodiments, the pro-inflammatory peptide is a chemokine or a cytokine. In some embodiments, the chemokine is selected from the group consisting of: IL-1, IL-3, IL-5, IL-6, IL-8, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL-17, IP-10, RANTES, and interferon. In some embodiments, the cytokine is selected from the group consisting of: IL-1, IL-3, IL-5, IL-6, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL-17, IP-10, RANTES, and interferon.

[0197] In some embodiments, an intracellular signaling domain from an intracellular adaptor protein known to be highly active in innate immune defense is incorporated into the chimeric receptor protein. In some embodiments, one or more mutations are introduced into one or more intracellular domains to reduce the response of the intracellular domain to intracellular stimuli that are characteristic of the native intracellular adaptor protein domain, without impairing the effectiveness of the chimeric protein. In some embodiments, such effectiveness is referred to as enhanced phagocytic potential compared to the same cells that do not express the chimeric protein. In some embodiments, such effectiveness is referred to as enhanced inflammatory potential compared to the same cells that do not express the chimeric protein. In some embodiments, such effectiveness is referred to as enhanced NF-κB activation or interferon activation in cells that express the chimeric protein compared to the same cells that do not express the chimeric protein.

[0198] Delivery medium - nanoparticles

[0199] In some embodiments, myeloid cells are specifically targeted for delivery. Specialized biodegradable polymers such as PLGA (poly(lactic-co-glycolic acid)) and / or polyvinyl alcohol (PVA) can be used to target myeloid cells. In some embodiments, one or more compounds are selectively incorporated into such polymer structures to influence myeloid cell function. In some embodiments, the targeting structure is multilayered, for example, consisting of one or more PLGA layers and one or more PVA layers. In some embodiments, the targeting structure is assembled in order of layered activity. In some embodiments, the targeting polymer structure is organized with components of a specific shape, such as an unstable structure that can adhere to the surface of myeloid cells and deliver one or more components (such as growth factors and cytokines) to maintain myeloid cells in a microenvironment that imparts a specific polarization. In some embodiments, the polymer structure is designed to prevent phagocytosis by myeloid cells, but it can remain adhered to the surface. In some embodiments, the one or more growth factors can be M1 polarization factors such as cytokines. In some embodiments, the one or more growth factors can be M2 polarization factors such as cytokines. In some embodiments, the one or more growth factors can be macrophage-activating cytokines such as IFNγ. In some embodiments, the polymer structure is capable of sustained release of the one or more growth factors in an in vivo environment, such as in solid tumors.

[0200] In some embodiments, the recombinant nucleic acid contains a sequence encoding a homeostatic regulator of inflammation. In some embodiments, the homeostatic regulator of inflammation is a sequence in the untranslated region (UTR) of the mRNA. In some embodiments, the sequence in the UTR is a sequence that binds to an RNA-binding protein. In some embodiments, translation is inhibited or prevented when the RNA-binding protein binds to the sequence in the UTR. In some embodiments, the RNA has a poly A tail. In some embodiments, the RNA (mRNA) has a poly A tail containing 200-1000 A (adenosine) residues. In some embodiments, the mRNA has a poly A tail containing about 200-800 A residues. In some embodiments, the mRNA has a poly A tail containing about 800-1200 A residues. In some embodiments, the mRNA has a poly A tail containing about or at most 1500 A residues.

[0201] In some embodiments, the target cell is a mammalian cell. In some embodiments, the target cell is a human cell. In some embodiments, the target cell comprises cells infected by a pathogen. In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell is a cancer cell that is a lymphocyte. In some embodiments, the target cell is a cancer cell that is an ovarian cancer cell. In some embodiments, the target cell is a cancer cell that is a breast cell. In some embodiments, the target cell is a cancer cell that is a pancreatic cell. In some embodiments, the target cell is a cancer cell that is a glioblastoma cell.

[0202] In some embodiments, the recombinant nucleic acid is DNA. In some embodiments, the recombinant nucleic acid is RNA. In some embodiments, the recombinant nucleic acid is mRNA. In some embodiments, the recombinant nucleic acid is unmodified mRNA. In some embodiments, the recombinant nucleic acid is modified mRNA. In some embodiments, the recombinant nucleic acid is circRNA. In some embodiments, the recombinant nucleic acid is tRNA. In some embodiments, the recombinant nucleic acid is microRNA.

[0203] This document also provides a vector comprising a recombinant nucleic acid sequence encoding the CFP described herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector or a lentiviral vector. In some embodiments, the vector further comprises a promoter operatively linked to at least one nucleic acid sequence encoding one or more polypeptides. In some embodiments, the vector is polycistronic. In some embodiments, each of the at least one nucleic acid sequence is operatively linked to a separate promoter. In some embodiments, the vector further comprises one or more internal ribosome entry sites (IRES). In some embodiments, the vector further comprises a 5' UTR and / or a 3' UTR flanked by the at least one nucleic acid sequence encoding one or more polypeptides. In some embodiments, the vector further comprises one or more regulatory regions.

[0204] This article also provides a polypeptide encoded by a recombinant nucleic acid of the composition described herein.

[0205] This document provides a composition comprising a recombinant nucleic acid sequence encoding a CFP, said CFP comprising a phagocytic or tethering receptor (PR) subunit (e.g., a phagocytic receptor fusion protein (PFP)), said CFP comprising: a PR subunit comprising: a transmembrane domain and an intracellular domain comprising an intracellular signal transduction domain; and an extracellular domain comprising an antigen-binding domain specific to antigens of target cells; wherein the transmembrane domain and the extracellular domain are operatively connected; and wherein, when the CFP binds to an antigen of a target cell, myeloid cells (e.g., neutrophils, monocytes, myeloid dendritic cells) expressing CFP show a higher binding rate compared to cells not expressing CFP. Increase in cytotoxic or phagocytic activity of mDCs, mast cells, or macrophages by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000%.

[0206] Table 1B. Exemplary chimeric fusion protein signal peptide sequences

[0207]

[0208] Table 1C. Extracellular (ECD) / hinge domains of exemplary chimeric fusion proteins

[0209]

[0210] Table 1D. Exemplary transmembrane domains (TMDs) of chimeric fusion proteins

[0211]

[0212] Table 2. Intracellular signal transduction domains (ICDs) of exemplary chimeric fusion proteins

[0213]

[0214]

[0215] Table 3. Intracellular domains of exemplary chimeric fusion proteins

[0216]

[0217] Table 4. Exemplary chimeric fusion protein sequences

[0218]

[0219]

[0220]

[0221] Table 5 - Exemplary TROP2 binding sequences (CDR sequences are underlined)

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228] This document provides a composition comprising a recombinant nucleic acid sequence encoding a CFP, said CFP comprising a phagocytic or tethered receptor (PR) subunit (e.g., a phagocytic receptor fusion protein (PFP)), said subunit comprising: an extracellular domain comprising an antigen-binding domain specific to an antigen on a target cell; a transmembrane domain; and an intracellular domain comprising an intracellular signal transduction domain; wherein the transmembrane domain and the extracellular domain are operatively connected; and wherein, when the CFP binds to an antigen on a target cell, cells expressing CFP show a higher expression rate compared to cells not expressing CFP. CFP increases the cytotoxic or phagocytic activity of myeloid cells (such as neutrophils, monocytes, myeloid dendritic cells (mDCs), mast cells, or macrophages) by at least 1.1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 75, or 100 times.

[0229] This article provides a recombinant nucleic acid sequence encoding CFP as described in the preceding paragraph, wherein the intracellular domain includes at least one innate immune activation intracellular domain, such as a pattern recognition receptor intracellular signal transduction domain, a TLR intracellular signal transduction domain, an FcR intracellular signal transduction domain, an intracellular adaptor protein signal transduction domain, or a fragment thereof, which can activate the innate immune response of myeloid cells, activate their phagocytic potential, activate inflammatory cytokine and chemokine responses, antigen presentation and T cell activation of myeloid cells expressing CFP, and upon contact with their target antigen, for example, when the antigen-binding domain binds to the target antigen.

[0230] In some embodiments, the pro-inflammatory signaling domain includes intracellular signaling domains derived from: TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, IFN receptor, STING, MAVS, TRIF or TASL intracellular domain, NLRP family members NLRP1-14, NOD1, NOD2, Pyrin, AIM2, NLRC4, FCGR3A, FCERIG, IL-1, IL3, IL5, IL-6, IL-12, IL-13, IL-23, TNF, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10 or RANTES.

[0231] In some embodiments, the CFP includes an intracellular signal transduction domain comprising a sequence of a protein derived from an interferon-activating response transcription factor IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, or IRF9.

[0232] In some embodiments, the CFP includes an intracellular signaling domain comprising a sequence derived from an intracellular adaptor protein. In some embodiments, the adaptor protein may include a transmembrane component that anchors it to organelles such as mitochondria, endoplasmic reticulum, or lysosomal compartments. In some embodiments, the intracellular adaptor protein is a cytoplasmic protein.

[0233] As described herein, a CFP may include the antigen-binding domain of Table 1A, the extracellular / hinge domain of Table 1C, and one or more intracellular signal transduction domains of Table 2. Optionally, a CFP as described herein may include the signal peptide sequence of Table 1B.

[0234] In some embodiments, the CFP may comprise a sequence having at least 85% sequence identity with the sequences in Table 4. For example, the CFP may comprise a sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequences in Table 4. In some embodiments, the CFP further comprises a signal peptide sequence, such as the signal peptide sequences from Table 1B.

[0235] In some embodiments, the intracellular signal transduction domain comprises an intracellular signal transduction domain derived from a TRIF intracellular domain, wherein the TRIF intracellular domain has an amino acid sequence of any of SEQ ID NO: 19-22 or has at least 85% sequence identity with any of SEQ ID NO: 19-22. In some embodiments, the intracellular signal transduction domain comprises a sequence having at least 86%, or at least 87%, or at least 88%, or at least 89% sequence identity with any of SEQ ID NO: 19-22. In some embodiments, the intracellular signal transduction domain comprises an intracellular signal transduction domain derived from a TRIF intracellular signal transduction domain, wherein the TRIF intracellular signal transduction domain has at least 90% sequence identity with any of SEQ ID NO: 19-22. In some embodiments, the intracellular signal transduction domain comprises a sequence having at least 91%, or at least 92%, or at least 93%, or at least 94% sequence identity with any of SEQ ID NO: 19-22. In some embodiments, the intracellular signal transduction domain comprises an intracellular signal transduction domain derived from a TRIF intracellular signal transduction domain, wherein the TRIF intracellular signal transduction domain has at least 95% sequence identity with any of SEQ ID NO: 19-22. In some embodiments, the intracellular domain of the CFP comprises an intracellular signal transduction domain derived from a TRIF intracellular signal transduction domain, wherein the TRIF intracellular signal transduction domain has at least 90% sequence identity with any of SEQ ID NO: 19-22; wherein the CFP comprises an extracellular binding domain capable of binding to TROP2 molecules on target cells, a CD89 transmembrane domain, and / or one or more additional intracellular signal transduction domains.

[0236] In some embodiments, the exemplary anti-TROP2 binding CFP described herein comprises an extracellular antigen-binding domain having a sequence of any of SEQ ID NO: 1-3, or a heavy chain variable domain comprising a CDR3 sequence of GGFGSSYWYFDV and / or a light chain variable domain comprising a CDR3 sequence of QQHYITPLT, and further comprises an intracellular domain of any of the sequences in Table 2 or Table 3. In some embodiments, the exemplary anti-TROP2 binding CFP described herein comprises a sequence having at least 80-100% sequence identity with any of SEQ ID NO: 26-33.

[0237] In some embodiments, this document provides a pharmaceutical product comprising a recombinant mRNA encoding a chimeric antigen receptor comprising: an anti-TROP2 binding scFv extracellular domain; and a CD89 transmembrane domain; wherein the pharmaceutical product is formulated in an aqueous formulation for systemic delivery. In some embodiments, the pharmaceutical composition comprises an scFv comprising a heavy chain and a light chain, the heavy chain comprising a CDR3 having the sequence GGFGSSYWYFDV, and the light chain comprising a CDR3 having the sequence QQHYITPLT. In some embodiments, the heavy chain further comprises a CDR1 sequence of NYGMN and a CDR2 sequence of WINTYTGEPTYTDDFKG; and the light chain further comprises a CDR1 sequence of KASQDVSIAVA and a CDR2 sequence of SASYRYT. The above pharmaceutical composition comprises a CD89 transmembrane domain having the sequence LIRMAVAGLVLVALLAILV.

[0238] An exemplary second-generation anti-TROP2 chimeric fusion protein (e.g., a drug) comprises an anti-TROP2 scFv, said anti-TROP2 scFv comprising the sequence (CDR highlighted according to Kabat nomenclature convention) QVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAP GQGLKWMG WINTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR G GFGSSYWYFDV Heavy-chain variable structural domains of WGQGSLVTVSSG. An exemplary TROP2 ScFv complete construct has the following sequence: (SEQ ID NO: 41). Bold letters indicate the signal peptide sequence. Mature proteins may lack this sequence, and those skilled in the art can interpret the protein sequence expressed as if in the absence of the signal peptide sequence. The underlined region is the CDR sequence of scFV, which binds to TROP2 in the CDR1, CDR2, and CDR3 of the heavy and light chains. Italic letters indicate the amino acid sequence of CD89 TMD.

[0239] Chimeric protein with TLR intracellular domain, TLR intracellular signaling pathway, and NF-κB activation:

[0240] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a TLR protein. In some embodiments, a CFP designed to include an intracellular signaling domain derived from a TLR intracellular signaling domain can activate NF-κB when the extracellular domain of the receptor binds to its target. In some embodiments, the intracellular domain may comprise an intracellular signaling domain of an endosomal TLR (e.g., TLR3, TLR7, TLR8, or TLR9). In some embodiments, the intracellular signaling domain may be derived from the TLR3 protein. In some embodiments, the intracellular signaling domain may be derived from the TLR7, 8, or 9 protein. In some embodiments, the intracellular domain may comprise intracellular signaling domains of cell surface TLRs 1, 2, 4, 5, 6, and 10. In some embodiments, the cytoplasmic domains for inflammatory responses include intracellular signal transduction domains of TLR3, TLR4, TLR9, MYD88, TRIF, RIG-1, MDA5, CD40, IFN receptor, NLRP-1, NLRP-2, NLRP-3, NLRP-4, NLRP-5, NLRP-6, NLRP-7, NLRP-8, NLRP-9, NLRP-10, NLRP-11, NLRP-12, NLRP-13, NLRP-14, NOD1, NOD2, Pyrin, AIM2, NLRC4, and / or CD40.

[0241] In some embodiments, the phagocytic scavenger receptor (PR) fusion protein (PFP) includes a pro-inflammatory cytoplasmic domain for activating the IL-1 signaling cascade.

[0242] In some embodiments, the cytoplasmic portion of a chimeric receptor (e.g., a phagocytic receptor (PR) fusion protein (PFP)) includes cytoplasmic domains derived from toll-like receptors, such as intracellular signaling domains of toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), and toll-like receptor 9 (TLR9).

[0243] In some embodiments, the intracellular domains mentioned herein (e.g., MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, TASL) may be adjusted or modified to pair with or be incorporated together with another structural domain (e.g., a transmembrane domain). In some embodiments, the transmembrane domain is a CD68 domain. In some embodiments, the transmembrane domain is a CD64 domain. In some embodiments, the transmembrane domain is a CD89 domain.

[0244] For the purposes of this disclosure, any pathway, signaling intermediate, or activating motif discussed in the preceding paragraphs may be considered activatable or functional when applied to a disclosed CFP containing an intracellular signaling domain of a TLR as disclosed herein. Similarly, the CFP disclosed herein may be used to target any applicable target described in the discussed pathways. Any pathway or portion thereof that can be readily known to those skilled in the art from the prior literature, involving signaling domains, signaling pathways, signaling intermediates, or transcription factors activating genes, should be understood to be within the scope of this disclosure.

[0245] In some embodiments, compared with cells that do not express CFP, the cytotoxic activity of cells expressing CFP is increased by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% when CFP binds to the antigen of the target cell. In some embodiments, when CFP is expressed in cells, CFP is functionally incorporated into the cell membrane. In some embodiments, compared with cells that do not express CFP, the cytotoxic activity of cells expressing CFP is increased by at least 1.1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 75-fold, or 100-fold when CFP binds to the antigen of the target cell.

[0246] Therapeutic Composition

[0247] On the one hand, TROP2, which targets myeloid cells, can be generated in vitro, and the resulting cell therapy products can be administered to subjects in need. However, generating cell therapy products can be expensive and time-consuming.

[0248] Alternatively, on the other hand, recombinant polynucleotides can be prepared as therapeutic components that can be administered to subjects in need, wherein the recombinant polynucleotides are designed to be specifically taken up and expressed in myeloid cells; wherein the expression of the recombinant polynucleotides activates myeloid cells and induces phagocytosis and killing of target cells.

[0249] A. Therapeutic myeloid cell composition

[0250] In some embodiments, the pharmaceutical composition comprises a cell population comprising a therapeutically effective dose of myeloid cells. In some embodiments, the cell population: differentiates into effector cells in a subject after administration; infiltrates or migrates to a diseased site in a subject after administration; and / or has a lifespan of at least 5 days in the subject after administration.

[0251] In some embodiments, myeloid cells can be further modified or manipulated to develop therapeutically effective myeloid cells. Isolated cells can be manipulated by expressing genes or fragments thereof in the cells without altering their function, developmental plasticity, differential potential, and cell viability.

[0252] In some embodiments, myeloid cells can be further modified or manipulated by expressing non-endogenous polynucleotides into cells to develop therapeutically effective myeloid cells. Non-endogenous polynucleotides may encode proteins or peptides. Alternatively, non-endogenous polypeptides may be non-coding sequences, such as repressive RNA or morpholinonucleotides.

[0253] In some embodiments, myeloid cells can be further modified or manipulated to develop therapeutically effective myeloid cells by stably altering the genomic sequence of the cells. In some embodiments, myeloid cells are manipulated by editing the myeloid cell genome using a CRISPR-CAS system. In some embodiments, one or more genes can be edited to silence gene expression. In some embodiments, myeloid cells are manipulated to delete genes. In some embodiments, one or more genes can be edited to enhance gene expression. In some embodiments, genetic material is introduced into myeloid cells in the form of messenger RNA, wherein the messenger RNA encodes a protein or peptide, thereby making myeloid cell therapy effective. In some embodiments, naked DNA or messenger RNA (mRNA) can be used to introduce nucleic acids into the myeloid cells.

[0254] B. Therapeutic mRNA compositions

[0255] In some embodiments, DNA or mRNA encoding a chimeric antigen receptor is introduced into phagocytes via a nucleic acid delivery medium. In some embodiments, the nucleic acid delivery medium may be nanoparticles encapsulating nucleic acid cargo. The nanoparticles may be polymer nanoparticles. For example, specialized biodegradable polymers such as PLGA (poly(lactic-co-glycolic acid) and / or polyvinyl alcohol (PVA)) can be used to target myeloid cells. In some embodiments, one or more compounds may be selectively incorporated into such polymer structures to influence myeloid cell function. In some embodiments, the targeting structure is multilayered, for example, consisting of one or more PLGA layers and one or more PVA layers. In some embodiments, the targeting structure is assembled in order of layered activity. In some embodiments, the targeting polymer structure is organized with components of a specific shape, such as an unstable structure that can adhere to the surface of myeloid cells and deliver one or more components (such as growth factors and cytokines) to maintain myeloid cells in a microenvironment that imparts a specific polarization. In some embodiments, the polymer structure is designed to prevent phagocytosis by myeloid cells, but it can remain adhered to the surface. In some embodiments, the delivery medium may contain lipids. In some embodiments, the phagocytes are in vivo.

[0256] In some embodiments, the mRNA is single-stranded and may be codon-optimized. In some embodiments, the mRNA may contain one or more modified or non-natural bases such as 5'-methylcytosine, pseudouridine, or methylpseudouridine. In some embodiments, about 50% or more of the uridine ('U') residues in the mRNA may be converted to methylpseudouridine. In some embodiments, the length of the mRNA may be 50-10,000 bases. On one hand, the transgene is delivered as mRNA. The mRNA may contain more 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, or 10,000 bases. In some embodiments, the length of the mRNA may be greater than 10,000 bases. In some embodiments, the length of the mRNA may be about 11,000 bases. In some embodiments, the length of the mRNA may be about 12,000 bases. In some embodiments, the mRNA contains a transgenic sequence encoding a fusion protein. LNP-encapsulated DNA or RNA can be used to transfect macrophages or administered to a subject. In some embodiments, the mRNA is incorporated into an effector myeloid cell population via transient transfection. In some embodiments, the transient transfection method includes electroporation of the mRNA.

[0257] In some embodiments, the therapeutically effective dose is in the range of 1-5,000 μg / ml mRNA per subject. In some embodiments, 1-5,000 μg / ml mRNA may be delivered using a suitable protocol for the methods described above. In some embodiments, 1-2,000 μg / ml mRNA may be delivered. In some embodiments, 1-1,000 μg / ml mRNA may be delivered. In some embodiments, 1-1,000 μg / ml mRNA may be delivered. In some embodiments, 1-500 μg / ml mRNA may be delivered. In some embodiments, 1-250 μg / ml mRNA may be delivered. In some embodiments, about 500 μg / ml mRNA or less may be delivered. In some embodiments, about 250 μg / ml mRNA or less may be delivered. In some embodiments, about 10 μg / ml mRNA may be delivered. In some embodiments, about 20 μg / ml mRNA may be delivered. In some embodiments, about 30 μg / ml mRNA may be delivered. In some embodiments, about 40 μg / ml mRNA may be delivered. In some embodiments, about 50 micrograms / ml of mRNA is used. In some embodiments, about 60 micrograms / ml of mRNA may be delivered. In some embodiments, about 80 micrograms / ml of mRNA may be delivered. In some embodiments, about 100 micrograms / ml of mRNA is used. In some embodiments, about 150 micrograms / ml of mRNA may be delivered. In some embodiments, about 200 micrograms / ml of mRNA may be delivered. In some embodiments, 20, 50, 100, 150, 200, 250, 300, 400, 500, or about 1000 micrograms / ml of mRNA are used as a therapeutically effective dose for human subjects.

[0258] The mRNA constructs can be thawed on ice and gently pipetted into monocytes and premixed. In some embodiments, the mRNA is electroporated into the cells. The panned cells can be pooled, centrifuged, and electroporated with the mRNA using a MaxCyte ATX system optimized for the purpose described above. In some embodiments, optimized electroporation buffer, cell density, and / or mRNA concentration are used for each protocol for each construct.

[0259] In some embodiments, the polynucleotide can be introduced into myeloid cells in the form of circular RNA (circRNA). In circRNA, the 3' and 5' ends are covalently linked. circRNA can be delivered intracellularly using LNPs.

[0260] In some embodiments, stable integration of transgenes into macrophages and other phagocytic cells can be achieved by using transposases and transposition elements, particularly mRNA-encoded transposases. In one embodiment, long dispersive element-1 (L1) RNA can be considered for retrotransposition of transgenes and stable integration into macrophages or phagocytic cells. Retrotransposons can be used for the stable integration of recombinant nucleic acids encoding phagocytic or tethered receptor (PR) fusion proteins (PFPs).

[0261] In some embodiments, myeloid cells can be modified by expressing the transgene in a transient expression vector. In some embodiments, the expression of the transgene can be temporally regulated by extracellular regulatory factors. Examples include the Tet-on Tet-off system, in which the expression of the transgene is regulated by the presence or absence of tetracycline.

[0262] In some embodiments, myeloid cells can be modified by contacting cells with a compound to develop therapeutically effective cells, said compound being an inhibitor or activator of proteins or enzymes within the myeloid cells.

[0263] In some embodiments, a polynucleotide encoding a chimeric antigen receptor may be introduced into isolated myeloid cells obtained by the methods described in the foregoing sections, wherein expression of the chimeric antigen receptor in the myeloid cells enhances the innate immune response of the myeloid cells. In some embodiments, chimeric antigen receptor expression may guide myeloid cells to specific targets in vivo or in vitro. In some embodiments, the chimeric antigen receptor may increase the phagocytic potential of myeloid cells. In some embodiments, the chimeric antigen receptor may increase the immunogenicity of myeloid cells. In some embodiments, the chimeric antigen receptor may enhance intracellular signaling. In some embodiments, the chimeric antigen receptor may act in cooperation with one or more intracellular proteins. In some embodiments, the chimeric antigen receptor may dimerize or polymerize with a second receptor or transmembrane protein within the myeloid cells, wherein the second receptor or transmembrane protein is an endogenous protein. In some embodiments, cells are briefly cultured in vitro after thawing or after incorporation of nucleic acids. In some embodiments, in vitro culture is performed in the presence of a suitable culture medium, which may contain a regulated serum component, such as human serum albumin (HSA). In some embodiments, in vitro culture and manipulation may be performed in a low-serum culture medium. In some embodiments, serum is specifically treated for complementary inactivation. In some embodiments, myeloid cells can be cultured in vitro in the presence of M-CSF as described above. In some embodiments, myeloid cells can be cultured in vitro in the presence of GM-CSF as described above. In some embodiments, myeloid cells can be cultured in the presence of one or more cytokines. In some embodiments, myeloid cells can be cultured or manipulated in vitro for a period of time in the absence of growth factors or cytokines. In some embodiments, the methods provided herein involve isolating, enriching, and manipulating myeloid cells for less than 72 hours, 70 hours, 65 hours, 60 hours, 55 hours, 50 hours, 45 hours, 40 hours, or 35 hours, or 30 hours, or 28 hours, or 26 hours, or 24 hours. In some embodiments, myeloid cells can be cultured for less than 24 hours, or less than 20 hours, or less than 16 hours, or less than 14 hours, or less than 12 hours, or less than 10 hours, or less than 8 hours, or less than 6 hours, or less than about 4 hours. After isolation, enrichment, and manipulation, myeloid cells can be briefly cultured and frozen until further use. In some embodiments, myeloid cells are thawed once or at most twice.

[0264] In some embodiments, the therapeutically capable cells are cells that have been electroporated, frozen and thawed, and cultured stably for less than 24 hours with recombinant nucleic acids encoding polypeptides, and wherein the cells in the cell population exhibit (i) greater than 70% viability, (ii) greater than 50% CD14+ and CD16- cells; and / or greater than 50% CD11b+ / CD14+ / CD16- cells; (iii) less than 5% CD3+ cells, less than 5% CD19+ cells, less than about 10% CD56+ cells, less than about 10% CD42b+ cells; and (iv) greater than 50% of cells expressing polypeptides encoded by the electroporated nucleic acids. In some embodiments, the therapeutically capable cells are cells that have been electroporated with recombinant nucleic acids encoding peptides, cultured stably for less than 24 hours, frozen and thawed, and wherein the cells in the cell population exhibit (i) greater than 70% viability, (ii) greater than 50% CD14+ and CD16- cells; and / or greater than 50% CD11b+ / CD14+ / CD16- cells; (iii) less than 5% CD3+ cells, less than 5% CD19+ cells, less than about 10% CD56+ cells, less than about 10% CD42b+ cells; and (iv) greater than 50% of cells expressing peptides encoded by the electroporated nucleic acids. In some embodiments, therapeutically capable cells are cells that have been cultured and stabilized for less than 24 hours, electroporated with recombinant nucleic acids encoding peptides, and frozen and thawed, wherein the cells in the cell population exhibit (i) greater than 70% viability, (ii) greater than 50% CD14+ and CD16- cells; and / or greater than 50% CD11b+ / CD14+ / CD16- cells; (iii) less than 5% CD3+ cells, less than 5% CD19+ cells, less than about 10% CD56+ cells, less than about 10% CD42b+ cells; and (iv) greater than 50% of cells expressing peptides encoded by the electroporated nucleic acids. The cells must be pathogen-free. In the above embodiments, therapeutically capable cells may have been frozen and thawed no more than twice, preferably once, and may be administered within 24 hours, 18 hours, 8 hours, or 2 hours after thawing. Prior to administration, the quality assurance of the cells is tested to meet the standards described herein.

[0265] This document provides a method for treating cancer in a subject using a pharmaceutical composition comprising engineered phagocytes (particularly macrophages) expressing a recombinant nucleic acid encoding a phagocytic receptor (PR) fusion protein (PFP) specifically designed to target, attack, and kill cancer cells. PFP is also designated as a chimeric antigen receptor (CAR-P) performing phagocytosis, and these two terms are used interchangeably herein. In the description herein, engineered phagocytes are also designated as CAR-P cells.

[0266] Cancers include, but are not limited to, T-cell lymphoma, cutaneous lymphoma, B-cell cancer (e.g., multiple myeloma, Waldenstrom's macroglobulinemia), heavy chain diseases (e.g., alpha chain disease, gamma chain disease, and μ chain disease), benign monoclonal globulinosis and immune cell amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer (e.g., metastatic hormone-resistant prostate cancer), pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, bile duct cancer, small bowel or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, and hematologic malignancies. Other non-limiting examples of cancer types suitable for the methods covered in this disclosure include human sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, and Wilms' tumor. Cancer, including tumors, cervical cancer, bone cancer, brain tumors, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, meningioma, angioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemia, such as acute lymphoblastic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); as well as polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia and heavy chain disease. In some embodiments, the cancer is epithelial cancer, such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney 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, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancer can be characterized in various other ways, including but not limited to serous, endometrioid, mucinous, clear cell, or undifferentiated.In some embodiments, this disclosure is used for the treatment, diagnosis, and / or prognosis of lymphoma or its subtypes, including but not limited to mantle cell lymphoma. Lymphoproliferative disorders are also considered proliferative diseases.

[0267] Typically, cell immunotherapy involves providing a patient with a drug containing live cells. In some aspects, the patient or subject with cancer is treated with autologous cells, the method comprising: isolating or enriching PBMC-derived macrophages; modifying macrophages in vitro to generate highly phagocytic macrophages capable of tumor lysis by introducing a recombinant nucleic acid encoding a chimeric antigen receptor into the macrophages, the chimeric antigen receptor acting as a phagocytic receptor fusion protein (PFP); and administering the modified macrophages to the patient or subject.

[0268] In some aspects, a pharmaceutical composition comprising one or more doses is administered to a subject, the pharmaceutical composition comprising therapeutic phagocytes, wherein the cells are allogeneic. HLA is matched for compatibility with the subject and the cells do not contribute to graft-versus-host disease (GVHD). Subjects arriving at the clinic are HLA-generated to determine the HLA antigens expressed by the subject before a therapeutic agent or treatment regimen is determined.

[0269] In some embodiments, the therapeutically effective dose for a single infusion is in the range of 10^7 to 10^12 myeloid cells. The cell count can vary depending on age, weight, and other subject-related parameters, and can be determined by a physician. In some embodiments, the therapeutically effective dose is about 10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 2 x 10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 3 x 10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 4 x 10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 5 x 10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 6 x 10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 7 x 10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 8 x 10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 9 x 10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 10^8 myeloid cells. In some embodiments, the effective therapeutic dose is about 2 x 10^8 myeloid cells. In some embodiments, the effective therapeutic dose is about 3 x 10^8 myeloid cells. In some embodiments, the effective therapeutic dose is about 4 x 10^8 myeloid cells. In some embodiments, the effective therapeutic dose is about 5 x 10^8 myeloid cells. In some embodiments, the effective therapeutic dose is about 6 x 10^8 myeloid cells. In some embodiments, the effective therapeutic dose is about 7 x 10^8 myeloid cells. In some embodiments, the effective therapeutic dose is about 8 x 10^8 myeloid cells. In some embodiments, the effective therapeutic dose is about 9 x 10^8 myeloid cells. In some embodiments, the effective therapeutic dose is about 10^9 myeloid cells. In some embodiments, the effective therapeutic dose is about 2 x 10^9 myeloid cells. In some embodiments, the effective therapeutic dose is about 3 x 10^9 myeloid cells. In some embodiments, the effective therapeutic dose is about 4 x 10^9 myeloid cells. In some embodiments, the effective therapeutic dose is about 5 x 10^9 myeloid cells. In some embodiments, the effective therapeutic dose is about 6 x 10^9 myeloid cells. In some embodiments, the effective therapeutic dose is about 7 x 10^9 myeloid cells. In some embodiments, the effective therapeutic dose is about 8 x 10^9 myeloid cells. In some embodiments, the effective therapeutic dose is about 9 x 10^9 myeloid cells. In some embodiments, the effective therapeutic dose is about 10^10 myeloid cells. In some embodiments, the effective therapeutic dose is about 5 x 10^10 myeloid cells. In some embodiments, the effective therapeutic dose is about 10^11 myeloid cells. In some embodiments, the effective therapeutic dose is about 5 x 10^11 myeloid cells.In some embodiments, the therapeutically effective dose is approximately 10^12 myeloid cells.

[0270] Methods for generating novel chimeric receptor fusion protein (CFP) constructs

[0271] On the one hand, this article provides a method for generating novel chimeric receptor proteins, the method comprising, for example, identifying novel domains that can be used to enhance myeloid cell function such that, when the fusion receptor is expressed in myeloid cells, it acts as the effector myeloid cell as described herein. The generation of the fusion protein as described herein can be performed using well-known molecular cloning techniques, and the sequence can be verified after the generation of the recombinant nucleic acid.

[0272] Preparation of recombinant nucleic acids encoding chimeric antigen receptors: A recombinant nucleic acid construct encoding a chimeric antigen receptor (CAR) is prepared, the CAR being engineered for expression in myeloid cells and incorporated into a plasmid vector for amplification and / or testing of expression in eukaryotic cells. The recombinant CAR is constructed using molecular cloning techniques known in the art. The recombinant CAR protein comprises an intracellular domain, a transmembrane domain, and an extracellular domain. Each domain or sub-segment of the domain may be encoded by a nucleic acid sequence generated from a heterologous sequence via PCR and spliced ​​together by cloning individually into a vector, or ligated into a longer nucleic acid that is then inserted into a multiple cloning site of a suitable plasmid or vector with an appropriate promoter and 3'-regulatory element for amplification. In short, an exemplary CAR is prepared by incorporating a nucleic acid sequence encoding one or more signal transduction domains (e.g., a PI3 kinase recruitment domain), a nucleic acid sequence encoding a CD8 hinge and transmembrane domain, and a nucleic acid sequence encoding an extracellular domain, the nucleic acid sequence having a sequence encoding a target antigen-binding scFv at its extracellular end. Some constructs include a FLAG peptide sequence at the extracellular end, which is designed so as not to impede the binding of scFv to its target antigen. These components are linked together to form a sequence encoding a fully functional transmembrane CAR. Nucleic acid subunits encoding individual domains of the recombinant protein are designed to include a short, flexible linker sequence between the two domains. The construct is linked to a plasmid having a promoter and a 3' stable structural unit. In one variant, the construct is placed within an Alu retrotransposon element encoding ORF2p and having corresponding 5'-UTR and 3'-UTR sequences and a CMV promoter. The plasmid is amplified in *E. coli* and verified by sequencing or stored at -80°C.

[0273] Phagocytosis assay:

[0274] Antigen-linked silica or polystyrene beads in the range of 1 nm, 5 nm, or 10 nm in diameter are used for macrophage screening. Inert beads can be coated in a supporting lipid bilayer, and the antigen can be linked to the lipid bilayer. J774 macrophage cell lines can be prepared, each expressing a cloned recombinant plasma membrane protein. The recombinant plasma membrane protein can also express a fluorescent tag. The cell lines can be maintained and propagated in complete RPMI medium with heat-inactivated serum and antibiotics (penicillin / streptomycin). On the day of assay, cells can be seeded at a density of 1 x 10^6 cells / ml / well in 6-well plates, or in 12-well or 24-well plates at a relative ratio, and incubated for 2–6 hours. The cells are then washed once in phosphate-buffered saline, and the beads can be added to serum-depleted or complement-depleted nutrient medium. Cells can be visualized by optical microscopy at 30 min and 2 h after bead addition. Immunofluorescence reactions can be performed using labeled antibodies, and fluorescence confocal microscopy is used to detect the interaction and co-localization of cellular proteins during phagocytosis. The confidence level can be determined using the Kruskal-Wallis test and Dunn's multiple comparison correction.

[0275] In some instances, dye-loaded tumor cells can be fed into macrophage cell lines, and phagocytosis can be assessed using a microscope.

[0276] Cytokine production:

[0277] Macrophage cell lines can be cultured as described above. In one assay, each J774 cell line expressing a plasma membrane protein is seeded in a multi-well container and challenged with antigen-binding beads, and cytokine production is measured by collecting the supernatant at 4 and 24 hours. Cytokines can be measured from the supernatant by ELISA. In another fraction, cells can be collected at 4 and 24 hours after incubation with the beads, and flow cytometry can be performed to detect cytokines. In each case, multiple cytokines can be measured in multiplex formats, and these multiple cytokines can be selected from: IL-1α, IL-1β, IL-6, IL-12, IL-23, TNF-α, GMCSF, CXCL1, CXCL3, CXCL9, CXCL-10, MIP1-α, and MIP-2. A Macrophage Inflammatory Cytokine Array Kit (R&D Systems) is used.

[0278] Intracellular signaling pathways activated by inflammatory genes and cytokines can be identified by Western blot analysis of phosphorylation of MAP kinase, JNK, Akt signaling pathways, phosphorylation of STAT-1, and activation of interferon activation pathways.

[0279] Functional testing

[0280] Inflammator activation assay:

[0281] Activation of the NLRP3 inflammasome was determined by ELISA detection of increased IL-1 production and by Western blotting to detect caspase-1 activation, thereby detecting the cleavage of caspaseogen to produce shorter caspase. In microplate reuse settings, caspase-Glo (Promega Corporation) was used for faster readout of caspase 1 activation.

[0282] iNOS activation assay:

[0283] The activation of oxidative burst potential can be measured by iNOS activation and NO production using the AbCAM fluorescence assay kit.

[0284] Cancer cell killing assay:

[0285] Raji B cells can be used as cancer antigen-presenting cells. Raji cells can be incubated with whole-cell crude extracts of cancer cells and co-incubated with J774 macrophages. Macrophages can destroy cells 1 hour after infection, which can be detected by microscopy or by cell death assays.

[0286] Methods for preparing myeloid cells from subjects

[0287] Isolation of myeloid cells from PBMCs:

[0288] Peripheral blood mononuclear cells (PBMCs) can be isolated from the brown-yellow layer of normal donor erythrocyte sedimentation rate using density centrifugation with a Histopaque 1077 (Sigma). After washing, CD14+ PBMCs can be isolated from the PBMC fraction using CliniMACS GMP-grade CD14 microbeads and an LS separation magnetic column (Miltenyi Biotec). In short, cells can be resuspended in PEA buffer (phosphate-buffered saline [PBS] with 2.5 mmol / L EDTA and human serum albumin [0.5% Alburex 20% final volume, Octopharma]) to the appropriate concentration, incubated with CliniMACS CD14 beads according to the manufacturer's instructions, then washed and passed through a magnetized LS column. After washing, the purified PBMCs can be eluted from the demagnetized column, washed, and resuspended in the appropriate culture medium for culture. CD14+ cells were isolated using leukocyte ablation: PBMCs were collected from cirrhotic donors who provided informed consent for participation in the study via leukocyte ablation. Leukocyte ablation of peripheral blood mononuclear cells (MNCs) was performed using the Optia apheresis system with aseptic collection. A blood volume of 2.5 was processed using standard collection procedures for MNCs. CD14+ cells were isolated using a GMP-compliant functional closed system (CliniMACS Prodigy system, Miltenyi Biotechnology). In short, the leukocyte ablation product was sampled for cell counting, and aliquots were taken for pre-isolation flow cytometry. The percentage and absolute number of mononuclear cells (CD14+) could be determined, and the volume was adjusted to meet the required selection criteria (≤ 20 x 10⁻⁶) if necessary. 9 Total white blood cell count: < 400 x 10⁻⁶ 6 White blood cells / mL; ≤ 3.5 x 10 9 CD14 cells (50-300 mL volume) were isolated and separated using CliniMACS Prodigy with CliniMACS CD14 microbeads (Medical Device Class III), TS510 tubing, and the LP-14 procedure. At the end of the procedure, selected CD14+ positive monocytes were washed in PBS / EDTA buffer (CliniMACS buffer, Miltenyi) containing pharmaceutical grade 0.5% human albumin (Alburex) and then resuspended in TexMACS (or comparative) medium for culture.

[0289] Cell count and purity:

[0290] Total MNCs and isolated monocyte fractions were counted using a Sysmex XP-300 automated analyzer (Sysmex). Absolute cell counts were determined by flow cytometry using TruCount tubes (Becton Dickinson), as Sysmex consistently underestimates monocyte counts. Purity of the isolates was assessed using flow cytometry (FACSCanto II, BD Biosciences) with a panel of antibodies against human leukocytes (CD45-VioBlue, CD15-FITC, CD14-PE, CD16-APC), and product quality was assessed by determining the amount of neutrophil contamination (CD45int, CD15pos).

[0291] Cell culture - Culturing with healthy donor samples

[0292] The optimal culture medium for macrophage differentiation was studied, and three candidates could be tested using cell products. Additionally, the effects of monocyte cryopreservation on the therapeutic use of myeloid-derived cells and macrophages were examined. Functional assays could be performed to quantify the phagocytic capacity and additional polarization capacity of myeloid cells and macrophages, as well as their phagocytic potential as described elsewhere in this disclosure.

[0293] Full-scale process validation using subject samples

[0294] In a culture bag (MACS GMP differentiation bag, Miltenyi Biotechnology) equipped with GMP-grade TexMACS (Meticlin Biotechnology) and 100 ng / mL M-CSF, monocytes cultured from Prodigy after leukocyte removal can be cultured at a rate of 1 cm⁻¹. 2 and 2 x 10 per mL 6 Mononuclear cells were cultured. The mononuclear cells were cultured using GMP-compliant recombinant human M-CSF (R&D Systems). Cells were cultured for 7 days at 37°C in a humid atmosphere with 5% CO2. During culture (days 2 and 4), two 50% volume replenishments of culture medium were performed, in which 50% of the medium was removed and then fresh medium supplemented with 200 ng / mL M-CSF was added (to restore the final concentration of 100 ng / mL).

[0295] Cell collection:

[0296] For macrophages from normal donors, cells can be removed from the wells on day 7 using cell dissociation buffer (Gibco, Thermo Fisher) and pipettes. Cells can be resuspended in PEA buffer and counted, and then approximately 1 x 10⁻⁶ cells can be used per test. 6 Cells were stained for flow cytometry. On day 7, leukocytes were removed from the culture bag to remove leukocyte-derived macrophages using PBS / EDTA buffer (CliniMACS buffer, Miltenyi Biotechnology) containing pharmaceutical-grade 0.5% human albumin (HAS; Alburex) from serum. The collected cells could be resuspended in an excipient consisting of two licensed products: 0.9% infusion saline (Baxter) and 0.5% human albumin (Alburex).

[0297] Flow cytometry characterization:

[0298] The expression of surface markers on monocytes and macrophages can be analyzed using FACSCanto II (BD Biosciences) or MACSQuant 10 (Mitengene). Typically, approximately 20,000 events can be obtained per sample. Cell surface expression of leukocyte markers in freshly isolated and day 7 mature cells is performed by incubating cells with a specific antibody (final dilution 1:100). Cells are incubated with an FcR block (Mitengene) for 5 minutes, followed by incubation at 4°C with the antibody mixture for 20 minutes. Cells can be washed in PEA and the dead cell exclusion dye DRAQ7 (BioLegend) is added at a 1:100 dilution. Cells can be stained against a range of surface markers, including CD45-VioBlue, CD14-PE or CD14-PerCP-Vio700, CD163-FITC, CD169-PE, and CD16-APC (all from Miltenyi Biotechnology), CCR2-BV421, CD206-FITC, CXCR4-PE, and CD115-APC (all from eBioscience), as well as 25F9-APC and CD115-APC (eBioscience). Both monocytes and macrophages can be gated using forward and side scattering and the DRAQ7 dead cell detector (eBioscience) to exclude debris, duplexes, and dead cells, and analysis can be performed using FlowJo software (Tree Star). Based on the initial detailed phenotypic analysis, a set of release criteria (CD45-VB / CD206-FITC / CD14-PE / 25F9APC / DRAQ7) was developed, defining the development of functional macrophages derived from monocytes. Macrophages were identified as having a mean fluorescence intensity (MFI) five times that of day 0 monocytes for both 25F9 and CD206. A second set of markers was developed, evaluating other biomarkers as part of an extended set (consisting of CCR2-BV421 / CD163-FITC / CD169-PE / CD14-PerCP-Vio700 / CD16-APC / DRAQ7), but not as part of the release criteria for the cell product.

[0299] The erythrocyte sedimentation rate (ESR) layer formed in the sucrose gradient centrifugation sample of isolated peripheral blood cells was extracted, from which monocytes and macrophages could be isolated. Phagocytosis by CD14 cells could be assessed using pHRodo beads, which fluoresce only upon entering acidic endosomes. Briefly, monocytes or macrophages were cultured for 1 hour with 1–2 μL of pHRodo E. coli bioparticles (Life Technologies, Thermo Fisher Scientific), followed by removal of the culture medium and washing of the cells to remove non-phagocytic particles. Phagocytosis was assessed using an EVOS microscope (Thermo Fisher Scientific), images were captured, and cellular uptake of the beads was quantified using ImageJ software (NIH). The ability to polarize toward defined differentiated macrophages was examined by treating macrophages for 48 hours on day 7 with IFNγ (50 ng / mL) or IL-4 (20 ng / mL) to induce polarization toward the M1 or M2 phenotype (or M[IFNγ] versus M[IL-4], respectively). After 48 hours, cells were visualized using an EVOS bright-field microscope, and then collected and subjected to phenotypic analysis as described previously. Further analysis of the macrophage cytokine and growth factor secretion profiles was performed post-production and in response to inflammatory stimuli. Macrophages could be generated from the erythrocyte sedimentation rate (ESR) tannin layer of healthy donors as described previously, either untreated or stimulated with TNFα (50 ng / mL, Peprotech) and polyinosine:polycytidylic acid (poly I:C, a viral homolog bound to TLR3, 1 g / mL, Sigma) to mimic conditions present in inflamed liver, or stimulated with lipopolysaccharide (LPS, 100 ng / mL, Sigma) plus IFNγ (50 IU / mL, Peprotech) to induce maximum macrophage activation. On day 7, macrophages could be incubated overnight, and the supernatant was collected and decelerated by rotation to remove debris, then stored at -80°C until testing. Secretome analysis was performed using the 27-plex human cytokine kit and the 9-plex matrix metalloproteinase kit, which were run on the Magpix BioRad multiplex ELISA reader.

[0300] Product stability:

[0301] Various excipients can be tested during process development, including PBS / EDTA buffer; PBS / EDTA buffer with 0.5% HAS (Alburex); 0.9% saline alone; or saline with 0.5% HAS. 0.9% saline (Baxter) with 0.5% HAS excipient was found to maintain optimal cell viability and phenotype (data not shown). The stability of macrophages from cirrhotic donors after collection was investigated in three process optimization runs, and a more limited time point range (n = 3) was evaluated in a process validation run. After collection and resuspending in the excipients (0.9% infusion saline, 0.5% human serum albumin), the bags can be stored at ambient temperature (21–22°C), and samples can be collected at 0, 2, 4, 6, 8, 12, 24, 30, and 48 hours post-collection. The release standard antibody set was run on each sample, and the activity and mean fold change from day 0 were measured according to the geometrical MFI of 25F9 and CD206. For mRNA products, all excipients and equipment must be satisfactorily RNase-treated and RNase-free.

[0302] Statistical analysis:

[0303] Results can be expressed as mean ± SD. Use the unpaired two-tailed t-test of GraphPad Prism 6 whenever possible to assess the statistical significance of differences. A result is considered statistically significant when the p-value is < 0.05.

[0304] This document also provides a cell comprising the compositions, carriers, or peptides described herein. In some embodiments, the cell is a phagocyte. In some embodiments, the cell is a stem cell-derived cell, myeloid cell, macrophage, dendritic cell, lymphocyte, mast cell, monocyte, neutrophil, microglia, or astrocyte. In some embodiments, the cell is an autologous cell. In some embodiments, the cell is an allogeneic cell. In some embodiments, the cell is an M1 cell. In some embodiments, the cell is an M2 cell. In some embodiments, the cell is an M1 macrophage. In some embodiments, the cell is an M2 macrophage. In some embodiments, the cell is an M1 myeloid cell. In some embodiments, the cell is an M2 myeloid cell.

[0305] This document also provides a method for treating a disease in a subject in need, the method comprising: administering a pharmaceutical composition described herein to the subject. In some embodiments, the disease is cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is selected from the group consisting of: ovarian cancer, suitable cancers including ovarian cancer, kidney cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, and lung cancer. In some embodiments, the cancer is a liquid-filled tumor. In some embodiments, the liquid-filled tumor is leukemia or lymphoma. In some embodiments, the liquid-filled tumor is T-cell lymphoma. In some embodiments, the disease is a T-cell malignancy.

[0306] In some embodiments, administration includes infusion or injection. In some embodiments, administration includes direct application to solid tumors. In some embodiments, administration includes circRNA-based delivery procedures, mRNA-encapsulated nanoparticle-based delivery procedures, mRNA delivery procedures, particle-based delivery procedures, liposome-based delivery procedures, or exosome-based delivery procedures.

[0307] In some embodiments, a CD4+ T cell response or a CD8+ T cell response is elicited in the subject.

[0308] This document provides a method for administering a therapeutic agent comprising any of the above-described compositions. In some embodiments, the therapeutic agent is administered via a parenteral route.

[0309] In some embodiments, the pharmaceutical substance is mRNA. In some embodiments, the pharmaceutical product is an aqueous or organic solution containing mRNA. In some embodiments, the pharmaceutical substance is in an aqueous excipient. In some embodiments, the pharmaceutical product may contain a lipid component. In some embodiments, the pharmaceutical product may contain delivery nanoparticles encapsulating mRNA. In some embodiments, the mRNA is delivered systemically to a human subject in need. Concentration and total volume are determined based on several factors, such as age, weight, etc. In some embodiments, the pharmaceutical composition, such as the pharmaceutical product, is delivered systemically, for example, via intravenous, skin, subcutaneous, inhalation, oral, intramuscular, or other routes.

[0310] In some embodiments, the therapeutic agent is administered via intravenous administration. In some embodiments, the therapeutic agent is administered via subcutaneous administration. In some embodiments, the therapeutic agent is administered via intramuscular administration.

[0311] This document also provides a method for preparing a pharmaceutical composition comprising one or more recombinant nucleic acids described herein and lipids in an aqueous composition described herein. In some embodiments, the composition comprises a carrier described herein. In some embodiments, the lipids comprise lipid nanoparticles.

[0312] Pharmaceutical Composition

[0313] In some embodiments, the pharmaceutical composition comprises the composition provided herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is an aqueous formulation. In some embodiments, the pharmaceutical composition is an aqueous pharmaceutical formulation having a stable shelf life of at least 6 months. In some embodiments, the aqueous pharmaceutical formulation is lyophilizable. In some embodiments, the lyophilized composition of the aqueous pharmaceutical formulation further comprises a lyophilization protectant. In some embodiments, the lyophilization protectant is sucrose. In some embodiments, the lyophilized composition has a stable shelf life of at least 6 months.

[0314] In some embodiments, this document provides a pharmaceutical composition comprising a dose of recombinant mRNA. In some embodiments, the engineered RNA (which may be interchangeably referred to as recombinant mRNA) encodes TROP2-binding CFP. In some embodiments, this document provides a pharmaceutical composition comprising a dose of engineered RNA (engineered mRNA) encoding anti-TROP2 CFP. In some embodiments, the pharmaceutical composition provided herein comprises a dose of recombinant mRNA, wherein the recombinant mRNA comprises a sequence encoding a chimeric fusion protein (CFP) comprising (i) an extracellular domain comprising a TROP2-binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain. In some embodiments, the transmembrane domain comprises a CD89 transmembrane domain. In some embodiments, the transmembrane domain is operatively linked to the extracellular domain. In some embodiments, the intracellular domain comprises a CD89 intracellular domain. Unless otherwise stated, each dose measurement indicates the amount of RNA in the pharmaceutical composition, in mg. The RNA is mRNA, which is engineered mRNA. For example, a 1 mg / kg drug product (DP), such as anti-TROP2-CD89 RNA: for a subject who has received a single dose of LNP, the LNP contains 1 mg of RNA per kilogram of body weight. A reported 1.0 mg / mL is 1.0 mg mRNA / mL mRNA-LNP product. For example, a dose of 0.10 mg / kg is “0.10 mg mRNA / kg patient body weight”.

[0315] In some embodiments, the pharmaceutical composition comprises a dose of engineered RNA, wherein the engineered RNA (e.g., engineered mRNA) contains a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (i) an extracellular domain comprising an anti-TROP2 binding domain; and (ii) a CD89 transmembrane domain operatively connected to the extracellular domain.

[0316] In some embodiments, the dose is about 0.0005 mg / kg to about 0.5 mg / kg of engineered mRNA.

[0317] In some embodiments, the dose is about 0.0005 to about 0.001, about 0.001 to about 0.005, about 0.005 to about 0.01, about 0.01 to about 0.05, about 0.05 to about 0.1, or about 0.1 to about 0.5 mg / kg of recombinant mRNA. In some embodiments, the dosage is about 0.001 mg / kg to about 0.0015 mg / kg, about 0.0015 mg / kg to about 0.002 mg / kg, about 0.002 mg / kg to about 0.0025 mg / kg, about 0.0025 mg / kg to about 0.003 mg / kg, about 0.003 mg / kg to about 0.0035 mg / kg, about 0.0035 mg / kg to about 0.004 mg / kg, about 0.004 mg / kg to about 0.0045 mg / kg, about 0.0045 mg / kg to about 0.005 mg / kg, about 0.0055 mg / kg to about 0.006 mg / kg, about 0.006 mg / kg to about 0.0065 mg / kg, or about 0.0065 mg / kg to about 0.007 mg / kg. Engineered mRNA at concentrations of mg / kg, about 0.007 mg / kg to about 0.0075 mg / kg, about 0.0075 mg / kg to about 0.008 mg / kg, about 0.008 mg / kg to about 0.0085 mg / kg, about 0.0085 mg / kg to about 0.009 mg / kg, about 0.009 mg / kg to about 0.0095 mg / kg, or about 0.0095 mg / kg to about 0.01 mg / kg.In some embodiments, the dosage is about 0.01 mg / kg to about 0.015 mg / kg, about 0.015 mg / kg to about 0.02 mg / kg, about 0.02 mg / kg to about 0.025 mg / kg, about 0.025 mg / kg to about 0.03 mg / kg, about 0.03 mg / kg to about 0.035 mg / kg, about 0.035 mg / kg to about 0.04 mg / kg, about 0.04 mg / kg to about 0.045 mg / kg, about 0.045 mg / kg to about 0.05 mg / kg, about 0.05 mg / kg to about 0.055 mg / kg, about 0.055 mg / kg to about 0.06 mg / kg, about 0.06 mg / kg to about 0.065 mg / kg, about 0.065 mg / kg to about 0.07 mg / kg, about 0.07 mg / kg to about 0.075 mg / kg, about 0.075 mg / kg. Engineered mRNA at a concentration of mg / kg to about 0.08 mg / kg, about 0.08 mg / kg to about 0.085 mg / kg, about 0.085 mg / kg to about 0.09 mg / kg, about 0.09 mg / kg to about 0.095 mg / kg, or about 0.095 mg / kg to about 0.1 mg / kg.

[0318] In some embodiments, the recombinant mRNA is administered at doses of about 0.001 mg / kg, about 0.0015 mg / kg, about 0.002 mg / kg, about 0.0025 mg / kg, about 0.003 mg / kg, about 0.0035 mg / kg, about 0.004 mg / kg, about 0.0045 mg / kg, about 0.005 mg / kg, about 0.0055 mg / kg, about 0.006 mg / kg, about 0.0065 mg / kg, about 0.007 mg / kg, about 0.0075 mg / kg, about 0.008 mg / kg, about 0.0085 mg / kg, about 0.009 mg / kg, about 0.0095 mg / kg, or about 0.01 mg / kg. In some embodiments, the engineered mRNA is present in doses of about 0.01 mg / kg, about 0.015 mg / kg, about 0.02 mg / kg, about 0.025 mg / kg, about 0.03 mg / kg, about 0.035 mg / kg, about 0.04 mg / kg, about 0.045 mg / kg, about 0.05 mg / kg, about 0.055 mg / kg, about 0.06 mg / kg, about 0.065 mg / kg, about 0.07 mg / kg, about 0.075 mg / kg, about 0.08 mg / kg, about 0.085 mg / kg, about 0.09 mg / kg, about 0.095 mg / kg, or about 0.1 mg / kg.

[0319] In some embodiments, the dose is at least about 0.001 mg / kg, at least about 0.0015 mg / kg, at least about 0.002 mg / kg, at least about 0.0025 mg / kg, at least about 0.003 mg / kg, at least about 0.0035 mg / kg, at least about 0.004 mg / kg, at least about 0.0045 mg / kg, at least about 0.005 mg / kg, at least about 0.0055 mg / kg, at least about 0.006 mg / kg, at least about 0.0065 mg / kg, at least about 0.007 mg / kg, at least about 0.0075 mg / kg, at least about 0.008 mg / kg, at least about 0.0085 mg / kg, at least about 0.009 mg / kg, at least about 0.0095 mg / kg, or at least about 0.01 mg / kg of recombinant mRNA. In some embodiments, the dose is at least about 0.01 mg / kg, at least about 0.015 mg / kg, at least about 0.02 mg / kg, at least about 0.025 mg / kg, at least about 0.03 mg / kg, at least about 0.035 mg / kg, at least about 0.04 mg / kg, at least about 0.045 mg / kg, at least about 0.05 mg / kg, at least about 0.055 mg / kg, at least about 0.06 mg / kg, at least about 0.065 mg / kg, at least about 0.07 mg / kg, at least about 0.075 mg / kg, at least about 0.08 mg / kg, at least about 0.085 mg / kg, at least about 0.09 mg / kg, at least about 0.095 mg / kg, or at least about 0.1 mg / kg of engineered mRNA.

[0320] In some embodiments, when stored in a container, the concentration of recombinant mRNA in the pharmaceutical composition is between about 1.5 mg / mL and about 0.5 mg / mL, or between about 1.3 mg / mL and about 0.7 mg / mL. In some embodiments, the container is a single-use or reusable vial.

[0321] In some embodiments, the pharmaceutical composition is formulated for intravenous delivery.

[0322] method

[0323] This document provides a method for delivering in vivo chimeric antigen receptors (CARs) of innate immune cells as polynucleotides, such as ribonucleic acid (e.g., mRNA) constructs. In some embodiments, lipid nanoparticles (LNPs) encapsulate engineered mRNA constructs for in vivo delivery. In some embodiments, the mRNA constructs are delivered systemically via LNPs. In some embodiments, systemic delivery of mRNA encoding CARs against tumor-associated antigens results in the generation of CAR myeloid cells in vivo and leads to antigen-specific activation and tumor cell killing.

[0324] In some embodiments, the engineered mRNA is primarily expressed in myeloid cells in vivo. In some embodiments, the engineered mRNA is expressed in vascularized tissue of the subject after infusion. In some embodiments, expression of the sequence encoded by the engineered mRNA is detectable in cells of the subject approximately 72 hours after infusion. In some embodiments, administration of the pharmaceutical composition does not produce a cytokine response.

[0325] This article provides a method for treating cancer in a subject of need, the method comprising administering to the subject an effective amount of the pharmaceutical composition provided herein. In some embodiments, the subject is a human being. In some embodiments, administration comprises intravenous administration to the subject.

[0326] In some embodiments, the subject is pretreated with a drug, medicament, immunomodulator, or immunosuppressant to prepare the therapy described herein. For example, a specific dose of fludarabine and / or cycloheximide may be administered to the subject at least once before initiating treatment with a therapeutic agent described herein delivered by an LNP (e.g., engineered RNA containing a sequence encoding a CAR). In some embodiments, a dose of fludarabine and / or cycloheximide may be administered to the subject at specific intervals before the first or subsequent administration of a therapeutic agent containing engineered RNA (containing a sequence encoding a CAR). In some embodiments, an anti-inflammatory drug, antiemetic drug, antipyretic drug, antihistamine, H2 blocker (H2 receptor antagonist), β-adrenergic blocker, or anticancer drug may be administered to the subject, as determined by a medically trained person in charge of the subject, or as instructed in the protocol. In some embodiments, cytokines may be administered to the subject. In some embodiments, cytokines may be administered at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, or more as needed, and as determined by a medically trained person. In some embodiments, the cytokine is colony-stimulating factor (CSF). In some embodiments, the cytokine is granulocyte colony-stimulating factor (GCSF or CSF3). In some embodiments, the cytokine is granulocyte colony-stimulating factor (MCSF or CSF1). In some embodiments, the cytokine is granulocyte-macrophage colony-stimulating factor (GMCSF). In some embodiments, the cytokine is IL1. In some embodiments, the cytokine is IL2. In some embodiments, the cytokine is TNF-α. In some embodiments, the cytokine is interferon-γ (IFN-γ).

[0327] In some embodiments, the effective amount of the pharmaceutical composition ranges from 0.01 mg / kg / dose to 3.0 mg / kg / dose, where milligrams (mg) refer to the amount of engineered mRNA (encoding CAR) in a unit dose of the drug administered per kilogram of subject body weight. A dose generally refers to the amount (absolute or relative) administered to the subject in a single administration. Administration can be, for example, by injection or, for example, by infusion. An absolute value of a dose may be expressed in the example: x mg, which indicates that a total of x mg is administered to the subject in a single administration. A relative value of a dose may be expressed in the example: x mg / kg, which indicates that x mg / kg of the subject's body weight is administered in a single administration; for example, if the subject weighs 80 kg, the subject receives 80 mg of the drug in a single administration. In some embodiments, the effective amount of the pharmaceutical composition ranges from 0.05 mg / kg / dose to 2.5 mg / kg / dose. In some embodiments, the effective amount of the pharmaceutical composition ranges from 0.1 mg / kg / dose to 1.0 mg / kg / dose.

[0328] In some embodiments, the effective amount of the pharmaceutical composition comprises about 0.1 mg / kg / dose, 0.15 mg / kg / dose, 0.2 mg / kg / dose, 0.22 mg / kg / dose, 0.25 mg / kg / dose, 0.275 mg / kg / dose, 0.3 mg / kg / dose, 0.32 mg / kg / dose, 0.33 mg / kg / dose, 0.34 mg / kg / dose, 0.35 mg / kg / dose, 0.36 mg / kg / dose, 0.38 mg / kg / dose, 0.4 mg / kg / dose, 0.45 mg / kg / dose, 0.475 mg / kg / dose, 0.5 mg / kg / dose, 0.525 mg / kg / dose, 0.55 mg / kg / dose, 0.575 mg / kg / dose, 0.6 mg / kg / dose, 0.7 mg / kg / dose, 0.8 mg / kg / dose, 0.9 mg / kg / dose, or 1.0 mg / kg / dose. mg / kg / dosage.

[0329] In some embodiments, an effective amount of the pharmaceutical composition is administered via intravenous (IV) infusion over 60 minutes per dose.

[0330] In some embodiments, an effective amount of the pharmaceutical composition is administered in 2, 3, 4, 5, 6, 7, 8, 9, 10 or more IV infusion doses.

[0331] In some embodiments, an effective amount of the pharmaceutical composition is administered at weekly intervals.

[0332] In some embodiments, an effective amount of the pharmaceutical composition is administered at intervals of once every 10 days.

[0333] In some embodiments, an effective amount of the pharmaceutical composition is administered at intervals of once every two weeks.

[0334] In some embodiments, an effective amount of the pharmaceutical composition is administered in at least five doses.

[0335] In some embodiments, an effective amount of the pharmaceutical composition is administered at a dose of 1 mg / kg on days 1, 8, 15, 29, 36, and 43.

[0336] This article provides a method for treating cancer in a subject of need, the method comprising administering to the subject a pharmaceutical composition comprising recombinant mRNA, wherein the recombinant mRNA comprises a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: an extracellular domain comprising a TROP2 binding domain; and a CD89 transmembrane domain operatively linked to the extracellular domain. In some embodiments, the method comprises administering the pharmaceutical composition to the subject for at least one treatment cycle. In some embodiments, the method comprises administering the pharmaceutical composition to the subject for at least two treatment cycles. In some embodiments, the method comprises administering the pharmaceutical composition to the subject for at least three treatment cycles. In some embodiments, the method comprises administering the pharmaceutical composition to the subject for at least four treatment cycles. In some embodiments, the method comprises administering the pharmaceutical composition to the subject for at least five treatment cycles. In some embodiments, the method comprises administering the pharmaceutical composition to the subject for at least six treatment cycles.

[0337] In some embodiments, for each treatment cycle, the pharmaceutical composition is administered to the subject approximately once a week (QW), approximately once every 2 weeks (Q2W), approximately once every 3 weeks (Q3W), approximately once every 4 weeks (Q4W), approximately once every 5 weeks (Q5W), approximately once every 6 weeks (Q6W), approximately once every 7 weeks (Q7W), approximately once every 8 weeks (Q8W), approximately once every 9 weeks (Q9W), approximately once every 10 weeks (Q10W), approximately once every 11 weeks (Q11W), or approximately once every 12 weeks (Q12W). In some embodiments, the method comprises administering the pharmaceutical composition to the subject at QW, Q2W, or Q4W for each treatment cycle. In some embodiments, the method comprises administering the pharmaceutical composition to the subject at the same frequency in each treatment cycle. In some embodiments, the method comprises administering the pharmaceutical composition to the subject at different frequencies for at least two treatment cycles. In some embodiments, the method comprises administering the pharmaceutical composition to the subject at QW or Q2W in the first treatment cycle.

[0338] In some embodiments, the method includes administering the pharmaceutical composition to the subject at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least eleven times, or at least twelve times during the first treatment cycle. In some embodiments, the method includes administering the pharmaceutical composition to the subject at least once to at least three times, at least three times to at least six times, at least six times to at least nine times, or at least nine times to at least twelve times during the first treatment cycle. In some embodiments, the method includes administering the pharmaceutical composition to the subject at most once, at most twice, at most three times, at most four times, at most five times, at most six times, at most seven times, at most eight times, at most nine times, at most ten times, at most eleven times, or at most twelve times during the first treatment cycle. In some embodiments, the method includes administering the pharmaceutical composition to the subject up to 1 to 3 times, up to 3 to 6 times, up to 6 to 9 times, or up to 9 to 12 times. In some embodiments, a first cycle includes administering the pharmaceutical composition to the subject between 1 and 12 times during a first treatment cycle.

[0339] In some embodiments, the method includes administering the pharmaceutical composition to the subject between about 1 and about 3 times, about 3 and about 6 times, about 6 and about 9 times, or about 9 and about 12 times during the first treatment cycle.

[0340] In some embodiments, the method includes administering the pharmaceutical composition to the subject three times during the first treatment cycle.

[0341] In some embodiments, the second cycle follows the first cycle. In some embodiments, the method further includes administering the pharmaceutical composition to the subject during the second cycle.

[0342] In some embodiments, during the second cycle, the pharmaceutical composition is administered to the subject at approximately QW, approximately Q2W, approximately Q3W, approximately Q4W, approximately Q5W, approximately Q6W, approximately Q7W, approximately Q8W, approximately Q9W, approximately Q10W, approximately Q11W, or approximately Q12W. In some embodiments, the method includes administering the pharmaceutical composition to the subject at approximately Q4W during the second cycle.

[0343] In some embodiments, the method includes administering the pharmaceutical composition to the subject at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least eleven times, or at least twelve times during the second cycle. In some embodiments, the method includes administering the pharmaceutical composition to the subject at least once to at least three times, at least three times to at least six times, at least six times to at least nine times, or at least nine times to at least twelve times during the second cycle. In some embodiments, the method includes administering the pharmaceutical composition to the subject at most once, at most twice, at most three times, at most four times, at most five times, at most six times, at most seven times, at most eight times, at most nine times, at most ten times, at most eleven times, or at most twelve times during the second cycle. In some embodiments, the method includes administering the drug composition to the subject at most 1 to 3 times, at most 3 to 6 times, at most 6 to 9 times, or at most 9 to 12 times during the second cycle. In some embodiments, the method includes administering the drug composition to the subject between 1 and 12 times during the second cycle. In some embodiments, the method includes administering the drug composition to the subject between about 1 and about 3 times, about 3 and about 6 times, about 6 and about 9 times, or about 9 and about 12 times during the second cycle.

[0344] In some embodiments, the method includes administering the pharmaceutical composition to the subject three times during the second cycle.

[0345] In some embodiments, the method includes administering the pharmaceutical composition to the subject for three treatment cycles. In some embodiments, the method includes administering the pharmaceutical composition to the subject at the same dose for each of the three treatment cycles.

[0346] In some embodiments, the method comprises administering a drug composition to a subject for four treatment cycles. In some embodiments, the method comprises administering the drug composition to the subject every 2 weeks in the first cycle and every 4 weeks in the second, third, and fourth cycles. In some embodiments, the method comprises administering the drug composition at a dose of about 0.0050 to about 0.05 mg / kg or about 0.0075 to about 0.03 mg / kg. In some embodiments, the method comprises administering the drug composition to the subject once a week in the first cycle and once every 4 weeks in the second, third, and fourth cycles.

[0347] In some embodiments, the method comprises administering an effective amount of the pharmaceutical composition to a subject.

[0348] In some embodiments, the effective amount of the pharmaceutical composition is from about 0.001 mg / kg to about 0.0015 mg / kg, from about 0.0015 mg / kg to about 0.002 mg / kg, from about 0.002 mg / kg to about 0.0025 mg / kg, from about 0.0025 mg / kg to about 0.003 mg / kg, from about 0.003 mg / kg to about 0.0035 mg / kg, from about 0.0035 mg / kg to about 0.004 mg / kg, from about 0.004 mg / kg to about 0.0045 mg / kg, from about 0.0045 mg / kg to about 0.005 mg / kg, from about 0.0045 mg / kg to about 0.005 mg / kg, from about 0.0055 mg / kg to about 0.006 mg / kg, from about 0.006 mg / kg to about 0.0065 mg / kg, from about 0.0065 mg / kg to about 0.007 mg / kg. The range of engineered RNA (e.g., recombinant mRNA) is from about 0.007 mg / kg to about 0.0075 mg / kg, about 0.0075 mg / kg to about 0.008 mg / kg, about 0.008 mg / kg to about 0.0085 mg / kg, about 0.0085 mg / kg to about 0.009 mg / kg, about 0.009 mg / kg to about 0.0095 mg / kg, or about 0.0095 mg / kg to about 0.01 mg / kg.

[0349] In some embodiments, the effective amount of the pharmaceutical composition is from about 0.01 mg / kg to about 0.015 mg / kg, from about 0.015 mg / kg to about 0.02 mg / kg, from about 0.02 mg / kg to about 0.025 mg / kg, from about 0.025 mg / kg to about 0.03 mg / kg, from about 0.03 mg / kg to about 0.035 mg / kg, from about 0.035 mg / kg to about 0.04 mg / kg, from about 0.04 mg / kg to about 0.045 mg / kg, from about 0.045 mg / kg to about 0.05 mg / kg, from about 0.05 mg / kg to about 0.055 mg / kg, from about 0.055 mg / kg to about 0.06 mg / kg, from about 0.06 mg / kg to about 0.065 mg / kg, from about 0.065 mg / kg to about 0.07 mg / kg, from about 0.07 mg / kg to about 0.075 mg / kg. The range of engineered RNA is approximately 0.075 mg / kg to approximately 0.08 mg / kg, approximately 0.08 mg / kg to approximately 0.085 mg / kg, approximately 0.085 mg / kg to approximately 0.09 mg / kg, approximately 0.09 mg / kg to approximately 0.095 mg / kg, or approximately 0.095 mg / kg to approximately 0.1 mg / kg.

[0350] In some embodiments, the effective amount of the pharmaceutical composition comprises about 0.001 mg / kg, about 0.0015 mg / kg, about 0.002 mg / kg, about 0.0025 mg / kg, about 0.003 mg / kg, about 0.0035 mg / kg, about 0.004 mg / kg, about 0.0045 mg / kg, about 0.005 mg / kg, about 0.0055 mg / kg, about 0.006 mg / kg, about 0.0065 mg / kg, about 0.007 mg / kg, about 0.0075 mg / kg, about 0.008 mg / kg, about 0.0085 mg / kg, about 0.009 mg / kg, about 0.0095 mg / kg, or about 0.01 mg / kg of engineered RNA.

[0351] In some embodiments, the effective amount of the pharmaceutical composition comprises about 0.01 mg / kg, about 0.015 mg / kg, about 0.02 mg / kg, about 0.025 mg / kg, about 0.03 mg / kg, about 0.035 mg / kg, about 0.04 mg / kg, about 0.045 mg / kg, about 0.05 mg / kg, about 0.055 mg / kg, about 0.06 mg / kg, about 0.065 mg / kg, about 0.07 mg / kg, about 0.075 mg / kg, about 0.08 mg / kg, about 0.085 mg / kg, about 0.09 mg / kg, about 0.095 mg / kg, or about 0.1 mg / kg of engineered RNA.

[0352] This article provides a method for treating cancer in a subject in need, the method comprising administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising engineered RNA (mRNA), wherein the engineered mRNA comprises a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (i) an extracellular domain comprising an anti-TROP2 binding domain; and (ii) a CD89 transmembrane domain operatively linked to the extracellular domain; wherein the subject has cancer selected from the group consisting of: cervical cancer, colorectal cancer, esophageal cancer, gastric adenocarcinoma, HR+ / HER2- breast cancer, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic ductal adenocarcinoma, triple-negative breast cancer, and urothelial carcinoma.

[0353] In some embodiments, the cancer is advanced or metastatic. In some embodiments, the cancer comprises advanced or metastatic epithelial carcinoma.

[0354] In some embodiments, cancer includes tumors. In some embodiments, tumors are associated with cervical cancer, colorectal cancer, esophageal cancer, gastric adenocarcinoma, HR+ / HER2- breast cancer, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic ductal adenocarcinoma, triple-negative breast cancer, or urothelial carcinoma.

[0355] In some embodiments, administration of the pharmaceutical composition is associated with a reduction or improvement in tumors in the subject.

[0356] In some embodiments, the treatment includes reducing the tumor by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or at least 10% after treatment.

[0357] In some embodiments, the treatment includes alleviating at least one of the symptoms associated with cancer.

[0358] This article provides a method for treating cancer in a subject in need, the method comprising administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising engineered RNA, wherein the engineered RNA comprises a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (i) an extracellular domain comprising an anti-TROP2 binding domain; and (ii) a CD89 transmembrane domain operatively linked to the extracellular domain; wherein the subject does not have: active CNS metastases; carcinomatous meningitis; clinically significant heart disease; previous allogeneic bone marrow transplantation; previous solid organ transplantation; active autoimmune disease; active acute or chronic infection; liver tumor involvement greater than 50%; or no prior splenectomy. Typically, for first-time human studies, study participants (e.g., patients) are selected based on a number of inclusion and exclusion criteria determined at initial screening, including but not limited to the patient's disease symptoms and various physiological parameters as described below and more specifically in the examples. Typically, in most cases, a baseline for any health parameter, such as tumor size, stage, patient weight, body temperature, etc., is established during an initial period before the start of a study with the new drug, and this initial or baseline measurement can be compared with subsequent measurements taken during and after the progression of the treatment regimen using the new drug. However, the baseline for any health / medical parameter at any given time during or after the study can be an immediately earlier measurement or any previous measurement deemed appropriate and necessary by a medical expert in the art. In some embodiments, a treatment regimen or schedule can be described, which can be considered as a description of a schedule for administering a drug at predetermined intervals over a period of time at a specific dose or predetermined dose or dosage (e.g., amount, concentration). For example, Figure 10A and 10B The dosing regimen for the planned study is described.

[0359] Inclusion criteria: In some embodiments, if a subject meets the inclusion criteria, the pharmaceutical composition provided herein may be administered to him / her. In some embodiments, inclusion criteria may include the following: the subject is an adult ≥ 18 years of age; and the subject has signed an informed consent form (ICF); for example, additionally, the subject has histologically proven metastatic or advanced epithelial carcinoma, wherein the cancer is selected from the group consisting of: urothelial carcinoma, cervical cancer, ovarian epithelial carcinoma, triple-negative breast cancer, HR+ / HER2- breast cancer, pancreatic ductal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, non-small cell lung cancer, and colorectal cancer; the subject has progressive disease at baseline that is refractory or recurrent to standard of care; the subject has refused standard therapy; the subject has measurable disease based on the RECIST criteria v 1.1; the subject has an Eastern Cooperative Oncology Group (ECOG) performance status grade of 0 or 1; the subject has a life expectancy > 12 weeks; the subject has an echocardiogram (ECHO) or multigated acquisition scan showing an ejection fraction ≥ 50%; the subject has no clinically significant abnormalities at screening, or shows a mean QTc interval < 1 in males. Subjects with an ECG of 450 msec or less, and <470 msec in females (<480 msec for participants with bundle branch block); subjects with an oxygen saturation of ≥90% in room air as measured by pulse oximetry; subjects with adequate organ function as defined at screening by the following laboratory values: a. Hemoglobin ≥9.0 g / dL without transfusion support within 2 weeks prior to screening; b. Platelet count ≥100,000 / µL without transfusion support within 2 weeks prior to screening; c. Absolute neutrophil count >1000 / mm3 (without granulocyte colony-stimulating factor support within 2 weeks prior to screening); d. Creatinine clearance >45 mL / min or creatinine <1.5 x ULN as calculated using the Cockcroft-Gault equation; e. ALT <2.5 x ULN; ALT <5.0 if known liver disease due to cancer. f. AST < 2.5 x ULN; if liver disease due to cancer is known, then AST < 5.0 x ULN; g. Serum bilirubin < 1.5 x ULN (≤ 3.0 x ULN if the participant has been diagnosed with Gilbert's syndrome); or h. INR or prothrombin time (PT) ≤ 1 unless the participant is receiving anticoagulant therapy and the INR or PT is within the expected or therapeutic range of the intended use of the anticoagulant.5. ULN (Underlying University Number); subjects willing and able to provide written informed consent; subjects willing to perform and comply with all study procedures, including study-related biopsies and attending clinic visits as scheduled; male subjects who must abstain from sperm donation during study treatment or for 4 months after the last dose of study treatment; male subjects willing to use highly effective contraception or female subjects who may become pregnant.

[0360] If clear progression has been confirmed in such lesions, lesions located in previously irradiated areas can be considered measurable.

[0361] Either the Fridericia or Bazett formula can be used to correct the QT interval.

[0362] Exclusion criteria: Exclusion criteria can refer to the conditions or standards that would lead to a subject being excluded from the study when they occur in prospective subjects.

[0363] In some embodiments, exclusion criteria may include, for example, prospective subjects with the following: active CNS metastases; carcinomatous meningitis; clinically significant heart disease; previous allogeneic bone marrow transplantation; previous solid organ transplantation; active autoimmune disease; active acute or chronic infection; liver tumor involvement greater than 50%; or no previous splenectomy.

[0364] In some embodiments, if a subject does not meet the exclusion criteria, the pharmaceutical composition provided herein is administered to the subject. In some embodiments, the exclusion criteria may be the following: subjects with known active CNS metastases and / or carcinomatous meningitis; subjects with previously treated brain metastases, provided they are not radioactively stable (i.e., have demonstrated no progression for at least 4 weeks by repeated imaging), clinically stable, or have not required steroid treatment for at least 14 days prior to the first dose of the study intervention; pregnant or lactating subjects; subjects > 28 days after major surgery (including hepatectomy or joint replacement); subjects with a history of allogeneic bone marrow transplantation or solid organ transplantation; subjects with spinal cord compression not clearly treated with surgery and / or radiation; subjects with uncontrolled pleural effusion, pericardial effusion, or ascites requiring repeated drainage procedures; or subjects with any acute illness within 7 days prior to Day 1, including fever (> 100.4℉ or > 100℉). Subjects with a temperature of 38°C or higher; subjects with an active systemic bacterial, fungal, or viral infection within 7 days prior to Day 1; subjects with an active infection of any of the following human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV): a. HIV-1 positive serology; b. HCV positive serology and not having received a detectable, documented curative therapy or quantitative RNA PCR; c. Positive hepatitis B surface antigen or IgM, HBV core antibody unless quantitative DNA PCR is undetectable and the patient is receiving stable antiviral prophylaxis against HBV reactivation; 10. Other primary malignancies, except: a. adequately treated basal cell carcinoma or squamous cell carcinoma, b. cervical or bladder carcinoma in situ, cured and proven to be relapse-free for at least 2 years prior to the study, or c. Subjects who have had complete resection and complete remission of a primary malignancy for at least 2 years; subjects with a history of (non-infectious) pneumonia / interstitial lung disease requiring steroids or currently have pneumonia / interstitial lung disease; subjects with a history of immune-related AEs of grade >3, such as pneumonia, colitis, hepatitis, or nephritis; subjects with active autoimmune diseases unrelated to prior therapy for a primary malignancy requiring systemic therapy within the past year; subjects with a history of symptomatic congestive heart failure (New York Heart Association Class II-IV) or severe active arrhythmia or other clinically significant cardiac events within 12 months of enrollment; subjects who have experienced toxicity from prior anticancer therapy defined as toxicity (other than hair loss, or the above laboratory values) that has not yet subsided to NCI CTCAE v5.0 grade ≤ 1 or baseline; and subjects who have received: a. radiation therapy within 2 weeks of the first administration of the drug composition provided herein; b.a. Cytotoxic chemotherapy administered within 21 days or 5 half-lives, whichever is shorter of the administration of the drug composition provided herein; b. Immunotherapy for primary malignancies (e.g., monoclonal antibody therapy, checkpoint inhibitors) within 21 days or 5 half-lives, whichever is shorter of the first administration of the drug composition provided herein; c. Anticancer vaccines administered within 12 weeks of the first administration of the drug composition provided herein; e. COVID-19 mRNA vaccines administered within 6 weeks of the first administration of the drug composition provided herein; subjects who have received a live vaccine ≤ 6 weeks prior to the first administration of the drug composition provided herein; subjects who have received a transfusion of packed red blood cells or platelets within 2 weeks prior to the first administration of the drug composition provided herein; subjects with a history of allergic reactions to any excipient; subjects enrolled in another interventional clinical trial within 21 days or 5 half-lives of the drug, whichever is shorter of the first administration of the drug composition provided herein; or subjects with any other medical condition that the investigator deems unsuitable for participation or unable to comply with the study requirements.

[0365] In some embodiments, the subject did not test positive for COVID-19 within 7 days prior to Day 1, and if the subject tested positive within 7 days prior to Day 1, the subject was excluded from the study.

[0366] In some embodiments, subjects with chronic grade 2 toxicity (e.g., peripheral neuropathy, laboratory values) may be eligible, depending on the decision of the investigator and medical monitor.

[0367] Replacement therapies such as thyroxine, insulin, or physiological steroid replacement therapy for adrenal or pituitary insufficiency are not considered systemic treatments.

[0368] Baseline can be considered as the most recent assessment performed prior to the first dose of the study treatment. Baseline assessment can be performed within the time period defined in the protocol eligibility criteria.

[0369] Measurable lesions: In addition to lymph nodes as described below, measurable lesions are defined as those lesions that can be accurately measured as ≥ 10 mm in at least one dimension (the maximum diameter to be recorded) on a CT scan (if the CT scan slice thickness is greater than 5 mm, the minimum size of a measurable lesion is twice the slice thickness).

[0370] When assessed by CT scan (with a recommended slice thickness of no more than 5 mm), lymph nodes must be ≥ 15 mm along the short axis to be considered pathologically enlarged and measurable. Only the short axis will be measured and recorded at baseline and follow-up.

[0371] MRI can replace contrast-enhanced CT for lesions in some anatomical locations, but not for lesions in the lungs. The minimum measurable size is the same as for CT (10 mm), provided the scan is performed with a 5 mm slice thickness and without gaps. If MRI is performed with thicker slices, the baseline measurable lesion size should be twice the slice thickness. If interslice gaps exist, this should also be considered when determining the baseline measurable lesion size.

[0372] Unmeasurable lesions: In some embodiments, all other lesions (or disease sites) besides the measurable lesions discussed above, including small lesions (pathological lymph nodes with a maximum diameter < 10 mm or a short axis ≥ 10 mm to < 15 mm), may be considered unmeasurable. Lymph nodes with a short axis < 10 mm may be considered non-pathological and may not be recorded or tracked. Bone lesions, meningeal diseases, ascites, pleural / pericardial effusions, cutaneous lymphangitis / pneumonia, and abdominal masses (not subsequently treated with CT or MRI) may be considered unmeasurable. After a CR timepoint response, non-target lymph node lesions and new lymph node lesions may be measured to determine whether they are or become pathological in size.

[0373] Target lesions: In some embodiments, target lesions are measurable lesions of up to two lesions per organ, and represent a total of five lesions across all relevant organs that can be identified as target lesions and measured and recorded at baseline. Target lesions can be selected based on their size (the largest diameter lesion), representing all relevant organs, and those suitable for reproducible, repeatable measurements. Sometimes, the largest lesion may not be suitable for reproducible measurement; in such cases, the next largest lesion that can be reproducibly measured should be selected. Target lesions (the longest axis for non-lymph node lesions and the shortest axis for measurable malignant lymph node lesions) can be measured at each evaluation.

[0374] Non-target lesions: In some embodiments, in addition to those discussed under target lesions above, all non-measurable lesions (including pathological lymph nodes with a short axis of ≥ 10 mm to < 15 mm) and other lesions (or disease sites) above and above all measurable lesions of the five target lesions may be identified as non-target lesions and recorded at baseline. These lesions are generally not required to be measured, but the presence, absence, or, in rare cases, clear progression of each lesion is recorded throughout follow-up. Lymph nodes with a short axis < 10 mm may be considered non-pathological and may not be recorded or tracked. After a CR timepoint response, non-target lymph node lesions and new lymph node lesions may be measured to determine whether they are or become pathological in size.

[0375] In some embodiments, for progression of a non-target lesion to be considered as a development of measurable disease, definitive progression can be defined as substantial worsening of the non-target disease, such that the overall tumor burden has increased sufficiently to warrant discontinuation of therapy, even in cases of SD or PR within the target disease. Cystic lesions other than metastatic cystic lesions may not be considered for measurability. Osteolytic lesions or mixed lytic-fibroblastic lesions that can be assessed by cross-sectional imaging techniques such as CT or MRI can be considered measurable lesions if the soft tissue component meets the above definition of measurability. Fibroblastic bone lesions are not measurable. Chest X-rays cannot be used for response assessment in this study. The minimum size of a measurable lesion is twice the slice thickness when the CT scan slice thickness is greater than 5 mm. MRI is acceptable in certain cases, except for the lungs (e.g., for body scans). Bone scan and PET scan It can be used for bone lesion monitoring, but may not be used for RECIST evaluation. CT or MRI scans can be used to confirm the results of bone scans performed for this purpose. MRI is the preferred method for confirmation. In this study, tumor markers cannot be used to determine PD.

[0376] Ascites and effusion: New or worsening ascites or effusion is considered malignant unless cytological or histological findings suggest it is unlikely to be of non-tumor origin.

[0377] Time point assessment

[0378] The frequency and schedule for tumor assessments are defined in the protocol. The schedule should be maintained regardless of whether the study treatment is reduced, paused, delayed, or stopped.

[0379] At baseline, tumors and lymph nodes can be classified and documented as target lesions or non-target lesions as described above. It is possible to document multiple non-target lesions involving the same organ as a single item (e.g., 'multiple liver metastases'). In all post-baseline (follow-up) assessments, the baseline classification (target, non-target) can be maintained, and the lesions can be documented and described in a consistent manner over time (e.g., in the same order on the source file).

[0380] At each assessment, the sum of the diameters of all target lesions (the longest axis for non-lymph node lesions and the short axis for lymph node lesions) can be calculated and included in the source file. The baseline sum of diameters (SoD) can be used as a reference to further characterize any objective tumor regression in the measurable dimensions of the disease. The lowest SoD (minimum point) since (and including) the baseline value can be used as a reference for assessing progression.

[0381] After baseline, the actual size of the target lesion can be recorded, if possible, even if the lesion becomes very small. If the radiologist believes the lesion may have disappeared, 0 mm can be recorded. If the lesion is present but too small to be measured, the 'too small to be measured' indicator can be included in the source file.

[0382] In some embodiments, for target lesions, measurements can be taken and recorded in metric notation. Non-target lesions (presence, regression, or definite progression) can be qualitatively assessed, and new lesions can be recorded separately if any. At each assessment, the progression status will be determined based on the time-point status of target lesions, non-target lesions, and new lesions. The discovery of new lesions should not be attributed to differences in scanning techniques, changes in imaging modalities, or findings that are thought to represent something other than a tumor. Necrosis of pre-existing lesions as part of a treatment response should be excluded before defining a 'new' cystic lesion. Lesions identified in follow-up studies in anatomical locations not scanned at baseline are generally considered new lesions. If a new lesion is not clearly defined due to its small size, a repeat scan is required to confirm its presence, and progression should be declared using the date of the initial scan.

[0383] In some embodiments, time-point progression may not be based solely on bone scan findings. If necessary, bone scans will be used to guide confirmatory CT / MRI imaging. CT / MRI findings can then be used to determine progression.

[0384] Example

[0385] Example 1. The first Phase I study in humans

[0386] This case describes a Phase I, open-label, first-in-human, multiple-escalation study to investigate the safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of a second-generation construct expressing TROP2 in adults with TROP2+ metastatic colorectal cancer. In this study, the construct was programmed to deliver the anti-TROP2-Fc-α fusion receptor in the form of mRNA encoding the construct encapsulated in lipid nanoparticles.

[0387] Number of participants planned: up to 20 participants for safety and efficacy evaluation.

[0388] Adults aged 18 years or older will be screened. Subjects who provide written informed consent and meet all inclusion and exclusion criteria will be enrolled in the trial. The study will be divided into two parts. Part A will be multiple escalation doses to determine the safety, tolerability, and pharmacokinetics (PK) of the anti-TROP2 second-generation chimeric fusion protein (anti-TROP2-CD89 CAR) in subjects with TROP2+ LM; Part B will be a dose extension to determine additional safety, tolerability, PK, and preliminary efficacy in patients with TROP2+ CRLM.

[0389] An effective amount of the formulation of mRNA, which encodes an anti-TROP2 second-generation chimeric fusion protein in lipid nanoparticles, will be administered intravenously to the subject within 60 minutes. The starting dose and dosing regimen will be determined after the completion of pharmacokinetic and safety studies in non-human primates.

[0390] Baseline assessments within 4 weeks of the planned start of treatment include patient history, physical examination with vital signs and performance status, CT or MRI scan, CBC differential and platelet count, routine serum chemistry, urinalysis, INR / PTT, EKG, and serum samples for human anti-human antibody (HAHA). In women of childbearing potential, urine or serum b-HCG is also required within one week of treatment. TROP2 expression will be confirmed by immunohistochemistry in archived biopsy samples obtained within the past 6 months or fresh biopsy samples obtained during the screening period at a central laboratory.

[0391] All subjects will receive weekly infusions of the anti-TROP2 second-generation chimeric fusion protein for the first 12 weeks. Dosing may continue until week 48 if there is no disease progression or unacceptable toxicity. After week 12, patients may continue to participate in the study until week 48, but the dosing regimen will be modified to once every two weeks. If a patient's disease progresses while receiving once-every-two-week dosing, dosing may be gradually increased to weekly. If a complete responder discontinues therapy at any time during the study and develops progressive disease, the dose and dosing schedule may be resumed upon discontinuation. If a patient has a partial response (PR) and surgical resection of CRLM is possible, the patient may discontinue treatment and opt for tumor resection. Patients undergoing surgical resection will be followed up until week 48. If a patient develops progressive disease, the dose and dosing schedule may be resumed upon discontinuation.

[0392] All patients should be closely monitored during treatment. NCI CTCAE version 5.0 will be used to grade all adverse events and provide guidelines for dose reduction, delay, or discontinuation in the event of treatment-related toxicities. All patients will also undergo CT / MRI scans throughout the study to assess disease progression and response to the investigational drug.

[0393] If any Grade 2 or 3 treatment-related toxicity occurs on the scheduled treatment day, treatment will be delayed by one week. If the toxicity has subsided to Grade 1 or lower by this time, treatment may continue with a 25% dose reduction. If toxicity recurs, treatment may continue with a 50% reduction from the initial dose, and if the toxicity worsens, treatment will be permanently discontinued. The decision to continue or discontinue treatment is entirely at the physician's discretion. In the event of any Grade 4 treatment-related toxicity, the patient should be permanently discontinued.

[0394] In Part A of the study, the Continuous Reassessment Method (CRM) will be used to determine the recommended dose for Part B. The primary assessment of safety and tolerability will be performed on day 28. The primary efficacy assessment will be performed at week 12. Subjects with stable disease (SD), partial response (PR), or complete response (CR) may be enrolled in the long-term extension study at week 48.

[0395] Part B will evaluate the safety, tolerability, and efficacy of the maximum acceptable dose (MAD) from Part A in patients with CRLM. The primary efficacy assessment will be the objective response rate (ORR) at week 12.

[0396] If treatment is discontinued due to unacceptable toxicity, the patient will continue to participate in the study until disease progression occurs, at which point a study termination assessment will be conducted, and further follow-up will be required until any treatment-related toxicity subsides or stabilizes. The survival of all patients will be monitored.

[0397] To ensure the safety of study participants, the first two participants in each dose group will be staggered by 14 days. If no safety issues are found, the remaining participants can be enrolled simultaneously at the stated dose level.

[0398] The pharmaceutical composition comprises an scFv, the scFv comprising a heavy chain and a light chain, the heavy chain comprising a CDR3 having the sequence GGFGSSYWYFDV, and the light chain comprising a CDR3 having the sequence QQHYITPLT. In some embodiments, the heavy chain further comprises a CDR1 sequence of NYGMN and a CDR2 sequence of WINTYTGEPTYTDDFKG; and the light chain further comprises a CDR1 sequence of KASQDVSIAVA and a CDR2 sequence of SASYRYT. The pharmaceutical composition comprises a CD89 transmembrane domain having the sequence LIRMAVAGLVLVALLAILV.

[0399] An exemplary second-generation anti-TROP2 chimeric fusion protein (e.g., a drug) comprises an anti-TROP2 scFv, said anti-TROP2 scFv comprising the sequence (CDR highlighted according to Kabat nomenclature convention) QVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAP GQGLKWMG WINTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR G GFGSSYWYFDV Heavy chain variable structural domain of WGQGSLVTVSSG.

[0400] An exemplary anti-TROP2 chimeric fusion protein containing anti-TROP2 scFv includes a light chain variable domain having the sequence DIQLTQSPSSLSASVGDRVSIT C KASQDVSIAVA WYQQKPGKAPKLLIY SASYRY T GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLT FGAGTKVEIKR.

[0401] The exemplary TROP2 scFv construct has the following sequence,

[0402] Bold letters indicate the signal peptide sequence. Mature proteins may lack this sequence, and those skilled in the art can interpret the protein sequence expressed in the absence of the signal peptide sequence. The underlined region is the CDR sequence of scFV, which binds to TROP2 in the CDR1, CDR2, and CDR3 of the heavy and light chains. Italic letters indicate the amino acid sequence of CD89 TMD.

[0403] Exemplary TROP2 scFv products for in vivo studies may contain the sequence QVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMG WINTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSSGGGGGSGGGGSGGGGSDIQLTQSPSSSLSASVGDRVSITC KASQDVSIAVA WYQQKPGKAPKLLIY SASYRYTGVPDRFSGSSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRSGGGGAAADYKDDDDKGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK.

[0404] Exemplary TROP2 scFv products for in vivo studies may contain a sequence

[0405] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSSLSASVGDRVSITCK ASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRGSGGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK.

[0406] This method takes advantage of the natural tendency for intravenous (iv) delivery.

[0407] The proprietary mRNA construct encodes a receptor consisting of a TROP2-targeting scFv (derived from the complementarity-determining region (CDR) of sacituzumab) and the transmembrane domain and cytoplasmic tail of CD89. While LNPs are uptaken by many cell types after administration of an anti-TROP2 binder, the functional CAR is expressed only on the surface of cells that also express the common γ-chain of the Fc receptor, primarily myeloid cells. The common γ-chain includes the activation motif (ITAM) domain, which is essential for cell signaling based on tyrosine-based immune receptors. Upon TROP2 scFV recognition, the intracellular signaling domain is activated, leading to tumor cell phagocytosis, inflammatory cytokine production, and tumor antigen presentation to T cells.

[0408] Based on the observed adverse events (AEs) including all potential dose-limiting toxicities (DLTs), the primary objective of this study was to evaluate the efficacy of the anti-TROP2 second-generation chimeric fusion protein in patients with TROP2-related adverse events. +Safety and tolerability in subjects with metastatic colorectal cancer, and to establish the maximum acceptable dose (MAD) and recommended phase 2 dose (RP2D).

[0409] The secondary objectives of this study were to determine: (i) pharmacokinetics, (ii) the objective response to the second-generation TROP2 chimeric fusion protein [time range: up to 12 months], and (iii) the duration of the response.

[0410] Secondary objectives further include:

[0411] • Area under the curve [Time range: up to 12 months]

[0412] • Maximum plasma concentration [Time range: up to 12 months]

[0413] • Time to peak plasma concentration [Time range: up to 12 months]

[0414] • Half-life [Time range: up to 12 months]

[0415] • Objective response to anti-TROP2 second-generation chimeric fusion protein [Time range: up to 12 months]

[0416] • Response duration

[0417] The exploratory objectives of this study were to determine (a) preliminary efficacy, including progression-free survival (PFS) and overall survival (OS), (b) conversion to surgical candidates, and (c) relevant biomarker studies.

[0418] Typically, the objectives of exploration include:

[0419] • Progression-free survival (PFS)

[0420] • Overall lifespan (OS)

[0421] • Converted to surgical candidate

[0422] • Development of antidrug antibodies (ADAs) targeting the second-generation chimeric fusion protein of TROP2

[0423] • To identify potential response biomarkers by assessing TROP2 expression levels in tumors.

[0424] • Therapeutic effects of blood cytokines and chemokines, TCR amplification, and cell phenotypes to identify potential response biomarkers.

[0425] • Assess treatment-related impacts on tumor architecture and cell phenotype to identify potential response biomarkers.

[0426] The overall study design includes a multicenter, open-label, phase 1, first-in-human study with dose-group expansion to evaluate the safety, tolerability, pharmacokinetics (PK), and efficacy of a second-generation anti-TROP2 binder in subjects with TROP2+ colorectal liver metastases. Screening will be conducted on adults aged 18 years or older. Subjects who provide written informed consent and meet all inclusion and exclusion criteria will be enrolled in the trial. The study will be divided into two parts. Part A will consist of multiple escalating doses to determine the safety, tolerability, and PK of the second-generation anti-TROP2 chimeric fusion protein in subjects with TROP2+ liver metastases (LM); Part B will consist of a dose expansion to further determine the safety, tolerability, PK, and preliminary efficacy in patients with TROP2+ colorectal liver metastases (CRLM).

[0427] The main qualification criteria are described below:

[0428] Inclusion criteria If a participant meets the following criteria, they are eligible to participate in the study:

[0429] 1. Dose escalation portion of the trial (Part A): Histologically confirmed metastatic TROP2+ LM with progressive disease at baseline, refractory to standard care, or refusing standard therapy.

[0430] 2. The extended cohort of the trial (Part B): Histologically confirmed metastatic TROP2+ CRLM with progressive disease at baseline that is refractory to standard care or has refused standard therapy.

[0431] 3. Tumors with a TROP2+ score of 2+ or 3+, as determined by immunohistochemistry (IHC). This will be performed in the central laboratory.

[0432] 4. Measurable diseases based on RECIST criteria v 1.1.

[0433] Exclusion criteria: If any of the following criteria are met, the participant is excluded from the study:

[0434] 1. Known active CNS metastases and / or carcinomatous meningitis.

[0435] 2. Previous allogeneic bone marrow transplant or solid organ transplant.

[0436] 3. Active autoimmune diseases.

[0437] 4. Active acute or chronic infection.

[0438] 5. Liver tumor involvement rate greater than 50%.

[0439] 6. No prior splenectomy. Patients with a history of partial splenic artery embolism may be eligible, depending on the investigator's decision.

[0440] Treatment duration: There will be a total of 3 cycles. Each cycle consists of weekly dosing for 4 weeks. After 3 cycles, if there is no disease progression, the patient may continue to participate in the study until week 48, at the investigator's discretion, but the dosing regimen will be changed to once every two weeks. If the patient develops progressive disease, the dosing may be gradually increased to weekly. If the patient's condition progresses during weekly dosing, treatment will be discontinued.

[0441] Dose-limiting toxicity definition:

[0442] DLT is defined using the Common Terminology Standard for Adverse Events (CTCAE) v5.0 as specified. All toxicities will be considered at least “probably” related to the anti-TROP2 second-generation chimeric fusion protein unless they are not time-related to the administration of the anti-TROP2 second-generation chimeric fusion protein but are related to other causes such as concomitant drugs or symptoms or the subject’s underlying disease. For this study, the following will be considered DLT if they occur within 28 days of the first dose (day 28) unless otherwise noted below:

[0443] •die

[0444] • Any CTCAE Class 4 toxicity

[0445] • CTCAE Grade 3 toxicity in vital organs (central nervous system [CNS], heart, and lungs)

[0446] • CTCAE Grade 3 toxicity that does not decrease to £2 level within 72 hours under maximal supportive care (e.g., nausea, vomiting, diarrhea), except for abnormalities in kidney and liver laboratories.

[0447] • For renal and hepatic laboratory abnormalities, if the toxicity does not decrease to a £2 level within 7 days, it is classified as CTCAE Grade 3 toxicity.

[0448] • Any grade 3 infusion-related response lasting >24 hours, and the patient has been pre-medicated with an H1 antagonist, H2 antagonist, and corticosteroid.

[0449] • Exceptions to the DLT Level 3 or 4 standard include:

[0450] ○ Laboratory values ​​not considered clinically significant by researchers

[0451] Study the stopping rules :

[0452] At any dose level, if a subject experiences a DLT as listed above, further dosing and enrollment will be temporarily suspended, and the Steering Committee will be immediately notified and a meeting convened to review the safety data. Following a review of the safety data, the Steering Committee will recommend whether to proceed based on the frequency of DLTs:

[0453] • Continue the study as planned, including administering additional doses.

[0454] • Expand the dose groups to obtain additional safety information

[0455] • Reduce the dose to either the previous lower or moderate dose.

[0456] • Eliminate the administration of additional doses

[0457] • Terminate the study and monitor the safety of all participants.

[0458] Statistical considerations:

[0459] Dosage progression will be determined by a continuous substitution model.

[0460] Evaluation endpoint / criteria:

[0461] Security:

[0462] • AE, IR, severe AE (SAE). Significantly abnormal physical examination findings.

[0463] The findings and laboratory results will be reported as AE.

[0464] • Vital signs

[0465] • Serum chemistry, hematology, T cell count, and plasma cytokines

[0466] Anti-TROP2 binder cell kinetics: pharmacokinetic parameters of the drug / product in blood after each qPCR infusion (e.g., Cmax, Tmax, T1 / 2).

[0467] ADA: The proportion of subjects with ADA, as measured by ELISA.

[0468] Clinical response:

[0469] •RECIST v1.1

[0470] • Converted to surgical candidate

[0471] Relevant response markers:

[0472] • Tumor penetration of cells via IHC imaging mass spectrometry

[0473] • Recruiting and transporting other immune cells using mass spectrometry cytometry imaging.

[0474] • Upregulate cytokine and chemokine production through transcriptional analysis

[0475] Table 6. Event Timeline

[0476]

[0477]

[0478] Example 2. A multicenter, open-label, phase 1 first-in-human study evaluating safety and tolerability in advanced epithelial cancer.

[0479] The pharmaceutical composition comprises an scFv, the scFv comprising a heavy chain and a light chain, the heavy chain comprising a CDR3 having the sequence GGFGSSYWYFDV, and the light chain comprising a CDR3 having the sequence QQHYITPLT. In some embodiments, the heavy chain further comprises a CDR1 sequence of NYGMN and a CDR2 sequence of WINTYTGEPTYTDDFKG; and the light chain further comprises a CDR1 sequence of KASQDVSIAVA and a CDR2 sequence of SASYRYT. The pharmaceutical composition comprises a CD89 transmembrane domain having the sequence LIRMAVAGLVLVALLAILV.

[0480] The study had four cohorts. Each cohort had four cycles. For cohorts 1-3, the dosing regimen was three doses every 14 days, followed by three doses every 28 days. For cohort 4, the dosing regimen was modified. Participants received one dose of the anti-TROP2 chimeric antigen receptor investigational drug weekly for three doses, followed by three additional doses every 28 days.

[0481] The conditions treated in this clinical study included malignant epithelial tumors.

[0482] The study type is interventional, phase 1.

[0483] The research model is a single-group assignment. Number of groups: 1.

[0484] The study is expected to enroll 48 patients.

[0485] Key outcome measures:

[0486] 1. Assess the safety and tolerability of the anti-TROP2 chimeric antigen receptor investigational drug by the incidence of adverse events. Adverse events will be graded according to NCI-CTCAE version 5.0 [time range: up to week 20].

[0487] 2. Establish the maximum tolerated dose (MTD) based on dose-limiting toxicity (DLT) [time range: up to week 20].

[0488] 3. Establish the maximum tolerated dose (MTD) based on the recommended phase 2 dose (RP2D) [time range: up to week 20].

[0489] Secondary outcome measures:

[0490] 4. The safety of the anti-trop2 chimeric antigen receptor investigational drug will be further characterized by the incidence of adverse events. Adverse events will be graded according to NCI-CTCAE version 5.0 [time range: up to week 20].

[0491] 5. Pharmacokinetics (PK) of anti-trop2 chimeric antigen receptor research drugs to determine PK parameters: plasma concentration [time range: up to week 20].

[0492] 6. Pharmacokinetics (PK) of anti-trop2 chimeric antigen receptor research drugs to determine PK parameters: area under the curve [time range: up to week 20].

[0493] 7. Pharmacokinetics (PK) of anti-trop2 chimeric antigen receptor research drugs to determine PK parameters: maximum observed plasma concentration [time range: up to week 20].

[0494] 8. Pharmacokinetics (PK) for determining the PK parameters of anti-trop2 chimeric antigen receptor research drugs: time to maximum observed plasma concentration [time range: up to week 20].

[0495] 9. Pharmacokinetics (PK) for determining the PK parameters of anti-trop2 chimeric antigen receptor research drugs: apparent terminal half-life [time range: up to week 20].

[0496] 10. Pharmacokinetics (PK) of anti-trop2 chimeric antigen receptor investigational drugs: plasma clearance [time range: up to week 20].

[0497] 11. Pharmacokinetics (PK) of anti-trop2 chimeric antigen receptor research drugs to determine PK parameters: volume of distribution [time range: up to week 20].

[0498] 12. Pharmacokinetics (PK) of anti-trop2 chimeric antigen receptor research drugs to determine PK parameters: mean residence time [time range: up to week 20].

[0499] 13. Pharmacokinetic (PK) parameters for determining anti-trop2 chimeric antigen receptor research drugs: terminal rate constant [time range: up to week 20].

[0500] 14. Determine the rate of ICANS - For the grading of potential immune effector cell-associated neurotoxic syndromes (ICANS), use the 10-point immune effector cell-associated encephalopathy (ICE) screening tool [time range: up to week 20].

[0501] 15. Determine the rate of CRS for grades 3-5 [time range: up to week 20].

[0502] Eligibility criteria: The following criteria were followed for enrolled patients.

[0503] Minimum age: 18 years old; Gender: All

[0504] The study does not accept healthy volunteers.

[0505] Inclusion criteria:

[0506] 1. Adults aged 18 years or older (inclusive) at the time of signing the Informed Consent Form (ICF).

[0507] 2. Histologically confirmed metastatic or advanced epithelial carcinoma, including the following cancer types:

[0508] a. Urothelial carcinoma

[0509] b. Cervical cancer

[0510] c. Ovarian epithelial cancer

[0511] d. Triple-negative breast cancer

[0512] e. HR+ / HER2- breast cancer

[0513] f. Pancreatic ductal adenocarcinoma

[0514] g. Gastric adenocarcinoma

[0515] h. Esophageal cancer

[0516] i. Non-small cell lung cancer

[0517] j. Colorectal cancer

[0518] 3. At baseline, the patient had a progressive disease that was refractory or relapsed to standard care, or had refused standard therapy.

[0519] 4. Measurable diseases based on the Responsive Evaluation Criteria for Solid Tumors (RECIST) v 1.1.

[0520] 5. The Eastern Cooperative Oncology Group (ECOG) performance status level is 0 or 1.

[0521] 6. Life expectancy > 12 weeks.

[0522] 7. Displays an echocardiogram (ECHO) or multi-gated acquisition scan with an ejection fraction greater than or equal to 50%.

[0523] 8. No clinically significant abnormalities were observed at screening, or an electrocardiogram (ECG) showing a mean QTc interval <450 msec in males and <470 msec in females (<480 msec for participants with bundle branch block). Either the Fridericia or Bazett formula may be used to correct for the QT interval.

[0524] 9. The oxygen saturation in indoor air is greater than or equal to 90%, as measured by pulse oximetry.

[0525] 10. Sufficient organ function as defined by laboratory values ​​during screening.

[0526] 11. Willing and able to provide written informed consent.

[0527] 12. Willing to perform and comply with all research procedures, including conducting research-related biopsies and attending scheduled clinic visits.

[0528] 13. Men must refrain from donating sperm during the study treatment period or for four months after the last dose of the study treatment.

[0529] 14. Both the man and the WOCBP must be willing to use an effective method of contraception.

[0530] Exclusion criteria:

[0531] 1. Known active CNS metastases and / or carcinomatous meningitis. Participants with previously treated brain metastases may participate if they are not radioactively stable (i.e., have demonstrated no progression for at least 4 weeks by repeat imaging), are clinically stable, and have not required steroid treatment for at least 14 days prior to the first dose of the study intervention.

[0532] 2. Pregnant or breastfeeding women.

[0533] 3. Must be > 28 days after major surgery (including liver resection or joint replacement).

[0534] 4. Previous allogeneic bone marrow transplant or solid organ transplant.

[0535] 5. Spinal cord compression that has not been clearly treated with surgery and / or radiation.

[0536] 6. Uncontrolled pleural effusion, pericardial effusion, or ascites requiring repeated drainage procedures.

[0537] 7. Any acute illness within 7 days prior to day 1, including fever (>100.4℉ or >38℃).

[0538] 8. Active systemic bacterial, fungal, or viral infection within 7 days prior to Day 1. Participants tested positive for COVID-19 within 7 days prior to Day 1.

[0539] 9. Active infection with human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV).

[0540] 10. Other primary malignancies, except: a. adequately treated basal cell carcinoma or squamous cell carcinoma, b. cervical or bladder carcinoma in situ, cured and proven to be relapse-free for at least 2 years prior to the study, or c. primary malignancies that have been completely removed and in complete remission for at least 2 years.

[0541] 11. History of (non-infectious) pneumonia / interstitial lung disease requiring steroids or current pneumonia / interstitial lung disease.

[0542] 12. Previous immune-related adverse events (AEs) of grade > 3, such as pneumonia, colitis, hepatitis, nephritis; previous dermatitis and endocrine disorders are permissible, provided that corticosteroids are no longer required, endocrine replacement therapy is stable, and previous therapy has been discontinued.

[0543] 13. Active autoimmune diseases unrelated to prior therapy for primary malignancies requiring systemic therapy within the past year.

[0544] 14. A history of symptomatic congestive heart failure (New York Heart Association Class II-IV), severe active arrhythmia, or other clinically significant heart disease within the 12 months of enrollment.

[0545] 15. Toxicity from prior anticancer therapy, defined as toxicity (other than hair loss, or the aforementioned laboratory values) that has not regressed to NCI CTCAE v5.0 grade ≤ 1 or baseline. Participants with chronic grade 2 toxicity (e.g., peripheral neuropathy, laboratory values) may be eligible, subject to the determination of the investigator and medical monitor.

[0546] 16. The following have been accepted:

[0547] a. Radiotherapy within 2 weeks of the first administration of an anti-trop2 chimeric antigen receptor research drug.

[0548] b. Cytotoxic chemotherapy administered within 21 days or 5 half-lives of the anti-TROP2 chimeric antigen receptor investigational drug, whichever is shorter.

[0549] c. Immunotherapy for primary malignancies within 21 days or 5 half-lives (e.g., monoclonal antibody therapy, checkpoint inhibitors), whichever is shorter of the first administration of an anti-trop2 chimeric antigen receptor investigational drug.

[0550] d. Cancer vaccines within 12 weeks of the first administration of an investigational drug against the trop2 chimeric antigen receptor. e. COVID-19 mRNA vaccines within 6 weeks of the first administration of an investigational drug against the trop2 chimeric antigen receptor.

[0551] 17. The patient had received a live vaccine ≤ 6 weeks prior to the first administration of the anti-trop2 chimeric antigen receptor investigational drug.

[0552] 18. The patient received a transfusion of packed red blood cells or platelets within 2 weeks prior to the first administration of the anti-trop2 chimeric antigen receptor investigational drug.

[0553] 19. History of allergic reactions to any excipient.

[0554] 20. Enroll in another interventional clinical trial within 21 days or 5 half-lives of the drug, whichever is shorter of the first administration of the anti-trop2 chimeric antigen receptor investigational drug.

[0555] 21. Any other medical condition that the researchers believe would make the participant unfit to participate in the study or unable to comply with the study requirements.

[0556] Example 3. Non-clinical study: Data from a mouse gp75+ tumor model

[0557] The aim of these studies was to evaluate the antitumor efficacy of myeloid cells programmed with anti-GP75-CD89 LNP. The anti-GP75 CD89 mRNA construct comprises a TA99-derived anti-gp75 scFv fused with a truncated CD89 having transmembrane and cytoplasmic domains. The efficacy of myeloid CARs targeting the cancer antigen was investigated in C57BL / 6 mice as an alternative to mouse B16 / F10-Ova melanoma tumors (subcutaneous, SC). Mouse model is An alternative B16 / F10 syngeneic animal model was established. In this alternative model, GP75+ B16 / F10 melanoma tumors were induced and treated with an LNP composition containing anti-GP75 CAR mRNA. The anti-GFP-CAR is structurally similar to the anti-TROP2 CAR, except that the extracellular antigen-binding domain is an scFv that binds to the GP75 antigen, rather than an scFv that binds to the TROP2 antigen. Figure 1 The general structure is described in the figure. C57BL / 6 mice (n = 5 mice / group) were inoculated with 2 x 10⁻⁶ nauplii in the right flank on day 0. 5 GP75+ B16 / F10-OVA cells / mouse. Treatment was initiated in all experiments when the tumor reached a volume > 30 mm³. Anti-GP75CAR mRNA-LNP was intravenously (IV) injected into immunocompetent C57BL / 6 mice carrying GP75+ B16 / F10-Ova melanoma tumors at intervals ranging from 4 days (Q4D) to 1 week (Q7D), and tumor volume was monitored. On days 8, 12, and 16 post-tumor inoculation, mice received mediator control, LNPs loaded with GP75-CD89 mRNA, and LNPs at doses of 0.5 or 2 mg / kg / dose (data in...). Figure 2 (As shown in the figure). Compared with the mediator, both 0.5 mg / kg and 2 mg / kg showed statistically significant slower / arrested tumor growth. This indicates that CAR has antitumor activity and can slow down and even stop tumor progression.

[0558] In one study, tumor-bearing C57BL / 6 mice were treated with a mordant (PBS), empty LNP (2 mg / kg), or GP75-CD89 LNP (2 mg / kg) on ​​days 9, 11, 13, and 15. Tumors were harvested 24 hours after the fourth dose. Single-cell suspensions were obtained from the tumors, and CD45+CD11b+Ly6Chi (monocytes) and CD45+CD11b+Ly6C were also identified. lo In a (resident myeloid cell) population, CAR expression was assessed by FACS using anti-FLAG Ab. Error bars represent the standard error of the mean. Statistical significance was established by ordinary two-dimensional ANOVA relative to empty LNP. Anti-GP75 CAR expression was detected in up to 15% of CD11b+ Ly6C+ cells in tumors in mice treated with 2 mg / kg / dose of gp75 LNP. Figure 3 ).

[0559] On days 9, 11, 13, and 15, C57BL / 6 mice carrying tumors were treated with PBS, empty LNP (2 mg / kg), or gp75-CD89 LNP (2 mg / kg). Tumors were collected 24 hours after the fourth dose. Single-cell suspensions were obtained from the tumors. Figure 4A Plotting dot and bar graphs of activation and depletion (TIM3 / PD-1), Figure 4B Plotting dot and bar graphs of proliferation (Ki-67), Figure 4CDot and bar plots were used to depict cell lysis activity (granzyme B). Dots in the bar plots represent individual animals. Error bars represent the standard error of the mean. Statistical significance was established by comparing empty LNPs with gp75-CD89 LNPs using the Mann-Whitney test. Treatment and proliferation (Ki-67+) Figure 4B ) and cell lysis (granzyme B+) Figure 4C The increase is associated with CD8+ T cells exhibiting an activated phenotype (where PD-1 expression levels are reduced). Figure 4A ). Figures 4A-4C The points on the bar chart represent individual animals. The error bars represent the standard error of the mean. Statistical significance was established using the Mann-Whitney test by comparing the empty LNP with the gp75-CD89 LNP.

[0560] Serum analysis revealed an increase in chemokines and cytokines associated with the induction of adaptive immune responses, including CXCL10 (IP-10), CCL5 (RANTES), CCL2 (MCP-1), CCL7 (MCP-3), CCL3 (MIP-1-α), CCL4 (MIP-1-β), CXCL2 (MIP-2), TNF-α, GM CSF, IFN-γ, and IL-17a, which was most significant at 2 mg / kg.

[0561] Example 4. Non-clinical study: Data from a mouse triple-negative breast cancer model

[0562] This study demonstrates the activity of the test composition (anti-TROP2-CD89 mRNA encapsulated in LNP) against human TROP2-expressing tumors in an immunodeficient mouse xenograft model. The aim of these studies was to evaluate the antitumor efficacy of the test composition under different dosing regimens in a TROP2+ HCC-1954 human epithelial breast cancer tumor xenograft model in NSG and NCG mice. Because these animals do not possess a fully developed immune system, these studies only address the direct antitumor activity of programmed myeloid cells. Studies of other downstream immune elements were not possible. Unlike endogenic models, where other immune cells, particularly adaptive compartments (T cells and B cells), can contribute to antitumor activity upon activation by CAR myeloid cells, clinical efficacy in xenograft models depends solely on CAR myeloid cells, which require continuous delivery to the tumor site. Despite this caveat, treatment of NSG and NCG mice carrying HC-1954 xenografts expressing TROP2 with the test composition was associated with significant antitumor activity.

[0563] On day 0, NSG or NCG mice (n = 5 mice / group) were inoculated with 2 x 10^6 TROP2+ HCC-1954 cells (triple-negative breast cancer) per mouse via subcutaneous (SC) injection into the right flank. When the tumor volume reached 50-100 mm... 3 Mice were administered a mediator (DPBS), empty LNP, or test composition at doses of 0.1, 0.5, 1, or 2 mg / kg. Mice were given intravenous (IV) injections according to various dosing regimens, including once every 4 days (Q4D) for a total of 5 injections, once weekly (QW) for a total of 3 or 4 injections, or once every other week (Q2W) for a total of 4 injections.

[0564] CFP expression was examined in various cell populations (Ly6C+, Ly6G+) from blood, spleen, bone marrow, and tumors of tumor-bearing animals at 6, 12, and 24 hours after infusion of the test composition. In all examined tissues (e.g., whole blood), CFP expression was immediately limited to myeloid lineage cells, particularly Ly6C+ (monocytes), at 6 hours post-infusion. Figures 5A-5D The expression of anti-TROP2 CFP in myeloid cells derived from whole blood was depicted in a TROP2+ HCC-1954 subcutaneous xenograft model in NCG mice following intravenous infusion of the test composition. Expression was assessed by FACS. Figure 5A Total Ly6C+ cells were depicted; Figure 5B Ly6C+ CD11b+ cells were depicted; Figure 5C Ly6C+ CD11c+ cells were depicted; and Figure 5D Ly6G+ cells were depicted. Statistical significance was determined at each time point using standard two-dimensional ANOVA followed by Sidak multiple comparisons test between mice treated with the test composition and those treated with the causative agent (n = 3 mice / group / time point). CFP expression in blood and spleen persisted for up to 24 hours, even with changes in myeloid cell migration patterns and phenotypic changes. Figure 5E A set of results consistent with the above is shown, in which CAR expression was found in monocytes and dendritic cells (DCs). Expression was depicted as the percentage of the total number of isolated cells with the specific markers described in the figure, which were positive for CAR expression. The data show that CAR is expressed in myeloid subpopulations.

[0565] Compared with the carcass and empty LNP control, the antitumor efficacy was dose-dependent in animals treated with the test composition at ≥0.5 mg / kg / dose, where the antitumor activity was significant. Significant antitumor efficacy was observed with the test composition at 2 mg / kg when IV Q4D was administered, and was observed up to 11 days after treatment interruption. Figure 6The TROP2 in NSG mice was depicted + In the HCC-1954 subcutaneous xenograft model, mean tumor volume was measured in each group following intravenous administration of the test composition every four days (Q4D) or weekly. Starting 22 days post-grafting, the mediator (DPBS) or the test composition (2 mg / kg) was administered IV every four days (Q4D) or weekly (QW). Statistical significance was determined using the Mann-Whitney test, followed by a two-stage false discovery method. *p < 0.05; **p < 0.01; ***p < 0.001. Red and green asterisks define statistical significance of Q4D and QW treatments of the test composition compared to the mediator, respectively. Significant efficacy was also observed with the test composition when administered IV at 0.5 mg / kg and 1 mg / kg at QW or Q2W. Treatment with empty LNPs was not associated with any antitumor efficacy, thus confirming antigen specificity of the antitumor effect. Treatments appeared to be well tolerated; no visible adverse events were reported, no weight loss was observed, and no changes in physical condition were noted in mice treated with the test composition. Similar results consistent with the above are shown in Figure 7 In the study described herein, the dosing schedule of the test composition after tumor inoculation is shown by arrows.

[0566] Example 5. Non-clinical research: Non-human primate studies

[0567] Adult primates (cynomolgus monkeys) express TROP2. The following studies were conducted to evaluate the presence and specificity of anti-TROP2 CFP in non-human primate blood leukocytes after infusion of the test composition. Flow cytometry was used to evaluate CD66abce+ granulocytes, CD14+CD11b+ monocytes, CD11b+CD14- myeloid cells, CD3+ T cells, CD20+ B cells, and CD16+ NK cells in the blood of cynomolgus monkeys treated with the test composition.

[0568] The results across studies were consistent, demonstrating the presence of an anti-TROP2 CAR+ myeloid cell population, including monocytes, CD11b+ myeloids, and granulocytes, following the first IV infusion of the test composition (dose 1). The number of CAR-positive cells in the blood was dose-dependent, with the highest expression observed in the monocyte population 12 hours after IV administration of the test composition at 1 mg / kg / day. The number of CFP-positive cells also significantly increased after IV administration of the test composition at 0.5 mg / kg / day. In all myeloid cell types, including monocytes, CD11b+ myeloid cells, and granulocytes, the number of CFP-positive cells in the blood peaked at 12 hours after the first infusion of the test composition, and decreased at 24 hours after infusion in monocytes (mean 0.92% + / - SD 1.98%), CD11b+ myeloid cells (mean 0.78% + / - SD 1.16%), and granulocytes (mean 0.48% + / - SD 0.84%). In CD11b+ myeloid cells and granulocytes, CAR expression at 24 hours after infusion of the test composition was only slightly above the aforementioned background. No significant CFP expression was observed in non-myeloid cell populations, including T cells, B cells, and NK cells. The anti-TROP2 CAR expression profile was similar between males and females in all cell types tested.

[0569] Figure 8 The frequency of anti-TROP2 CFP monocytes in whole blood following intravenous infusion of the test composition in cynomolgus monkeys was characterized. Cynomolgus monkeys were administered the carrier, 1 mg / kg / dose of empty LNP, or 0.1, 0.5, or 1 mg / kg / dose of the test composition via intravenous infusion. The frequency of anti-TROP2 CARs was assessed by flow cytometry at 12 hours after the first infusion. Statistical analysis was performed by two-way ANOVA followed by Dunnett's multiple comparison test, comparing each treatment group to the carrier.

[0570] The effects of the test composition on the central nervous and cardiovascular systems were evaluated in cynomolgus monkeys. The test composition was administered via intravenous infusion over 60 minutes to 4 (2 males, 2 females) to 10 (5 males, 5 females) cynomolgus monkeys, either as a carrier, 1.0 mg / kg / dose of empty LNP, or 0.1 mg / kg / dose of the test composition (days 1, 8, 15, 29, 36, and 43), or 0.5 or 1.0 mg / kg / dose of the test composition (days 1, 8, 15, and 29). See Table 7.

[0571] In cynomolgus monkeys, no test composition-related effects were observed in neurological examinations on day 8 following administration of the test composition at doses of 0.1, 0.5, or 1.0 mg / kg / day over 60 minutes, or on day 36 following administration of the test composition at doses of 0.1 mg / kg / day over 60 minutes. In cynomolgus monkeys, or in any dose group after a 30-day recovery period, no test compound-related effects were observed in electrocardiogram (ECG) examinations on day 43 following administration of the test composition at doses of 0.1 mg / kg / day over 60 minutes.

[0572] In the following study, anti-TROP2-CD89 CAR:LNP was used to assess distribution in a model in which the on-target antitumor activity of the drug could be monitored. mRNA distribution was assessed in highly perfused tissues (liver, lung, heart, brain, kidney, ovary / testis) from cynomolgus monkeys in the recovery group of the GLP toxicology study 30 days after drug administration. The no-adverse-effects-level (NOAEL) in non-human primates was 0.1 mg / kg.

[0573] Pharmacokinetic studies in nonhuman primates showed that, for group 1, the observed half-lives for the two lipids were approximately 8–10 hours and 35–55 hours, respectively. Therefore, the Q14D dosing interval allows for complete drug clearance before repeated doses. Hypersensitivity observed in NHPs cannot predict hypersensitivity in patients.

[0574] The human equivalent dose (HED) for hypersensitivity was observed to be 0.16 mg / kg in NHP toxicology studies.

[0575] Secondary pharmacodynamics and studies from similar pharmacological products

[0576] In vivo safety pharmacology was tested, and the effects of anti-TROP2-CD89 CAR:LNP on the central nervous and cardiovascular systems were evaluated as part of a GLP-compliant repeated-dose toxicology study. This study was conducted in groups of 4 to 10 (5 M, 5 F) cynomolgus monkeys administered empty LNP at 1 mg / kg / dose, or anti-TROP2-CD89 CAR:LNP at 0.1 mg / kg / dose (days 1, 8, 15, 29, 36, and 43), or anti-TROP2 CAR:LNP at 0.5 or 1.0 mg / kg / dose, or anti-TROP2 CAR:LNP at 2.0 mg / kg / dose (days 1, 8, 15, 22, or 29, or as indicated). Results are presented in Table 7.

[0577]

[0578]

[0579] Example 6. In vitro expression of anti-TROP2 CAR

[0580] Direct tumor cell killing and cytokine production were evaluated in PBMCs transfected with the TROP2-CD89 CAR construct. These cells showed elevated TROP2-positive tumor cell killing upon contact (…). Figure 9 (Left bar chart). These cells also exhibited high levels of cytokines, such as those depicting TNF production. Figure 9 As shown in the figure on the right.

[0581] Example 7. Design and progress of the first human Phase I study

[0582] This example describes a Phase 1, open-label, non-randomized, first-in-human dose-escalation study to investigate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of the composition in adults with advanced or metastatic epithelial carcinoma. This was a multicenter study that included previously treated patients with metastatic epithelial cancer, including: urothelial carcinoma, cervical cancer, ovarian epithelial carcinoma, triple-negative breast cancer, HR+ / HER2- breast cancer, pancreatic ductal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, non-small cell lung cancer, and colorectal cancer.

[0583] The study is designed to screen adult patients aged 18 years or older. Patients who provide written informed consent and meet all inclusion and exclusion criteria will be enrolled in the trial.

[0584] Screening assessments 28 days (4 weeks) prior to the planned start of treatment included participant medical history, physical examination with vital signs and performance status, computed tomography (CT) or magnetic resonance imaging (MRI) scans, complete blood count (CBC) plus differential and platelet count, routine serum chemistry, urinalysis, international normalized ratio (INR) / PTT, EKG, and serum samples for human anti-human antibodies (both anti-TROP2 CAR and anti-PEG antibodies). In women of childbearing potential, serum β-HCG was also required within one week of treatment.

[0585] The study consists of four cycles. For groups 1–3, during cycle 1, all participants will receive an infusion of the test compound every 14 days for a total of three doses. For group 4, all participants will receive an infusion of the test compound every 7 days for a total of three doses. Participants will continue to participate in the study until week 18 if there are no unacceptable toxicities, but for all groups, the dosing regimen will be modified to once every 28 days for cycles 2–4. If a participant has stable disease (SD) or a partial response (PR), the participant will continue to receive monthly dosing until week 48, until progressive disease or complete response. Once participants discontinue dosing, they will continue to be followed up, with assessments every 3 months, until the end of the study or until disease progression.

[0586] All patients should be closely monitored during treatment, and guidelines for dose reduction, delay, or discontinuation should be provided in the event of treatment-related toxicities. NCI CTCAE version 5.0 will be used to grade all adverse events. All patients will also undergo CT / MRI scans throughout the study to assess disease progression and response to the investigational drug.

[0587] If no archived biopsy (< 6 months) is available, a baseline tumor biopsy will be obtained during screening. For groups 1 and 2, biopsies at week 12 and at EOT are optional. Biopsies are required for groups 3 and 4. The biopsy will be used to determine whether myeloid cells programmed by the test compound have migrated to the tumor and to assess changes in the tumor microenvironment, including T-cell and NK-cell infiltration.

[0588] Whole blood and serum will be obtained for anti-drug antibody (ADA) assays, cell phenotype analysis, cell kinetics analysis, and TCR sequencing.

[0589] Table 8. Summary of Dose Elevation and Study Groups

[0590]

[0591] Figure 10A This section summarizes the clinical trial design. For cohorts 1-3, the dosing regimen in human studies will be three doses administered every 14 days, followed by three doses every 28 days. For cohort 4, the dosing regimen will be modified so that participants will receive one dose of the test compound weekly for three doses, followed by three doses every 28 days.

[0592] This example describes results to date from an ongoing Phase I, open-label, first-in-human dose-escalation study investigating the safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of an anti-TROP2-CD89 CAR mRNA-LNP drug in adults with the described advanced or metastatic epithelial tumors. As of May 2024, nine patients have been administered up to 0.03 mg / kg of the drug without dose-limiting toxicities (DLTs). In this study, cohort 1 (0.005 mg / kg) and cohort 2 (0.015 mg / kg) have been completed, and cohort 3 (0.03 mg / kg) was completed at the time of preparation of this application and has been reported to be well-tolerated. Therefore, the results are very encouraging.

[0593] Example 8. First-in-human Phase I dose-escalation update: A Phase 1, open-label, first-in-human dose-escalation study investigating the safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of anti-TROP2-CD89 CAR mRNA-LNP in adults with advanced or metastatic epithelial tumors.

[0594] This example illustrates the progress of FIH studies, including another group with higher doses ( Figure 10B The starting dose was based on the highest non-serious toxic dose (HNSTD), which is equivalent to the no-observed adverse event level (NOAEL) in the non-human primate (NHP) safety study. The HNSTD in NHP was 0.1 mg / kg. Using a conversion based on body surface area, the human equivalent dose (HED) of 0.1 mg / kg in NHP was 0.032 mg / kg. For this calculation, 0.1 mg / kg in NHP was multiplied by a scaling factor of 0.32 to obtain the HED in mg / kg. The initial dose of 0.005 mg / kg in Group 1 is 1 / 6 of the HED of the HNSTD in the NHP safety study, and there will be 3 dose levels (Table 9).

[0595] For groups 1-3, the dosing regimen in the human studies was once every 14 days (Q14D) for a total of 3 doses, followed by once every 28 days (Q28D) for a total of 3 doses. For group 4, the dosing regimen was modified. Participants received one dose of anti-TROP2-CD89 CAR mRNA-LNP weekly for a total of 3 doses (Q7D), followed by once every 28 days (Q28D) for 3 additional doses. The initial doses will elicit an innate immune response, allowing for short-term control of tumor growth; all six doses will initiate and enhance an adaptive immune response, allowing for long-term control of tumor growth.

[0596] To advance the safety and efficacy assessment of anti-TROP2-CD89 CAR mRNA-LNP, this protocol will further escalate the dose to levels above the hemorheologically indicated (HED) of the no-anaphylaxis effect (NOAEL) found in NHP (0.032 mg / kg). Specifically, doses of 0.06 mg / kg, 0.10 mg / kg, and 0.15 mg / kg are planned; all of these doses are lower than the HED (0.16 mg / kg) observed in the NHP toxicology study (Example 5). The justification for these additional dose levels can be based on the initial safety profile observed at doses up to 0.03 mg / kg. Continuously enhanced monitoring of potential hypersensitivity reactions and the addition of hypersensitivity treatment algorithms will be used to optimize patient safety. Figure 11 This is a schematic overview of the dosing and schedule.

[0597] In toxicology studies of the anti-TROP2-CD89 CAR mRNA-LNP (DP) NHP, some animals receiving doses higher than 0.16 mg / kg HED experienced severe or fatal reactions. These reactions in NHP were characterized by a syndrome consistent with previously observed NHP type III hypersensitivity reactions, confirmed by complement activation, and associated with the development of antidrug antibodies (ADA) in 75% of the animals (see IB for more information). Empty LNP (1 mg / kg) showed no toxicity. ADA in monkeys targets a single-chain variable fragment (scFv) of the CAR. The incidence of immunogenicity or immune response to human protein therapeutics in NHP has limited correlation with the incidence / response of immunogenicity in humans (Vahle 2018, van Meer 2013). Therefore, immunogenicity-related risks in humans cannot be reliably predicted from NHP (i.e., immunogenicity-related risks observed in NHP with human therapeutics do not typically translate into immunogenicity-related risks in humans).

[0598] Groups 1 (0.005 mg / kg) and 2 (0.015 mg / kg) have been completed; enrollment for Group 3 (0.030 mg / kg) is ongoing as of May 1, 2024. No dose-limiting toxicities (DLTs) or ADAs have been observed to date. All AEs assessed by the investigator as related to DP were Grade 1, except for one transient Grade 2 sweating reported in one patient (Group 2). Notably, a confirmed partial response at 0.015 mg / kg was observed in patients with hormone receptor-positive breast cancer (according to RECIST 1.1). Furthermore, peripheral changes in the cytokine environment and T-cell receptor repertoire indicate potential biological activity for the drug development candidate. Overall, the favorable safety profile and potential activity observed as of May 1, 2024 support continued clinical studies at higher doses.

[0599] For doses ranging from 0.06 mg / kg to 0.15 mg / kg in groups 5–7, the dosing frequency will be modified to one dose every 14 days (Q14D) to assess safety on a consistent dosing schedule (e.g., two doses per 28-day cycle). The proposed dosing schedule is supported by initial pharmacokinetic (PK) data from the ongoing anti-TROP2-CD89 CAR mRNA-LNP study. For group 1, the observed half-lives for ionizable lipids and PEGylated lipids were approximately 8–10 hours and 35–55 hours, respectively. Lipid clearance prior to repeated doses is consistent with data collected in the anti-TROP2-CD89 CAR mRNA-LNP GLPNHP toxicology study and thus supports the Q14D dosing interval.

[0600] During the proposed additional dose escalation, patients will be closely monitored for hypersensitivity reactions, and any emerging symptoms will be treated immediately based on appropriate disclosure and education by investigators, the nursing team, and participants. Ongoing comprehensive evaluations of inflammatory markers and ADA will continue, along with a 16-day sentinel observation period for the first patient treated at each dose level, allowing for staggered safety assessments of repeated dosing across patients.

[0601] The proposed measured dose assessment and patient management approach was supported by experts who reviewed NHP hypersensitivity data (Vahle 2018) in the context of predicting human risks.

[0602] Table 9. Summary of Dose Elevation and Study Groups

[0603]

[0604] Updated overall research design

[0605] This is a phase 1, first-in-human, non-randomized, open-label, multicenter, dose-escalation study evaluating the safety, tolerability, pharmacokinetics, and pharmacodynamics of anti-TROP2-CD89 CAR mRNA-LNP.

[0606] Some exemplary terms used in abbreviations in this article and throughout the literature can be summarized in the following table:

[0607] Table 10. List of Non-Exclusive Abbreviations and Terminology Definitions

[0608]

[0609]

[0610]

[0611]

[0612] Screening assessments 28 days prior to the start of planned treatment included participants’ medical and medication history, physical examination with vital signs and performance status, computed tomography (CT) or magnetic resonance imaging (MRI) scans, complete blood count (CBC) plus differential and platelet count, routine serum chemistry, urinalysis, international normalized ratio (INR) / partial thromboplastin time (PTT) and electrocardiogram (ECG). For women of childbearing potential (WOCBP), serum β-HCG was also required within one week of treatment.

[0613] The primary assessment of DLT will include the first 30 days following the first dose of the study drug. Efficacy and safety assessments will continue until a participant withdraws from the study or the study concludes, whichever comes first. Dosing intervals may be increased in the context of ongoing clinical benefit and discussed with the medical monitor (e.g., one dose every 28 days).

[0614] If treatment is discontinued due to unacceptable toxicity, participants may continue to participate in the study until disease progression or treatment for the primary malignancy is initiated. At this point, a study termination assessment will be conducted, and further follow-up will be required until any treatment-related toxicity subsides or stabilizes. Overall survival of all participants will be monitored.

[0615] To ensure the safety of study participants, the first two participants in each cohort will be staggered by 16 days. This 16-day interval allows for two days of follow-up after either the third dose of the study drug (for cohort 4) or the second dose of the study drug (for cohorts 5–7). If no safety issues arise, participants can continue to be enrolled in cohorts simultaneously. Cohorts 4 and 5 can be enrolled concurrently.

[0616] The BOIN design will be used to identify the recommended dose for DP amplification. Dosage escalation / decrease decisions will be based on events occurring during the DLT assessment period (30 days) and will be guided by the BOIN algorithm. Participants who have undergone DLT or completed the DLT assessment period without undergoing DLT will be considered evaluable for the purpose of making dose escalation decisions; participants who did not complete the DLT assessment period for reasons other than safety will not be considered evaluable.

[0617] In this study, the target toxicity probability of the anti-TROP2-CD89 CAR mRNA-LNP MTD / RP2D was set at a target toxicity rate of 25% (pT = 0.25) and a boundary of [0.197, 0.298]. Doses with toxicity probabilities between 0.197 and 0.298 were considered acceptable MTDs, and below these doses, except for 1 / 3 DLT, the BOIN design retained the current dose.

[0618] To advance the safety and efficacy assessment of DP, version 3.0 seeks further dose escalations to include three additional doses. The justification for these dose levels is based on the safety profile observed in the first three cohorts. Based on emerging clinical data, intermediate dose levels and / or alternative dosing regimens may be evaluated in accordance with the SRC committee.

[0619] Future modifications could include further increasing the dose above the NOAEL pending safety results, restricting enrollment in the expanded cohort to specific groups, such as breast cancer or non-small cell lung cancer, or adding checkpoint inhibitors or other immune-modifying agents.

[0620] Participant pool: Screening will be conducted for adult participants aged 18 or older. Participants who provide written informed consent and meet all inclusion and exclusion criteria will be enrolled in the trial.

[0621] Dose-limiting toxicities (DLTs): DLTs can be understood using the Common Terminology Standards for Adverse Events (CTCAE) as specified by the National Cancer Institute (NCI). All toxicities will be considered “probably” related to anti-TROP2-CD89 CAR mRNA-LNP unless they are not time-related to DP administration but are related to other causes such as concomitant drugs or symptoms or the patient’s underlying disease. For this study, the following conditions are considered DLTs if they occur within 30 days of the first dose (day 30):

[0622] •die

[0623] • Any CTCAE Class 4 toxicity

[0624] • CTCAE Grade 3 toxicity in vital organs (central nervous system [CNS], heart, and lungs)

[0625] • CTCAE Grade 3 toxicity that does not decrease to £2 within 72 days, except for infection or abnormal renal / hepatic laboratory findings.

[0626] • For infections or abnormal kidney / liver laboratory results, if the toxicity does not decrease to £2 within 7 days, it is classified as CTCAE Grade 3 toxicity.

[0627] • Any grade 3 infusion-related response lasting > 24 hours

[0628] • Exceptions to the DLT Level 3 or 4 standard include:

[0629] Laboratory values ​​not considered clinically significant by researchers

[0630] Individuals may discontinue subsequent infusions if grade 4 toxicity, grade 3 DLT, or type III hypersensitivity occurs after infusion. Once the toxicity subsides to grade 1 or lower, the reduced dose of the drug may be resumed after a grade 3 DLT, and if a DLT recurs, the infusion may be permanently discontinued.

[0631] Sample size

[0632] In the dose-escalation cohort, the estimated number of participants to be enrolled in this Phase 1 study evaluating anti-TROP2-CD89 CAR mRNA-LNP in participants with advanced solid tumors was approximately up to 42. An additional 18 subjects may be added to the fill cohort at the funder's discretion. The number of participants per dose-escalation cohort was determined based on a BOIN design. Participants were accumulated into the dosing cohort at a cohort size of 3, and the number of participants at each dose level could be expanded to up to 12, depending on the observed number of participants with DLT.

[0633] Inclusion criteria

[0634] Participants are eligible to participate in the research if they meet the following criteria:

[0635] Adults aged 18 years or older (inclusive) at the time of signing the Informed Consent Form (ICF).

[0636] Histologically confirmed metastatic or advanced epithelial carcinoma, including the following cancer types: urothelial carcinoma, cervical cancer, ovarian epithelial carcinoma, triple-negative breast cancer, HR+ / HER2- breast cancer, pancreatic ductal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, non-small cell lung cancer, and colorectal cancer.

[0637] Other tumor types will be considered pending discussion with the medical supervisor.

[0638] At baseline, the patient had a progressive disease that was refractory or relapsed to standard care, or had refused standard therapy.

[0639] Measurable disease is defined based on the Responsive Evaluation Criteria for Solid Tumors (RECIST) v 1.1. Lesions located in previously irradiated areas are considered measurable if clear progression has been confirmed in such lesions. Lesions intended for biopsy should not be identified as target lesions. If no measurable disease is found, evaluable disease will be considered in consultation with the medical supervisor.

[0640] 1. The Eastern Cooperative Oncology Group (ECOG) performance status level is 0 or 1.

[0641] 2. Life expectancy > 12 weeks.

[0642] 3. Displays an echocardiogram (ECHO) or multi-gated acquisition scan with an ejection fraction of ≥50%.

[0643] 4. No clinically significant abnormalities were observed at screening, or ECGs showing a mean QTc interval <450 msec in males and <470 msec in females (<480 msec for participants with bundle branch block). Either the Fridericia or Bazett formula may be used to correct for the QT interval.

[0644] 5. The oxygen saturation in indoor air measured by pulse oximetry is ≥90%.

[0645] 6. Sufficient organ function as defined by the following laboratory values ​​at the time of screening:

[0646] a. Hemoglobin ≥9.0 g / dL within 2 weeks prior to screening without transfusion support.

[0647] b. Platelet count ≥ 100,000 / µL in the two weeks prior to screening without transfusion support.

[0648] c. Absolute neutrophil count > 1000 / mm 3 (Without granulocyte colony-stimulating factor support within 2 weeks prior to screening)

[0649] d. Creatinine clearance calculated using the CKD-EPI equation > 45 mL / min or creatinine < 1.5 x ULN

[0650] e. ALT < 2.5 x ULN; if liver disease due to cancer is known, then ALT < 5.0 x ULN.

[0651] f. AST < 2.5 x ULN; if liver disease is known to be caused by cancer, then AST < 5.0 x ULN.

[0652] g. Serum bilirubin < 1.5 x ULN (if the participant has been diagnosed with Gilbert's syndrome) < 3.0x ULN)

[0653] h. Unless the participant is receiving anticoagulant therapy and the INR or PT is within the expected or therapeutic range for the intended use of the anticoagulant, an INR or prothrombin time (PT) ≤ 1.5 ULN

[0654] 7. Willing and able to provide written informed consent.

[0655] 8. Willing to perform and comply with all research procedures, including conducting research-related biopsies and attending scheduled clinic visits.

[0656] 9. Men must refrain from donating sperm during the study treatment period or for 4 months after the last dose of the study treatment.

[0657] 10. Both the man and the WOCBP must be willing to use a highly effective method of contraception. Effective contraceptive options include:

[0658] a. Double barrier (a diaphragm, condom, or cervical cap used in conjunction with spermicide foam, gel, or cream)

[0659] b. Abstinence (If sexually active during the study, consent to the double-barrier approach must be given)

[0660] c. Use of implanted or intrauterine contraceptive devices, with or without hormones, for at least 6 consecutive months prior to study administration and throughout the entire study duration.

[0661] d. Hormonal contraceptives (continuous use of oral, injectable, implantable, transdermal, or vaginal hormonal devices for at least 3 consecutive months prior to the screening visit and throughout the study duration).

[0662] e. Male partners must undergo surgical sterilization (vasectomy) at least 6 months prior to the screening visit.

[0663] f. Same-sex partners

[0664] Exclusion criteria

[0665] Participants are excluded from the study if they meet any of the following criteria:

[0666] 1. Known active CNS metastases and / or carcinomatous meningitis. Participants with previously treated brain metastases may participate if they are not radioactively stable (e.g., demonstrated no progression by repeat imaging for at least 4 weeks), are clinically stable, and have not required steroid treatment for at least 14 days prior to the first dose of the study intervention.

[0667] 2. Pregnant or breastfeeding women.

[0668] 3. Must be performed after major surgery (including liver resection or joint replacement). > 28 days.

[0669] 4. Previous allogeneic bone marrow transplant or solid organ transplant.

[0670] 5. Spinal cord compression that has not been clearly treated with surgery and / or radiation.

[0671] 6. Uncontrolled pleural effusion, pericardial effusion, or ascites requiring repeated drainage procedures.

[0672] 7. Any acute illness within 7 days prior to day 1, including fever (>100.4℉ or >38℃).

[0673] 8. Active systemic bacterial, fungal, or viral infection within 7 days prior to Day 1. Participants tested positive for COVID-19 within 7 days prior to Day 1.

[0674] 9. Active infection with human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV), as defined below:

[0675] a. Serological test positive for HIV-1

[0676] b. Serologically positive for HCV and has not received a detectable, documented curative treatment or quantitative RNA PCR.

[0677] c. Positive hepatitis B surface antigen or IgM, HBV core antibody unless quantitative DNA PCR is undetectable and the patient is receiving stable antiviral prophylaxis against HBV reactivation;

[0678] 10. Other primary malignant tumors, except:

[0679] a. Well-treated basal cell carcinoma or squamous cell carcinoma

[0680] b. Cervical or bladder carcinoma in situ, cured and proven to be recurrence-free for at least 2 years prior to the study, or

[0681] c. Primary malignant tumor that has been completely removed and in complete remission for at least 2 years.

[0682] 11. History of (non-infectious) pneumonia / interstitial lung disease requiring steroids or current pneumonia / interstitial lung disease.

[0683] 12. Previous Levels > Grade 3 immune-related adverse events, such as pneumonia, colitis, hepatitis, nephritis; pre-existing dermatitis and endocrine disorders are permitted, provided that corticosteroids are no longer needed and endocrine replacement therapy is stable and previous therapy has been discontinued.

[0684] 13. Active autoimmune diseases unrelated to prior therapy for primary malignancies requiring systemic treatment within the past year. Replacement therapies such as thyroxine, insulin, or physiological steroid replacement therapy for adrenal or pituitary insufficiency are not considered systemic treatments. Celiac disease is permissible if adequately controlled by diet.

[0685] 14. A history of symptomatic congestive heart failure (New York Heart Association Class II-IV), severe active arrhythmia, or other clinically significant heart disease within the 12 months of enrollment.

[0686] 15. Toxicity from prior anticancer therapy, defined as toxicity (other than hair loss, or the aforementioned laboratory values) that has not regressed to NCI CTCAE v5.0 grade ≤ 1 or baseline. Participants with chronic grade 2 toxicity (e.g., peripheral neuropathy, laboratory values) may be eligible, subject to the determination of the investigator and medical monitor.

[0687] 16. The following have been accepted:

[0688] a. Radiotherapy within 2 weeks of the first administration of anti-TROP2-CD89 CAR mRNA-LNP.

[0689] b. Cytotoxic chemotherapy for the treatment of primary malignant tumors within 28 days or 5 half-lives, whichever is shorter of the administration of anti-TROP2-CD89 CAR mRNA-LNP.

[0690] c. Immunotherapy for primary malignancies (e.g., monoclonal antibody therapy, checkpoint inhibitors) within 28 days or 5 half-lives, whichever is shorter of the first administration of anti-TROP2-CD89 CAR mRNA-LNP.

[0691] d. For targeted therapy of primary malignant tumors within 28 days or 5 half-lives, whichever is shorter of the first administration of anti-TROP2-CD89 CAR mRNA-LNP.

[0692] e. Anticancer therapy within 12 weeks of the first administration of anti-TROP2-CD89 CAR mRNA-LNP.

[0693] f. COVID-19 mRNA vaccine within 6 weeks of the first administration of TROP2-CD89 CAR mRNA-LNP.

[0694] 17. Live vaccine was administered ≤ 6 weeks prior to the first administration of anti-TROP2-CD89 CAR mRNA-LNP.

[0695] 18. The patient had received a transfusion of packed red blood cells or platelets within 2 weeks prior to the first administration of anti-TROP2-CD89 CAR mRNA-LNP.

[0696] 19. History of allergic reactions to any excipient.

[0697] 20. Enroll in another interventional clinical trial within 28 days or 5 half-lives of the drug, whichever is shorter of the first administration of anti-TROP2-CD89CAR mRNA-LNP.

[0698] 21. Any other medical condition that the researchers believe would make the participant unfit to participate in the study or unable to comply with the study requirements.

[0699]

[0700]

[0701]

[0702] Related to Table 11A above,

[0703] a Patients with SD or PR will continue cycle 5-12 until PD or CR. Dosing days are D141, D169, D197, D225, D253, D281, D309, and D337, with a window of ±4 days. Follow-up visits must be performed on the day of dosing; planned visit days are D142, D170, D198, D226, D254, D282, D310, and D338. Treatment evaluation concludes on D351 ± 7 days.

[0704] b The patient will be hospitalized for 24 hours of observation with dose 1.

[0705] c EOT assessment will be conducted 14 days after the last treatment or in the event of early termination of the study.

[0706] d For patients who have not progressed or have started a new line of therapy, follow-up (including MRI / CT, ​​complete blood cfDNA, and survival status) is required every 3 months (±14 days) for up to 2 years, or only until any treatment-related abnormalities following patient progression have subsided. For patients with PD or who have started a new line of therapy, survival status only will be required until the study is completed.

[0707] e If the screening visit is less than 14 days before C1D1, the baseline visit is optional. If the screening visit is more than 14 days before C1D1, the baseline visit is optional. If a baseline lab is available, these visits do not need to be repeated at C1D1.

[0708] f If medication is delayed, subsequent follow-up visits will also be delayed. For cycle 1, follow-up visits must be performed one day and two days after medication. For cycles 2-4, follow-up visits are performed immediately after medication.

[0709] g Administer once every 28 days (D57, D85, D113).

[0710] h After the infusion of cycle 1 begins, blood pressure (BP), heart rate (HR), temperature, respiratory rate (RR), and pulse oximetry are measured before administration (-15 minutes), at 15 ± 5 minutes, 30 ± 5 minutes, 1 hour, 2 hours, 4 hours, and 6 hours (± 10 minutes). After the infusion of cycles 2-12 begins, BP, HR, temperature, and pulse oximetry are measured before administration (-15 minutes), at 15 ± 5 minutes, 30 ± 5 minutes, 1 hour, and 2 hours (± 10 minutes). Height is measured at screening.

[0711] I Weight will be measured before administration (from the date of administration until 8 days prior to administration).

[0712] j ECGs prior to dose 1 and dose 2 can be obtained up to 3 days before the administration visit.

[0713] k ECHO / MUGA can be repeated during the study period as directed by clinical instructions.

[0714] l An MRI / CT scan performed after the subject's last line of treatment and before signing a consent form, and within a 6-month period, can be used to determine eligibility. However, another MRI / CT scan may be performed within the screening window.

[0715] m MRI / CT scans should be performed on day 50 ± 7 days, day 127 ± 7 days, and then every 12 weeks ± 14 days until the end of the study. Additionally, an MRI / CT scan is required 4 weeks after the first occurrence of any partial response (PR) or complete remission (CR) to confirm the response.

[0716] n Bone scans are required at screening (before MRI / CT) in participants with a previously documented or suspected bone metastasis and in participants with advanced breast cancer.

[0717] o Serum chemistry is performed locally (up to 3 days prior to administration on the day of administration). Serum chemistry includes sodium, potassium, magnesium, phosphorus, chloride, bicarbonate, urea, creatinine, glucose, calcium, total protein, albumin, total bilirubin, AST (SGOT), ALT (SGPT), GGT, and alkaline phosphatase.

[0718] p Laboratories that do not meet the screening criteria should be reported to the medical supervisor for review of their continued eligibility for research.

[0719] q Refer to the procedure for evaluating tumor biomarkers. If baseline levels are within the normal range for C1D1, no additional testing is required. If baseline levels are outside the normal range, samples should be collected as described above on the day of administration (-3 days) and within 7 da...

Claims

1. A pharmaceutical composition comprising a polynucleotide encoding a chimeric antigen receptor comprising an anti-TROP2 binding scFv extracellular domain and a CD89 transmembrane domain, wherein the polynucleotide is formulated in an aqueous formulation for systemic delivery in a therapeutically effective amount and time interval suitable for treating a cancer in a subject.

2. The pharmaceutical composition of claim 1, wherein the polynucleotide is an engineered RNA.

3. The pharmaceutical composition of claim 2, wherein the engineered RNA is an engineered mRNA.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the anti-TROP2 binding scFv comprises a heavy chain and a light chain, wherein the heavy chain comprises a CDR3 having the sequence GGFGSSYWYFDV and the light chain comprises a CDR3 having the sequence QQHYITPLT.

5. The pharmaceutical composition of claim 4, wherein the heavy chain further comprises a CDR1 sequence of NYGMN and a CDR2 sequence of WINTYTGEPTYTDDFKG; and the light chain further comprises a CDR1 sequence of KASQDVSIAVA and a CDR2 sequence of SASYRYT.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the CD89 transmembrane domain comprises the sequence LIRMAVAGLVLVALLAILV.

7. The pharmaceutical composition of any one of claims 1 to 6, further comprising a lipid nanoparticle delivery vehicle.

8. The pharmaceutical composition of claim 7, wherein the lipid nanoparticle delivery vehicle comprises a cationic lipid, a non-cationic lipid, a neutral lipid, a PEGylated lipid, or a combination thereof.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the subject is a human subject.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the cancer is an epithelial cancer.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein the subject has an epithelial cancer.

12. The pharmaceutical composition of claims 1 to 11, wherein the cancer is selected from the group consisting of urothelial carcinoma, cervical cancer, ovarian epithelial cancer, triple negative breast cancer, HR+ / HER2- breast cancer, pancreatic ductal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, non-small cell lung cancer, and colorectal cancer.

13. The pharmaceutical composition of claim 12, wherein the cancer is metastatic.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the pharmaceutical composition is formulated for systemic delivery.

15. The pharmaceutical composition of any one of claims 1 to 14, wherein the pharmaceutical composition is formulated for intravenous delivery.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the pharmaceutical composition is formulated for intravenous injection or infusion.

17. The pharmaceutical composition of any one of claims 1-16, wherein a dose of the therapeutically effective amount comprises 1-5000 micrograms / microliter of the engineered RNA.

18. The pharmaceutical composition of any one of claims 1-17, comprising the engineered RNA in an amount of about 0.0005 mg / kg to about 0.001 mg / kg, about 0.001 mg / kg to about 0.005 mg / kg, about 0.005 mg / kg to about 0.01 mg / kg, about 0.01 mg / kg to about 0.05 mg / kg, about 0.05 mg / kg to about 0.1 mg / kg, or about 0.1 mg / kg to about 0.5 mg / kg per dose of the pharmaceutical composition.

19. The pharmaceutical composition of any one of claims 1-18, comprising the engineered RNA in an amount of about 0.001 mg to about 0.0015 mg, about 0.0015 mg to about 0.002 mg, about 0.002 mg to about 0.0025 mg, about 0.0025 mg to about 0.003 mg, about 0.003 mg to about 0.0035 mg, about 0.0035 mg to about 0.004 mg, about 0.004 mg to about 0.0045 mg, about 0.0045 mg to about 0.005 mg, about 0.005 mg to about 0.0055 mg, about 0.0055 mg to about 0.006 mg, about 0.006 mg to about 0.0065 mg, about 0.0065 mg to about 0.007 mg, about 0.007 mg to about 0.0075 mg, about 0.0075 mg to about 0.008 mg, about 0.008 mg to about 0.0085 mg, about 0.0085 mg to about 0.009 mg, about 0.009 mg to about 0.0095, or about 0.0095 mg to about 0.01 mg per dose of the pharmaceutical composition per kilogram of body weight of the subject (mg / kg).

20. The pharmaceutical composition of any one of claims 1-19, comprising the engineered RNA in an amount of about 0.01 mg / kg to about 0.015 mg / kg, about 0.015 mg / kg to about 0.02 mg / kg, about 0.02 mg / kg to about 0.025 mg / kg, about 0.025 mg / kg to about 0.03 mg / kg, about 0.03 mg / kg to about 0.035 mg / kg, about 0.035 mg / kg to about 0.04 mg / kg, about 0.04 mg / kg to about 0.045 mg / kg, about 0.045 mg / kg to about 0.05 mg / kg, about 0.05 mg / kg to about 0.055 mg / kg, about 0.055 mg / kg to about 0.06 mg / kg, about 0.06 mg / kg to about 0.065 mg / kg, about 0.065 mg / kg to about 0.07 mg / kg, about 0.07 mg / kg to about 0.075 mg / kg, about 0.075 mg / kg to about 0.08 mg / kg, about 0.08 mg / kg to about 0.085 mg / kg, about 0.085 mg / kg to about 0.09 mg / kg, about 0.09 mg / kg to about 0.095 mg / kg, or about 0.095 mg / kg to about 0.1 mg / kg per dose of the pharmaceutical composition.

21. The pharmaceutical composition of any one of claims 1-20, comprising the engineered RNA in an amount of about 0.001 mg / kg, about 0.0015 mg / kg, about 0.002 mg / kg, about 0.0025 mg / kg, about 0.003 mg / kg, about 0.0035 mg / kg, about 0.004 mg / kg, about 0.0045 mg / kg, about 0.005 mg / kg, about 0.0055 mg / kg, about 0.006 mg / kg, about 0.0065 mg / kg, about 0.007 mg / kg, about 0.0075 mg / kg, about 0.008 mg / kg, about 0.0085 mg / kg, about 0.009 mg / kg, about 0.0095 mg / kg, or about 0.01 mg / kg per dose of the pharmaceutical composition.

22. The pharmaceutical composition of any one of claims 1-21, comprising the engineered RNA at a dose of about 0.01 mg / kg, about 0.015 mg / kg, about 0.02 mg / kg, about 0.025 mg / kg, about 0.03 mg / kg, about 0.035 mg / kg, about 0.04 mg / kg, about 0.045 mg / kg, about 0.05 mg / kg, about 0.055 mg / kg, about 0.06 mg / kg, about 0.065 mg / kg, about 0.07 mg / kg, about 0.075 mg / kg, about 0.08 mg / kg, about 0.085 mg / kg, about 0.09 mg / kg, about 0.095 mg / kg, or about 0.1 mg / kg per dose of the pharmaceutical composition.

23. The pharmaceutical composition of any one of claims 1-22, comprising the engineered RNA at an amount of at least about 0.001 mg / kg, at least about 0.0015 mg / kg, at least about 0.002 mg / kg, at least about 0.0025 mg / kg, at least about 0.003 mg / kg, at least about 0.0035 mg / kg, at least about 0.004 mg / kg, at least about 0.0045 mg / kg, at least about 0.005 mg / kg, at least about 0.0055 mg / kg, at least about 0.006 mg / kg, at least about 0.0065 mg / kg, at least about 0.007 mg / kg, at least about 0.0075 mg / kg, at least about 0.008 mg / kg, at least about 0.0085 mg / kg, at least about 0.009 mg / kg, at least about 0.0095 mg / kg, or at least about 0.01 mg / kg per dose of the pharmaceutical composition.

24. The pharmaceutical composition according to any one of claims 1 to 23, comprising, per dose of the pharmaceutical composition, the following amounts of the engineered RNA: at least about 0.01 mg / kg, at least about 0.015 mg / kg, at least about 0.02 mg / kg, at least about 0.025 mg / kg, at least about 0.03 mg / kg, at least about 0.035 mg / kg, at least about 0.04 mg / kg, at least about 0.045 mg / kg, at least about 0.05 mg / kg, at least about 0.055 mg / kg, at least about 0.06 mg / kg, at least about 0.065 mg / kg, at least about 0.07 mg / kg, at least about 0.075 mg / kg, at least about 0.08 mg / kg, at least about 0.085 mg / kg, at least about 0.09 mg / kg, at least about 0.095 mg / kg, or at least about 0.1 mg / kg, or at least about 0.2 mg / kg. mg / kg, or at least about 0.3 mg / kg, or at least about 0.4 mg / kg, or at least about 0.5 mg / kg, or at least about 0.6 mg / kg, or at least about 0.7 mg / kg, or at least about 0.8 mg / kg, or at least about 0.9 mg / kg, or at least about 1 mg / kg.

25. The pharmaceutical composition according to any one of claims 2 to 24, wherein, when stored in a container, the concentration of the engineered RNA is between about 0.5 mg / mL and about 1.5 mg / mL or between about 0.7 mg / mL and about 1.3 mg / mL.

26. The pharmaceutical composition of claim 25, wherein the container is a disposable or reusable vial.

27. The pharmaceutical composition according to any one of claims 1 to 26, wherein the total volume of the aqueous formulation is about 1.0 mL to about 3.0 mL, about 1.5 mL to about 2.5 mL, or about 2.0 mL.

28. The pharmaceutical composition according to any one of claims 1 to 27, wherein for three doses, the dosing interval is 14 days, and subsequently for three doses, and optionally another three, four, five, six, seven or eight doses, the interval is 28 days.

29. A method of treating a subject with cancer, the method comprising: administering to the subject a pharmaceutical composition according to any one of claims 1 to 28.

30. The method of claim 29, wherein the subject is over 18 years of age.

31. The method of claim 29 or 30, wherein the subject exhibits progressive disease at baseline, or exhibits refractory disease or relapse in response to standard care.

32. The method according to any one of claims 29 to 31, wherein the subject is not pregnant or has not donated sperm.

33. The method according to any one of claims 29 to 32, wherein the subject does not have CNS metastases or carcinomatous meningitis.

34. The pharmaceutical composition of any one of claims 1-28 or the method of claim 32, wherein treatment comprises reducing a tumor by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or at least 10% following the treatment.

35. The pharmaceutical composition of any one of claims 1-28 or the method of claim 32, wherein treatment comprises reducing at least one symptom associated with the cancer.

36. The pharmaceutical composition of any one of claims 1-28, 34, or 35, comprising an engineered RNA encapsulated in a lipid nanoparticle, wherein the engineered RNA comprises a sequence encoding a chimeric antigen receptor (CAR) having: an extracellular antigen binding domain comprising an scFv that binds to TROP2, the scFv having a heavy chain comprising a CDR1 sequence of NYGMN, a CDR2 sequence of WINTYTGEPTYTDDFKG, and a CDR3 sequence of GGFGSSYWYFDV; and the light chain further comprising a CDR1 sequence of KASQDVSIAVA, a CDR2 sequence of SASYRYT, and a CDR3 sequence of QQHYITPLT; a transmembrane domain and an intracellular domain from CD89 having the sequence DSIHQDYTTQNLIRMAVAGLVLVALLAILVENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK, wherein the pharmaceutical composition has no dose limiting toxicity (DLT) in a human subject in need thereof at doses at least up to about 0.03 mg / kg when administered at a dosing regimen of at least twice per week for about 18 weeks.

37. The pharmaceutical composition of any one of claims 1 to 28, 34, 35, or 36, wherein the extracellular antigen binding domain comprises a sequence at least 90% identical to QVQLQQSGSELKKPGASVKVSCKASGYTF TNYGM NWVKQAPGQGLKWMG WINTYTGEP TYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITC KASQDVSIA VAWYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLTF GAGTKVEIKR (SEQ ID NO: 3).

38. The pharmaceutical composition of any one of claims 1-28, 34, 35, 36, or 37, wherein the engineered CAR comprises a sequence that is at least 95% identical to the sequence of SEQ ID NO:

3.

39. The pharmaceutical composition of any one of claims 1-28, 34, 35, 36, 37, or 38, wherein the engineered CAR comprises a sequence that is at least 99% identical to the sequence of SEQ ID NO:

3.

40. The pharmaceutical composition of any one of claims 1-28, 34, 35, 36-39, wherein the engineered CAR comprises a sequence that is the sequence of SEQ ID NO:

3.

41. The pharmaceutical composition of any one of claims 1-28, 34, 35, 36-40, wherein the engineered CAR comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 26 or SEQ ID NO:

41.

42. The pharmaceutical composition of any one of claims 1-28, 34, 35, 36-40, wherein the engineered CAR comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 27 or SEQ ID NO:

34.

43. The pharmaceutical composition of any one of claims 1-28, 34, 35, 36-40, wherein the engineered CAR comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 28 or SEQ ID NO:

35.

44. The pharmaceutical composition of any one of claims 1-28, 34, 35, 36-40, wherein the engineered CAR comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 29 or SEQ ID NO:

36.

45. The pharmaceutical composition of any one of claims 1-28, 34, 35, 36-40, wherein the engineered CAR comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 30 or SEQ ID NO:

37.

46. The pharmaceutical composition of any one of claims 1-28, 34, 35, 36-40, wherein the engineered CAR comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 31 or SEQ ID NO:

38.

47. The pharmaceutical composition of any one of claims 1-28, 34, 35, 36-40, wherein the engineered CAR comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 32 or SEQ ID NO:

39.

48. The pharmaceutical composition of any one of claims 1-28, 34, 35, 36-40, wherein the engineered CAR comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 33 or SEQ ID NO:

40.

49. The pharmaceutical composition of any one of claims 1-28, 34, 35, 36-40, wherein the engineered CAR comprises a sequence that is at least 95% identical to any one of the sequences of SEQ ID NOs: 26-41.

50. The method of any one of claims 29-33, further comprising administering to the subject one or more therapeutic agents in combination with or in addition to administering the pharmaceutical composition.

51. The method of claim 50, wherein administering the one or more therapeutic agents comprises administering a therapeutic agent prior to administering the pharmaceutical composition.

52. The method of claim 50 or 51, wherein administering the one or more therapeutic agents comprises administering a therapeutic agent concurrently with administering the pharmaceutical composition.

53. The method of any one of claims 50-52, wherein administering the one or more therapeutic agents comprises administering a therapeutic agent after administering the pharmaceutical composition.

54. The method of any one of claims 29-33 and 50-53, wherein the method comprises administering the pharmaceutical composition to the subject for at least one cycle.

55. The method of any one of claims 29-33 and 50-54, wherein the method comprises administering the pharmaceutical composition to the subject for at least 2, 3, or 4 cycles.

56. The method of any one of claims 29-33 and 50-55, wherein during a first cycle, the pharmaceutical composition 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.

57. The method of any one of claims 29-33 and 50-56, wherein the method comprises administering the pharmaceutical composition to the subject once a week or once every 2 weeks during the first cycle.

58. The method of any one of claims 29-33 and 50-57, wherein the method comprises administering the pharmaceutical composition to the subject at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, or at least 12 times for each cycle.

59. The method of any one of claims 29-33 and 50-58, wherein the method comprises administering the pharmaceutical composition to the subject about 1 to about 3 times, about 3 to about 6 times, about 6 to about 9 times, or about 9 to about 12 times for each cycle.

60. The method of any one of claims 29-33 and 50-59, wherein the method comprises administering the pharmaceutical composition to the subject up to 1 time, up to 2 times, up to 3 times, up to 4 times, up to 5 times, up to 6 times, up to 7 times, up to 8 times, up to 9 times, up to 10 times, up to 11 times, or up to 12 times for each cycle.

61. The method of any one of claims 29-33 and 50-60, wherein the method comprises administering the pharmaceutical composition to the subject between 1 and 12 times for each cycle.

62. The method of any one of claims 29-33 and 50-61, wherein the method comprises administering the pharmaceutical composition to the subject between about 1 and about 3 times, about 3 and about 6 times, about 6 and about 9 times, or about 9 and about 12 times for each cycle.

63. The method of any one of claims 29-33 and 50-62, wherein the method comprises administering the pharmaceutical composition to the subject 3 times in a first cycle.

64. The method of any one of claims 29-33 and 50-63, wherein a second cycle follows the first cycle.

65. The method of claim 64, wherein the method further comprises administering the pharmaceutical composition to the subject during the second cycle.

66. The method of claim 65, wherein during the second period, the pharmaceutical composition is administered to the subject about once every 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.

67. The method of claim 66, wherein the method comprises administering the pharmaceutical composition to the subject once every 4 weeks during the second period.

68. The method of any one of claims 65-67, wherein the method comprises administering the pharmaceutical composition to the subject at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, or at least 12 times in the second period.

69. The method of any one of claims 65-68, wherein the method comprises administering the pharmaceutical composition to the subject about 1 to about 3 times, about 3 to about 6 times, about 6 to about 9 times, or about 9 to about 12 times in the second period.

70. The method of any one of claims 65-69, wherein the method comprises administering the pharmaceutical composition to the subject up to 1 time, up to 2 times, up to 3 times, up to 4 times, up to 5 times, up to 6 times, up to 7 times, up to 8 times, up to 9 times, up to 10 times, up to 11 times, or up to 12 times in the second period.

71. The method of any one of claims 65-70, wherein the method comprises administering the pharmaceutical composition to the subject between 1 and 12 times in the second period.

72. The method of any one of claims 65-71, wherein the method comprises administering the pharmaceutical composition to the subject between about 1 and about 3 times, about 3 and about 6 times, about 6 and about 9 times, or about 9 and about 12 times in the second period.

73. The method of any one of claims 65-72, wherein the method comprises administering the pharmaceutical composition to the subject 3 times in the second period.

74. The method of any one of claims 29-33 and 50-72, wherein the method comprises administering the pharmaceutical composition, wherein the therapeutically effective dose comprises an amount of 0.01 mg / kg to 1 mg / kg per dose at an interval of once every two weeks to once every week over a period of about 18 weeks to about 50 weeks.

75. A pharmaceutical composition comprising an engineered mRNA, the engineered mRNA comprising the sequence of SEQ ID NO: 41, and wherein the pharmaceutical composition comprises a dose of about 0.005 mg / kg to about 0.15 mg / kg of the engineered mRNA, wherein the dose is measured based on the body weight of the subject.

76. The pharmaceutical composition of claim 75, wherein the pharmaceutical composition comprises a dose of the engineered mRNA of about 0.005 mg / kg, 0.015 mg / kg, 0.03 mg / kg, 0.06 mg / kg, 0.10 mg / kg, or 0.15 mg / kg.

77. The pharmaceutical composition of claim 75 or 76, wherein the engineered mRNA is encapsulated in a lipid nanoparticle.

78. A method for treating cancer in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an engineered mRNA to the subject, the engineered mRNA comprising a sequence of SEQ ID NO: 41, at a dose of about 0.005 mg / kg to about 0.15 mg / kg of the engineered mRNA, wherein the dose is measured based on the subject’s body weight.

79. The method of claim 78, wherein the engineered mRNA is encapsulated in a lipid nanoparticle.

80. The method of claim 78 or 79, wherein the dose of the engineered mRNA is about 0.005 mg / kg, 0.015 mg / kg, 0.03 mg / kg, 0.06 mg / kg, 0.10 mg / kg, or 0.15 mg / kg.

81. The method of any one of claims 78-80, wherein the pharmaceutical composition is administered to the subject once every 7 days, once every 14 days, or once every 28 days.

82. The method of claim 80, wherein the pharmaceutical composition is administered to the subject for at least 4 cycles.

83. The method of claim 82, wherein the pharmaceutical composition is administered at a dose of about 0.05 mg / kg, 0.15 mg / kg, or 0.03 mg / kg, and wherein the pharmaceutical composition is administered every 14 days in cycle 1 and every 28 days in cycles 2-4.

84. The method of claim 82, wherein the pharmaceutical composition is administered at a dose of about 0.03 mg / kg, and wherein the pharmaceutical composition is administered every 7 days in cycle 1 and every 28 days in cycles 2-4.

85. The method of claim 82, wherein the pharmaceutical composition is administered at a dose of about 0.06 mg / kg, 0.10 mg / kg, or 0.15 mg / kg, and wherein the pharmaceutical composition is administered every 14 days.

86. The method of any one of claims 82-85, wherein a cycle comprises at least 28 days, 42 days, or 56 days.

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