Engineered chimeric fusion protein compositions and methods of use thereof

Abstract

Compositions and methods for making and using engineered cells, such as engineered myeloid cells expressing chimeric fusion proteins having binding domains capable of binding surface molecules on target cells, such as diseased cells.

Description

[0001] Cross-reference

[0002] This application claims the priority of the provisional application US 63 / 409,193, filed on September 22, 2022; the provisional application is incorporated herein by reference in its entirety. Background Art

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

[0004] Understanding the limitations of CAR-T cells is key to leveraging the technology and continuing to innovate better immunotherapy modalities. Specifically, in T cell malignancies, CAR-T cells appear to face a major problem. CAR-T cells and malignant T cells share surface antigens in most T cell lymphomas (TCLs), and thus, CAR-T cells are subject to cytotoxicity in the same way as cancer cells. In some cases, CAR-T products may be contaminated with malignant T cells. Additionally, due to the long-term persistence of CAR-T cells, T cell hypoplasia is a potential problem. Other limitations include the poor ability of CAR-T cells to penetrate solid tumors, and the effective tumor microenvironment can downregulate their anti-tumor potential. CAR-T cell function is also adversely affected by the immunosuppressive tumor microenvironment (TME) that leads to 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 that enter the bloodstream and can migrate to tissues. Some of their main functions include phagocytosis, activation of T cell responses, and clearance of cell debris and extracellular matrix. They also play important roles in maintaining homeostasis and initiating and resolving inflammation. In addition, myeloid cells can differentiate into many downstream cells, including macrophages, which can exhibit different responses ranging from pro-inflammatory to anti-inflammatory, depending on the type of stimuli they receive from the surrounding microenvironment. Moreover, tissue macrophages have been shown to play a wide range of regulatory and activating roles on other immune cell types, including CD8+ and CD4+ T effector cells, NK cells, and T regulatory cells. Macrophages have been shown to be the major immune infiltrates in malignancies and have been shown to have a wide range of 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 therapies, which can be engineered to have numerous therapeutic effects. This disclosure relates to immunotherapies using myeloid cells of the immune system (e.g., CD14+ cells), particularly phagocytes. Many therapeutic indications can be considered for the use of myeloid cells. For example, myeloid cell immunotherapies may be extremely important in treating cancer, autoimmune, fibrotic diseases, and infections. This disclosure relates to immunotherapies 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 use. For example, the object of the invention disclosed herein is to utilize the phagocytic activity of myeloid cells (including engineered myeloid cells and myeloid cells modified in vivo to express chimeric fusion proteins) for therapeutic use. For example, the object of the invention disclosed herein is to utilize the ability of myeloid cells (including engineered myeloid cells and myeloid cells modified in vivo to express chimeric fusion proteins) 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 and myeloid cells modified in vivo to express chimeric fusion proteins) to promote the secretion of anti-tumor molecules. For example, the object of the invention disclosed herein is to utilize the ability of myeloid cells (including engineered myeloid cells and myeloid cells modified in vivo to express chimeric fusion proteins) to promote the recruitment and trafficking of immune cells and molecules. In one aspect, this disclosure provides novel and useful chimeric constructs that, when expressed in myeloid cells, can drive the targeted attack and phagocytosis of myeloid cells of molecules, molecular assemblies, objects, or cells containing a target (e.g., a target antigen, such as a target antigen on the surface of a target cell). One of many aspects of this disclosure is (i) enhancing the phagocytic ability of myeloid cells (e.g., engineered myeloid cells expressing novel and improved chimeric constructs); helping to initiate a coordinated and sustained immune response against a target (e.g., a target antigen). This disclosure provides innovative methods and compositions that express chimeric fusion proteins in myeloid cells (e.g., myeloid cells in vivo) of a subject suffering from a disease (e.g., cancer). One improved strategy described herein is to induce an inflammatory phenotype in myeloid cells to generate effector myeloid cells. One strategy is to generate effector myeloid cells capable of producing an inflammatory phenotype upon engagement with a target.

[0007] The present disclosure provides compositions comprising a recombinant polynucleotide, wherein the recombinant polynucleotide comprises a sequence encoding a chimeric fusion protein (CFP) comprising: (i) an extracellular domain comprising an antigen-binding domain, (ii) a transmembrane domain operably linked to the extracellular domain; and (iii) an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains are selected from the intracellular signaling domain from Fcε receptor Ig (FCER1G), a PI3K recruitment domain, the intracellular signaling domain from CD40, and the intracellular signaling domain from TRIF.

[0008] In some embodiments, the antigen-binding domain is a GPC3-binding domain or a TROP2-binding domain.

[0009] In some embodiments, the antigen-binding domain has the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the antigen-binding domains described in Table 1A.

[0010] In some embodiments, the antigen-binding domain has a VH having at least 80% sequence identity to the VH of the antigen-binding domain of Table 1A and a VL having at least 80% sequence identity to the VL of the antigen-binding domain described in Table 1A.

[0011] In some embodiments, the CFP further comprises a signal peptide.

[0012] In some embodiments, the signal peptide has the sequence of the signal peptide shown in Table 1B.

[0013] In some embodiments, the extracellular domain comprises a sequence having at least 80% sequence identity to the sequence of Table 1C.

[0014] In some embodiments, the transmembrane domain comprises a sequence having at least 80% sequence identity to the sequence of Table 1D.

[0015] In some embodiments, each of the at least two intracellular signaling domains has a sequence having at least 80% sequence identity to the sequence shown in Table 2.

[0016] In some embodiments, the intracellular domain has a sequence having at least 80% sequence identity to the sequence shown in Table 3.

[0017] In some embodiments, the CFP has a sequence having at least 80% sequence identity to the sequence shown in Table 4.

[0018] The present invention also provides a composition comprising a recombinant polynucleotide, wherein the recombinant polynucleotide comprises a sequence encoding a chimeric fusion protein (CFP), and wherein the CFP has a sequence with at least 95% sequence identity to the sequence shown in Table 4.

[0019] In some embodiments, the CFP has a sequence with at least 98%, 99% or 100% sequence identity to the sequence shown in Table 4.

[0020] In some embodiments, the intracellular domain comprises at least one additional intracellular signaling domain.

[0021] In some embodiments, the antigen-binding domain comprises an antibody or a fragment thereof.

[0022] In some embodiments, the antigen-binding domain comprises a scFv.

[0023] In some embodiments, the antigen-binding domain comprises an extracellular domain that comprises a hinge domain linking the antigen-binding domain and the transmembrane domain.

[0024] In some embodiments, the recombinant polynucleotide is an mRNA.

[0025] In some embodiments, the recombinant polynucleotide is associated with one or more lipids.

[0026] In some embodiments, the recombinant polynucleotide is encapsulated in a liposome.

[0027] In some embodiments, the liposome is a lipid nanoparticle.

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

[0029] The present invention also provides a composition comprising a nanoparticle delivery vehicle and the recombinant polynucleotide described herein, wherein the recombinant polynucleotide is associated with or within the nanoparticle delivery vehicle.

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

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

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

[0033] In some embodiments, the recombinant polynucleotide is mRNA, and the lipid nanoparticle encapsulates the mRNA.

[0034] In some embodiments, the lipid nanoparticle comprises a polar lipid and a nonpolar lipid.

[0035] In some embodiments, the lipid nanoparticle has a diameter of 100 to 300 nm.

[0036] Also provided herein are compositions comprising cells that contain the recombinant polynucleotides described herein.

[0037] In some embodiments, the cells are immune cells.

[0038] In some embodiments, the cells are myeloid cells, lymphoid cells, precursor cells, stem cells, or induced pluripotent cells.

[0039] In some embodiments, the cells are CD14+CD16− cells.

[0040] Also provided herein are pharmaceutical compositions comprising the compositions described herein; and a pharmaceutically acceptable excipient.

[0041] Also provided herein is a method of treating cancer in a subject, comprising: administering to the subject the pharmaceutical composition described herein. In one aspect, provided herein are compositions comprising a recombinant polynucleotide and a polynucleotide delivery vehicle, wherein the recombinant polynucleotide comprises a sequence encoding a chimeric fusion protein (CFP) comprising: (i) an extracellular domain comprising an antigen-binding domain, (ii) a transmembrane domain operably linked to the extracellular domain of the CFP and that multimerizes with an Fc receptor protein endogenously expressed in a cell; and (iii) an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains are selected from the intracellular signaling domain from Fcε receptor Ig (FCER1G), a PI3K recruitment domain, the intracellular signaling domain from CD40, and the intracellular signaling domain from TRIF, wherein the antigen-binding domain comprises an antibody or fragment thereof having a heavy chain variable domain (VH) that comprises a CDR3 sequence selected from the sequences GGFGSSYWYFDV or FYSYTY.

[0042] In some embodiments, the antigen-binding domain comprises a GPC3-binding domain or a TROP2-binding domain. In some embodiments, the antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), the VH comprising VH CDR1, CDR2, and CDR3, and the VL comprising VL CDR1, CDR2, and CDR3; wherein the VH CDR1, CDR2, and CDR3 comprise the sequences listed in Table 1A; and the VL CDR1, CDR2, and CDR3 comprise the sequences listed in Table 1A. In some embodiments, the antigen-binding domain has a VH having at least 80% sequence identity to the VH of the antigen-binding domain of Table 1A and a VL having at least 80% sequence identity to the VL of the antigen-binding domain of Table 1A. In some embodiments, the CFP further comprises a signal peptide. In some embodiments, the signal peptide comprises the sequence MWLQSLLLLGTVACSIS (SEQ ID NO:7). In some embodiments, the extracellular domain comprises a sequence having at least 90% sequence identity to the sequence of Table 1C. In some embodiments, the CFP comprises a CD89 transmembrane domain, the CD89 transmembrane domain comprising a sequence having at least 90% sequence identity to SEQ ID NO:11 (Table 1D). In some embodiments, the intracellular signaling domain in the at least two intracellular signaling domains comprises a sequence having at least 80% sequence identity to the sequence of Table 2. In some embodiments, the CFP comprises an intracellular domain, the intracellular domain comprising a sequence having at least 80% sequence identity to the sequence of Table 3. In some embodiments, the CFP comprises a sequence having at least 80% sequence identity to the sequence of Table 4.

[0043] In one aspect, provided herein are compositions comprising a recombinant polynucleotide, wherein the recombinant polynucleotide comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP has a sequence having at least 95% sequence identity to the sequence of Table 4. In some embodiments, the CFP has a sequence having at least 98%, 99%, or 100% sequence identity to the sequence of Table 4. In some embodiments, the intracellular domain comprises at least one additional intracellular signaling domain. In some embodiments, the antigen-binding domain comprises an antibody or a fragment thereof. In some embodiments, the antigen-binding domain comprises an scFv. In some embodiments, the antigen-binding domain comprises an extracellular domain, the extracellular domain comprising a hinge domain that connects the antigen-binding domain and the transmembrane domain. In some embodiments, the recombinant polynucleotide is mRNA. In some embodiments, the recombinant polynucleotide is associated with one or more lipids. In some embodiments, the recombinant polynucleotide is encapsulated in a liposome.

[0044] In some embodiments, the liposome is a lipid nanoparticle. In some embodiments, the recombinant polynucleotide is a vector.

[0045] In one aspect, the present disclosure provides a composition comprising a nanoparticle delivery vehicle and a composition as described in any of the foregoing embodiments, wherein the recombinant polynucleotide is associated with or within the nanoparticle delivery vehicle. In some embodiments, the transmembrane domain is a transmembrane domain from a protein that dimerizes with an endogenous FcR-γ receptor in myeloid cells. In some embodiments, the transmembrane domain comprises a transmembrane domain from CD16a, CD64, CD68, or CD89. In some embodiments, the recombinant polynucleotide is associated with or within a delivery vehicle, wherein the delivery vehicle comprises lipid nanoparticles. In some embodiments, the recombinant polynucleotide is mRNA, and the lipid nanoparticles encapsulate the mRNA. In some embodiments, the lipid nanoparticles comprise polar lipids and nonpolar lipids. In some embodiments, the lipid nanoparticles have a diameter of 100 to 300 nm.

[0046] In some embodiments, the recombinant polynucleotide comprises (i) a sequence encoding a polypeptide having at least 80% sequence identity with SEQ ID NO: 35. In some embodiments, the recombinant polynucleotide comprises (i) a sequence encoding a polypeptide having at least 80% sequence identity with SEQ ID NO: 36. In some embodiments, the recombinant polynucleotide comprises (i) a sequence encoding a polypeptide having at least 80% sequence identity with SEQ ID NO: 151. In some embodiments, the recombinant polynucleotide comprises (i) a sequence encoding a polypeptide having at least 80% sequence identity with SEQ ID NO: 152. In some embodiments, a recombinant polynucleotide comprising a sequence encoding a polypeptide having at least 80% sequence identity with SEQ ID NO: 36 or 35 or 152 or 151 is encapsulated in an LNP and formulated in a pharmaceutical composition for systemic administration to a subject having cancer, wherein the cancer is a GPC3-positive cancer; wherein the cancer is lung cancer, liver cancer, prostate cancer, lymphoma, or hepatocellular carcinoma, non-small cell lung cancer (NSCLC), breast cancer, cervical cancer, colon cancer, gastric cancer, bladder cancer, thyroid cancer, osteosarcoma, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma, and urothelial carcinoma.

[0047] In some embodiments, the recombinant polynucleotide comprises (i) a sequence encoding a polypeptide having at least 80% sequence identity with SEQ ID NO:143. In some embodiments, the recombinant polynucleotide comprises (i) a sequence encoding a polypeptide having at least 80% sequence identity with any one of SEQ ID NO:144 - 150. In some embodiments, a recombinant polynucleotide comprising a sequence encoding a polypeptide having at least 80% sequence identity with any one of SEQ ID NO:143, 144, 145, 146, 147, 148, 149, or 150 is encapsulated in an LNP and formulated in a pharmaceutical composition for systemic administration to a subject having cancer, wherein the cancer is a TROP2-positive cancer; wherein the cancer is lung cancer, liver cancer, prostate cancer, lymphoma, or hepatocellular carcinoma, non-small cell lung cancer (NSCLC), breast cancer, cervical cancer, colon cancer, gastric cancer, bladder cancer, thyroid cancer, osteosarcoma, squamous cell carcinoma of the lung, ovarian yolk sac tumor, melanoma, and urothelial carcinoma.

[0048] In some embodiments, the recombinant polynucleotide comprises (i) a sequence having at least 80% sequence identity with SEQ ID NO:141 or a portion of SEQ ID NO:141 without the 5'UTR and / or 3'UTR, or (ii) a sequence having at least 80% sequence identity with SEQ ID NO:142 or a portion of SEQ ID NO:142 without the 5'UTR and / or 3'UTR. In some embodiments, the recombinant polynucleotide is encapsulated in an LNP and formulated in a pharmaceutical composition for systemic administration to a subject having cancer.

[0049] In one aspect, provided herein is a composition comprising cells, wherein the cells comprise the recombinant polynucleotide of the composition of any one of the foregoing embodiments. In some embodiments, the cells are immune cells. In some embodiments, the cells are myeloid cells, lymphoid cells, progenitor cells, stem cells, or induced pluripotent cells. In some embodiments, the cells are CD14+ / CD16-. In one aspect, provided herein is a pharmaceutical composition comprising the composition of any one of the foregoing embodiments; and a pharmaceutically acceptable excipient.

[0050] In one aspect, the present disclosure provides a method of treating cancer in a subject, comprising: administering to the subject the pharmaceutical composition described herein. In some embodiments, the cancer is a cancer that expresses TROP2. In some embodiments, the cancer is a cancer that expresses GPC3. In some embodiments, the cancer is lymphoma, melanoma, liver cancer, lung cancer, hepatic lymphoma, or epithelial cell carcinoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), breast cancer, cervical cancer, colon cancer, gastric cancer, bladder cancer, prostate cancer, thyroid cancer, osteosarcoma. In one embodiment, the cancer is any one of lung cancer, liver cancer, prostate cancer, lymphoma, or hepatocellular carcinoma, non-small cell lung cancer (NSCLC), breast cancer, cervical cancer, colon cancer, gastric cancer, bladder cancer, thyroid cancer, osteosarcoma, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma, and urothelial carcinoma.

[0051] In one aspect, the present disclosure provides a composition comprising: (i) a lipid delivery vehicle; and (ii) a recombinant polynucleotide comprising a sequence encoding a chimeric fusion protein (CFP), wherein the CFP comprises (a) an extracellular antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain, wherein the extracellular antigen-binding domain comprises a scFv or VHH that binds a cancer antigen; and the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 11; and wherein the recombinant polynucleotide is expressed substantially in myeloid cells when contacted with cells. In one embodiment, the recombinant polynucleotide is not expressed substantially in T cells. In one embodiment, the extracellular antigen-binding domain comprises a CDR3 sequence of any one of the CDR3 sequences shown in column 4 of Table 4. In one embodiment, the extracellular antigen-binding domain comprises a VHH domain, and the VHH domain comprises the CDR3 sequence of Table 5. In one embodiment, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences shown in any one row of Table 5. In one embodiment, the CFP comprises an extracellular antigen-binding domain that binds GPC3 and comprises a sequence having at least 80% amino acid sequence identity to the sequences shown in Tables 5 and 6B. In one embodiment, the CFP comprises an extracellular antigen-binding domain that binds GPC3 and comprises a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 34-42, 135, 136, 137, 151, and 152. In one embodiment, the recombinant polynucleotide is an mRNA. In one embodiment, the mRNA comprises a 5' UTR, a 3' UTR, a 5' cap, and a polyA tail. In one embodiment, the recombinant polynucleotide is codon-optimized. In one embodiment, the recombinant polynucleotide is a fully human polynucleotide. In one embodiment, the extracellular antigen-binding domain encoded by the recombinant polynucleotide is at least a human sequence between a murine sequence and a human sequence. In one embodiment, the CFP comprises an extracellular antigen-binding domain that binds TROP2 and comprises a sequence having at least 80% identity to the sequences shown in Table 6A.

[0052] In one aspect, the present disclosure provides a composition comprising a recombinant polynucleotide, wherein: (a) the recombinant polynucleotide is RNA; (b) the recombinant polynucleotide is associated with or within a lipid nanoparticle; (c) the recombinant polynucleotide comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP is an anti-TROP2 CFP and has a sequence with at least 95% sequence identity to SEQ ID NO: 36 or 152, and a sequence with at least 95% sequence identity to SEQ ID NO: 26 or 143; and (d) the recombinant polynucleotide comprises (i) a sequence with at least 80% sequence identity to SEQ ID NO: 141 or a portion of SEQ ID NO: 141 without the 5' UTR and / or 3' UTR. The present disclosure also provides a pharmaceutical composition comprising a recombinant polynucleotide, wherein: (a) the recombinant polynucleotide is RNA; (b) the recombinant polynucleotide is associated with or within a lipid nanoparticle; (c) the recombinant polynucleotide comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP is an anti-TROP2 CFP and has a sequence with at least 95% sequence identity to SEQ ID NO: 36 or 152, and a sequence with at least 95% sequence identity to SEQ ID NO: 26 or 143; and (d) the recombinant polynucleotide comprises (i) a sequence with at least 80% sequence identity to SEQ ID NO: 141 or a portion of SEQ ID NO: 141 without the 5' UTR and / or 3' UTR. In one aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject at least the pharmaceutical composition described herein and elsewhere, wherein the cancer is any one of lung cancer, liver cancer, prostate cancer, lymphoma, or hepatocellular carcinoma, non-small cell lung cancer (NSCLC), breast cancer, cervical cancer, colon cancer, gastric cancer, bladder cancer, thyroid cancer, osteosarcoma, squamous cell carcinoma of the lung, ovarian yolk sac tumor, melanoma, and urothelial cancer.

[0053] In another aspect, the present disclosure provides a composition comprising a recombinant polynucleotide, wherein: (a) the recombinant polynucleotide is RNA; (b) the recombinant polynucleotide is associated with or within a lipid nanoparticle; (c) the recombinant polynucleotide comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP is an anti-GPC3 CFP and has a sequence with at least 95% sequence identity to SEQ ID NO: 36 or 152; and (d) the recombinant polynucleotide comprises a sequence with at least 80% sequence identity to SEQ ID NO: 142 or a portion of SEQ ID NO: 142 without the 5' UTR and / or 3' UTR. The present disclosure also provides a pharmaceutical composition comprising a recombinant polynucleotide, wherein: (a) the recombinant polynucleotide is RNA; (b) the recombinant polynucleotide is associated with or within a lipid nanoparticle; (c) the recombinant polynucleotide comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP is an anti-GPC3 CFP and has a sequence with at least 95% sequence identity to SEQ ID NO: 36 or 152; and (d) the recombinant polynucleotide comprises a sequence with at least 80% sequence identity to SEQ ID NO: 142 or a portion of SEQ ID NO: 142 without the 5' UTR and / or 3' UTR. In one aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition described herein and elsewhere in this specification, wherein the cancer is any one of lung cancer, liver cancer, prostate cancer, lymphoma, or hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), breast cancer, cervical cancer, colon cancer, gastric cancer, bladder cancer, thyroid cancer, osteosarcoma, squamous cell carcinoma of the lung, ovarian yolk sac tumor, melanoma, and urothelial cancer.

[0054] In one aspect, the present disclosure provides a method of in vivo expressing a CFP specific for myeloid cell expression, the method comprising (i) preparing a therapeutically acceptable aqueous formulation of a composition comprising any one of the compositions described above; (ii) systemically administering the therapeutically acceptable aqueous formulation to a subject; wherein the expression of the CFP is detected in myeloid cells in the tissue sample after separating and testing a suitable tissue sample from the subject at a suitable time after administration. In some embodiments, the suitable tissue sample can be peripheral blood. In some embodiments, the suitable time for testing can be 2, 3, or 4 days after administration.

[0055] Incorporated by reference

[0056] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The novel features of the present invention are particularly set forth in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description of illustrative embodiments and the drawings, in which the principles of the present invention are utilized.

[0058] Figure 1 FIG. depicts the structure of a chimeric fusion protein (CFP) receptor and myeloid cell-targeted expression, the CFP receptor comprising (i) an extracellular domain comprising a binding domain (e.g., scFv) for a cancer cell surface antigen, (ii) a transmembrane domain that oligomerizes with an endogenously expressed myeloid cell-specific transmembrane protein (e.g., the Fc receptor common gamma chain) and is thus expressed only in cells that endogenously express the Fc receptor common gamma chain (e.g., myeloid cells in vivo); and (iii) one or more intracellular signaling domains. The chimeric fusion protein is also referred to in the figure as an Fcα fusion construct because an exemplary transmembrane domain of the chimeric fusion protein is the Fcα transmembrane domain. The armed Fcα construct depicted in the figure is a construct that is expressed in myeloid cells and that participates in oligomerization with the endogenous Fcγ transmembrane receptor and is thus functionalized (e.g., armed). The figure also depicts a recombinant polynucleotide (e.g., mRNA) construct encoding a CFP having the described structural features that is designed for in vivo delivery, encapsulated in a lipid nanoparticle (LNP), which, upon entry into myeloid cells, is expressed on the surface of myeloid cells via oligomerization with the endogenous Fcγ transmembrane receptor, which is functionalized and armed to kill tumor cells by phagocytosis of tumor cells by the myeloid cells that activate and express the CFP via the CFP receptor. However, when taken up in vivo by cells that do not express the endogenous Fcγ transmembrane receptor (e.g., T cells), the construct will not be expressed on the membrane and will not be functional.

[0059] Figure 2Depicts different CFP constructs for in vivo delivery, each construct comprising a cancer cell-specific extracellular antigen-binding domain (ECD, e.g., an scFV against a target cancer antigen), a CD89 TMD, and one or more intracellular signaling domains, which one or more intracellular signaling domains constitute a next-generation in vivo receptor that incorporates additional signaling domains, e.g., for enhancing the activation of myeloid cells expressing the CFP to actively phagocytose and kill tumor cells targeted by the ECD of the CFP. The intracellular signaling domains are the intracellular signaling domain of FcRγ (FcRg), the PI3 kinase recruitment domain (PI3K), the intracellular signaling domain of TRIF (TRIF), and the intracellular domain of CD40 (CD40) or portions thereof, and combinations of these, as indicated by the arrows. Collectively, these constructs (including transmembrane domain types capable of polymerizing with cell-specific endogenous proteins to be expressed and function in specific cell types, and further comprising intracellular domains or combinations of intracellular domains that enhance intracellular signaling in cells expressing the CFP and activate effective phagocytosis and killing of target cells) are generally named second-generation CFP constructs (or Gen 2 or variants thereof) in the present disclosure.

[0060] Figure 3 The upper inset depicts a schematic view of different CFP designs as transmembrane proteins that were developed and tested for expression and activity in myeloid cells (e.g., in monocytes), showing the location of the CFP expressed on the cell membrane (e.g., on the lipid bilayer shown in a cell slice). Figure 3 The lower inset shows the expression data of each of the CFP constructs in a monocyte cell line. These data show that the CFP with additional intracellular signaling domains (e.g., FcR, CD40, FcR-PI3K, TRIF, and FcR-TRIF) is well tolerated and well expressed.

[0061] Figure 4A and Figure 4B Depicts pro-inflammatory cytokines and chemokines produced by transfecting monocytes expressing the indicated next-generation receptor (Gen 2) for in vivo expression. The data show a side-by-side comparison of the first-generation (Gen 1) cell-specific CFP construct lacking an intracellular signaling domain with the second-generation CFP construct in terms of cell activation and IL-12p70 and IFN cytokine production. Detailed Description

[0062] T cell therapies have revolutionized cancer treatment for many patients. However, for the majority of patients with advanced solid tumors, durable clinical benefit has not been achieved. Unlike T cells, myeloid cells readily accumulate in tumors and, in some cases, contribute up to 50% of the tumor mass. Myeloid cells can be specifically engineered to become highly effective anti-tumor cells, called Activated, Targeted, Attack, and Kill (ATAK) cells, which specifically target, engulf, and lyse tumor cells and coordinate immune activation against tumor cells in the body.

[0063] All terms are intended to be understood as would be 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.

[0064] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0065] While various features of the disclosure may be described in the context of a single embodiment, these features may also be provided singly or in any suitable combination. Conversely, while the disclosure may be described herein in the context of separate embodiments for clarity, the disclosure may also be implemented in a single embodiment.

[0066] References in the specification to "some embodiments", "an embodiment", "one embodiment" or "other embodiments" mean that the features, structures or characteristics described in connection with the embodiments are included in at least some embodiments of the disclosure, but not necessarily in all embodiments.

[0067] As used in this specification and the claims, the words "comprising" (and any form of "comprising"), "having" (and any form of "having"), "including" (and any form of "including") or "containing" (and any form of "containing") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure and vice versa. Additionally, the compositions of the disclosure can be used to implement the methods of the disclosure.

[0068] When referring to measurable values (such as parameters, amounts, time intervals, etc.), the term "about" or "approximately" as used herein can refer to variations of + / −30% or less, + / −20% or less, + / −10% or less, + / −5% or less, or + / −1% or less of the specified value, so long as such variations are appropriate in the context of the disclosure. It is understood that the value itself to which the modifier "about" or "approximately" refers is also specifically disclosed.

[0069] "Agent" can refer to any cell, small molecule compound, antibody or fragment thereof, nucleic acid molecule or polypeptide.

[0070] "Alteration" or "change" can refer to an increase or a decrease. For example, an alteration can be an increase or a 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, an alteration can be an increase or a decrease by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, or 40-fold, 50-fold, 60-fold, or even up to 70-fold, 75-fold, 80-fold, 90-fold or 100-fold.

[0071] As used herein, "antigen presenting cell" or "APC" can include professional antigen presenting cells (e.g., B lymphocytes, macrophages, monocytes, dendritic cells, Langerhans cells), and other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes, thymic epithelial cells, thyroid epithelial cells, glial cells (brain), pancreatic beta 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. Professional antigen presenting cells, especially dendritic cells, play a key role in stimulating naive T cells. Non-professional antigen presenting cells, such as fibroblasts, can also contribute to the process. APCs can also cross-present peptide antigens by processing exogenous antigens and presenting the processed antigens on class I MHC molecules. Antigens that generate proteins recognized in association with class I MHC molecules are usually proteins that are produced intracellularly and are processed and associated with class I MHC molecules.

[0072] "Biological sample" can refer to any tissue, cell, fluid or other material derived from an organism.

[0073] The term "epitope" can refer to any protein determinant capable of binding to an antibody or binding fragment thereof, T cell receptor and / or antibody-like molecule, such as a sequence or structure or amino acid residue. Epitope determinants usually consist of the chemically reactive surface groups of a molecule (e.g., amino acids or sugar side chains) and usually have specific three-dimensional structural features as well as specific charge features. "T cell epitope" can refer to a peptide or peptide-MHC complex recognized by a T cell receptor.

[0074] Engineered cells, such as engineered myeloid cells, can refer to cells that have at least one exogenous nucleic acid sequence in the cell, even if transiently expressed. Expression of the exogenous nucleic acid can be carried out by various methods described elsewhere and includes methods known in the art. The present disclosure relates to the preparation and use of engineered cells, such as, for example, engineered myeloid cells, such as engineered phagocytes. The present disclosure particularly relates to an engineered cell that comprises an exogenous nucleic acid encoding, for example, a chimeric fusion protein (CFP).

[0075] 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 affected by the regulation of T cell co-stimulation and NK cell co-stimulation. Exemplary immune responses include T cell responses, such as, for example, cytokine production and cytotoxicity. In addition, immune responses include immune responses that are indirectly affected by NK cell activation, B cell activation, and / or T cell activation, such as, for example, antibody production (humoral response) and activation of cytokine-responsive cells (e.g., macrophages). Immune responses include adaptive immune responses. The adaptive immune system can respond to foreign molecular structures (e.g., antigens of invading organisms). Different from the innate immune system, the adaptive immune system is highly specific for pathogens. Adaptive immunity can also provide long-lasting protection. Adaptive immune responses include humoral immune responses and cell-mediated immune responses. In a humoral immune response, antibodies secreted by B cells into the body fluid bind to antigens derived from pathogens, resulting in the elimination of pathogens through various mechanisms, such as, for example, complement-mediated lysis. In a cell-mediated immune response, T cells capable of destroying other cells are activated. For example, if disease-related proteins are present in cells, they can be proteolytically fragmented into peptides within the cells. Then, specific cellular proteins can attach themselves to the 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 the cells carrying these antigens.

[0076] A "ligand" can refer to a molecule that is 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, a receptor has a specific ligand. In some embodiments, a receptor can have promiscuous binding to ligands, in which case it can bind several ligands that share at least a similarity in terms of their structural conformation, charge distribution, or any other physicochemical feature. A ligand can be a biomolecule. A ligand can be a non-biological material. 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 the CFP described herein, the extracellular binding domain can bind a ligand, which is also designated as the target of the binding domain. In some embodiments, the target is an antigen expressed on a diseased cell (e.g., a cancer cell), in which case the diseased cell is the target cell, meaning that the target cell expresses on its cell surface the target antigen that binds to the extracellular antigen-binding domain of the CFP. The anti-(target) binding domain or anti-(target) binding extracellular domain or anti-(target) CFP can often be used interchangeably in the present disclosure with terms such as, for example, the (target) binding domain or (target) binding extracellular domain or (target) CFP. For example, HER2 expressed on a cancer cell is the anti-HER2 binding extracellular domain of the CFP, or in other words, the antigen (ligand) that binds to the HER2 binding extracellular domain of the CFP.

[0077] The terms "major histocompatibility complex (MHC)", "MHC molecule", or "MHC protein" refer to proteins that are capable of binding antigenic peptides and presenting the antigenic peptides to T lymphocytes. Such antigenic peptides can represent T cell epitopes. The human MHC is also known as the HLA complex. Thus, the terms "human leukocyte antigen (HLA)", "HLA molecule", or "HLA protein" can be used interchangeably with the terms "major histocompatibility complex (MHC)", "MHC molecule", and "MHC protein". HLA proteins can be classified as class I HLA or class II HLA. The structures of the proteins of the two HLA types are 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 are loaded with antigens that typically originate from endogenous proteins or pathogens present within the cell and then presented to naive or cytotoxic T lymphocytes (CTLs). Class II HLA proteins are present on antigen-presenting cells (APCs), including but not limited to dendritic cells, B cells, and macrophages. They mainly present peptides processed from external antigen sources (e.g., outside the cell) to helper T cells.

[0078] In the HLA class II system, phagocytic cells (such as macrophages and immature dendritic cells) can take up entities into phagosomes by phagocytosis - although B cells show a more general uptake into endosomes by endocytosis - which fuse with lysosomes, and the acidic enzymes of the lysosomes cleave the ingested proteins into many different peptides. Autophagy is another source of HLA class II peptides. The most studied class II HLA genes are: HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1.

[0079] The presentation of peptides by HLA class II molecules to CD4+ helper T cells can lead to an immune response against 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 that activate and induce the differentiation of other immune cells. HLA class II molecules are usually heterodimers of an α and a β chain, which interact to form a peptide-binding groove that is more open than that of class I peptide-binding grooves.

[0080] HLA alleles are usually expressed in a co-dominant manner. For example, each person carries two alleles of each of the three class I genes (HLA-A, HLA-B, and HLA-C), and thus can express six different types of class II HLA. At the class II HLA locus, each person inherits a pair of HLA-DP genes (DPA1 and DPB1, which encode the α and β chains), HLA-DQ (DQA1 and DQB1, for the α and β chains), one gene HLA-DRα (DRA1), and one or more genes HLA-DRβ (DRB1 and DRB3, DRB4, or DRB5). HLA-DRB1, for example, has more than nearly 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. Thus, HLA genes are highly polymorphic; there are many different alleles present in different individuals within a population. The 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 of which has a specific number. In some embodiments, class I HLA alleles are HLA-A*02:01, HLA-B*14:02, HLA-A*23:01, HLA-E*01:01 (atypical). 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.

[0081] "Myeloid cells" can broadly refer to cells of the myeloid lineage of the hematopoietic cell system and can exclude, for example, the lymphocytic lineage. Myeloid cells include cells of, for example, the granulocyte lineage and the monocyte lineage. Myeloid cells are a major cellular compartment of the immune system and include monocytes, dendritic cells, tissue macrophages, and granulocytes. In recent years, models of the cellular ontogeny, activation, differentiation, and tissue-specific functions of myeloid cells have been re-investigated and have yielded surprising results. However, their great plasticity and heterogeneity during homeostasis and disease are far from understood. Despite the many functions of myeloid cells, including phagocytosis and their ability to activate T cells, the use of these functions for therapeutic purposes remains poorly understood. Thus, there is a search for updated approaches to develop improved therapeutic agents using other cell types, including but not limited to T cell malignancies.

[0082] Myeloid cells typically differentiate from common progenitors derived from hematopoietic stem cells in the bone marrow. Commitment to the myeloid cell lineage can be controlled by the activation of different transcription factors, and thus myeloid cells can be characterized as cells with a level of plasticity that 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 various cytokines and chemokines.

[0083] Myeloid cells can be, for example, cells derived from hematopoietic stem cells in the bone marrow under the influence of one or more cytokines and chemokines (such as G-CSF, GM-CSF, Flt3L, CCL2, VEGF, and S100A8 / 9). In some embodiments, the myeloid cells are precursor cells. In some embodiments, the myeloid cells can be cells characterized by having a common myeloid progenitor or granulocyte progenitor, myeloblast, or monocyte-dendritic cell progenitor, or a combination thereof. Myeloid cells can include granulocytes or monocytes or their precursors. Myeloid cells can include immature granulocytes, immature monocytes, immature macrophages, immature neutrophils, and immature dendritic cells. Myeloid cells can include monocytes or pre-monocytic cells or monocyte precursors. In some cases, myeloid cells as used herein can refer to monocytes having an M0 phenotype, M1 phenotype, or M2 phenotype. Myeloid cells can include dendritic cells (DCs), mature DCs, monocyte-derived DCs, plasmacytoid DCs, pre-dendritic cells, or precursors of DCs. Myeloid cells can include neutrophils, which can be mature neutrophils, neutrophil precursors, or polymorphonuclear cells (PMNs). Myeloid cells can include macrophages, monocyte-derived macrophages, tissue macrophages, macrophages of the M0, M1, or M2 phenotype. 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 (commonly referred to as physiological states). In some cases, macrophages can transition from one polarization state to another. For example, macrophages can polarize into classically activated (M1) and alternatively activated (M2) macrophages. M2 macrophages are divided 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 a phenotype in which the cells express TLR-2, TLR-4, CD80, CD86, iNOS, and MHC-II on the surface. These cells release various cytokines and chemokines, such as TNF-α, IL-1α, IL-1β, IL-6, IL-12, CXCL9, and CXCL10, and generally exhibit activation of transcription factors (such as NF-kB, STAT1, STAT5, IRF3, and IRF5) that regulate M1 gene expression. It is believed that NF-κB and STAT1 are two major pathways involved in M1 macrophage polarization. The M1 phenotype is associated with the microbicidal and antitumor functions of macrophages, exhibiting high phagocytic and inflammatory functions. On the other hand, tumor-associated macrophages exposed to an immunosuppressive environment typically become more M2 polarized. Myeloid cells can include tumor-infiltrating monocytes (TIMs).Myeloid cells can include tumor-associated monocytes (TAM). Myeloid cells can include myeloid-derived suppressor cells (MDSC). Myeloid cells can include tissue-resident macrophages. Myeloid cells can include tumor-associated DC (TADC). Thus, myeloid cells can express one or more cell surface markers, e.g., 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 can be characterized by high or low expression of one or more of the cell surface markers, e.g., 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, activation of M1 polarization of macrophages using the methods described herein is desirable.

[0084] "Phagocytosis" can be used interchangeably with "engulfment" and can refer to the process by which a cell engulfs a particle (e.g., a cancer cell or an infected cell). The process can result in an internal compartment (phagosome) containing the particle. The process can be used for the uptake and / or removal of particles, e.g., cancer cells or infected cells from the body. Phagocytic receptors can be involved in the process of phagocytosis. The process of phagocytosis can be closely related to immune responses and antigen presentation. The processing of exogenous antigens occurs after they are taken up into professional antigen-presenting cells by some types of endocytic events. Phagocytosis can also promote antigen presentation. For example, antigens from phagocytosed cells or pathogens, including cancer antigens, can be processed and presented on the cell surface of APCs.

[0085] "Polypeptide" can refer to a molecule containing amino acids linked together via peptide bonds, e.g., glycoproteins, lipoproteins, cellular proteins, or membrane proteins. A polypeptide can comprise one or more subunits of a protein. A polypeptide can be encoded by a recombinant nucleic acid. In some embodiments, a polypeptide can contain more than one peptide sequence in a single amino acid chain, which can be separated by a spacer, linker, or peptide-cleavable sequence. A polypeptide can be a fusion polypeptide. A polypeptide can comprise one or more domains, modules, or portions.

[0086] "Receptor" can refer to a chemical structure composed of a polypeptide that transduces signals, such as a polypeptide that transduces extracellular signals into a cell. A receptor can be used to transmit information in a cell, cell formation, or organism. A receptor comprises at least one receptor unit and can contain two or more receptor units, where each receptor unit contains a protein molecule, e.g., a glycoprotein molecule. A receptor can contain a structure that binds a ligand and can form a complex with the ligand. Signal transduction information can be transmitted through conformational changes in the receptor after binding to a ligand on the cell surface.

[0087] The term "antibody" refers 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 not limited to full-length antibodies, single-chain antibodies, single-domain antibodies (sdAbs), and antigen-binding fragments thereof. Antigen-binding antibody fragments include but not limited to Fab, Fab’, and F(ab’)2, Fd (composed of V H and C H 1), single-chain variable fragments (scFv), single-chain antibodies, disulfide-linked variable fragments (dsFv), and fragments containing V L and / or V H domains. Antibodies can be from any animal source. Antigen-binding antibody fragments, including single-chain antibodies, can contain a variable region alone or in combination with one or more of a hinge region, CH1 domain, CH2 domain, and CH3 domain. Also included are any combinations of variable regions and hinge regions, CH1, CH2, and CH3 domains. Antibodies can be monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies that, for example, specifically bind HLA-related polypeptides or HLA peptide complexes.

[0088] The term "recombinant nucleic acid" refers to a nucleic acid prepared, expressed, produced, or isolated by recombinant means. Recombinant nucleic acids can contain nucleotide sequences that do not occur naturally. Recombinant nucleic acids can be synthesized in the laboratory. Recombinant nucleic acids can be prepared by using recombinant DNA techniques, e.g., enzymatic modification of DNA such as 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 ex vivo, e.g., to generate messenger RNA (mRNA). Recombinant mRNA can be isolated, purified, and used to transfect cells. Recombinant nucleic acids can encode proteins or polypeptides. Throughout the specification, the nucleic acid sequences described can contain deoxynucleotides (DNA), ribonucleotides (RNA), or in some embodiments, modified deoxynucleotides or modified ribonucleotides. For example, modified nucleotides can be 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 7-methylguanosine, pseudouridine, dihydrouridine, etc. A person skilled in the art can readily determine the RNA sequence, e.g., the mRNA sequence, from a given polynucleotide sequence. The sequence can be codon-optimized.

[0089] The process of introducing or incorporating nucleic acids into cells can be via 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, which take up extracellular DNA from the environment. Some bacterial species are naturally competent under certain environmental conditions, but competence is induced artificially in a laboratory setting. Transfection is the introduction of small molecules (e.g., DNA, RNA) or antibodies into eukaryotic cells. Transfection can also refer to the introduction of bacteriophages into bacterial cells. "Transduction" is mainly used to describe the introduction of recombinant viral vector particles into target cells, while "infection" refers to the natural infection of humans or animals with wild-type viruses.

[0090] The term "vector" can refer to a nucleic acid molecule that is capable of autonomous replication in a host cell and permits cloning of a nucleic acid molecule. As is known to those skilled in the art, vectors include, but are not limited to, plasmids, cosmids, phagemids, viral vectors, phage vectors, yeast vectors, mammalian vectors, etc. For example, the vector for exogenous gene transformation can be a plasmid. In certain embodiments, the vector comprises a nucleic acid sequence containing an origin of replication and other elements necessary for replication and / or maintenance of the nucleic acid sequence in the host cell. In some embodiments, the vectors or plasmids provided herein are expression vectors. An expression vector is capable of directing the expression of a gene and / or nucleic acid sequence operably linked thereto. In some embodiments, the expression vector or plasmid is in the form of a circular double-stranded DNA molecule. The vector or plasmid may or may not integrate into the genome of the host cell. In some embodiments, the nucleic acid sequence of the plasmid does not integrate 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, e.g., a gene or open reading frame carried by 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 the host cell. In some embodiments, the nucleic acid sequence of the plasmid integrates into the genome or chromosome of the host cell 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, phages, or viral vectors. The vector can be a DNA or RNA vector. In some embodiments, the vectors provided herein are RNA vectors that are capable of integrating into the host cell genome upon introduction into the host cell (e.g., via reverse transcription), e.g., retroviral vectors or lentiviral vectors. Other forms of expression vectors known to those skilled in the art with equivalent functions can also be used, e.g., self-replicating extrachromosomal vectors or vectors capable of integrating into the host genome. Exemplary vectors are vectors capable of autonomous replication and / or expression of nucleic acids linked thereto.

[0091] In some embodiments, the nucleic acid 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 can contain polar lipids. In some embodiments, the lipid nanoparticles contain cationic lipids. In some embodiments, the lipid nanoparticles contain cationic lipids and non-cationic lipids. In some embodiments, the lipid nanoparticles contain neutral lipids. In some embodiments, the lipid nanoparticles contain PEGylated lipids.

[0092] Alternatively, in some embodiments, the nucleic acid can be electroporated ex vivo in a living cell to prepare a cell therapy, wherein the cell is a myeloid cell.

[0093] The term "spacer" or "linker" as used in reference to a fusion protein refers to a peptide sequence that links two other peptide sequences of the fusion protein. In some embodiments, the linker or spacer has no specific biological activity other than to link or maintain some minimal distance or other spatial relationship between the protein or RNA sequences. In some embodiments, the constituent amino acids of the spacer can be selected to affect some properties of the molecule, such as the folding, flexibility, net charge, or hydrophobicity of the molecule. Suitable linkers for use in the embodiments of the present 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, a linker is used to separate two or more polypeptides (e.g., two antigenic peptides) by a distance sufficient to ensure proper folding of each antigenic peptide. Exemplary peptide linker sequences adopt a flexible extended conformation and do not exhibit a tendency to form ordered secondary structures. The amino acids in the flexible linker protein region can 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, can also be used in the linker sequence.

[0094] The term "treatment" refers to alleviating, preventing, or ameliorating a disorder and / or symptoms associated therewith (e.g., neoplasia or tumor or infectious pathogen or autoimmune disease). "Treatment" can refer to administering a therapy to a subject after the onset or suspected onset of a disease (e.g., cancer or infection by an infectious pathogen or autoimmune disease). "Treatment" encompasses the concept of "mitigation", which can refer to reducing the frequency or severity of occurrence or recurrence of any symptoms or other adverse effects associated with the disease and / or side effects associated with the treatment. The term "treatment" also encompasses the concept of "management", which refers to reducing the severity of a patient's disease or disorder, e.g., prolonging the life or viability of a patient with a disease, or delaying its recurrence, e.g., prolonging the remission period of a patient with a disease. It is understood that, although not excluded, treating a disorder or condition does not require complete elimination of the disorder, condition, or symptoms associated therewith. As used herein, the terms "prevention" and their grammatical equivalents can refer to avoiding or delaying the onset of symptoms associated with a disease or condition in a subject that do not develop upon initiation of administration of an agent or compound. In certain embodiments, treating a subject or patient as described herein includes administering a therapeutic composition, such as a drug, metabolite, prophylactic component, nucleic acid, peptide, or protein that encodes or otherwise forms a drug, metabolite, or prophylactic component. In some embodiments, treatment includes administering a cell or population of cells to a subject in need thereof. In some embodiments, treatment includes administering one or more of the engineered cells described herein to a subject, e.g., one or more engineered myeloid cells, such as phagocytes. Treatment includes treating a disease or disorder or syndrome, which can be a pathologic disease, disorder, or syndrome, or a latent disease, disorder, or syndrome. In some instances, treatment as used herein can include administering a therapeutic vaccine. In some embodiments, engineered phagocytes are administered to a patient or subject. In some embodiments, the cells administered to a human subject result in reduced immunogenicity. For example, engineered phagocytes can result in no or reduced graft-versus-host disease (GVHD) or fratricide. In some embodiments, the engineered cells administered to a human subject are immunocompatible with the subject (i.e., have a matching HLA subtype that is naturally expressed in the subject). The subject-specific HLA alleles or HLA genotype of a subject can be determined by any method known in the art.In an exemplary embodiment, the method includes determining a polymorphic genotype, which can include generating an alignment of reads extracted from a sequencing data set with a gene reference set that includes allelic variants of a polymorphic gene, determining a first posterior probability or a posterior probability-derived score for each allelic variant in the alignment, identifying as a first allelic variant the allelic variant having the greatest first posterior probability or posterior probability-derived score, identifying one or more overlapping reads that align with the first allelic variant and one or more other allelic variants, determining a second posterior probability or a posterior probability-derived score for the one or more other allelic variants using a weighting factor, identifying as a second allelic variant the allelic variant having the greatest second posterior probability or posterior probability-derived score, the first and second allelic variants defining the genotype of the polymorphic gene, and providing an output of the first and second allelic variants.

[0095] A "fragment" can 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 the reference protein or nucleic acid.

[0096] The terms "isolated," "purified," "biologically pure," and their grammatical equivalents can refer to materials that are, to varying degrees, free of the components that normally accompany them as they occur in their native state. "Isolated" represents a degree of separation from the original source or environment. "Purified" represents a degree of separation that is greater than isolation. A "purified" or "biologically pure" protein can be sufficiently free of other materials such that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of the present disclosure can be purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are generally determined using analytical chemistry techniques (e.g., polyacrylamide gel electrophoresis or high performance liquid chromatography). The term "purified" can indicate that a nucleic acid or protein produces substantially a single band on an electrophoretic gel. For proteins that can be modified (e.g., phosphorylated or glycosylated), different modifications can result in different isolated proteins, which can be purified separately.

[0097] The term "neoplasia" or "cancer" refers to any disease caused or resulting from inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Glioblastoma is a non-limiting example of neoplasia or cancer. The term "cancer" or "tumor" or "hyperproliferative disorder" refers to the presence of cells having the typical characteristics of cells that cause cancer, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and certain characteristic morphological features. Cancer cells are typically in the form of tumors, but such cells can exist alone in an animal or can be non-tumorigenic cancer cells, such as leukemia cells.

[0098] The term "vaccine" is understood to mean a composition that generates immunity for the prevention and / or treatment of diseases (e.g., neoplasia / tumor / infectious pathogen / autoimmune disease). Thus, a vaccine as used herein is a medicament comprising a recombinant nucleic acid, or a cell comprising and expressing a recombinant nucleic acid, and is intended for use in humans or animals to generate specific defensive and protective substances by vaccination. A "vaccine composition" may include a pharmaceutically acceptable excipient, carrier, or diluent. Aspects of the present disclosure relate to the use of the technology in the preparation of phagocyte-based vaccines.

[0099] The term "pharmaceutically acceptable" means approved or approvable by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals, including humans. A "pharmaceutically acceptable excipient, carrier, or diluent" means an excipient, carrier, or diluent that can be administered to a subject together with a medicament and that, when administered in a dose sufficient to deliver a therapeutically effective amount of the medicament, does not destroy the pharmacological activity and is non-toxic.

[0100] Nucleic acid molecules useful in the methods of the present disclosure include, but are not limited to, any nucleic acid molecule having activity or encoding a polypeptide. Polynucleotides having substantial identity to an endogenous sequence are generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridization" refers to the pairing of nucleic acid molecules under various stringency conditions to form a double-stranded molecule between complementary polynucleotide sequences or portions thereof. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507). For example, stringent salt concentrations can generally be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents (e.g., formamide), while high stringency hybridization can be achieved in the presence of at least about 35% formamide or at least about 50% formamide. Stringent temperature conditions can generally include temperatures of at least about 30°C, at least about 37°C, or at least about 42°C. Different additional parameters (e.g., hybridization time), concentrations of detergents (e.g., sodium dodecyl sulfate (SDS)), and the inclusion or exclusion of carrier DNA are well known to those of skill in the art. Various levels of stringency are achieved by combining these different conditions as needed. In an exemplary embodiment, hybridization can occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another exemplary embodiment, hybridization can occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In another exemplary embodiment, hybridization can occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be apparent to those of skill in the art. For most applications, the stringency of the wash step after hybridization can also vary. Wash stringency conditions can be defined by salt concentration and by temperature. As described above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, the salt concentration for a stringent wash 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 temperature conditions for a stringent wash 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 wash step can occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS.In other exemplary embodiments, the washing step can occur at 42 °C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the washing step can occur at 68 °C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Other 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 Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0101] "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 can be at least 60%, 80%, or 85%, 90%, 95%, 96%, 97%, 98%, or even 99% or more identical to the sequence being compared at the amino acid level or nucleic acid level. Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, Biotechnology Center, University of Wisconsin, 1710 University Avenue, Madison, Wisconsin 53705, the BLAST, BESTFIT, GAP, or PILEUP / PRITYBOX programs). Such software matches identical or similar sequences by specifying the degree of homology of various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, the BLAST program can be used, where a probability score between e-3 and e-m° indicates closely related sequences. "Reference" is the standard for comparison. It will be understood that the numbering of specific positions or residues in the corresponding sequences depends on the particular protein and numbering scheme used. For example, the numbering may be different in the precursor of a mature protein and the mature protein itself, and sequence differences between species may affect the numbering. Those skilled in the art will be able to identify any homologous proteins and the corresponding residues in the corresponding coding nucleic acids by methods well known in the art, e.g., by sequence alignment with a reference sequence and determination of homologous residues.

[0102] The term "subject" or "patient" can refer to an organism, e.g., an animal (e.g., a human) that is the subject of treatment, observation, or experiment. By way of example only, subjects include, but are not limited to, mammals, including, but not limited to, humans or non-human mammals, such as non-human primates, rats, cows, horses, dogs, sheep, or cats.

[0103] The term "therapeutic effect" can refer to a degree of alleviation of one or more of a disease condition (e.g., tumor formation, tumor or infection by an infectious pathogen or an autoimmune disease) or its associated pathological symptoms. On the one hand, it can indicate a reduction in disease symptoms, e.g., a 10%, 20%, 30% etc. reduction in tumor mass after administration of a therapeutic composition. In another embodiment, it can relate to a partial or complete remission of one or more symptoms, or an improvement in the disease. As used herein, a "therapeutically effective amount" refers to an effective dosage of a drug that, upon single or multiple dosing to a cell or subject, exceeds what would be expected in the absence of such treatment in terms of prolonging the survival of a patient suffering from such a condition, reducing one or more signs or symptoms of the condition, preventing or delaying, etc. A "therapeutically effective amount" is intended to define the amount required to achieve a therapeutic effect. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe a "therapeutically effective amount" (e.g., ED50) of the required pharmaceutical composition.

[0104] Provided herein are engineered myeloid cells (including but not limited to neutrophils, monocytes, myeloid dendritic cells (mDCs), mast cells, and macrophages) that are designed to specifically bind to a target antigen. The target antigen may be expressed only on target cells, such as infected cells, damaged cells, malignant cells, leukemic cells, or tumor cells. The engineered myeloid cells can attack and kill target cells directly (e.g., by phagocytosis) and / or indirectly (e.g., by activating T cells). In some embodiments, the target cells are cancer cells.

[0105] Although cancer is an exemplary embodiment described in detail in the present disclosure, the methods and techniques contemplated herein can be used to target infected cells or other diseased cells in vivo. Similarly, therapeutic and vaccine compositions using engineered cells are described herein.

[0106] Myeloid effector cells can be generated from isolated myeloid cells from a human biological sample and modified ex vivo using methods that engineer such cells to prepare cells for therapeutic purposes such that the modification does not alter the plasticity of these cells. Monocytic lineage cells have phagocytic capabilities and are efficient antigen-presenting cells. In one aspect, the present invention stems from the important discovery that engineered myeloid cells can be an efficient therapeutic modality in treating a variety of diseases including cancer. Myeloid cells can be engineered to express a chimeric antigen receptor that enhances the immune function of the myeloid cells, wherein the cells have a high phagocytic capacity and can attack and kill diseased or infected cells in the body. The chimeric antigen receptor is a recombinant construct that is designed and specifically modified as described herein to (a) have a high target specificity of the extracellular antigen-binding domain, specifically designed to bind to a target antigen, and (b) an intracellular domain that is highly specialized to activate myeloid cells to obtain an activated phagocytic cell phenotype. For example, the highly specialized intracellular domain is designed to generate a chimeric receptor that, when activated by binding of the extracellular region of the receptor to the target, can generate a signaling cue within the cell that activates the intracellular interferon signaling cascade and a transcription factor, namely, the transcription factor IRF (IFN regulatory factor). In addition, the methods and compositions described herein can also be used for gene therapy, wherein a recombinant nucleic acid encoding a chimeric antigen receptor is administered locally or systemically to a subject in need such that the recombinant nucleic acid is specifically expressed in vivo in myeloid cells, thereby generating activated myeloid cells with therapeutic capabilities. In some embodiments, the nucleic acid is mRNA. In some embodiments, the mRNA is delivered in an LNP.

[0107] Phagocytes are the natural sentinels of the immune system, forming the first line of defense in the body. They engulf pathogens, pathogen-infected cells, foreign substances, 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 overt infection or disease. This can involve receptor-mediated uptake via the clathrin-coated pit system, pinocytosis, and particularly macropinocytosis due to membrane ruffling and phagocytosis. Thus, phagocytes can be activated by a variety of non-self (and self) elements and exhibit a certain level of plasticity upon recognition of their "targets". Most phagocytes express scavenger receptors on their surface, which are pattern recognition molecules and can bind a wide range of foreign particles in the body, as well as dead cells, debris, and unwanted particles. On the one hand, recombinant nucleic acids encoding chimeric antigen receptors (CARs) can be expressed in cells. CARs can be designed differently to attack specific tumor cells, and myeloid effector cells expressing CARs can be activated to phagocytose and kill tumor cells. CARs can be designed to generate phagocytic receptors that are specifically activated in response to target engagement, and the phagocytic potential of macrophages is enhanced by the specific engineering of the intracellular domain of the receptor. The CAR platform for myeloid cells as described herein is designed such that upon in vivo administration, or at any time prior to engagement of the myeloid cell with its target via the CAR, no tonic signaling is detected in the myeloid cell. This is typically tested ex vivo. Meanwhile, myeloid cells expressing CARs can be further differentiated into M0, M1, or M2 phenotypes in the presence of appropriate stimuli and maintain cellular plasticity at least upon administration. In addition, myeloid effector cells expressing CARs can migrate to lymph nodes and cross-present antigens to naive T cells in the lymph nodes, thereby activating an adaptive response.

[0108] In some embodiments, compositions and methods are disclosed herein for generating myeloid cells that are isolated from a biological sample and engineered ex vivo to express a recombinant protein and formulated as a pharmaceutical composition such that the myeloid cells of the composition are “effector” myeloid cells that are effective at inducing immune activation in vivo. In some embodiments, the myeloid cells of the composition are referred to as “ATAK” myeloid cells, wherein the cells are myeloid cells that are effective at attacking and destroying target cells. The ATAK myeloid cells disclosed herein are engineered myeloid cells that express a recombinant protein (e.g., a chimeric receptor, e.g., a chimeric antigen receptor) that comprises at least one intracellular signaling domain derived from an interferon-induced protein in immune cells. In some embodiments, the methods and compositions described herein are designed to cause the engineered myeloid cells to exhibit an effector phenotype. In some embodiments, the engineered myeloid cells (e.g., 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 designed to target tumor cells, the M1 phenotype exhibited by the engineered cells renders the cells highly tumoricidal.

[0109] When it is indicated that a polynucleotide is expressed substantially in a certain cell type, it can be understood to exhibit higher expression in that cell type relative to another cell type or other cell types. For example, the concepts disclosed herein are used for compositions comprising a recombinant polynucleotide that is expressed “substantially” in a myeloid cell type, which can be understood to be expressed preferentially in the myeloid cell type relative to other cells (e.g., hepatocytes, neuronal cells, or T cells). This can mean that after a certain time following introduction of the polynucleotide into the cell, the protein encoded by the polynucleotide can be readily detected in the myeloid cells and not readily detected in non-myeloid cells. In some embodiments, the term can be intended to mean a higher number of the preferred cell type express the protein encoded by the polynucleotide, in which case, for example, a higher number of myeloid cells express the protein encoded by the polynucleotide compared to neutrophils, T cells, or hepatocytes or neurons.

[0110] Compositions and methods are provided herein for treating a disease or condition (e.g., 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 tissue, by 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, reduction of regulatory T cells, and phagocytosis. For example, myeloid cells can be used to maintain an immune response against cancer cells.

[0111] The applicant previously described compositions that comprise a recombinant nucleic acid encoding a chimeric fusion protein (CFP), such as a phagocytic receptor (PR) fusion protein (PFP), a scavenger receptor (SR) fusion protein (SFP), an integrin receptor (IR) fusion protein (IFP), or a caspase recruitment receptor (caspase-CAR) fusion protein. The CFP encoded by the recombinant nucleic acid can comprise an extracellular domain (ECD) that contains an antigen-binding domain that binds a target cell antigen. The extracellular domain can be fused to a hinge domain or an extracellular domain derived from a receptor such as CD2, CD8, CD28, CD68, a phagocytic receptor, a scavenger receptor, or an integrin receptor. The CFP encoded by the recombinant nucleic acid can further comprise a transmembrane domain, such as a transmembrane domain derived from CD2, CD8, CD28, CD68, a phagocytic receptor, a scavenger receptor, or an integrin receptor. In some embodiments, the CFP encoded by the recombinant nucleic acid further comprises an intracellular domain that contains an intracellular signaling domain, such as an intracellular signaling domain derived from a phagocytic receptor, a scavenger receptor, or an integrin receptor. For example, the intracellular domain can comprise one or more intracellular signaling domains derived from a phagocytic receptor, a scavenger receptor, or an integrin receptor. For example, the intracellular domain can comprise one or more intracellular signaling domains that promote phagocytic activity, an inflammatory response, nitric oxide production, integrin activation, enhanced effector cell migration (e.g., via chemokine receptor expression), antigen presentation, and / or enhanced cross-presentation. In some embodiments, the CFP is a phagocytic receptor fusion protein (PFP). In some embodiments, the CFP is a phagocytic scavenger receptor fusion protein (PFP). In some embodiments, the CFP is an integrin receptor fusion protein (IFP). In some embodiments, the CFP is an inflammatory receptor fusion protein. In some embodiments, the CFP encoded by the recombinant nucleic acid further comprises an intracellular domain that contains a recruitment domain. For example, the intracellular domain can comprise one or more PI3K recruitment domains, caspase recruitment domains, or caspase activation and recruitment domains (CARDs).

[0112] The present disclosure provides improved immunogenic CAR compositions, e.g., recombinant nucleic acids encoding chimeric fusion proteins (CFPs, interchangeably referred to as chimeric antigen receptors, CARs) that include intracellular domains that activate the interferon response in cells expressing the CAR. The present disclosure provides immunogenic CFPs that include at least one intracellular domain that includes a pLxIS motif. The recombinant nucleic acid can be DNA or RNA. The recombinant nucleic acid encoding the CAR can be included in a vector. When expressed in a cell, the recombinant CAR activates type I interferon production in the cell. Such a cell is a mammalian cell that is capable of activating a type I interferon response. Such a cell is an immune cell, e.g., a lymphocyte or a myeloid cell.

[0113] In some embodiments, the recombinant nucleic acid encoding the chimeric receptor includes a specific sequence encoding a pro-inflammatory intracellular domain of the chimeric receptor therein. In some embodiments, the chimeric receptor protein described herein includes an intracellular domain capable of activating an interferon response gene or signaling cascade that results in the induction of type I interferon production in a cell that expresses the chimeric antigen receptor upon engagement of a target at its extracellular domain. In some embodiments, the chimeric receptor protein described herein includes a domain from an innate immune pathway adapter protein, such as mitochondrial antiviral-signaling protein (MAVS), stimulator of interferon genes (STING), Toll / IL-1R domain-containing adapter inducing IFN (TRIF), and TLR adapter that interacts with the endolysosomal SLC15A4 protein (TASL), or a portion thereof. In some embodiments, a domain or fragment of an innate immune pathway adapter protein (e.g., MAVS, STING, TRIF, or TASL protein) can be incorporated into the intracellular domain of the CFP or CAR described herein by recombinant DNA techniques, wherein the domain or fragment includes a pLxIS motif (where p represents a hydrophilic residue, x represents any residue, and S represents a phosphorylation site) that is phosphorylated by TBK1 or IKKε and mediates the recruitment of IRF-3 to the signaling complex.

[0114] In some embodiments, the chimeric receptor protein described herein includes an intracellular domain capable of activating a nuclear factor κB response gene or signaling cascade that results in the induction of an NF-κB response in a cell that expresses the chimeric antigen receptor upon engagement of a target at its extracellular domain.

[0115] Effector myeloid cells and interferon activation

[0116] 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, and all of these subtypes utilize the same cell surface receptor, IFNαR, which is a heterodimer composed of the IFNαR1 and IFNαR2 proteins. Type II IFN is represented by IFNγ. These two types of IFNs bind to distinct cell surface receptors expressed by nearly all cells to trigger signal transduction events and elicit different cellular responses. Myeloid cells are key targets of interferons. During the early immune response to intracellular bacterial infection, activated natural killer (NK) cells and T cells are sources of IFNγ production. During the early stages of infection, the production of the cytokines interleukin (IL)-12 and IL-18 drives the production of antigen-nonspecific IFNγ by these lymphocyte populations. Antigen-specific CD4 + and CD8 +T cells can also produce IFNγ in response to these pathogens. There are a large number of individual type I IFNs, including approximately 20 IFNα proteins and a single IFNβ. Each of these type I IFNs signals to host cells by binding to the conserved cell surface type I IFN receptor IFNαR. Ligation of the cell surface IFNαR induces the expression of many antiviral immune-stimulatory gene (ISG) products and thus protects the host from certain viral infections (Sadler AJ, Interferon-inducible antiviral effectors. (Review) Nat Rev Immunol. July 2008; 8(7):559-68). However, responsiveness to type I IFN is 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. September - October 2010; 1(5):418-22), including Listeria monocytogenes, Mycobacterium tuberculosis, Fransicella tularensis, etc. IFNγ is secreted as a homodimer and acts on host cells by ligating cell surface receptors. Each IFNγ receptor is a heterodimer composed of two type I integral membrane subunits, IFNγR1 and IFNγR2. Binding of the IFNγ homodimer to the cell results in the aggregation of two receptor complexes such that there are two IFNγR1 subunits and two IFNγR2 subunits as well as additional signaling components. Although both subunits are required for signal transduction, the actual binding site of IFNγ is located on IFNγR1 (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). When IFNγ interacts with the IFNγR1 subunit, IFNγ induces conformational changes that allow for a closer association of the IFNγR1 and IFNγR2 subunits. These rearrangements in the receptor induce autophosphorylation and cross-phosphorylation of Janus-associated kinases (JAKs) that are constitutively associated with the receptor.IFNγR1 contains a binding motif for JAK1, and IFNγR2 contains a binding motif for JAK2. Phosphorylation of the JAK proteins stimulates their catalytic activity, which then phosphorylates tyrosine residues (Y. 440 ) at the C-terminus of IFNγR1. This phosphorylated tyrosine residue provides a docking site for the SH2 domain on the signal transducer and activator of transcription-1 (STAT-1) protein. Since each receptor complex contains two IFNγR1 subunits, two STAT-1 proteins can bind to the receptor. JAK1 and JAK2 remain receptor-associated and phosphorylate each recruited STAT-1 protein at tyrosine residue 701 (Y 701 ). This phosphorylation allows the STAT-1 monomers to be released from the receptor and form homodimers. The STAT-1 homodimers translocate to the nucleus and bind to the gamma-activated sequence (GAS) in the promoter DNA of IFN-stimulated genes (ISGs), resulting in increased transcription of them. Type I IFNs signal through the canonical JAK / STAT pathway, similar to the pathway activated by IFNγ. The ligand binding to IFNαR induces dimerization of the two receptor subunits and transphosphorylation of their associated TYK2 and JAK1 kinases. The kinases phosphorylate residues in the cytoplasmic tail regions of IFNαR1 and IFNαR2 to recruit STAT1 and STAT2 proteins via their SH2 domains. The docking of these STAT proteins to the receptor subunits allows them to be phosphorylated by the activated JAK proteins at Y 701 on STAT-1 and Y 690 on STAT-2. Phosphorylation of the STAT monomers releases them from the docking sites, allowing dimerization and binding to IRF9 in the form of homodimers or heterodimers to generate the transcription factor ISG factor 3 (ISGF3). ISGF3 translocates into the nucleus to identify ISGs and induce their transcription. ISGs induced by type I IFN signaling usually contain interferon-stimulated response elements (ISREs) or gamma-activated sequence (GAS) elements in their promoters, although there is a clear preference for genes containing ISREs. Some examples of ISGs transcribed as a result of type I IFN are ISREs containing the genes ISG15, IP-10, IRF-7, and PKR

[66] , and GASs containing the genes 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).

[0117] Recombinant chimeric receptor protein

[0118] The present disclosure provides a class of phagocytic or binding receptor (PR) subunits (e.g., phagocytic receptor fusion protein (PFP)), the subunit comprising: (i) a transmembrane domain, and (ii) an intracellular domain comprising a phagocytic receptor intracellular signaling domain; and an antigen-binding domain specific for an antigen (e.g., an antigen of a target cell or an antigen presented on a target cell); wherein the transmembrane domain and the antigen-binding domain are operably linked such that binding of the antigen-binding domain of the fusion receptor to the antigen of the target activates the intracellular signaling domain of the phagocytic receptor.

[0119] In some embodiments, the extracellular domain of the CFP comprises an Ig-binding domain. In some embodiments, the extracellular domain comprises an IgA, IgD, IgE, IgG, IgM, FcRγI, FcRγIIA, FcRγIIB, FcRγIIC, FcRγIIIA, FcRγIIIB, FcRn, TRIM21, FcRL5-binding domain. In some embodiments, the extracellular domain of the CFP comprises an FcR extracellular domain. In some embodiments, the extracellular domain of the CFP comprises an FcRα, FcRβ, FcRε or FcRγ extracellular domain. In some embodiments, the extracellular domain comprises an FcRα (FCAR) extracellular domain. In some embodiments, the extracellular domain comprises an FcRβ extracellular domain. In some embodiments, the extracellular domain comprises an FCER1A extracellular domain. In some embodiments, the extracellular domain comprises an FDGR1A, FCGR2A, FCGR2B, FCGR2C, FCGR3A or FCGR3B extracellular domain. In some embodiments, the extracellular domain comprises an integrin domain or an integrin receptor domain. In some embodiments, the extracellular domain comprises one or more integrin α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7 or β8 domains.

[0120] In some embodiments, the CFP further comprises an extracellular domain that includes an antigen-binding domain operably linked to a transmembrane domain. In some embodiments, the extracellular domain further comprises the extracellular domain of a receptor, a hinge, a spacer, and / or a linker. In some embodiments, the extracellular domain comprises the extracellular portion of a phagocytic receptor. In some embodiments, the extracellular portion of the CFP is derived from the same receptor from which the intracellular signaling domain is derived. In some embodiments, the extracellular domain comprises the extracellular domain of a scavenger receptor. In some embodiments, the extracellular domain comprises an immunoglobulin domain. In some embodiments, the immunoglobulin domain comprises the extracellular domain of an immunoglobulin or an immunoglobulin hinge region. In some embodiments, the extracellular domain comprises a phagocytic engulfment domain. In some embodiments, the extracellular domain comprises a structure capable of multimeric assembly. In some embodiments, the extracellular domain comprises a scaffold for multimerization. In some embodiments, the length of the extracellular domain is at least 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 extracellular domain is at most 500, 400, 300, 200, or 100 amino acids. In some embodiments, the antigen-binding domain specifically binds an antigen of a target cell. In some embodiments, the antigen-binding domain comprises an antibody domain. In some embodiments, the antigen-binding domain comprises a receptor domain, an antibody domain, where the antibody domain comprises a functional antibody fragment, a single-chain variable fragment (scFv), Fab, a single-domain antibody (sdAb), a nanobody, a V H domain, a V L domain, a VNAR domain, a V HH domain, a bispecific antibody, a diabody, or a functional fragment or combination thereof. In some embodiments, the antigen-binding domain comprises a ligand, the extracellular domain of a receptor, or an adaptor. In some embodiments, the antigen-binding domain comprises a single antigen-binding domain specific for a single antigen. In some embodiments, the antigen-binding domain comprises at least two antigen-binding domains, where each of the at least two antigen-binding domains is specific for a different antigen.

[0121] In some embodiments, the antigen is a cancer-associated antigen, a lineage-associated antigen, a pathogenic antigen, or an autoimmune antigen. In some embodiments, the antigen comprises a viral antigen. In some embodiments, the antigen is a T lymphocyte antigen. In some embodiments, the antigen is an extracellular antigen. In some embodiments, the antigen is an intracellular antigen. In some embodiments, the antigen is selected from antigens from thymidine kinase (TK1), hypoxanthine-guanine phosphoribosyltransferase (HPRT), receptor tyrosine kinase-like orphan receptor 1 (ROR1), mucin-1, mucin-16 (MUC16), MUC1, epidermal growth factor receptor vIII (EGFRvIII), mesothelin, human epidermal growth factor receptor 2 (HER2), EBNA-1, LEMD1, phosphatidylserine, carcinoembryonic antigen (CEA), B cell maturation antigen (BCMA), glypican 3 (GPC3), follicle-stimulating hormone receptor, fibroblast activation protein (FAP), erythropoietin-producing hepatocellular carcinoma A2 (EphA2), EphB2, natural killer group 2D (NKG2D) ligand, disialoganglioside 2 (GD2), CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD45, CD56, CD79b, CD97, CD117, CD123, CD133, CD138, CD171, CD179a, CD213A2, CD248, CD276, PSCA, CS-1, CLECL1, GD3, PSMA, FLT3, TAG72, EPCAM, IL-1, integrin receptor, PRSS21, VEGFR2, PDGFRβ, SSEA-4, EGFR, NCAM, prostate enzyme, PAP, ELF2M, GM3, TEM7R, CLDN6, TSHR, GPRC5D, ALK, Dsg1, Dsg3, IGLL1, and combinations thereof. In some embodiments, the antigen is an antigen of a protein selected from CD2, CD3, CD4, CD5, CD7, CCR4, CD8, CD30, CD45, and CD56. In some embodiments, the antigen is an ovarian cancer antigen or a T lymphoma antigen. In some embodiments, the antigen is an antigen of an integrin receptor. In some embodiments, the antigen is an antigen of an integrin receptor selected from α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, and β8 or an integrin. In some embodiments, the antigen is an antigen of an integrin receptor ligand. In some embodiments, the antigen is an antigen of fibronectin, vitronectin, collagen, or laminin.In some embodiments, the antigen-binding domain can bind two or more different antigens.

[0122] In some embodiments, the antigen-binding domain comprises the sequence of an antigen-binding domain provided herein, such as the sequence of an antigen-binding domain in the tables provided herein.

[0123] In some embodiments, the target protein is CD70. In some embodiments, the antigen-binding domain comprises an anti-CD70 antibody or a binding fragment thereof, wherein the antigen-binding domain comprises a heavy-chain variable domain (VH), the VH comprises a heavy-chain complementarity-determining region 3 (CDR3), and the CDR3 is the CDR3 of any one of the VH sequences selected from the following amino acid sequences: QVQLQESGGGLVQAGGSLRLSCAAPRSIFSINAMGWYRQAPGKQR ELVAAITSGGSPTYADSVKGRFTISRDNAKNTVYLQMNSLKAEDT AVYYCATGPYGLDNALDAWGQGTQVTVSS (SEQ ID NO:43) and QVQLQESGGGLVQTGGSLRLACTASGFTFDDYAIAWFRQAPGKER EFVAAISWSGGTTHYADSVKGRFTISRDNAKNTLYLQMSSLKPED TAVYFCAKSLRSSPSSRWFGSRGQGTQVTVSS (SEQ ID NO:44).

[0124] In some embodiments, the anti-CD70 binding domain comprises a heavy-chain complementarity-determining region 3 (CDR3) that is the CDR3 of the VH sequence of SEQ ID NO:43. In some embodiments, the anti-CD70 VH domain CDR3 has the following sequence: GPYGLDNALDA. In some embodiments, the VH of the anti-CD70 antibody or its binding fragment comprises a heavy-chain complementarity-determining region 1 (CDR1) that is the CDR1 of the VH sequence of SEQ ID NO:43. In some embodiments, the anti-CD70 VH domain CDR1 has the following sequence: INAMG. In some embodiments, the VH of the anti-CD70 antibody or its binding fragment comprises a heavy-chain complementarity-determining region 2 (CDR2) that is the CDR2 of the VH sequence of SEQ ID NO:43. In some embodiments, the anti-CD70 VH domain CDR2 has the following sequence: AITSGGSPTYADSVKG.

[0125] In some embodiments, the anti-CD70 binding domain comprises a heavy chain complementarity determining region 3 (CDR3) that is the CDR3 of the VH sequence of SEQ ID NO:44. In some embodiments, the anti-CD70 VH domain CDR3 has the following sequence: SLRSSPSSRWFGS. In some embodiments, the VH of the anti-CD70 antibody or a binding fragment thereof comprises a heavy chain complementarity determining region 1 (CDR1) that is the CDR1 of the VH sequence of SEQ ID NO:44. In some embodiments, the anti-CD70 VH domain CDR1 has the following sequence: DYAIA. In some embodiments, the VH of the anti-CD70 antibody or a binding fragment thereof comprises a heavy chain complementarity determining region 2 (CDR2) that is the CDR2 of the VH sequence of SEQ ID NO:44. In some embodiments, the anti-CD70 VH domain CDR2 has the following sequence: AISWSGGTTHYADSVKG.

[0126] The following table lists the corresponding CDRs according to three numbered CDR formats (Chothia, Kabat, and IMGT):

[0127]

[0128] In some embodiments, the VH of the anti-CD70 antigen-binding fragment has at least about 70% sequence identity with any one of the sequences selected from SEQ ID NOs: 43 and 44. In some embodiments, the VH of the anti-CD70 antigen-binding fragment has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:43 or SEQ ID NO:44. In some embodiments, the VH is a single-domain antibody domain. In some embodiments, the VH is a VHH.

[0129] In some embodiments, the target protein is GPC3. The glypicans belong to the heparan sulfate proteoglycan family with similar structures, which includes: a core protein of 60-70 kD, which is anchored to the surface of the cell membrane by a glycosylphosphatidylinositol (GPI) anchor; and the carboxyl terminus is modified with heparan sulfate side chains. The GPC3 gene is located at position 26 on the long arm of the X chromosome and contains 11 exons. The transcript is 2130 bp, encoding 580 amino acids, and the molecular weight of the protein is about 70 kDa. It is involved in various liver cancers. The expression of GPC3 is associated with the prognosis of hepatocellular carcinoma (HCC). In addition, GPC3 is highly expressed in lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma and urothelial carcinoma. In one aspect, the present disclosure provides CFP, which targets GPC+ cells in cancer and can be developed as a therapeutic agent for HCC, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma and urothelial carcinoma. In some embodiments, the antigen-binding domain comprises an anti-GPC3 antibody or its antigen-binding domain or fragment, wherein the antigen-binding domain comprises a heavy-chain variable domain (VH), and the VH comprises a heavy-chain complementarity-determining region 3 (HC CDR3) selected from any one of the following sequences: ATACADTTQYAYDY, ATACADTTLYEYDY, ATACVDTTQYEYDY, ATACADATQHEYDY, ATACADTTQYDYDY, ATACADTTQYEYDY, ATACADTTHYEYDY, ATACVITTLYEYDY, ATACAETTLYEYDY, ATACADTTQHEYDY, ATACVDTTHYEYDY, ATACASTTLYEYDY, ATACVVTTLYEYDY, ATACGGATGPYDY, ATACAGAIGPYDY, ATACVVVGDQNDY, ATACVVVGDRNDY, ATDCAGGTSTPYDY, ATDCAGGTATPYDY, ATACVVADRNEYDY, ATSCVVVTKNEYDY, ATACSGLTHEYDY, ATTCSGLTHEYDY, ATACANWSSLGPYDY, ATACANWSTLGPYDY, ATACSDPRVYEYDY, ATTCASPEKYEYDY, ATHCGGTSWGTSYDY, ATHCGGSSWSNEYDY, YARYSGRTY, ASSAWPAGPKHQVEYDY, ATACGSLVGMYDY, ATACGSAVHEYDY, ATDCVGFGSNWFDY, ATACASPVIYEYDY, ATDCAGGVGHEYDY, ATDCSLHGSDYPYDY and AVRIYSGSFDNTLAYDY.In some embodiments, the VH of an anti-GPC3 antibody or its binding fragment further comprises a heavy chain complementarity determining region 1 (HC CDR1) selected from any of the sequences: GFPLAYYA, GFSLDYYA, GFPLDYYA, GFTLDYYA, GFSLNYYA, GFTLAYYA, GFTLGYYA, GFPLNYYA, GFPLHYYA, GFSLGYYA, GFPLGYYA, GFPLEYYA, GSDFRADA, GRTFSSYG, GFSLAYYA, and GLTFRSVG. In some embodiments, the VH of an anti-GPC3 antibody or its binding fragment further comprises a heavy chain complementarity determining region 2 (HC CDR2) selected from any of the sequences: ISNSDGST, ISASDGST, ISSSDGST, ISSSDGNT, ISSADGST, ISSSGGST, ISSGDGST, ISAGDGNT, ISSSDDST, ISSNDGST, ISSPDGST, ISSRTGGT, ISAGDGSST, ISSSDGSSSDGNT, ISSGDGNT, ISSGDGKT, ISSSDGGT, ISSRTGST, ISSRTGNT, ISSSDGHSST, ISSSSDGNT, ISASNGNT, ISSGSDGNT, ISASDGNT, IDSITSI, ISWSGGSTIAASVGST, ISSSDGSDGNT, and ASPSGVIT. In some embodiments, the VH of an anti-GPC3 antibody or its binding fragment has 70%-100% sequence identity with any of the sequences selected from SEQ ID NOs: 45-134 listed in Table 5. In some embodiments, the VH is a single domain antibody domain. In some embodiments, the VH is a VHH. Some exemplary anti-GPC3 VHH domain sequences are listed in Table 5.

[0130] In some embodiments, the anti-GPC3 antigen-binding domain comprises a VH domain having CDR1, CDR2, and CDR3, wherein CDR1 has the following amino acid sequence: DYEMH; CDR2 has the following amino acid sequence: ALDPKTGDTAYSQKFKG; and CDR3 has the following amino acid sequence: FYSYTY.

[0131] In some embodiments, the anti-GPC3 antigen-binding domain is an scFv having a VH domain and a VL domain, the VH domain comprising CDR1, CDR2, and CDR3 domains, wherein the CDR1 sequence is DYEMH; the CDR2 sequence is: ALDPKTGDTAYSQKFKG; the CDR3 sequence is: FYSYTY; the VL domain comprising CDR1, CDR2, and CDR3 domains, wherein the CDR1 sequence is RSSQSLVHSNRNTYLH; the CDR2 sequence is: KVSNRFS; the CDR3 sequence is: SQNTHVPPT.

[0132] In some embodiments, provided herein is a CFP having an antigen-binding domain, a transmembrane domain, and an intracellular domain comprising CDR1, CDR2, and CDR3 as described above, wherein the transmembrane domain comprises the sequence of CD89 TMD, such as SEQ ID NO:11; and one or more of the intracellular domains described herein are, for example, in Table 2. Exemplary CFP sequences can be found in Table 4. In addition, the CFP domains can be exchanged or rearranged by using general molecular biology skills and techniques utilizing the exemplary guidance from Table 4.

[0133] Thus, in some embodiments, the antigenic target on the cancer cell is CD5. A CFP capable of binding the CD5 antigen can comprise an extracellular antigen-binding domain having the following sequence:

[0134] EIQLVQSGGGLVKPGGSVRISCAASGYTFT NYGMN WVRQAPGKGLEWMG WINTHTGEPTYADSFKG RFTFSLDDSKNTAYLQINSLRAEDTAVYFCTR RGYDWYFDV WGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC RASQDINSYLS WFQQKPGKAPKTLIY RANRLES GVPSRFSGSGSGTDYTLTISSLQYEDFGIYYC QQYDESPWT FGGGTKLEIK or a sequence having at least about 90% identity to the above sequence. The CDR sequences according to the Kabat numbering scheme are underlined, for example, the VH CDR1 sequence is NYGMN, the CDR2 sequence is WINTHTGEPTYADSFKG, and the CDR3 sequence is RGYDWYFDV; and the VL CDR1 sequence is RASQDINSYLS, the CDR2 sequence is RANRLES, and the CDR3 sequence is QQYDESPWT.

[0135] In some embodiments, the antigenic target on the cancer cell is HER2. The CFP capable of binding to the HER2 antigen may comprise an extracellular antigen-binding domain having the following sequence:

[0136] DIQMTQSPSSLSASVGDRVTITC RASQDVNTAVA WYQQKPGKAPKLLIY SASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHYTTPPT FGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIK DTYIH WVRQAPGKGLEWVA RIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDV WGQGTLVTVSS or a sequence having at least 90% identity to the above sequence. The CDR sequences according to the Kabat numbering scheme are underlined. For example, the VL CDR1 sequence is RASQDVNTAVA, the CDR2 sequence is SASFLYS, and the CDR3 sequence is QQHYTTPPT; and the VH CDR1 sequence is DTYIH, the CDR2 sequence is RIYPTNGYTRYADSVKG, and the CDR3 sequence is WGGDGFYAMDV.

[0137] In some embodiments, the antigenic target on cancer cells is trophoblast cell surface antigen 2 (TROP2). TROP2 is a membrane glycoprotein and regulates cell growth, proliferation, self-renewal, survival, and invasion. It is highly upregulated in various cancers, including non-small cell lung cancer (NSCLC), breast cancer, cervical cancer, colon cancer, gastric cancer, bladder cancer, prostate cancer, thyroid cancer, osteosarcoma, etc. The FDA and EPA have approved sacituzumab govitan for the treatment of patients with triple-negative breast cancer. The transmembrane glycoprotein trophoblast cell surface antigen-2 (Trop-2) is widely expressed in various epithelial cancers as well as in certain normal tissues. Trop-2 is also known as tumor-associated calcium signal transducer 2 (TACSTD2), membrane component 1 chromosome surface marker 1 (M1S1), gastrointestinal antigen 733-1 (GA733-1), and epithelial glycoprotein-1 (EGP-1). TROP2 is an attractive therapeutic target in various cancers. This patent application focuses on methods of using engineered myeloid cells to target TROP2 and eradicate TROP2+ cancer cells by enhancing phagocytosis and further by activating immune cells against TROP2+ cells. Provided herein is a CFP that can bind to the TROP2 antigen and can comprise an extracellular antigen-binding domain having the following sequence: QVQLQQSGSELKKPGASVKVSCKASGYTF TNYGM NWVKQAPGQGLKWMG WINTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITC KASQD VSIA VAWYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLTF GAGTKVEIKR or a sequence having at least 90% identity to the above sequence.

[0138] Any of the above sequences can comprise an N-terminal signal peptide sequence, such as MWLQSLLLLGTVACSIS, and the mature protein expressed on myeloid cells may or may not comprise this signal sequence.

[0139] In addition, any of the recombinant nucleic acids can comprise one or more linker sequences, one or more self-cleaving peptide sequences (such as T2A or P2A).

[0140] In some embodiments, the target antigen is an autoantigen or a fragment thereof, such as Dsg1 or Dsg3. In some embodiments, the antigen-binding domain comprises a receptor domain or an antibody domain, wherein the antibody domain binds to an autoantigen, such as Dsg1 or Dsg3.

[0141] In some embodiments, the transmembrane domain and the antigen-binding domain are operably linked by a linker. In some embodiments, the transmembrane domain and the antigen-binding domain are operably linked by a linker (such as the hinge region of CD8α, IgG1 or IgG4).

[0142] In some embodiments, the extracellular domain comprises a multimerization scaffold.

[0143] In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD68 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD2 transmembrane domain. In some embodiments, the transmembrane domain comprises an FcR transmembrane domain. In some embodiments, the transmembrane domain comprises an FcRγ transmembrane domain. In some embodiments, the transmembrane domain comprises an FcRα transmembrane domain. In some embodiments, the transmembrane domain comprises an FcRβ transmembrane domain. In some embodiments, the transmembrane domain comprises an FcRε transmembrane domain. In some embodiments, the transmembrane domain comprises a transmembrane domain from a synaptotagmin (such as synaptotagmin 3 or synaptotagmin 4 or synaptotagmin 5). In some embodiments, when CFP is expressed in a cell, the transmembrane domain oligomerizes with the transmembrane domain of an endogenous receptor. In some embodiments, when CFP is expressed in a cell, the transmembrane domain oligomerizes with the transmembrane domain of an exogenous receptor. In some embodiments, when CFP is expressed in a cell, the transmembrane domain dimerizes with the transmembrane domain of an endogenous receptor. In some embodiments, when CFP is expressed in a cell, the transmembrane domain dimerizes with the transmembrane domain of an exogenous receptor. In some embodiments, the transmembrane domain is derived from a protein different from the protein from which the intracellular signaling domain is derived. In some embodiments, the transmembrane domain is derived from a protein different from the protein from which the extracellular domain is derived. In some embodiments, the transmembrane domain comprises the transmembrane domain of a phagocytic receptor. In some embodiments, the transmembrane domain and the extracellular domain are derived from the same protein. In some embodiments, the transmembrane domain is derived from the same protein as the intracellular signaling domain. In some embodiments, the recombinant nucleic acid encodes a DAP12 recruitment domain. In some embodiments, the transmembrane domain comprises a transmembrane domain that oligomerizes with DAP12.

[0144] In some embodiments, the transmembrane domain has a length of 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 transmembrane domain has a length of 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.

[0145] In some embodiments, the intracellular domain comprises an intracellular domain derived from a phagocytic receptor. In some embodiments, the intracellular domain comprises an intracellular domain derived from a T cell receptor (such as a CD3 molecule, such as CD3ζ). In some embodiments, the intracellular domain comprises an intracellular domain derived from a phagocytic receptor other than a phagocytic receptor selected from Megf10, MerTk, FcRα, or Bai1. In some embodiments, the intracellular domain comprises an intracellular signaling domain derived from a receptor selected from TNFR1, MDA5, CD40, lectin, 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, Fc-α receptor I, CR1, CD35, CD3ζ, CR3, CR4, Tim-1, Tim-4, and CD169. In some embodiments, the intracellular signaling domain comprises a PI3K recruitment domain. In some embodiments, the intracellular domain does not comprise a PI3K recruitment domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a scavenger receptor. In some embodiments, the intracellular domain comprises a CD47 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, the Cdc42 inhibitory domain, or the GTPase inhibitory domain inhibits Rac, Cdc42, or GTPase at the phagocytic cup of a cell expressing PFP. In some embodiments, the intracellular domain comprises an F-actin degrading 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 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more ITAM domains.In some embodiments, the intracellular domain comprises at least one immunoreceptor tyrosine-based activation motif (ITAM) domain selected from CD3ζ, CD3ε, CD3γ, CD3δ, the Fcε receptor 1 chain, the Fcε receptor 2 chain, the Fcγ receptor 1 chain, the Fcγ receptor 2a chain, the Fcγ receptor 2b1 chain, the Fcγ receptor 2b2 chain, the Fcγ receptor 3a chain, the Fcγ receptor 3b chain, the Fcβ receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and ITAM domains having at least one but no more than 20 modifications to their amino acid sequences. In some embodiments, at least one ITAM domain comprises a Src family kinase phosphorylation site. In some embodiments, 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.

[0146] In some embodiments, the intracellular domain does not comprise a domain derived from the CD3ζ intracellular domain. In some embodiments, the intracellular domain does not comprise a domain derived from the MerTK intracellular domain. In some embodiments, the intracellular domain does not comprise a domain derived from the TLR4 intracellular domain. In some embodiments, the intracellular domain comprises a CD47 inhibitory domain. In some embodiments, the intracellular signaling domain comprises a domain that activates integrins, such as the intracellular region of PSGL-1.

[0147] In some embodiments, the intracellular signaling domain comprises a domain that activates the Rap1 GTPase, such as domains 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 comprises a phagocytosis enhancement domain.

[0148] 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, pyroptosis protein, AIM2, NLRC4, FCGR3A, FCERIG, CD40, Tank1-binding kinase (TBK), caspase domain, procaspase-binding domain, or any combination thereof.

[0149] In some embodiments, the intracellular domain comprises a signaling domain derived from a connexin (Cx), such as an intracellular signaling domain. For example, the intracellular domain may comprise a signaling domain derived from Cx43, Cx46, Cx37, Cx40, Cx33, Cx50, Cx59, Cx62, Cx32, Cx26, Cx31, Cx30.3, Cx31.1, Cx30, Cx25, Cx45, Cx47, Cx31.3, Cx36, Cx31.9, Cx39, Cx40.1, or Cx23, such as an intracellular signaling domain. For example, the intracellular domain may comprise a signaling domain derived from Cx43, such as an intracellular signaling domain.

[0150] In some embodiments, the intracellular domain comprises a signaling domain derived from a SIGLEC protein, such as an intracellular signaling domain. For example, the intracellular domain can comprise a signaling domain derived from Siglec-1 (sialoadhesin), Siglec-2 (CD22), Siglec-3 (CD33), Siglec-4 (MAG), Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-12, Siglec-13, Siglec-14, Siglec-15, Siglec-16, or Siglec-17, such as an intracellular signaling domain. In some embodiments, a recombinant polynucleotide encoding SIGLEC is co-administered with CFP for expression; wherein SIGLEC is an engineered SIGLEC molecule lacking an ITIM domain. The construct is designed as a decoy to block anti-phagocytic signaling by myeloid cells that take up and express the recombinant polynucleotide and to promote the pro-phagocytic cell function of CFP. In some embodiments, CFP and the engineered SIGLEC construct can be encoded by the same polycistronic polynucleotide to ensure uptake and co-expression of both in the same cell. In some embodiments, CFP (such as anti-TROP2 CFP or anti-GPC3 CFP) and the SIGLEC construct are encoded in different polynucleotides but are loaded in LNPs that ensure co-delivery.

[0151] In some embodiments, the intracellular domain comprises a signaling domain derived from a TLR protein, such as an intracellular signaling domain. In some embodiments, the intracellular domain can comprise the intracellular signaling domain of an endolysosomal TLR (e.g., TLR3, TLR7, TLR8, or TLR9). In some embodiments, the intracellular signaling domain can be derived from the TLR3 protein. In some embodiments, the intracellular signaling domain can be derived from the TLR7, TLR8, or TLR9 protein. In some embodiments, the intracellular domain can comprise the intracellular signaling domain of cell surface TLRs 1, 2, 4, 5, 6, and 10.

[0152] In some embodiments, the intracellular signaling domain is specifically paired 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 comprising CD64 TM or a portion thereof can be specifically paired with an intracellular signaling domain comprising a natural immune adaptor protein ICD or a PI3 kinase recruitment domain (PI3K) or both. In some embodiments, the combination of domains of the chimeric receptor intracellular domain and / or transmembrane domain is designed to maximize the phagocytosis index of cells (e.g., myeloid cells) expressing the construct. In some embodiments, the combination of domains of the chimeric receptor intracellular domain and / or transmembrane domain is designed to maximize the inflammatory potential of cells expressing the construct such that the cells are capable of lysing target cells and activating immune response pathways to provide long-term immune responsiveness. In some embodiments, the combination of domains of the chimeric receptor intracellular domain and / or transmembrane domain is designed to minimize or eliminate any tonic signaling of cells expressing the chimeric protein. In some embodiments, the combination of domains of the chimeric receptor intracellular domain and / or transmembrane domain is designed to maximize the specificity of the immune response.

[0153] In some embodiments, the intracellular domain comprises a signaling domain derived from a C-type lectin protein, such as an intracellular signaling domain. For example, the intracellular domain can comprise a signaling domain derived from a mannose receptor protein, such as an intracellular signaling domain. For example, the intracellular domain can comprise a signaling domain derived from an asialoglycoprotein receptor protein, such as an intracellular signaling domain. For example, the intracellular domain can comprise a signaling domain derived from macrophage galactose-type lectin (MGL), DC-SIGN (CLEC4L), Langerin (CLEC4K), myeloid DAP12-associated lectin (MDL)-1 (CLEC5A), DC-associated C-type lectin 1 (Dectin 1) subfamily proteins, dectin 1 / CLEC7A, DNGR1 / CLEC9A, myeloid C-type lectin-like receptor (MICL) (CLEC12A), CLEC2 (CLEC1B), CLEC12B, DC immunoreceptor (DCIR) subfamily proteins, DCIR / CLEC4A, dectin 2 / CLEC6A, blood DC antigen 2 (BDCA2) (CLEC4C), Mincle (macrophage-inducible C-type lectin) (CLEC4E), NOD-like receptor proteins, NOD-like receptor MHC class II transactivator (CIITA), IPAF, BIRC1, RIG-I-like receptor (RLR) proteins, RIG-I, MDA5, LGP2, NAIP5 / Birc1e, NLRP proteins, NLRP1, NLRP2, NLRP3, NLRP4, NLRP5, NLRP6, NLRP7, NLRP89, NLRP9, NLRP10, NLRP11, NLRP12, NLRP13, NLRP14, NLR proteins, NOD1 or NOD2 or any combination thereof, such as an intracellular signaling domain.

[0154] In some embodiments, the intracellular domain comprises a signaling domain derived from a cell adhesion molecule, such as an intracellular signaling domain. For example, the intracellular domain can comprise a signaling domain derived from an IgCAM, cadherin, integrin, C-type lectin-like domain protein (CTLD), and / or proteoglycan molecule, such as an intracellular signaling domain. For example, the intracellular domain can comprise a signaling domain derived from E-cadherin, P-cadherin, N-cadherin, R-cadherin, B-cadherin, T-cadherin, or M-cadherin, such as an intracellular signaling domain. For example, the intracellular domain can comprise a signaling domain derived from a selectin (such as E-selectin, L-selectin, or P-selectin), such as an intracellular signaling domain.

[0155] In some embodiments, the CFP does not contain a full-length intracellular signaling 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.

[0156] In some embodiments, the recombinant nucleic acid encodes an extracellular domain of the FcRα chain, a transmembrane domain of the FcRα chain, and / or an intracellular domain of the FcRα chain. In some embodiments, the recombinant nucleic acid encodes an extracellular domain of the FcRβ chain, a transmembrane domain of the FcRβ chain, and / or an intracellular domain of the FcRβ chain. In some embodiments, when expressed in a cell, the FcRα chain or the FcRβ chain forms a complex with FcRγ. In some embodiments, when expressed in a cell, the FcRα chain or the FcRβ chain forms a complex with endogenous FcRγ. In some embodiments, the FcRα chain or the FcRβ chain is not incorporated 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 of TREM, a transmembrane domain of TREM, and / or an intracellular domain of TREM. In some embodiments, TREM is TREM1, TREM2, or TREM3.

[0157] In some embodiments, the recombinant nucleic acid contains 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, e.g., ATP, ADP, UTP, UDP, and / or UDP-glucose.

[0158] Intracellular interferon response domain

[0159] Most TLR activation involves the adaptor protein MyD88, which activates the transcription factor protein NF-κB, which drives the expression of pro-inflammatory genes as part of the immune response. A subset of TLRs (TLR3 and TLR4) can engage the protein TRIF, which acts as a scaffold enabling kinases to add phosphate groups to the transcription factor IRF3. This phosphorylation activates IRF3, a member of the transcription factor family called interferon regulatory factors (IRFs), which activates a broad gene expression program. A hallmark of these programs is the production of type I interferon molecules. Interferons are potent drivers of the branch of the immune system called the adaptive immune response, and thus their presence poses a risk of autoimmunity. To prevent such attacks by the host's own immune system, the interferon response must be tightly regulated. As a safety measure, a specific amino acid residue sequence, the pLxIS motif, in TRIF must be phosphorylated before IRF3 can be activated. This control mechanism provides a "licensing step" that is specific not only for TRIF as an adaptor protein in TLR signaling but is a general hallmark of sensing pathways that engage IRF3 or the related protein IRF7 to drive interferon expression. It has previously been shown that every identified innate sensing pathway that links nucleic acid recognition to type I interferon production (with one exception) signals through one of three adaptor proteins known thus far to contain the pLxIS motif: TRIF, MAVS, and STING. Thus, adaptor proteins containing the pLxIS motif specifically hard-wire nucleic acid recognition to antiviral defense. In some embodiments, the intracellular signaling domain of CFP contains the ICD of an innate immune response protein.In some embodiments, the innate immune response proteins are selected from the intracellular signaling 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, pyroprotein, AIM2, NLRC4, FCGR3A, FCERIG, CD40, Tank1-binding kinase (TBK), TNFR1, chemokines, MHC class II transactivator (CIITA), IPAF, BIRC1, RIG-I-like receptor (RLR) proteins, macrophage galactotype lectin (MGL), DC-SIGN (CLEC4L), Langerin (CLEC4K), myeloid DAP12-associated lectin (MDL)-1 (CLEC5A), DC-associated C-type lectin 1 (Dectin 1) subfamily proteins, dectin 1 / CLEC7A, DNGR1 / CLEC9A, myeloid C-type lectin-like receptor (MICL) (CLEC12A), CLEC2 (CLEC1B), CLEC12B, DC immunoreceptor (DCIR) subfamily proteins, DCIR / CLEC4A, Dectin 2 / CLEC6A, blood DC antigen 2 (BDCA2) (CLEC4C), and Mincle (macrophage-inducible C-type lectin) (CLEC4E). In some embodiments, the CFP comprises at least one intracellular signaling domain that comprises the amino acid sequence motif pLxIS.

[0160] In some embodiments, the composition further comprises a pro-inflammatory polypeptide. In some embodiments, the pro-inflammatory polypeptide is a chemokine, cytokine. In some embodiments, the chemokines are selected from IL-1, IL3, IL5, IL-6, il8, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, RANTES, and interferon. In some embodiments, the cytokines are selected from IL-1, IL3, IL,5, IL-6, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, RANTES, and interferon.

[0161] In some embodiments, an intracellular signaling domain from an intracellular adaptor protein known to have high activity in innate immune defense is incorporated into a chimeric receptor protein. In some embodiments, one or more mutations are introduced into one or more intracellular domains to reduce the responsiveness of the intracellular domain to intracellular stimuli that are characteristic of the native intracellular adaptor protein domain without compromising the effectiveness of the chimeric protein. In some embodiments, this effectiveness is referred to as enhanced phagocytic potential compared to the same cells that do not express the chimeric protein. In some embodiments, this effectiveness is referred to as enhanced inflammatory potential compared to the same cells that do not express the chimeric protein. In some embodiments, this effectiveness is referred to as enhanced NF-κB activation or interferon activation in cells expressing the chimeric protein compared to the same cells that do not express the chimeric protein.

[0162] 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 can be selectively incorporated into such polymer structures to affect myeloid cell function. In some embodiments, the targeting structure is multilayered, for example, one or more PLGA and one or more PVA layers. In some embodiments, the targeting structure is assembled in an order of hierarchical activity. In some embodiments, the targeted polymer structure is organized into 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), for example, to maintain myeloid cells in a microenvironment that confers a specific polarization. In some embodiments, the polymer structures are such that they are not phagocytosed by myeloid cells, but they can remain adhered to the surface. In some embodiments, one or more growth factors can be M1 polarization factors, such as cytokines. In some embodiments, one or more growth factors can be M2 polarization factors, such as cytokines. In some embodiments, 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 one or more growth factors in an in vivo environment (such as in a solid tumor).

[0163] In some embodiments, the recombinant nucleic acid comprises a sequence encoding a homeostatic regulator of inflammation. In some embodiments, the homeostatic regulator of inflammation is a sequence in the mRNA untranslated region (UTR). In some embodiments, the sequence in the UTR is a sequence that binds an RNA-binding protein. In some embodiments, translation is inhibited or blocked when the RNA-binding protein binds to the sequence in the untranslated region (UTR). In some embodiments, the sequence in the UTR comprises the consensus sequence WWWU(AUUUA)UUUW, where W is A or U. In some embodiments, the recombinant nucleic acid is expressed on a bicistronic vector.

[0164] 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 a pathogen-infected cell. 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 cancer cell. In some embodiments, the target cell is a cancer cell that is a pancreatic cancer cell. In some embodiments, the target cell is a cancer cell that is a glioblastoma cell.

[0165] 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.

[0166] Also provided herein is a vector that comprises a recombinant nucleic acid sequence encoding CFP as 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 operably 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 sequences is operably 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 flanking at least one nucleic acid sequence encoding one or more polypeptides. In some embodiments, the vector further comprises one or more regulatory regions.

[0167] The present disclosure also provides a polypeptide encoded by the recombinant nucleic acid of the composition described herein.

[0168] The present disclosure provides a composition comprising a recombinant nucleic acid sequence encoding a CFP that comprises a phagocytic or binding receptor (PR) subunit (e.g., a phagocytic receptor fusion protein (PFP)), the CFP comprising: a PR subunit comprising: a transmembrane domain and an intracellular domain comprising an intracellular signaling domain; and an extracellular domain comprising an antigen-binding domain specific for a target cell antigen; wherein the transmembrane domain and the extracellular domain are operably linked; and wherein upon antigen binding of the CFP to the target cell, the killing or phagocytic activity of myeloid cells (e.g., neutrophils, monocytes, myeloid dendritic cells (mDCs), mast cells, or macrophages) expressing the CFP is increased by at least greater than 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% compared to cells not expressing the CFP.

[0169] Table 1A shows exemplary sequences of chimeric fusion protein domains and / or fragments thereof, which sequences are intended to be non-limiting of the present disclosure. Underlining indicates the CDR sequences of the corresponding heavy and light chains in the order of CDR1, CDR2, and CDR3 according to the Kabat numbering system. The rightmost columns show the CDR1, CDR2, and CDR3 sequences determined by the Chothia, Kabat, and IMGT systems, respectively.

[0170] Table 1A. Antigen-Binding Domains of Exemplary Chimeric Fusion Proteins

[0171]

[0172]

[0173]

[0174]

[0175] Table 1B. Signal Peptide Sequences of Exemplary Chimeric Fusion Proteins

[0176]

[0177] Table 1C. Extracellular / Hinge Domains of Exemplary Chimeric Fusion Proteins

[0178]

[0179] Table 1D. Transmembrane Domains of Exemplary Chimeric Fusion Proteins

[0180]

[0181] Table 2. Intracellular Signaling Domains of Exemplary Chimeric Fusion Proteins

[0182]

[0183]

[0184]

[0185] Table 3. Intracellular Domains of Exemplary Chimeric Fusion Proteins

[0186]

[0187] Table 4. Exemplary Chimeric Fusion Protein Sequences

[0188] Exemplary sequences of chimeric fusion protein domains and / or fragments thereof are shown, which are intended to be non-limiting to the present disclosure. Underlining indicates the CDR sequences of the corresponding variable heavy chain (VH) and variable light chain (VL) in the order of CDR1, CDR2, and CDR3 according to the Kabat numbering system.

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203] In some embodiments, an exemplary chimeric fusion protein (CFP) can comprise a sequence having at least 80% identity to any one of the sequences of SEQ ID NOs: 26-42, 135-137 of Table 4. In some embodiments, an exemplary chimeric fusion protein (CFP) can comprise a sequence having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% identity to any one of the sequences of SEQ ID NOs: 26-42, 135-137. In some embodiments, an exemplary chimeric fusion protein (CFP) can comprise a sequence having 90% identity to any one of the sequences of SEQ ID NOs: 26-42, 135-137 of Table 4. In some embodiments, an exemplary chimeric fusion protein (CFP) can comprise a sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the sequences of SEQ ID NOs: 26-42, 135-137.

[0204] In some embodiments, provided herein are chimeric fusion proteins that comprise (i) an extracellular antigen-binding domain capable of binding to a TROP2 antigen; further comprising the TMD of CD89; wherein the extracellular antigen-binding domain is a scFv and comprises a variable heavy chain CDR3 having the sequence GGFGSSYWYFDV, and wherein optionally further comprises a variable light chain CDR3 having the sequence QQHYITPLT.

[0205] In some embodiments, provided herein are chimeric fusion proteins that comprise (i) an extracellular antigen-binding domain capable of binding to a GPC3 antigen, further comprising the TMD of CD89; wherein the extracellular antigen-binding domain is a scFv and comprises a variable heavy chain (VH) CDR3 having the sequence FYSYTY.

[0206] In some embodiments, an exemplary chimeric fusion protein (CFP) can comprise a binding domain that binds to the cancer antigen GPC3, wherein the binding domain is a VHH and can comprise any of the variable domains listed in Table 5. The CFP can comprise a binding domain that is a scFv that binds the GPC3 antigen and comprises any of the variable domains listed in the following table (Table 5). An exemplary CFP can comprise an extracellular antigen-binding domain fused to a transmembrane domain (TMD) as described in this specification, the extracellular antigen-binding domain comprising any one or more of the variable domains listed in Table 5. In some embodiments, the CFP comprises a GPC3-binding domain (comprising one or more VH domains listed in Table 5), a TMD having the sequence of, for example, CD89 (as described elsewhere in this specification), and any intracellular domain or combination of intracellular domains as described herein. In some embodiments, the CFP comprises a GPC3-binding domain (comprising one or more VH domains listed in Table 5), a hinge domain, a TMD having the sequence of, for example, CD89 (as described elsewhere in this specification), and one or more intracellular signaling domains or combination of intracellular signaling domains as described herein (e.g., but not limited to, CD40ICD, TRIF ICD, PI3K recruitment domain ICD, etc.). Those skilled in the art can obtain constructs that fall within the scope contemplated herein using standard molecular biology cloning techniques and basic reverse engineering of recombinant techniques.

[0207] Table 5: Exemplary anti-GPC3 binding variable heavy chain (VH) domain sequences of the extracellular domain of CFP.

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235] The present disclosure provides a composition comprising a recombinant nucleic acid sequence encoding a CFP, the CFP comprising a phagocytic or tethering receptor (PR) subunit (e.g., a phagocytic receptor fusion protein (PFP)), the subunit comprising: an extracellular domain comprising an antigen-binding domain specific for a target cell antigen; a transmembrane domain; and an intracellular domain comprising an intracellular signaling domain; wherein the transmembrane domain and the extracellular domain are operably linked; and wherein upon binding of the CFP to an antigen on a target cell, the killing or phagocytic activity of myeloid cells (e.g., neutrophils, monocytes, myeloid dendritic cells (mDCs), mast cells or macrophages) expressing the 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 compared to cells not expressing the CFP.

[0236] The present disclosure provides a recombinant nucleic acid sequence encoding a CFP as described in the previous paragraph, wherein the intracellular domain comprises at least one intracellular domain that activates innate immunity, e.g., an intracellular signaling domain of a pattern recognition receptor, an intracellular signaling domain of a TLR, an intracellular signaling domain of an FcR, an intracellular adapter protein signaling domain or a fragment thereof, which is capable of activating the innate immune response of myeloid cells, activating its phagocytic potential, activating the inflammatory cytokine and chemokine response, antigen presentation and T cell activation of myeloid cells expressing the CFP upon contact with its target antigen, e.g., upon engagement of the antigen-binding domain with the target antigen.

[0237] 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, MAVS, TRIF or TASL intracellular domain, NLRP family members, NLRP1-14, NOD1, NOD2, pyroptosis proteins, 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.

[0238] In some embodiments, the CFP comprises an intracellular signaling domain that comprises a sequence from a protein derived from an activated interferon response transcription factor IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, or IRF9.

[0239] In some embodiments, the CFP comprises an intracellular signaling domain that comprises a sequence from an intracellular adaptor protein. In some embodiments, the adaptor protein may comprise a transmembrane portion that anchors it to an organelle (e.g., mitochondrion, endoplasmic reticulum, or lysosomal compartment). In some embodiments, the intracellular adaptor protein is a cytoplasmic protein.

[0240] The CFP as described herein may comprise an antigen-binding domain of Table 1A, an extracellular / hinge domain of Table 1C, and one or two or more intracellular signaling domains of Table 2. Optionally, the CFP as described herein may comprise a signal peptide sequence of Table 1B.

[0241] In some embodiments, the CFP may comprise a sequence having at least 85% sequence identity to a sequence 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 to a sequence in Table 4. In some embodiments, the CFP further comprises a signal peptide sequence, such as a signal peptide sequence from Table 1B.

[0242] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the intracellular domain of TRIF, the intracellular domain of TRIF having the amino acid sequence of any one of SEQ ID NOs: 19-22 or having at least 85% sequence identity with any one of SEQ ID NOs: 19-22. In some embodiments, the intracellular signaling domain comprises a sequence having at least 86% or at least 87% or at least 88% or at least 89% sequence identity with any one of SEQ ID NOs: 19-22. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the intracellular signaling domain of TRIF, the intracellular signaling domain of TRIF having at least 90% sequence identity with any one of SEQ ID NOs: 19-22. In some embodiments, the intracellular signaling domain comprises a sequence having at least 91% or at least 92% or at least 93% or at least 94% sequence identity with any one of SEQ ID NOs: 19-22. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the intracellular signaling domain of TRIF, the intracellular signaling domain of TRIF having at least 95% sequence identity with any one of SEQ ID NOs: 19-22. In some embodiments, the intracellular domain of CFP comprises an intracellular signaling domain derived from the intracellular signaling domain of TRIF, the intracellular signaling domain of TRIF having at least 90% sequence identity with any one of SEQ ID NOs: 19-22; wherein CFP comprises an extracellular binding domain that is capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell.In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the intracellular signaling domain of TRIF, which TRIF intracellular signaling domain has at least 90% sequence identity with any one of SEQ ID NOs: 19-22; wherein the CFP comprises an extracellular binding domain that is capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell; a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD68 transmembrane domain or a CD64 transmembrane domain or a CD16 transmembrane domain, or a CD89 transmembrane domain, and a hinge domain. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from the intracellular signaling domain of TRIF, which TRIF intracellular signaling domain has at least 90% sequence identity with any one of SEQ ID NOs: 19-22; wherein the CFP comprises an extracellular binding domain that is capable of binding to a CD5 molecule on a target cell, a HER2 molecule on a target cell, a CD19 molecule on a target cell, a TROP2 molecule on a target cell, a GPC3 molecule on a target cell, a CD70 molecule on a target cell, a CD137 molecule on a target cell, a CD7 molecule on a target cell, a Claudin molecule on a target cell, a CD22 molecule on a target cell, or a GP75 molecule on a target cell; a CD8 transmembrane domain or a CD28 transmembrane domain, or a CD64 transmembrane domain or a CD16 transmembrane domain, or a CD89 transmembrane domain, and one or more additional intracellular signaling domains (such as a PI3 kinase recruitment domain or a CD40 intracellular signaling domain).

[0243] In some embodiments, the exemplary anti-TROP2 binding CFP described herein comprises an extracellular antigen-binding domain and further comprises an intracellular domain of any one of the sequences in Table 2 or Table 3, the extracellular antigen-binding domain having a sequence of any one of SEQ ID NOs: 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. In some embodiments, the exemplary anti-TROP2 binding CFP described herein comprises a sequence having at least 80%-100% sequence identity with any one of SEQ ID NOs: 26-33. IRF-inducing proteins and IRF activation pathways

[0244] Type I IFNs are key cytokines that mediate innate antiviral immunity and thus drive pro-inflammatory responses. Type I IFNs are readily induced by cGMP-AMP synthase, retinoic acid-inducible gene I (RIG-I)-like receptors, and Toll-like receptors that recognize microbial double-stranded (ds) DNA, dsRNA, and LPS. These signaling pathways converge on the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-β) mediate the recruitment of IRF-3 via the conserved pLxIS motif. The pLxIS motifs of phosphorylated STING, MAVS, and TRIF generally bind IRF-3 in a similar manner, while residues upstream of the motif confer specificity. Type I IFNs (e.g., IFN-α and IFN-β) are the major family of cytokines that mediate antiviral immunity. Microbial dsDNA in the cytosol binds and activates the enzyme cGAS (cGMP-AMP synthase), which catalyzes the synthesis of the cyclic dinucleotide cGAMP (cyclic [G(2’,5’)pA(3’,5’)p]). As a second messenger, cGAMP binds to the adaptor protein STING (stimulator of IFN genes) located on the endoplasmic reticulum (ER) membrane and directs the activation of the transcription factor IRF-3 (IFN regulatory factor 3) via the protein kinase TBK1 (TANK-binding kinase 1). Phosphorylated IRF-3 dimerizes and translocates to the nucleus to initiate transcription of the IFN-β gene. In contrast, viral dsRNA in the cytosol is sensed by RLRs [retinoic acid-inducible gene I (RIG-I)-like receptors] to activate IRF-3 via the adaptor protein MAVS (mitochondrial antiviral signaling). In addition, TLRs (Toll-like receptors) TLR3 and TLR4, which recognize viral dsRNA in endosomes and LPS in the bacterial cell wall components, respectively, also mediate the induction of type I IFNs and inflammatory cytokines (1). These two TLRs use the adaptor protein TRIF (TIR domain-containing adaptor inducing IFN-β) to mediate the recruitment and activation of IRF-3. Notably, the signaling pathways of these three innate immune sensor families converge on the activation of TBK1 and IRF-3. Mechanistically, the adaptor proteins STING, MAVS, and TRIF contain a conserved motif pLxIS (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 signaling complex. The induced proximity between TBK1 and IRF-3 results in IRF-3 phosphorylation and activation. In addition, IRF-3 itself also contains a pLxIS motif, which is crucial for phosphorylation-induced dimerization and activation of IRF-3.Mutation of the serine residue at the phosphorylation site in the pLxIS motif of STING, MAVS, and TRIF abolishes the induction of type I IFN in their respective signaling pathways. However, the exact molecular mechanism of IRF-3 recruitment and activation remains unknown. To elucidate the structural basis for the recruitment of IRF-3 by phosphorylated STING (pSTING), MAVS (pMAVS), and TRIF (pTRIF), we expressed peptides containing the pLxIS motif from the three adaptor proteins, phosphorylated them in vitro with TBK1, and determined the crystal structures of their complexes with the C-terminal domain (CTD) of IRF-3.

[0245] Mechanisms of IRF-3 recruitment by pMAVS and pTRIF.

[0246] In contrast to dsDNA sensing via the cGAS-STING pathway, RLR senses dsRNA in the cytosol and activates IRF-3 via the adaptor MAVS, while TLR3 and TLR4 recruit IRF-3 using the adaptor TRIF. Phosphorylation of the pLxIS motif of MAVS or TRIF is required for the recruitment and activation of IRF-3.

[0247] TRAF interacting protein:

[0248] The presence of the TRAF domain (a protein interaction domain of approximately 180 amino acids) is a unique feature of TRAF family proteins, and by this criterion, six of the seven TRAF proteins in this family (TRAF1 - TRAF6) are identified as mammalian TRAF family. The TRAF domain can be subdivided into two distinct regions: the TRAF-N domain and the TRAF-C domain. Various receptors bind to the TRAF-C domain, while various intracellular signaling molecules bind to the TRAF-N domain. Although the structures of the TRAF domains are similar, each TRAF protein has specific biological functions that are specific to its interaction partners (upstream receptors and downstream effector molecules). The structure of the TRAF domain of TRAF2 was first reported by Dr. Wu's team around 1999, and 3 years later the same team reported the structure of the TRAF domain of TRAF6. Since then, the structures of the TRAF domains of TRAF3, TRAF5, TRAF4, and TRAF1 have been reported. The TRAF structure reveals that the TRAF-N domain is a coiled-coil structure and TRAF-C consists of seven to eight antiparallel β-sheet folds. Structural alignments of all six TRAF family members show a well-aligned TRAF-C domain, while the position and length of TRAF-N vary among TRAF family members. Sequence analysis indicates that the length of TRAF-N varies in the family, while the length of the TRAF-C domain is conserved: the TRAF-N of TRAF4 and TRAF6 is relatively short, while that of TRAF3 and TRAF5 is relatively long. Although the overall structures are nearly identical, distinct structural differences have been observed. For example, the length and position of some loops in the TRAF domains of TRAF4 and TRAF6 are different from those of the loops in other TRAF family members. TRAF4 contains a more negatively charged surface in the middle of the receptor-binding region, while TRAF6 contains a more positively charged surface in the receptor-binding region. Because surface features usually determine the mode of its interaction with its partners, the similar electrostatic surfaces of the TRAF domains in TRAF1, TRAF2, TRAF3, and TRAF5, i.e., different charged surfaces, have been shown to be important for accommodating different receptors with similar interaction modes in the same binding pocket. In contrast, the different features on the binding surfaces of the functionally distinct TRAFs (TRAF4 and TRAF6) suggest that TRAF4 and TRAF6 can accommodate different receptors with different interaction modes.

[0249] For the purposes of the present disclosure, any pathway, signaling intermediate, or activating moiety discussed in the above paragraphs can be considered activatable or functional when inducing the CFP disclosed herein. Similarly, the CFP disclosed herein can be used to target any of the applicable targets described in the pathways discussed. Any pathway or portion thereof involving signaling domains, signaling pathways, signaling intermediates, transcription factors of activating genes that are readily known to those of skill in the art as of the date of the present literature should be understood to be within the scope of the present disclosure.

[0250] Chimeric proteins having an intracellular domain of TLR, an intracellular signaling pathway of TLR, and NF-κB activation:

[0251] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a TLR protein. In some embodiments, a CFP designed to comprise an intracellular signaling domain derived from an intracellular signaling domain of TLR can activate NF-κB when the extracellular domain of the receptor engages its target. In some embodiments, the intracellular domain can comprise the intracellular signaling domain of an endolysosomal TLR (e.g., TLR3, TLR7, TLR8, or TLR9). In some embodiments, the intracellular signaling domain can be derived from the TLR3 protein. In some embodiments, the intracellular signaling domain can be derived from the TLR7, TLR8, or TLR9 protein. In some embodiments, the intracellular domain can comprise the intracellular signaling domain of cell surface TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10. In some embodiments, the cytoplasmic domain for an inflammatory response comprises the intracellular signaling domain 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, pyroptosis protein, AIM2, NLRC4, and / or CD40.

[0252] In some embodiments, a phagocytic scavenger receptor (PR) fusion protein (PFP) comprises a pro-inflammatory cytoplasmic domain for activating the IL-1 signaling cascade.

[0253] In some embodiments, the cytoplasmic portion of a chimeric receptor (e.g., a phagocytic receptor (PR) fusion protein (PFP)) comprises the cytoplasmic domain from a toll-like receptor, such as the intracellular signaling domain of toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), or toll-like receptor 9 (TLR9).

[0254] Generally, TLRs have different cellular localizations, production pathways, activation, recognition, and modes of action. TLRs are expressed in innate immune cells (e.g., dendritic cells (DCs) and macrophages) as well as non-immune cells (e.g., fibroblasts and epithelial cells). Cell surface TLRs mainly recognize microbial membrane components, such as lipids, lipoproteins, and proteins. TLR4 recognizes bacterial lipopolysaccharide (LPS). TLR2, together with TLR1 or TLR6, recognizes a variety of PAMPs, including lipoproteins, peptidoglycan, lipoteichoic acid, zymosan, mannan, and tGPI-mucin. TLR5 recognizes bacterial flagellin. Due to the insertion of a stop codon, TLR10 is a pseudogene in mice, but human TLR10 cooperates with TLR2 to recognize ligands from Listeria. TLR10 can also sense influenza A virus infection.

[0255] Intracellular TLRs recognize nucleic acids derived from bacteria and viruses and also recognize self-nucleic acids in disease conditions such as autoimmunity. TLR3 recognizes viral double-stranded RNA (dsRNA), small interfering RNA, and self-RNA from damaged cells. TLR7 is mainly expressed in plasmacytoid DCs (pDCs) and recognizes single-stranded (ss) RNA from viruses. It also recognizes RNA from Streptococcus B bacteria in conventional DCs (cDCs). Human TLR8 responds to viral and bacterial RNAs. Structural analysis has revealed that unstimulated human TLR8 exists as a preformed dimer, and although the Z-loop between LRR14 and LRR15 is cleaved, the N-terminal and C-terminal portions still associate with each other and participate in ligand recognition and dimerization. Ligand binding induces reorganization of the dimer to bring the two C-termini into close proximity. TLR13 recognizes bacterial 23S rRNA and an unknown component of vesicular stomatitis virus. TLR9 recognizes bacterial and viral DNA rich in unmethylated CpG-DNA motifs; it also recognizes hemozoin, an insoluble crystalline byproduct produced by Plasmodium falciparum during the detoxification process following digestion of host hemoglobin. TLR11 is localized in endolysosomes and recognizes flagellin or an unknown protein component of uropathogenic Escherichia coli (UPEC) as well as an inhibitor-like molecule derived from Toxoplasma gondii. TLR12 is mainly expressed in myeloid cells, is highly similar to TLR11, and recognizes an inhibitor from Toxoplasma gondii. TLR12 functions as a homodimer or as a heterodimer with TLR11. All TLRs are synthesized in the ER, transported to the Golgi apparatus, and recruited to the cell surface or intracellular compartments such as endosomes. The intracellular localization of TLRs is thought to be crucial for ligand recognition and for preventing TLRs from coming into contact with self-nucleic acids, which can cause autoimmunity.

[0256] Individual TLRs differentially recruit members of a group of TIR domain-containing adaptors, such as MyD88, TRIF, TIRAP / MAL, or TRAM. MyD88 is utilized by all TLRs and activates NF-κB and MAPK to induce inflammatory cytokine genes. TIRAP is a sorting adaptor that recruits MyD88 to cell surface TLRs (such as TLR2 and TLR4). TIRAP is also involved in signaling through endosomal TLRs (such as TLR9). The lipid-binding domain of TIRAP binds PI(4,5)P2 on the plasma membrane and PI(3)P on endosomes, which mediates the formation of functional TLR4 and TLR9 signaling complexes at their respective sites. Thus, TIRAP associates with cell surface and endosomal TLRs by binding different lipids. TRIF is recruited to TLR3 and TLR4 and promotes an alternative pathway leading to the activation of IRF3, NF-κB, and MAPK to induce type I IFN and inflammatory cytokine genes. TRAM is selectively recruited to TLR4 but not TLR3 to link between TRIF and TLR4. TLR3 directly interacts with TRIF, and this interaction requires phosphorylation of two tyrosine residues in the cytoplasmic domain of TLR3 by the epidermal growth factor ErbB1 and Btk. After TLR engagement, MyD88 forms a complex with IRAK kinase family members. IRAK4 activates IRAK1, which then autophosphorylates at several sites and dissociates from MyD88. IRAK1 associates with the RING-domain E3 ubiquitin ligase TRAF6. TRAF6, together with the ubiquitin-conjugating enzyme UBC13 and UEV1A, promotes K63-linked polyubiquitination of both TRAF6 itself and the TAK1 protein kinase complex. TAK1 is a member of the MAPKKK family and forms a complex with the regulatory subunits TAB1, TAB2, and TAB3, which interact with the polyubiquitin chain generated by TRAF6 to drive TAK1 activation. Then TAK1 activates two different pathways, leading to the activation of the IKK complex-NF-κB pathway and the MAPK pathway. The IKK complex consists of the catalytic subunits IKKα and IKKβ and the regulatory subunit NEMO (also known as IKKγ). TAK1 binds the IKK complex through the ubiquitin chain, phosphorylates it, and activates IKKβ. The IKK complex phosphorylates the NF-κB inhibitory protein IκBα, which undergoes proteasomal degradation, allowing NF-κB to translocate into the nucleus to induce pro-inflammatory gene expression. TAK1 activation also leads to the activation of MAPK family members (such as ERK1 / 2, p38, and JNK), which mediate the activation of AP-1 family transcription factors or the stabilization of mRNA to regulate the inflammatory response.

[0257] In some embodiments, the intracellular domains described herein can be specifically paired with another domain, e.g., a structural domain, such as another intracellular domain, a transmembrane domain, or an extracellular domain; a functional domain, such as a signaling domain. In some embodiments, the intracellular domains described in this section of the present document, such as MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, TASL, or any other domain mentioned herein, can be regulated or modified to pair with another domain or its components. Pairing refers to including the domain under consideration, a part of the domain, or a fragment or component thereof in the CFP design as any part of the CFP protein molecular structure. In some embodiments, the regulation can be the structural alignment or placement of the domains within the CFP molecule, e.g., the two domains under consideration are juxtaposed, or separated by one or more domains therebetween, or separated by one or more amino acids therebetween. One or more amino acids can be a linker. One or more amino acids can provide a structural distance between two adjacent domains, provide flexibility between two adjacent domains, or confer a three-dimensional orientation of the molecular structure that is superior to that of a domain that does not include one or more amino acids. In some embodiments, the regulation or modification can include modifications within the domain, such as mutations.

[0258] In some embodiments, the intracellular domains described in this section of the present document, such as MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, TASL, or any other domain mentioned herein, can be regulated or modified to pair with or be included together with another intracellular domain within the CFP, such as a kinase recruitment domain, such as a PI3 kinase recruitment domain. In some embodiments, the PI3 kinase recruitment domain is modified to mask tonic signaling. In some embodiments, one of the other intracellular domains, such as any one or more of the MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, TASL domains or fragments thereof, is modified to reduce or eliminate tonic signaling.

[0259] In some embodiments, the intracellular domains described in this section of the present document, such as MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, TASL, or any other domain mentioned herein, can be regulated or modified to pair with or be included together with another structural domain (such as 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.

[0260] For the purposes of the present disclosure, any pathway, signaling intermediate, or activating moiety discussed in the above paragraphs can be considered to be activatable or functional when inducing the disclosure of a CFP comprising an intracellular signaling domain of a TLR as disclosed herein. Similarly, the CFP disclosed herein can be used to target any of the suitable targets described in the pathways discussed. Any pathway or portion thereof involving a signaling domain, signaling pathway, signaling intermediate, or transcription factor of an activating gene that is readily known to those skilled in the art as of the date of the present disclosure should be understood to be within the scope of the present disclosure.

[0261] Therapeutic composition

[0262] In one aspect, the present disclosure provides a composition comprising a polynucleotide, such as a recombinant polynucleotide encoding a CFP as described in the present specification, the CFP comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises a scFv or VHH capable of binding to a specific cancer antigen expressed on a cancer cell; wherein the CFP is designed to be specifically expressed in myeloid cells such as CD14+ cells, CD14+ / CD16− cells, CD14+ / CD16+ cells, CD14− / CD16+ cells, CD14− / CD16− cells, dendritic cells, M0 macrophages, M2 macrophages, M1 macrophages, or chimeric myeloid cell / macrophage / dendritic cells. The composition is designed to be administered, for example, via a systemic administration route to a subject in need thereof, and thereby generate engineered myeloid cells in vivo, e.g., because the polynucleotide (e.g., mRNA encoding the CFP) in the composition is expressed substantially in myeloid cells and substantially not in non-myeloid cells. Exemplary non-myeloid cells are lymphoid cells such as T cells, B cells, etc. For example, the CFP encoded by the polynucleotide described herein is “expressed substantially in myeloid cells” because when tested at a given time, expression of the CFP can be detected in greater than 50% of the myeloid cells in a mixed population of myeloid and non-myeloid cells in vitro, while less than 5% or less than 4% or less than 3% or less than 2% of the T cells or B cells express the CFP. The design related to myeloid cell-specific expression involves having a transmembrane domain capable of dimerizing or multimerizing with an endogenous protein in myeloid cells such that the dimerization or multimerization confers expression, stability, and functionality of the CFP on the membrane of myeloid cells and does not result in significant expression in non-myeloid cells (e.g., T cells). In some embodiments, the composition is formulated for in vivo delivery of the polynucleotide. In some embodiments, the polynucleotide is RNA. In some embodiments, the polynucleotide is mRNA. In some embodiments, the delivery vehicle comprises a lipid. In some embodiments, the delivery vehicle comprises a lipid nanoparticle (LNP).

[0263] In some embodiments, provided herein are therapeutic compositions comprising chimeric fusion proteins such as chimeric fusion receptor proteins (CFPs), the CFPs comprising: (a) an extracellular domain comprising: (i) an scFv or VHH that specifically binds to any one of the targets disclosed herein, e.g., a TROP2 or GPC3 antigen-binding domain; (ii) a hinge domain derived from the CD89 hinge sequence, (b) a CD89 transmembrane domain, and (c) an intracellular domain comprising one or more intracellular signaling domains, wherein the one or more intracellular signaling domains include any one of intracellular signaling domains derived from FcRγ or FcRε, a PI3 kinase recruitment domain, a CD40 intracellular signaling domain; or an intracellular signaling domain from an inflammatory signaling molecule, e.g., an intracellular signaling domain derived from TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, IFN receptor, STING, MAVS, TRIF or TASL intracellular domain, an NLRP family member, NLRP1-14, NOD1, NOD2, pyroprotein, 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.

[0264] In some embodiments, myeloid cells can be further modified or engineered to generate therapeutically effective myeloid cells. Isolated cells can be engineered by expressing a gene or a fragment thereof in the cells without altering their function and developmental plasticity, differentiation potential, and cell viability.

[0265] In some embodiments, myeloid cells can be further modified or engineered by expressing a non-endogenous polynucleotide in the cells to generate therapeutically effective myeloid cells. The non-endogenous polynucleotide can encode a protein or a peptide. Alternatively, the non-endogenous polynucleotide can be a non-coding sequence, e.g., inhibitory RNA or morpholino.

[0266] In some embodiments, myeloid cells can be further modified or engineered by stably altering the genomic sequence of the cells to produce therapeutically effective myeloid cells. In some embodiments, myeloid cells are engineered 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 engineered to delete a gene. 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, where the messenger RNA encodes a protein or peptide, such that the myeloid cells are therapeutically effective. In some embodiments, nucleic acids can be introduced into myeloid cells using naked DNA or messenger RNA (mRNA). In some embodiments, DNA or mRNA encoding a chimeric antigen receptor is introduced into phagocytes by lipid nanoparticle (LNP) encapsulation. The mRNA is single-stranded and can be codon-optimized. In some embodiments, the mRNA can contain one or more modified or unnatural bases, such as 5'-methylcytosine or pseudouridine or methylpseudouridine. In some embodiments, greater than or about 50% of the uridine (“U”) residues in the mRNA can be converted to methylpseudouridine. In some embodiments, the length of the mRNA is 2 kb - 20,000 kb. In some embodiments, the length of the mRNA can be 50 - 10,000 bases. In one aspect, the transgene is delivered in the form of mRNA. The mRNA can contain greater than about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 bases. In some embodiments, the length of the mRNA can exceed 10,000 bases. In some embodiments, the length of the mRNA can be about 11,000 bases. In some embodiments, the length of the mRNA can be about 12,000 bases. In some embodiments, the mRNA contains a transgene sequence encoding a fusion protein. In some embodiments, mRNA encoding CFP as described anywhere in the present disclosure can contain a 5' untranslated region (UTR). In some embodiments, mRNA encoding CFP can further contain a 3' UTR. In some embodiments, the 5' UTR as described herein can contain the following sequence:

[0267] GGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUUGCCAC C(SEQ ID NO:138); or a sequence having at least 90% identity to SEQ ID NO:138. In some embodiments, the 5'UTR as described herein may comprise the following sequence: GGGAGACCCAAGCUGGCUAGCGCCACC(SEQ ID NO:139).

[0268] In some embodiments, the mRNA may comprise the 3'UTR sequence CUCGAGUCUAGAGGGCCCGUUUAAACCCGCUGAUCAGCCUCGACUGUGCCUUCUAGUUGCCAGCCAUCUGUUGUUUGCCCCUCCCCCGUGCCUUCCUUGACCCUGGAAGGUGCCACUCCCACUGUCCUUUCCUAAUAAAAUGAGGAAAUUGCAUCGCAUUGUCUGAGUAGGUGUCAUUCUAUUCUGGGGGGUGGGGUGGGGCAGGACAGCAAGGGGGAGGAUUGGGAAGACAAUAGC(SEQ ID NO:140). In some embodiments, the recombinant polynucleotide comprises one or more elements for stabilizing the structure. For long mRNAs, 5' cap protection is particularly important. In some embodiments, the RNA product required for generating engineered myeloid cells for therapeutic purposes is generated in vitro, with a 5' cap and a 3'-polyA tail. In some embodiments, the 5' cap is modified from a natural mRNA 5' cap.

[0269] In some embodiments, the recombinant mRNA encoding CFP as described anywhere in the present disclosure may comprise an effective cap structure using a suitable capping analog. In some embodiments, the RNA product prepared from IVT mRNA is co-transcriptionally capped with an m7GpppN-derived dinucleotide. In some embodiments, the 5'-cap is a trinucleotide cap analog. In some embodiments, the first nucleobase after the 5'-cap plays a key role in mRNA stability. In some embodiments, the base is adenine (A). In some embodiments, the base is cytosine (C). In some embodiments, the base is guanine (G). In some embodiments, the base is uracil (U). In some embodiments, the cap analog is m 7 GpppApG. In some embodiments, the cap analog is m 7 GpppAmpG. In some embodiments, the cap analog is m 7 Gpppm 6 ApG. In some embodiments, the cap analog is m7 Gpppm 6 A m pG. In some embodiments, the cap analog is m 7 GpppCpG. In some embodiments, the cap analog is m 7 GpppC m pG. In some embodiments, the cap analog is m 7 GpppGpG. In some embodiments, the cap analog is m 7 GpppG m pG. In some embodiments, the cap analog is m 7 GpppUpG. In some embodiments, the cap analog is m 7 GpppU m pG. In some embodiments, the 5'-cap is an anti-reverse cap analog (ARCA) with a chemical modification at the 3-O or 2-O position of m7G(m2 7,3-O GpppG). In some embodiments, the 5'-cap is N7-methylguanosine 5-diphosphate (m7GDPIm). In some embodiments, capping involves post-transcriptional enzymatic capping of 5-triphosphate RNA using the vaccinia virus capping complex. In some embodiments, 5'-capping confers mRNA stability and translation efficiency. In some embodiments, the cap-proximal sequence is crucial for mRNA stability and function.

[0270] In some embodiments, the recombinant mRNA encoding CFP described herein may comprise a polyA sequence comprising from about 80 to about 150 adenosine (A) residues. In some embodiments, the polyA sequence comprises about 120 A residues. In some embodiments, the polyA sequence comprises about 110 polyA residues. In some embodiments, the polyA sequence comprises about 100 polyA residues.

[0271] LNP-encapsulated DNA or RNA can be used to transfect macrophages or can be administered to a subject. In some embodiments, mRNA is incorporated into an effector myeloid cell population by transient transfection. In some embodiments, the transient transfection method includes electroporation of the mRNA. In some embodiments, transient transfection includes chemical transfection. In some embodiments, 1 - 5,000 micrograms / ml of mRNA can be used for transfection using a suitable protocol of the methods described above. In some embodiments, 1 - 2,000 micrograms / ml of mRNA can be used for transfection. In some embodiments, 1 - 1,000 micrograms / ml of mRNA can be used for transfection. In some embodiments, 1 - 1,000 micrograms / ml of mRNA can be used for transfection. In some embodiments, 1 - 500 micrograms / ml of mRNA can be used for transfection. In some embodiments, 1 - 250 micrograms / ml of mRNA can be used for transfection. In some embodiments, about 500 micrograms / ml or less of mRNA can be used for transfection. In some embodiments, about 250 micrograms / ml or less of mRNA can be used for transfection. In some embodiments, about 10 micrograms / ml of mRNA is used. In some embodiments, about 20 micrograms / ml of mRNA is used. In some embodiments, about 30 micrograms / ml of mRNA is used. In some embodiments, about 40 micrograms / ml of mRNA is used. In some embodiments, about 50 micrograms / ml of mRNA is used. In some embodiments, about 60 micrograms / ml of mRNA is used. In some embodiments, about 80 micrograms / ml of mRNA is used. In some embodiments, about 100 micrograms / ml of mRNA is used. In some embodiments, about 150 micrograms / ml of mRNA is used. In some embodiments, about 200 micrograms / ml of mRNA is used. In some embodiments, 20, 50, 100, 150, 200, 250, 300, 400, 500, or about 1,000 micrograms / ml of mRNA is used. A suitable cell density for transfection is selected based on the methods and the manufacturer's instructions for the instrument and / or reagents, or as is known to those of skill in the art.

[0272] Exemplary mRNA sequences encoding two CFPs described herein are provided below:

[0273] The mRNA sequence encoding the exemplary anti-TROP2 CFP described herein GGGAGACCCAAGCUGGCUAGCGUUUAA ACUUAAGCUUGCCACCCUCGAGUCUAGAGGGCCCG UUUAAACCCGCUGAUCAGCCUCGACUGUGCCUUCUAGUUGCCAGCCAUCUGUUGUUUGCCCCUCCCCCGUGCCUUC CUUGACCCUGGAAGGUGCCACUCCCACUGUCCUUUCCUAAUAAAAUGAGGAAAUUGCAUCGCAUUGUCUGAGUAGG UGUCAUUCUAUUCUGGGGGGUGGGGUGGGGCAGGACAGCAAGGGGGAGGAUUGGGAAGACAAUAGC (SEQ ID NO:141). The underlined sequences represent the 5’UTR and 3’UTR sequences, respectively. In some embodiments, the mRNA encoding anti-TROP2 CFP comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:141. In some embodiments, the mRNA encoding anti-TROP2 CFP comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the portion of SEQ ID NO:141 that does not contain the 5’UTR and / or 3’UTR.

[0274] The mRNA sequence encoding anti-GPC3 CFP

[0275] GGGAGACCCAAGCUGGCUAGCGCCACCCUCGAGUCUAGAGGGCCCGUU UAAACCCGCUGAUCAGCCUCGACUGUGCCUUCUAGUUGCCAGCCAUCUGUUGUUUGCCCCUCCCCCGUGCCUUCCU UGACCCUGGAAGGUGCCACUCCCACUGUCCUUUCCUAAUAAAAUGAGGAAAUUGCAUCGCAUUGUCUGAGUAGGUG UCAUUCUAUUCUGGGGGGUGGGGUGGGGCAGGACAGCAAGGGGGAGGAUUGGGAAGACAAUAGC (SEQ ID NO:142). The underlined sequences represent the 5’UTR and 3’UTR sequences, respectively. In some embodiments, the mRNA encoding anti-GPC3 CFP comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:142. In some embodiments, the mRNA encoding anti-GPC3 CFP comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a portion of SEQ ID NO:142 that does not include the 5’UTR and / or 3’UTR.

[0276] In some embodiments, the recombinant nucleic acid is mRNA. The mRNA construct can be thawed on ice, gently pipetted into monocytes and pre-mixed. In some embodiments, the mRNA is electroporated into the cells. The panned cells can be pooled, centrifuged, and electroporated with mRNA using the MaxCyte ATX system optimized for the purpose. In some embodiments, optimized electroporation buffers, cell densities, and / or mRNA concentrations are used for each protocol of each construct.

[0277] In some embodiments, the polynucleotide can be introduced into myeloid cells in the form of circular RNA (circRNA). In circular RNA (circRNA), the 3’ and 5’ ends are covalently linked. LNP can be used for intracellular delivery of circRNA.

[0278] In some embodiments, stable integration of the transgene into macrophages and other phagocytic cells can be achieved via the use of transposases and transposon elements, particularly mRNA-encoded transposases. In one embodiment, long interspersed element-1 (L1) RNA can be considered for retrotransposition and stable integration of the transgene into macrophages or phagocytic cells. Retrotransposons can be used for stable integration of the recombinant nucleic acid encoding CFP.

[0279] In some embodiments, myeloid cells can be modified to express a transgene by incorporating the transgene into a transient expression vector. In some embodiments, the expression of the transgene can be temporarily regulated by a modulator from outside the cell. Examples include the Tet-on and Tet-off systems, in which the expression of the transgene is regulated by the presence or absence of tetracycline.

[0280] In some embodiments, myeloid cells can be modified by contacting the cells with a compound to produce a therapeutically effective cell, and the compound can be an inhibitor or activator of a protein or enzyme within the myeloid cells.

[0281] In some embodiments, a polynucleotide encoding a chimeric antigen receptor can be introduced into isolated myeloid cells obtained by the methods described in the foregoing sections, wherein the chimeric antigen receptor, when expressed in the myeloid cells, enhances the innate immune response function of the myeloid cells. In some embodiments, chimeric antigen receptor expression can direct myeloid cells to a specific target in vivo or in vitro. In some embodiments, the chimeric antigen receptor can increase the phagocytic potential of myeloid cells. In some embodiments, the chimeric antigen receptor increases the immunogenicity of myeloid cells. In some embodiments, the chimeric antigen receptor can increase intracellular signaling. In some embodiments, the chimeric antigen receptor can cooperate with one or more proteins within the cell. In some embodiments, the chimeric antigen receptor can dimerize or multimerize with a second receptor or transmembrane protein within the myeloid cell, wherein the second receptor or transmembrane protein is an endogenous protein.

[0282] In some embodiments, provided herein are therapeutic compositions comprising chimeric fusion proteins such as chimeric fusion receptor proteins (CFPs), the CFPs comprising: (a) an extracellular domain comprising: (i) an scFv that specifically binds to any one of the targets disclosed herein, and (ii) at least a portion of a hinge domain derived from CD8, a hinge domain derived from CD28, or an extracellular domain from CD68; (b) a CD8 transmembrane domain, a CD28 transmembrane domain, a CD2 transmembrane domain, or a CD68 transmembrane domain; and (c) an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains include: (i) a first intracellular signaling domain derived from FcRγ or FcRε, (ii) a second intracellular signaling domain, e.g., an intracellular signaling domain derived from TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, IFN receptor, STING, MAVS, TRIF, or TASL intracellular domain, an NLRP family member, NLRP1-14, NOD1, NOD2, pyroprotein, 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, and a second intracellular signaling domain comprising a PI3K recruitment domain or a domain derived from CD40.

[0283] In some embodiments, the pharmaceutical composition comprises a cell population that comprises 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 of a subject after administration; and / or has a lifespan of at least 5 days in a subject after administration. In some embodiments, provided herein are therapeutic compositions comprising at least 20%, at least 30%, at least 40%, or at least 50% CD14+ cells. In some embodiments, the therapeutic composition comprises at least 20%, at least 30%, at least 40%, or at least 50% CD14+ / CD16− cells. In some embodiments, provided herein are therapeutic compositions comprising less than 20%, less than 15%, less than 10%, or less than 5% dendritic cells. The myeloid cells for use in the therapeutic compositions described herein comprise a recombinant nucleic acid encoding a chimeric fusion protein that encodes a CFP receptor protein or an engaging protein as described herein. The myeloid cells for use in the therapeutic compositions described herein express a CFP encoded by the recombinant nucleic acid described herein or express an engaging protein encoded by the recombinant nucleic acid described herein.

[0284] In some embodiments, the cells are cultured ex vivo briefly after thawing or after nucleic acid incorporation. In some embodiments, the ex vivo culture is carried out in the presence of a suitable medium, which may comprise conditioned serum components, e.g., human serum albumin (HSA). In some embodiments, the ex vivo culture and manipulation can be carried out in a medium containing low serum. In some embodiments, the serum is specifically treated to inactivate complement. In some embodiments, the myeloid cells can be cultured ex vivo in the presence of M-CSF as described above. In some embodiments, the myeloid cells can be cultured ex vivo in the presence of GM-CSF as described above. In some embodiments, the myeloid cells can be cultured in the presence of one or more cytokines. In some embodiments, the myeloid cells can be cultured or manipulated ex vivo for a period of time in the absence of growth factors or cytokines. In some embodiments, the methods provided herein comprise isolating or enriching and manipulating myeloid cells in less than 72 hours, 70 hours, 65 hours, 60 hours, 55 hours, 50 hours, 45 hours, 40 hours, 35 hours, 30 hours, 28 hours, 26 hours, or 24 hours. In some embodiments, the myeloid cells can be cultured for less than 24 hours, less than 20 hours, or less than 16 hours, less than 14 hours, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, or less than about 4 hours. The myeloid cells after isolation or enrichment and manipulation can be cultured briefly and frozen until further use. In some embodiments, the myeloid cells are thawed once or at most twice.

[0285] In some embodiments, the therapeutic competent cells are cells that have been electroporated with a recombinant nucleic acid encoding a polypeptide, frozen and thawed, and cultured stably for less than 24 hours, and wherein the cells in the cell population at the time of administration exhibit (i) viability greater than at least 70%, (ii) greater than at least 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, (iv) greater than 50% of the cells expressing the polypeptide encoded by the electroporated nucleic acid. In some embodiments, the therapeutic competent cells are cells that have been electroporated with a recombinant nucleic acid encoding a polypeptide, cultured stably for less than 24 hours, frozen and thawed, and wherein the cells in the cell population at the time of administration exhibit (i) viability greater than at least 70%, (ii) greater than at least 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, (iv) greater than 50% of the cells expressing the polypeptide encoded by the electroporated nucleic acid. In some embodiments, the therapeutic competent cells are cells that have been cultured stably for less than 24 hours, electroporated with a recombinant nucleic acid encoding a polypeptide, and frozen and thawed, and wherein the cells in the cell population at the time of administration exhibit (i) viability greater than at least 70%, (ii) greater than at least 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, (iv) greater than 50% of the cells expressing the polypeptide encoded by the electroporated nucleic acid. The cells must be pathogen-free. In the above embodiments, the therapeutic competent cells may have been frozen and thawed no more than twice, preferably once, and may be administered within 24 hours of thawing, within 18 hours of thawing, within 8 hours of thawing, or within 2 hours of thawing. The quality of the cells is tested for quality assurance prior to administration to meet the criteria described herein in the present disclosure.

[0286] Provided herein are methods of treating cancer in a subject with a pharmaceutical composition comprising engineered phagocytic cells (particularly macrophages) expressing a recombinant nucleic acid encoding CFP, the CFP being specifically designed to target, attack, and kill cancer cells. CFP may alternatively be referred to as a chimeric antigen receptor (CAR), particularly a CAR for phagocytosis (CAR-P), and the two terms may be used interchangeably herein. Engineered phagocytic cells are also referred to as CAR-P cells in the description herein.

[0287] In some embodiments, the recombinant polynucleotide composition comprises a lipid nanoparticle (LNP). In some embodiments, the recombinant polynucleotide composition further comprises a nucleic acid delivery vehicle that includes a cationic lipid, a non-cationic lipid, a neutral lipid, cholesterol, or a polyethylene glycol (PEG)-lipid. In some embodiments, the lipid nanoparticle comprises a polar lipid. In some embodiments, the lipid nanoparticle comprises a non-polar lipid. In some embodiments, the lipid nanoparticle has a diameter of 100 to 300 nm. In some embodiments, the lipid nanoparticle comprises 4-(dimethylamino)butyric acid-(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA; MC3). In some embodiments, the lipid nanoparticle comprises (a) a nucleic acid; (b) a cationic lipid; (c) a non-cationic lipid; and (d) a conjugated lipid that inhibits particle aggregation. In some embodiments, the recombinant polynucleotide composition comprises a polymeric nucleic acid delivery vehicle.

[0288] Cancers include, but are not limited to, T-cell lymphoma, cutaneous lymphoma, B-cell carcinomas (e.g., multiple myeloma, Waldenström macroglobulinemia), heavy chain diseases (e.g., alpha chain disease, gamma chain disease, and mu chain disease), benign monoclonal gammopathy and immunocyte amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer (e.g., metastatic hormone-refractory 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, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, blood tissue cancer, etc. Other non-limiting examples of cancer types applicable to the methods encompassed by the present disclosure include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, 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, hepatoma, cholangiocarcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphoblastic leukemia and acute myeloid leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia); chronic leukemias (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, and heavy chain diseases. In some embodiments, the cancer is an epithelial carcinoma, 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 other embodiments, the epithelial carcinoma is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial carcinomas can be characterized in various other ways, including but not limited to serous, endometrioid, mucinous, clear cell, or undifferentiated. In some embodiments, the present disclosure is used 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.In some embodiments, the indications treatable with the CFP disclosed herein may include, but are not limited to, lung cancer, liver cancer, prostate cancer, lymphoma or hepatocellular carcinoma, non-small cell lung cancer (NSCLC), breast cancer, cervical cancer, colon cancer, gastric cancer, bladder cancer, thyroid cancer, osteosarcoma, squamous cell carcinoma of the lung, ovarian yolk sac tumor, melanoma, and urothelial carcinoma.

[0289] Generally, adoptive cell therapy involves providing a patient with a drug product containing live cells. In some aspects, treating a patient or subject having cancer with autologous cells, the method comprising: isolating or enriching PBMC-derived macrophages, ex vivo modifying the macrophages by introducing a recombinant nucleic acid encoding a chimeric antigen receptor for phagocytosis (phagocytic receptor fusion protein (PFP)) into the macrophages to generate highly phagocytic macrophages capable of lysing tumors, and administering the modified macrophages to the patient or subject.

[0290] In one aspect, administering to a subject one or more doses of a pharmaceutical composition comprising therapeutic phagocytes, wherein the cells are allogeneic. The HLA may be compatibility-matched to the subject such that the cells do not cause graft-versus-host disease (GVHD). HLA typing of a clinical subject is performed prior to determining a therapeutic agent or treatment regimen to determine the HLA antigens expressed by the subject.

[0291] In some embodiments, for a single infusion, the therapeutically effective dose ranges from 10^7 cells to 10^12 myeloid cells. The cell number 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 2×10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 3×10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 4×10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 5×10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 6×10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 7×10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 8×10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 9×10^7 myeloid cells. In some embodiments, the therapeutically effective dose is about 10^8 myeloid cells. In some embodiments, the therapeutically effective dose is about 2×10^8 myeloid cells. In some embodiments, the therapeutically effective dose is about 3×10^8 myeloid cells. In some embodiments, the therapeutically effective dose is about 4×10^8 myeloid cells. In some embodiments, the therapeutically effective dose is about 5×10^8 myeloid cells. In some embodiments, the therapeutically effective dose is about 6×10^8 myeloid cells. In some embodiments, the therapeutically effective dose is about 7×10^8 myeloid cells. In some embodiments, the therapeutically effective dose is about 8×10^8 myeloid cells. In some embodiments, the therapeutically effective dose is about 9×10^8 myeloid cells. In some embodiments, the therapeutically effective dose is about 10^9 myeloid cells. In some embodiments, the therapeutically effective dose is about 2×10^9 myeloid cells. In some embodiments, the therapeutically effective dose is about 3×10^9 myeloid cells. In some embodiments, the therapeutically effective dose is about 4×10^9 myeloid cells. In some embodiments, the therapeutically effective dose is about 5×10^9 myeloid cells. In some embodiments, the therapeutically effective dose is about 6×10^9 myeloid cells. In some embodiments, the therapeutically effective dose is about 7×10^9 myeloid cells. In some embodiments, the therapeutically effective dose is about 8×10^9 myeloid cells. In some embodiments, the therapeutically effective dose is about 9×10^9 myeloid cells. In some embodiments, the therapeutically effective dose is about 10^10 myeloid cells. In some embodiments, the therapeutically effective dose is about 5×10^10 myeloid cells. In some embodiments, the therapeutically effective dose is about 10^11 myeloid cells. In some embodiments, the therapeutically effective dose is about 5×10^11 myeloid cells. In some embodiments, the therapeutically effective dose is about 10^12 myeloid cells.

[0292] In one aspect, the present disclosure provides one or more recombinant polynucleotides encoding one or more recombinant proteins that can be chimeric fusion proteins, such as receptors or conjugates as described herein. In some embodiments, the recombinant polynucleotide is mRNA. In some embodiments, the recombinant polynucleotide comprises circRNA. In some embodiments, the recombinant polynucleotide is contained within a viral vector. In some embodiments, the recombinant polynucleotide is delivered via a viral vector.

[0293] In some embodiments, the present disclosure provides a therapeutic composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (such as a chimeric fusion receptor protein (CFP)), the CFP comprising: (a) an extracellular domain comprising: (i) an scFv that specifically binds any one of the targets described herein, and (ii) a hinge domain derived from CD8; a hinge domain derived from CD28 or at least a portion of the extracellular domain of CD68; (b) a CD8 transmembrane domain, a CD28 transmembrane domain, a CD2 transmembrane domain, or a CD68 transmembrane domain; and (c) an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise: (i) a first intracellular signaling domain derived from FcRγ or FcRε, an interferon-inducible domain, and / or (ii) a third intracellular signaling domain that: (A) comprises a PI3K recruitment domain, or (B) is derived from CD40.

[0294] In some embodiments, the present disclosure provides a therapeutic composition comprising a recombinant nucleic acid encoding a bispecific or trispecific conjugate as disclosed herein.

[0295] Other therapeutic compositions for co-administration

[0296] In some embodiments, the therapeutic composition further comprises an additional therapeutic agent selected from CD47 agonists, agents that inhibit Rac, agents that inhibit Cdc42, agents that inhibit GTPases, agents that promote F-actin disassembly, agents that promote PI3K recruitment to the CFP, agents that promote PI3K activity, agents that promote phosphatidylinositol 3,4,5-triphosphate production, agents that promote ARHGAP12 activity, agents that promote ARHGAP25 activity, agents that promote SH3BP1 activity, agents that promote lymphocyte sequestration in primary and / or secondary lymphoid organs, agents that increase the concentration of naive T cells and central memory T cells in secondary lymphoid organs, and any combination thereof.

[0297] In some embodiments, the myeloid cells further comprise: (a) an endogenous peptide or protein that dimerizes with CFP, (b) a non-endogenous peptide or protein that dimerizes with CFP; and / or (c) a second recombinant polynucleotide sequence, wherein the second recombinant polynucleotide sequence comprises a sequence encoding a peptide or protein that interacts with CFP; wherein the dimerization or interaction enhances phagocytosis of the myeloid cells that express CFP as compared to myeloid cells that do not express CFP.

[0298] In some embodiments, the myeloid cells exhibit an increase in (i) effector activity, cross-presentation, respiratory burst, ROS production, iNOS production, inflammatory mediators, extracellular vesicle production, phosphatidylinositol 3,4,5-trisphosphate production, trogocytosis with antigen-expressing target cells, resistance to CD47-mediated inhibition of phagocytosis, resistance to LILRB1-mediated inhibition of phagocytosis, or any combination thereof; and / or (ii) expression of IL-1, IL3, IL-6, IL-10, IL-12, IL-13, IL-23, TNFα, cytokines of the TNF family, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL-17, IP-10, RANTES, interferons, MHC class I proteins, MHC class II proteins, CD40, CD48, CD58, CD80, CD86, CD112, CD155, TRAIL / TNF family death receptors, TGFβ, B7-DC, B7-H2, LIGHT, HVEM, TL1A, 41BBL, OX40L, GITRL, CD30L, TIM1, TIM4, SLAM, PDL1, MMP (e.g., MMP2, MMP7, and MMP9), or any combination thereof.

[0299] In some embodiments, the intracellular signaling domain is derived from a phagocytic or tethering receptor, or wherein the intracellular signaling domain comprises a phagocytosis activation domain. In some embodiments, the intracellular signaling domain is derived from a receptor other than a phagocytic receptor selected from Megf10, MerTk, FcR-α, or Bai1. In some embodiments, the intracellular signaling domain is derived from a protein, such as a receptor (e.g., a phagocytic receptor), selected from TNFR1, MDA5, CD40, lectin, 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, complement receptor, CR3, CR4, Tim-1, Tim-4, and CD169. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain.

[0300] In some embodiments, the intracellular signaling domain is derived from a receptor containing an ITAM domain.

[0301] Provided herein is a composition comprising a recombinant nucleic acid encoding a CFP (e.g., a phagocytic or tethering receptor (PR) fusion protein (PFP)), the CFP comprising: a PR subunit comprising: a transmembrane domain and an intracellular domain comprising an intracellular signaling domain; and an extracellular domain comprising an antigen-binding domain specific for a target cell antigen; wherein the transmembrane domain and the extracellular domain are operably linked; and wherein the intracellular signaling domain is derived from a phagocytic receptor other than a phagocytic receptor selected from Megf10, MerTk, FcRα, or Bai1.

[0302] In some embodiments, when the CFP binds to an antigen on a target cell, the killing activity of cells expressing CFP is increased by at least greater than 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% compared to cells that do not express CFP. In some embodiments, when CFP is expressed in a cell, the CFP is functionally incorporated into the cell's cell membrane. In some embodiments, when the CFP binds to an antigen on a target cell, the killing 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 compared to cells that do not express CFP.

[0303] In some embodiments, the intracellular signaling domain is derived from a receptor, such as a phagocytic receptor, selected from TNFR1, MDA5, CD40, lectin, 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 signaling domain comprises a pro-inflammatory signaling domain.

[0304] The present disclosure provides a composition comprising a recombinant nucleic acid encoding a CFP (such as a phagocytic or binding receptor (PR) fusion protein (PFP)), the CFP comprising: a PR subunit comprising: a transmembrane domain and an intracellular domain comprising an intracellular signaling domain; and an extracellular domain comprising an antigen-binding domain specific for a target cell antigen; wherein the transmembrane domain and the extracellular domain are operably linked; and wherein the intracellular signaling domain is derived from a receptor, such as a phagocytic receptor, selected from TNFR1, MDA5, CD40, lectin, dectin1, 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.

[0305] In some embodiments, upon antigen binding of the CFP to a target cell, the killing activity of cells expressing the CFP is increased by at least greater than 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% compared to cells not expressing the CFP. In some embodiments, the intracellular signaling domain is derived from a phagocytic receptor other than a phagocytic receptor selected from Megf10, MerTk, FcR-α, or Bai1. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain. In some embodiments, the intracellular signaling domain comprises a PI3K recruitment domain, such as a PI3K recruitment domain derived from CD19. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain.

[0306] In some embodiments, cells expressing CFP exhibit increased phagocytosis of antigen-expressing target cells compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit at least 1.1-fold increased phagocytosis of antigen-expressing target cells compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, or 50-fold increased phagocytosis of antigen-expressing target cells compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased production of cytokines compared to cells not expressing CFP. In some embodiments, the cytokines are selected from IL-1, IL3, IL-6, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, RANTES, interferons, and combinations thereof. In some embodiments, cells expressing CFP exhibit increased effector activity compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased cross-presentation compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of MHC class II proteins compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD80 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD86 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of MHC class I proteins compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of TRAIL / TNF family death receptors compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of B7-H2 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of LIGHT compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of HVEM compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD40 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of TL1A compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of 41BBL compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of OX40L compared to cells not expressing CFP.In some embodiments, cells expressing CFP exhibit an increase in the expression of the GITRL death receptor compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in the expression of CD30L compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in the expression of TIM4 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in the expression of TIM1 ligand compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in the expression of SLAM compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in the expression of CD48 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in the expression of CD58 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in the expression of CD155 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in the expression of CD112 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in the expression of PDL1 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in the expression of B7-DC compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in respiratory burst compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in ROS production compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in iNOS production compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in iNOS production compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in extracellular vesicle production compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in trogocytosis with antigen-expressing target cells compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in resistance to CD47-mediated phagocytosis inhibition compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in resistance to LILRB1-mediated phagocytosis inhibition compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in the production of phosphatidylinositol 3,4,5-trisphosphate.

[0307] The present disclosure also provides a pharmaceutical composition comprising a composition described herein, such as a recombinant nucleic acid described herein, a vector described herein, a polypeptide described herein, or a cell described herein; and a pharmaceutically acceptable excipient. The engineered cell can be a myeloid cell. In one aspect, a pharmaceutical composition is disclosed that comprises a recombinant nucleic acid encoding or comprising any one of the sequences of SEQ ID NOs: 26-42, or a cell comprising a recombinant nucleic acid encoding or comprising any one of the sequences of SEQ ID NOs: 26-42, or an engineered cell comprising a recombinant nucleic acid encoding or comprising any one of the sequences of SEQ ID NOs: 26-42; and a pharmaceutically acceptable excipient. In one embodiment, the cell comprises a recombinant nucleic acid encoding an amino acid sequence comprising at least one of the sequences selected from SEQ ID NOs: 26-42. In one embodiment, the cell is a myeloid cell. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a primary human cell. In one embodiment, the cell is a human primary immune cell. In some embodiments, the cell is a progenitor cell or a stem cell, or an undifferentiated cell. In some embodiments, the cell is obtained from a biological sample of a human subject. In some embodiments, the cell is isolated from a biological sample of a human subject and selected for a phenotype (e.g., expression of cell surface markers). In one embodiment, the isolated cell is a progenitor cell, a progenitor myeloid cell, a cell characterized as CD14+ / CD16-.

[0308] In one embodiment, the isolated cell or engineered cell is CD14+ / CD16-.

[0309] In one aspect, the pharmaceutical composition comprises an engineered cell, wherein the engineered cell is CD14+ / CD16-.

[0310] In one aspect, the pharmaceutical composition comprises a cell population, wherein at least 50% of the cells are CD14+ / CD16- and less than 10% of the cells are dendritic cells. In one embodiment, the cells exhibit high expression of CCR2. In one embodiment, the cells do not exhibit tonic signaling and de novo activation and exhibit M0, M1, or M2 differentiation upon activation.

[0311] The present disclosure provides a method of treating cancer or viral infection in a subject, comprising: administering to the subject a pharmaceutical composition comprising a recombinant nucleic acid encoding or comprising any one of the sequences of SEQ ID NOs: 26-42, or a cell comprising a recombinant nucleic acid encoding or comprising any one of the sequences of SEQ ID NOs: 26-42, or an engineered cell comprising a recombinant nucleic acid encoding or comprising any one of the sequences of SEQ ID NOs: 26-42; and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a cell population, wherein at least 50% of the cells are CD14+ / CD16−, less than 10% of the cells are dendritic cells; and the cells exhibit high expression of CCR2. In one embodiment, the cells do not exhibit tonic signaling and de novo activation and exhibit M0, M1 or M2 differentiation upon activation.

[0312] In some embodiments, the present disclosure provides a therapeutic composition comprising a cell comprising a recombinant nucleic acid as described anywhere within this specification. In some embodiments, the therapeutic composition comprises a recombinant nucleic acid encoding a chimeric protein as described anywhere herein. In some embodiments, the myeloid cell is a CD14+ cell, a CD14+ / CD16− cell, a CD14+ / CD16+ cell, a CD14− / CD16+ cell, a CD14− / CD16− cell, a dendritic cell, an M0 macrophage, an M2 macrophage, an M1 macrophage, or a chimeric myeloid cell / macrophage / dendritic cell.

[0313] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from a CD47 agonist, an agent that inhibits Rac, an agent that inhibits Cdc42, an agent that inhibits a GTPase, an agent that promotes F-actin disassembly, an agent that promotes PI3K recruitment to PFP, an agent that promotes PI3K activity, an agent that promotes phosphatidylinositol 3,4,5-trisphosphate production, an agent that promotes ARHGAP12 activity, an agent that promotes ARHGAP25 activity, an agent that promotes SH3BP1 activity, and any combination thereof. In some embodiments, the pharmaceutically acceptable excipient comprises a serum-free medium, a lipid, or a nanoparticle.

[0314] In some embodiments, the therapeutic agent is a recombinant nucleic acid that encodes or comprises any one of the sequences of SEQ ID NOs: 26-42, 135, 136, or 137, or a sequence having at least 80% identity to the sequences of SEQ ID NOs: 26-42, 135, 136, or 137, and the therapeutic agent is injected systemically or locally into a subject. In some embodiments, the recombinant nucleic acid comprises at least one sequence encoding a sequence selected from SEQ ID NOs: 26-42, 135, 136, or 137. In some embodiments, the recombinant nucleic acid is mRNA. In some embodiments, the recombinant nucleic acid is associated with one or more lipid components in a pharmaceutical composition. The lipid components can be associated in the form of liposomes or lipid nanoparticles. The lipid components can comprise at least one cationic lipid. One or more lipids of the pharmaceutical composition can be conjugated or modified.

[0315] Methods for generating novel chimeric receptor fusion protein (CFP) constructs

[0316] In one aspect, provided herein is a method for generating a novel chimeric receptor protein, which includes, 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 serves as an effector myeloid cell as described in the present specification. Generation of the fusion proteins as described herein can be performed using well-known molecular cloning techniques, and the sequence can be verified after generating the recombinant nucleic acid.

[0317] Preparation of recombinant nucleic acids encoding chimeric antigen receptors:Prepare a recombinant nucleic acid construct encoding a chimeric antigen receptor (CAR) designed for expression in myeloid cells and incorporate it into a plasmid vector for amplification and / or testing of expression in eukaryotic cells. Construct the recombinant CAR 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-part of a domain can be encoded by a nucleic acid sequence generated by PCR from a heterologous source sequence and spliced together by separately cloning into a vector, or ligated to a longer nucleic acid and then inserted into the multiple cloning site of a suitable plasmid or vector with a suitable promoter and 3'-regulatory elements for amplification. Briefly, prepare an exemplary CAR by incorporating a nucleic acid sequence encoding one or more signaling 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 having a sequence encoding a target antigen-binding scFv at the extracellular terminus. Certain constructs include a FLAG peptide sequence at the extracellular terminus, which is designed such that it does not impede the binding of the scFv to its target antigen. These components are ligated together into a sequence encoding a fully functional transmembrane CAR. The nucleic acid subunits encoding the individual domains of the recombinant protein are designed to include a short flexible linker sequence inserted between the two domains. The construct is ligated into a plasmid having a promoter and a 3' stabilizing unit. In one variant, the construct is placed within an Alu retrotransposon element encoding ORF2p and has the corresponding 5'- and 3'-UTR sequences, namely the CMV promoter. The plasmid is amplified in Escherichia coli, verified by sequencing, or stored at (-)80 °C.

[0318] mRNA preparation : mRNA can be prepared by in vitro transcription using the digested plasmid as a template and purified to remove contaminating DNA and polyadenylation. Purify the RNA product, resuspend it in RNase-free water to 1 mg / ml, and store it in a freezer tube.

[0319] The useful CFP ECD, TM, ICD, and antigen-binding domains for generating novel CFP can be identified using the methods described herein. Briefly, a large number of potential candidate proteins can be screened to enhance phagocytic properties and their corresponding intracellular signaling related to phagocytosis. Then, the useful domains can be used to generate novel CFP. The screening can be divided into two parts: A. Screening of the phagocytic receptor (PR) domain; B. Screening of the antigen-binding domain. Screening of PR domains:

[0320] In one embodiment, the phagocytic potential of approximately 5,800 plasma membrane proteins is screened according to the general methods described herein. J774 macrophages can be transiently transfected with a library of 5,800 plasma proteins. High-throughput multiplex assays (ranging from 6-well plate assay setups to up to 384-well plate assays, with robotic operation) can be established to evaluate various potential functions of the plasma membrane. Exemplary assays include, but are not limited to, phagocytosis assays, cytokine production assays, inflammasome activation assays, and iNOS activation assays. Exemplary simplified methods can be described in the following paragraphs. Variations of each method can also be used, and those skilled in the art can understand these variations. Variations of each method can also be used, and those skilled in the art can understand these variations. Exemplary intracellular signaling domains tested include, but are not limited to, CD40-FcRγ; FcRγ-CD40; NLRP3; FcRγ-SH2-procaspase; FcRγ-Myd88; FcRγ-IFN receptor; FcR-TNFR1; FcRγ-TNFR2; FcR-AIM2; FcRγ-TRIFN; FcRγ-procaspase; TRIFC; RIG1; MDA5; TBK; CD64; CD16a; CD89; FcRε; SIRPβ; (two consecutive intracellular domains can be represented as a hyphenated term, e.g., FcRγ-Myd88 refers to an intracellular domain containing the FcRγ intracellular signaling domain as signaling domain 1; and the Myd88 intracellular signaling domain as signaling domain 2). Extracellular linker domains screened include, but are not limited to, CD64, CD16A, CD89, SIRPα, FcRε, CD8 hinge. Transmembrane domains tested include, but are not limited to, CD8, CD64, CD16A, CD89, FcRε, SIRPα, TNFR1, and CD40. The MDA5 domain was also screened.

[0321]

[0322] Antigen-conjugated silica or polystyrene beads with a diameter range of 1 nm, 5 nm, or 10 nm are used to screen macrophages. Inert beads can be coated in a supported lipid bilayer, and the antigen can be conjugated 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 containing heat-inactivated serum and antibiotics (penicillin / streptomycin). On the day of the assay, the cells can be plated in 6-well plates at a density of 1×10^6 cells / ml / well or plated in 12- 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. At 30 minutes and 2 hours after the addition of the beads, the cells are observed by light microscopy. 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 engulfment. The confidence level can be determined by the Kruskal-Wallis test with Dunn's multiple comparison correction.

[0323] In some instances, dye-loaded tumor cells can be fed to the macrophage cell line, and phagocytosis can be evaluated by microscopy.

[0324] Phagocytosis assay:

[0325] The macrophage line can be cultured as described above. In one assay, each J774 cell line expressing a plasma membrane protein is plated in a multi-well plate and challenged with antigen-conjugated beads, and cytokine production is measured by collecting the supernatant at 4 hours and 24 hours. The cytokines in the supernatant can be measured by ELISA. In another fraction, the 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 form, which 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. Use a macrophage inflammatory cytokine array kit (R&D Systems).

[0326] Inflammatory genes and intracellular signaling pathways activated by cytokines can be identified by Western blot analysis of phosphorylation of MAP kinases, JNK, Akt signaling pathways, and interferon-activated pathways including STAT-1 phosphorylation and activation.

[0327] Cytokine production:

[0328] Functional assay

[0329] Activation of the NLRP3 inflammasome was determined by detecting increased IL-1 production by ELISA and caspase-1 activation and cleavage of procaspase to generate shorter caspases by Western blotting. In a microplate multiplex setting, Caspase-Glo (Promega Corporation) was used for a faster readout of caspase 1 activation.

[0330] Inflammasome activation assay:

[0331] Activation of the oxidative burst potential can be measured by iNOS activation and NO production using a fluorescence assay NOS activity assay kit (AbCAM).

[0332] iNOS activation assay:

[0333] 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 the J774 macrophage cell line. Macrophages can destroy the cells 1 hour after infection, which can be detected by microscopy or by cell death assays.

[0334] Cancer cell killing assay:

[0335] The variable domains of the antibody light and heavy chains of cancer ligands can be screened to generate the extracellular binding domain of CFP. Human full-length antibodies or scFv libraries can be screened. Potential ligands can also be used to immunize llamas to develop novel immunoglobulin-binding domains in llamas and prepare single-domain antibodies.

[0336] Then, the specific useful domains identified from the screening can be reverse transcribed and cloned into a lentiviral expression vector to generate the CFP construct. One or more domains from the extracellular, TM, and cytoplasmic regions of the highly phagocytic receptors generated from the screening can be used to generate the recombinant nucleic acid encoding CFP. Briefly, plasma membrane receptors that display high activators of pro-inflammatory cytokine production and inflammasome activation can be identified. Bioinformatics studies can be performed to identify functional domains, including extracellular activation domains, transmembrane domains, and intracellular signaling domains, such as specific kinase activation sites, SH2 recruitment sites. Then, these screened functional domains can be cloned in a modular structure to generate novel CFP. These can be candidate CFP, and each of these chimeric constructs can be tested for enhanced phagocytosis, production of cytokines and chemokines, and / or tumor cell killing in vitro and / or in vivo. A particle-based phagocytosis assay is used to examine changes in phagocytosis. Briefly, streptavidin-conjugated fluorescent polystyrene particles (6 μm in diameter) can be conjugated with biotinylated recombinant expressed and purified cancer ligands. Myeloid cells expressing the novel CFP can be incubated with ligand-coated particles for 1-4 h, and the amount of phagocytosis can be analyzed and quantified using flow cytometry. Then, plasmids or lentiviral constructs of the designed CFP can be prepared and tested for cancer cell lysis in macrophages.

[0337] Screening for high-affinity antigen-binding domains:

[0338] Methods for generating ex vivo myeloid cells from a subject

[0339] Peripheral blood mononuclear cells can be separated from the buffy coat of normal donors by density centrifugation using Histopaque 1077 (Sigma). After washing, CD14+ monocytes can be isolated from the mononuclear cell fraction using CliniMACS GMP-grade CD14 microbeads and an LS separation magnetic column (Miltenyi Biotec). Briefly, the cells can be resuspended at an appropriate concentration in PEA buffer (phosphate-buffered saline [PBS] plus 2.5 mmol / L ethylenediaminetetraacetic acid [EDTA] and human serum albumin [20% Alburex, Octopharma at 0.5% final volume]), incubated with CliniMACS CD14 beads according to the manufacturer's instructions, and then washed and passed through a magnetized LS column. After washing, the purified monocytes can be eluted from the demagnetized column, washed, and resuspended in the relevant medium for culture. Isolation of CD14+ cells from leukapheresis: PBMC can be collected from cirrhotic donors who have given informed consent to participate in this study by leukapheresis. Leukapheresis of peripheral blood mononuclear cells (MNC) is performed by aseptic collection using an Optia apheresis system. Using the standard collection procedure for MNC, 2.5 blood volumes are processed. CD14 cell isolation is performed using a GMP-compliant functional closure system (CliniMACS Prodigy system, Miltenyi Biotec). Briefly, the leukapheresis product is taken for cell counting, and an aliquot is taken for pre-separation flow cytometry. The percentage and absolute cell number of monocytes (CD14+) can be determined, and if necessary, the volume is adjusted to meet the required selection criteria (≤20×10 9 total white blood cells; <400×10 6 white blood cells / mL; ≤3.5×10 9 CD14 cells, volume 50 - 300 mL). CD14 cell isolation is performed using CliniMACS Prodigy (Class III medical device), a TS510 tube set, and an LP-14 program with CliniMACS CD14 microbeads. At the end of the procedure, the selected CD14+ positive monocytes can be washed in PBS / EDTA buffer (CliniMACS buffer, Miltenyi) containing pharmaceutical-grade 0.5% human albumin (Alburex) and then resuspended in TexMACS (or control) medium for culture.

[0340] Cell counting and purity:

[0341] Total MNC and cell counts of the separated monocyte fraction can be performed using a Sysmex XP-300 automated analyzer (Sysmex). Assessment of macrophage numbers was performed by flow cytometry using TruCount tubes (Becton Dickinson) to determine absolute cell numbers, as Sysmex consistently underestimates monocyte numbers. Isolation purity was evaluated 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).

[0342] Cell culture - Development of healthy donor sample cultures

[0343] The optimal medium for macrophage differentiation was investigated, and three candidates could be tested using cell products. In addition, the effect of monocyte cryopreservation on derived myeloid cells and macrophages for therapeutic use was examined. Functional assays could be performed to quantify the phagocytic capacity of myeloid cells and macrophages and their ability to further polarize, as well as the phagocytic potential as described elsewhere in this disclosure.

[0344] Full process validation of subject samples

[0345] Monocytes cultured from leukapheresis of Prodigy isolates can be cultured in culture bags (MACS GMP differentiation bags, Miltenyi) containing GMP-grade TexMACS (Miltenyi) and 100 ng / mL M-CSF at 2×10 6 monocytes / cm 2 and / mL. Monocytes can be cultured with 100 ng / mL GMP-compliant recombinant human M-CSF (R&D Systems). Cells can be cultured for 7 days in a humid atmosphere at 37 °C and 5% CO2. During the culture period (days 2 and 4), two 50% volume medium replenishments are performed, removing 50% of the medium and then feeding with fresh medium supplemented with 200 ng / mL M-CSF (to restore a final concentration of 100 ng / mL).

[0346] Cell harvest:

[0347] For macrophages from normal donor sources, cells can be removed from the wells on day 7 using cell dissociation buffer (Gibco, ThermoFisher) and a pastette. Cells can be resuspended in PEA buffer and counted, and then approximately 1×10 6Cell staining for flow cytometry. On day 7, leukapheresis-derived macrophages can be removed from the culture bag using PBS / EDTA buffer (CliniMACS buffer, Miltenyi) containing pharmaceutical grade 0.5% human serum albumin (HAS; Alburex). The harvested cells can be resuspended in an excipient consisting of two licensed products: 0.9% saline for infusion (Baxter) containing 0.5% human albumin (Alburex).

[0348] Flow cytometry characterization:

[0349] FACSCanto II (BD Biosciences) or MACSQuant 10 (Miltenyi) flow cytometry can be used to analyze the surface marker expression of monocytes and macrophages. Typically, nearly 20,000 events can be obtained for each sample. Cell surface expression of leukocyte markers was performed in freshly isolated and day 7 mature cells by incubating the cells with specific antibodies (final dilution 1:100). The cells were incubated with an FcR blocker (Miltenyi) for 5 min and then with the antibody mixture at 4 °C for 20 min. The cells can be washed in PEA and the dead cell exclusion dye DRAQ7 (BioLegend) was added at 1:100. The following series of surface markers of the cells can be stained: CD45-VioBlue, CD14-PE or CD14-PerCP-Vio700, CD163-FITC, CD169-PE, and CD16-APC (all from Miltenyi), CCR2-BV421, CD206-FITC, CXCR4-PE, and CD115-APC (all from BioLegend), as well as 25F9-APC and CD115-APC (eBioscience). Forward and side scatter and the DRAQ7 dead cell discriminator (BioLegend) can be used to gate monocytes and macrophages to exclude debris, doublets, and dead cells, and analysis was performed using FlowJosoftwcan be (Tree Star). Based on the initially detailed phenotypic analysis, a set was developed as the release criteria (CD45-VB / CD206-FITC / CD14-PE / 25F9 APC / DRAQ7), which defines the development of functional macrophages from monocytes. The mean fluorescence intensity (MFI) of macrophages can be determined to be five-fold higher than the level of day 0 monocytes for 25F9 and CD206. A second set was developed, which evaluated additional markers as part of an extended set and consisted of CCR2-BV421 / CD163-FITC / CD169-PE / CD14-PerCP-Vio700 / CD16-APC / DRAQ7, but was not used as part of the release criteria for the cell product.

[0350] Monocytes and macrophages can be isolated from the buffy coat formed in the sucrose gradient centrifugation sample of extracted and separated peripheral blood cells. The phagocytic uptake of CD14 cells can be tested using pHRodo beads, which fluoresce only when internalized into acidic endosomes. Briefly, monocytes or macrophages can be cultured with 1 - 2 μL of pHRodo Escherichia coli bioparticles (Life Technologies, Thermo Fisher) for 1 h, then the medium is removed and the cells are washed to remove unphagocytosed particles. Phagocytosis is evaluated using an EVOS microscope (Thermo Fisher), images are captured, and the cellular uptake of beads is quantified using ImageJ software (NIH). The ability to polarize towards defined differentiated macrophage phenotypes is examined by treating day 7 macrophages with IFNγ (50 ng / mL) or IL-4 (20 ng / mL) for 48 h to induce polarization into M1 or M2 phenotypes (or M[IFNγ] and M[IL-4] respectively). After 48 h, cells can be observed by EVOS brightfield microscopy, then harvested and phenotypically analyzed as described previously. Further analysis of the cytokine and growth factor secretion profiles of macrophages at the time of generation and in response to inflammatory stimuli is performed. Macrophages can be generated from healthy donor buffy coats as described previously and left untreated or stimulated with TNFα (50 ng / mL, Peprotech) and polyinosinic acid:polycytidylic acid (poly I:C, a viral mimic that binds TLR3, 1 μg / mL, Sigma) to mimic conditions present in the inflamed liver, or lipopolysaccharide (LPS, 100 ng / mL, Sigma) plus IFNγ (50 IU / mL, Peprotech) to produce maximal macrophage activation. Day 7 macrophages can be incubated overnight, the supernatant is collected and centrifuged to remove debris, then stored at -80 °C until testing. Secretome analysis is performed on a Magpix multiplex ELISA reader (BioRad) using a 27-plex human cytokine kit and a 9-plex matrix metalloproteinase kit.

[0351] Product stability:

[0352] During the process opening, various excipients can be tested, including PBS / EDTA buffer; PBS / EDTA buffer (Alburex) containing 0.5% HAS, 0.9% saline alone, or saline containing 0.5% HAS. 0.9% saline (Baxter) containing 0.5% HAS excipient was found to maintain optimal cell viability and phenotype (data not shown). The stability of cirrhotic donor macrophage harvest was studied in three process optimization runs, and a more limited range of time points (n = 3) was evaluated in the process validation run. After harvesting and resuspending in excipient (0.9% saline for infusion, 0.5% human serum albumin), the bags can be stored at ambient temperature (21 - 22°C) and sampled at 0, 2, 4, 6, 8, 12, 24, 30, and 48 h post-harvest. A release standard antibody panel was run on each sample, and viability and mean fold change from day 0 were measured based on the geometric MFI of 25F9 and CD206.

[0353] Statistical analysis:

[0354] Results are presented as mean ± SD. Whenever possible, GraphPad Prism 6 was used to evaluate the statistical significance of differences using an unpaired two-tailed t-test. Results were considered statistically significant when the P value < 0.05.

[0355] The present invention also provides a cell comprising the composition, the vector, or the polypeptide described herein. In some embodiments, the cell is a phagocyte. In some embodiments, the cell is a cell derived from a stem cell, a myeloid cell, a macrophage, a dendritic cell, a lymphocyte, a mast cell, a monocyte, a neutrophil, a microglial cell, or an 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 phagocyte. In some embodiments, the cell is an M2 phagocyte. In some embodiments, the cell is an M1 myeloid cell. In some embodiments, the cell is an M2 myeloid cell.

[0356] The present invention also provides a method for treating a disease in a subject in need thereof, which comprises administering to the subject the pharmaceutical composition described herein. In some embodiments, the disease is cancer. In some embodiments, the cancer is solid cancer. In some embodiments, the solid cancer is selected from ovarian cancer, and suitable cancers include ovarian cancer, renal cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, lung cancer. In some embodiments, the cancer is liquid cancer. In some embodiments, the liquid cancer is leukemia or lymphoma. In some embodiments, the liquid cancer is T cell lymphoma. In some embodiments, the disease is T cell malignancy.

[0357] In some embodiments, the method further comprises administering to the subject an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from CD47 agonists, agents that inhibit Rac, agents that inhibit Cdc42, agents that inhibit GTPase, agents that promote F-actin disassembly, agents that promote PI3K recruitment to PFP, agents that promote PI3K activity, agents that promote phosphatidylinositol 3,4,5-trisphosphate production, agents that promote ARHGAP12 activity, agents that promote ARHGAP25 activity, agents that promote SH3BP1 activity, and any combination thereof.

[0358] In some embodiments, the administration comprises infusion or injection. In some embodiments, the administration comprises direct administration to the solid cancer. In some embodiments, the administration comprises a circRNA-based delivery procedure, a non-particle-encapsulated mRNA-based delivery procedure, an mRNA-based delivery procedure, a virus-based delivery procedure, a particle-based delivery procedure, a liposome-based delivery procedure, or an exosome-based delivery procedure. In some embodiments, a CD4+ T cell response or a CD8+ T cell response is elicited in the subject.

[0359] The present invention also provides a method for preparing cells, which comprises contacting the cells with the composition described herein, the vector described herein, or the polypeptide described herein. In some embodiments, the contacting comprises transduction. In some embodiments, the contacting comprises chemical transfection, electroporation, nucleofection, or viral infection or transduction.

[0360] The present invention provides a method for administering a therapeutic agent comprising any one of the above compositions. In some embodiments, the therapeutic agent is administered via a parenteral administration route.

[0361] In some embodiments, the therapeutic agent is administered via an intramuscular administration route. In some embodiments, the therapeutic agent is administered via an intravenous administration route. In some embodiments, the therapeutic agent is administered via a subcutaneous administration route.

[0362] The present invention also provides a method for preparing a pharmaceutical composition, which pharmaceutical composition comprises one or more recombinant nucleic acids as described herein and a lipid in an aqueous composition as described herein. In some embodiments, the composition comprises a vector as described herein. In some embodiments, the lipid comprises a lipid nanoparticle-forming lipid.

[0363] Examples

[0364] Example 1. Chimeric antigen receptor protein constructs with intracellular interferon activation domains

[0365] In this example, the amino acid and nucleic acid sequences of various CFP constructs with intracellular domains for inflammatory signal transduction as described in Table 4 herein are disclosed. The nucleic acid sequences detailed below can be readily understood by those skilled in the art as DNA and mRNA sequences to provide guidance in preparing and using suitable constructs or their variants using common molecular cloning techniques. Isolation of myeloid cells from PBMCs: The general principle of the expression and mode of action of the chimeric fusion protein is illustrated. The transmembrane domains (TMDs) selected for constructing the fusion protein are those that can dimerize or multimerize with endogenous myeloid cell transmembrane proteins for successful expression and functioning of the chimeric fusion protein. In an exemplary construct, such a TM domain is the CD89 TMD. By utilizing a transmembrane domain that multimerizes with endogenous Fcγ (which mediates myeloid cell-targeted expression Figure 1 of the chimeric fusion protein shown), Figure 1 different constructs are shown, which have been prepared using a TROP2-binding domain in the extracellular region, a transmembrane domain from the CD89 TMD, and an intracellular signaling domain as Figure 2 shown. In some embodiments, the CFP comprising the CD89 TMD is designed to comprise the intracellular domain of CD89 or a fragment thereof, without additional intracellular signaling domains; the idea being that the mature CFP would be expressed in the cell membrane and associate with endogenous FcRγ, and that once the extracellular domain of the CFP binds to the target antigen on the cancer cell, this association would ensure the stability of the CFP in the cell and trigger intracellular signaling via the intracellular domain of FcRγ. In some embodiments, constructs comprising the CD89 transmembrane domain can be designed to comprise an intracellular domain of an FcR, such as an intracellular signaling domain. In some embodiments, the CFP can comprise the intracellular domain of CD40.

[0366] In some embodiments, the CFP can include more than one intracellular signaling domain; for example, the FcR intracellular signaling domain and the PI3 kinase intracellular signaling domain. Additional intracellular domains can include the TRIF intracellular signaling domain and the CD40 intracellular signaling domain. These constructs are referred to as second-generation (or next-generation) chimeric fusion receptor constructs for in vivo myeloid cell delivery and expression. Some of the various sequences that have been tested are provided below. An exemplary anti-TROP2-CD89 sequence (with an extracellular TROP2-binding scFv fused to the CD89 TMD, and segments of the CD89 extracellular and intracellular regions) is shown below, with the construct name anti-TROP2-CD89, which has the following sequence: MWLQSLLLLGTVACSISQVQLQQSGSELKKPGASVKVSCKASGYTFT Figure 2 WVKQAPGQGLKWMG NYGMN RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR WINTYTGEPTYTDDFKG WGQGSLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITC GGFGSSYWYFDV VAWYQQKPGKAPKLLIY KASQDVSIA SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC Q QHYITPLTF GAGTKVEIKRGSGGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK (SEQ ID NO:143). (The corresponding CDR 1, 2, and 3 of VH and VL are underlined in order, Kabat numbering system). Table 6A shows the individual domains.

[0367] Another exemplary anti-TROP2-CD89 sequence is provided below: MWLQSLLLLGTVACSISQVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMG WINTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITC KASQDVS IAVA WYQQKPGKAPKLLIY SASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLT FGAGTKVEIKRSGGGGAAADYKDDDDKGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK(SEQ ID NO:144). (The corresponding CDR 1, 2, and 3 of VH and VL are underlined in order, Kabat numbering system). Table 6A shows each domain.

[0368] Exemplary anti-TROP2-CD89-FcR with an additional FcRICD compared to SEQ ID NO:143 is:

[0369] MWLQSLLLLGTVACSISQVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMG WI NTYTGEPTYTDDFK GRFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLT FGAGTKVEIKRSGGGGAAADYKDDDDKGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ(SEQ ID NO:145). (The corresponding CDR 1, 2, and 3 of VH and VL are underlined in order, Kabat numbering system). Table 6A shows each domain.

[0370] Exemplary anti-TROP2 binding CFP sequence is anti-TROP2-CD89-FcR-PI3K sequence:

[0371] MWLQSLLLLGTVACSISQVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMG WI NTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDVWGQGSLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITC KASQDVSIAVA WYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLT FGAGTKVEIKRSGGGGAAADYKDDDDKGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQGSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENM(SEQ ID NO:146). (The corresponding CDR 1, 2, and 3 of VH and VL are underlined in sequence, Kabat numbering system). Table 6A shows each domain.

[0372] The amino acid sequence of anti-TROP2-CD89-CD40 is provided below: MWLQSLLLLGTVACSISQVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMG WINTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITC KASQ DVSIAVA WYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLT FGAGTKVEIKRSGGGGAAADYKDDDDKGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ(SEQ ID NO:147). (The corresponding CDR 1, 2, and 3 of VH and VL are underlined in sequence, Kabat numbering system). Table 6A shows each domain.

[0373] The amino acid sequence of anti-TROP2-CD89-CD40-FcR is provided below: MWLQSLLLLGTVACSISQVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMG WINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITC KASQDVSIAVA WYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLT FGAGTKVEIKRSGGGGAAADYKDDDDKGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQGSRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ(SEQ ID NO:148). (The corresponding CDR 1, 2, and 3 of VH and VL are underlined in sequence, Kabat numbering system). Table 6A shows each domain.

[0374] The amino acid sequence of anti-TROP2-CD89-TRIF is provided below: MWLQSLLLLGTVACSISQVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMG WINTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITC KASQ DVSIAVA WYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLTFGAGTKVEIKRSGGGGAAADYKDDDDKGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVRGSIRTLQSNLGCLPPSSALPSGTRSLPRPIDGVSDWSQGCSLRSTGSPASLASNLEISQSPTMPFLSLHRSPHGPSKLCDDPQASLVPEPVPGGCQEPEEMSWPPSGEIASPPELPSSPPPGLPEVAPDATSTGLPDTPAAPETSTNYPVECTEGSAGPQSLPLPILEPVKNPCSVKDQTPLQLSVEDTTSPNTKPCPPTPTTPETSPPPPPPPPSSTPCSAHLTPSSLFPSSLE(SEQ ID NO:149). (The corresponding CDR 1, 2, and 3 of VH and VL are underlined in order, Kabat numbering system). Table 6A shows the respective domains.

[0375] The amino acid sequence of anti-TROP2-CD89-FcR-TRIF is provided below: MWLQSLLLLGTVACSISQVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMG WINTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITC KASQDVSIAVA WYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLTFGAGTKVEIKRSGGGGAAADYKDDDDKGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQGSGSIRTLQSNLGCLPPSSALPSGTRSLPRPIDGVSDWSQGCSLRSTGSPASLASNLEISQSPTMPFLSLHRSPHGPSKLCDDPQASLVPEPVPGGCQEPEEMSWPPSGEIASPPELPSSPPPGLPEVAPDATSTGLPDTPAAPETSTNYPVECTEGSAGPQSLPLPILEPVKNPCSVKDQTPLQLSVEDTTSPNTKPCPPTPTTPETSPPPPPPPPPPSSTPCSAHLTPSSLFPSSLE(SEQ ID NO:150). (The corresponding CDR 1, 2, and 3 of VH and VL are underlined in order, Kabat numbering system). Table 6A shows the respective domains.

[0376] Table 6A - First and Second Generation Sequences of Exemplary TROP2 Conjugates

[0377]

[0378]

[0379]

[0380]

[0381]

[0382] The following shows an exemplary anti - GPC3 - CD89 CFP sequence:

[0383] The exemplary anti - GPC3 - CD89 has the following amino acid sequence: MWLQSLLLLGTVACSISQVQLVQSGAEVKKPGASVKVSCKASGYTFT DYEMH WVRQAPGQGLEWMG ALDPKTGDTAYSQKFKG KATLTADKSTSTAYMELSSLTSEDTAVYYCTR FYSYTY WGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISC RSSQSLVHSNRNTY LH WYLQKPGQSPQLLIY KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQNTHVPPT FGQGTKLEIKGSGGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK(SEQ ID NO:151). (The corresponding CDR 1, 2, and 3 of VH and VL are underlined in sequence, Kabat numbering system). Table 6B shows each domain.

[0384] Exemplary anti-GPC3-CD89 has the following amino acid sequence: MWLQSLLLLGTVACSISQVQLVQSGAEVKKPGASVKVSCKASGYTFT DYEMH WVRQAPGQGLEWMG ALDPKTGDTAYSQKFKG RVTLTADKSTSTAYMELSSLTSEDTAVYYCTR FYSYTY WGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISC RSSQSLVHSNRNTY LH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQNTHVPPT FGQGTKLEIKGSGGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK(SEQ ID NO:152). (The corresponding CDR 1, 2, and 3 of VH and VL are underlined in sequence, Kabat numbering system). Table 6B shows each domain.

[0385] Table 6B - First and second generation sequences of exemplary GPC3 conjugates

[0386]

[0387]

[0388] Example 2. Functional assays for testing CFP constructs

[0389] Test the functional properties of the targeting constructs. Transfect THP-1 cells or CD14+ / CD16- monocytes isolated from apheresis samples with polynucleotide constructs encoding the corresponding CFP.

[0390] Methods for cell transfection and detection of transfection efficiency: Harvest THP-1 cells and wash them once with MaxCyte electroporation buffer. Resuspend the cells at a density of 10 million cells / ml and add them to 100 μg of ATAK-receptor RNA in an Eppendorf tube. Mix twice and load into a MaxCyte processing assembly (OC-25x3). Electroporate the cells using the THP-1 program on MaxCyte. After electroporation, incubate the cells at 37 °C for 10 min to recover in the processing assembly, then transfer them to a plate containing pre-warmed medium at a density of 500,000 cells / ml.

[0391] After overnight incubation, evaluate the expression of ATAK-receptor in electroporated monocytes by flow cytometry. Use an anti-Fab-Alexa Fluor-647 antibody (1:50 dilution) to detect the expression of the scFv of the ATAK receptor. Obtain the stained samples on a Cytek Northernlights flow cytometer and calculate the percentage of conjugate-positive cells based on the increase in anti-Fab intensity compared to a mock-transfected control.

[0392] Methods for phagocytosis assay: Label target tumor cells (SKOV3) with pHrodo-Red dye (final concentration of 700 ng / ml) according to the Sartorius Incucyte pHrodo-Red labeling kit protocol. After labeling, resuspend the SKOV3 cells at a density of 500,000 cells / ml using medium. Isolate CD14-positive monocytes from donor leukopak and electroporate them with 100 μg / ml of ATAK-receptor RNA using MaxCyte. After electroporation, recover the cells overnight at a density of 2 million cells / mL in 37 °C medium. The next day, count the cells using an NC-200 and resuspend the cells at a density of 2.5 million cells / ml using medium. In a low-adhesion U-bottom 96-well plate, add 50 μL of tumor cells (a total of 50,000 cells) at an E:T ratio of 5:1 to 50 μL of ATAK-receptor-transfected monocytes (a total of 125,000 cells). Mix the cells and incubate overnight at 37 °C.

[0393] On the second day, the cells were stained with CD45-Alexa Fluor 700 that specifically labels monocytes. Then samples were obtained on a Cytek Northern lights to detect the pHrodo-Red and CD45 signal intensities. Phagocytosis was measured as the percentage of the phagocytosis index and the specific increase in pHrodo-Red intensity in monocytes. The phagocytosis index was calculated as the percentage of monocytes with a high pHrodo-red signal normalized to the total number of monocytes. The phagocytic activity of ATAK-monocytes was compared with that of mock-transfected controls to determine the efficacy of the ATAK-receptor.

[0394] Phagocytosis can be tested using labeled tumor cells.

[0395] Method for SKOV3 cell killing assay: SKOV3-luciferase cells were used to test the tumor-killing activity of ATAK receptor-transfected monocytes. CD14-positive monocytes were isolated from donor leukopak and electroporated with 100 μg / ml of ATAK-receptor RNA using MaxCyte. After electroporation, the cells were recovered in medium at 37 °C for 2 hours. After recovery, the cells were resuspended in medium at a density of 2.5 million cells / ml. SKOV3-luciferase cells were harvested and resuspended at a density of 250,000 cells / mL, and 100 μl of the cell suspension (a total of 25,000 cells per well) was added to a 96-well flat-bottom plate. 100 μL of ATAK-receptor-transfected monocytes were added to the same wells at E:T ratios of 1:1 and 10:1. The cells were mixed and incubated at 37 °C for 3 days.

[0396] On day 3, the supernatant was collected and frozen to measure the cytokines and chemokines secreted by ATAK-monocytes. The cells were lysed and the SKOV3 luciferase level was measured using a luminometer. A decrease in the luciferase level of the sample containing ATAK-monocytes compared to the mock-transfected control indicated the SKOV3 killing activity of ATAK cells.

[0397] The polarization potential of myeloid cells was tested using the following method. These effector myeloid cells were electroporated with a polynucleic acid construct and frozen for subsequent use and testing. Upon thawing, the cells were cultured in polarization stimuli, such as in separate aliquots, with (i) GMCSF, (ii) IL4, IL10, and TGFβ (M2 stimulation), (iii) activated T cell conditioned medium (TCM), and (iv) MCSF. The cells were analyzed by flow cytometry at 24, 48, and 72 hours and cytokine analysis was performed by Luminex.

[0398] Method for detecting the activation of NF-κB and IFN pathways using THP1-Dual cells: THP1-Dual cells have an NF-κB response element upstream of secreted alkaline phosphatase and an IFN-stimulated response element upstream of secreted luciferase. Measuring the level of alkaline phosphatase in the supernatant indicates the activation of the NF-κB signaling pathway, while the level of luciferase in the supernatant indicates the activation of the IFN signaling pathway. Electroporate THP1-Dual cells with 100 ug / ml of ATAK-receptor RNA using MaxCyte. After electroporation, recover the cells in medium at 37 °C for 2 hours. After recovery, resuspend the cells in medium at a density of 5 million cells / ml. Harvest SKOV3 cells and resuspend them at a density of 5 million cells / mL. In a 96-well plate, add 100 ul of the SKOV3 cell suspension (a total of 50,000 cells per well) and 100 uL of the ATAK-THP1-Dual cells (a total of 50,000 cells) to a 96-well flat-bottom plate at a 1:1 E:T ratio. Mix the cells and incubate them at 37 °C for 24 hours. After 24 hours, centrifuge the cells and collect the supernatant.

[0399] To detect the activation of the NF-κB pathway, add the QUANTI-Blue solution (Invivogen) to the supernatant and incubate it at 37 °C for 2 hours, then measure the OD using an absorbance microplate reader. An increase in absorbance indicates the activation of NF-κB signaling, and the OD value of the ATAK-transfected THP1-Dual cells can be compared with the OD value of the mock-transfected control to determine the activity of the ATAK-receptor.

[0400] To detect the activation of the IFN pathway, add the QUANTI-Luc solution (Invivogen) to the supernatant and measure the luciferase level using a luminometer. An increase in the luciferase level means the activation of IFN signaling, and the ATAK-transfected THP1-Dual cells can be compared with the mock-transfected control.

[0401] Figure 2 The expression of TROP2 conjugates in the monocyte cell line is exemplified. Figure 3 Data indicating the successful expression of each construct are shown. The results show that greater than at least 50% of the cells express each construct. The results also show that the addition of multiple intracellular signaling domains is well tolerated and the constructs are well expressed.

[0402] In addition, these second-generation in vivo receptors showed high levels of pro-inflammatory cytokine and chemokine production. Comparison of the first-generation constructs for cytokine production (with CD89 TMD and lacking multiple intracellular signaling domains) with second-generation constructs (i.e., TROP2-CD89-FcR, TROP2-CD89-FcR-PI3K, TROP2-CD89-TRIF, TROP2-CD89-CD40, TROP2-CD89-CD40-FcR) is shown in Figure 4A and 4B . Compared to a representative first-generation construct, the second-generation constructs showed higher production of IL-12p70 and IFNβ ( Figure 4A ). Additionally, similar results were found for IP-10, TNFα, and IL-6 production ( Figure 4B ). These results indicate that the second-generation anti-TROP2 constructs are successfully expressed in monocytes, are highly functional, and release pro-inflammatory cytokines; this activates the monocytes expressing the constructs and renders them pro-inflammatory. Thus, these cells are activated phagocytes against TROP2-expressing cancer cells.

[0403] Example 3. Study in Humans

[0404] This example describes a Phase I, open-label, first-in-human, multi-escalating dose 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 administer an anti-TROP2-Fc-α fusion receptor in the form of mRNA encoding the construct encapsulated in lipid nanoparticles.

[0405] Number of planned subjects and treatment information

[0406] Up to 20 subjects evaluable for safety and efficacy will be recruited. Adults 18 years of age 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 a multi-escalating dose study to determine the safety, tolerability, and pharmacokinetics (PK) of the second-generation anti-TROP2 chimeric fusion protein in subjects with TROP2+ liver metastases (LM); Part B will be a dose expansion to determine further safety, tolerability, and PK as well as preliminary efficacy in patients with TROP2-positive (TROP2+) colorectal liver metastases (CRLM).

[0407] Administer an effective amount of a formulation of mRNA encoding an anti-TROP2 second-generation chimeric fusion protein in lipid nanoparticles intravenously to the subject over 60 minutes. After completion of PK and safety studies in non-human primates, the starting dose and dosing regimen will be determined.

[0408] Baseline evaluations within 4 weeks of planned treatment initiation include patient history, physical examination with vital signs and performance status, CT or MRI scans, CBC differential and platelet counts, routine serum chemistries, urinalysis, INR / PTT, EKG, and serum samples for human anti-human antibodies (HAHA). For women of childbearing potential, urine or serum β-HCG testing is also required within 1 week of treatment. TROP2 expression will be confirmed by immunohistochemistry on archived biopsy specimens obtained within the past 6 months or fresh biopsy specimens during screening at the central laboratory.

[0409] All subjects will receive infusions of the anti-TROP2 second-generation chimeric fusion protein weekly for the first 12 weeks. In the absence of disease progression or unacceptable toxicity, dosing may continue up to week 48. After week 12, patients may continue in the study until week 48, but the dosing regimen will be modified to once every two weeks. If a patient experiences progression during once-every-two-week dosing, dosing may be escalated to once weekly. If a complete responder discontinues treatment at any time during the study and disease progression occurs, dosing may be restarted at the dose and dosing regimen at the time of discontinuation. If a patient has a partial response (PR) and CRLM is surgically resectable, the patient may discontinue treatment and elect tumor resection. Patients who undergo surgical resection will be followed up to week 48. If a patient experiences disease progression, dosing may be restarted at the dose and dosing regimen at the time of discontinuation.

[0410] All patients should be closely monitored during treatment. All adverse events will be graded using the National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE), version 5.0, and guidelines for dose reduction, delay, or discontinuation will be provided in the event of treatment-related toxicity. All patients will also undergo CT / MRI scans throughout the study to assess disease progression and response to the study drug.

[0411] The occurrence of any grade 2 or 3 treatment-related toxicity at the time of planned treatment will delay treatment for 1 week. If the toxicity has resolved to grade 1 or lower at that time, treatment may continue at a 25% dose reduction. If the toxicity reappears, treatment may continue at a 50% reduction from the initial dose, and if the toxicity worsens, treatment will be permanently discontinued. The decision to continue or discontinue treatment depends solely on the judgment of the physician. If any grade 4 treatment-related toxicity occurs, the patient should be permanently withdrawn from the drug.

[0412] In part A of the study, the Continuous Reassessment Method (CRM) will be used to determine the recommended dose for part B. The initial assessment of safety and tolerance will be conducted on day 28. The initial assessment of efficacy will be conducted at 12 weeks. If the subject has stable disease (SD), partial response (PR), or complete response (CR), they may be enrolled in the long-term extension study at 48 weeks.

[0413] In part B, the safety, tolerance, and efficacy of the maximum acceptable dose (MAD) from part A will be evaluated in patients with CRLM. The initial assessment of efficacy will be the objective response rate (ORR) at 12 weeks.

[0414] If treatment is terminated due to unacceptable toxicity, the patient will continue in the study until disease progression occurs, at which time end-of-study evaluations will be conducted and additional follow-up will be required until any treatment-related toxicity has resolved or stabilized. All patients will be followed for survival.

[0415] To ensure the safety of study subjects, the first two subjects in each dose cohort will be staggered by 14 days. If there are no safety issues, the remaining subjects may enter at the same dose level simultaneously.

[0416] Exemplary anti-TROP2 second-generation chimeric fusion proteins (e.g., drug products) containing anti-TROP2 scFv have the following sequences:

[0417]

[0418] Bold letters indicate the signal peptide sequence. The mature protein lacks this signal sequence, and those skilled in the art can interpret the protein sequence as being expressed without the signal peptide sequence, as disclosed below:

[0419]

[0420] The underlined regions are the CDR sequences of the scFV that bind TROP2, in order being the CDR1, CDR2, and CDR3 of the heavy and light chains. Italic letters indicate the amino acid sequence of the CD89 TMD.

[0421] The anti-TROP2 conjugate is designed as an mRNA-lipid nanoparticle (LNP) formulation for systemic intravenous delivery. This approach takes advantage of the natural tendency of LNPs administered intravenously (i.v.) to accumulate in the liver. The lipid component of the anti-TROP2 conjugate also protects the mRNA encoding the CAR from degradation by plasma and tissue nucleases. The proprietary mRNA construct encodes a receptor consisting of a TROP2-targeting scFv (complementary determining regions (CDRs) derived from sacituzumab) and the transmembrane domain and cytoplasmic tail region of CD89. After administration of the anti-TROP2 conjugate, the LNPs are taken up by multiple cell types; however, functional CAR can only be expressed on the surface of cells that also express the Fc receptor common gamma chain (primarily myeloid cells). The common gamma chain contains an immunoreceptor tyrosine-based activation motif (ITAM) domain that is essential for cellular signaling. After the scFV recognizes TROP2, the intracellular signaling domain is activated, resulting in tumor cell phagocytosis, production of inflammatory cytokines, and presentation of tumor antigens to T cells.

[0422] Study objectives

[0423] The primary objective of this study was to evaluate the safety and tolerability of the anti-TROP2 second-generation chimeric fusion protein in subjects with TROP2+ metastatic colorectal cancer and to determine the maximum acceptable dose (MAD) and recommended phase 2 dose (RP2D) based on the observed adverse events (AEs), including all possible dose-limiting toxicities (DLTs).

[0424] The secondary objectives of this study were to determine (i) the pharmacokinetics, (ii) the objective response (time frame up to 12 months) of the anti-TROP2 second-generation chimeric fusion protein, and (iii) the duration of response.

[0425] The secondary objectives of this study also included the evaluation of the area under the curve (up to 12 months), maximum plasma concentration (up to 12 months), time to reach maximum plasma concentration (up to 12 months), half-life (up to 12 months), objective response (up to 12 months) of the anti-TROP2 second-generation chimeric fusion protein, and the duration of response.

[0426] The exploratory objectives of this study are to determine preliminary efficacy, including progression-free survival (PFS) and overall survival (OS), conversion to surgical candidates, and related biomarker studies. Additionally, exploratory objectives may generally include developing anti-drug antibodies (ADA) to the anti-TROP2 second-generation chimeric fusion protein, assessing TROP2 expression levels in tumors, identifying potential biomarkers of response, and the treatment-related effects on cytokine and chemokine production, TCR amplification, cellular phenotypes in the blood, tumor architecture, and tumor cell phenotypes of potential biomarkers for identifying response.

[0427] The overall study design includes a multicenter, open-label, phase 1 first-in-human study with dose cohort expansion to evaluate the safety, tolerability, pharmacokinetics (PK), and efficacy of the anti-TROP2 second-generation conjugate in subjects with TROP2+ colorectal liver metastases. Adults 18 years of age 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 a multiple ascending dose to determine the safety, tolerability, and PK of the anti-TROP2 second-generation chimeric fusion protein in subjects with TROP2+ liver metastases (LM); Part B will be dose expansion to determine further safety, tolerability, and PK as well as preliminary efficacy in patients with TROP2+ colorectal liver metastases (CRLM).

[0428] Inclusion criteria

[0429] Subjects are eligible to enter the ascending dose part (Part A) of the study if they are diagnosed with histologically confirmed metastatic TROP2+ LM, have progressive disease at baseline, are refractory to standard of care, or have refused standard treatment.

[0430] Subjects are eligible to enter the cohort expansion part (Part B) of the study if they are diagnosed with histologically confirmed metastatic TROP2+ CRLM, have progressive disease at baseline, are refractory to standard of care, or have refused standard treatment. Additional criteria include the presence of a TROP2+ tumor with a score of 2+ or 3+ as determined by immunohistochemistry (IHC) performed at a central laboratory and measurable disease based on Response Evaluation Criteria in Solid Tumors (RECIST) v1.1.

[0431] Exclusion criteria

[0432] An object will be excluded from the study if it meets any of the following criteria: (1) having known active CNS metastases and / or carcinomatous meningitis, (2) previous allogeneic bone marrow transplantation or solid organ transplantation, (3) active autoimmune disease, (4) active acute or chronic infection, (5) hepatic tumor involvement greater than 50%, (6) previous splenectomy (patients who have undergone previous partial splenic artery embolization may be eligible at the discretion of the investigator).

[0433] Treatment duration

[0434] There are a total of 3 cycles. Each cycle involves once-weekly dosing for 4 weeks. After 3 cycles, in the absence of disease progression, at the discretion of the investigator, patients may continue to participate in the study until week 48, but the dosing schedule will be modified to once every two weeks. If a patient experiences disease progression, the patient may escalate to once-weekly dosing. If a patient progresses during once-weekly dosing, treatment will be discontinued.

[0435] Definition of dose-limiting toxicity

[0436] The Common Terminology Criteria for Adverse Events (CTCAE) v5.0 is used to define dose-limiting toxicity (DLT). All toxicities will be considered at least "possibly" related to the anti-TROP2 second-generation chimeric fusion protein, unless they are not temporally related to the administration of the anti-TROP2 second-generation chimeric fusion protein but are related to other etiologies (such as concomitant medications or medical conditions, or the subject's underlying disease).

[0437] For this study, unless otherwise stated, if any of the following occur within 28 days (day 28) of the first dose, it will be considered DLT: (1) death, (2) any CTCAE grade 4 toxicity, (3) CTCAE grade 3 toxicity in vital organs (central nervous system [CNS], heart, and lungs), (4) CTCAE grade 3 toxicity that does not increase to ≤ grade 2 within 72 hours under maximum supportive care (e.g., nausea, vomiting, diarrhea), excluding renal and hepatic laboratory abnormalities, (5) CTCAE grade 3 toxicity that does not decrease to ≤ grade 2 within 7 days (for renal and hepatic laboratory abnormalities), and (6) any grade 3 infusion-related reaction lasting > 24 hours, and the patient receives premedication with a histamine H1 receptor antagonist, a histamine H2 receptor antagonist, and a corticosteroid. Exceptions to the grade 3 or 4 DLT criteria include laboratory values that the investigator deems not clinically significant.

[0438] Study stopping rules

[0439] At any dose level, if a subject experiences a DLT as listed above, further dosing and enrollment of the subject will be temporarily halted, and the Steering Committee will be immediately notified and a meeting will be convened to review the safety data. After reviewing the safety data, the Steering Committee will recommend, based on the frequency of DLTs, whether to (1) continue the study as planned, including administering additional doses, (2) expand the dose cohort to obtain additional safety information, (3) reduce the dose to a previous lower or intermediate dose, (4) discontinue administering additional doses, and / or (5) terminate the study and follow all subjects for safety.

[0440] Statistical considerations

[0441] Dose escalation will be determined by the Continuous Replacement Model.

[0442] Endpoints / evaluation criteria

[0443] Endpoints and evaluation criteria regarding safety include adverse events (AEs), IRs, serious AEs (SAEs) (clinically significant abnormal physical examination findings, and laboratory events will be reported as AEs), vital signs, serum chemistry, hematology, T-cell counts, and plasma cytokines.

[0444] Endpoints and evaluation criteria regarding the pharmacokinetics of the anti-TROP2 conjugate include the pharmacokinetic parameters of the drug / product in the blood after each infusion (e.g., maximum plasma concentration (C max ), time to reach maximum plasma concentration (T max ), half-life (T 1 / 2 )) as quantified by qPCR.

[0445] Endpoints and evaluation criteria regarding anti-drug antibodies (ADAs) include measuring the proportion of subjects in whom ADAs occur, as measured by ELISA.

[0446] Endpoints and evaluation criteria regarding clinical response include measuring Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 and transition to surgical candidates.

[0447] Endpoints and evaluation criteria regarding relevant markers of response include tumor infiltration of cells as determined by IHC Image Mass Cytometry, recruitment and trafficking of other immune cells as determined by mass cytometry imaging, and upregulation of cytokine and chemokine production as determined by transcriptional analysis. The event schedule is presented in the table below.

[0448] Table 7. Schedule of events

[0449]

[0450]

Claims

1. A composition comprising a recombinant polynucleotide and a polynucleotide delivery vehicle, wherein the recombinant polynucleotide comprises a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (i) an extracellular domain comprising an antigen-binding domain, (ii) a transmembrane domain operably linked to the extracellular domain of the CFP and multimerized with an Fc receptor protein endogenously expressed in a cell; and (iii) an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains are selected from the intracellular signaling domain from Fc epsilon receptor Ig (FCER1G), a PI3K recruitment domain, the intracellular signaling domain from CD40, and the intracellular signaling domain from TRIF, wherein the antigen-binding domain comprises an antibody or a fragment thereof having a heavy chain variable domain (VH), the VH comprising a heavy chain CDR3 (HCDR3) sequence selected from GGFGSSYWYFDV and FYSYTY.

2. The composition according to claim 1, wherein the antigen-binding domain comprises a GPC3-binding domain or a TROP2-binding domain.

3. The composition according to claim 1 or 2, wherein the antigen-binding domain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), the VH comprising HCDR1, HCDR2, and HCDR3, the VL comprising light chain CDR1 (LCDR1), LCDR2, and LCDR3; wherein the HCDR1, HCDR2, and HCDR3 comprise the HCDR1, HCDR2, and HCDR3 sequences listed in Table 1A; and the LCDR1, LCDR2, and LCDR3 comprise the LCDR1, LCDR2, and LCDR3 sequences listed in Table 1A.

4. The composition according to claim 3, wherein the antigen-binding domain has a VH having at least 80% sequence identity with the VH of Table 1A and a VL having at least 80% sequence identity with the VL of Table 1A.

5. The composition according to any one of claims 1-4, wherein the CFP further comprises a signal peptide.

6. The composition according to claim 5, wherein the signal peptide comprises the sequence MWLQSLLLLGTVACSIS (SEQ ID NO:7).

7. The composition according to any one of claims 1-6, wherein the extracellular domain comprises a sequence having at least 90% sequence identity with the extracellular domain sequence of Table 1C.

8. The composition according to any one of claims 1-7, wherein the CFP comprises a CD89 transmembrane domain, the CD89 transmembrane domain comprising a sequence having at least 90% sequence identity with SEQ ID NO:

11.

9. The composition according to any one of claims 1-8, wherein the intracellular signaling domain of the at least two intracellular signaling domains comprises a sequence having at least 80% sequence identity with the sequence of Table 2.

10. The composition according to claim 1, wherein the CFP comprises an intracellular domain, and the intracellular domain comprises a sequence having at least 80% sequence identity with the sequence of Table 3.

11. The composition according to claim 1, wherein the CFP comprises a sequence having at least 80% sequence identity with any one of the sequences of Table 4.

12. A composition comprising a recombinant polynucleotide, wherein the recombinant polynucleotide comprises a sequence encoding a chimeric fusion protein (CFP), and the CFP has a sequence having at least 95% sequence identity with SEQ ID NO: 36 or 152, a sequence having at least 95% sequence identity with SEQ ID NO: 26 or 143, or a sequence having at least 95% sequence identity with the sequences of Table 4.

13. The composition according to claim 12, wherein the CFP has a sequence having at least 98%, 99% or 100% sequence identity with SEQ ID NO: 36 or 152, a sequence having at least 98%, 99% or 100% sequence identity with SEQ ID NO: 26 or 143, or a sequence having at least 98%, 99% or 100% sequence identity with the sequences of Table 4.

14. The composition according to any one of claims 1-13, wherein the intracellular domain comprises at least one additional intracellular signaling domain.

15. The composition according to any one of claims 1-14, wherein the antigen-binding domain comprises an antibody or a fragment thereof.

16. The composition according to any one of claims 1-15, wherein the antigen-binding domain comprises a scFv.

17. The composition according to any one of claims 1-16, wherein the antigen-binding domain comprises an extracellular hinge domain connecting the antigen-binding domain and the transmembrane domain.

18. The composition according to any one of claims 1-17, wherein the recombinant polynucleotide is mRNA.

19. The composition according to claims 1-18, wherein the recombinant polynucleotide is associated with one or more lipids.

20. The composition according to any one of the foregoing claims, wherein the recombinant polynucleotide is encapsulated in a liposome.

21. The composition according to claim 20, wherein the liposome is a lipid nanoparticle.

22. The composition according to any one of claims 12-21, wherein the recombinant polynucleotide comprises (i) a sequence having at least 80% sequence identity with SEQ ID NO: 141 or a portion of SEQ ID NO: 141 without the 5' UTR and / or 3' UTR, or (ii) a sequence having at least 80% sequence identity with SEQ ID NO: 142 or a portion of SEQ ID NO: 142 without the 5' UTR and / or 3' UTR.

23. A composition comprising a nanoparticle delivery vehicle and a recombinant polynucleotide according to any one of claims 1-22, wherein the recombinant polynucleotide is associated with or within the nanoparticle delivery vehicle.

24. The composition according to any one of claims 1-23, wherein the transmembrane domain is a transmembrane domain from a protein that dimerizes with the endogenous FcR-γ receptor in myeloid cells.

25. The composition according to any one of claims 1-24, wherein the transmembrane domain comprises a transmembrane domain from CD16a, CD64, CD68 or CD89.

26. The composition according to any one of claims 1-25, wherein the recombinant polynucleotide is associated with or within the delivery vehicle, wherein the delivery vehicle comprises a lipid nanoparticle.

27. The composition according to any one of claims 1-26, wherein the recombinant polynucleotide is mRNA and the lipid nanoparticle encapsulates the mRNA.

28. The composition according to any one of claims 1-27, wherein the lipid nanoparticle comprises polar lipids and nonpolar lipids.

29. The composition according to any one of claims 1-28, wherein the lipid nanoparticle has a diameter of 100 to 300 nm.

30. A composition comprising a cell comprising the recombinant polynucleotide of the composition according to any one of claims 1-29.

31. The composition according to claim 30, wherein the cell is an immune cell.

32. The composition according to claim 30, wherein the cell is a myeloid cell, a lymphoid cell, a progenitor cell, a stem cell or an induced pluripotent cell.

33. The composition according to claim 30, wherein the cell is CD14+ / CD16-.

34. A pharmaceutical composition comprising the composition according to any one of claims 1-33; and a pharmaceutically acceptable excipient.

35. A method of treating cancer in a subject, comprising: Administering to the subject the pharmaceutical composition according to claim 34.

36. The method according to claim 35, wherein the cancer is a cancer expressing TROP2.

37. The method according to claim 35, wherein the cancer is a cancer expressing GPC-3.

38. A composition comprising: (i) a lipid delivery vehicle; and (ii) a recombinant polynucleotide comprising a sequence encoding a chimeric fusion protein (CFP), wherein the CFP comprises (a) an extracellular domain comprising an antigen-binding domain; (ii) a transmembrane domain and (iii) an intracellular domain, wherein the antigen-binding domain comprises a scFv or VHH that binds a cancer antigen; and the transmembrane domain comprises the amino acid sequence of SEQ ID NO:11; and wherein when contacted with a cell, the recombinant polynucleotide is expressed substantially in myeloid cells.

39. The composition according to claim 38, wherein the recombinant polynucleotide is not expressed substantially in T cells.

40. The composition according to claim 38, wherein the extracellular antigen-binding domain comprises a CDR3 sequence of any one of the CDR3 sequences shown in column 4 of Table 4.

41. The composition according to claim 38, wherein the extracellular antigen-binding domain comprises a VHH domain, and the VHH domain comprises the CDR3 sequences of Table 5.

42. The composition according to claim 41, wherein the VHH domain comprises the CDR1, CDR2, and CDR3 sequences shown in any single row of Table 5.

43. The composition according to claim 38, wherein the CFP comprises an extracellular antigen-binding domain that binds to GPC3 and comprises a sequence having at least 80% amino acid sequence identity with the sequences shown in Table 5 and Table 6B.

44. The composition according to claim 38, wherein the CFP comprises an extracellular antigen-binding domain that binds to GPC3 and comprises a sequence having at least 80% identity with a sequence selected from SEQ ID NO: 34 - 42, 135, 136, 137, 151, and 152.

45. The composition according to any one of claims 38 - 44, wherein the recombinant polynucleotide is mRNA.

46. The composition according to any one of claims 38 - 45, wherein the recombinant polynucleotide is codon-optimized.

47. The composition according to any one of claims 38 - 45, wherein the recombinant polynucleotide is mRNA and comprises 5' and 3' UTRs, as well as a 5'-cap and a 3'-polyA tail.

48. The composition according to any one of claims 38 - 45, wherein the recombinant polynucleotide is a fully human polynucleotide.

49. The composition according to any one of claims 38 - 45, wherein the extracellular antigen-binding domain encoded by the recombinant polynucleotide is at least a human sequence between a murine sequence and a human sequence.

50. The composition according to claim 38, wherein the CFP comprises an extracellular antigen-binding domain that binds to TROP2 and comprises a sequence having at least 80% identity with the sequences shown in Table 6A.

51. A method of expressing a chimera in in vivo myeloid cells, the method comprising (i) preparing a therapeutically acceptable aqueous formulation of a composition, the composition comprising a composition according to any one of the compositions of claims 1-30 and 38-49; (ii) Systemically administering the therapeutically acceptable aqueous formulation to a subject; wherein the expression of the CFP is detected in myeloid cells in the tissue sample after separating and testing a suitable tissue sample from the subject at a suitable time after administration.

52. Use of the composition according to any one of claims 1 - 30, 38 - 50 for the preparation of a medicament for treating cancer in a subject.

53. The use according to claim 52, wherein the cancer is selected from lung cancer, liver cancer, prostate cancer, lymphoma, or hepatocellular carcinoma, non-small cell lung cancer (NSCLC), breast cancer, cervical cancer, colon cancer, gastric cancer, bladder cancer, thyroid cancer, osteosarcoma, squamous cell carcinoma of the lung, ovarian yolk sac tumor, melanoma, and urothelial carcinoma.

54. A composition comprising a recombinant polynucleotide, wherein: (a) The recombinant polynucleotide is RNA; (b) The recombinant polynucleotide is associated with or within a lipid nanoparticle; (c) The recombinant polynucleotide comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP is an anti-TROP2 CFP and has a sequence with at least 95% sequence identity to SEQ ID NO: 36 or 152, a sequence with at least 95% sequence identity to SEQ ID NO: 26 or 143; and (d) The recombinant polynucleotide comprises (i) a sequence with at least 80% sequence identity to SEQ ID NO: 141 or a portion of SEQ ID NO: 141 without the 5' UTR and / or 3' UTR.

55. A pharmaceutical composition comprising the composition according to claim 54.

56. A method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition according to claim 55.

57. The method according to claim 56, wherein the cancer is lung cancer, liver cancer, prostate cancer, lymphoma or hepatocellular carcinoma, non-small cell lung cancer (NSCLC), breast cancer, cervical cancer, colon cancer, gastric cancer, bladder cancer, thyroid cancer, osteosarcoma, squamous cell carcinoma of the lung, ovarian yolk sac tumor, melanoma and urothelial carcinoma.

58. A composition comprising a recombinant polynucleotide, wherein: (a) The recombinant polynucleotide is RNA; (b) The recombinant polynucleotide is associated with or within a lipid nanoparticle; (c) The recombinant polynucleotide comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP is an anti-GPC3 CFP and has a sequence with at least 95% sequence identity to SEQ ID NO: 36 or 152; and (d) The recombinant polynucleotide comprises a sequence with at least 80% sequence identity to SEQ ID NO: 142 or a portion of SEQ ID NO: 142 without the 5' UTR and / or 3' UTR.

59. A pharmaceutical composition comprising the composition according to claim 58.

60. A method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition according to claim 59.

61. The method according to claim 60, wherein the cancer is lung cancer, liver cancer, prostate cancer, lymphoma or hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), breast cancer, cervical cancer, colon cancer, gastric cancer, bladder cancer, thyroid cancer, osteosarcoma, squamous cell carcinoma of the lung, ovarian yolk sac tumor, melanoma and urothelial carcinoma.

Images