Engineered chimeric fusion protein compositions and methods of use thereof

Abstract

Compositions and methods for making and using engineered cells, such as engineered bone marrow cells, that express a chimeric fusion protein having a binding domain capable of binding to a surface molecule on a target cell, such as a diseased cell.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 409,193, filed September 22, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002]

[0002] Cellular immunotherapy is a promising new technology for combating difficult-to-treat diseases, such as cancer and persistent infections, as well as certain diseases that are refractory to other forms of treatment. The discovery of CAR-T cells and their potential use in immunotherapy has resulted in a major breakthrough. CAR-T cells are T lymphocytes that express chimeric antigen receptors (CARs), which help target T cells to specific diseased cells, such as cancer cells. Depending on the intracellular domain employed and the immunosuppressive cytokines coexpressed, CAR-T cells can either induce a cytotoxic response designed to kill the targeted cancer cells or induce immunosuppression and / or immune tolerance. While CAR-T cells remain a promising tool for cancer treatment, several limitations along the way have slowed their progress and limited their promise in clinical trials.

[0003] Understanding the limitations of CAR-T cells is key to harnessing the technology and sustaining innovation toward successful immunotherapy models. Specifically, CAR-T cells face significant challenges in T-cell malignancies. Because CAR-T cells and malignant T cells share surface antigens in most T-cell lymphomas (TCLs), CAR-T cells, like cancer cells, are subject to cytotoxic effects. In some cases, CAR-T products may be contaminated by malignant T cells. In addition, T-cell aplasia is a potential problem for prolonged CAR-T cell survival. Other limitations include the poor ability of CAR-T cells to penetrate solid tumors and the potent tumor microenvironment, which acts to downregulate their antitumor potential. CAR-T cell function is also negatively affected by the immunosuppressive tumor microenvironment (TME), which leads to the inactivation and exhaustion of endogenous T cells.

[0004]

[0004] Myeloid cells, including macrophages, are derived from the myeloid lineage and belong to the innate immune system. They originate from bone marrow stem cells, which escape into the blood and migrate to tissues. Some of their primary functions include phagocytosis, activation of T cell responses, and removal of cellular debris and extracellular matrix. Myeloid cells also play important roles in maintaining homeostasis and in the initiation and resolution of inflammation. Furthermore, myeloid cells can differentiate into numerous downstream cells, including macrophages, which can display distinct responses ranging from pro-inflammatory to anti-inflammatory, depending on the type of stimuli they receive from the surrounding microenvironment. Furthermore, tissue macrophages have been shown to play a broad regulatory and activating role on other immune cell types, including CD8+ and CD4+ T effector cells, NK cells, and regulatory T cells. Macrophages have been shown to be the primary immune infiltrate within malignant tumors and to exert a broad immunosuppressive effect on effector immune infiltration and immune function. Summary of the Invention

[0005] The diverse functionalities of bone marrow cells make them ideal candidates for cellular therapy, which can be engineered to exert numerous therapeutic effects. The present disclosure relates to immunotherapy using bone marrow cells of the immune system (e.g., CD14+ cells), particularly phagocytes. Numerous therapeutic applications using bone marrow cells may be envisioned. For example, immunotherapy with bone marrow cells may be crucial in cancer, autoimmune, fibrotic diseases, and infectious diseases. The present disclosure relates to immunotherapy using phagocytes of the immune system, particularly bone marrow cells, including monocytes. Harnessing one or more of these functions of bone marrow cells for therapeutic use is an object of the present invention. For example, harnessing the phagocytic activity of bone marrow cells, including engineered bone marrow cells and bone marrow cells modified in vivo to express chimeric fusion proteins, for therapeutic use is an object of the present invention. For example, harnessing the ability of bone marrow cells, including engineered bone marrow cells and bone marrow cells modified in vivo to express chimeric fusion proteins, to promote T cell activation is an object of the present invention. It is an object of the invention disclosed herein to utilize the ability of bone marrow cells, including, for example, engineered bone marrow cells and bone marrow cells modified in vivo to express chimeric fusion proteins, to promote the secretion of tumor-killing molecules. It is an object of the invention disclosed herein to utilize the ability of bone marrow cells, including, for example, engineered bone marrow cells and bone marrow cells modified in vivo to express chimeric fusion proteins, to promote the recruitment and trafficking of immune cells and immune molecules. In one aspect, the present disclosure provides new and useful chimeric constructs that, when expressed in bone marrow cells, can drive the targeted attack and phagocytosis by the bone marrow cells of a molecule, molecular assembly, object, or target, e.g., a cell containing a target antigen, such as a target antigen on the surface of a target cell. One of the many aspects of the present disclosure is (i) to enhance the phagocytic ability of bone marrow cells (e.g., engineered bone marrow cells expressing new and improved chimeric constructs) and help to elicit an organized and sustained immune response against a target (e.g., a target antigen).The present disclosure provides novel methods and compositions for expressing chimeric fusion proteins in bone marrow cells, such as bone marrow cells, in vivo in subjects with diseases such as cancer. One strategy for the improvement described herein is to induce the inflammatory phenotype of bone marrow cells to generate effector bone marrow cells. One strategy is to generate effector bone marrow cells that can take on an inflammatory phenotype upon engagement with a target.

[0006]

[0006] Provided herein is a composition comprising a recombinant polynucleic acid, the recombinant polynucleic acid comprising 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, and (iii) an intracellular domain comprising at least two intracellular signaling domains, the at least two intracellular signaling domains being selected from the group consisting of an intracellular signaling domain derived from Fc epsilon receptor Ig (FCER1G), a PI3K recruitment domain, an intracellular signaling domain derived from CD40, and an intracellular signaling domain derived from TRIF.

[0007]

[0007] In some embodiments, the antigen-binding domain is a GPC3-binding domain or a TROP2-binding domain.

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

[0008]

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

[0009]

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

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

[0010]

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

[0013] In some embodiments, the transmembrane domain comprises a sequence having at least 80% sequence identity to a sequence in Table 1D.

[0011]

[0014] In some embodiments, each of the intracellular signaling domains of the at least two intracellular signaling domains has a sequence with at least 80% sequence identity to a sequence shown in Table 2.

[0012]

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

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

[0013]

[0017] Also provided herein are compositions comprising a recombinant polynucleic acid, wherein the recombinant polynucleic acid comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP has a sequence having at least 95% sequence identity to a sequence set forth in Table 4.

[0014]

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

[0019] In some embodiments, the intracellular domain comprises at least one additional intracellular signaling domain.

[0015]

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

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

[0022] In some embodiments, the extracellular domain comprising the antigen-binding domain comprises a hinge domain connecting the antigen-binding domain and the transmembrane domain.

[0016]

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

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

[0017]

[0025] In some embodiments, the recombinant polynucleic acid is encapsulated in a liposome.

[0026] In some embodiments, the liposome is a lipid nanoparticle.

[0027] In some embodiments, the recombinant polynucleic acid is a vector.

[0018]

[0028] Also provided herein are compositions comprising a nanoparticle delivery vehicle and a recombinant polynucleic acid described herein, wherein the recombinant polynucleic acid is associated with or within the nanoparticle delivery vehicle.

[0019]

[0029] In some embodiments, the transmembrane domain is a transmembrane domain derived from a protein that dimerizes with endogenous FcR-gamma receptors in myeloid cells.

[0030] In some embodiments, the transmembrane domain comprises a transmembrane domain from CD16a, CD64, CD68, or CD89.

[0020]

[0031] In some embodiments, the recombinant polynucleic acid is associated with or within a nanoparticle delivery vehicle, and the nanoparticle delivery vehicle comprises a lipid nanoparticle.

[0032] In some embodiments, the recombinant polynucleic acid is mRNA and the lipid nanoparticles encapsulate the mRNA.

[0021]

[0033] In some embodiments, the lipid nanoparticles comprise a polar lipid and a non-polar lipid.

[0034] In some embodiments, the lipid nanoparticles are 100-300 nm in diameter.

[0035] Also provided herein are compositions comprising cells containing a recombinant polynucleic acid described herein.

[0022]

[0036] In some embodiments, the cell is an immune cell.

[0037] In some embodiments, the cells are myeloid cells, lymphoid cells, progenitor cells, stem cells, or induced pluripotent cells.

[0023]

[0038] In some embodiments, the cells are CD14+CD16- cells.

[0039] Also provided herein is a pharmaceutical composition comprising a composition described herein; and a pharmaceutically acceptable excipient.

[0024]

[0040] Also provided herein is a method for treating cancer in a subject, comprising administering to the subject a pharmaceutical composition described herein.In one aspect of the present specification, a composition is provided, comprising a recombinant polynucleic acid and a polynucleotide delivery vehicle, wherein the recombinant polynucleic acid comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP comprises: (i) an extracellular domain comprising an antigen-binding domain; (ii) a transmembrane domain operably linked to the extracellular domain of the CFP, and 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 group consisting of an intracellular signaling domain derived from Fc epsilon receptor Ig (FCER1G), a PI3K recruitment domain, an intracellular signaling domain derived from CD40, and an intracellular signaling domain derived from TRIF, and the antigen-binding domain comprises an antibody or a fragment thereof having a heavy chain variable domain (VH) comprising a CDR3 sequence selected from the sequence GGFGSSYWYFDV or FYSYTY.

[0025]

[0041] 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) comprising a VH CDR1, CDR2, and CDR3, and a light chain variable domain (VL) comprising a VL CDR1, CDR2, and CDR3; the VH CDR1, CDR2, and CDR3 comprise a sequence listed in Table 1A; and the VL CDR1, CDR2, and CDR3 comprise a sequence 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 in Table 1A, and a VL having at least 80% sequence identity to the VL of the antigen-binding domain in 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 a sequence in Table 1C. In some embodiments, the CFP comprises a CD89 transmembrane domain comprising a sequence having at least 90% sequence identity to SEQ ID NO:11 (Table ID). In some embodiments, the intracellular signaling domain of the at least two intracellular signaling domains comprises a sequence having at least 80% sequence identity to a sequence in Table 2. In some embodiments, the CFP comprises an intracellular domain comprising a sequence having at least 80% sequence identity to a sequence in Table 3. In some embodiments, the CFP comprises a sequence having at least 80% sequence identity to a sequence in Table 4.

[0026]

[0042] In one aspect herein, a composition is provided comprising a recombinant polynucleic acid, wherein the recombinant polynucleic acid comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP has a sequence having at least 95% sequence identity to a sequence in Table 4. In some embodiments, the CFP has a sequence having at least 98%, 99%, or 100% sequence identity to a sequence in 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 fragment thereof. In some embodiments, the antigen-binding domain comprises an scFv. In some embodiments, the extracellular domain comprising the antigen-binding domain comprises a hinge domain connecting the antigen-binding domain and the transmembrane domain. In some embodiments, the recombinant polynucleic acid is mRNA. In some embodiments, the recombinant polynucleic acid is associated with one or more lipids. In some embodiments, the recombinant polynucleic acid is encapsulated in a liposome.

[0027]

[0043] In some embodiments, the liposome is a lipid nanoparticle. In some embodiments, the recombinant polynucleic acid is a vector.

[0044] In one aspect of the present specification, a composition is provided comprising a nanoparticle delivery vehicle and the composition of any one of the preceding embodiments, wherein a recombinant polynucleic acid is associated with or within the nanoparticle delivery vehicle. In some embodiments, the transmembrane domain is a transmembrane domain derived from a protein that dimerizes with endogenous FcR-gamma receptors in myeloid cells. In some embodiments, the transmembrane domain comprises a transmembrane domain derived from CD16a, CD64, CD68, or CD89. In some embodiments, the recombinant polynucleic acid is associated with or within the delivery vehicle, and the delivery vehicle comprises a lipid nanoparticle. In some embodiments, the recombinant polynucleic acid is mRNA, and the lipid nanoparticle encapsulates the mRNA. In some embodiments, the lipid nanoparticle comprises a polar lipid and a non-polar lipid. In some embodiments, the lipid nanoparticle is 100 to 300 nm in diameter.

[0028]

[0045] In some embodiments, a recombinant polynucleic acid comprises a sequence that (i) encodes a polypeptide having at least 80% sequence identity to SEQ ID NO: 35. In some embodiments, a recombinant polynucleic acid comprises a sequence that (i) encodes a polypeptide having at least 80% sequence identity to SEQ ID NO: 36. In some embodiments, a recombinant polynucleic acid comprises a sequence that (i) encodes a polypeptide having at least 80% sequence identity to SEQ ID NO: 151. In some embodiments, a recombinant polynucleic acid comprises a sequence that (i) encodes a polypeptide having at least 80% sequence identity to SEQ ID NO: 152. In some embodiments, a recombinant polynucleic acid comprising a sequence encoding a polypeptide having at least 80% sequence identity to SEQ ID NO: 36 or 35 or 152 or 151 is encapsulated in an LNP and formulated into a pharmaceutical composition for systemic administration to a subject having cancer, wherein the cancer is a GPC3-positive cancer; or 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, or urothelial carcinoma.

[0029]

[0046] In some embodiments, the recombinant polynucleic acid comprises a sequence that encodes a polypeptide having at least 80% sequence identity to SEQ ID NO: 143. In some embodiments, the recombinant polynucleic acid comprises a sequence that encodes a polypeptide having at least 80% sequence identity to any one of SEQ ID NOs: 144-150. In some embodiments, a recombinant polynucleic acid comprising a sequence that encodes a polypeptide having at least 80% sequence identity to any one of SEQ ID NOs: 143, 144, 145, 146, 147, 148, 149, or 150 is encapsulated in an LNP and formulated into a pharmaceutical composition for systemic administration to a subject having cancer, wherein the cancer is a TROP2-positive cancer; 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.

[0030]

[0047] In some embodiments, the recombinant polynucleic acid comprises (i) a sequence having 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, or (ii) a sequence having 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 some embodiments, the recombinant polynucleic acid is encapsulated in LNPs and formulated into a pharmaceutical composition for systemic administration to a subject with cancer.

[0031]

[0048] In one aspect of the present specification, there is provided a composition comprising a cell comprising the recombinant polynucleic acid of any one of the compositions of the preceding embodiments. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a myeloid cell, a lymphoid cell, a progenitor cell, a stem cell, or an induced pluripotent cell. In some embodiments, the cell is CD14+ / CD16-. In one aspect of the present specification, there is provided a pharmaceutical composition comprising the composition of any one of the preceding embodiments; and a pharmaceutically acceptable excipient.

[0032]

[0049] In one aspect of the present disclosure, a method for treating cancer in a subject is provided, comprising administering to the subject a pharmaceutical composition described herein. In some embodiments, the cancer is a TROP2-expressing cancer. In some embodiments, the cancer is a GPC3-expressing cancer. In some embodiments, the cancer is lymphoma, melanoma, liver cancer, lung, hepatic lymphoid or epithelial cancer. 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, or 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.

[0033]

[0050] In one aspect of the present specification, a composition is provided comprising: (i) a lipid delivery vehicle; and (ii) a recombinant polynucleic acid 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 an scFv or VHH that binds to a cancer antigen; and the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 11; and when contacted with a cell, the recombinant polynucleic acid is substantially expressed in a myeloid cell. In one embodiment, the recombinant polynucleic acid is not substantially expressed in a T cell. In one embodiment, the extracellular antigen-binding domain comprises any one of the CDR3 sequences set forth in column 4 of Table 4. In one embodiment, the extracellular antigen-binding domain comprises a VHH domain, and the VHH domain comprises a CDR3 sequence of Table 5. In one embodiment, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences set forth in any one row of Table 5. In one embodiment, the CFP comprises an extracellular antigen-binding domain that binds to GPC3 and comprises a sequence having at least 80% amino acid sequence identity to a sequence set forth in Tables 5 and 6B. In one embodiment, the CFP comprises an extracellular antigen-binding domain that binds to GPC3 and comprises a sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 34-42, 135, 136, 137, 151, and 152. In one embodiment, the recombinant polynucleic acid is 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 polynucleic acid is codon-optimized. In one embodiment, the recombinant polynucleic acid is fully human. In one embodiment, at least the extracellular antigen-binding domain encoded by the recombinant polynucleic acid is human, between mouse and human sequences. In one embodiment, the CFP comprises an extracellular antigen-binding domain that binds to TROP2 and comprises a sequence having at least 80% identity to a sequence set forth in Table 6A.

[0034]

[0051] In one aspect herein, a composition is provided comprising a recombinant polynucleic acid, wherein (a) the recombinant polynucleic acid is RNA; (b) the recombinant polynucleic acid is associated with or within a lipid nanoparticle; (b) the recombinant polynucleic acid comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP is an anti-TROP2 CFP and has a sequence having at least 95% sequence identity to SEQ ID NO: 36 or 152, a sequence having at least 95% sequence identity to SEQ ID NO: 26 or 143; and (d) the recombinant polynucleic acid comprises (i) a sequence having at least 80% sequence identity to SEQ ID NO: 141 or a portion of SEQ ID NO: 141 not including the 5' UTR and / or the 3' UTR. Also provided herein are pharmaceutical compositions comprising a recombinant polynucleic acid, wherein (a) the recombinant polynucleic acid is RNA; (b) the recombinant polynucleic acid is associated with or within a lipid nanoparticle; (b) the recombinant polynucleic acid comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP is an anti-TROP2 CFP and has a sequence having at least 95% sequence identity to SEQ ID NO: 36 or 152, a sequence having at least 95% sequence identity to SEQ ID NO: 26 or 143; and (d) the recombinant polynucleic acid comprises (i) a sequence having at least 80% sequence identity to SEQ ID NO: 141 or a portion of SEQ ID NO: 141 not including the 5' UTR and / or 3' UTR. In one aspect herein, there is provided a method of treating cancer in a subject in need thereof, the method comprising the step of administering to the subject at least a pharmaceutical composition described in this paragraph 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, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma, and urothelial carcinoma.

[0035]

[0052] In another aspect herein, there is provided a composition comprising a recombinant polynucleic acid, wherein (a) the recombinant polynucleic acid is RNA; (b) the recombinant polynucleic acid is associated with or within a lipid nanoparticle; (c) the recombinant polynucleic acid comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP is an anti-GPC3 CFP and has a sequence having at least 95% sequence identity to SEQ ID NO: 36 or 152; and (d) the recombinant polynucleic acid comprises a sequence having at least 80% sequence identity to SEQ ID NO: 142 or a portion of SEQ ID NO: 142 not including the 5' UTR and / or 3' UTR. Also provided herein are pharmaceutical compositions comprising a recombinant polynucleic acid, wherein (a) the recombinant polynucleic acid is RNA; (b) the recombinant polynucleic acid is associated with or within a lipid nanoparticle; (c) the recombinant polynucleic acid comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP is an anti-GPC3 CFP and has a sequence having at least 95% sequence identity to SEQ ID NO: 36 or 152; and (d) the recombinant polynucleic acid comprises a sequence having at least 80% sequence identity to SEQ ID NO: 142 or a portion of SEQ ID NO: 142 not including the 5' UTR and / or 3' UTR. In one aspect herein, there is provided a method of treating cancer in a subject in need thereof, the method comprising the step of administering to the subject at least a pharmaceutical composition described in this paragraph and elsewhere, 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, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma, and urothelial carcinoma.

[0036]

[0053] In one aspect of the present specification, a method for expressing CFP specific to bone marrow cell expression in vivo is provided, the method comprises: (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, and isolating and testing a suitable tissue sample from the subject at an appropriate time after administration, and detecting the expression of CFP in bone marrow cells in the tissue sample.In some embodiments, the suitable tissue sample can be peripheral blood.In some embodiments, the appropriate time for testing can be 2 days, 3 days, or 4 days after administration.

[0037] Incorporation by Reference

[0054] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0038]

[0055] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0039]

Figure 1

[0056]

[0023] Figure 1 shows an illustration of the structure and myeloid cell-targeted expression of a chimeric fusion (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 multimerizes with an endogenously expressed myeloid cell-specific transmembrane protein, e.g., the Fc receptor common gamma chain, and is therefore 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. Because an exemplary transmembrane domain of the chimeric fusion protein is the Fcα transmembrane domain, the chimeric fusion protein is also referred to as an Fcα fusion construct in the figure. The functionally enhanced Fcα construct shown in the figure is expressed in myeloid cells and multimerizes with endogenous Fcγ transmembrane receptors, and is therefore functionalized (e.g., functionally enhanced). This figure also shows that a recombinant polynucleotide (e.g., mRNA) encoding a CFP with the described structural features is designed for in vivo delivery encapsulated in a lipid nanoparticle (LNP), and upon entry into myeloid cells, is expressed and functionalized on the surface of the myeloid cells via multimerization with endogenous Fc gamma transmembrane receptors, and is functionalized to kill tumor cells via activation of the CFP receptor and phagocytosis of tumor cells by CFP-expressing myeloid cells. However, when taken up in vivo by cells (e.g., T cells) that do not express endogenous Fc gamma transmembrane receptors, the construct is not expressed on the membrane and is not functional.

Figure 2

[0057] Different CFP constructs for in vivo delivery are shown, each comprising a cancer cell-specific extracellular antigen-binding domain (ECD, e.g., scFV against a target cancer antigen), a CD89 TMD, and one or more intracellular signaling domains that constitute next-generation in vivo receptors incorporating additional signaling domains, e.g., FcR gamma (FcRγ) intracellular signaling domain, PI3 kinase recruitment domain (PI3K), TRIF intracellular signaling domain (TRIF), and CD40 intracellular domain (CD40), or portions thereof, and combinations thereof, indicated after the arrow, to enhance activation of CFP-expressing myeloid cells to actively phagocytose and kill tumor cells targeted by the CFP's ECD. Collectively, these constructs, which contain a type of transmembrane domain that can multimerize with cell-specific endogenous proteins for expression and function in specific cell types, and further contain an intracellular domain(s) or combination of intracellular domains that enhance intracellular signaling in cells expressing the CFP and activate efficient phagocytosis and killing of target cells, are often designated throughout this disclosure as second generation CFP constructs (or 2ndgen or variants thereof).

Figure 3

[0058] The upper panel of Figure 3 shows a perspective view of different CFP designs as transmembrane proteins that have been developed and tested for expression and activity in myeloid cells, e.g., monocytic cells. Shown here is the location of the expressed CFP on the cell membrane (e.g., on the lipid bilayer shown in the cross-section of the cell). The lower panel of Figure 3 shows expression data for each CFP construct in monocytic cell lines. These data indicate that CFPs with additional intracellular signaling domains (e.g., FcR, CD40, FcR-PI3K, TRIF, and FcR-TRIF) are well tolerated and expressed well.

Figure 4A

[0059] Figure 1 shows pro-inflammatory cytokine and chemokine production by transfected monocytic cells expressing next-generation receptors (2ndGen) designated for in vivo expression. Data show a side-by-side comparison of a first-generation cell-specific CFP construct (1stGen) lacking the intracellular signaling domain with a second-generation CFP construct in cell activation and production of IL-12p70 and IFN cytokines.

Figure 4B

[0040]

[0060] T cell therapy has transformed cancer treatment for many patients. However, sustained clinical benefit has not been achieved for the majority of patients with advanced solid tumors. Unlike T cells, myeloid cells readily accumulate within tumors, sometimes contributing up to 50% of the tumor mass. Myeloid cells can be specifically engineered to become highly effective antitumor cells, termed Activate, Target, Attack & Kill (ATAK) cells, that specifically target, phagocytose, and lyse tumor cells and orchestrate in vivo immune activation against tumor cells.

[0041]

[0061] All terms are intended to be understood as they would be understood by one of ordinary skill in the art. Unless otherwise specified, 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.

[0042]

[0062] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0063] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may, for clarity, be described herein in the context of separate embodiments, the present disclosure may also be practiced in a single embodiment.

[0043]

[0064] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments, but not necessarily all embodiments, of the present disclosure.

[0044]

[0065] As used in this specification and claims, the terms "comprising" (and any form of "comprising," such as "comprise," "comprises," and "comprised"), "having" (and any form of "having," such as "have" and "has"), "including" (and any form of "including," such as "include" and "includes"), or "containing" (and any form of "containing," such as "contain" and "contains") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein may be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure may be used to achieve the methods of the disclosure. The methods of the present invention can be achieved.

[0045]

[0066] As used herein, the terms "about" or "approximately" when referring to a measurable value, such as a parameter, amount, time duration, etc., may be intended to encompass variations of ±30% or less, ±20% or less, ±10% or less, ±5% or less, or ±1% or less of the specified value, and variations therefrom, to the extent such variations are appropriate for practice in this disclosure. It may be understood that values ​​referred to by the modifier "about" or "approximately" may also be specifically disclosed.

[0046]

[0067] An "agent" may refer to any cell, small molecule compound, antibody or fragment thereof, nucleic acid molecule, or polypeptide.

[0068] "Alteration" or "change" can refer to an increase or decrease. For example, the alteration can be a 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or 40%, 50%, 60%, or even up to 70%, 75%, 80%, 90%, or 100% increase or decrease. For example, the alteration can be a 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 increase or decrease.

[0047]

[0069] As used herein, "antigen-presenting cells" or "APCs" may include professional antigen-presenting cells (e.g., B lymphocytes, macrophages, monocytes, dendritic cells, and Langerhans cells) as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes, thymic epithelial cells, thyroid epithelial cells, glial cells (brain), pancreatic beta cells, and vascular endothelial cells). APCs can express major histocompatibility complex (MHC) molecules and present MHC-complexed antigens on their surface, which can be recognized by T cells and induce T cell activation and an immune response. Professional antigen-presenting cells, particularly dendritic cells, play a key role in stimulating naive T cells. Non-professional antigen-presenting cells, such as fibroblasts, can also contribute to this process. APCs can also cross-present peptide antigens by processing exogenous antigens and presenting the processed antigens on class I MHC molecules. Antigens that result in proteins that are recognized upon association with class I MHC molecules are generally proteins produced intracellularly, which are processed and associated with class I MHC molecules.

[0048]

[0070] A "biological sample" may refer to any tissue, cell, body fluid, or other material derived from an organism.

[0071] The term "epitope" can refer to any protein determinant, such as a sequence or structure or amino acid residue, capable of binding to an antibody or binding fragment thereof, a T cell receptor, and / or an antibody-like molecule. Epitope determinants typically consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. A "T cell epitope" can refer to a peptide or peptide-MHC complex recognized by a T cell receptor.

[0049]

[0072] Engineered cells, such as engineered bone marrow cells, may refer to cells that have at least one exogenous nucleic acid sequence within them, even if transiently expressed. Expression of the exogenous nucleic acid may be performed by a variety of methods, as described elsewhere, and encompasses methods known in the art. The present disclosure relates to the preparation and use of engineered cells, e.g., engineered bone marrow cells, such as engineered phagocytes. The present disclosure relates, inter alia, to engineered cells that contain an exogenous nucleic acid, e.g., encoding a chimeric fusion protein (CFP).

[0050]

[0073] The term "immune response" includes, but is not limited to, T cell-mediated immune responses, NK cell-mediated immune responses, and / or B cell-mediated immune responses. These responses can be influenced by modulation of T cell costimulation and NK cell costimulation. Exemplary immune responses include T cell responses, e.g., cytokine production and cell-mediated cytotoxicity. In addition, immune responses include immune responses indirectly influenced by NK cell activation, B cell activation, and / or T cell activation, e.g., antibody production (humoral response), and activation of cytokine-responsive cells, e.g., macrophages. Immune responses include adaptive immune responses. The adaptive immune system can react to foreign molecular structures, such as antigens of invading organisms. Unlike 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 and cell-mediated immune responses. In humoral immune responses, antibodies secreted by B cells into bodily fluids bind to pathogen-derived antigens and eliminate the pathogen through various mechanisms, such as complement-mediated lysis. In cell-mediated immune responses, T cells capable of destroying other cells are activated. For example, when disease-related proteins are present within cells, they are proteolytically fragmented into peptides within the cell. Specific cellular proteins then bind to the antigens or peptides thus formed and transport them to the cell surface, where they are presented by molecular defense mechanisms such as T cells. Cytotoxic T cells can recognize these antigens and kill cells bearing these antigens.

[0051]

[0074] A "ligand" may refer to a molecule capable of binding to or forming a complex with another molecule, such as a receptor. Ligands may include, but are not limited to, proteins, glycoproteins, carbohydrates, lipoproteins, hormones, fatty acids, phospholipids, or any entity that binds to a receptor. In some embodiments, a receptor has a specific ligand. In some embodiments, a receptor may bind to a ligand globally, in which case the receptor may bind to several ligands that share at least one similarity in structural organization, charge distribution, or any other physicochemical characteristic. A ligand may be a biomolecule. A ligand may be a non-biological material. For example, a ligand may be a ligand for a negatively charged particle, such as the scavenger receptor MARCO. For example, a ligand may be a ligand for TiO2, such as the scavenger receptor SRA1. In the context of the CFPs described herein, the extracellular binding domain may bind to a ligand, also referred to as the target of the binding domain. In some embodiments, the target is an antigen expressed on diseased cells such as cancer cells, which are target cells in the sense that the target cells express the target antigen that the extracellular antigen-binding domain of CFP binds to on their cell surface.In the present disclosure, anti-(target) binding domain, or anti-(target) binding extracellular domain, or anti-(target)CFP are often used interchangeably with terms such as (target) binding domain, or (target) binding extracellular domain, or (target)CFP, respectively.For example, HER2 expressed on cancer cells is the antigen (ligand) that the anti-HER2 binding extracellular domain of CFP binds to; or alternatively, it is the antigen (ligand) that is also stated to be bound by the HER2 binding extracellular domain of CFP.

[0052]

[0075] The terms "major histocompatibility complex (MHC)," "MHC molecule," or "MHC protein" refer to proteins capable of binding to antigenic peptides and presenting them to T lymphocytes. Such antigenic peptides may represent T cell epitopes. Human MHC is also sometimes referred to as an HLA complex. Thus, the terms "human leukocyte antigen (HLA)," "HLA molecule," or "HLA protein" are used interchangeably with the terms "major histocompatibility complex (MHC)," "MHC molecule," and "MHC protein." HLA proteins are sometimes classified as HLA class I or HLA class II. Although the proteins of the two HLA classes are structurally very similar, they have completely 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 usually loaded with antigens derived from endogenous proteins or pathogens present in cells, and then presented to naive or cytotoxic T-lymphocytes (CTLs).HLA class II proteins are present on antigen-presenting cells (APCs), including but not limited to dendritic cells, B cells, and macrophages.HLA class II proteins mainly present peptides processed from external, for example, external antigen sources to helper T cells.

[0053]

[0076] In the HLA class II system, phagocytes such as macrophages and immature dendritic cells can engulf entities into phagosomes (although B cells exhibit a more general endocytosis into endosomes) that fuse with lysosomes, where acidic enzymes cleave the ingested protein into many different peptides. Autophagy is another source of HLA class II peptides. The most well-studied subclass II HLA genes are HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1.

[0054]

[0077] Presentation of peptides by HLA class II molecules to CD4+ helper T cells results in an immune response to foreign antigens. Upon activation, 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 typically heterodimers of an α chain and a β chain that interact to form a peptide-binding groove that is more open than the class I peptide-binding groove.

[0055]

[0078] HLA alleles are typically expressed in a codominant manner. For example, each person possesses 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 (DPAl and DPB1, encoding 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 about 400 known alleles. This means that a heterozygous individual can inherit six or eight functional class II HLA alleles: three or more alleles from each parent. Thus, HLA genes are highly polymorphic; many different alleles exist in different individuals within a population. Genes encoding HLA proteins have many possible variations, allowing each person's immune system to respond to a wide range of foreign invaders. Hundreds of versions (alleles) of some HLA genes have been identified, 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, and HLA-E*01:01 (non-classical). In some embodiments, class II HLA alleles are HLA-DRB*01:01, HLA-DRB*01:02, HLA-DRB*11:01, HLA-DRB*15:01, and HLA-DRB*07:01.

[0056]

[0079] "Myeloid cells" broadly refers to cells of the myeloid lineage within the hematopoietic cell lineage, and may exclude, for example, the lymphoid lineage. Myeloid cells include, for example, cells of the granulocytic and monocytic lineages. Myeloid cells are a major cellular compartment of the immune system, including monocytes, dendritic cells, tissue macrophages, and granulocytes. Models of myeloid cell ontogeny, activation, differentiation, and tissue-specific functions have been reexamined over the past few years, yielding surprising results. However, their enormous plasticity and heterogeneity in both homeostasis and disease remain far from being understood. Myeloid cells possess many functions, including phagocytosis and their ability to activate T cells, yet harnessing these functions for therapeutic use remains elusive. Thus, novel approaches to use other cell types for the development of improved therapeutics, including, but not limited to, T cell malignancies, are needed.

[0057]

[0080] Myeloid cells differentiate from a common progenitor cell, typically derived from hematopoietic stem cells in the bone marrow. Myeloid lineage commitment may be governed by the activation of distinct transcription factors; thus, myeloid cells may be characterized as cells with a level of plasticity, which can be described as the ability to further differentiate into terminal cell types based on extracellular and intracellular stimuli. Myeloid cells can be rapidly recruited to local tissues via diverse chemokine receptors on their surface. Myeloid cells are responsive to a variety of cytokines and chemokines.

[0058]

[0081] Myeloid cells can be cells derived from hematopoietic stem cells under the influence of one or more cytokines and chemokines, such as G-CSF, GM-CSF, Flt3L, CCL2, VEGF, and S100A8 / 9, within the bone marrow. In some embodiments, myeloid cells are progenitor cells. In some embodiments, myeloid cells can be cells with characteristics of common myeloid progenitor cells, or granulocyte progenitors, myeloblasts, or monocyte-dendritic cell progenitors, or combinations thereof. Bone marrow can contain granulocytes or monocytes, or their progenitors. Bone marrow can contain immature granulocytes, immature monocytes, immature macrophages, immature neutrophils, and immature dendritic cells. Bone marrow can contain monocytes or promonocytic cells or monocyte precursors. In some cases, myeloid cells, as used herein, can refer to monocytes with an M0 phenotype, an M1 phenotype, or an M2 phenotype. Bone marrow may contain dendritic cells (DCs), mature DCs, monocyte-derived DCs, plasmacytoid DCs, pre-dendritic cells, or DC precursors. Bone marrow may contain neutrophils, which may be mature neutrophils, neutrophil precursors, or polymorphonuclear cells (PMNs). Bone marrow may contain macrophages, monocyte-derived macrophages, tissue macrophages, and macrophages of the M0, M1, or M2 phenotype. Monocytes or macrophages exhibit polarization. As used herein, "polarization" may refer to the process by which macrophages exhibit distinct functional phenotypes in response to specific microenvironmental stimuli and signals, often referred to as physiological states. In some cases, macrophages may transition from one polarization state to another. For example, macrophages may be polarized into classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages). M2 macrophages are divided into subtypes, M2a, M2b, M2c, and M2d, which 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 their surface.These cells release a variety of cytokines and chemokines, such as TNF-α, IL-1α, IL-1β, IL-6, IL-12, CXCL9, and CXCL10, and typically exhibit activation of transcription factors such as NF-κB, STAT1, STAT5, IRF3, and IRF5, which regulate M1 gene expression. NF-κB and STAT1 are thought to be the two major pathways involved in M1 macrophage polarization. The M1 phenotype is associated with macrophage bactericidal and tumor-killing functions and exhibits high phagocytic and proinflammatory functions. On the other hand, tumor-associated macrophages in an immunosuppressive environment generally exhibit a greater degree of M2 polarization. Bone marrow can contain tumor-infiltrating monocytes (TIMs). Bone marrow can contain tumor-associated monocytes (TAMs). Bone marrow can contain myeloid-derived suppressor cells (MDSCs). Bone marrow can contain tissue-resident macrophages. Bone marrow may contain tumor-associated DCs (TADCs). Thus, bone marrow cells may express one or more cell surface markers, such as CD11b, CD14, CD15, CD16, CD38, CCR5, CD66, Lox-1, CD11c, CD64, CD68, CD163, CCR2, CCR5, HLA-DR, CD1c, CD83, CD141, CD209, MHC-II, CD123, CD303, CD304, SIGLEC family proteins, and CLEC family proteins. In some cases, myeloid cells can be characterized by high or low expression of one or more cell surface markers, such as CD11b, CD14, CD15, CD16, CD66, Lox-1, CD11c, CD64, CD68, CD163, CCR2, CCR5, HLA-DR, CD1c, CD83, CD141, CD209, MHC-II, CD123, CD303, CD304, or a combination thereof. In one embodiment, it is desired to activate M1 polarization of macrophages using the methods described herein.

[0059]

[0082] "Phagocytosis" is used interchangeably with "pinocytosis" and can refer to the process by which cells engulf particles, such as cancer cells or infected cells. This process can result in an internal compartment (phagosome) containing the particle. This process can be used to ingest particles, such as cancer cells or infected cells, and / or remove them from the body. Phagocytic receptors can be involved in the phagocytic process. The phagocytic process can be closely linked to immune responses and antigen presentation. Processing of exogenous antigens follows their uptake into professional antigen-presenting cells through some types of endocytic events. Phagocytosis can also facilitate antigen presentation. For example, antigens derived from phagocytosed cells or pathogens, including cancer antigens, can be processed and presented on the cell surface of APCs.

[0060]

[0083] A "polypeptide" may refer to a molecule containing amino acids linked together via peptide bonds, such as a glycoprotein, lipoprotein, cellular protein, or membrane protein. A polypeptide may comprise one or more subunits of a protein. A polypeptide may be encoded by a recombinant nucleic acid. In some embodiments, a polypeptide may comprise more than one peptide sequence within a single amino acid chain, which may be separated by a spacer, linker, or peptide cleavage sequence. A polypeptide may be a fusion polypeptide. A polypeptide may comprise one or more domains, modules, or moieties.

[0061]

[0084] A "receptor" may refer to a chemical structure composed of a signal-transmitting polypeptide, such as a polypeptide that transmits an extracellular signal into a cell. Receptors are used to transmit information within a cell, the formation of a cell, or an organism. A receptor includes at least one receptor unit and may contain two or more receptor units, where each receptor unit includes a protein molecule, e.g., a glycoprotein molecule. A receptor may contain a structure that can bind to and form a complex with a ligand. Signaling information may be transmitted by a conformational change in the receptor following binding to a ligand on the surface of a cell.

[0062]

[0085] The term "antibody" refers to the class of proteins commonly known as immunoglobulins, including, but not limited to, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, IgM, and IgY. The term "antibody" includes, but is not limited to, full-length antibodies, single-chain antibodies, single-domain antibodies (sdAbs), and antigen-binding fragments thereof. Antigen-binding antibody fragments include Fab, Fab', and F(ab')2, Fd(V ... H and C H 1), single chain variable fragments (scFv), single chain antibodies, disulfide-linked variable fragments (dsFv), and V L Domain and / or V HAntibody fragments include, but are not limited to, fragments comprising the variable region(s). Antibodies can be derived from any animal. Antigen-binding antibody fragments, including single-chain antibodies, can comprise the variable region(s) alone or in combination with one or more of the hinge region, CH1 domain, CH2 domain, and CH3 domain. Any combination of the variable region(s) with the hinge region, CH1, CH2, and CH3 domain is also included. Antibodies can be, for example, monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies that specifically bind to an HLA-associated polypeptide or HLA-peptide complex.

[0063]

[0086] The term "recombinant nucleic acid" refers to a nucleic acid prepared, expressed, created, or isolated by recombinant means. Recombinant nucleic acids may contain a nucleotide sequence that is not naturally occurring. Recombinant nucleic acids may be synthesized in a laboratory. Recombinant nucleic acids may be prepared by using recombinant DNA techniques, such as enzymatic modification of DNA, such as enzymatic restriction digestion, ligation, and DNA cloning. Recombinant nucleic acids may be DNA, RNA, analogs thereof, or combinations thereof. Recombinant DNA may be transcribed ex vivo or in vitro, such as to produce messenger RNA (mRNA). Recombinant mRNA may be isolated, purified, and used to transfect cells. Recombinant nucleic acids may encode proteins or polypeptides. Throughout this specification, nucleic acid sequences are described that may include deoxyribonucleotides (DNA), ribonucleotides (RNA), or, in some embodiments, modified deoxyribonucleotides or modified ribonucleotides. For example, modified nucleotides can be 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 7-methylguanosine, pseudouridine, dihydrouridine, etc. Those skilled in the art can easily determine the RNA sequence, e.g., mRNA sequence, from a given polynucleotide sequence. The sequence may be codon-optimized.

[0064]

[0087] The process of introducing or incorporating nucleic acids into cells can be via transformation, transfection, or transduction. Transformation is the process of uptake of foreign nucleic acids by bacterial cells. This process is adapted for plasmid DNA propagation, protein production, and other applications. Transformation introduces recombinant plasmid DNA into competent bacterial cells that take up extracellular DNA from the environment. Some bacterial species are naturally competent under certain environmental conditions, but competence can be artificially induced in laboratory settings. Transfection is the introduction of small molecules, such as DNA, RNA, or antibodies, into eukaryotic cells. Transfection can also refer to the introduction of bacteriophage into bacterial cells. "Transduction" is often 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.

[0065]

[0088] The term "vector" may refer to a nucleic acid molecule capable of autonomous replication in a host cell and allowing for the cloning of a nucleic acid molecule. As 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, a vector for transforming an exogenous gene may be a plasmid. In certain embodiments, a vector comprises a nucleic acid sequence containing an origin of replication and other elements necessary for the replication and / or maintenance of a nucleic acid sequence in a host cell. In some embodiments, the vectors or plasmids provided herein are expression vectors. Expression vectors are capable of directing the expression of genes and / or nucleic acid sequences to which they are operably linked. In some embodiments, an expression vector or plasmid is in the form of a circular double-stranded DNA molecule. A vector or plasmid may or may not integrate into the genome of a host cell. In some embodiments, the nucleic acid sequence of a plasmid is not integrated into the genome or chromosome of the host cell after introduction. For example, a plasmid may contain elements for transient or stable expression of a nucleic acid sequence, e.g., a gene or open reading frame carried by the plasmid, in a host cell. In some embodiments, the vector is a transient expression vector. In some embodiments, the vector is a stable expression vector that replicates autonomously in a host cell. In some embodiments, the nucleic acid sequence of the plasmid is integrated into the genome or chromosome of the host cell when introduced into the host cell. Expression vectors that can be used in the methods disclosed herein include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, or viral vectors. The vector can be a DNA vector or an RNA vector. In some embodiments, the vector provided herein is an RNA vector, such as a retroviral vector or a lentiviral vector, that can be integrated into the genome of the host cell (e.g., via reverse transcription) when introduced into the host cell.Other forms of expression vectors which serve equivalent functions and are known to those skilled in the art, such as self-replicating extrachromosomal vectors or vectors capable of integrating into a host genome, can also be used. Exemplary vectors are those capable of autonomous replication and / or autonomous expression of nucleic acids to which they are linked.

[0066]

[0089] In some embodiments, nucleic acids can be delivered to biological systems in the form of nanoparticles. The nucleic acid sequences disclosed herein can be delivered in vivo via suitable nanoparticles, such as liposomes, lipid nanoparticles, or polymer nanoparticles. The lipid nanoparticles can comprise polar lipids. In some embodiments, the lipid nanoparticles comprise cationic lipids. In some embodiments, the lipid nanoparticles comprise cationic lipids and non-cationic lipids. In some embodiments, the lipid nanoparticles comprise neutral lipids. In some embodiments, the lipid nanoparticles comprise PEGylated lipids.

[0067]

[0090] Alternatively, in some embodiments, the nucleic acid may be electroporated into living cells ex vivo, in preparation for cell therapy, where the cells are bone marrow cells.

[0068]

[0091] The term "spacer" or "linker," when used in reference to a fusion protein, refers to a peptide sequence that connects two other peptide sequences of the fusion protein. In some embodiments, the linker or spacer has no specific biological activity other than connecting protein or RNA sequences or preserving some minimum distance or other spatial relationship between the protein or RNA sequences. In some embodiments, the amino acids that are components of the spacer can be selected to affect some molecular property, such as molecular folding, flexibility, net charge, or hydrophobicity. Linkers suitable for use in embodiments of the present disclosure are well known to those of skill in the art and include, but are not limited to, linear or branched 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 toward the development of ordered secondary structures. The amino acids in the flexible linker protein region can include any permutation of Gly, Asn, and Ser, or 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.

[0069]

[0092] The terms "treat," "treated," "treating," "treatment," and the like are intended to refer to reducing, preventing, or ameliorating a disorder and / or its associated symptoms (e.g., a neoplasm or tumor, or an infectious agent, or an autoimmune disease). "Treating" 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 agent, or an autoimmune disease). "Treating" can refer to reducing the frequency or severity of any symptoms or other morbidity associated with a disease and / or the occurrence or recurrence of side effects associated with a treatment. The term "treating" also encompasses the concept of "alleviating." Included is the concept of "managing," which refers to reducing the severity of a disease or disorder in a patient, e.g., increasing the lifespan or survival of a patient with a disease, or delaying its recurrence, e.g., extending the period of remission in a patient who has previously suffered from a disease. It is understood that treating a disorder or condition does not require, but does not preclude, the complete disappearance of the disorder, condition, or symptoms associated therewith. As used herein, the terms "prevent," "preventing," "prevention," and their grammatical equivalents may refer to avoiding or delaying the onset of symptoms associated with a disease or condition in a subject who does not experience such symptoms at the time administration of an agent or compound begins. In certain embodiments, treatment of a subject or patient described herein includes administration of a therapeutic composition, such as a drug, metabolite, preventative component, nucleic acid, peptide, or protein, that encodes or otherwise forms the drug, metabolite, or preventative component. In some embodiments, treatment includes administering a cell or cell population to a subject in need thereof. In some embodiments, treatment includes administering to the subject one or more of the engineered cells described herein, e.g., bone marrow cells, such as one or more engineered phagocytes.Treatment includes treatment of a disease, condition, or syndrome, which may be a pathological disease, condition, or syndrome, or a latent disease, condition, or syndrome. In some cases, treatment as used herein may 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, the engineered phagocytes may not result in or may reduce graft-versus-host disease (GVHD) or fratricide effects. In some embodiments, the engineered cells administered to a human subject are immunocompatible with the subject (i.e., have a match with an HLA subtype naturally expressed in the subject). Subject-specific HLA alleles, or the subject's HLA genotype, may be determined by any method known in the art. In an exemplary embodiment, the method includes determining genetic polymorphisms, generating an alignment of reads extracted from sequencing a dataset against a genetic reference set that includes allelic variants having the genetic polymorphisms; determining a first posterior probability or a posterior probability-derived score for each allelic variant in the alignment; identifying the allelic variant with the highest first posterior probability or posterior probability-derived score as the first allelic variant; aligning the first allelic variant and one or more other allelic variants; the method may include steps of identifying one or more overlapping reads that have been identified using a weighting factor; determining a second posterior probability or a score derived from the posterior probability for one or more other allelic variants using a weighting factor; identifying the second allelic variant by selecting the allelic variant with the largest second posterior probability or score derived from the posterior probability, where the first allelic variant and the second allelic variant define a genotype of the polymorphism; and providing an output for the first allelic variant and the second allelic variant.

[0070]

[0093] A "fragment" may refer to a portion of a protein or nucleic acid. In some embodiments, a 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.

[0071]

[0094] The terms "isolated," "purified," "biologically pure," and their grammatical equivalents can refer to material that is free, to varying degrees, from components that normally accompany it as found in its natural state. "Isolated from" refers to a degree of isolation from the original source or environment. "Purified from" refers to a degree of isolation that is greater than isolation. A "purified" or "biologically pure" protein can be sufficiently free from other materials such that 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 when it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA methodologies, or when it is substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry methods, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can refer to a nucleic acid or protein that yields essentially one band in an electrophoretic gel. For proteins that can be subjected to modifications, such as phosphorylation or glycosylation, different modifications can yield different isolated proteins that can be individually purified.

[0072]

[0095] The term "neoplasm" or "cancer" refers to any disease caused by or resulting from inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Glioblastoma is one non-limiting example of a neoplasm or cancer. The term "cancer" or "tumor" or "hyperproliferative disorder" refers to the presence of cells that possess characteristics typical of cancer-causing cells, such as uncontrolled growth, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells often take the form of tumors, but such cells can also exist alone in animals or be non-tumorigenic cancer cells, such as leukemia cells.

[0073]

[0096] The term "vaccine" should be understood to mean a composition for generating immunity for the prevention and / or treatment of disease (e.g., neoplasm / tumor / infectious agent / autoimmune disease). Thus, as used herein, a vaccine is a pharmaceutical agent that includes a recombinant nucleic acid or a cell that contains and expresses a recombinant nucleic acid and is intended for use in humans or animals to generate specific defense and protection by vaccination. A "vaccine composition" may include a pharmaceutically acceptable excipient, carrier, or diluent. Aspects of the present disclosure relate to the use of technology in the preparation of phagocyte-based vaccines.

[0074]

[0097] The term "pharmaceutically acceptable" refers to approved or approvable by a federal or state regulatory agency for use in animals, including humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopeia. A "pharmaceutically acceptable excipient, carrier, or diluent" refers to an excipient, carrier, or diluent that can be administered to a subject together with a drug, does not destroy its pharmacological activity, and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the drug.

[0075]

[0098] The nucleic acid molecules useful in the method of the present disclosure include, but are not limited to, any nucleic acid molecules that have activity or encode polypeptides.Polynucleotides that have substantial identity to endogenous sequences can typically hybridize with at least one strand of a double-stranded nucleic acid molecule.The term "hybridize" refers to the situation where nucleic acid molecules pair with complementary polynucleotide sequences or their portions to form a double-stranded molecule under various stringency conditions (see, for example, Wahl, GM and S L. Berger (1987), Methods Enzymol., 152:399; Kimmel, AR (1987), Methods Enzymol., 152:507). For example, stringent salt concentrations can typically be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents, such as 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 typically include a temperature of at least about 30°C, at least about 37°C, or at least about 42°C. Those skilled in the art are familiar with the variation of additional parameters, such as hybridization time, detergent concentration, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA. Various levels of stringency can be achieved by combining these various conditions as needed. In an exemplary embodiment, hybridization may occur in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30° C. In another exemplary embodiment, hybridization may occur in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C.In another exemplary embodiment, hybridization may occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations in these conditions will be readily apparent to those of skill in the art. In most applications, the wash step following hybridization may also vary in stringency. Stringency conditions for washes may be defined by salt concentration and temperature. As noted above, wash stringency may be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentrations for wash steps may be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for wash steps may 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 steps may occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the wash steps may occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations in these conditions will be readily apparent to those of skill in the art.Hybridization methods 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.

[0076]

[0099] "Substantially homologous" can refer to a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Such a sequence may be at least 60%, 80%, or 85%, 90%, 95%, 96%, 97%, 98%, or even 99% identical at the amino acid or nucleic acid level to the sequence used for comparison. Sequence homology is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST program, BESTFIT program, GAP program, or PILEUP / PRETTYBOX program). Such software matches identical or similar sequences by assigning degrees of homology to 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. An exemplary method for determining the degree of identity may use a BLAST program, and a probability score between e-3 and em may indicate a closely related sequence. The "reference" is the basis for comparison. It will be understood that the numbering of specific positions or residues within each sequence depends on the particular protein and the numbering scheme used. For example, the numbering may differ between a precursor to a mature protein and the mature protein itself, and sequence differences between species may affect the numbering.One skilled in the art will be able to identify the respective residues in any homologous proteins and in the nucleic acids encoding each by methods well known in the art, for example, by sequence alignment against a reference sequence and determination of homologous residues.

[0077]

[0100] The term "subject" or "patient" can refer to an organism, such as an animal (e.g., a human), that is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.

[0078]

[0101] The term "therapeutic effect" may refer to some degree of relief of one or more of the symptoms of a disorder (e.g., a neoplasm, tumor, or infection by an infectious agent, or an autoimmune disease) or its related pathologies. Alternatively, "therapeutic effect" may refer to a reduction in disease symptoms, e.g., a 10%, 20%, 30%, etc. reduction in tumor mass, following administration of a therapeutic composition. In another embodiment, "therapeutic effect" may relate to partial or complete remission of one or more symptoms, or amelioration of the disease. As used herein, "therapeutically effective amount" refers to the amount of agent, upon administration to a cell or subject, in a single or multiple doses, that is effective to prolong survival of a patient with such a disorder, alleviate one or more signs or symptoms of the disorder, prevent or delay the disorder beyond that expected in the absence of such treatment. "Therapeutically effective amount" is intended to quantify the amount required to achieve a therapeutic effect. An attending physician or veterinarian having ordinary skill in the medical arts can readily determine and prescribe the "therapeutically effective amount" (eg, ED50) of the pharmaceutical composition required.

[0079]

[0102] 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, leukemia cells, or tumor cells. The engineered myeloid cells may attack and kill the 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.

[0080]

[0103] Although cancer is one exemplary embodiment described in detail in this disclosure, it is envisioned that the methods and techniques described herein will be useful in targeting infected or otherwise diseased cells within the body. Also described herein are therapeutic and vaccine compositions that use engineered cells.

[0081]

[0104] Myeloid effector cells can be isolated from human biological samples and modified ex vivo using methods to engineer such cells to prepare cells for therapeutic purposes, but the modifications do not alter the plasticity of these cells. Cells of the monocyte lineage are phagocytic and effective antigen-presenting cells. In one aspect, the present invention is based on the important finding that engineered myeloid cells can be a highly effective therapeutic modality in the treatment of numerous diseases, including cancer. Myeloid cells can be engineered to express chimeric antigen receptors (CARs), which enhance the immune function of myeloid cells; in this case, the cells are highly phagocytic and can attack and kill diseased or infected cells in the body. Chimeric antigen receptors are recombinant constructs designed and specifically engineered as described herein to be (a) highly target-specific, possessing an extracellular antigen-binding domain specifically oriented to bind to a target antigen, and (b) possessing a highly specialized intracellular domain to activate myeloid cells and achieve activation of the phagocyte phenotype. For example, a highly specialized intracellular domain is designed to create a chimeric receptor that, upon activation by target binding of the extracellular region of the receptor, can generate a signaling cue inside the cell that activates the intracellular interferon signaling cascade and transcription factors, i.e., directs the activation of transcription factors IRF (IFN regulatory factor). In addition, the methods and compositions described herein are also useful in gene therapy, in which a recombinant nucleic acid encoding a chimeric antigen receptor is administered locally or systemically to a subject in need thereof, so that the recombinant nucleic acid is specifically expressed in bone marrow cells in vivo, thereby generating activated bone marrow cells with therapeutic potential. In some embodiments, the nucleic acid is mRNA. In some embodiments, the mRNA is delivered by LNP.

[0082]

[0105] Phagocytes are natural sentinels of the immune system and form the body's first line of defense. Phagocytes engulf pathogens, infected cells, foreign bodies, or cancerous cells and remove them from the body. Most potential pathogens are rapidly neutralized by the phagocytic system, for example, before they can cause significant infection or disease. This can involve pinocytosis, a receptor-mediated uptake via clathrin-coated pit systems, and in particular macropinocytosis, a consequence of membrane ruffling and engulfment. Thus, phagocytes are activated by a variety of non-self (and self) elements and exhibit a level of plasticity in recognizing their "targets." Most phagocytes express scavenger receptors on their surface, which are pattern recognition molecules that can bind to a wide range of foreign particles, as well as dead cells, cell debris, and unwanted particles within the body. In one embodiment, a recombinant nucleic acid encoding a chimeric antigen receptor (CAR) can be expressed intracellularly. CARs can be designed in various ways to attack specific tumor cells, and CAR-expressing myeloid effector cells can be activated to phagocytose and kill tumor cells. CARs can be designed to create phagocytic receptors that are activated specifically in response to target engagement, and the phagocytic potential of macrophages is enhanced by specifically engineered intracellular domains of the receptor. The CAR platform for myeloid cells described herein is designed so that tonic signaling is not detected in the myeloid cells at the time of administration into the body or at any time before the myeloid cells engage their target via the CAR. This is often examined ex vivo. At the same time, CAR-expressing myeloid cells can further differentiate into an M0, M1, or M2 phenotype in the presence of appropriate stimuli, and can retain cellular plasticity to differentiate at least at the time of administration. In addition, CAR-expressing myeloid effector cells can migrate to lymph nodes and cross-present antigens to naive T cells in the lymph nodes, thereby activating adaptive responses.

[0083]

[0106] In some embodiments, disclosed herein are compositions and methods for producing bone marrow cells that are isolated from a biological sample, engineered ex vivo to express a recombinant protein, and formulated into a pharmaceutical composition such that the bone marrow cells of the composition are "effector" bone marrow cells that are effective in inducing immune activation in vivo. In some embodiments, the bone marrow cells of the composition are referred to as "ATAK" bone marrow cells, which are bone marrow cells that are effective in attacking and destroying target cells. The ATAK bone marrow cells disclosed herein are engineered bone marrow recombinant proteins that express, for example, a chimeric receptor, e.g., a chimeric antigen receptor, that includes at least one intracellular signaling domain derived from an interferon-inducible protein in immune cells. In some embodiments, the methods and compositions described herein are directed to causing the engineered bone marrow cells to exhibit an effector phenotype. In some embodiments, the engineered bone marrow cells, e.g., monocytes, are monocytes of an M0 or M1 phenotype, and activation of the chimeric antigen receptor expressed in the bone marrow cells causes the cells to exhibit an M1 phenotype. The M1 phenotype exhibited by the engineered cells makes them highly tumoricidal when designed to be targeted to tumor cells.

[0084]

[0107] When a polynucleic acid is said to be substantially expressed in a particular cell type, it can be understood to indicate higher expression in the particular cell type compared to another cell type or other cell types. For example, although the concepts disclosed herein relate to compositions comprising a recombinant polynucleic acid that is "substantially" expressed in a myeloid cell type, this can be understood to indicate preferential expression in the myeloid cell type compared to other cells, such as hepatocytes, neurons, or T cells. This can mean that the protein encoded by the polynucleic acid is readily detectable in myeloid cells and not readily detectable in non-myeloid cells after a specific time period following introduction of the polynucleic acid into the cells. In some embodiments, this term can refer to the expression of the protein encoded by the polynucleic acid in greater numbers in myeloid cells than in a preferred cell type, in this case, for example, neutrophils, T cells, hepatocytes, or neurons.

[0085]

[0108] Provided herein are compositions and methods for treating diseases or conditions such as cancer. The compositions and methods provided herein utilize human bone marrow cells, including but not limited to neutrophils, monocytes, myeloid dendritic cells (mDCs), mast cells, and macrophages, to target diseased cells, such as cancer cells. The compositions and methods provided herein can be used to eliminate diseased cells, such as cancer cells, and / or diseased tissues through various mechanisms, including activation and recruitment of T cells, activation of effector immune cells (e.g., activation of CD8 T cells and NK cells), antigen cross-presentation, enhancement of inflammatory responses, reduction of regulatory T cells, and phagocytosis. For example, bone marrow cells can be used to maintain an immune response against cancer cells.

[0086]

[0109] The present applicants have previously described compositions comprising recombinant nucleic acids encoding chimeric fusion proteins (CFPs), such as phagocytic receptor (PR) fusion proteins (PFPs), scavenger receptor (SR) fusion proteins (SFPs), integrin receptor (IR) fusion proteins (IFPs), or caspase-recruiting receptor (caspase-CAR) fusion proteins. The CFPs encoded by the recombinant nucleic acids can include an extracellular domain (ECD) comprising an antigen-binding domain that binds to an antigen on a target cell. The extracellular domain can be fused to a hinge domain or an extracellular domain derived from a receptor, such as a hinge domain or an extracellular domain. The CFPs encoded by the recombinant nucleic acids 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 CFPs encoded by the recombinant nucleic acids further comprise an intracellular domain comprising an intracellular signaling domain, such as an intracellular signaling domain derived from a phagocytic receptor, a scavenger receptor, or an integrin receptor. For example, the intracellular domain may include one or more intracellular signaling domains derived from a phagocytic receptor, a scavenger receptor, or an integrin receptor. For example, the intracellular domain may include 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 expression of a chemokine receptor), antigen presentation, and / or enhanced cross-presentation. In some embodiments, the CFP is a phagocytic receptor fusion protein (PFP). In some embodiments, the CFP is a phagocytic scavenger receptor fusion protein (PFP). In some embodiments, the CFP is an integrin receptor fusion protein (IFP). In some embodiments, the CFP is an inflammatory receptor fusion protein. In some embodiments, the CFP encoded by the recombinant nucleic acid further includes an intracellular domain comprising a recruitment domain. For example, the intracellular domain may include one or more PI3K recruitment domains, caspase recruitment domains, or caspase activation / recruitment domains (CARDs).

[0087]

[0110] Provided herein are recombinant nucleic acids encoding improved immunogenic CAR compositions, such as chimeric fusion proteins (CFPs; alternatively referred to as CARs, chimeric antigen receptors), that contain an intracellular domain that activates an interferon response in cells expressing the CAR. Provided herein are immunogenic CFPs that contain at least one intracellular domain containing a pLxIS motif. The recombinant nucleic acid may be DNA or RNA. The recombinant nucleic acid encoding the CAR may be incorporated into a vector. When expressed in a cell, the recombinant CAR activates the production of type I interferon in the cell. Such cells are mammalian cells capable of type I interferon response. Such cells are immune cells, such as lymphocytes or myeloid cells.

[0088]

[0111] In some embodiments, the recombinant nucleic acid encoding the chimeric receptor comprises a specific sequence encoding the pro-inflammatory intracellular domain of the chimeric receptor. In some embodiments, the chimeric receptor proteins described herein comprise an intracellular domain that, upon engagement with its target in the extracellular domain, is capable of activating a signaling cascade that leads to the induction of interferon response genes or the production of type I interferon in cells expressing the chimeric antigen receptor. In some embodiments, the chimeric receptor proteins described herein comprise a domain derived from an innate immune pathway adaptor protein, such as mitochondrial antiviral signaling protein (MAVS), stimulator of interferon genes (STING), Toll / IL-1R domain-containing adaptor-inducing IFN (TRIF), and TLR adaptor interacting with endolysosomal SLC15A4 protein (TASL), or a portion thereof. In some embodiments, a domain or fragment of an innate immune pathway adaptor protein, e.g., a MAVS, STING, TRIF, or TASL protein, is incorporated by recombinant DNA techniques into the intracellular domain of a CFP or CAR described herein, where the domain or fragment contains a pLxIS motif (p represents a hydrophilic residue, x represents any residue, and S represents a phosphorylation site), which is phosphorylated by TBK1 or IKKε and mediates recruitment of IRF-3 to the signaling complex.

[0089]

[0112] In some embodiments, the chimeric receptor proteins described herein comprise an intracellular domain that, upon engagement with its target at the extracellular domain, is capable of activating a signaling cascade that results in a nuclear factor kappa B-responsive gene, or NF-kappa B response, in cells expressing the chimeric antigen receptor.

[0090] Effector myeloid cells and interferon activation

[0113] Type I and type II interferons (IFNs) play important roles in regulating immune responses in infection and cancer. Type I is represented by multiple subtypes, including numerous IFNα family members: IFNβ, IFNδ, IFNε, IFNκ, IFNτ, and IFNω, all of which utilize the same cell surface receptor, the heterodimeric IFNαR, composed of the IFNαR1 and IFNαR2 proteins. Type II IFNs are represented by IFNγ. These two IFN types are expressed by nearly all cells and bind to distinct cell surface receptors that trigger signaling events and elicit diverse cellular responses. Myeloid cells are key targets for interferons. In the early immune response to intracellular bacterial infection, activated natural killer (NK) cells and activated T cells are the source of IFNγ production. During the early stages of infection, production of the cytokines interleukin (IL) 12 and IL-18 drives antigen-nonspecific IFNγ production by these lymphocyte populations. Antigen-specific CD4+ and CD8+ T cells can also produce IFNγ in response to these pathogens. There are numerous distinct 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 a conserved cell surface type I IFN receptor, the IFNαR. Ligation of the cell surface IFNαR induces the expression of numerous antiviral immune-stimulated gene (ISG) products, thereby protecting 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 IFNs also dramatically correlates with increased susceptibility to numerous intracellular bacterial infections, including Listeria monocytogenes, Mycobacterium tuberculosis, Fransicella tularensis, and other bacteria (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). 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 cells triggers aggregation of the two receptor complexes, resulting in the presence of 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 for 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, it induces conformational changes that allow for tight association of the IFNγR1 and IFNγR2 subunits. These rearrangements within the receptor induce autophosphorylation and cross-phosphorylation of Janus-associated kinases (JAKs), which are constitutively associated with the receptor. IFNγR1 contains a binding motif for JAK1, and IFNγR2 contains a binding motif for JAK2.Phosphorylation of JAK proteins stimulates their catalytic activity, which then phosphorylates a tyrosine residue (Y440) in 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. Because each receptor complex contains two IFNγR1 subunits, two STAT-1 proteins can bind to the receptor. JAK1 and JAK2 maintain receptor association and phosphorylate each recruited STAT-1 protein at tyrosine residue 701 (Y701). This phosphorylation allows the release of STAT-1 monomers from the receptor and their subsequent formation into homodimers. STAT-1 homodimers translocate to the nucleus and bind to gamma-activating sequences (GAS) within the promoter DNA of IFN-inducible genes (ISGs), resulting in increased transcription. Type I IFNs, like those activated by IFNγ, signal through the canonical JAK / STAT pathway. Ligand binding to the IFNαR triggers dimerization of the two receptor subunits and transfer of phosphorylation to the associated TYK2 and JAK1 kinases. The kinases phosphorylate residues in the cytoplasmic tails of IFNαR1 and IFNαR2, recruiting the proteins STAT1 and STAT2 via their SH2 domains. Docking of these STAT proteins to the receptor subunits allows them to be phosphorylated by activated JAK proteins at Y701 on STAT-1 and Y690 on STAT-2. Phosphorylation of STAT monomers releases them from their docking sites, allowing them to dimerize and combine with IRF9 in homodimeric or heterodimeric forms to produce the transcription factor ISG factor 3 (ISGF3). ISGF3 translocates to the nucleus, identifies ISGs, and induces their transcription. ISGs induced by type I IFN signaling typically contain interferon-stimulated response elements (ISREs) or gamma-activated sequence (GAS) elements within their promoters, with a clear preference for genes containing ISREs.Some examples of ISGs that are transcribed as a result of type I IFNs are the ISRE containing genes ISG15, IP-10, IRF-7, and PKR

[66] , and the GAS containing 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., 2013 March;55(0):187-200).

[0091] Recombinant chimeric receptor proteins

[0114] Provided herein are classes of phagocytic or tethered receptor (PR) subunits (e.g., phagocytic receptor fusion proteins (PFPs)) that include (i) a transmembrane domain and (ii) an intracellular domain that includes a phagocytic receptor intracellular signaling domain; and an extracellular antigen-binding domain that is specific for an antigen, e.g., an antigen of a target cell or an antigen displayed on a target cell, wherein the transmembrane domain and the antigen-binding domain are operatively linked such that antigen binding to the target by the antigen-binding domain of the fusion receptor activates the intracellular signaling domain of the phagocytic receptor.

[0092]

[0115] 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, or 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 the extracellular domain of FCGR1A, FCGR2A, FCGR2B, FCGR2C, FCGR3A, or FCGR3B. 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 domains: 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.

[0093]

[0116] In some embodiments, the CFP further comprises an extracellular domain comprising an antigen-binding domain operably linked to the 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 an immunoglobulin extracellular domain or an immunoglobulin hinge region. In some embodiments, the extracellular domain comprises a phagocytic domain. In some embodiments, the extracellular domain comprises a structure capable of multimer assembly. In some embodiments, the extracellular domain comprises a scaffold for multimerization. In some embodiments, 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 length. In some embodiments, the extracellular domain is at most 500, 400, 300, 200, or 100 amino acids in length. In some embodiments, the antigen-binding domain specifically binds to an antigen on 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 is a single-chain variable fragment (scFv), Fab, single-domain antibody (sdAb), nanobody, V H Domain, V L domain, VNAR domain, V HHIn some embodiments, the antigen-binding domain comprises a functional antibody fragment that is a domain, a bispecific antibody, a diabody, or a functional fragment or combination thereof. In some embodiments, the antigen-binding domain comprises a ligand, an extracellular domain of a receptor, or an adaptor. In some embodiments, the antigen-binding domain comprises a single antigen-binding domain that is 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.

[0094]

[0117] 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 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 2D (NKG2D) family ligand, disialoganglioside 2 (GD2), CD2 , CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD45, CD56CD79b, CD97, CD117, CD123, CD133, CD138, CD171, CD179a, CD213A2, CD248, CD276, PSCA, CS-1, CLECL1, GD3, PSMA, FLT3, TAG72, EPCAM, IL-1, integrin receptors, PRSS21, VEGFR2, PDGFRβ, SSEA-4, EGFR, NCAM, prostase, PAP, ELF2M, GM3, TEM7R, CLDN6, TSHR, GPRC5D, ALK, Dsg1, Dsg3, IGLL1, and combinations thereof. In some embodiments, the antigen is a protein antigen selected from the group consisting of 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 integrin receptor antigen.In some embodiments, the antigen is an antigen of an integrin receptor or integrin selected from the group consisting of 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. 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 to two or more different antigens.

[0095]

[0118] In some embodiments, the antigen-binding domain comprises a sequence of an antigen-binding domain presented herein, such as a sequence of an antigen-binding domain in the tables presented herein.

[0119] In some embodiments, the target protein is CD70. In some embodiments, the antigen-binding domain comprises an anti-CD70 antibody or binding fragment thereof, wherein the antigen-binding domain comprises a heavy chain variable domain (VH) comprising a heavy chain complementarity-determining region 3 (CDR3), that is a CDR3, of any one of the VH sequences selected from the group consisting of the amino acid sequences: QVQLQESGGGLVQAGGSLRLSCAAPRSIFSINAMGWYRQAPGKQRELVAAITSGGSPTYADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCATGPYGLDNALDAWGQGTQVTVSS (SEQ ID NO: 43) and QVQLQESGGGLVQTGGSLRLACTASGFTFDDYAIAWFRQAPGKEREFVAAISWSGGTTHYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYFCAKSLRSSPSSRWFGSRGQGTQVTVSS (SEQ ID NO: 44).

[0096]

[0120] 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 sequence: GPYGLDNALDA. In some embodiments, the VH of an anti-CD70 antibody or binding fragment thereof 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 sequence: INAMG. In some embodiments, the VH of an anti-CD70 antibody or binding fragment thereof 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 sequence: AITSGGSPTYADSVKG.

[0097]

[0121] 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 sequence: SLRSSPSSRWFGS. In some embodiments, the VH of an anti-CD70 antibody or 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 sequence: DYAIA. In some embodiments, the VH of an anti-CD70 antibody or 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 sequence: AISWSGGTTHYADSVKG.

[0098]

[0122] The following table lists each CDR according to the three numbering CDR formats of Chothia, Kabat and IMGT:

[0099] [Table 1]

[0123] In some embodiments, the VH of the anti-CD70 antigen-binding fragment comprises at least about 70% sequence identity to any one of the sequences selected from the group consisting of SEQ ID NOs: 43 and 44. In some embodiments, the VH of the anti-CD70 antigen-binding fragment comprises 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 to SEQ ID NO: 43 or SEQ ID NO: 44. In some embodiments, the VH is a domain of a single-domain antibody. In some embodiments, the VH is a VHH.

[0100]

[0124] In some embodiments, the target protein is GPC3. Glypicans belong to the heparan sulfate proteoglycan family, which share a similar structure and contain a 60-70 kD core protein linked to the cell membrane surface by a glycosylphosphatidylinositol (GPI) anchor, with the carboxy terminus modified with heparan sulfate side chains. The GPC3 gene is located at position 26 on the long arm of chromosome X and contains 11 exons. The transcript is 2130 bp long, encodes 580 amino acids, and the protein has a molecular weight of approximately 70 kDa. It is involved in various liver cancers. GPC3 expression correlates with the prognosis of hepatocellular carcinoma (HCC). Furthermore, GPC3 is highly expressed in lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma, and urothelial carcinoma. In one aspect of the present specification, a CFP targeting GPC+ cells in cancer is provided 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 an antigen-binding domain or fragment thereof, and the antigen-binding domain is selected from the group consisting of ATACADTTQYAYDY, ATACADTTLYEYDY, ATACVDTTQYEYDY, ATACADATQHEYDY, ATACADTTQYDYDY, ATACADTTQYEYDY, ATACADTTHYEYDY, ATACVITTLYEYDY, ATACAETTLYEYDY, ATACADTTQHEYDY, ATACVDTTHYEYDY, ATACASTTLYEYDY, ATACVVTTLYEYDY, ATACGGATGPYDY, ATACAGAIGPYDY, ATACVVVGDQNDY, ATACVVVGDRNDY, ATDCAGGTSTPYDY, ATDCAGG The heavy chain variable domain (VH) comprises a heavy chain complementarity determining region 3 (HC CDR3) of any one of the sequences selected from the group consisting of TATPYDY, 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 the anti-GPC3 antibody or binding fragment thereof further comprises a heavy chain complementarity determining region 1 (HC CDR1) of any one of the sequences selected from the group consisting of GFPLAYYA, GFSLDYYA, GFPLDYYA, GFTLDYYA, GFSLNYYA, GFTLAYYA, GFTLGYYA, GFPLNYYA, GFPLHYYA, GFSLGYYA, GFPLGYYA, GFPLEYYA, GSDFRADA, GRTFSSYG, GFSLAYYA, and GLTFRSVG.In some embodiments, the VH of the anti-GPC3 antibody or binding fragment thereof is selected from the group consisting of ISNSDGST, ISASDGST, ISSSDGST, ISSSDGNT, ISSADGST, ISSSGGST, ISSGDGST, ISSGDGNT, ISSSDDST, ISSNDGST, ISSPDGST, ISSRTGGT, ISAGDGSST, ISSSDGSSSDGNT, ISSGDGNT, ISSGDGKT, ISSSDGGT, ISSRTGST, ISSRTGNT, ISSSDGHSST, ISSSSDGNT, ISASNGNT, ISSGSDGNT, ISASDGNT, IDSITSI, ISWSGGSTIAASVGST, ISSSDGSDGNT, and ASPSGVIT. Further comprising any one of the heavy chain complementarity determining regions 2 (HC CDR2). In some embodiments, the VH of the anti-GPC3 antibody or binding fragment thereof has 70-100% sequence identity to any one of the sequences selected from the group consisting of SEQ ID NOs: 45-134 listed in Table 5. In some embodiments, the VH is a domain of a single domain antibody. In some embodiments, the VH is a VHH. Some exemplary anti-GPC3 VHH domain sequences are listed in Table 5.

[0101]

[0125] In some embodiments, the anti-GPC3 antigen-binding domain comprises a VH domain having CDR1, CDR2 and CDR3, wherein CDR1 has the amino acid sequence: DYEMH, CDR2 has the amino acid sequence: ALDPKTGDTAYSQKFKG, and CDR3 has the amino acid sequence: FYSYTY.

[0102]

[0126] In some embodiments, the anti-GPC3 antigen binding domain is a VH domain comprising CDR1, CDR2 and CDR3 domains in which the CDR1 sequence is DYEMH; the CDR2 sequence is ALDPKTGDTAYSQKFKG; and the CDR3 sequence is FYSYTY; and a VL domain comprising CDR1, CDR2 and CDR3 domains in which the CDR1 sequence is RSSQSLVHSNRNTYLH; the CDR2 sequence is KVSNRFS; and the CDR3 sequence is SQNTHVPPT. An scFv having a VL domain.

[0103]

[0127] In some embodiments herein, provided is a CFP having an antigen-binding domain comprising CDR1, CDR2, and CDR3, a transmembrane domain, and an intracellular domain, as described above, wherein the transmembrane domain comprises the sequence of a CD89 TMD, e.g., SEQ ID NO: 11; and a CFP having one or more intracellular domains described herein, e.g., in Table 2. Exemplary CFP sequences can be found in Table 4. Furthermore, CFP domains can be exchanged or rearranged using common molecular biology skills and techniques, using exemplary guidance from Table 4.

[0104]

[0128] Thus, in some embodiments, the antigen target on cancer cells is CD5. A CFP capable of binding to the CD5 antigen has the sequence: EIQLVQSGGGLVKPGGSVRISCAASGYTFT NYGMN WVRQAPGKGLEWMG WINTHTGEPTYADSFKG RFTFSLDDSKNTAYLQINSLRAEDTAVYFCTR RGYDWYFDV WGQGTTVTVSSGGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC RASQDINSYLS WFQQKPGKAPKTLIY RANRLES GVPSRFSGSGSGTDYTLTISSLQYEDFGIYYC QQYDESPWT The extracellular antigen-binding domain may comprise an extracellular antigen-binding domain having a sequence of FGGGTKLEIK, or a sequence at least 90% identical to the above sequence. CDR sequences according to the Kabat numbering scheme are underlined, e.g., the VH CDR1 sequence is NYGMN, the CDR2 sequence is WINTHTGEPTYADSFKG, and the CDR3 sequence is RGYDWYFDV, the VL CDR1 sequence is RASQDINSYLS, the CDR2 sequence is RANRLES, and the CDR3 sequence is QQYDESPWT.

[0105]

[0129] In some embodiments, the antigen target on cancer cells is HER2. A CFP capable of binding to the HER2 antigen has the sequence: DIQMTQSPSSLSASVGDRVTITC RASQDVNTAVAWYQQKPGKAPKLLIY SASFLYS GVPSRFSGSRSGTDFLTISSLQPEDFATYYC QQHYTTPPT FGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIK DTYIH WVRQAPGKGLEWVA RIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDV The extracellular antigen-binding domain may comprise WGQGTLVTVSS, or a sequence at least 90% identical to the above sequence. CDR sequences according to the Kabat numbering scheme are underlined, e.g., the VL CDR1 sequence is RASQDVNTAVA, the CDR2 sequence is SASFLYS, and the CDR3 sequence is QQHYTTPPT, the VH CDR1 sequence is DTYIH, the CDR2 sequence is RIYPTNGYTRYADSVKG, and the CDR3 sequence is WGGDGFYAMDV.

[0106]

[0130] In some embodiments, the antigen target on cancer cells is trophoblast cell surface antigen 2 (TROP2). TROP2 is a membrane glycoprotein that regulates cell growth, proliferation, self-renewal, survival, and invasion. It is highly upregulated in a variety of cancers, including non-small cell lung cancer (NSCLC), breast cancer, cervical cancer, colon cancer, gastric cancer, bladder cancer, prostate cancer, thyroid cancer, and osteosarcoma, among others. The FDA and EPA have approved sacituzumab govitanin for the treatment of patients with triple-negative breast cancer. Trop-2, a transmembrane glycoprotein, is widely expressed in various epithelial cancers as well as certain normal tissues. Trop-2 is also known as tumor-associated calcium signal transducer 2 (TACSTD2), membrane component chromosome 1 surface marker 1 (M1S1), gastrointestinal antigen 733-1 (GA733-1), and epithelial glycoprotein-1 (EGP-1). TROP2 is an attractive therapeutic target in a variety of cancers. This patent application focuses on an approach that employs engineered bone marrow cells to target TROP2, eradicate TROP2+ cancer cells through enhanced phagocytosis, and activate immune cells against TROP2+ cells. Provided herein is a CFP capable of binding the TROP2 antigen, with the sequence: QVQLQQSGSELKKPGASVKVSCKASGYTF TNYGM NWVKQAPGQGLKWMG WINTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSSGGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITC KASQDVSIA VAWYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFLTISSLQPEDFAVYYC QQHYITPLTF GAGTKVEIKR, or an extracellular antigen-binding domain having a sequence that is at least 90% identical to the above sequence.

[0107]

[0131] Any one of the above sequences may include an N-terminal signal peptide sequence, eg, MWLQSLLLLGTVACSIS, and the mature protein expressed on the myeloid cells may or may not include the signal sequence.

[0108]

[0132] Additionally, any one of the recombinant nucleic acids may include one or more linker sequences, one or more self-cleaving peptide sequences such as T2A, or P2A.

[0133] 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, where the antibody domain binds to an autoantigen, such as Dsg1 or Dsg3.

[0109]

[0134] In some embodiments, the transmembrane domain and the antigen-binding domain are operably linked via a linker, such as the hinge region of CD8α, IgG1, or IgG4.

[0110]

[0135] In some embodiments, the extracellular domain comprises a multimerization scaffold.

[0136] 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 derived from a syntaxin, such as syntaxin 3 or syntaxin 4 or syntaxin 5. In some embodiments, the transmembrane domain oligomerizes with the transmembrane domain of an endogenous receptor when the CFP is expressed in a cell. In some embodiments, the transmembrane domain oligomerizes with the transmembrane domain of an exogenous receptor when the CFP is expressed in a cell. In some embodiments, the transmembrane domain dimerizes with the transmembrane domain of an endogenous receptor when the CFP is expressed in a cell. In some embodiments, the transmembrane domain dimerizes with the transmembrane domain of an exogenous receptor when the CFP is expressed in a cell. 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.

[0111]

[0137] In some embodiments, the transmembrane domain is at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids in length, hi some embodiments, the transmembrane domain is at most 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids in length.

[0112]

[0138] In some embodiments, the intracellular transduction domain comprises an intracellular transduction 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, e.g., 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 transduction domain comprises an intracellular domain 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, SSC5 In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a receptor selected from the group consisting of CD, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fc alpha 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, Cdc42 inhibitory domain, or GTPase inhibitory domain inhibits Rac, Cdc42, or GTPase in the phagocytic cup of cells expressing PFP. In some embodiments, the intracellular domain comprises an F-actin disassembly 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 ITAM domain selected from CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and ITAM domains of these amino acid sequences having at least one but not more than 20 modifications thereof. In some embodiments, at least one ITAM domain comprises a phosphorylation site for a Src family kinase. 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.

[0113]

[0139] In some embodiments, the intracellular domain does not include a domain derived from the CD3 zeta intracellular domain. In some embodiments, the intracellular domain does not include a domain derived from the MerTK intracellular domain. In some embodiments, the intracellular domain does not include a domain derived from the TLR4 intracellular domain. In some embodiments, the intracellular domain includes a CD47 inhibitory domain. In some embodiments, the intracellular signaling domain includes a domain that activates integrins, such as the intracellular region of PSGL-1.

[0114]

[0140] In some embodiments, the intracellular signaling domain comprises a domain that activates the GTPase Rap1, such as domains derived from EPAC and C3G. In some embodiments, the intracellular signaling domain is derived from paxillin. In some embodiments, the intracellular signaling domain activates focal adhesion kinase. In some embodiments, the intracellular signaling domain is derived from a single phagocytic receptor. In some embodiments, the intracellular signaling domain is derived from a single scavenger receptor. In some embodiments, the intracellular domain comprises a phagocytosis-enhancing domain.

[0115]

[0141] In some embodiments, the intracellular domain comprises a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain comprises a kinase activation domain or a kinase binding domain. In some embodiments, the pro-inflammatory signaling domain comprises an IL-1 signaling cascade activation domain. In some embodiments, the pro-inflammatory signaling domain comprises an intracellular signaling domain derived from TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, IFN receptor, STING, NLRP family members NLRP1-14, NOD1, NOD2, pyrin, AIM2, NLRC4, FCGR3A, FCERIG, CD40, Tank1-binding kinase (TBK), a caspase domain, a pro-caspase binding domain, or any combination thereof.

[0116]

[0142] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a connexin (Cx) protein. For example, the intracellular domain may comprise a signaling domain, such as an intracellular 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. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from Cx43.

[0117]

[0143] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a SIGLEC protein. For example, the intracellular domain can comprise a signaling domain, such as an intracellular signaling domain derived from Siglec-1 (sialoadesin), 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. In some embodiments, a recombinant polynucleic acid encoding SIGLEC is co-administered with CFP for expression, and SIGLEC is an engineered SIGLEC molecule that lacks ITIM domain. This construct is designed to act as a decoy to block anti-phagocyte signaling and promote the phagocyte-promoting function of CFP by bone marrow cells that take up and express the recombinant polynucleotide. In some embodiments, the CFP and the engineered SIGLEC construct can be encoded by the same polycistronic polynucleic acid to ensure both are taken up and co-expressed in the same cell. In some embodiments, the CFP, for example, anti-TROP2 CFP or anti-GPC3 CFP; and the SIGLEC construct are encoded by different polynucleotides but loaded into LNPs to ensure co-delivery.

[0118]

[0144] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a TLR protein. In some embodiments, the intracellular domain may comprise the intracellular signaling domain of an endolysosomal TLR, e.g., TLR3, TLR7, TLR8, or TLR9. In some embodiments, the intracellular signaling domain may be derived from a TLR3 protein. In some embodiments, the intracellular signaling domain may be derived from a TLR7 protein, a TLR8 protein, or a TLR9 protein. In some embodiments, the intracellular domain may comprise the intracellular signaling domain of cell surface TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10.

[0119]

[0145] In some embodiments, the intracellular signaling domain is specifically paired with another intracellular domain or transmembrane domain to maximize the efficacy and phagocytic potential of myeloid cells expressing the construct. For example, in some embodiments, a transmembrane domain comprising CD64 TM or a portion thereof may be specifically paired with an intracellular signaling domain comprising the ICD or PI3 kinase recruitment domain (PI3K) of the innate immune adaptor protein, or both. In some embodiments, the combination of domains with chimeric receptor intracellular domain(s) and / or transmembrane domain is directed toward maximizing the phagocytic index of the cells expressing the construct, e.g., myeloid cells. In some embodiments, the combination of domains with chimeric receptor intracellular domain(s) and / or transmembrane domain is directed toward maximizing the inflammatory potential of the cells expressing the construct, such that the cells can lyse target cells and activate immune response pathways to render the immune responsive for extended periods of time. In some embodiments, the domain combinations of the chimeric receptor intracellular domain(s) and / or transmembrane domains are directed toward minimizing or eliminating tonic signaling by cells expressing the chimeric protein, hi some embodiments, the domain combinations of the chimeric receptor intracellular domain(s) and / or transmembrane domains are directed toward maximizing the specificity of the immune response.

[0120]

[0146] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a C-type lectin protein. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from a mannose receptor protein. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from an asialoglycoprotein receptor protein. For example, the intracellular domain is a protein that encodes 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 and / or any combination thereof.

[0121]

[0147] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain derived from a cell adhesion molecule. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from the molecules IgCAM, cadherin, integrin, C-type lectin-like domain protein (CTLD), and / or proteoglycan. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from E-cadherin, P-cadherin, N-cadherin, R-cadherin, B-cadherin, T-cadherin, or M-cadherin. For example, the intracellular domain may comprise a signaling domain, such as an intracellular signaling domain derived from a selectin, such as E-selectin, L-selectin, or P-selectin.

[0122]

[0148] In some embodiments, CFP does not comprise full-length intracellular signaling domain.In some embodiments, intracellular domain is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400 or 500 amino acid long.In some embodiments, intracellular domain is at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400 or 500 amino acid long.

[0123]

[0149] In some embodiments, the recombinant nucleic acid encodes an FcR α chain extracellular domain, an FcR α chain transmembrane domain, and / or an FcR α chain intracellular domain. In some embodiments, the recombinant nucleic acid encodes an FcR β chain extracellular domain, an FcR β chain transmembrane domain, and / or an FcR β chain intracellular domain. In some embodiments, the FcR α chain or FcR β chain, when expressed intracellularly, forms a complex with FcRγ. In some embodiments, the FcR α chain or FcR β chain, when expressed intracellularly, forms a complex with endogenous FcRγ. In some embodiments, the FcR α chain or FcR β chain is not incorporated into the membrane of cells that do not express FcRγ. In some embodiments, the CFP does not comprise an FcR α chain intracellular signaling domain. In some embodiments, the CFP does not comprise an FcR β chain intracellular signaling domain. In some embodiments, the recombinant nucleic acid encodes a TREM extracellular domain, a TREM transmembrane domain, and / or a TREM intracellular domain. In some embodiments, the TREM is TREM1, TREM2, or TREM3.

[0124]

[0150] In some embodiments, the recombinant nucleic acid comprises a sequence encoding a pro-inflammatory polypeptide. In some embodiments, the composition further comprises a pro-inflammatory nucleotide or nucleotides, e.g., ATP, ADP, UTP, UDP, and / or UDP-glucose, within the recombinant nucleic acid.

[0125] Intracellular interferon-responsive domain

[0151] Most TLRs activate an adaptor protein called 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 that allows kinase enzymes to add phosphate groups to the transcription factor IRF3. This phosphorylation activates IRF3, a member of a family of transcription factors called interferon regulatory factors (IRFs), which activate a wide range of gene expression programs. A manifestation of these programs is the production of type I interferon molecules. Because interferons are potent drivers of a branch of the immune system called the adaptive immune response, their presence risks contributing to autoimmunity. To prevent such attacks by the host's own immune system, the interferon response must be tightly regulated. As a safety valve, a specific sequence of amino acid residues within TRIF, the pLxIS motif, must be phosphorylated before IRF3 can be activated. This control mechanism is not specific to TRIF as an adaptor protein for TLR signaling, but rather involves IRF3 or the related protein IRF7, resulting in a "licensing step," a general manifestation of sensing pathways that drive interferon expression. With one exception, all identified innate sensing pathways that link nucleic acid recognition to type I interferon production have been shown to signal through one of three adaptor proteins known to contain the pLxIS motif: TRIF, MAVS, and STING. Thus, pLxIS motif-containing adaptor proteins specifically connect nucleic acid recognition to antiviral defense. In some embodiments, the intracellular signaling domain of a CFP comprises the ICD of an innate immune response protein.In some embodiments, the innate immune response protein is TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, IFN-receptor, STING, MAVS, TRIF, TASL, NLRP1, NLRP2, NLRP3, NLRP4, NLRP5, NLRP6, NLRP7, NLRP89, NLRP9, NLRP10, NLRP11, NLRP12, NLRP13, NLRP1-14, NOD1, NOD2, Pyrin, AIM2, NLRC4, FCGR3A, FCERIG, CD40, Tank1-binding kinase (TBK), TNFR1, chemokine, MHC class II transactivator (CIITA), IPAF, BIRC1, RIG-I-like receptor (RLR) proteins. In some embodiments, the CFP comprises at least one intracellular signaling domain comprising the amino acid sequence motif pLxIS.

[0126]

[0152] In some embodiments, the composition further comprises a pro-inflammatory polypeptide. In some embodiments, the pro-inflammatory polypeptide is a chemokine or cytokine. In some embodiments, the chemokine is selected from the group consisting of IL-1, IL3, IL5, IL-6, IL-8, 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 cytokine is selected from the group consisting of IL-1, IL3, IL5, 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.

[0127]

[0153] In some embodiments, an intracellular signaling domain derived from an intracellular adaptor protein known to be highly active in innate immune defense is incorporated into the chimeric receptor protein. In some embodiments, one or more mutations are introduced into one or more intracellular domains to reduce the responsiveness of the intracellular domain to intracellular stimuli characteristic of the natural intracellular adaptor protein domain without impairing the efficacy of the chimeric protein. In some embodiments, this efficacy is referred to as enhanced phagocytic potential compared to the same cells that do not express the chimeric protein. In some embodiments, this efficacy is referred to as enhanced inflammatory potential compared to the same cells that do not express the chimeric protein. In some embodiments, this efficacy is referred to as enhanced NF-kappa B activation or interferon activation in cells that express the chimeric protein compared to the same cells that do not express the chimeric protein.

[0128]

[0154] In some embodiments, bone marrow cells are specifically targeted for delivery. Bone marrow cells can be targeted using specialized biodegradable polymers such as PLGA (poly(lactic-co-glycolic acid)) and / or polyvinyl alcohol (PVA). In some embodiments, one or more compounds can be selectively incorporated into such polymer structures to affect bone marrow cell function. In some embodiments, the targeting structures are multilayered, e.g., with one or more PLGA layers and one or more PVA layers. In some embodiments, the targeting structures are assembled in an order for layering activity. In some embodiments, the targeting polymer structures are organized into specifically shaped components, such as flexible structures, that can attach to the bone marrow cell surface and deliver one or more components, such as growth factors and cytokines, to maintain the bone marrow cells in a microenvironment that confers specific polarization. In some embodiments, the polymer structures are configured such that they are not phagocytosed by bone marrow cells and can remain attached on the surface. In some embodiments, the one or more growth factors can be M1 polarization factors, such as cytokines. In some embodiments, the one or more growth factors can be M2-polarizing factors, such as cytokines. In some embodiments, the one or more growth factors can be macrophage-activating cytokines, such as IFNγ. In some embodiments, the polymeric structure is capable of sustained release of the one or more growth factors in an in vivo environment, such as within a solid tumor.

[0129]

[0155] 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 within an untranslated region (UTR) of an mRNA. In some embodiments, the sequence within the UTR is a sequence that binds to an RNA-binding protein. In some embodiments, translation is inhibited or blocked upon binding of the RNA-binding protein to the sequence within the untranslated region (UTR). In some embodiments, the sequence within 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.

[0130]

[0156] 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 cell infected by a pathogen. In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell is a lymphocyte cancer cell. In some embodiments, the target cell is an ovarian cancer cell. In some embodiments, the target cell is a breast cell cancer cell. In some embodiments, the target cell is a pancreatic cell cancer cell. In some embodiments, the target cell is a glioblastoma cell cancer cell.

[0131]

[0157] 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 a circRNA. In some embodiments, the recombinant nucleic acid is a tRNA. In some embodiments, the recombinant nucleic acid is a microRNA.

[0132]

[0158] Also provided herein is a vector comprising a recombinant nucleic acid sequence encoding a CFP described herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector or a lentiviral vector. In some embodiments, the vector further comprises a promoter operably linked to at least one nucleic acid sequence encoding one or more polypeptides. In some embodiments, the vector is a polycistronic vector. In some embodiments, each of the at least one nucleic acid sequence 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 adjacent to at least one nucleic acid sequence encoding one or more polypeptides. In some embodiments, the vector further comprises one or more regulatory regions.

[0133]

[0159] Also provided herein are polypeptides encoded by the recombinant nucleic acids of the compositions described herein.

[0160] Provided herein is a composition comprising a recombinant nucleic acid sequence encoding a CFP (e.g., a phagocytic receptor fusion protein (PFP)) comprising a phagocytic or tethered receptor (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 an antigen of a target cell, wherein the transmembrane domain and the extracellular domain are operably linked; and upon binding of the CFP to the antigen of the target cell, killing or phagocytosis of myeloid cells, such as neutrophils, monocytes, myeloid dendritic cells (mDCs), mast cells, or macrophages, that express the CFP. Compositions are provided in which activity is increased by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or more than 1000% compared to cells that do not express CFP.

[0134]

[0161] Table 1A shows exemplary sequences of chimeric fusion protein domains and / or fragments thereof, which are intended to be non-limiting for the disclosure. The underlines indicate the CDR sequences in the order of CDR1, CDR2, and CDR3 for each heavy and light chain according to the Kabat numbering system. The rightmost column shows the sequences of CDR1, CDR2, and CDR3, respectively, as determined by the Chothia, Kabat, and IMGT systems.

[0135] [Table 2-1]

[0136] [Table 2-2]

[0137] [Table 2-3]

[0138] [Table 3]

[0139] [Table 4]

[0140] [Table 5]

[0141] [Table 6-1]

[0142] [Table 6-2]

[0143] [Table 7]

[0162] Exemplary sequences of chimeric fusion protein domains and / or fragments thereof, which are intended to be non-limiting for the disclosure, are shown below. The underlines indicate the CDR sequences in the order of CDR1, CDR2, and CDR3 for each variable heavy (VH) and variable light (VL) chain according to the Kabat numbering system.

[0144] [Table 8-1]

[0145] [Table 8-2]

[0146] [Table 8-3]

[0147]

Table 8-4

[0148]

Table 8-5

[0149]

Table 8-6

[0150]

Table 8-7

[0151]

Table 8-8

[0152]

Table 8-9

[0153]

Table 8-10

[0163] In some embodiments, an exemplary chimeric fusion protein (CFP) may comprise a sequence that is at least 80% identical to any one of the sequences of SEQ ID NOs: 26-42, 135-137 in Table 4. In some embodiments, an exemplary chimeric fusion protein (CFP) may comprise a sequence that is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% identical to any one of the sequences of SEQ ID NOs: 26-42, 135-137 in Table 4. In some embodiments, an exemplary chimeric fusion protein (CFP) may comprise a sequence that is 90% identical to any one of the sequences of SEQ ID NOs: 26-42, 135-137 in Table 4. In some embodiments, an exemplary chimeric fusion protein (CFP) may comprise a sequence that is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the sequences of SEQ ID NOs: 26-42, 135-137.

[0154]

[0164] In some embodiments herein, there is provided a chimeric fusion protein that includes (i) an extracellular antigen-binding domain capable of binding to a TROP2 antigen, and further includes a TMD of CD89, wherein the extracellular antigen-binding domain is an scFv and includes a variable heavy chain CDR3 having the sequence GGFGSSYWYFDV; and optionally further includes a variable light chain CDR3 having the sequence QQHYITPLT.

[0155]

[0165] In some embodiments herein, provided are chimeric fusion proteins that include (i) an extracellular antigen-binding domain capable of binding to a GPC3 antigen, and further include a TMD of CD89, wherein the extracellular antigen-binding domain is an scFv and includes a variable heavy chain (VH) CDR3 having the sequence FYSYTY.

[0156]

[0166] In some embodiments, an exemplary chimeric fusion protein (CFP) comprises a binding domain that binds to the cancer antigen GPC3, where the binding domain is a VHH and may comprise any one of the variable domains listed in Table 5. The CFP may comprise a binding domain that is an scFv that binds to the GPC3 antigen and comprises any one of the variable domains listed in the table below (Table 5). An exemplary CFP may comprise an extracellular antigen-binding domain comprising any one or more variable domains listed in Table 5 fused to a transmembrane domain (TMD) described herein. In some embodiments, a CFP comprising a GPC3-binding domain comprises one or more VH domains listed in Table 5, a TMD described elsewhere herein, e.g., having the sequence of CD89, and any intracellular domain or combination of intracellular domains described herein. In some embodiments, the CFP comprises one or more VH domains listed in Table 5, a hinge domain, a TMD described elsewhere herein, e.g., having the sequence of CD89, and a GPC3-binding domain comprising one or more intracellular signaling domains or combinations of intracellular signaling domains described herein, e.g., but not limited to, CD40 ICD, TRIF ICD, PI3K recruitment domain ICD, etc. One of skill in the art can use basic reverse engineering of standard molecular biology cloning and recombinant techniques to arrive at constructs encompassed within the scope contemplated herein.

[0157]

[0167]

[0158] [Table 9-1]

[0159] [Table 9-2]

[0160] [Table 9-3]

[0161]

Table 9-4

[0162]

Table 9-5

[0163]

Table 9-6

[0164]

Table 9-7

[0165]

Table 9-8

[0166]

Table 9-9

[0167]

Table 9-10

[0168]

Table 9-11

[0169]

Table 9-12

[0170]

Table 9-13

[0171]

Table 9-14

[0172]

Table 9-15

[0173]

Table 9-16

[0174]

Table 9-17

[0175]

Table 9-18

[0176]

Table 9-19

[0177]

Table 9-20

[0178]

Table 9-21

[0179]

Table 9-22

[0180]

Table 9-23

[0181]

Table 9-24

[0182] [Table 9-25] Provided herein is a composition comprising a recombinant nucleic acid sequence encoding a CFP (e.g., a phagocytic receptor fusion protein (PFP)) comprising a phagocytic or tethering receptor (PR) subunit, the composition comprising an extracellular domain comprising an antigen-binding domain specific for an antigen of a target cell; a transmembrane domain; and an intracellular domain comprising an intracellular signaling domain, wherein the transmembrane domain and the extracellular domain are operably linked; and the CFP is expressed by neutrophils, monocytes, myeloid dendritic cells, or the like, upon binding of the CFP to the antigen of the target cell. Compositions are provided in which the killing or phagocytic activity of bone marrow cells, such as mDCs, mast cells, or macrophages, 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.

[0183]

[0169] Provided herein is a recombinant nucleic acid sequence encoding a CFP described in the immediately preceding paragraph, which includes at least one innate immune-activating intracellular domain, e.g., a pattern recognition receptor intracellular signaling domain, a TLR intracellular signaling domain, an FcR intracellular signaling domain, an intracellular adaptor protein signaling domain, or a fragment thereof, wherein the intracellular domain, upon contact with its target antigen, e.g., upon engagement of the antigen-binding domain with the target antigen, is capable of activating the innate immune response of myeloid cells, activating their phagocytic potential, activating inflammatory cytokine and chemokine responses, antigen presentation and T cell activation of myeloid cells expressing the CFP.

[0184] 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, NLRP family members NLRP1-14, NOD1, NOD2, pyrin, AIM2, NLRC4, FCGR3A, FCERIG, IL-1, IL3, IL5, IL-6, IL-12, IL-13, IL-23, TNF, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, or RANTES.

[0185]

[0171] In some embodiments, the CFP comprises an intracellular signaling domain comprising a sequence derived from a protein that activates interferon-responsive transcription factor IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, or IRF9.

[0186] In some embodiments, the CFP comprises an intracellular signaling domain comprising a sequence derived from an intracellular adaptor protein. In some embodiments, the adaptor protein may comprise a transmembrane protein that tethers the CFP to an organelle, such as a mitochondrial, endoplasmic reticulum, or lysosomal compartment. In some embodiments, the intracellular adaptor protein is a cytosolic protein.

[0187] A CFP described herein can 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, a CFP described herein can comprise a signal peptide sequence of Table 1B.

[0188] In some embodiments, a CFP can comprise a sequence having at least 85% sequence identity to a sequence in Table 4. For example, a CFP can 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, a CFP further comprises a signal peptide sequence, such as a signal peptide sequence in Table 1B.

[0189] In some embodiments, the intracellular signaling domain comprises the amino acid sequence of any one of SEQ ID NOs: 19-22, or an intracellular signaling domain derived from the TRIF intracellular domain having at least 85% sequence identity to 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 to any one of SEQ ID NOs: 19-22. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 90% sequence identity to 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 to any one of SEQ ID NOs: 19-22. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from the TRIF intracellular signaling domain having at least 95% sequence identity to any one of SEQ ID NOs: 19-22. In some embodiments, the intracellular domain of the CFP comprises an intracellular signaling domain derived from a TRIF intracellular signaling domain having at least 90% sequence identity to any one of SEQ ID NOs: 19-22, and the CFP comprises an extracellular binding domain 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 a TRIF intracellular signaling domain having at least 90% sequence identity to any one of SEQ ID NOs: 19-22; in this case, the CFP comprises an extracellular binding domain 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, together with 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 a TRIF intracellular signaling domain having at least 90% sequence identity to any one of SEQ ID NOs: 19-22, and the CFP comprises an extracellular binding domain 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, and comprises one or more additional intracellular signaling domains, such as 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 a PI3 kinase recruitment domain, or a CD40 intracellular signaling domain.

[0190] In some embodiments, exemplary anti-TROP2-binding CFPs described herein comprise an extracellular antigen-binding domain having the sequence of any one of SEQ ID NOs: 1-3, or a heavy chain variable domain comprising the CDR3 sequence of GGFGSSYWYFDV and / or a light chain variable domain comprising the CDR3 sequence of QQHYITPLT, and further comprise an intracellular domain of any one of the sequences in Table 2 or Table 3. In some embodiments, exemplary anti-TROP2-binding CFPs described herein comprise a sequence having at least 80-100% sequence identity to any one of SEQ ID NOs: 26-33.

[0191] IRF-induced proteins and IRF-activated pathways Type I IFNs are key cytokines mediating innate antiviral immunity and thus driving proinflammatory responses. Type I IFNs are readily induced by cGMP-AMP synthase, retinoic acid-inducible protein 1 (RIG-I)-like receptors, and Toll-like receptors, which recognize microbial double-stranded (ds) DNA, dsRNA, and LPS. These signaling pathways converge with 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-inducible IFN-β) mediate IRF-3 recruitment via a conserved pLxIS motif. While the pLxIS motifs of phosphorylated STING, MAVS, and TRIF generally bind to IRF-3 in a similar manner, residues upstream of the motif confer specificity. Type I IFNs, such as IFN-α and IFN-β, are a major cytokine family mediating antiviral immunity. Microbial dsDNA in the cytosol binds to and activates cGAS (cGMP-AMP synthase), an enzyme that 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, directing 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, translocates to the nucleus, and initiates IFN-β gene transcription. In contrast, in the cytosol, viral dsDNA is sensed by RLRs [retinoic acid-inducible protein 1 (RIG-I)-like receptors], which activate IRF-3 through the adaptor protein MAVS (mitochondrial antiviral signaling).Furthermore, the Toll-like receptors (TLRs), TLR3 and TLR4, which recognize viral dsRNA in endosomes and LPS, a bacterial cell wall component, also mediate the induction of type I IFN and proinflammatory cytokines, respectively (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 upon the activation of TBK1 and IRF-3. Mechanistically, the adaptor proteins STING, MAVS, and TRIF contain a conserved motif, pLxIS (p represents a hydrophilic residue, x represents any residue, and S represents the phosphorylation site), which is phosphorylated by TBK1 or IKKε and mediates the recruitment of IRF-3 to the signaling complex. The induction of proximity between TBK1 and IRF-3 results in the phosphorylation and activation of IRF-3. Furthermore, IRF-3 itself also contains a pLxIS motif, which is crucial for phosphorylation-induced dimerization / activation of IRF-3. Mutation of the serine phosphorylation site within the pLxIS motif of STING, MAVS, and TRIF abolishes the induction of type I IFN in their respective signaling pathways. However, the precise molecular mechanism of IRF-3 recruitment and activation remains unknown. To elucidate the structural basis of IRF-3 recruitment by phosphorylated STING (pSTING), MAVS (pMAVS), and TRIF (pTRIF), we expressed peptides containing the pLxIS motif from the three adaptor proteins, phosphorylated them with TBK1 in vitro, and determined the crystal structure of their complex with the C-terminal domain (CTD) of IRF-3.

[0192] Mechanism of IRF-3 recruitment by pMAVS and pTRIF. In contrast to dsDNA sensing via the cGAS-STING pathway, RLRs sense dsDNA in the cytosol and activate IRF-3 via the adaptor MAVS, whereas TLR3 and TLR4 recruit IRF-3 using the adaptor TRIF. Phosphorylation of the pLxIS motif of MAVS or TRIF is required for IRF-3 recruitment and activation.

[0193] TRAF-interacting proteins: The presence of a TRAF domain, a protein-interaction domain of approximately 180 amino acids, is a distinctive feature of TRAF family proteins. In mammals, six of the seven TRAF proteins in the family (TRAF1-TRAF6) conform to this criterion and have been identified as part of the TRAF family. The TRAF domain can be subdivided into two distinct regions: the TRAF-N domain and the TRAF-C domain. Diverse receptors bind to the TRAF-C domain, whereas diverse intracellular signaling molecules bind to the TRAF-N domain. Despite the structural similarity of the TRAF domains, each TRAF protein has a distinct biological function with specificity for its interaction partners: upstream receptors and downstream effector molecules. The structure of the TRAF2 TRAF domain was first reported by Dr. Wu's research group around 1999, and the structure of the TRAF6 TRAF domain was reported by the same research group three years later. Since then, the structures of the TRAF domains of TRAF3, TRAF5, TRAF4, and TRAF1 have also been reported. The TRAF structures revealed that the TRAF-N domain is a coiled-coil structure, while TRAF-C is composed of seven to eight antiparallel β-sheet folds. Structural alignment of all six TRAF family members indicates that the TRAF-C domains are well aligned, whereas the position and length of TRAF-N vary among TRAF family members. Sequence analysis indicates that while the length of TRAF-N varies within the family, the length of the TRAF-C domain is conserved: TRAF-N in TRAF4 and TRAF6 is relatively short, whereas TRAF-N in TRAF3 and TRAF5 is relatively long. Although the overall structures are nearly identical, clear structural differences were observed. For example, the length and position of some loops within the TRAF domains of TRAF4 and TRAF6 differ from those in other TRAF family members. TRAF4 contains a more negatively charged surface within the receptor-binding region, whereas TRAF6 contains a more positively charged surface within the receptor-binding region.Because surface features often determine their mode of interaction with partners, the electrostatic surface of TRAF domains, which is similar among TRAF1, TRAF2, TRAF3, and TRAF5, i.e., the diverse charged surfaces, have been shown to be important for accepting diverse receptors into the same binding pocket in a similar interaction mode. In contrast, the different features on the binding surfaces of functionally distinct TRAFs, TRAF4 and TRAF6, indicate that TRAF4 and TRAF6 accept different receptors in different interaction modes.

[0194] For purposes of this disclosure, any pathway, signaling intermediate, or activating moiety discussed in the preceding paragraphs may be considered activatable or functional upon induction of a CFP disclosed herein, depending on the application. Similarly, the CFPs disclosed herein may be useful in targeting any of the applicable targets described within the pathways discussed. It is understood that any pathway or portion thereof readily known to one of skill in the art as of the date of existing literature regarding signaling domains, signaling pathways, signaling intermediates, and transcription factors of activated genes is within the contemplation of this disclosure.

[0195] Chimeric proteins with TLR intracellular domains, TLR intracellular signaling pathways, and activation of NF-kappa B: 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 a TLR intracellular signaling domain can activate NF-kappaB upon engagement of the extracellular domain of the receptor with 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 a TLR3 protein. In some embodiments, the intracellular signaling domain can be derived from a TLR7 protein, a TLR8 protein, or a 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, an IFN receptor, NLRP-1, NLRP-2, NLRP-3, NLRP-4, NLRP-5, NLRP-6, NLRP-7, NLRP-8, NLRP-9, NLRP-10, NLRP-11, NLRP-12, NLRP-13, NLRP-14, NOD1, NOD2, Pyrin, AIM2, NLRC4, and / or CD40.

[0196] In some embodiments, the phagocytic scavenger receptor (PR) fusion protein (PFP) comprises a pro-inflammatory cytoplasmic domain for activation of the IL-1 signaling cascade.

[0197]

[0183] In some embodiments, the cytoplasmic portion of the chimeric receptor (e.g., phagocytic receptor (PR) fusion protein (PFP)) comprises a cytoplasmic domain derived from a toll-like receptor, such as the intracellular signaling domains 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).

[0198]

[0184] In general, TLRs have diverse subcellular localizations, developmental pathways, activation, recognition, and modes of action. TLRs are expressed in innate immune cells such as dendritic cells (DCs) and macrophages, as well as non-immune cells such as fibroblasts and epithelial cells. Cell surface TLRs primarily recognize microbial membrane components such as lipids, lipoproteins, and proteins. TLR4 recognizes bacterial lipopolysaccharide (LPS). TLR2, together with TLR1 or TLR6, recognizes a wide variety of PAMPs, including lipoproteins, peptidoglycan, lipoteichoic acid, zymosan, mannan, and tGPI-mucin. TLR5 recognizes bacterial flagellin. TLR10 is a pseudogene in mice due to the insertion of a stop codon, but human TLR10 recognizes ligands derived from Listeria monocytogenes in cooperation with TLR2. TLR10 also senses viral infection by influenza A virus.

[0199] Intracellular TLRs recognize nucleic acids derived from bacteria and viruses, and in disease states such as autoimmunity, they also recognize self-nucleic acids. TLR3 recognizes viral double-stranded RNA (dsRNA), small interfering RNA, and self-RNA derived from damaged cells. TLR7 is primarily expressed in plasmacytoid DCs (pDCs) and recognizes single-stranded (ss) RNA derived from viruses. TLR7 also recognizes RNA derived from group B streptococcus in conventional DCs (cDCs). Human TLR8 responds to viral and bacterial RNA. Structural analysis revealed that unstimulated human TLR8 exists as a preformed dimer, and the Z loop between LRR14 and LRR15 is cleaved into N- and C-terminal halves but maintains association with each other and participates in ligand recognition and dimerization. Ligand binding induces reorganization of the dimer, bringing 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 by-product produced by Plasmodium falciparum during the detoxification process after host hemoglobin is digested. TLR11 is localized within endolysosomes and recognizes flagellin or an unknown protein component of uropathogenic Escherichia coli (UPEC), as well as a profilin-like molecule from Toxoplasma gondii. TLR12 is expressed primarily in myeloid cells and recognizes profilin from T. gondii, which is highly similar to TLR11. TLR12 functions as a homodimer or 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 as well as for preventing TLRs from coming into contact with self-nucleic acids, which can lead to autoimmunity.

[0200] Individual TLRs differentially recruit members of a set 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 for the induction of inflammatory cytokine genes. TIRAP is a selective adaptor that recruits MyD88 to cell surface TLRs, such as TLR2 and TLR4. TIRAP also participates in signaling through endosomal TLRs, such as TLR9. The lipid-binding domain of TIRAP binds PI(4,5)P2 at the plasma membrane and PI(3)P on endosomes, mediating the formation of functional TLR4 / TLR9 signaling complexes at the respective sites. Thus, TIRAP associates with both cell surface and endosomal TLRs by binding to different lipids. TRIF is recruited to TLR3 and TLR4, promoting an alternative pathway that leads to the activation of IRF3, NF-κB, and MAPK for the induction of type I IFN and proinflammatory cytokine genes. TRAM is selectively recruited to TLR4, but not to TLR3, to link TRIF and TLR4. TLR3 interacts directly with TRIF, and this interaction requires phosphorylation of two tyrosine residues in the cytoplasmic domain of TLR3 by epidermal growth factor ErbB1 and Btk. After TLR engagement, MyD88 forms a complex with members of the IRAK kinase family. IRAK4 activates IRAK1, which is then autophosphorylated at several sites and released from MyD88. IRAK1 associates with TRAF6, a RING domain E3 ubiquitin ligase. TRAF6, together with the ubiquitin-conjugating enzymes 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 TRAF6-generated polyubiquitin chains and drive TAK1 activation.TAK1 then activates two distinct pathways, the IKK complex-NF-κB pathway and the IKK complex-MAPK pathway. The IKK complex is composed of catalytic subunits IKKα and IKKβ and a regulatory subunit, NEMO (also called IKKγ). TAK1 binds to the IKK complex via ubiquitin chains, allowing the IKK complex to phosphorylate and activate IKKβ. The IKK complex phosphorylates the NF-κB inhibitor protein IκBα, which undergoes proteasomal degradation, allowing NF-κB to translocate to the nucleus and induce pro-inflammatory gene expression. TAK1 activation also results in the activation of MAPK family members, such as ERK1 / 2, p38, and JNK, which mediate the activation of AP-1 family transcription factors or mRNA stabilization, thereby regulating inflammatory responses.

[0201] 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; or a functional domain such as a signaling domain. In some embodiments, the intracellular domains described in this section, such as MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, TASL, or any other domain mentioned herein, can be adjusted or modified to pair with another domain or component thereof. Pairing is intended to mean the incorporation into the CFP design of a portion of one or more domains under consideration, or a fragment or component thereof, as any part of the molecular structure of the CFP protein. In some embodiments, adjustment can be the structural alignment or positioning of domains within a CFP molecule, e.g., two domains under consideration are juxtaposed or separated by one or more domains between them, or separated by one or more amino acids between them. The one or more amino acids may be linkers. The one or more amino acids may provide structural separation between two adjacent domains, may provide flexibility between two adjacent domains, or may impart more three-dimensional orientation to a molecular structure, including domains without one or more amino acids. In some embodiments, the adjustment or alteration may include modifications within the domains, such as mutations.

[0202] In some embodiments, an intracellular domain described in a section herein, such as MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, TASL, or any other domain mentioned herein, can be tailored or modified for pairing with or incorporation into another intracellular domain within a 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 block tonic signaling. In some embodiments, one of the other intracellular domains, such as any one or more of the domains MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, TASL, or fragments thereof, is modified to reduce or eliminate tonic signaling.

[0203] In some embodiments, an intracellular domain described in a section herein, such as MyD88, TRIF, TIRAP / MAL, TLR, MAVS, MDA5, STING, RIG1, TASL, or any other domain mentioned herein, may be adjusted or modified for pairing with or incorporation into 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.

[0204] For purposes of this disclosure, any pathway, signaling intermediate, or activating moiety discussed in the paragraphs above may be considered activatable or functional, depending on the application, upon induction of the disclosed CFPs, including the TLR intracellular signaling domains disclosed herein. Similarly, the CFPs disclosed herein may be useful in targeting any of the applicable targets described within the pathways discussed. It is understood that any pathway, or portion thereof, readily known to one of skill in the art as of the date of existing literature regarding signaling domains, signaling pathways, signaling intermediates, and transcription factors of activated genes is within the contemplation of this disclosure.

[0205] therapeutic composition

[0191] In one aspect herein, a composition is provided comprising a polynucleic acid, e.g., a recombinant polynucleic acid encoding a CFP described herein, comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises an scFv or VHH capable of binding to a specific cancer antigen expressed on cancer cells, and the CFP is designed for specific expression 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 mosaic myeloid cells / macrophages / dendritic cells. The composition is designed to be administered to a subject in need thereof, e.g., via a systemic administration route, thereby producing myeloid cells engineered in vivo, e.g., such that the polynucleic acid (e.g., mRNA encoding the CFP) in the composition is substantially expressed in myeloid cells and substantially not expressed in non-myeloid cells. Exemplary non-myeloid cells include lymphoid cells, such as T cells and B cells. For example, a CFP encoded by a polynucleic acid described herein is "substantially expressed" in myeloid cells when CFP expression is detected in 50% or more of myeloid cells in a mixed population of myeloid and non-myeloid cells tested in vitro at a given time, and less than 5%, or less than 4%, or less than 3%, or less than 2% of T cells or B cells may express CFP. A design for myeloid cell-specific expression involves having a transmembrane domain capable of dimerizing or multimerizing with endogenous proteins in myeloid cells, such that dimerization or multimerization confers expression, stability, and functionality of the CFP on the membrane of myeloid cells without significant expression in non-myeloid cells (e.g., T cells). In some embodiments, the composition is formulated for in vivo delivery of the polynucleic acid. In some embodiments, the polynucleic acid is RNA. In some embodiments, the polynucleic acid is mRNA. In some embodiments, the delivery vehicle comprises a lipid. In some embodiments, the delivery vehicle comprises a lipid nanoparticle (LNP).

[0206]

[0192] In some embodiments herein, therapeutic compositions are provided that include a chimeric fusion protein, such as a chimeric fusion receptor protein (CFP), wherein the CFP includes: (a) (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) an extracellular domain that includes a hinge domain derived from a CD89 hinge sequence, (b) a CD89 transmembrane domain, and (c) an intracellular domain that includes one or more intracellular signaling domains, wherein the one or more intracellular signaling domains are selected from the group consisting of an intracellular signaling domain derived from FcRγ or FcRε, a PI3-kinase recruitment domain, a CD40 intracellular signaling domain, and a CD40 intracellular signaling domain. or an intracellular signaling domain derived from an inflammatory signaling molecule, for example, any one of the intracellular signaling domains derived from TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, an IFN receptor, STING, MAVS, TRIF or TASL intracellular domain, an NLRP family member, NLRP1-14, NOD1, NOD2, pyrin, AIM2, NLRC4, FCGR3A, FCERIG, IL-1, IL3, IL5, IL-6, IL-12, IL-13, IL-23, TNF, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, or RANTES.

[0207] In some embodiments, bone marrow cells can be further modified or engineered to generate therapeutically effective bone marrow cells. The isolated cells can be engineered by expressing genes, or fragments thereof, within the cells without altering their functional / developmental plasticity, differentiation potential, and cell viability.

[0208] In some embodiments, bone marrow cells can be further modified or engineered to generate therapeutically effective bone marrow cells by expressing a non-endogenous polynucleotide into the cells. The non-endogenous polynucleotide encodes a protein or peptide. Alternatively, the non-endogenous polypeptide can be a non-coding sequence, such as an inhibitory RNA or a morpholino.

[0209] In some embodiments, bone marrow cells can be further modified or engineered to generate therapeutically effective bone marrow cells by stably altering the genomic sequence of the cells. In some embodiments, bone marrow cells can be engineered by editing the bone marrow cell genome using a CRISPR-CAS system. In some embodiments, one or more genes can be edited to silence gene expression. In some embodiments, bone marrow cells can be engineered to delete genes. In some embodiments, one or more genes can be edited to enhance genes. In some embodiments, genetic material is introduced into bone marrow cells in the form of messenger RNA, where the messenger RNA encodes a protein or peptide, thereby rendering the bone marrow cells therapeutically effective. In some embodiments, naked DNA or messenger RNA (mRNA) can be used to introduce nucleic acids into bone marrow cells. In some embodiments, DNA or mRNA encoding a chimeric antigen receptor is introduced into phagocytic cells by encapsulation in lipid nanoparticles (LNPs). The mRNA is single-stranded and may be codon-optimized. In some embodiments, the mRNA may include one or more modified or unnatural bases, such as 5'-methylcytosine, or pseudouridine or methylpseudouridine. In some embodiments, about 50% or more of the uridine ("U") residues in the mRNA may be converted to methylpseudouridine. In some embodiments, the mRNA is between 2 kb and 20,000 kb in length. In some embodiments, the mRNA may be between 50 and 10,000 bases in length. In one aspect, the transgene is delivered as mRNA. The mRNA may contain more than about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 bases. In some embodiments, the mRNA may be more than 10,000 bases in length. In some embodiments, the mRNA may be about 11,000 bases in length.In some embodiments, the mRNA may be about 12,000 bases in length. In some embodiments, the mRNA comprises a transgene sequence encoding a fusion protein. In some embodiments, an mRNA encoding a CFP described elsewhere in this disclosure may comprise a 5' untranslated region (UTR). In some embodiments, an mRNA encoding a CFP may further comprise a 3' UTR. In some embodiments, a 5' UTR described herein may comprise the sequence: GGGAGACCCAAGCUGGCUAGCGUUAAACUUAAGCUUGCCACC (SEQ ID NO: 138); or a sequence at least 90% identical to SEQ ID NO: 138. In some embodiments, a 5' UTR described herein may comprise the sequence: GGGAGACCCAAGCUGGCUAGCGCCACC (SEQ ID NO: 139).

[0210] In some embodiments, the mRNA has the 3'UTR sequence CUCGAGUCUAGAGGGCCCGUUUAAACCCGCUGAUCAGCCUCGACUGUGCCUUCUAGUUGCCAGCCAUCUGUUGUUUGCCCCUCCCCCGUGCCUUCCUUGACCCUGGAAGGUGCCACUCCCACUGUCCUUUCCUAAUAAAAUGAGGAAAUUGCAUCGCAUUGUCUGAGUAGGUGUCAUUCUAUUCUGGGGGGUGGGGUGGGGCAGGACAGCAAGGGGGAGGAUUGGGAAGACAAUAGC (SEQ ID NO: 140). In some embodiments, the recombinant polynucleic acid comprises one or more elements for stabilizing the structure. Of particular importance for long mRNAs is having 5' cap protection. In some embodiments, the RNA products required to generate therapeutically engineered bone marrow cells are generated in vitro and are 5' capped and 3' poly-A tailed. In some embodiments, the 5' cap is modified from the 5' cap of the native mRNA.

[0211] In some embodiments, recombinant mRNAs encoding CFPs described elsewhere in this disclosure can contain an efficient cap structure using an appropriate capping analog. In some embodiments, RNA products prepared by IVT mRNA are co-transcriptionally capped with a dinucleotide derived from m7GpppN. In some embodiments, the 5'-cap is a trinucleotide cap analog. In some embodiments, the first nucleotide base following the 5'-cap plays an important 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 In some embodiments, the cap analog is m 7 In some embodiments, the cap analog is m 7 Gpppm 6 In some embodiments, the cap analog is m 7 Gpppm 6 In some embodiments, the cap analog is m 7 In some embodiments, the cap analog is m 7 In some embodiments, the cap analog is m 7 In some embodiments, the cap analog is m 7 In some embodiments, the cap analog is m 7 In some embodiments, the cap analog is m 7 In some embodiments, the 5'-cap is GpppUmpG. 7 G(m2 7,3-OThe 5'-cap is an anti-reverse cap analog (ARCA) with a chemically modified 3-O or 2-O position of 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 stability and translation efficiency to mRNA. In some embodiments, the cap-proximal sequence is important for mRNA stability and function.

[0212] In some embodiments, a recombinant mRNA described herein encoding a CFP can include a polyA sequence comprising 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.

[0213]

[0199] DNA or RNA encapsulated in LNPs may be used to transfect macrophages or administered to a subject. In some embodiments, mRNA is incorporated into effector myeloid cell populations by transient transfection. In some embodiments, the transient transfection method comprises electroporation of mRNA. In some embodiments, the transient transfection comprises chemical transfection. In some embodiments, 1 to 5,000 micrograms of mRNA per ml may be used for transfection using protocols suitable for the methods described above. In some embodiments, 1 to 2,000 micrograms of mRNA per ml may be used for transfection. In some embodiments, 1 to 1,000 micrograms of mRNA per ml may be used for transfection. In some embodiments, 1 to 1,000 micrograms of mRNA per ml may be used for transfection. In some embodiments, 1 to 500 micrograms of mRNA per ml may be used for transfection. In some embodiments, 1-250 micrograms of mRNA per ml may be used for transfection. In some embodiments, about 500 micrograms or less of mRNA per ml may be used for transfection. In some embodiments, about 250 micrograms or less of mRNA per ml may be used for transfection. In some embodiments, about 10 micrograms of mRNA per ml is used. In some embodiments, about 20 micrograms of mRNA per ml is used. In some embodiments, about 30 micrograms of mRNA per ml is used. In some embodiments, about 40 micrograms of mRNA per ml is used. In some embodiments, about 50 micrograms of mRNA per ml is used. In some embodiments, about 60 micrograms of mRNA per ml is used. In some embodiments, about 80 micrograms of mRNA per ml is used.In some embodiments, about 100 micrograms of mRNA per ml is used. In some embodiments, about 150 micrograms of mRNA per ml is used. In some embodiments, about 200 micrograms of mRNA per ml is used. In some embodiments, about 20, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 micrograms of mRNA per ml is used. An appropriate cell density is selected for transfection based on the manufacturer's instructions for the method, instrument, and / or reagent, or as would be known to one skilled in the art.

[0214]

[0200] Exemplary mRNA sequences encoding the two CFPs described herein are provided below. mRNA sequences encoding exemplary anti-TROP2 CFPs described herein GGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUUGCCACCCUCGAGUCUAGAGGGCCCGUUUAAACCCGCUGAUCAGCCUCGACUGUGCCUUCUAGUUGCCAGCCAUCUGUUGUUUGCCCCUCCCCCGUGCCUUCCUUGACCCUGGAAGGUGCCACUCCCACUGUCCUUUCCUAAUAAAAUGAGGAAAUUGCAUCGCAUUGUCUGAGUAGGUGUCAUUCUAUUCUGGGGGGUGGGGUGGGGCAGGACAGCAAGGGGGAGGAUUGGGAAGACAAUAGC (SEQ ID NO: 141). The underlined sequences represent the 5'UTR and 3'UTR sequences, respectively. In some embodiments, the mRNA encoding the 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 the anti-TROP2 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: 141 not including the 5'UTR and / or 3'UTR.

[0215]

[0202] mRNA sequence encoding anti-GPC3 CFP GGGAGACCCAAGCUGGCUAGCGCCACCCUCGAGUCUAGAGGGCCCGUUUAAACCCGCUGAUCAGCCUCGACUGUGCCUUCUAGUUGCCAGCCAUCUGUUGUUUGCCCCUCCCCCGUGCCUUCCUUGACCCUGGAAGGUGCCACUCCCACUGUCCUUUCCUAAUAAAAUGAGGAAAUUGCAUCGCAUUGUCUGAGUAGGUGUCAUUCUAUUCUGGGGGGUGGGGUGGGGCAGGACAGCAAGGGGGAGGAUUGGGAAGACAAUAGC (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 not including the 5'UTR and / or 3'UTR.

[0216] In some embodiments, the recombinant nucleic acid is mRNA. The mRNA construct can be thawed on ice and gently pipetted into monocytes and premixed. In some embodiments, the mRNA is electroporated into the cells. After elutriation, the cells can be pooled, centrifuged, and electroporated with the mRNA using a MaxCyte ATX system optimized for this purpose. In some embodiments, electroporation buffer, cell density, and / or mRNA concentration optimized for each protocol for each construct are used.

[0217] In some embodiments, polynucleotides can be introduced into bone marrow cells in the form of circular RNAs (circRNAs), in which the 3' and 5' ends are covalently linked. CircRNAs can be delivered into cells using LNPs.

[0218] In some embodiments, stable integration of transgenes into macrophages and other phagocytes can be achieved through the use of transposases and transposable elements, particularly mRNA-encoded transposases. In one embodiment, long interspersed element-1 (L1) RNA can be envisioned for retrotransposition of transgenes and stable integration into macrophages or phagocytes. Retrotransposons can be used for stable integration of recombinant nucleic acids encoding CFPs.

[0219] In some embodiments, bone marrow cells can be modified by expressing a transgene via incorporation of the transgene into a transient expression vector. In some embodiments, expression of the transgene can be temporally regulated from outside the cell by a regulatory factor. Examples include the Tet-on / Tet-off system, in which transgene expression is regulated via the presence or absence of tetracycline.

[0220]

[0207] In some embodiments, bone marrow cells can be modified to produce therapeutically effective cells by contacting the cells with a compound, which may be a protein, enzyme, inhibitor, or activator within the bone marrow cells.

[0221] In some embodiments, a polynucleotide encoding a chimeric antigen receptor may be introduced into isolated bone marrow cells obtained by the methods described in the preceding paragraphs, wherein the chimeric antigen receptor, upon expression in the bone marrow cells, enhances the innate immune response function of the bone marrow cells. In some embodiments, expression of the chimeric antigen receptor may direct bone marrow cells to specific targets in vivo or in vitro. In some embodiments, the chimeric antigen receptor may increase the phagocytic potential of bone marrow cells. In some embodiments, the chimeric antigen receptor increases the immunogenicity of bone marrow cells. In some embodiments, the chimeric antigen receptor may enhance intracellular signaling. In some embodiments, the chimeric antigen receptor may function cooperatively with one or more proteins within a cell. In some embodiments, the chimeric antigen receptor may dimerize or multimerize with a second receptor or transmembrane protein within the bone marrow cells, where the second receptor or transmembrane protein is an endogenous protein.

[0222]

[0209] In some embodiments herein, therapeutic compositions are provided that include a chimeric fusion protein, such as a chimeric fusion receptor protein (CFP), the CFP comprising: (a) (i) an scFv that specifically binds to any one of the targets disclosed herein, and (ii) an extracellular domain comprising at least a portion of a hinge domain derived from CD8, a hinge domain derived from CD28, or an extracellular domain derived 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, the at least two intracellular signaling domains being: (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, an IFN receptor, STING, MAVS, TRIF, or TASL intracellular domain, an NLRP family member, NLRP1-14, NOD1, NOD2, pyrin, AIM2, NLRC4, FCGR3A, FCERIG, IL-1, IL3, IL5, IL-6, IL-12, IL-13, IL-23, TNF, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, or RANTES, and a PI3K recruitment domain, or a domain derived from CD40.

[0223] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a cell population comprising bone marrow cells. In some embodiments, the cell population differentiates into effector cells in a subject after administration; infiltrates or migrates to an affected site in a subject after administration; and / or has a lifespan of at least 5 days in a subject after administration. 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. Some embodiments provided herein are therapeutic compositions comprising less than 20%, less than 15%, less than 10%, or less than 5% dendritic cells. The bone marrow cells for the therapeutic compositions described herein comprise a recombinant nucleic acid encoding a chimeric fusion protein, wherein the recombinant nucleic acid encodes a CFP receptor protein or an engager protein, as described herein. Bone marrow cells for the therapeutic compositions described herein express a CFP encoded by a recombinant nucleic acid or express an engager protein encoded by a recombinant nucleic acid described herein.

[0224] In some embodiments, cells are cultured ex vivo immediately after thawing or after nucleic acid incorporation. In some embodiments, ex vivo culture is performed in the presence of an appropriate medium, which may include regulated serum components, such as human serum albumin (HSA). In some embodiments, ex vivo culture and manipulation may be performed in a medium with a low serum content. In some embodiments, the serum is specifically treated for complement inactivation. In some embodiments, bone marrow cells may be cultured ex vivo as described above in the presence of M-CSF. In some embodiments, bone marrow cells may be cultured ex vivo as described above in the presence of GM-CSF. In some embodiments, bone marrow cells may be cultured in the presence of one or more cytokines. In some embodiments, bone marrow cells may 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 involve the separation or enrichment and manipulation of bone marrow 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 bone marrow cells may be cultured for less than 24 hours, or less than 20 hours, or less than 16 hours, or less than 14 hours, or less than 12 hours, or less than 10 hours, or less than 8 hours, or less than 6 hours, or less than about 4 hours. After separation or enrichment and manipulation, the bone marrow cells may be cultured for a short period of time and frozen until further use. In some embodiments, the bone marrow cells are thawed once or at most twice.

[0225]

[0212] In some embodiments, therapeutically competent cells are cells electroporated with a recombinant nucleic acid encoding a polypeptide, frozen and thawed, and the culture stabilized for less than 24 hours, wherein the cells in the cell population at the time of administration exhibit (i) at least 70% viability, (ii) at least 50% CD14+ / CD16− cells; and / or more than 50% CD11b+ / CD14+ / CD16− cells; (iii) less than 5% CD3+ cells, less than 5% CD19+ cells, less than about 10% CD56+ cells, and less than about 10% CD42b+ cells; and (iv) more than 50% cells expressing the polypeptide encoded by the electroporated nucleic acid. In some embodiments, therapeutically competent cells are cells electroporated with a recombinant nucleic acid encoding a polypeptide, the culture stabilized for less than 24 hours, frozen, and thawed, wherein the cells in the cell population at the time of administration exhibit (i) greater than 70% viability, (ii) at least greater than 50% CD14+ / 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, and less than about 10% CD42b+ cells; and (iv) greater than 50% cells expressing the polypeptide encoded by the electroporated nucleic acid. In some embodiments, therapeutically competent cells are cells whose culture has been stabilized for less than 24 hours, electroporated with a recombinant nucleic acid encoding a polypeptide, and then frozen and thawed, wherein the cells in the cell population at the time of administration exhibit (i) at least 70% viability, (ii) at least 50% CD14+ / CD16- cells; and / or more than 50% CD11b+ / CD14+ / CD16- cells; (iii) less than 5% CD3+ cells, less than 5% CD19+ cells, less than about 10% CD56+ cells, and less than about 10% CD42b+ cells; and (iv) more than 50% cells expressing the polypeptide encoded by the electroporated nucleic acid. The cells must be pathogen-free.In the above embodiments, the therapeutically competent cells may be 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. Prior to administration, the cells are inspected for quality assurance to meet the standards described herein in this disclosure.

[0226]

[0213] Provided herein is a method for treating cancer in a subject using a pharmaceutical composition comprising engineered phagocytes, particularly macrophages, expressing a recombinant nucleic acid encoding a CFP, specifically designed to target, attack, and kill cancer cells. CFPs are also alternatively referred to as chimeric antigen receptors (CARs), particularly CARs for phagocytosis (CAR-Ps), and either term can be used interchangeably herein. In the description herein, engineered phagocytes are also referred to as CAR-P cells.

[0227] In some embodiments, the recombinant polynucleic acid composition comprises a lipid nanoparticle (LNP). In some embodiments, the recombinant polynucleic acid composition further comprises a nucleic acid delivery vehicle comprising 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 is 100-300 nm in diameter. In some embodiments, the lipid nanoparticle comprises (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA; MC3). In some embodiments, the lipid nanoparticle comprises (a) nucleic acid; (b) cationic lipid; (c) non-cationic lipid; and (d) a co-conjugated lipid that inhibits particle aggregation. In some embodiments, the recombinant polynucleic acid composition comprises a polymeric nucleic acid delivery vehicle.

[0228]

[0215] Cancers include, but are not limited to, B-cell cancers (e.g., multiple myeloma, Waldenstrom's macroglobulinemia), heavy chain diseases (e.g., alpha chain diseases, gamma chain diseases, and mu chain diseases), benign monoclonal gammopathy, and immune cell amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer (e.g., metastatic prostate cancer, 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, bile duct cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, hematopoietic tissue cancer, and the like. Other non-limiting examples of cancer types amenable to methods encompassed by the present disclosure include human sarcomas and human carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chondroma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, 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 carcinoma, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemia, e.g., acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia); chronic leukemia (chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is an epithelial cancer such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancer may also be characterized in various other forms, including, but not limited to, serous carcinoma, endometrial carcinoma, mucinous carcinoma, clear cell carcinoma, or undifferentiated carcinoma. In some embodiments, the present disclosure is used in the treatment, diagnosis, and / or prognosis of lymphoma or subtypes thereof, including, but not limited to, mantle cell lymphoma. Lymphoproliferative disorders are also considered to be proliferative diseases. In some embodiments, indications for treatment with the CFPs 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, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma, and urothelial carcinoma.

[0229]

[0216] Generally, cellular immunotherapy involves administering to a patient a medication containing live cells. In some aspects, a patient or subject with cancer is treated with autologous cells, the method comprising isolating or enriching PBMC-derived macrophages, modifying the macrophages ex vivo by introducing into the macrophages a recombinant nucleic acid encoding a chimeric antigen receptor for phagocytosis, which is a phagocytic receptor fusion protein (PFP), to generate highly phagocytic macrophages capable of tumor lysis, and administering the modified macrophages to the patient or subject.

[0230] In one embodiment, a subject is administered one or more doses of a pharmaceutical composition comprising therapeutic phagocytic cells, where the cells are allogeneic. HLA can be matched for compatibility with the subject and to prevent the cells from causing graft-versus-host disease (GVHD). Subjects presenting to a clinic undergo HLA genotyping to determine the HLA antigens expressed by the subject before determining a therapeutic agent or treatment regimen.

[0231] In some embodiments, a therapeutically effective dose is 10 cells / infusion. 7 10 bone marrow cells 12 The number of cells may vary according to age, weight, and other parameters related to the subject and can be determined by a medical professional. In some embodiments, a therapeutically effective dose ranges between about 10 bone marrow cells and about 10 7 In some embodiments, the therapeutically effective dose is about 2 x 10 bone marrow cells. 7 In some embodiments, the therapeutically effective dose is about 3 x 10 bone marrow cells. 7 In some embodiments, the therapeutically effective dose is about 4 x 10 bone marrow cells. 7 In some embodiments, the therapeutically effective dose is about 5 x 10 bone marrow cells. 7 In some embodiments, a therapeutically effective dose is about 6 x 10 bone marrow cells. 7 In some embodiments, the therapeutically effective dose is about 7 x 10 bone marrow cells. 7 In some embodiments, a therapeutically effective dose is about 8 x 10 bone marrow cells. 7 In some embodiments, a therapeutically effective dose is about 9 x 10 bone marrow cells. 7 In some embodiments, a therapeutically effective dose is about 10 bone marrow cells. 8 In some embodiments, the therapeutically effective dose is about 2 x 10 bone marrow cells. 8 In some embodiments, the therapeutically effective dose is about 3 x 10 bone marrow cells. 8 In some embodiments, the therapeutically effective dose is about 4 x 10 bone marrow cells. 8 In some embodiments, the therapeutically effective dose is about 5 x 10 bone marrow cells. 8 In some embodiments, a therapeutically effective dose is about 6 x 10 bone marrow cells. 8 In some embodiments, the therapeutically effective dose is about 7 x 10 bone marrow cells. 8 In some embodiments, a therapeutically effective dose is about 8 x 10 bone marrow cells. 8 In some embodiments, a therapeutically effective dose is about 9 x 10 bone marrow cells. 8In some embodiments, a therapeutically effective dose is about 10 bone marrow cells. 9 In some embodiments, the therapeutically effective dose is about 2 x 10 bone marrow cells. 9 In some embodiments, the therapeutically effective dose is about 3 x 10 bone marrow cells. 9 In some embodiments, the therapeutically effective dose is about 4 x 10 bone marrow cells. 9 In some embodiments, the therapeutically effective dose is about 5 x 10 bone marrow cells. 9 In some embodiments, a therapeutically effective dose is about 6 x 10 bone marrow cells. 9 In some embodiments, the therapeutically effective dose is about 7 x 10 bone marrow cells. 9 In some embodiments, a therapeutically effective dose is about 8 x 10 bone marrow cells. 9 In some embodiments, a therapeutically effective dose is about 9 x 10 bone marrow cells. 9 In some embodiments, a therapeutically effective dose is about 10 bone marrow cells. 10 In some embodiments, the therapeutically effective dose is about 5 x 10 bone marrow cells. 10 In some embodiments, a therapeutically effective dose is about 10 bone marrow cells. 11 In some embodiments, the therapeutically effective dose is about 5 x 10 bone marrow cells. 11 In some embodiments, a therapeutically effective dose is about 10 bone marrow cells. 12 There are individuals.

[0232]

[0219] In one aspect herein, there are provided one or more recombinant polynucleic acids encoding one or more recombinant proteins, which may be chimeric fusion proteins, such as receptors or engagers, as described herein. In some embodiments, the recombinant polynucleic acid is an mRNA. In some embodiments, the recombinant polynucleic acid comprises a circRNA. In some embodiments, the recombinant polynucleic acid is incorporated into a viral vector. In some embodiments, the recombinant polynucleic acid is delivered via a viral vector.

[0233]

[0220] In some embodiments herein, therapeutic compositions are provided that comprise a chimeric fusion protein, such as a chimeric fusion receptor protein (CFP), wherein the CFP comprises: (a) (i) an scFv that specifically binds to any one of the targets disclosed herein, and (ii) an extracellular domain comprising at least a portion of a hinge domain derived from CD8, a hinge domain derived from CD28; or an extracellular domain derived from CD68; (b) a CD8 transmembrane domain, a CD28 transmembrane domain, a CD2 transmembrane domain, or a CD68 transmembrane domain; and (c) 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) an intracellular domain comprising a third intracellular signaling domain, wherein (A) comprises a PI3K recruitment domain or (B) is derived from CD40.

[0234]

[0221] In some embodiments, provided herein are therapeutic compositions comprising recombinant nucleic acids encoding the bispecific or trispecific engagers disclosed herein.

[0235] Other Therapeutic Compositions for Co-Administration In some embodiments, the therapeutic composition further comprises an additional therapeutic agent selected from the group consisting of a CD47 agonist, an agent that inhibits Rac, an agent that inhibits Cdc42, an agent that inhibits GTPase, an agent that promotes F-actin disassembly, an agent that promotes the recruitment of PI3K to CFP, an agent that promotes the activity of PI3K, an agent that promotes the production of phosphatidylinositol 3,4,5-trisphosphate, an agent that promotes the activity of ARHGAP12, an agent that promotes the activity of ARHGAP25, an agent that promotes the activity of SH3BP1, an agent that promotes the sequestration of lymphocytes in primary and / or secondary lymphoid organs, an agent that increases the concentration of naive T cells and central memory T cells in secondary lymphoid organs, and any combination thereof.

[0236]

[0223] In some embodiments, the bone marrow 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 polynucleic acid sequence, wherein the second recombinant polynucleic acid sequence comprises a sequence encoding a peptide or protein that interacts with CFP; and the dimerization or interaction enhances phagocytosis by bone marrow cells that express CFP compared to bone marrow cells that do not express CFP.

[0237] In some embodiments, the bone marrow cells exhibit (i) increased effector activity, cross-presentation, respiratory burst, ROS production, iNOS production, inflammatory mediators, extracellular vesicle production, phosphatidylinositol 3,4,5-triphosphate production, engagement 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) increased expression of IL-1, IL-3, IL-6, IL-10, IL-12, IL-13, IL-23, TNFα, the TNF family of cytokines, CCL2, CXCL9, CXCL10, CXC and exhibit increased expression of L11, 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, death receptors of the TRAIL / TNF family, TGFβ, B7-DC, B7-H2, LIGHT, HVEM, TL1A, 41BBL, OX40L, GITRL, CD30L, TIM1, TIM4, SLAM, PDL1, MMPs (e.g., MMP2, MMP7, and MMP9), or any combination thereof.

[0238] In some embodiments, the intracellular signaling domain is derived from a phagocytic or tethering receptor (PR), or the intracellular signaling domain comprises a phagocytic activation domain. In some embodiments, the intracellular signaling domain is derived from a receptor other than a phagocytic receptor selected from Megf10, MerTk, FcR alpha, or Bai1. In some embodiments, the intracellular signaling domain is derived 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 (e.g., a phagocytic receptor). 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.

[0239]

[0226] In some embodiments, the intracellular signaling domain is derived from a receptor that contains an ITAM domain.

[0227] Provided herein are compositions comprising a recombinant nucleic acid encoding a CFP, such as a phagocytic or tethered receptor (PR) fusion protein (PFP), comprising a PR subunit including a transmembrane domain, and an intracellular domain including an intracellular signaling domain; and an extracellular domain including an antigen-binding domain specific to an antigen of a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain is derived from a phagocytic receptor other than a phagocytic receptor selected from Megf10, MerTk, FcRα, or Bai1.

[0240] In some embodiments, upon binding of the CFP to an antigen on a target cell, the killing activity of cells expressing the CFP is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133 In some embodiments, the CFP is functionally integrated into the cell membrane of a cell when the CFP is expressed in the cell. In some embodiments, upon binding of the CFP to an antigen on a target cell, the killing activity of cells 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 that do not express the CFP.

[0241] In some embodiments, the intracellular signaling domain is selected from the group consisting of 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, S The intracellular signaling domain is derived from a receptor such as a phagocytic receptor selected from the group consisting of RCRB4D, 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 CD 169. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain.

[0242]

[0230] As used herein, a PR subunit includes a transmembrane domain, and an intracellular domain including an intracellular signaling domain; and an extracellular domain including an antigen-binding domain specific to an antigen of a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; and the intracellular signaling domain is selected from the group consisting of TNFR1, MDA5, CD40, lectin, dectin-1, CD206, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, Compositions are provided that include a recombinant nucleic acid encoding a CFP, such as a phagocytic or tethered receptor (PR) fusion protein (PFP), derived from a receptor, such as a phagocytic receptor selected from the group consisting of 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.

[0243] In some embodiments, upon binding of the CFP to an antigen on a target cell, the killing activity of cells expressing the CFP is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, In some embodiments, the intracellular signaling domain is increased by more than 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%. In some embodiments, the intracellular signaling domain is derived from a phagocytic receptor other than a phagocytic receptor selected from Megf10, MerTk, FcRα, 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.

[0244] In some embodiments, cells expressing CFP exhibit increased phagocytosis of target cells expressing an antigen compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit at least a 1.1-fold increase in phagocytosis of target cells expressing an antigen compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit at least a 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 increase in phagocytosis of target cells expressing an antigen compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased cytokine production compared to cells that do not express CFP. In some embodiments, the cytokine is selected from the group consisting of 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, interferon, 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, CFP-expressing cells exhibit increased expression of CD86 compared to cells that do not express CFP. In some embodiments, CFP-expressing cells exhibit increased expression of MHC class I proteins compared to cells that do not express CFP. In some embodiments, CFP-expressing cells exhibit increased expression of death receptors of the TRAIL / TNF family compared to cells that do not express CFP. In some embodiments, CFP-expressing cells exhibit increased expression of B7-H2 compared to cells that do not express CFP. In some embodiments, CFP-expressing cells exhibit increased expression of LIGHT compared to cells that do not express 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 increased expression of GITRL death receptor compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD30L compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of TIM4 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of a ligand for TIM1 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of SLAM compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD48 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD58 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD155 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of CD112 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of PDL1 compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased expression of B7-DC compared to cells not expressing CFP. In some embodiments, cells that express CFP exhibit an increased respiratory burst compared to cells that do not express CFP. In some embodiments, cells that express CFP exhibit an increased production of ROS compared to cells that do not express CFP.In some embodiments, cells expressing CFP exhibit increased iNOS production compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased iNOS production compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased extracellular vesicle production compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased engagement with antigen-expressing target cells compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased resistance to CD47-mediated inhibition of phagocytosis compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased resistance to LILRB1-mediated inhibition of phagocytosis compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased production of phosphatidylinositol 3,4,5-trisphosphate.

[0245] Also provided herein are pharmaceutical compositions 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 bone marrow cell. In one aspect, a pharmaceutical composition is disclosed comprising a recombinant nucleic acid encoding or comprising any one of the sequences set forth in SEQ ID NOs: 26-42, or a cell comprising a recombinant nucleic acid encoding or comprising any one of the sequences set forth in SEQ ID NOs: 26-42, or an engineered cell comprising a recombinant nucleic acid encoding or comprising any one of the sequences set forth in 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 bone marrow 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 primary human immune cell. In some embodiments, the cells are progenitor or stem cells, or undifferentiated cells. In some embodiments, the cells are obtained from a biological sample of a human subject. In some embodiments, the cells are isolated from the biological sample of a human subject and selected for a phenotype, such as the expression of cell surface markers. In one embodiment, the isolated cells are characterized as progenitor cells, myeloid progenitor cells, or CD14+ / CD16-.

[0246]

[0234] In one embodiment, the isolated or engineered cells are CD14+ / CD16-.

[0235] In one embodiment, the pharmaceutical composition comprises an engineered cell, wherein the engineered cell is CD14+ / CD16-.

[0247] In one aspect, the pharmaceutical composition comprises a population of cells, 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 tonal signaling and de novo activation, and upon activation, exhibit M0, M1, or M2 differentiation.

[0248] Provided herein are methods for 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, 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 population of cells, wherein at least 50% of the cells are CD14+ / CD16- and 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 tonal signaling and de novo activation, and upon activation, exhibit M0, M1, or M2 differentiation.

[0249]

[0238] In some embodiments, provided herein are therapeutic compositions comprising cells, cells comprising recombinant nucleic acids, as described elsewhere herein. In some embodiments, the therapeutic compositions comprise recombinant nucleic acids expressing chimeric proteins, as described elsewhere herein. In some embodiments, the myeloid cells are CD14+ cells, CD14+ / CD16- cells, CD14+ / CD16+ cells, CD14- / CD16+ cells, CD14- / CD16- cells, dendritic cells, M0 macrophages, M2 macrophages, M1 macrophages, or mosaic myeloid cells / macrophages / dendritic cells.

[0250] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from the group consisting of a CD47 agonist, an agent that inhibits Rac, an agent that inhibits Cdc42, an agent that inhibits GTPase, an agent that promotes F-actin disassembly, an agent that promotes PI3K recruitment to PFPs, 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 serum-free medium, a lipid, or a nanoparticle.

[0251] In some embodiments, the therapeutic agent is a recombinant nucleic acid that encodes or includes any one of the sequences set forth in SEQ ID NOs: 26-42, 135, 136, or 137, or a sequence that is at least 80% identical to the sequence set forth in SEQ ID NOs: 26-42, 135, 136, or 137, and is injected systemically or locally in a subject. In some embodiments, the recombinant nucleic acid includes at least one of the sequences 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 component may be associated with the lipid component in the form of a liposome or lipid nanoparticle. The lipid component may include at least one cationic lipid. The one or more lipids of the pharmaceutical composition may be conjugated or modified.

[0252] Methods for generating novel chimeric receptor fusion protein (CFP) constructs

[0241] In one aspect herein, methods are provided for generating novel chimeric receptor proteins, comprising the identification of novel domains that may be useful, for example, in enhancing myeloid cell function, such that the fusion receptor, when expressed in myeloid cells, functions as an effector myeloid cell as described herein. The generation of the fusion proteins described herein may be performed using well-known molecular cloning methods, and the sequence may be verified after generating the recombinant nucleic acid.

[0253] Preparation of recombinant nucleic acids encoding chimeric antigen receptors: Recombinant nucleic acid constructs encoding chimeric antigen receptors (CARs) designed for expression in myeloid cells and incorporated into plasmid vectors for amplification and / or expression testing in eukaryotic cells are prepared. Recombinant CARs are constructed using molecular cloning methods known in the art. The recombinant CAR protein comprises an intracellular domain, a transmembrane domain, and an extracellular domain. Each domain, or subsection of a domain, may be encoded by a nucleic acid sequence generated by PCR from a heterologous source sequence and individually joined by cloning into a vector, or ligated into a long nucleic acid chain that is then inserted into the multiple cloning site of an appropriate plasmid or vector along with a promoter and 3' regulatory elements suitable for amplification. Briefly, an exemplary CAR is prepared by incorporating a nucleic acid sequence encoding one or more signal transduction domains (e.g., a PI3 kinase recruitment domain), a nucleic acid sequence encoding a CD8 hinge domain and a CD8 transmembrane domain, and a nucleic acid sequence encoding an extracellular domain with a sequence encoding a target antigen-binding scFv at the extracellular end. Certain constructs include a FLAG peptide sequence at the extracellular end, designed to not sterically interfere with the scFv binding to the target antigen. These components are ligated together to form 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 flexible short linker sequence between the two domains. The construct with the promoter and 3' stabilization structural unit is ligated into a plasmid. In one variant, the construct is placed within a retrotransposon Alu element encoding ORF2p, with its respective 5'-UTR and 3'-UTR sequences and a CMV promoter. Plasmids are amplified in E. coli and verified by sequencing or stored at -80°C.

[0254] Preparation of mRNA: mRNA can be prepared by in vitro transcription using digested plasmid as a template, purified to remove contaminating DNA, and polyadenylated. The RNA product is purified, resuspended in RNase-free water to 1 mg / ml, and stored in cryovials.

[0255] Identification of CFP ECD, TM, ICD, and antigen-binding domains useful for generating novel CFPs can be achieved using the methods described herein. Briefly, a large number of potential candidate proteins can be screened for phagocytic properties and enhancement of their respective phagocytosis-related intracellular signaling. Useful domains can then be used to generate novel CFPs. The screen can be divided into two parts: A. screening for phagocytic receptor (PR) domains; B. screening for antigen-binding domains.

[0256] Screening for PR domains: In one embodiment, approximately 5,800 cell membrane proteins were screened for their phagocytic potential according to the general method described herein. J774 macrophage cells were transiently transfected with a library of 5,800 cell membrane proteins. High-throughput multiplex assays (ranging from 6-well plate assay setups to 384-well plate assays with robotic manipulation) can be set up to assess the diverse potential functions of cell membranes. 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 will be understood by those skilled in the art. Exemplary intracellular signaling domains to be tested include, but are not limited to, CD40-FcRγ; FcRγ-CD40; NLRP3; FcRγ-SH2-Pro-caspase; FcRγ-Myd88; FcRγ-IFN receptor; FcR-TNFR1; FcRγ-TNFR2; FcR-AIM2; FcRγ-TRIFN; FcRγ-Pro-caspase; TRIFC; RIG1; MDA5; TBK; CD64; CD16A; CD89; FcRε; SIRPβ (two consecutive intracellular domains may be represented by a hyphenated term, e.g., FcRγ-Myd88 refers to an intracellular domain comprising the FcRγ intracellular signaling domain as signaling domain 1; and the Myd88 intracellular signaling domain as signaling domain 2). The extracellular linker domains screened include, but are not limited to, CD64, CD16A, CD89, SIRPα, FcRε, and CD8 hinge. The transmembrane domains tested include, but are not limited to, CD8, CD64, CD16A, CD89, FcRε, SIRPα, TNFR1, and CD40. The MDA5 domain was also screened.

[0257] Phagocytosis assay: Antigen-linked silica or polystyrene beads ranging from 1 nm, 5 nm, or 10 nm in diameter were used for macrophage screens. Inert beads can be coated with a supported lipid bilayer, and antigens can be ligated to the lipid bilayer. J774 macrophage cell lines can be prepared, each expressing a cloned recombinant cell membrane protein. The recombinant cell membrane protein can also express a fluorescent tag. Cell lines can be maintained and propagated in complete RPMI medium with heat-inactivated serum and antibiotics (penicillin / streptomycin). On the day of the assay, cells were plated at 1 x 10 cells per ml per well in a 6-well plate. 6 Cells can be seeded at a density of 1000 μg / well or a proportional density in 12- or 24-well plates and incubated for 2-6 minutes. Cells are then washed once in phosphate-buffered saline, and beads can be added in serum-depleted or complement-depleted nutrient medium. Cells can be visualized by light microscopy 30 minutes and 2 hours after addition of beads. Immunofluorescence reactions can be performed using tagged antibodies, and fluorescence confocal microscopy is used to detect interactions and colocalization of cellular proteins during pinocytosis. Confidence levels can be determined by Kruskal-Wallis test with Dunnett's multiple comparisons correction.

[0258]

[0246] In some instances, dye-loaded tumor cells can be fed into a macrophage cell line and assessed microscopically for phagocytosis. Cytokine production: Macrophage cell lines can be cultured as described above. In one assay, each J774 cell line expressing a cell membrane protein was assayed for cytokine production by seeding it into multiwell plates, challenging it with antigen-linked beads, and collecting the supernatant after 4 and 24 hours. Cytokines can be assayed from the supernatant by ELISA. In another fraction, cells can be collected with beads after 4 and 24 hours of incubation, and flow cytometry is performed to detect cytokines. In each case, multiple cytokines can be assayed in a multiplex format, 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. A macrophage inflammatory cytokine array kit (R&D Systems) is used.

[0259]

[0248] Intracellular signaling pathways for inflammatory gene and cytokine activation can be identified by Western blot analysis for the interferon activation pathway, including phosphorylation of MAP kinase, the JNK signaling pathway, the Akt signaling pathway, and the phosphorylation and activation of STAT-1.

[0260] Functional assays Inflammasome activation assay: NLRP3 inflammasome activation is assayed by detecting increased IL-1 production by ELISA and activation of caspase-1, which detects cleavage of procaspases to generate short-chain caspases, by Western blot. In a multiplexed microwell plate setup, Caspase-Glo (Promega Corporation) is used for rapid readout of caspase-1 activation.

[0261] iNOS activation assay:

[0250] Activation of oxidative burst potential can be measured by activation of iNOS and production of NO using the fluorometric assay NOS Activity Assay Kit (AbCAM).

[0262] Cancer cell killing assay:

[0251] Raji B cells can be used as cancer antigen-presenting cells. Raji cells can be incubated with crude whole cell extracts of cancer cells and co-incubated with the J774 macrophage cell line. One hour after infection, the macrophages can destroy the cells, which can be detected by microscopy or cell death assays.

[0263] Screening for high affinity antigen-binding domains: To generate extracellular binding domains for CFP, cancer ligands can be screened against antibody light and heavy chain variable domains. Human full-length antibody libraries or human scFv libraries can be screened. Potential ligands can also be used to develop novel immunoglobulin-binding domains in llamas and to immunize llamas for the preparation of single-domain antibodies.

[0264] Specific useful domains identified from the screen can then be reverse transcribed and cloned into lentiviral expression vectors to generate CFP constructs. One or more domains derived from the extracellular, transmembrane, and cytoplasmic regions of the highly phagocytic receptors generated from the screen can be used to generate recombinant nucleic acids encoding CFPs. Briefly, cell membrane receptors that exhibit 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 and SH2 recruitment sites. These screened functional domains can then be cloned into modular constructs to generate novel CFPs. These may be candidate CFPs, and each of these chimeric constructs is tested for enhanced phagocytosis, cytokine production, and chemokine and / or tumor cell killing in vitro and / or in vivo. Alterations in phagocytosis were examined using a microparticle-based phagocytosis assay. Briefly, streptavidin-coupled fluorescent polystyrene microparticles (6 μm diameter) can be conjugated with biotinylated recombinantly expressed / purified cancer ligands. Bone marrow cells expressing novel CFPs are incubated with the ligand-coated microparticles for 1-4 hours and analyzed and quantified for phagocytosis using flow cytometry. Plasmid or lentiviral constructs containing the designer CFP can then be prepared and examined for cancer cell lysis in macrophages.

[0265] Method for producing bone marrow cells ex vivo from a subject Isolation of bone marrow cells from PBMCs: Peripheral blood mononuclear cells can be isolated from normal donor buffy coats 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 magnetic separation column (Miltenyi Biotec). Briefly, cells are resuspended to the appropriate concentration in PEA buffer (phosphate-buffered saline [PBS] + 2.5 mM ethylenediaminetetraacetic acid [EDTA] per liter, and human serum albumin [0.5% final volume of 20% Alburex, Octopharma]) according to the manufacturer's instructions, incubated with CliniMACS CD14 beads, and then washed and passed through a magnetized LS column. After washing, purified monocytes can be eluted from the demagnetized column, washed, and resuspended in culture-related medium. Isolation of CD14+ cells from leukapheresis: PBMCs can be collected by leukapheresis from cirrhotic donors who provide informed consent to participate in the study. Leukapheresis of peripheral blood for mononuclear cells (MNCs) is performed by sterile collection using Optia apheresis. A standard collection program for MNCs is used, processing 2.5 times the blood volume. CD14 cell isolation is performed using a GMP-compliant closed system (CliniMACS Prodigy system, Miltenyi Biotec). Briefly, the leukapheresis product is sampled for cell counting, and an aliquot is taken for pre-separation flow cytometry. The percentage of monocytes (CD14+) and absolute cell numbers can be determined, and if required, the volume is adjusted to the required criteria for selection (total leukocytes 20 x 10 9 ≤400 x 10 white blood cells per mL 6 CD14 cells: 3.5 x 10 9The volume is adjusted to fill the 50-300 mL (maximum of 100 mL). CD14 cell isolation and separation are performed using the CliniMACS Prodigy with CliniMACS CD14 MicroBeads (Medical Device Classification III), the TS510 tubing set, and the LP-14 program. At the end of the process, the selected CD14+ positive monocytes can be washed in PBS / EDTA buffer (CliniMACS buffer, Miltenyi) containing 0.5% pharmaceutical-grade human albumin (Alburex) and then resuspended in TexMACS (or equivalent) medium for culture.

[0266] Cell counting and purity: Cell counts of total MNC and separated monocyte fractions can be performed using a Sysmex XP-300 automated analyzer (Sysmex). Because the Sysmex consistently underestimated monocyte numbers, assessment of macrophage numbers is performed by flow cytometry with TruCount tubes (Becton Dickinson) to determine absolute cell numbers. Flow cytometry (FACSCanto II, BD Biosciences) is used with a panel of antibodies against human leukocytes (CD45-VioBlue, CD15-FITC, CD14-PE, CD16-APC) to assess the purity of the isolation and the quality of the product by determining the amount of neutrophil contamination (CD45int, CD15pos).

[0267] Cell culture: Cultivation of cultures from healthy donor samples

[0256] The optimal culture medium for macrophage differentiation was explored, and three candidates were examined for use in cell production. In addition, the effect of cryopreservation of monocytes on the derivation of bone marrow cells and macrophages for therapeutic use was examined. Functional assays, as described elsewhere in this disclosure, were performed to quantify the phagocytic capacity of bone marrow cells and macrophages, as well as their capacity for further polarization and phagocytic potential.

[0268] Full-scale process verification with targeted samples Monocytes cultured from leukapheresis derived from Prodigy isolation were cultured at 1 cm per mL in culture bags (MACS GMP differentiation bags, Miltenyi) with GMP-grade TexMACS (Miltenyi) and 100 ng / mL M-CSF. 2 2 x 10 monocytes per 6 Monocytes can be cultured at 100 ng / mL GMP-compliant recombinant human M-CSF (R&D Systems). Cells can be cultured for 7 days at 37°C in a humidified atmosphere with 5% CO2. Two 50% volume medium changes are performed during the culture period (days 2 and 4) by removing 50% of the culture medium and then feeding fresh medium supplemented with 200 ng / mL M-CSF (to restore a final concentration of 100 ng / mL).

[0269] Cell harvesting: For normal donor-derived macrophages, cells can be harvested from the wells on day 7 using cell dissociation buffer (Gibco, ThermoFisher) and a pastette. Cells are resuspended in PEA buffer, counted, and then harvested at approximately 1 x 10 cells per test for flow cytometry. 6 The cells can be stained. On day 7, leukapheresis-derived macrophages can be harvested from the culture bag using PBS / EDTA buffer (CliniMACS buffer, Miltenyi) containing 0.5% pharmaceutical-grade human albumin (HAS; Alburex) derived from serum. The harvested cells can be resuspended in a vehicle consisting of two approved products: 0.9% saline for infusion (Baxter) with 0.5% human albumin (Alburex).

[0270] Flow cytometric characterization:

[0259] Monocyte and macrophage cell surface marker expression can be analyzed using a flow cytometer, FACSCanto II (BD Biosciences) or MACSQuant 10 (Miltenyi). Typically, approximately 20,000 events can be collected for each sample. Cell surface expression of leukocyte markers in freshly isolated and mature day 7 cells is performed by incubating the cells with specific antibodies (final dilution: 1:100). Cells are incubated with FcR blocking agent (Miltenyi) for 5 minutes, followed by incubation with the antibody cocktail for 20 minutes at 4°C. Cells can be washed in PEA, and the dead cell exclusion dye, DRAQ7 (BioLegend), is added at 1:100. Cells can be stained for a range of surface markers: 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), and 25F9-APC and CD115-APC (eBioscience). Both monocytes and macrophages can be gated using forward and side scatter and a DRAQ7 dead cell discriminator (BioLegend) to exclude debris, doublets, and dead cells, and analyzed using FlowJo software (Tree Star). From initial detailed phenotypic analysis, a panel defining the generation of functional macrophages from monocytes was developed as the shipping criteria (CD45-VB / CD206-FITC / CD14-PE / 25F9 APC / DRAQ7). Macrophages can be defined as cells with mean fluorescence intensity (MFI) levels higher than monocytes on day 0 for both 25F9 and CD206 at five time points.A second panel will also be developed in which other markers will be evaluated as part of an expanded panel (consisting of CCR2-BV421 / CD163-FITC / CD169-PE / CD14-PerCP-Vio700 / CD16-APC / DRAQ7), but will not be used as part of the release criteria for the cell product.

[0271] Monocytes and macrophages can be isolated by collecting the buffy coat layer formed in sucrose gradient centrifugation samples of isolated peripheral blood cells. CD14 cells can be examined for phagocytic uptake 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 Scientific) for 1 hour, after which the medium can be collected and the cells washed to remove unengulfed particles. Phagocytosis was assessed using an EVOS microscope (Thermo Fisher Scientific), and images were captured and intracellular bead uptake quantified using ImageJ software (NIH). Day 7 macrophages are treated with IFNγ (50 ng / mL) or IL-4 (20 ng / mL) for 48 hours to induce polarization toward M1 or M2 phenotypes (or M[IFNγ] vs. M[IL-4] phenotypes, respectively) to examine their ability to polarize into defined differentiated macrophages. After 48 hours, cells can be visualized using an EVOS brightfield microscope and then harvested and phenotyped as described above. Further analysis of the cytokine / growth factor secretion profiles of macrophages after generation and in response to inflammatory stimuli is performed. As before, macrophages were generated from healthy donor buffy coats and either left untreated or stimulated with TNFα (50 ng / mL; Peprotech) and polyinosinic:polycytidylic acid (polyI:C, a viral homolog that binds to TLR3, 1 g / mL; Sigma) to mimic conditions present in an inflamed liver, or lipopolysaccharide (LPS, 100 ng / mL; Sigma) plus IFNγ (50 IU / mL; Peprotech) to maximize macrophage activation. Day 7 macrophages were incubated overnight, and the supernatant was collected, spun down to remove debris, and stored at -80°C until testing.Secretome analysis will be performed using a 27-plex human cytokine kit and a 9-plex matrix metalloproteinase kit run on a Magpix multiplex enzyme immunoassay plate reader (BioRad).

[0272] Product stability: During process development, various excipients can be investigated, including PBS / EDTA buffer; PBS / EDTA buffer with 0.5% HAS (Alburex), 0.9% saline alone, or saline with 0.5% HAS. 0.9% saline (Baxter) with 0.5% HAS excipient is found to maintain optimal cell viability and phenotype (data not shown). The post-harvest stability of macrophages from cirrhotic donors was explored in three process optimization trials, with a more limited range of time points evaluated in process validation trials (n=3). After harvest and resuspension in excipients (0.9% saline for injection, 0.5% human serum albumin), bags can be stored at ambient temperature (21°C–22°C) and sampled at 0, 2, 4, 6, 8, 12, 24, 30, and 48 hours after harvest. A panel of antibodies for release criteria will be run on each sample and viability and mean fold change from day 0 will be measured by geometric MFI for 25F9 and CD206.

[0273] Statistical analysis:

[0262] Results may be expressed as mean ± SD. Differences are assessed for statistical significance using GraphPad Prism 6, where possible by unpaired two-tailed t-test. Results may be considered statistically significant if the P value is <0.05.

[0274] Also provided herein are cells comprising a composition described herein, a vector described herein, or a polypeptide described herein. In some embodiments, the cell is a phagocyte. In some embodiments, the cell is a stem cell-derived cell, a bone marrow cell, a macrophage, a dendritic cell, a lymphocyte, a mast cell, a monocyte, a neutrophil, a microglia, 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 macrophage cell. In some embodiments, the cell is an M2 macrophage cell. In some embodiments, the cell is an M1 myeloid cell. In some embodiments, the cell is an M2 myeloid cell.

[0275]

[0264] Also provided herein are methods of treating a disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition described herein. In some embodiments, the disease is cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the solid cancer is ovarian cancer, and suitable cancers include ovarian cancer, kidney cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, and lung cancer. In some embodiments, the cancer is a liquid 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 a T-cell malignancy.

[0276] In some embodiments, the method further includes administering to the subject an additional therapeutic agent, in some embodiments, the additional therapeutic agent is selected from the group consisting of a CD47 agonist, an agent that inhibits Rac, an agent that inhibits Cdc42, an agent that inhibits GTPase, an agent that promotes F-actin disassembly, an agent that promotes the recruitment of PI3K to PFPs, an agent that promotes the activity of PI3K, an agent that promotes the production of phosphatidylinositol 3,4,5-trisphosphate, an agent that promotes the activity of ARHGAP12, an agent that promotes the activity of ARHGAP25, an agent that promotes the activity of SH3BP1, and any combination thereof.

[0277] In some embodiments, the administering step comprises infusion or injection. In some embodiments, the administering step comprises direct administration into the solid tumor. In some embodiments, the administering step 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.

[0278] Also provided herein are methods of preparing a cell, the methods comprising contacting the cell with a composition described herein, a vector described herein, or a 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.

[0279]

[0268] Provided herein are methods for administering a therapeutic agent comprising any one of the compositions described above. In some embodiments, the therapeutic agent is administered via a parenteral route of administration.

[0280] In some embodiments, the therapeutic is administered via an intramuscular route of administration. In some embodiments, the therapeutic is administered via an intravenous route of administration. In some embodiments, the therapeutic is administered via a subcutaneous route of administration.

[0281] Also provided herein are methods for preparing pharmaceutical compositions comprising one or more recombinant nucleic acids described herein and a lipid in an aqueous composition described herein. In some embodiments, the composition comprises a vector described herein. In some embodiments, the lipid comprises the formation of lipid nanoparticles. [Example]

[0282] Example 1. Chimeric antigen receptor protein constructs with intracellular interferon-activating domains

[0271] In this example, the amino acid and nucleic acid sequences of various CFP constructs, described herein in Table 4, having an inflammatory signaling intracellular domain are disclosed. The nucleic acid sequences detailed below can be readily interpreted by those skilled in the art for DNA and mRNA sequences to provide guidance for creating and using appropriate constructs or variants therefrom using commonly used molecular cloning methods. Figure 1 illustrates the general principles of chimeric fusion protein expression and function. The transmembrane domain (TMD) selected for fusion protein construction is one that can dimerize or multimerize with endogenous myeloid cell transmembrane proteins for successful expression and function of the chimeric fusion protein. In an exemplary construct, such a TM domain is the CD89 TMD. By utilizing the transmembrane domain that multimerizes with endogenous Fc gamma-mediated myeloid cell targeting of the chimeric fusion protein shown in Figure 1, different constructs prepared using a TROP2 binder domain in the extracellular region, a transmembrane domain derived from the CD89 TMD, and an intracellular signaling domain are shown in Figure 2. In some embodiments, a CFP comprising a CD89 TMD is designed to include the intracellular domain of CD89 or a fragment thereof, without adding an intracellular signaling domain; the idea is that mature CFP is expressed at the cell membrane, associates with endogenous FcR gamma, the association stabilizes the CFP intracellularly, and when the extracellular domain of the CFP binds to a target antigen on a cancer cell, intracellular signaling is triggered via the FcR gamma intracellular domain. In some embodiments, a construct comprising a CD89 transmembrane domain can be designed to include an FcR intracellular domain, e.g., an intracellular signaling domain. In some embodiments, a CFP can include a CD40 intracellular domain.

[0283] In some embodiments, a CFP may comprise more than one intracellular signaling domain; for example, an FcR intracellular signaling domain and a PI3-kinase intracellular signaling domain. Additional intracellular domains may include a TRIF intracellular signaling domain and a CD40 intracellular signaling domain. These constructs are referred to as second-generation (or next-generation) chimeric fusion receptor constructs for delivery to and expression in myeloid cells in vivo. Some of the various sequences tested are provided below. An exemplary anti-TROP2-CD89 sequence (having an extracellular TROP2-binding scFv fused to an anti-CD89 TMD and having segments of the CD89 extracellular and intracellular regions) is shown below, with the construct name anti-TROP2-CD89 and the sequence: MWLQSLLLLGTVACSISQVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMG WINTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSSGGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITC KASQDVSIA VAWYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFLTISSLQPEDFAVYYC QQHYITPLTF GAGTKVEIKRGSGGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK (SEQ ID NO: 143). (CDRs 1, 2, and 3 of VH and VL, respectively, are underlined sequentially; Kabat numbering system). Table 6A shows the individual domains.

[0284] Another exemplary anti-TROP2-CD89 sequence is provided below: MWLQSLLLLGTVACSISQVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMG WINTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDVWGQGSLVTVSSGGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITC KASQDVSIAVA WYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFLTISSLQPEDFAVYYC QQHYITPLT FGAGTKVEIKRSGGGGAAADYKDDDDKGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVENWHSHTALNKEASADVAEPSWSQQMCQPGLTFARTPSVCK (SEQ ID NO: 144). (CDRs 1, 2, and 3 of VH and VL, respectively, are underlined sequentially. Kabat numbering system). Table 6A shows the individual domains.

[0285] An exemplary anti-TROP2-CD89-FcR with an additional FcR ICD compared to SEQ ID NO: 143 is MWLQSLLLLGTVACSISQVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMG WINTYTGEPTYTDDFK GRFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSSGGGGSGGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFLTISSLQPEDFAVYYC QQHYITPLT FGAGTKVEIKRSGGGGAAADYKDDDDKGSDSIHQDYTTQNLIRMAVAGLVLVALLAILVRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO: 145). (CDRs 1, 2, and 3 of VH and VL, respectively, are underlined sequentially. Kabat numbering system). Table 6A shows the individual domains.

[0286] An exemplary anti-TROP2 binding CFP sequence is the anti-TROP2-CD89-FcR-PI3K sequence

[0287] [ka] (SEQ ID NO: 146). (CDR1, 2, and 3 of VH and VL, respectively, are underlined sequentially; Kabat numbering system). Table 6A shows the individual domains.

[0288] The amino acid sequence of anti-TROP2-CD89-CD40 is provided below:

[0289] [ka] (SEQ ID NO: 147). (CDR1, 2, and 3 of VH and VL, respectively, are underlined sequentially. Kabat numbering system). Individual domains are shown in Table 6A.

[0290] The amino acid sequence of anti-TROP2-CD89-CD40-FcR is provided below:

[0291] [ka] (SEQ ID NO: 148). (CDR1, 2, and 3 of VH and VL, respectively, are underlined sequentially. Kabat numbering system). Individual domains are shown in Table 6A.

[0292] The amino acid sequence of anti-TROP2-CD89-TRIF is provided below:

[0293] [ka] (SEQ ID NO: 149). (CDR1, 2, and 3 of VH and VL, respectively, are underlined sequentially. Kabat numbering system). Individual domains are shown in Table 6A.

[0294] The amino acid sequence of anti-TROP2-CD89-FcR-TRIF is provided below:

[0295] [ka] (SEQ ID NO: 150). (CDR1, 2, and 3 of VH and VL, respectively, are underlined sequentially. Kabat numbering system). Individual domains are shown in Table 6A.

[0296]

[0280]

[0297] [Table 10-1]

[0298] [Table 10-2]

[0299] [Table 10-3]

[0300] [Table 10-4]

[0281] An exemplary anti-GPC3-CD89 CFP sequence is shown below: An exemplary anti-GPC3-CD89 has the amino acid sequence:

[0301] [ka] (SEQ ID NO: 151). (CDR1, 2, and 3 of VH and VL, respectively, are underlined sequentially; Kabat numbering system). Individual domains are shown in Table 6B.

[0302] An exemplary anti-GPC3-CD89 has the amino acid sequence:

[0303] [ka] (SEQ ID NO: 152). (CDR1, 2, and 3 of VH and VL, respectively, are underlined sequentially; Kabat numbering system). Individual domains are shown in Table 6B.

[0304]

[0284]

[0305] [Table 11] Example 2. Functional assays to investigate CFP constructs

[0285] Targeting constructs are examined for functional properties. THP-1 cells or CD14+ / CD16- monocytes isolated from leukapheresis samples were transfected with polynucleotide constructs encoding each CFP.

[0306] Method for cell transfection and detection of transfection efficiency: THP-1 cells were harvested and washed once with MaxCyte Electroporation buffer. Cells were resuspended at a density of 10 million cells per ml and added to 100 μg of ATAK receptor RNA in an Eppendorf tube, mixed twice, and loaded into a MaxCyte processing assembly (OC-25x3). Cells were electroporated using the THP-1 program on the MaxCyte. After electroporation, cells were incubated at 37°C for 10 minutes, collected in the MaxCyte processing assembly, and then transferred to plates containing preheated medium at a density of 0.5 million cells per ml.

[0307] After overnight incubation, ATAK receptor expression in electroporated monocytes was assessed by flow cytometry. Anti-Fab-Alexa Fluor-647 antibody (diluted 1:50) was used to detect the expression of the ATAK receptor scFv. Stained samples were collected on a Cytek Northern lights cytometer, and the percentage of binder-positive cells was calculated based on the increase in anti-Fab intensity compared to mock-transfected controls.

[0308]

[0288] Method for phagocytosis assay: Target tumor cells (SKOV3) are labeled with pHrodo-Red dye (final concentration 700ng / ml) according to the Sartorius Incucyte pHrodo-Red labeling kit protocol. After labeling, SKOV3 cells are resuspended at a density of 0.5 million cells per ml using culture medium. CD14-positive monocytes were isolated from donor Leukopaks and electroporated with 100µg / ml of ATAK receptor RNA using MaxCyte. After electroporation, cells were allowed to recover overnight at 37°C in culture medium at a density of 2 million cells per ml. The next day, cells were counted using NC-200 and resuspended at a density of 2.5 million cells per ml using culture medium. In a low-adhesion U-bottom 96-well plate, 50 μL of tumor cells (50,000 total cells) were added to 50 μL of ATAK receptor-transfected monocytes (125,000 total cells) at an E:T ratio of 5:1. The cells were mixed and incubated overnight at 37°C.

[0309] The next day, cells are stained with CD45-Alexa Fluor 700, which specifically labels monocytes. Samples are then collected on Cytek Northern lights to detect the signal intensity of pHrodo-Red and CD45. Phagocytosis is measured as the percent phagocytic index and the specific increase in pHrodo-Red intensity within monocytes. The phagocytic index is calculated as the percent of monocytes with a high pHrodo-red signal, normalized to the total monocyte number. The phagocytic activity of ATAK monocytes is compared with mock-transfected controls to determine the effectiveness of the ATAK receptor.

[0310]

[0290] Phagocytosis can be examined using labeled tumor cells.

[0291] Method for SKOV3 cell killing assay: SKOV3-luciferase cells are used to examine the tumor-killing activity of ATAK receptor-transfected monocytes. CD14-positive monocytes are isolated from donor Leukopak and electroporated with 100 μg / ml of ATAK receptor RNA using MaxCyte. After electroporation, cells are recovered for 2 hours in culture medium at 37°C. After recovery, cells are resuspended at a density of 2.5 million cells per ml using culture medium. SKOV3-luciferase cells are harvested and resuspended at a density of 2.5 million cells per ml, and 100 μl of the cell suspension (a density of 25,000 total cells per well) is added to a 96-well flat-bottom plate. 100 μL of ATAK receptor-transfected monocytes are added to the same well at E:T ratios of 1:1 and 10:1. The cells are mixed and incubated at 37° C. for 3 days.

[0311] On day 3, supernatants were collected and frozen to measure cytokines and chemokines secreted by ATAK monocytes. Cells were lysed and SKOV3 luciferase levels were measured using a luminescence plate reader. A decrease in luciferase levels for samples containing ATAK monocytes compared to mock-transfected controls indicates SKOV3-killing activity of ATAK cells.

[0312] The following method is used to examine the polarization potential of bone marrow cells. These effector bone marrow cells are electroporated with a nucleic acid construct and frozen for subsequent use and testing. Then, upon thawing, the cells are cultured under polarization stimuli in separate aliquot cultures, e.g., with (i) GMCSF, (ii) IL4, IL10, and TGF-beta (M2 stimulation), (iii) activated T cell conditioned medium (TCM), and (iv) MCSF. Cells are analyzed by flow cytometry after 24, 48, and 72 hours, and cytokine analysis is performed by Luminex.

[0313] Method for detecting activation of the NF-κB / IFN pathway 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. Measurement of supernatant levels of alkaline phosphatase indicates activation of the NF-κB signaling pathway, while supernatant levels of luciferase indicate activation of the IFN signaling pathway. THP1-Dual cells are electroporated with 100 μg / ml of ATAK receptor RNA using MaxCyte. After electroporation, cells are allowed to recover in culture medium at 37°C for 2 hours. After recovery, cells are resuspended at a density of 0.5 million cells per ml using culture medium. SKOV3 cells are harvested and resuspended at a density of 0.5 million cells per ml. In a 96-well plate, 100 ul of SKOV3 cell suspension (at a density of 50,000 total cells per well) and 100 ul of ATAK-THP1-Dual cells (at 50,000 total cells) are added to a 96-well flat-bottom plate at an E:T ratio of 1:1. The cells are mixed and incubated at 37°C for 24 hours. After 24 hours, the cells are centrifuged and the supernatant is collected.

[0314] To detect activation of the NF-κB pathway, QUANTI-Blue solution (Invivogen) was added to the supernatant and incubated at 37° C. for 2 hours, after which the OD was measured using an absorbance plate reader. An increase in absorbance indicates activation of NF-κB signaling, and the OD values ​​for ATAK-transfected THP1-Dual cells can be compared with those of mock-transfected controls to determine the activity of the ATAK receptor.

[0315] To detect activation of the IFN pathway, QUANTI-Luc solution (Invivogen) is added to the supernatant and luciferase levels are measured using a luminescence plate reader. An increase in luciferase levels, indicating activation of IFN signaling, can be compared between ATAK-transfected THP1-Dual cells and mock-transfected controls.

[0316] Expression of the TROP2-binding agents illustrated in Figure 2 in monocytic cell lines. Figure 3 shows data demonstrating successful expression of each construct. The results show that at least 50% of the cells express each construct. The results also demonstrate that the addition of multiple intracellular signaling domains is well tolerated and the constructs are well expressed.

[0317] Furthermore, these second-generation in vivo receptors demonstrated higher levels of proinflammatory cytokine and chemokine production. A comparison of cytokine production between first-generation constructs (with CD89 TMDs lacking multiple intracellular signaling domains) and second-generation constructs (i.e., TROP2-CD89-FcR, TROP2-CD89-FcR-PI3K, TROP2-CD89-TRIF, TROP2-CD89-CD40, and TROP2-CD89-CD40-FcR) is shown in Figures 4A and 4B. The second-generation constructs demonstrated higher IL-12p70 and IFN-beta production compared with representative first-generation constructs (Figure 4A). Furthermore, similar results were found for IP-10, TNF-α, and IL-6 production (Figure 4B). These results demonstrate that the second-generation anti-TROP2 construct is well expressed and highly functional in monocytes, releasing pro-inflammatory cytokines that activate the construct-expressing monocytic cells, rendering them pro-inflammatory. These cells are therefore phagocytes activated against TROP2-expressing cancer cells.

[0318]

[0299] Example 3. Human studies This example describes a phase I, open-label, first-in-human, multiple ascending-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. The study was designed to administer an anti-TROP2-Fc-alpha fusion receptor in the form of lipid nanoparticles encapsulating mRNA encoding the construct.

[0319] Planned number of subjects and treatment information 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 study. The study is divided into two parts. Part A is a repeat ascending dose study to determine the safety, tolerability, and pharmacokinetics (PK) of an anti-TROP2 second-generation chimeric fusion protein in subjects with TROP2+ liver metastases (LM). Part B is a dose expansion study to further determine safety, tolerability, PK, and preliminary efficacy in patients with TROP2-positive (TROP2+) colorectal liver metastases (CRLM).

[0320] Subjects will receive an effective amount of mRNA encoding a second-generation anti-TROP2 chimeric fusion protein in lipid nanoparticles administered intravenously over 60 minutes. The starting dose and dosing regimen will be determined after completion of PK and safety studies in non-human primates.

[0321]

[0303] Baseline evaluation within 4 weeks of the planned start of treatment will include patient history, physical examination with vital signs and performance status, CT or MRI scan, CBC with differential and platelet count, routine serum chemistries, urinalysis, INR / PTT, EKG, and a serum sample for human anti-human antibodies (HAHA). In women of childbearing potential, urine or serum b-HCG will also be required within 1 week of treatment. TROP2 expression will be confirmed immunohistochemically at a central laboratory on archived biopsy specimens obtained within the past 6 months or fresh biopsy specimens during screening.

[0322] All subjects will receive a weekly dose of the anti-TROP2 second-generation chimeric fusion protein for the first 12 weeks. In the absence of disease progression or unacceptable toxicity, treatment may continue through week 48. After week 12, patients may remain on the study until week 48, but the dosing regimen will be changed to once every two weeks. If a patient progresses while on the once every two weeks regimen, they may be titrated to weekly dosing. Complete responders may discontinue treatment at any time during the study and resume treatment at the dose and dosing schedule at which they were discontinued if they develop progressive disease. If a patient demonstrates a partial response (PR) and surgical resection of the CRLM is feasible, they may discontinue treatment and elect to undergo tumor resection. Patients who undergo surgical resection will be followed until week 48. If a patient develops progressive disease, treatment may be resumed at the dose and dosing schedule at which treatment was discontinued.

[0323] All patients should be closely monitored during treatment. The NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 will be used to assess all adverse events and provide guidelines for dose reduction, delay, or discontinuation in the event of treatment-related toxicity. All patients will also undergo CT / MRI scans during the study to assess disease progression and response to study medication.

[0324]

[0306] If any grade 2 or 3 treatment-related toxicity occurs on the scheduled day of treatment, treatment will be postponed for one week. At that time, if toxicity has resolved to grade 1 or less, treatment may be continued at a 25% dose reduction. If toxicity recurs, treatment may be continued at a 50% reduction from the initial dose, or if toxicity worsens, treatment will be permanently discontinued. The decision to continue or discontinue treatment is solely at the physician's discretion. If grade 4 treatment-related toxicity occurs, patients should permanently discontinue treatment.

[0325] Part A of this study will use a continuous reassessment method (CRM) to determine the recommended dose for Part B. The primary assessment of safety and tolerability is at Day 28. The primary efficacy assessment is at Week 12. Subjects may enroll in a long-term extension study at Week 48 if they achieve either stable disease (SD), partial response (PR), or complete response (CR).

[0326] In Part B, the maximum tolerated dose (MAD) obtained in Part A will be evaluated for safety, tolerability, and efficacy in patients with CRLM. The primary efficacy outcome will be the objective response rate (ORR) at 12 weeks.

[0327] If treatment is discontinued due to unacceptable toxicity, patients will remain on study until disease progression occurs, at which point an end-of-study evaluation will occur and additional follow-up will be required until any treatment-related toxicity has resolved or stabilized. All patients will be followed for survival.

[0328] To ensure the safety of study subjects, the first two subjects in each dose cohort will be enrolled 14 days apart. If there are no safety concerns, the remaining subjects may be enrolled simultaneously at that dose.

[0329] An exemplary anti-TROP2 second generation chimeric fusion protein (e.g., pharmaceutical product) comprising an anti-TROP2 scFv has the sequence:

[0330] [ka] The bold text represents the signal peptide sequence. The mature protein lacks this signal sequence, which one skilled in the art can interpret as the protein sequence expressed without the signal peptide sequence, as disclosed below:

[0331] [ka] The underlined regions are the CDR sequences of the scFV that bind to TROP2, with the heavy and light chains being arranged in the order of CDR1, CDR2, and CDR3. The italicized letters represent the amino acid sequence of the CD89 TMD.

[0332] The anti-TROP2 binding agent is designed for systemic intravenous delivery as an mRNA-lipid nanoparticle (LNP) formulation. This approach takes advantage of the natural tendency of intravenously administered LNPs to accumulate in the liver. The lipid component of the anti-TROP2 binding agent also protects the CAR-encoding mRNA from degradation by plasma and tissue nucleases. The unique mRNA construct encodes a TROP2-targeting scFv (derived from the complementarity-determining regions (CDRs) of sacituzumab) and a receptor consisting of the transmembrane domain and cytoplasmic tail of CD89. Upon administration of the anti-TROP2 binding agent, the LNP is internalized by many cell types, but 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, which is necessary for intracellular signaling. Recognition of TROP2 by scFV activates the intracellular signaling domain, leading to phagocytosis of tumor cells, production of inflammatory cytokines, and presentation of tumor antigens to T cells.

[0333] Research purpose

[0313] The primary objective of this study is to evaluate the safety and tolerability of an anti-TROP2 second-generation chimeric fusion protein in subjects with TROP2+ metastatic colorectal cancer and to establish the maximum tolerated dose (MAD) and recommended phase 2 dose (RP2D) based on observed adverse events (AEs), including all potential dose-limiting toxicities (DLTs).

[0334]

[0314] The secondary objectives of this study are to determine (i) the pharmacokinetics, (ii) the objective response (up to a 12-month time frame) of the anti-TROP2 second-generation chimeric fusion protein, and (iii) the duration of response.

[0335]

[0315] Secondary objectives of this study further include assessment of the area under the curve (up to 12 months), maximum plasma concentration (up to 12 months), time to 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 duration of response.

[0336] The exploratory objectives of this study are to determine progression-free survival (PFS) and overall survival (OS), conversion to surgical candidacy, and preliminary efficacy, including correlative biomarker studies. Further exploratory objectives may generally include the development of anti-drug antibodies (ADAs) against the anti-TROP2 second-generation chimeric fusion protein, evaluation of TROP2 expression levels in tumors, identification of potential biomarkers of response, and treatment-related effects of cytokine and chemokine production, TCR expansion, blood cell phenotype, tumor architecture, and tumor cell phenotype to identify potential biomarkers of response.

[0337] 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 a second-generation anti-TROP2 conjugate in subjects with TROP2+ colorectal liver metastases. Adults aged 18 years or older will be screened. Subjects who provide written informed consent and meet all inclusion and exclusion criteria will be enrolled in the study. The study is divided into two parts: Part A is a repeat ascending dose study to determine the safety, tolerability, and PK of the second-generation anti-TROP2 chimeric fusion protein in subjects with TROP2+ liver metastases (LM), and Part B is a dose expansion study to further determine the safety, tolerability, and PK, as well as preliminary efficacy, in patients with TROP2+ colorectal liver metastases (CRLM).

[0338] Inclusion criteria

[0318] Subjects are eligible for the escalating dose portion of the study (Part A) if they are diagnosed with histologically proven metastatic TROP2+ LM with progressive disease at baseline and have not responded to or refused standard treatment.

[0339] Subjects are eligible for the cohort extension portion (Part B) of the study if they are diagnosed with histologically proven metastatic TROP2+ CRLM with progressive disease at baseline and have not responded to or refused standard treatment. Additional criteria include the presence of TROP2+ tumors 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.

[0340] Exclusion criteria

[0320] Subjects will be excluded from the study if any of the following criteria are met: (1) known active central nervous system metastases and / or carcinomatous meningitis, (2) history of allogeneic bone marrow transplant or solid organ transplant, (3) active autoimmune disease, (4) active acute or chronic infection, (5) liver tumor invasion greater than 50%, or (6) history of splenectomy (patients with a history of partial splenic artery embolization may be eligible at the investigator's discretion).

[0341] Treatment duration There will be a total of three cycles. Each cycle will be administered weekly for four weeks. After three cycles, if there is no disease progression, patients may continue on the study through week 48 at the investigator's discretion, but the dosing regimen will be changed to once every two weeks. If patients develop progressive disease, they may be titrated to weekly dosing. If patients progress on weekly dosing, treatment will be discontinued.

[0342] Definition of dose-limiting toxicity Dose-limiting toxicities (DLTs) are defined using the Common Terminology Criteria for Designated Adverse Events (CTCAE) v5.0. All toxicities are considered "possibly related" to at least the anti-TROP2 second-generation chimeric fusion protein, unless they are not temporally related to administration of the anti-TROP2 second-generation chimeric fusion protein but rather are related to other etiologies, such as concomitant medications or symptoms, or the subject's underlying disease.

[0343]

[0323] In this study, unless otherwise specified, the following were considered DLTs if they occurred within 28 days (Day 28) of the first dose: (1) death, (2) 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 despite maximal supportive care (e.g., nausea, vomiting, diarrhea) (excluding renal and hepatic laboratory abnormalities), (5) CTCAE Grade 3 toxicity with renal and hepatic laboratory abnormalities that does not decrease to Grade 2 within 7 days, and (6) any Grade 3 infusion-related reaction lasting more than 24 hours and in which the patient was premedicated with a histamine H1 receptor antagonist, a histamine H2 receptor antagonist, and a corticosteroid. Exceptions to the criteria for DLT grade 3 or 4 include laboratory values ​​that the investigator judges to be clinically insignificant.

[0344] Research Suspension Rules If a subject experiences a DLT listed above at any dose level, further dosing and enrollment of the subject will be temporarily halted, and the Steering Committee will be immediately notified and convened to review the safety data. After reviewing the safety data, the Steering Committee will recommend, based on the frequency of the DLT, whether to (1) continue the study as planned, including additional dosing, (2) expand the dose cohort to obtain additional safety information, (3) reduce the dose to the previous low or intermediate dose, (4) discontinue administration of additional dosing, and / or (5) terminate the study and follow all subjects for safety.

[0345] Statistical considerations

[0325] Dose progression is determined by a continuous substitution model. Evaluation items / evaluation criteria

[0326] Safety endpoints and criteria included adverse events (AEs), IRs, serious adverse events (SAEs) (clinically significant abnormal physical examination findings and laboratory values ​​are reported as AEs), vital signs, serum chemistry values, hematology values, T-cell counts, and plasma cytokines.

[0346]

[0327] Endpoints and criteria for anti-TROP2 binding cellular kinetics include pharmacokinetic parameters of drug / product in the blood after each infusion (e.g., maximum plasma concentration (Cmax), time to maximum plasma concentration (Tmax), half-life (T1 / 2)) as quantified by qPCR.

[0347]

[0328] Endpoints and criteria for anti-drug antibodies (ADA) include determining the proportion of subjects who develop ADA as measured by ELISA.

[0329] Clinical response endpoints and criteria include measurement of Response Evaluation Criteria in Solid Tumors (RECIST) criteria v1.1 and conversion to surgical candidacy.

[0348]

[0330] Endpoints and criteria for correlative markers of response included cellular tumor penetration by IHC imaging mass cytometry, recruitment and trafficking of other immune cells by mass cytometry imaging, and upregulation of cytokine and chemokine production by transcriptional analysis. The timeline of events is shown in the table below.

[0349] [Table 12-1]

[0350] [Table 12-2]

[0351]

Table 12-3

Claims

1. 1. A composition comprising a recombinant polynucleic acid and a polynucleotide delivery vehicle, wherein the recombinant polynucleic acid 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 a CFP that multimerizes with an endogenously expressed Fc receptor protein within a cell; and (iii) an intracellular domain comprising at least two intracellular signaling domains; wherein at least two intracellular signaling domains are selected from the group consisting of an intracellular signaling domain derived from Fc epsilon receptor Ig (FCER1G), a PI3K recruitment domain, an intracellular signaling domain derived from CD40, and an intracellular signaling domain derived from TRIF, and the antigen-binding domain comprises an antibody or fragment thereof having a heavy chain variable domain (VH) comprising a heavy chain CDR3 (HCDR3) sequence selected from the group consisting of GGFGSSYWYFDV and FYSYTY.

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

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

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

5. The composition of claim 1 , wherein the CFP further comprises a signal peptide.

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

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

8. 8. The composition of claim 1, wherein the CFP comprises a CD89 transmembrane domain comprising a sequence having at least 90% sequence identity to SEQ ID NO:

11.

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

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

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

12. 1. A composition comprising a recombinant polynucleic acid, wherein the recombinant polynucleic acid comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP has a sequence having at least 95% sequence identity to SEQ ID NO: 36 or 152, a sequence having at least 95% sequence identity to SEQ ID NO: 26 or 143, or a sequence having at least 95% sequence identity to a sequence in Table 4.

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

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

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

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

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

18. 18. The composition of any one of claims 1 to 17, wherein the recombinant polynucleic acid is mRNA.

19. 19. The composition of claims 1 to 18, wherein the recombinant polynucleic acid is associated with one or more lipids.

20. 10. The composition of any one of the preceding claims, wherein the recombinant polynucleic acid is encapsulated in a liposome.

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

22. 22. The composition of any one of claims 12 to 21, wherein the recombinant polynucleic acid comprises (i) a sequence having 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, or (ii) a sequence having 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.

23. A composition comprising a nanoparticle delivery vehicle and a recombinant polynucleic acid described in any one of claims 1 to 22, wherein the recombinant polynucleic acid is associated with or within the nanoparticle delivery vehicle.

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

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

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

27. 27. The composition of any one of claims 1 to 26, wherein the recombinant polynucleic acid is mRNA and the lipid nanoparticles encapsulate the mRNA.

28. 28. The composition of any one of claims 1 to 27, wherein the lipid nanoparticles comprise polar lipids and non-polar lipids.

29. 29. The composition of any one of claims 1 to 28, wherein the lipid nanoparticles are 100 to 300 nm in diameter.

30. 30. A composition comprising a cell comprising a recombinant polynucleic acid of the composition of any one of claims 1 to 29.

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

32. 31. The composition of claim 30, wherein the cell is a bone marrow cell, a lymphoid cell, a progenitor cell, a stem cell, or an induced pluripotent cell.

33. The composition of claim 30, wherein the cells are CD14+ / CD16-.

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

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

36. 36. The method of claim 35, wherein the cancer is a TROP2-expressing cancer.

37. 36. The method of claim 35, wherein the cancer is a GPC3-expressing cancer.

38. A composition comprising: (i) a lipid delivery vehicle; and (ii) a recombinant polynucleic acid 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 an scFv or VHH that binds to a cancer antigen; and the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 11; and when contacted with cells, the recombinant polynucleic acid is substantially expressed in bone marrow cells.

39. 39. The composition of claim 38, wherein the recombinant polynucleic acid is not substantially expressed in T cells.

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

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

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

43. The composition of 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 to a sequence shown in Tables 5 and 6B.

44. The composition of claim 38, wherein the CFP comprises an extracellular antigen-binding domain that binds to GPC3 and comprises a sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NOs: 34-42, 135, 136, 137, 151, and 152.

45. 45. The composition of any one of claims 38 to 44, wherein the recombinant polynucleic acid is mRNA.

46. 46. ​​The composition of any one of claims 38 to 45, wherein the recombinant polynucleic acid is codon optimized.

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

48. 46. ​​The composition of any one of claims 38 to 45, wherein the recombinant polynucleic acid is fully human.

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

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

51. A method for expressing a chimera in bone marrow cells in vivo, comprising the steps of: (i) preparing a therapeutically acceptable aqueous formulation of a composition comprising a composition described in any one of claims 1 to 30 and 38 to 49; and (ii) systemically administering the therapeutically acceptable aqueous formulation to a subject, wherein upon isolating and testing an appropriate tissue sample from the subject at an appropriate time after administration, expression of CFP is detected in bone marrow cells in the tissue sample.

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

53. 53. The use of 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, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma, and urothelial carcinoma.

54. 1. A composition comprising a recombinant polynucleic acid, (a) the recombinant polynucleic acid is RNA; (b) the recombinant polynucleic acid is associated with or within the lipid nanoparticle; (b) the recombinant polynucleic acid comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP is an anti-TROP2 CFP and has a sequence having at least 95% sequence identity to SEQ ID NO: 36 or 152, a sequence having at least 95% sequence identity to SEQ ID NO: 26 or 143; (d) a composition, wherein the recombinant polynucleic acid comprises (i) a sequence having at least 80% sequence identity to SEQ ID NO: 141 or a portion of SEQ ID NO: 141 not including the 5'UTR and / or 3'UTR.

55. 55. A pharmaceutical composition comprising the composition of claim 54.

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

57. 57. The method of 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, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma, and urothelial carcinoma.

58. 1. A composition comprising a recombinant polynucleic acid, (a) the recombinant polynucleic acid is RNA; (b) the recombinant polynucleic acid is associated with or within the lipid nanoparticle; (b) the recombinant polynucleic acid comprises a sequence encoding a chimeric fusion protein (CFP), wherein the CFP is an anti-GPC3 CFP and has a sequence having at least 95% sequence identity to SEQ ID NO: 36 or 152; (d) a composition, wherein the recombinant polynucleic acid comprises a sequence having at least 80% sequence identity to SEQ ID NO: 142 or a portion of SEQ ID NO: 142 not including the 5'UTR and / or 3'UTR.

59. 59. A pharmaceutical composition comprising the composition of claim 58.

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

61. 61. The method of 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, lung squamous cell carcinoma, ovarian yolk sac tumor, melanoma, and urothelial carcinoma.

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