CN116916940A - Engineered chimeric fusion protein compositions and methods of use thereof - Google Patents

Engineered chimeric fusion protein compositions and methods of use thereof Download PDF

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CN116916940A
CN116916940A CN202180089305.2A CN202180089305A CN116916940A CN 116916940 A CN116916940 A CN 116916940A CN 202180089305 A CN202180089305 A CN 202180089305A CN 116916940 A CN116916940 A CN 116916940A
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domain
composition
cell
binding
cells
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丹尼尔·盖茨
王宇枭
小布鲁斯·麦克里迪
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Merlot Biomedical Co
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Merlot Biomedical Co
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Priority claimed from PCT/US2021/058104 external-priority patent/WO2022098905A2/en
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Abstract

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

Description

Engineered chimeric fusion protein compositions and methods of use thereof
Cross reference
The present application claims U.S. provisional application No. 63/109,445 filed on 11/4 of 2020; U.S. provisional application No. 63/251,400 filed on day 1, 10, 2021; U.S. provisional application No. 63/196,994 filed on 6/4 of 2021; the benefits of U.S. provisional application No. 63/162,205 filed on day 17 of 3 months 2021; each of these applications is incorporated by reference herein in its entirety.
Background
Cellular immunotherapy is a promising new technique for combating difficult-to-treat diseases (e.g. cancer) and persistent infections as well as some other forms of treatment for difficult-to-cure diseases. The discovery of CAR-T cells and their potential use in immunotherapy has created a major breakthrough. CAR-T cells are T lymphocytes that express a chimeric antigen receptor that helps target T cells to specific diseased cells, such as cancer cells, and can induce a cytotoxic response or immunosuppression and/or tolerance that aims to kill the target cancer cells, depending on the intracellular domain employed and the co-expressed immunosuppressive cytokine. However, some limitations along the way slow the progression of CAR-T cells and impair their promise in clinical trials.
Knowing the limitations of CAR-T cells is key to utilizing this technology and continuing to innovate better immunotherapeutic patterns. In particular, CAR-T cells appear to face a significant problem in T cell malignancies. CAR-T cells and malignant T cells share surface antigens in most T Cell Lymphomas (TCLs), and therefore CAR-T cells undergo cytotoxicity in the same manner as cancer cells. In some cases, CAR-T products may be contaminated with malignant T cells. In addition, T cell hypoplasia is a potential problem due to the long-term presence of CAR-T cells. Other limitations include poor penetration of CAR-T cells into solid tumors, and the effective tumor microenvironment may down-regulate its anti-tumor potential. CAR-T cell function is also adversely affected by the immunosuppressive Tumor Microenvironment (TME) that leads to inactivation and depletion of endogenous T cells.
Myeloid cells (including macrophages) are cells derived from the myeloid lineage and belong to the innate immune system. They are derived from bone marrow stem cells that enter the blood and can migrate into the tissue. Some of their primary functions include phagocytosis, activation of T cell responses, and clearance of cell debris and extracellular matrix. They also play an important role in maintaining homeostasis, and in initiating and eliminating inflammation. In addition, myeloid cells can differentiate into many downstream cells, including macrophages, which can display different responses from pro-inflammatory to anti-inflammatory depending on the type of stimulus they receive from the surrounding microenvironment. In addition, tissue macrophages have been shown to have a broad regulatory and activating effect on other immune cell types, including CDT effector cells, NK cells, and T regulatory cells. Macrophages have been shown to be the primary immunoinfiltrate in malignant tumors and have been shown to have a broad immunosuppressive impact on effector immune infiltration and function.
Myeloid cells are the main cellular compartments of the immune system, comprising monocytes, dendritic cells, tissue macrophages and granulocytes. In recent years, models of cell ontogenesis, activation, differentiation and tissue-specific functions of myeloid cells have been re-studied and surprising results have been achieved. However, their great plasticity and heterogeneity during homeostasis and disease are far from understood. Although myeloid cells have many functions, including phagocytosis and their ability to activate T cells and produce soluble factors, the use of these functions for therapeutic uses remains elusive. Thus, other cell types are sought for developing newer approaches to improving therapeutic agents, including but not limited to T cell malignancies.
In vivo or ex vivo engineering of myeloid cells can also be short lived in vivo, phenotypically diverse, sensitive, plastic, and often found difficult to manipulate in vitro. For example, exogenous gene expression in monocytes is difficult compared to exogenous gene expression in non-hematopoietic cells. There are significant technical difficulties associated with transfecting myeloid cells (e.g., monocytes/macrophages). As professional phagocytes, myeloid cells (e.g. monocytes/macrophages) contain many potent degrading enzymes that can disrupt nucleic acid integrity and make gene transfer into these cells an inefficient process. This is especially true for activated macrophages whose physiology undergoes a significant change upon exposure to an immune or inflammatory stimulus. Viral transduction of these cells is hindered because macrophages are end-stage cells that do not normally divide; thus, some have had limited success depending on the integration of the vector into the replicating genome. In addition, macrophages are fully responsive to "danger signals", so several original viral vectors used for gene transfer induce potent antiviral responses in these cells, making these vectors unsuitable for gene delivery.
Disclosure of Invention
The diverse functions of myeloid cells make them ideal cell therapy candidates, which can be engineered to have many therapeutic effects. The present invention relates to immunotherapy using myeloid cells of the immune system (e.g., cd14+ cells), particularly phagocytes. Many therapeutic indications may contemplate the use of myeloid cells. For example, myeloid cell immunotherapy may be extremely important in cancer, autoimmune, fibrotic diseases and infections. The present invention relates to immunotherapy using myeloid cells (including phagocytes of the immune system), in particular macrophages. The invention disclosed herein is directed to utilizing one or more of these functions of myeloid cells for therapeutic use. For example, the invention disclosed herein is directed to utilizing phagocytic activity of myeloid cells (including engineered myeloid cells) for therapeutic use. For example, it is an object of the invention disclosed herein to take advantage of the ability of myeloid cells (including engineered myeloid cells) to promote T cell activation. For example, it is an object of the invention disclosed herein to take advantage of the ability of myeloid cells (including engineered myeloid cells) to promote secretion of tumoricidal molecules. For example, it is an object of the invention disclosed herein to take advantage of the ability of myeloid cells (including engineered myeloid cells) to promote immune cell and molecule recruitment and transport. The present invention provides innovative methods and compositions that can successfully transfect or transduce, or otherwise induce, genetic modifications in, myeloid cells with the aim of increasing functional aspects of the myeloid cells, and in addition, without compromising the differentiation capacity, maturation potential, and/or plasticity of the cells. The myeloid cells can reside in vivo or be engineered ex vivo.
The present invention relates to in vivo or ex vivo programming of myeloid cells, preparation and use of engineered myeloid cells (e.g., CD14+ cells, such as macrophages or other phagocytes) that can directly and/or indirectly attack and kill (ATAK) diseased cells, such as cancer cells and infected cells, engineered myeloid cells (e.g., macrophages and other phagocytes) can be prepared by using, e.g., ex vivo, recombinant nucleic acid techniques, synthetic nucleic acids, gene editing techniques (e.g., CRISPR), transduction (e.g., using viral constructs), electroporation or nuclear transfection, or in vivo using mRNA delivery techniques including but not limited to LNP techniques, incorporating nucleic acid sequences (e.g., mRNA, plasmids, viral constructs) encoding Chimeric Fusion Proteins (CFPs) into cells, which chimeric fusion proteins have extracellular binding domains specific for disease-related antigens (e.g., cancer antigens). The cells promote activation of T cells (e.g., T cells in a tumor microenvironment). The myeloid cells can be engineered to promote secretion of the tumoricidal molecules such that when CFP binds to an antigen on a target cell, the cells promote secretion of tumoricidal molecules from nearby cells. The myeloid cells can be engineered to promote the recruitment and transport of immune cells and molecules such that when CFPs bind to antigens on target cells, the cells promote the recruitment and transport of immune cells and molecules to the target cells or tumor microenvironment.
The invention is based on the following important findings: engineered myeloid cells overcome at least some of the limitations of CAR-T cells, including ease of recruitment to solid tumors; has a short and engineered survival duration, thus reducing the long-standing risk of causing hypoplasia and immunodeficiency; the myeloid cells are not contaminated by T cells; myeloid cells can avoid mutual killing (fratricide), at least because they do not express the same antigen as malignant T cells; and myeloid cells have excessive antitumor functions, which can be effectively utilized. In some aspects, engineering bone marrow-derived cells can be a safer immunotherapeutic tool to target and destroy diseased cells.
In addition, myeloid cells (e.g., macrophages) are ubiquitous in the tumor environment (TME), particularly the most abundant cells in some tumor types. As part of their role in the immune system, myeloid cells (e.g., macrophages) are naturally involved in clearing diseased cells. The invention also relates to the use of myeloid cell functions, in particular for targeting, killing and direct and/or indirect clearance of diseased cells and delivery of payloads (e.g. antigens and cytokines).
The invention is also based on the important finding that engineering myeloid cells can promote endogenous T cell activity. In addition to the programming of myeloid cells, the present invention relates to. Engineered myeloid cells can be achieved by in vivo incorporation of nucleic acid sequences (e.g., mRNA, plasmid, viral construct) encoding Chimeric Fusion Proteins (CFP) having extracellular binding domains specific for disease-associated antigens (e.g., cancer antigens) into cells using, for example, recombinant nucleic acid techniques ex vivo, synthetic nucleic acid, gene editing techniques (e.g., CRISPR), transduction (e.g., using viral constructs), electroporation or nuclear transfection, or in vivo using mRNA delivery techniques including, but not limited to, LNP techniques.
Engineered myeloid cells can also be short lived in vivo, phenotypically diverse, sensitive, plastic, and often found difficult to manipulate in vitro. For example, exogenous gene expression in monocytes is difficult compared to exogenous gene expression in non-hematopoietic cells. There are significant technical difficulties associated with transfecting myeloid cells (e.g., monocytes/macrophages). As professional phagocytes, myeloid cells (e.g. monocytes/macrophages) contain many potent degrading enzymes that can disrupt nucleic acid integrity and make gene transfer into these cells an inefficient process. This is especially true for activated macrophages whose physiology undergoes a significant change upon exposure to an immune or inflammatory stimulus. Viral transduction of these cells is hindered because macrophages are end-stage cells that do not normally divide; thus, some have had limited success depending on the integration of the vector into the replicating genome. In addition, macrophages are fully responsive to "danger signals", so several original viral vectors used for gene transfer induce potent antiviral responses in these cells, making these vectors unsuitable for gene delivery. The present invention provides innovative methods and compositions that can successfully transfect or transduce, or otherwise induce, genetic modifications in, myeloid cells with the aim of increasing functional aspects of the myeloid cells, and in addition, without compromising the differentiation capacity, maturation potential, and/or plasticity of the cells.
Provided herein are therapeutic agents for binding to certain antigens expressed on diseased cells (e.g., cancer cells), and the binding of the therapeutic agent (e.g., a targeted "conjugate") to a target antigen on a target diseased cell triggers a process of destruction of the target cell. The therapeutic agents described herein may be recombinant nucleic acids that can be expressed in a suitable cell (e.g., a mammalian cell, such as a human cell), wherein the suitable cell may be a myeloid cell. In some embodiments, the therapeutic agents described herein may be recombinant proteins that bind to a target antigen on a target cell. In some embodiments, a therapeutic agent described herein can be a cell, e.g., a myeloid cell, wherein the myeloid cell comprises a recombinant nucleic acid described herein, and/or expresses a recombinant protein described herein, such that the myeloid cell can target a diseased cell that expresses a target antigen on the cell surface; and the myeloid cells lyse or phagocytose the diseased cells.
In some embodiments, the therapeutic agent is a myeloid cell, e.g., as described herein. In some embodiments, the myeloid cell is a bone marrow precursor cell. In some embodiments, the myeloid cells are undifferentiated and/or unpolarized myeloid cells. In some embodiments, the myeloid cell is a phagocyte. In some embodiments, the myeloid cells are CD14+/CD 16-cells.
In some embodiments, the therapeutic agent is a recombinant or engineered nucleic acid. In some embodiments, the nucleic acid is RNA.
In some embodiments, the engineered nucleic acid is mRNA. In some embodiments, the recombinant or engineered nucleic acid therapeutic agent encodes a chimeric fusion receptor protein (CFP).
In some embodiments, the recombinant nucleic acid encodes an extracellular or soluble protein, also referred to as a "conjugate", that binds to a target antigen on a diseased cell with a target conjugate domain, at one end of the protein, for example; and is capable of binding to an effector cell, such as an effector myeloid cell, e.g., an active phagocyte having at least one other domain that binds to a molecule on the structure of the effector cell (e.g., myeloid cell). Exemplary conjugates are bispecific conjugates (bimes) or trispecific conjugates (tri mes) as described herein.
In one aspect, disclosed herein is a composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP) comprising: (a) Phagocytosis or binding receptor (PR) subunits comprising: (i) A transmembrane domain, or (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising a CD137 antigen binding domain that can specifically bind CD137 on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
In one aspect, disclosed herein is a composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first CD137 antigen binding domain that specifically binds to a CD137 antigen on a target cell, and (b) a second binding domain that specifically binds to a surfactant on a myeloid cell; wherein the first antigen binding domain binds to CD137 antigen on a target cell and the second binding domain binds to a surfactant on a myeloid cell.
In one aspect, disclosed herein is a composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP) comprising: (a) Phagocytosis or binding receptor (PR) subunits comprising: (i) A transmembrane domain, or (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising a Claudin18.2 antigen binding domain that can specifically bind Claudin18.2 on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
In one aspect, disclosed herein is a composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first Claudin18.2 antigen binding domain that specifically binds to a Claudin18.2 antigen on a target cell, and (b) a second binding domain that specifically binds to a surfactant on a myeloid cell; wherein the first antigen binding domain binds to Claudin18.2 antigen on the target cell and the second binding domain binds to a surfactant on the myeloid cell.
In one aspect, disclosed herein is a composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP) comprising: (a) Phagocytosis or binding receptor (PR) subunits comprising: (i) A transmembrane domain, or (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising a Claudin3 antigen binding domain that can specifically bind to Claudin3 on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
In one aspect, disclosed herein is a composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first Claudin3 antigen binding domain that specifically binds to a Claudin3 antigen on a target cell, and (b) a second binding domain that specifically binds to a surfactant on a myeloid cell; wherein the first antigen binding domain binds to Claudin 18.2 antigen on the target cell and the second binding domain binds to a surfactant on the myeloid cell.
In one aspect, disclosed herein is a composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP) comprising: (a) Phagocytosis or binding receptor (PR) subunits comprising: (i) A transmembrane domain, or (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising a CD70 antigen binding domain that can specifically bind CD70 on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
In one aspect, disclosed herein is a composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first CD70 antigen binding domain that specifically binds to a CD70 antigen on a target cell, and (b) a second binding domain that specifically binds to a surfactant on a myeloid cell; wherein the first antigen binding domain binds to a CD70 antigen on a target cell and the second binding domain binds to a surfactant on a myeloid cell.
In one aspect, disclosed herein is a composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP) comprising: (a) Phagocytosis or binding receptor (PR) subunits comprising: (i) A transmembrane domain, or (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising a TROP2 antigen binding domain that can specifically bind to TROP2 on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
In one aspect, disclosed herein is a composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first TROP2 antigen binding domain that specifically binds a TROP2 antigen on a target cell, and (b) a second binding domain that specifically binds a surfactant on a myeloid cell; wherein the first antigen binding domain binds to a TROP2 antigen on a target cell and the second binding domain binds to a surfactant on a myeloid cell.
In one aspect, disclosed herein is a composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP) comprising: (a) Phagocytosis or binding receptor (PR) subunits comprising: (i) A transmembrane domain, or (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising a TMPRSS antigen binding domain that can specifically bind TMPRSS on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
In one aspect, disclosed herein is a composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first TMPRSS antigen binding domain that specifically binds to a TMPRSS antigen on a target cell, and (b) a second binding domain that specifically binds to a surfactant on a myeloid cell; wherein the first antigen binding domain binds to TMPRSS antigen on a target cell and the second binding domain binds to a surfactant on a myeloid cell.
In one aspect, disclosed herein is a composition comprising a recombinant nucleic acid encoding a phagocytic or binding receptor (PR) fusion protein (PFP) comprising: (a) a PR subunit comprising: (i) A transmembrane domain, and (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising an antigen binding domain of any of the above, having a strong binding affinity for an antigen of a target cell; wherein the transmembrane domain and extracellular domain are operably linked; and wherein upon binding of the PFP to the antigen of the target cell, the killing or phagocytosis activity of the PFP-expressing cell is increased by at least more than 20% as compared to a cell not expressing the PFP.
In some embodiments, the intracellular signaling domain of any of the PFPs described herein is derived from a phagocytic or tethered receptor, or wherein the intracellular signaling domain comprises a phagocytosis activating domain.
In some embodiments, the intracellular signaling 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 intracellular signaling domain comprises a PI3 kinase recruitment 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, NLRP family member, NLRP1-14, NOD1, NOD2, thermal protein, AIM2, NLRC4, FCGR3A, FCERIG, CD40, caspase domain or pro-caspase binding domain, or any combination thereof.
In some embodiments, any of the PFPs described herein further comprise a transmembrane domain derived from a CD2, CD8, CD28, CD64, or CD68 protein TM domain.
In some embodiments, any of the PFPs described herein further comprise a hinge domain.
In some embodiments, the killing activity of the PFP-expressing cell is increased by at least 20% when the PFP binds to an antigen of the target cell, as compared to a cell that does not express the PFP.
In some embodiments, upon binding of PFP to an antigen of a target cell, the killing activity of the PFP-expressing cell is increased by at least a factor of 1.1 compared to a cell that does not express PFP.
In some embodiments, the composition comprises a first therapeutic agent, wherein the therapeutic agent comprises: a first binding domain, wherein the first binding domain is a first antibody or functional fragment thereof that specifically interacts with an antigen on a target cell, and a second binding domain, wherein the second binding domain is a second antibody or functional fragment thereof that specifically interacts with a myeloid cell; wherein (i) the first therapeutic agent is coupled to a first component, wherein the first component is an additional therapeutic agent or a third binding domain, or (ii) in some embodiments, the composition comprises an additional therapeutic agent.
In some embodiments, the therapeutic agent comprises: (a) a first binding domain that specifically interacts with an antigen of a target cell, (b) a second binding domain that specifically interacts with a myeloid cell, and (c) a third binding domain that specifically interacts with a myeloid cell.
In some embodiments, any of the binding domains of the therapeutic agent comprises a binding domain of an antibody, a functional fragment of an antibody, a variable domain thereof, V H Domain, V L Domain, VNAR domain, V HH A domain, a single chain variable fragment (scFv), a Fab, a single domain antibody (sdAb), a nanobody, a bispecific antibody, a diabody, or a functional fragment or combination thereof.
In some embodiments, the antigen on the target cell to which the first binding domain binds is a cancer antigen or a pathogenic antigen or an autoimmune antigen on the target cell.
In some embodiments, the first therapeutic agent comprises a polypeptide less than 1000 amino acids in length or 1000 nm. In some embodiments, the first therapeutic agent comprises a polypeptide less than 500 amino acids in length or 500nm in length. In some embodiments, the first therapeutic agent comprises a polypeptide that is 200-1000 amino acids in length or 200-1000nm in length.
In some embodiments, the binding domain of the first therapeutic agent is conjugated to a cancer cell.
In some embodiments, the second binding domain specifically interacts with a myeloid cell and promotes phagocytic activity of the myeloid cell.
In some embodiments, the second binding domain specifically interacts with and promotes inflammatory signaling by myeloid cells.
In some embodiments, the second binding domain specifically interacts with a myeloid cell or an adhesion molecule and promotes adhesion of the myeloid cell to the target cell.
In some embodiments, the second binding domain specifically interacts with a myeloid cell and inhibits the antiphagic activity of the myeloid cell mediated by the target cell.
In some embodiments, the second binding domain specifically interacts with a myeloid cell and inhibits anti-inflammatory activity of the myeloid cell mediated by the target cell.
In some embodiments, the second binding domain and/or the third binding domain promotes phagocytic activity of the myeloid cells.
In some embodiments, the second binding domain and/or the third binding domain promotes inflammatory signaling of myeloid cells.
In some embodiments, the second binding domain and/or the third binding domain specifically interact with a myeloid cell or an adhesion molecule and promote adhesion of the myeloid cell to the target cell.
In some embodiments, the second binding domain and/or the third binding domain inhibits antiphagic activity of the myeloid cells mediated by the target cells.
In some embodiments, the second binding domain and/or the third binding domain inhibits anti-inflammatory activity of the myeloid cells mediated by the target cells.
In some embodiments, the third binding domain or additional therapeutic agent comprises a CD47 antagonist, a CD47 blocker, an antibody, a chimeric CD47 receptor, a sialidase, a cytokine, a pro-inflammatory gene, a pro-caspase, or an anti-cancer agent.
In some embodiments, the third binding domain or additional therapeutic agent comprises a SIRP-a antagonist, a SIRPA blocker, an antibody, a chimeric SIRPA receptor, a cytokine, a pro-inflammatory gene, a pro-caspase, or an anti-cancer agent.
In some embodiments, the third binding domain or additional therapeutic agent comprises a PD1 antagonist, a PD1 blocker, an antibody, a chimeric PD1 receptor, a cytokine, a pro-inflammatory gene, a pro-caspase, or an anti-cancer agent.
In some embodiments, the second binding domain and the third binding domain bind different, non-identical target antigens.
In some embodiments, the first binding domain, the second binding domain, or the third binding domain is a ligand binding domain.
In some embodiments, the first binding domain, the second binding domain, or the third binding domain is operably linked by one or more linkers.
In some embodiments, the linker is a polypeptide. In some embodiments, the linker is a functional peptide. In some embodiments, the linker is a ligand for the receptor. In some embodiments, a ligand for a monocyte or macrophage receptor. In some embodiments, the linker activates the receptor. In some embodiments, the linker inhibits the receptor.
In some embodiments, the linker is a ligand for M2 macrophage receptor. In some embodiments, the linker is a ligand for a TLR receptor (e.g., TLR 4). In some embodiments, the linker activates TLR receptors. In some embodiments, the first, second, and/or third binding domains are associated with a mask that binds the binding domains.
In some embodiments, the mask is an inhibitor that inhibits the binding domain from interacting with its target while the mask remains associated with the corresponding binding domain. In some embodiments, the mask is associated with the binding domain via a peptide linker. In some embodiments, the linker comprises a cleavable moiety.
In some embodiments, the cleavable moiety is cleaved by a protein or enzyme that is selectively abundant in the cancer or tumor site.
In some embodiments, the therapeutic agent is a nucleic acid, e.g., an engineered nucleic acid, wherein the nucleic acid is RNA.
In some embodiments, the nucleic acid is mRNA.
In some embodiments, the nucleic acid is a self-replicating RNA designed to target myeloid cells.
In some embodiments, the nucleic acid, i.e., the engineered nucleic acid, is circRNA designed to target myeloid cells.
In some embodiments, the engineered recombinant nucleic acid is RNA designed to target myeloid cells.
In some embodiments, the engineered nucleic acid is mRNA designed to target myeloid cells.
In some embodiments, the engineered nucleic acid is a self-replicating RNA designed to target myeloid cells.
In some embodiments, the engineered nucleic acid is circRNA designed to target myeloid cells.
In some embodiments, the engineered recombinant nucleic acid is associated with one or more lipids.
In some embodiments, the recombinant nucleic acid is encapsulated in a liposome.
In some embodiments, the liposome is a nanoparticle.
In some embodiments, the recombinant nucleic acid is contained in a vector.
Provided herein is a pharmaceutical composition comprising any of the recombinant nucleic acids of the compositions of the above embodiments, and an acceptable excipient.
Provided herein is a pharmaceutical composition comprising a polypeptide encoded by a recombinant nucleic acid encoding any one of the recombinant proteins described above.
Provided herein is a cell comprising a recombinant nucleic acid encoding any one of the recombinant proteins described above.
In some embodiments, the cell is a myeloid cell.
In some embodiments, the cell is CD14+, CD16-.
Provided herein is a pharmaceutical composition comprising a population of cells comprising the recombinant nucleic acid of any of the above embodiments, wherein at least 50% of the cells are cd14+cd16-.
In some embodiments, less than 10% of the cells in the pharmaceutical composition are dendritic cells.
In some embodiments, the pharmaceutical composition further comprises a suitable excipient.
Provided herein is a method of preparing any of the compositions of any of the above embodiments.
In one aspect, provided herein is a method of treating cancer in a subject comprising administering to the subject a pharmaceutical composition of any of the above embodiments.
In one aspect, provided herein is a method of treating cancer in a subject comprising administering to the subject a pharmaceutical composition of any of the above embodiments.
In some embodiments, the cancer is selected from the group consisting of gastric cancer, ovarian cancer, renal cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, glioblastoma, and lung cancer.
In one aspect, provided herein is a method of preparing any of the compositions as described herein.
In one aspect, provided herein is a method of treating cancer in a subject comprising administering to the subject a pharmaceutical composition as described herein.
In one aspect, provided herein is a method of treating cancer in a subject comprising administering to the subject a pharmaceutical composition as described herein.
In some embodiments, the cancer is selected from the group consisting of gastric cancer, ovarian cancer, renal cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, glioblastoma, and lung cancer.
In one aspect, provided herein is a composition comprising a recombinant polynucleic acid comprising a sequence encoding a Chimeric Fusion Protein (CFP), the CFP comprising: (a) An extracellular domain comprising an antigen binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is a transmembrane domain from a protein that dimerizes with an endogenous FcR-gamma receptor in a myeloid cell; wherein the recombinant polynucleic acid is encapsulated by a nanoparticle delivery vehicle; and wherein CFP is expressed on the surface of myeloid cells of the human subject after administration of the composition to the human subject.
In some embodiments, the antigen binding domain comprises a Fab fragment, scFv domain, or sdAb domain. In some embodiments, the transmembrane domain is a transmembrane domain from CD8, CD16a, CD64, CD68 or CD 89.
In some embodiments, the extracellular domain further comprises a hinge domain derived from CD8, wherein the hinge domain is operably linked to the transmembrane domain and the antigen binding domain.
In some embodiments, the transmembrane domain is a transmembrane domain from a protein that dimerizes with an endogenous FcR-gamma receptor in a myeloid cell, monocyte, or macrophage; wherein CFP is specifically expressed in myeloid cells, monocytes or macrophages of the human subject after administration of the pharmaceutical composition to the human subject. In some embodiments, the transmembrane domain is a transmembrane domain from CD16a, CD64, CD68 or CD 89. In some embodiments, the CFP further comprises an intracellular domain, wherein the intracellular domain comprises one or more intracellular signaling domains, and wherein the one or more intracellular signaling domains comprise an intracellular signaling domain from fcγ R, fc a R, fc epsilon R, CD40 or CD3 zeta. In some embodiments, the one or more intracellular signaling domains further comprise a phosphoinositide 3 kinase (PI 3K) recruitment domain. In some embodiments, the PI3K recruitment domain comprises a sequence having at least 90% sequence identity to SEQ ID NO. 26. In some embodiments, the intracellular domain comprises an intracellular domain from CD16a, CD64, CD68, or CD 89.
In some embodiments, the intracellular domain of a CFP described herein comprises an immunoreceptor tyrosine based activation motif (ITAM) domain. In some embodiments, the intracellular domains of CFPs described herein comprise more than one ITAM domain. In some embodiments, the ITAM domain is from an intracellular domain of a protein or polypeptide selected from the group consisting of: the CD3 ζtcr subunit, CD3 εtcr subunit, CD3 γtcr subunit, CD3 δ TCR subunit, TCR ζ chain, fcε receptor 1 chain, fcε receptor 2 chain, fcγreceptor 1 chain, fcγreceptor 2a chain, fcγreceptor 2b 1 chain, fcγreceptor 2b2 chain, fcγreceptor 3a chain, fcγreceptor 3b chain, fcβreceptor 1 chain, TYROBP (DAP 12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and having at least one but no more than 20 modified amino acid sequences.
In some embodiments, at least one ITAM domain comprises a Src family kinase phosphorylation site.
In some embodiments, at least one ITAM domain comprises a Syk recruitment domain.
The composition of any one of claims 107-110, wherein the intracellular signaling subunit further comprises a DAP12 recruitment domain.
In some embodiments, the intracellular domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ITAM domains.
In some embodiments, the recombinant polynucleic acid is mRNA. In some embodiments, the mRNA is delivered into the cell via a nanoparticle delivery vehicle. In some embodiments, the nanoparticle delivery vehicle comprises a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a polar lipid. In some embodiments, the lipid nanoparticle comprises a non-polar lipid. In some embodiments, the lipid nanoparticle has a diameter of 100 to 300nm. In some embodiments, the antigen binding domain binds an antigen selected from the group consisting of TROP2, GPC3, CD5, HER2, CD137, CD70, claudin 3, claudin 18.2, TMPRSS, CD19, CD22, CD7, and GP 75.
Provided herein is a pharmaceutical composition comprising a composition as described above, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of a composition as described herein, wherein the composition is effective to inhibit the growth of cancer when administered to a human subject having cancer.
In one aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering a pharmaceutical composition described herein.
In one aspect, provided herein is a method of introducing a composition described herein into a myeloid cell, comprising: electroporating a myeloid cell in the presence of a recombinant polynucleic acid comprising a sequence encoding a Chimeric Fusion Protein (CFP), the CFP comprising: (a) An extracellular domain comprising an anti-TROP 2 binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the recombinant polynucleic acid is (i) present in a myeloid cell, or (ii) encapsulated by a nanoparticle delivery vehicle; wherein the recombinant polynucleic acid is configured for expression of the recombinant polynucleic acid in myeloid cells of a human subject.
In one aspect, provided herein is a composition comprising a recombinant polynucleic acid comprising a sequence encoding a Chimeric Fusion Protein (CFP), the CFP comprising: (a) An extracellular domain comprising an anti-TROP 2 binding domain, the extracellular domain comprising at least one of the sequences shown in SEQ ID No. 34 and SEQ ID No. 35, or a sequence at least 85% identical to SEQ ID No. 34 or SEQ ID No. 35; (b) A transmembrane domain operably linked to an extracellular domain, the transmembrane domain comprising a sequence from a transmembrane domain of an fcγr1 molecule (CD 64), an fcγriiia molecule (CD 16) or an fcαr1 molecule (CD 89); and (c) an optional hinge domain operably linked to the extracellular domain and the transmembrane domain, wherein the hinge domain comprises an amino acid sequence from a CD8 a hinge domain.
In some embodiments, the composition further comprises an intracellular domain comprising an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NOs 26, 27 or 28; or a sequence which is at least 80% identical to the amino acid sequence selected from the sequences shown in SEQ ID NO. 26, 27 or 28. In some embodiments, the recombinant polynucleic acid is mRNA.
Provided herein is a cell comprising a recombinant polynucleic acid comprising a sequence encoding a chimeric fusion protein having an extracellular domain comprising an anti-TROP 2 binding domain, wherein the cell is a cd14+ cell.
Provided herein is the use of any of the compositions described herein or a pharmaceutical composition herein or a cell as described herein in the treatment of a disease or disorder.
Provided herein is a use of a pharmaceutical composition described herein in the treatment of cancer in a subject, wherein the cancer is selected from the group consisting of gastric cancer, ovarian cancer, renal cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, glioblastoma, and lung cancer.
Provided herein is a use of any of the compositions described herein or a pharmaceutical composition described herein or a cell described herein in the manufacture of a medicament for treating cancer in a subject, wherein the cancer is selected from the group consisting of gastric cancer, ovarian cancer, renal cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, glioblastoma, and lung cancer.
Also provided herein is a composition comprising one or more recombinant nucleic acid sequences comprising: (a) a first nucleic acid sequence encoding an exogenous polypeptide; (B) A second nucleic acid sequence encoding a chimeric antigen receptor fusion protein (CFP), wherein the CFP comprises: (a) An intracellular signaling subunit comprising an intracellular signaling domain having one or more tyrosine residues that are phosphorylated upon receptor binding to an antigen; (b) A transmembrane domain, (c) an extracellular binding domain having binding specificity for a component on the surface of a target cell, wherein the extracellular binding domain is operably linked to the transmembrane domain and an intracellular signaling subunit; and (d) a transcriptional activator domain operably linked to the intracellular signaling subunit by a protease cleavage sequence, wherein the transcriptional activator domain facilitates transcription of the first nucleic acid sequence encoding the exogenous polypeptide; and (C) a third nucleic acid sequence encoding (i) a protease that cleaves a protease cleavage sequence operably linked to the transcriptional activator domain to the intracellular signaling subunit; (ii) A domain that binds to tyrosine residues that are phosphorylated upon CFP activation; wherein the protease that cleaves the protease cleavage sequence and the domain that binds to tyrosine residues are operably linked.
In some embodiments, the third nucleic acid sequence further encodes (iii) a stimulus responsive element.
In some embodiments, the stimulus-responsive element (iii) is fused to a domain that binds a phosphorylated tyrosine residue.
In some embodiments, the stimulus-responsive element (iii) is responsive to the microenvironment of the cell expressing the nucleic acid sequence.
In some embodiments, one or more recombinant nucleic acids are expressed in myeloid cells.
In some embodiments, the transcriptional activator domain further comprises a DNA binding domain.
In some embodiments, the DNA binding domain is selected from the group consisting of the DNA binding Domain (DB) of Gal4, ZFHD1, or tet-R.
In some embodiments, the transcriptional activator domain comprises a VP64 transactivation domain.
In some embodiments, the protease that cleaves a protease cleavage sequence that operably links the transcriptional activator domain to an intracellular signaling subunit is a Hepatitis C Virus (HCV) NS3 protease.
In some embodiments, the domain that binds to a tyrosine residue that is phosphorylated upon CFP activation is a phosphotyrosine binding (PTB) domain.
In some embodiments, the PTB is Shc PTB.
In some embodiments, (iii) is a degradation determinant operably linked to (ii).
In some embodiments, the degradation determinant is a HIF-1a degradation solution stator.
Provided herein is a pharmaceutical composition comprising the compositions listed in paragraphs [0114] to [00126], and a pharmaceutically acceptable excipient. Provided herein is a cell comprising the compositions listed in paragraphs [0114] to [00126 ]. In some embodiments, the cells are cd14+. Provided herein is a method of treating a disease in a subject comprising administering to the subject any one of: (i) a pharmaceutical composition; or (ii) the cells listed in paragraph [0127 ].
Incorporation by reference
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.
Drawings
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, together with the accompanying drawings, in which the principles of the invention are utilized.
FIG. 1 depicts a schematic diagram showing two exemplary CFPs, the left CFP containing an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, the right CFP containing an extracellular binding domain, a transmembrane domain, a first intracellular signaling domain, a second intracellular signaling domain, a third intracellular signaling domain, and one or more additional intracellular signaling domains. The signaling domain may be derived from other receptors and designed to elicit any number of cellular functions. An exemplary binding domain is the CD137 binding domain. An exemplary binding domain is a TROP2 binding domain.
Fig. 2A depicts a schematic diagram showing an exemplary CFP dimer containing an anti-CD 137 extracellular binding domain, a transmembrane domain, and an intracellular signaling domain containing an intracellular domain derived from fcrγ fused to a PI3K recruitment domain.
Fig. 2B depicts a schematic showing an exemplary CFP dimer comprising an extracellular anti-CD 137 antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a phagocytosis domain, a PI3K recruitment domain, and a pro-inflammatory domain.
FIG. 3 is a schematic drawing depicting an exemplary CFP homodimer in which each subunit contains an extracellular domain fused to a scFv that binds a single target (left), and an exemplary CFP heterodimer in which a first subunit of the heterodimer contains an extracellular domain fused to a scFv that binds a first target and a second subunit of the heterodimer subunit contains an extracellular domain fused to a scFv that binds a second target (right).
FIG. 4A is a schematic diagram depicting an exemplary recombinant nucleic acid encoding a CFP containing a signal peptide fused to an antigen-specific scFv fused to the extracellular domain (ECD), transmembrane domain (TMD) and intracellular domain (ICD) of a scavenger receptor having signaling domains 1, 2 and 3. In addition, exemplary recombinant nucleic acids are mRNAs with 5 'and 3' UTRs.
FIG. 4B shows data with viral gene delivery (left panel), some cell types show transduction and poor cell differentiation, which may adversely affect the potential role of therapeutic cell function and longevity; plasmid-mediated delivery (top right) can induce differentiation and cell death; whereas mRNA delivery is safe and causes almost negligible immune responses in the cells after delivery (bottom right).
Fig. 5 is exemplary data depicting the expected outcome of relative phagocytosis in human primary myeloid cells transduced with empty vector (control) or vector encoding CFP co-cultured with antigen coated beads, as depicted in the top panel. Phagocytosis was quantified using flow cytometry.
Fig. 6A is exemplary data depicting the expected outcome of relative phagocytosis in human primary myeloid cells transduced with empty vector (control) or vector encoding CFP co-cultured with antigen expressing target cells labeled with fluorescent dye, as depicted in the top panel. Phagocytosis was quantified using flow cytometry. The effect of different myeloid cell to target ratios is as expected.
Figure 6B shows data demonstrating the specificity of CFP binding domains (binders), cells expressing the CD19 binding CFP construct (CD 19-ATAK) show high phagocytosis only in the presence of a cd19+ target, and not in the presence of a cd22+ target carrying no CD19 on the surface, and vice versa.
FIG. 6C shows data showing successful chemotaxis and localization of myeloid cells expressing fluorescent CFP constructs in tumors (by imaging, left panel); the right panel shows chemotaxis in vitro in the presence of different concentrations of CCL 2.
FIG. 7A depicts an exemplary plot from a flow cytometry analysis depicting the performance of a flow cytometry analysis under different polarization conditions (shown in the above plot), e.g., in the presence of MCSF alone (M0 condition); or primary human monocytes expressing anti-CD 5 CFP incubated with IL-10, IL-4 and tgfβ (M2 conditions) or in the presence of IFN and LPS for 24 hours followed by H9T cell lymphoma cells, the results indicate that expression of CFP does not alter the ability of the cells to polarize under these conditions. In addition, incubation with tumor cells did not alter polarization. In addition, primary human monocytes expressing anti-CD 5 CFP were tested and demonstrated to have potent tumor cell phagocytosis and killing activity in M2 environment using the same culture conditions (not shown here). The same method can be used to test the in vitro efficacy of any of the conjugates described herein in an M1 or M2 promoting environment.
Fig. 7B depicts a schematic diagram of an exemplary experimental flow chart showing treatment of a peripheral T cell lymphoma animal model experiment. 1X 10≡6 CD5-HU9 Szeary syndrome tumor lineage cells were injected subcutaneously into mice. Treatment with indicated amounts of human primary monocytes expressing anti-CD 5 CFP was started on day 11 after injection of tumor line cells. The total infusion was administered 5 times, once every 3 days. The same method can be used to test the in vitro efficacy of any of the conjugates described herein in a suitable in vivo experimental tumor model.
Fig. 7C depicts a graph of relative tumor size (emissivity) at the indicated time points according to the experimental protocol described in fig. 7B.
Fig. 8 shows an exemplary modular design of CFP constructs for monocyte-specific expression (ecd=extracellular domain; h=hinge; tmd=transmembrane domain; icd=intracellular domain).
FIG. 9 shows a representative workflow of expression and functional analysis of exemplary CFP constructs including anti-GPC 3 CFP constructs and anti-TROP 2 CFP constructs having an anti-GPC 3 or anti-TROP 2 antibody domain, a FLAG extracellular domain, a human CD16 or human CD89 Transmembrane (TM) domain, and a human CD16 or human CD89 intracellular (cell) domain. As described, 200ug/mL RNA encoding constructs can be Electroporated (EP) into PBMCs and expression, killing of target tumor cells, and cytokine/chemokine production can be analyzed.
FIG. 10A depicts flow cytometry data analyzing expression of the indicated anti-TROP 2 CFP constructs in various cell types. As shown in the following figures, expression of the indicated anti-TROP 2 CFP construct was observed in cd14+ myeloid cells, but not in cd19+ B cells, cd3+ T cells or cd56+ NK cells.
FIG. 10B depicts flow cytometry data analyzing the expression of the indicated anti-GPC 3 CFP constructs in various cell types. As shown in the following figures, expression of the indicated anti-GPC 3 CFP construct was observed in cd14+ myeloid cells, but not in cd19+ B cells, cd3+ T cells, or cd56+ NK cells.
Figure 11A depicts data from a luciferase assay measuring killing of SKOV3 cells by PBMCs transfected with the indicated anti-TROP 2 CFP constructs. PBMC transfected with the indicated anti-TROP 2 CFP construct were co-cultured with SKOV3-Luc cells at a 5:1 effector to target ratio for 3 days. PBMCs transfected with the indicated anti-TROP 2 CFP constructs showed specific killing of SKOV3 cells compared to the control.
FIG. 11B depicts data for analysis of expression, tumor-specific phagocytosis and anti-tumor cytokine production using myeloid cells contacted with LNP containing mRNA encoding the indicated anti-TROP 2 CFP constructs. The left panel depicts flow cytometry data analyzing expression of the indicated anti-TROP 2 CFP constructs in various cell types contacted with LNPs containing mRNA encoding the indicated anti-TROP 2 CFP constructs. Expression of the indicated anti-TROP 2 CFP construct was observed in cd14+ myeloid cells, but not in cd19+ B cells, cd3+ T cells or cd56+ NK cells. The middle panel depicts a data plot of luciferase assays from PBMCs that measure killing of SKOV3 cells contacted with LNP containing mRNA encoding the indicated anti-TROP 2 CFP constructs. PBMCs contacted with LNP containing mRNA encoding the indicated anti-TROP 2 CFP constructs and co-cultured with SKOV3-Luc cells showed specific killing of SKOV3 cells compared to controls. The right panel depicts a plot of TNF- α production in the samples of the middle panel.
Figure 12A depicts data for cytokine production using myeloid cells contacted with LNP containing mRNA encoding the indicated anti-TROP 2 CFP constructs. Graphs showing IL-1 beta and IL-18 production in samples cultured alone or co-cultured with SKOV3 cells with PBMC contacted with LNP containing mRNA encoding the indicated anti-TROP 2 CFP constructs are shown.
Figure 12B depicts data for cytokine production using myeloid cells contacted with LNP containing mRNA encoding the indicated anti-TROP 2 CFP constructs. Figures showing CCL2 and TNF- α production in samples cultured alone or co-cultured with SKOV3 cells with PBMCs contacted with LNP containing mRNA encoding the indicated anti-TROP 2 CFP constructs.
Fig. 12C shows a schematic of successful utilization of CD89 (FcR alpha chain) to drive myeloid cell-specific in vivo programming. mRNA encoding CFP with scFv attached to CD89 was formulated in LNP and injected into mice. Percentage plots of CFP expressing myeloid cells and lymphocytes are shown. CFP expression was observed in cd14+ myeloid cells but not in lymphocytes, as compared to control.
FIG. 13A depicts an exemplary schematic of different VH and VL domain configurations of two exemplary anti-TROP 2 CFP constructs with scFv.
FIG. 13B depicts exemplary flow cytometry data showing the effect of different VH and VL domain configurations of two exemplary anti-TROP 2CFP constructs having scFv shown in FIG. 13A on expression in THP-1 cells electroporated with RNA encoding the indicated CFP constructs.
FIG. 13C depicts an exemplary graph showing phagocytosis of SKOV3 cells by THP-1 cells expressing the indicated constructs shown on the X-axis by increasing the quantitative amount of pHrhodo Red signal at the indicated effector to target cell (E: T) ratio.
Fig. 13D depicts exemplary flow cytometry data showing expression of the indicated anti-TROP 2CFP constructs with the indicated transmembrane and intracellular domain combinations. The mock transfected group and the anti-HER 2CFP construct transfected group served as negative and positive controls, respectively.
Fig. 13E depicts exemplary data showing expression of the indicated anti-HER 2 (right) and anti-TROP 2 (left) CFP constructs with the indicated transmembrane and intracellular domain combinations.
Fig. 14A depicts a schematic diagram of an exemplary anti-GP 75 (αgp 75) CFP construct. Exemplary anti-GP 75-FLAG-hCD8-Fcg-PI3K constructs (generation 1 ATAK receptor) are shown in the upper panel. Each of the other anti-GP 75 CFP constructs is depicted as containing a sequence encoding the indicated domain and a GFP encoding sequence separated from the sequence encoding the intracellular domain by a T2A peptide coding sequence. The left panel depicts the multimerization of the CD16 or CD89 transmembrane domain of the anti-GP 75 CFP construct with endogenous fcγ receptor proteins and the binding of the anti-GP 75 CFP construct to GP75 antigen on tumor cells.
Fig. 14B depicts an exemplary flow cytometry gating strategy for in vivo analysis of expression of a indicated CFP construct in various types of mouse lung cells after injection of LNP containing RNA encoding the indicated CFP construct into mice.
Fig. 14C depicts a graph of CFP construct expression as measured by GFP fluorescence positive cell percentage or flag expression measured by in vivo immunoassay in mouse lung cells of the type shown after single or double injection of LNP containing RNA encoding the CFP construct into mice and flow cytometry analysis according to the gating strategy described in fig. 14B. Expression was observed in myeloid cells of the lung, but not in lymphocytes or CD 45-cells.
Fig. 14D depicts an exemplary flow cytometry gating strategy for in vivo analysis of expression of the indicated CFP constructs in various types of mouse hepatocytes after injection of LNP containing RNA encoding the indicated CFP constructs into mice.
Figure 14E depicts a graph of CFP construct expression as measured by GFP fluorescence positive cell percentage or flag expression measured by in vivo immunoassay in mouse hepatocytes of the indicated type after single or double injection of LNP containing RNA encoding the CFP construct into mice and flow cytometry analysis according to the gating strategy described in figure 14D. Expression was observed in myeloid cells of the liver.
Fig. 14F depicts exemplary flow cytometry data showing expression of the indicated anti-GP 75 CFP construct in mouse monocytes in vitro contacted with LNP containing mRNA encoding the CFP construct.
FIG. 15A depicts an exemplary in vitro assay to measure phagocytosis of pHrhodoRed-labeled B16 cells by myeloid cells expressing the anti-GP 75 CFP construct. Myeloid cells have been contacted (e.g., electroporated) with LNP containing mRNA encoding the CFP construct. The flow cytometry data showed that CD11B positive monocytes phagocytose pHrhodoRed-labeled B16 cells.
FIG. 15B depicts exemplary data using monocytes expressing the indicated anti-GP 75 CFP construct following electroporation with LNPs containing mRNA encoding the CFP construct using the assay described in FIG. 15A. A graph of phagocytosis index and flow cytometry data is shown. MYL157 construct = αGP75-FLAG-mouse CD16 TMD-mouse CD16ICD-T2A-GFP (also referred to as GP75-CD16 a). MYL158 construct = αgp 75-FLAG-mouse CD89 TMD-mouse CD89 ICD-T2A-GFP (also referred to as GP75-CD 89).
FIG. 15C depicts exemplary data using monocytes expressing the indicated anti-GP 75 CFP construct following electroporation with LNPs containing RNA encoding the CFP construct using the assay described in FIG. 15A. The structural diagram is shown in the above figure. Data depicting phagocytic index based on flow cytometry analysis is shown. Constructs comprising CD8 hinges showed slightly better expression.
FIG. 15D depicts data for cytokine production by electroporated myeloid cells with LNP containing mRNA encoding the indicated anti-GP 75 CFP construct using the assay described in FIG. 15A. Figures showing TNF-a, CCL3, CCL4 and CCL7 production by B16 cells alone or by co-culture of monocytes contacted with LNP containing mRNA encoding the indicated anti-GP 75 CFP construct are shown.
Fig. 16 depicts an exemplary schematic of preparing LNPs containing mRNA encoding various CFP constructs for in vivo delivery.
Fig. 17A depicts an exemplary LNP dosing regimen for establishing and monitoring tumor growth in a B16 isogenic mouse model. Tumor cells were injected on day 0 and tumors were established in mice more than 10 days post injection. After tumor establishment, mice were injected multiple times with LNP preparations containing mRNA encoding CFP constructs and tumor size was monitored.
Fig. 17B depicts an exemplary schematic for preparing jet PEI mediated transfection complexes containing mRNA encoding various CFP constructs and for in vivo delivery of the complexes via injection.
Figure 18A depicts expression data for the indicated CFP constructs demonstrating positive expression of the indicated CFP constructs in vivo in myeloid cells of the lung, liver and spleen following tail vein injection of LNP containing mRNA encoding the CFP constructs. No expression was observed in the non-myeloid cell types of lung, liver and spleen.
Fig. 18B depicts an expression profile of an exemplary CFP construct (graphically depicted in the upper profile) as measured by percent GFP fluorescence or FLAG expression positive cells measured by in vivo immunoassays in lung and liver cells of the indicated types of mice after single or double injection of LNP containing RNA encoding the CFP construct into the mice and flow cytometry analysis according to the gating strategy described in fig. 14B or 14D.
Fig. 18C depicts GFP and FLAG shift histograms showing clear GFP expression in monocytes and neutrophils.
Figure 18D depicts flow cytometry data for CFP construct expression as measured by in vivo flow cytometry immunoassay in lung and liver cells of mice of the indicated type following injection of LNP containing RNA encoding the CFP construct into the mice and flow cytometry analysis according to the gating strategy described in figure 14B or figure 14D.
Figure 19 depicts histopathological analysis of tumors in B16 mice treated with LNP containing RNA encoding the anti-GP 75 construct. 2 out of 4 mice showed complete anti-tumor responses, no tumor was detected.
Figure 20A depicts cytokine-producing data from cells of naive mice or mouse tumor models treated with vehicle or myeloid cells expressing the anti-GP 75-ATAK CFP construct. A graph showing the production of CCL2 and IL-1 a by mice is shown. In treated mice, CCL7, which promotes tumor progression, was decreased, while IL-1 a, which was associated with tumor cell death, was increased. ATAK is an acronym for antigen targeting and killing.
Figure 20B depicts cytokine-producing data from cells of naive mice or mouse tumor models treated with vehicle or myeloid cells expressing the anti-GP 75 CFP construct. Graphs showing that mice produce IL23, eotaxin (CXCL 1, IL17a, G-CSF, and CXCL5, and demonstrate increased T-cell and myeloid cell activation.
Figure 20C shows recruitment of T cells by CAR-expressing monocytes upon stimulation. In an in vitro assay, CD5-ATAK cells induce T cell chemotaxis.
Figure 21 depicts cytokine-producing data from naive mice or from mice tumor models treated with vehicle or myeloid cells expressing the anti-GP 75 CFP construct. Flow cytometry data for IFNγ, TNF- α and IL-2 production by cells isolated from the corresponding mice are shown. Also shown are percentage plots of CD4+ T cells producing IFNγ, TNF- α and IL-2.
Figure 22 depicts a plot of tumor volume over time for mice treated with LNP containing mRNA encoding the indicated anti-GP 75 CFP construct at the indicated time points (shown by the arrow below the X axis). On day 9 post-treatment, 5 mice were taken from each group. The following organs were harvested for flow analysis: tumors, lung, liver and spleen. RNA samples from tumor samples were saved for NanoString gene expression analysis. T cell repeat stimulation assays were also performed with Ova peptide.
Figure 23 shows that LNP treatment alters immune cell tumor infiltration. A graph of the percentage of the cell types shown in all cd45+ immune cells in the tumor microenvironment of mice treated with LNP containing mRNA encoding the indicated anti-GP 75 CFP construct is depicted. Treatment resulted in a significant increase in inflammatory monocytes in the tumor, and a slight increase in macrophages and T cells.
Figure 24 shows that LNP treatment resulted in a significant increase in CD8 cytotoxic T cells and a decrease in Treg cells. A graph of the percentage of the cell types shown in all cd45+ immune cells of mice treated with LNP containing mRNA encoding the indicated anti-GP 75 CFP construct is depicted. Treatment increased the presence of cd8+ cytotoxic T cells while reducing immunosuppressive tregs.
Figure 25 shows that LNP treatment resulted in reduced expression of CD 8T cell immune checkpoints. Depicted is a graph of the indicated immune checkpoint molecule expression levels of cd8+ T cells from the tumor microenvironment of mice treated with LNP containing mRNA encoding the indicated anti-GP 75 CFP construct. Treatment resulted in decreased PD1 expression levels and slightly increased LAG3 expression.
Figure 26 shows the conversion of tumor microenvironment to an inflammatory phenotype upon in vivo administration of nucleic acid encoding CFP in an LNP composition.
FIG. 27 shows that LNP treatment results in reduced Ki-67 and perforin expression in CD8+ T cells. Depicted is a graph of the indicated levels of molecular expression of cd8+ T cells from the tumor microenvironment of mice treated with LNP containing mRNA encoding the indicated anti-GP 75 CFP construct.
Figure 28 shows flow cytometry data showing expression of CFP constructs in THP-1 cells electroporated with mRNA encoding CFP.
FIG. 29 shows a schematic diagram demonstrating the production of mouse anti-GP 75 (trp-1) CFP ATAK cells. anti-GP 75 CFP is efficiently expressed in mouse monocytes via electroporation of mRNA encoding anti-GP 75 CFP, and cells expressing anti-GP 75 CFP have phagocytic activity.
Figure 30 shows data demonstrating tumor inhibition by anti-GP 75 ATAK monocytes in a syngeneic B16 murine model. The data show a plot of tumor volume over time in a mouse tumor model that is refractory to CAR-T and checkpoint inhibitors after treatment with mouse monocytes expressing anti-GP 75 CFP. Mice were given 8 infusions of 2X 10≡6 cells relative to vehicle. Infusions were 4 times per day, resting for 3 days, 4 times per day +/-SD (Mann-Whitney test).
Figure 31 shows a graph demonstrating that after infusion into a mouse tumor model, mouse monocytes expressing anti-GP 75 CFP penetrate into the tumor, maintain expression of CFP and differentiate into effector cells (e.g., inflammatory cells, dendritic cells, and macrophages). The left panel shows that about 40% of mouse monocytes expressing anti-GP 75 CFP maintain CFP expression 5 days after infusion. The upper right panel shows that ATAK cells (CFP expressing cells) penetrate the tumor and spleen and become dendritic cells. The lower right panel shows that ATAK cells penetrate the tumor and spleen and become macrophages.
Figure 32 depicts cytokine-producing data from naive mice or from mice tumor models treated with vehicle or myeloid cells expressing the anti-GP 75 CFP construct. A graph of the cytokine/chemokine production is shown. The therapeutic response is related to the anti-tumor serum cytokine profile. Analysis included response animals in the treatment group at the end of the study.
Figure 33 depicts data showing cross-presentation of tumor antigens by mouse monocytes expressing anti-GP 75 CFP. Mouse monocytes expressing anti-GP 75 CFP are capable of phagocytosing, processing and presenting surrogate tumor neoantigens (in this case derived from OVA proteins) to T cells bearing homologous TCRs derived from OVA class I restriction peptides (SIINFEKL) and determining T cell activation by IL2 (left) or interferon (right) production; whereas monocytes from the same mice that do not express the ATAK receptor were unable. This demonstrates that mouse monocytes expressing anti-GP 75 CFP can process and cross-present antigens to adaptive immune cells, such as CD 8T cells. An ATAK-GP75 (also known as TRP-1 ATAK) construct.
Figure 34 depicts data showing that CFP expressing human monocytes utilize innate immunity and stimulate an adaptive anti-tumor immune response. The data show that CFP expressing human monocytes penetrated, accumulated in and recognized the tumor (upper left). The data show that CFP expressing human monocytes lead to myeloid cell activation, cytokine production, chemokine production and inflammatory polarization (upper right panel). The data show that CFP expressing human monocytes directly kill tumor cells via phagocytosis (lower left), cytokines and death receptor (CD 95L). The data show that CFP expressing human monocytes demonstrate long term control of tumors via antigen presentation, epitope spreading and T cell engagement (bottom right panel), ensuring complete activation of immune repertoires.
Fig. 35 depicts data showing that human monocytes expressing anti-CD 5 CFP exhibit potent activity in vitro inhibitory Tumor Microenvironment (TME) conditions. Human monocytes expressing anti-CD 5 CFP were incubated overnight in medium containing IL10, IL4 and TGF- β with or without homologous antigen. Phagocytic activity of the cells is shown in the upper left-most panel. Cytokines/chemokines as shown were measured in the culture supernatants.
Fig. 36 depicts data showing that human monocytes expressing anti-CD 5 CFP inhibit growth and prolong survival in a cd5+ctcl xenograft model despite the absence of an adaptive immune system.
FIG. 37 depicts a display of the contentLNP injection of RNA encoding anti-GP 75 CFP and CD89 (fcrα) chains into mice selectively programs myeloid cells in vivo, resulting in data for potent anti-tumor activity. Tumor mass (volume in mm) was observed in the established cold tumor model 3 Measured) 75% reduction.
Fig. 38 depicts data showing that injection of LNP containing RNA encoding anti-GP 75 CFP and CD89 (fcrα) chains into mice resulted in expression of CFP by inflammatory monocytes within TME. >15% of tumor myeloid cells were observed to express anti-GP 75 CFP.
Fig. 39 depicts data showing that injection of LNP containing RNA encoding anti-GP 75 CFP and CD89 (fcrα) chains into mice resulted in TME modification characterized by accumulation of inflammatory cells. Treatment induced tumor inflammatory monocytes (left) and dendritic cells (right) in vivo as measured after death of the treated animals. Treatment increases immune cells associated with bridging innate and adaptive immunity.
Figure 40 depicts data showing that injection of LNP containing RNA encoding anti-GP 75 CFP and CD89 (fcrα) chains into mice resulted in TME modification characterized by increased CTL and decreased Treg. Treatment promotes anti-tumor CD 8T cells and reduces tumor-associated tregs.
Figure 41 depicts data for the production of the indicated pro-inflammatory serum cytokines before, 24 hours after 2 injections and 72 hours after 4 injections of LNP containing RNA encoding the anti-GP 75 CFP and CD89 (fcrα) chains into mice.
Figure 42 depicts data for the generation of the indicated serum chemokines before, 24 hours after 2 injections and 72 hours after 4 injections of LNP containing RNA encoding the anti-GP 75 CFP and CD89 (fcrα) chains into mice.
Fig. 43 depicts experimental design and tumor growth. Tumors were inoculated on day 0 and grown to 75mm 3 . LNP-mRNA was administered at 2 mg/kg.
Figure 44 depicts a study of T cell subpopulations and phenotypes in tumors.
FIG. 45 depicts data for mRNA-LNP treatment inducing frequency changes in T cell subsets within tumors. Treatment resulted in a reduction of Treg% in tumors.
Fig. 46A depicts a graph showing flow cytometry analysis of CD8 depletion marker (PD 1) and CD8 maturation/effector T cell marker (CD 44) in% cd8+ T cells in mice injected with GP75-CD 89-LNP.
Fig. 46B depicts flow cytometry data for PD1 and CD44 expression in T cells.
FIG. 47A depicts a graph showing flow cytometry analysis of CD8 depletion markers (TIM 3 and TOX) in% CD8+ T cells in mice injected with GP75-CD 89-LNP.
FIG. 47B depicts flow cytometry data for TIM3 and TOX expression in T cells.
FIG. 48A depicts a graph showing flow cytometry analysis of the cell proliferation marker Ki67 and the T cell activation marker granzyme B in mice injected with GP75-CD 89-LNP.
FIG. 48B depicts flow cytometry data for Ki67 and granzyme B in CD8T cells from mice injected with GP75-CD 89-LNP.
Figure 49 shows data from a cell lineage study; the set of markers and fluorophores is shown on the left.
Figure 50 depicts data indicating that mRNA/LNP treatment does not induce a significant shift in immune cell populations.
Figure 51 shows data on receptor expression.
FIG. 52 shows a dot plot showing FLAG (i.e., receptor) expression in dendritic cells.
FIG. 53 shows a display Ly6C hi Dot plot of FLAG expression in inflammatory monocytes.
FIG. 54 shows a display Ly6C lo Dot plot of FLAG expression in resident myeloid cells.
FIG. 55 depicts a graph demonstrating that treatment in mice significantly increases Ly6C hi And CCR2 hi Data on CD40 and CD206 levels in inflammatory monocytes.
FIG. 56 depicts Ly6C lo And CCR2 lo PDL1, CD40, CD86 and CD206 levels in resident myeloid cells. The data indicate that treatment in mice significantly increases CD40 and CD206 levels.
FIG. 57 shows PDL1, CD40, CD86 and CD206 levels; the results indicate that treatment slightly reduces PDL1 levels in tumor resident macrophages.
Figure 58 shows that treatment significantly increases the DC activation markers, including mhc ii, CD86 and CD40, but also increases PD-L1 levels in the cd103+cd11b-dendritic cell phenotype in the spleen.
FIG. 59 shows that treatment significantly increases the DC activation markers, including MHCII, CD86, and CD40, but also increases the PD-L1 levels in the CD103-CD11b+ dendritic cell phenotype in the spleen.
FIG. 60 shows a schematic representation of an exemplary expression vector design for expressing in a myeloid cell a first vector encoding a chimeric receptor protein comprising a protease cleavable inducible gene transcription activator protein that is specifically activated upon contact of the myeloid cell with a cancer cell; and a second vector expressing a target gene inducible by the transcriptional activator of the gene encoded by the first vector.
FIG. 61A shows a graphical representation of an exemplary chimeric protein produced by expression of the vector of FIG. 60. FIG. 61A also specifically shows that proteases fused to PTB-HIF-degradation determinants are susceptible to degradation when PTB does not bind to the phosphotyrosine residue of the ITAM motif of the chimeric receptor intracellular domain, which is triggered by the degradation determinant complex fused to PTB.
FIG. 61B shows the expected mode of action of the chimeric protein of FIG. 61A resulting in expression of the target gene. Upon binding of the HIF-degrading determinant protease to the phosphorylated ITAM motif of the receptor, the protease is activated and cleaves the transcriptional activator GAL-VP64.
FIG. 62A shows a schematic representation of a nucleic acid construct encoding an inducible cytotoxic protein. Constructs are designed for expression and secretion by myeloid cells (e.g., macrophages). The construct comprises the acidic domain and the cytotoxic domain of the human eosinophil major basic protein. These two domains are interspersed with sequences that encode MMP recognition peptides; MMP recognition peptides can be cleaved by MMPs present in large numbers in the tumor microenvironment. In the absence of MMPs, secreted cytotoxic proteins are retained in inactive form by association with the acidic domain, held together by MMP recognition peptides.
Figure 62B shows a graphical representation of cytotoxic domain protein activation resulting in proteolytic cleavage of tumor cells by the cytotoxic domain when MMPs cleave MMP recognition sequences in the tumor microenvironment.
Detailed description of the preferred embodiments
All terms are intended to be interpreted as understood by those skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
While various features of the invention may be described in the context of a single embodiment, such features may also be provided separately or in any suitable combination. Conversely, although the invention may be described in the context of separate embodiments herein for clarity, the invention may also be practiced in a single embodiment.
Reference in the specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the invention.
As used in this specification and claims, the terms "comprises," comprising, "" and any form of "comprising," such as "comprises" and "includes," "having," "has," "including," or "containing," "including," are inclusive or open-ended, and do not exclude additional, unrecited elements or method steps. It is contemplated that any of the embodiments discussed in this specification may be implemented with respect to any of the methods or compositions of the invention, and vice versa. Furthermore, the compositions of the present invention may be used to carry out the methods of the present invention.
When referring to measurable values (e.g., parameters, amounts, time intervals, etc.), the term "about" or "approximately" as used herein is meant to include variations of +/-30% or less, +/-20% or less, +/-10% or less, +/-5% or less, or +/-1% or less of the specified value, so long as such variations are suitable for being made in the present invention. It is to be understood that the value itself to which the modifier "about" or "approximately" refers is also specifically disclosed.
An "agent" may refer to any cell, small molecule compound, antibody or fragment thereof, nucleic acid molecule, or polypeptide.
"change" or "change" may refer to an increase or decrease. For example, the change may be an increase or decrease of 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or 40%, 50%, 60%, or even up to 70%, 75%, 80%, 90%, or 100%. For example, the change may be an increase or decrease by a factor of 1, 2, 3, 4, 5, 10, 20, 30, or 40, 50, 60, or even up to a factor of 70, 75, 80, 90, or 100.
As used herein, "antigen presenting cells" or "APCs" include professional antigen presenting cells (e.g., B lymphocytes, macrophages, monocytes, dendritic cells, langerhans cells) and other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes, thymic epithelial cells, thyroid epithelial cells, glial cells (brain), pancreatic beta cells, and vascular endothelial cells). APCs can express Major Histocompatibility Complex (MHC) molecules and can display antigens on their surface that complex with MHC that can be recognized by T cells and trigger T cell activation and immune responses. Professional antigen presenting cells, particularly dendritic cells, play a critical role in stimulating primitive T cells. Non-professional antigen presenting cells, such as fibroblasts, may also assist in this process. APCs can also cross-present peptide antigens by treating exogenous antigens and presenting the treated antigens on MHC class I molecules. Antigens that produce proteins recognized in association with class I MHC molecules are typically proteins produced in cells, which are processed and associated with class I MHC molecules.
"biological sample" may refer to any tissue, cell, fluid, or other material derived from an organism.
The term "epitope" may 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 are generally composed of chemically active surface groupings of molecules (e.g., amino acids or sugar side chains) and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. "T cell epitope" may refer to a peptide or peptide-MHC complex recognized by a T cell receptor.
An engineered cell, such as an engineered myeloid cell, may refer to a cell that has at least one exogenous nucleic acid sequence in the cell, even though transiently expressed. Expression of the exogenous nucleic acid can be performed by various methods described elsewhere, and includes methods known in the art. The present invention relates to the preparation and use of engineered cells, e.g., engineered myeloid cells, e.g., engineered phagocytes. In particular, the invention relates to an engineered cell comprising an exogenous nucleic acid encoding, for example, a Chimeric Fusion Protein (CFP).
The term "immune response" includes, but is not limited to, T cell-mediated, NK cell-mediated, and/or B cell-mediated immune responses. These responses may be affected by the modulation of T cell co-stimulation and NK cell co-stimulation. Exemplary immune responses include T cell responses, e.g., cytokine production and cytotoxicity. In addition, immune responses include immune responses that are affected by NK cell activation, B cell activation, and/or T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells (e.g., macrophages). The immune response includes an adaptive immune response. The adaptive immune system may react to foreign molecular structures (e.g., antigens of invading organisms). Unlike the innate immune system, the adaptive immune system is highly specific for pathogens. Adaptive immunity may also provide durable protection. Adaptive immune responses include humoral immune responses and cell-mediated immune responses. In humoral immune responses, antibodies secreted into the body fluid by B cells bind pathogen-derived antigens, resulting in elimination of the pathogen by a variety of mechanisms, e.g., complement-mediated lysis. In a cell-mediated immune response, T cells capable of destroying other cells are activated. For example, if proteins associated with a disease are present in a cell, they may be proteolytically fragmented into peptides within the cell. Specific cellular proteins can then attach themselves to antigens or peptides formed in this way and transport them to the cell surface where they can be presented to molecular defense mechanisms, such as T cells. Cytotoxic T cells can recognize these antigens and kill cells carrying these antigens.
A "ligand" may refer to a molecule that is capable of binding or forming a complex with another molecule (e.g., a receptor). Ligands may include, but are not limited to, proteins, glycoproteins, carbohydrates, lipoproteins, hormones, fatty acids, phospholipids, or any component that binds to a receptor. In some embodiments, the receptor has a specific ligand. In some embodiments, the receptor may have promiscuous binding to a ligand, in which case it may bind several ligands sharing at least similarity in terms of structural configuration, charge distribution, or any other physicochemical characteristic. The ligand may be a biological molecule. The ligand may be a non-biological material. For example, the ligand may be a negatively charged particle, which is a ligand for the scavenger receptor MARCO. For example, the ligand may be TiO 2 Which is a ligand for the scavenger receptor SRA 1.
The term "Major Histocompatibility Complex (MHC)", "MHC molecule" or "MHC protein" refers to a protein capable of binding an antigen peptide and presenting the antigen peptide to T lymphocytes. Such antigenic peptides may represent T cell epitopes. Human MHC is also known as HLA complex. Thus, the terms "Human Leukocyte Antigen (HLA)", "HLA molecule" or "HLA protein" may be used interchangeably with the terms "Major Histocompatibility Complex (MHC)", "MHC molecule" and "MHC protein". HLA proteins can be classified as either HLA class I or HLA class II. The structure of the two HLA-type proteins is very similar; however, they have very different functions. HLA class I proteins are present on the surface of almost all cells of the body, including most tumor cells. HLA class I proteins are loaded with antigens typically derived from endogenous proteins or pathogens present within the cell and then presented to primitive or Cytotoxic T Lymphocytes (CTLs). HLA class II proteins are found on Antigen Presenting Cells (APCs) including, but not limited to, dendritic cells, B cells and macrophages. They primarily present peptides processed from external antigen sources (e.g., outside the cell) to helper T cells.
In HLA class II systems, phagocytes (e.g., macrophages and immature dendritic cells) can ingest entities into the phagosome by phagocytosis-although B cells appear to be more commonly taken up into the endosome by endocytosis-they fuse with lysosomes, the acid enzymes of which cleave the ingested protein into many different peptides. Autophagy is another source of HLA class II peptides. The most studied HLA class II genes are: HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA and HLA-DRB1.
The presentation of peptides by HLA class II molecules to cd4+ helper T cells can lead to an immune response to foreign antigens. Once activated, cd4+ T cells can promote B cell differentiation and antibody production, as well as cd8+ T Cell (CTL) responses. Cd4+ T cells can also secrete cytokines and chemokines that activate and induce differentiation of other immune cells. HLA class II molecules are typically heterodimers of the α -and β -chains that interact to form a peptide binding groove that is more open than a class I peptide binding groove.
HLA alleles are usually expressed in co-dominant fashion. For example, each person carries 2 alleles of each of 3 class I genes (HLA-A, HLA-B, and HLA-C), so six different types of HLA class II can be expressed. In the class II HLA locus, each person inherits a pair of HLA-DP genes (DPA 1 and DPB1, which encode the alpha and beta chains), HLA-DQ (DQA 1 and DQB1 for the alpha and beta chains), one gene HLA-DR alpha (DRA 1) and one or more genes HLA-DR beta (DRB 1 and DRB3, -4 or-5). HLA-DRB1, for example, has more than nearly 400 known alleles. This means that a heterozygous individual can inherit six or eight functional HLA class II alleles: three or more from each parent. Thus, HLA genes are highly polymorphic; there are many different alleles in different individuals within a population. The genes encoding HLA proteins have many possible variations, allowing the immune system of each individual to respond to a variety of foreign invaders. Some HLA genes have hundreds of identified versions (alleles), each of which has a specific number. In some embodiments, HLA class I alleles are HLA-A.times.02:01, HLA-B.times.14:02, HLA-A.times.23:01, HLA-E.times.01:01 (atypical). In some embodiments, the HLA class II alleles are HLA-DRB.times.01:01, HLA-DRB.times.01:02, HLA-DRB.times.11:01, HLA-DRB.times.15:01, and HLA-DRB.times.07:01.
"myeloid cells" can broadly refer to cells of the myeloid lineage of the hematopoietic cell system, and can exclude, for example, the lymphocyte lineage. Myeloid cells include, for example, cells of the granulocyte lineage and of the monocyte lineage. Myeloid cells are differentiated from common progenitor cells derived from hematopoietic stem cells in bone marrow. The typing of myeloid cell lineages can be controlled by the activation of different transcription factors, and thus myeloid cells can be characterized as cells with a level of plasticity that can be described as the ability to differentiate further into terminal cell types based on extracellular and intracellular stimuli. Myeloid cells can be rapidly recruited to local tissues via various chemokine receptors on their surface. Myeloid cells respond to a variety of cytokines and chemokines.
The myeloid cells may be, for example, cells derived from hematopoietic stem cells in bone marrow under the influence of one or more cytokines and chemokines (e.g., G-CSF, GM-CSF, flt3L, CCL2, VEGF, and S100A 8/9). In some embodiments, the myeloid cell is a precursor cell. In some embodiments, the myeloid cells can be cells that have characteristics of common myeloid progenitor cells or granulocyte progenitor cells, myeloblasts or monocyte-dendritic cell progenitor cells, or a combination thereof. The myeloid cells may comprise granulocytes or monocytes or precursor cells thereof. Myeloid cells can include immature granulocytes, immature monocytes, immature macrophages, immature neutrophils and immature dendritic cells. The myeloid cells may comprise monocytes or pre-monocytes (pre-monocytical cells) or monocyte precursors. In some cases, a myeloid cell as used herein may refer to a monocyte having an M0 phenotype, an M1 phenotype, or an M2 phenotype. Myeloid cells may include Dendritic Cells (DCs), mature DCs, monocyte-derived DCs, plasmacytoid DCs, pre-dendritic cells (pre-dendritic cells), or precursors of DCs. The myeloid cells may include neutrophils, which may be mature neutrophils, neutrophil precursors, or polymorphonuclear cells (PMNs). Myeloid cells can include macrophages, monocyte-derived macrophages, tissue macrophages, macrophages of M0, M1 or M2 phenotype. The myeloid cells may comprise tumor-infiltrating monocytes (TIMs). Myeloid cells can include tumor-associated monocytes (TAMs). Myeloid cells may include Myeloid Derived Suppressor Cells (MDSCs). Myeloid cells can include tissue resident macrophages. Myeloid cells can include tumor-associated DCs (TADCs). Thus, myeloid cells can express one or more cell surface markers, e.g., CD11b, CD14, CD15, CD16, CD38, CCR5, CD66, lox-1, CD11c, CD64, CD68, CD163, CCR2, CCR5, HLA-DR, CD1c, CD83, CD141, CD209, CD205, P-selectin, integrin, ICAMS, VCAMS, MHC-II, CD123, CD303, CD304, SIGLEC family proteins, and CLEC family proteins. In some cases, the myeloid cells can be characterized by high or low expression of one or more of the cell surface markers, e.g., CD11b, CD14, CD15, CD16, CD66, lox-1, CD11c, CD64, CD68, CD163, CCR2, CCR5, HLA-DR, CD1c, CD83, CD141, CD209, MHC-II, CD123, CD303, CD304, or a combination thereof.
"phagocytosis" is used interchangeably with "engulfment" and may refer to the process by which a cell engulfs particles (e.g., cancer cells or infected cells). This process can create an internal compartment (phagosome) containing particles. This process may be used to ingest and/or remove particles, such as cancer cells or infected cells from the body. Phagocytic receptors may be involved in the process of phagocytosis. The process of phagocytosis may be closely related to immune responses and antigen presentation. The processing of exogenous antigens is performed after they are taken up into professional antigen presenting cells by some type of endocytic event. Phagocytosis may also promote antigen presentation. For example, antigens from phagocytes or pathogens, including cancer antigens, may be processed and presented on the cell surface of APCs.
"polypeptide" may refer to a molecule, such as a glycoprotein, lipoprotein, cellular protein, or membrane protein, containing amino acids linked together via peptide bonds. The polypeptide may comprise one or more subunits of a protein. The polypeptide may be encoded by a recombinant nucleic acid. In some embodiments, a polypeptide may comprise more than one peptide sequence in a single amino acid chain, which may be separated by a spacer, linker, or peptide cleavable sequence. The polypeptide may be a fusion polypeptide. The polypeptide may comprise one or more domains, modules or portions.
A "receptor" may refer to a chemical structure consisting of a polypeptide that transduces a signal, e.g., a polypeptide that transduces extracellular light into a cell. Receptors may be used to transmit information in cells, cell formations, or organisms. The receptor comprises at least one receptor unit and may contain two or more receptor units, wherein each receptor unit comprises a protein molecule, e.g., a glycoprotein molecule. Receptors may contain structures that bind ligands and may form complexes with ligands. Signaling information can be transmitted through conformational changes of the receptor upon binding to a ligand on the cell surface.
The term "antibody" refers to a class of proteins commonly referred to as immunoglobulins, including but not limited to IgG1, igG2, igG3 and IgG4, igA (including IgA1 and IgA 2), 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, but are not limited to, fab 'and F (ab') 2, fd (from V) H And C H 1), single chain variable fragment (scFv), single chain antibody, disulfide-linked variable fragment (dsFv) and comprising V L And/or V H Fragments of the domains. The antibodies may be from any animal source. Antigen binding antibody fragments, including single chain antibodies, may comprise a variable region alone or in combination with one or more of a hinge region, a CH1 domain, a CH2 domain, and a CH3 domain. Also included are any combination of variable and hinge regions, CH1, CH2, and CH3 domains. Antibodies can be monoclonal, polyclonal, chimeric, humanized and human monoclonal and polyclonal antibodies, for example, that specifically bind to HLA-related polypeptides or HLA peptide complexes. As used herein, " A conjugate "refers to a polypeptide comprising a binding domain that can bind to a target, wherein the target of the conjugate can be a protein, e.g., a cancer antigen, glycoprotein, etc. Conjugates are typically used to represent CFPs, e.g., CARs, and are named by the target to which they bind, e.g., CD5 conjugates comprising a CD5 antigen binding domain. In some cases, it may alternatively or interchangeably be referred to as an anti-CD 5 conjugate or an anti-TROP 2 conjugate. As generally used, the term conjugate refers to any molecule having a binding domain. In some cases, the conjugate may be BiME or treme when described in the context.
The term "recombinant nucleic acid" refers to a nucleic acid that has been prepared, expressed, produced, or isolated by recombinant means. Recombinant nucleic acids may contain non-naturally occurring nucleotide sequences. Recombinant nucleic acids can be synthesized in the laboratory. Recombinant nucleic acids can be prepared by using recombinant DNA techniques, for example, enzymatic modification of DNA, such as enzymatic restriction digestion, ligation, and DNA cloning. The recombinant nucleic acid may be DNA, RNA, analogs thereof, or combinations thereof. Recombinant DNA may be transcribed ex vivo or in vitro, for example, to produce messenger RNA (mRNA). Recombinant mRNA can be isolated, purified and used to transfect cells. The recombinant nucleic acid may encode a protein or polypeptide.
The process of introducing or incorporating nucleic acid into a cell may be via transformation, transfection or transduction. Transformation is the process by which bacterial cells ingest foreign nucleic acids. The process is suitable for plasmid DNA propagation, protein production and other applications. Transformation recombinant plasmid DNA is introduced into competent bacterial cells, which uptake extracellular DNA from the environment. Some bacterial species are naturally competent under certain environmental conditions, but competent are induced artificially in laboratory environments. Transfection is the introduction of small molecules (e.g., DNA, RNA) or antibodies into eukaryotic cells. Transfection may also refer to the introduction of phage into bacterial cells. "transduction" is used primarily to describe the introduction of recombinant viral vector particles into target cells, while "infection" refers to the natural infection of a human or animal with wild-type virus.
The term "vector" may refer to a nucleic acid molecule capable of autonomous replication in a host cell and allowing cloning of the 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, and the like. For example, the vector for exogenous gene transformation may be a plasmid. In certain embodiments, the vector comprises a nucleic acid sequence comprising an origin of replication and other elements necessary for replication and/or maintenance of the nucleic acid sequence in a host cell. In some embodiments, the vector or plasmid provided herein is an expression vector. The expression vector is capable of directing expression of a gene and/or nucleic acid sequence to which it is operably linked. In some embodiments, the expression vector or plasmid is in the form of a circular double stranded DNA molecule. The vector or plasmid may or may not be integrated into the genome of the host cell. In some embodiments, the nucleic acid sequence of the plasmid is not integrated into the genome or chromosome of the host cell after introduction. For example, a plasmid may comprise elements for transiently expressing or stably expressing a nucleic acid sequence in a host cell, e.g., a gene or open reading frame carried by the plasmid. In some embodiments, the vector is a transient expression vector. In some embodiments, the vector is a stable expression vector that replicates autonomously in the host cell. In some embodiments, the nucleic acid sequence of the plasmid is integrated into the genome or chromosome of the host cell upon introduction into the host cell. Expression vectors that may be used in the methods as disclosed herein include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, phage or viral vectors. The vector may be a DNA or RNA vector. In some embodiments, the vectors provided herein are RNA vectors, e.g., retroviral vectors or lentiviral vectors, capable of integrating into the host cell genome upon introduction into the host cell (e.g., via reverse transcription). Other forms of expression vectors known to those of skill in the art to have equivalent functions, such as self-replicating extra-chromosomal vectors or vectors capable of integration into the host genome, may also be used. Exemplary vectors are vectors capable of autonomously replicating and/or expressing a nucleic acid to which they are linked.
The term "spacer" or "linker" as used in reference to a fusion protein refers to a peptide sequence that links two other peptide sequences of the fusion protein. In some embodiments, the linker or spacer has no specific biological activity other than to link or maintain some minimal distance or other spatial relationship between the protein or RNA sequences. In some embodiments, the constituent amino acids of the spacer may be selected to affect some property of the molecule, such as folding, flexibility, net charge, or hydrophobicity of the molecule. Suitable linkers for use in embodiments of the present invention are well known to those skilled in the art and include, but are not limited to, straight or branched chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. In some embodiments, a linker is used to separate two or more polypeptides (e.g., two antigenic peptides) a distance sufficient to ensure that each antigenic peptide folds correctly. Exemplary peptide linker sequences adopt a flexible extended conformation and do not exhibit a propensity to form ordered secondary structures. Amino acids in the flexible linker protein region can include Gly, asn and Ser, or any arrangement of amino acid sequences containing Gly, asn and Ser. Other near neutral amino acids, such as Thr and Ala, may also be used for the linker sequence.
The term "treating" refers to reducing, preventing or ameliorating a disorder and/or symptom associated therewith (e.g., neoplasia or tumor or infectious pathogen or autoimmune disease). "treating" can refer to administering therapy to a subject after the onset or suspected onset of a disease (e.g., an infection by a cancer or infectious pathogen or autoimmune disease). "treatment" includes the concept of "alleviation" which may refer to reducing the frequency or severity of occurrence or recurrence of any symptoms or other adverse effects associated with a disease and/or side effects associated with a treatment. The term "treatment" also includes the concept of "management" which refers to reducing the severity of a disease or disorder in a patient, e.g., extending the life span or increasing the viability of a patient suffering from a disease, or delaying their recurrence, e.g., extending the remission period of a patient suffering from a disease. It is to be understood that although not precluded, treating a disorder or condition does not require complete elimination of the disorder, condition, or symptom associated therewith. The term "preventing" and grammatical equivalents thereof as used herein may refer to avoiding or delaying the onset of symptoms associated with a disease or disorder in a subject that have not developed upon initiation of administration of an agent or compound. In certain embodiments, treating a subject or patient as described herein includes administering a therapeutic composition, e.g., a drug, metabolite, prophylactic component, nucleic acid, peptide, or protein encoding or otherwise forming a drug, metabolite, or prophylactic component. In some embodiments, the treatment comprises administering a cell or population of cells to a subject in need thereof. In some embodiments, the treatment comprises administering to the subject one or more of the engineered cells described herein, e.g., one or more engineered myeloid cells, e.g., phagocytes. Treatment includes treatment of a disease or 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 administration of a therapeutic vaccine. In some embodiments, the engineered phagocytes are administered to a patient or subject. In some embodiments, the cells administered to the human subject result in reduced immunogenicity. For example, engineering phagocytes may result in no or reduced Graft Versus Host Disease (GVHD) or mutual killing. In some embodiments, the engineered cells are administered to a human subject to be immunocompatible with the subject (i.e., have a matched HLA subtype naturally expressed in the subject). The subject-specific HLA allele or HLA genotype of the subject can be determined by any method known in the art. In an exemplary embodiment, the method includes determining a polymorphism genotype, which may include generating an alignment of reads extracted from the sequencing dataset with a reference set of genes comprising allelic variants of the polymorphism gene, determining a first posterior probability or posterior probability derivative score for each allelic variant in the alignment, identifying the allelic variant having the greatest first posterior probability or posterior probability derivative score as the first allelic variant, identifying one or more overlapping reads aligned with the first allelic variant and one or more other allelic variants, determining a second posterior probability or posterior probability derivative score for the one or more other allelic variants using a weighting factor, identifying the second allelic variant by selecting the allelic variant having the greatest second posterior probability or posterior probability derivative score, the first and second allelic variants defining the genotype of the polymorphism gene, and providing an output of the first and second allelic variants.
A "fragment" may refer to a portion of a protein or nucleic acid. In some embodiments, the fragment retains at least 50%, 75%, or 80%, or 90%, 95%, or even 99% of the biological activity of the reference protein or nucleic acid.
The terms "isolated", "purified", "biologically pure" and grammatical equivalents thereof refer to materials that are free to varying degrees of components that normally accompany them as they exist in their native state. "isolated" means separated from the original source or environment. "purification" means a degree of separation that is higher than separation. A "purified" or "biologically pure" protein is sufficiently free of other materials that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of the invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques (e.g., polyacrylamide gel electrophoresis or high performance liquid chromatography). The term "purified" may mean that the nucleic acid or protein substantially produces a band in the electrophoresis gel. For proteins that can be modified (e.g., phosphorylated or glycosylated), different modifications can result in different isolated proteins, which can be purified separately.
The term "neoplasia" or "cancer" refers to any disease caused or resulting from inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Glioblastomas are one non-limiting example of neoplasia or cancer. The term "cancer" or "tumor" or "hyperproliferative disorder" refers to the presence of cells that have characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and certain characteristic morphological features. The cancer cells are typically in the form of tumors, but such cells may be present in the animal body alone, or may be non-tumorigenic cancer cells, such as leukemia cells.
The term "vaccine" is understood to mean a composition that develops immunity for the prevention and/or treatment of a disease (e.g. neoplasia/tumor/infectious pathogen/autoimmune disease). Thus, a vaccine as used herein is a drug comprising a recombinant nucleic acid, or a cell comprising and expressing a recombinant nucleic acid, and is intended for use in humans or animals to produce specific defenses and protective substances by vaccination. The "vaccine composition" may include a pharmaceutically acceptable excipient, carrier or diluent. Aspects of the invention relate to the use of technology in the preparation of phagocyte-based vaccines.
The term "pharmaceutically acceptable" refers to those approved or that are approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans. By "pharmaceutically acceptable excipient, carrier or diluent" is meant an excipient, carrier or diluent that can be administered to a subject with a pharmaceutical agent that does not destroy the pharmacological activity and is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the pharmaceutical agent.
Nucleic acid molecules useful in the methods of the invention include, but are not limited to, any nucleic acid molecule that is active or encodes a polypeptide. Polynucleotides having substantial identity to an endogenous sequence are typically capable of hybridizing to at least one strand of a double stranded nucleic acid molecule. "hybridization" refers to pairing of nucleic acid molecules under various stringency conditions to form a double-stranded molecule between complementary polynucleotide sequences or portions thereof. (see, e.g., wahl, G.M. and S.L.Berger (1987) Methods enzymes 152:399; kimmel, A.R. (1987) Methods enzymes 152:507). For example, stringent salt concentrations can typically be less than about 750mM NaCl and 75mM trisodium citrate, less than about 500mM NaCl and 50mM trisodium citrate, or less than about 250mM NaCl and 25mM trisodium citrate. Low stringency hybridization can be achieved in the absence of an organic solvent (e.g., formamide), while high stringency hybridization can be achieved in the presence of at least about 35% formamide or at least about 50% formamide. Stringent temperature conditions may generally include temperatures of at least about 30 ℃, at least about 37 ℃, or at least about 42 ℃. The inclusion or exclusion of various additional parameters (e.g., hybridization time), the concentration of detergent (e.g., sodium Dodecyl Sulfate (SDS)), and vector DNA are well known to those skilled in the art. Various levels of stringency are achieved by combining these different conditions as needed. In an exemplary embodiment, hybridization can occur at 30℃in 750mM NaCl, 75mM trisodium citrate, and 1% SDS. In another exemplary embodiment, hybridization may occur at 37℃in 500mM NaCl, 50mM trisodium citrate, 1% SDS, 35% formamide, and 100. Mu.g/ml denatured salmon sperm DNA (ssDNA). In another exemplary embodiment, hybridization may occur at 42℃in 250mM NaCl, 25mM trisodium citrate, 1% SDS, 50% formamide, and 200. Mu.g/ml ssDNA. Useful variations of these conditions will be apparent to those skilled in the art. The stringency of the wash steps after hybridization can also vary for most applications. Wash stringency conditions can be defined by salt concentration and by temperature. As described above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, the stringent salt concentration of the washing step may be less than about 30mM NaCl and 3mM trisodium citrate, or less than about 15mM NaCl and 1.5mM trisodium citrate. Stringent temperature conditions for the washing step may include temperatures of at least about 25 ℃, at least about 42 ℃, or at least about 68 ℃. In an exemplary embodiment, the washing step may occur at 25 ℃ in 30mM NaCl, 3mM trisodium citrate, and 0.1% sds. In other exemplary embodiments, the washing step may occur at 42 ℃ in 15mM NaCl, 1.5mM trisodium citrate, and 0.1% sds. In another exemplary embodiment, the washing step may occur at 68 ℃ in 15mM NaCl, 1.5mM trisodium citrate, and 0.1% sds. Other variations of these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); grnstein 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: ALaboratory Manual, cold Spring Harbor Laboratory Press, new York.
"substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or a nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Such sequences may be at least 60%, 80% or 85%, 90%, 95%, 96%, 97%, 98%, or even 99% or more identical to the sequences used for comparison at the amino acid level or the nucleic acid level. Sequence identity is typically measured using sequence analysis software (e.g., sequence Analysis Software Package of the Genetics Computer Group (sequence analysis software package of genetics computer group), university of wisconsin biotechnology center, university of madison, wisconsin, lane 1710, postal code 53705, blast, BESTFIT, GAP, or PILEUP/pritox program). Such software matches identical or similar sequences by specifying the degree of homology for various substitutions, deletions and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine. In an exemplary method of determining the degree of identity, the BLAST program can be used, wherein the probability score between e-3 and e-m indicates closely related sequences. "reference" is a standard for comparison. It will be appreciated that the numbering of the positions or residues in the corresponding sequences depends on the particular protein and numbering scheme used. For example, in the precursor of the mature protein and the mature protein itself, numbering may be different, and sequence differences between species may affect numbering. Those skilled in the art will be able to identify the corresponding residues in any homologous protein and corresponding encoding nucleic acid by methods well known in the art, for example, by sequence alignment with a reference sequence and determination of homologous residues.
The term "subject" or "patient" refers to an organism, such as an animal (e.g., a human) that is the subject of treatment, observation, or experiment. By way of example only, subjects include, but are not limited to, mammals, including, but not limited to, humans or non-human mammals, such as non-human primates, mice, cows, horses, dogs, sheep, or cats.
The term "therapeutic effect" refers to a degree of alleviation of one or more of the symptoms of a disorder (e.g., a neoplasia, an infection by a tumor or infectious pathogen, or an autoimmune disease) or its associated pathological symptoms. As used herein, a "therapeutically effective amount" refers to an amount of an agent that, when administered to a cell or subject in a single dose or multiple doses, is effective in prolonging the survival of a patient suffering from such a condition, reducing one or more signs or symptoms of the condition, preventing or delaying, etc., beyond that which would be expected in the absence of such treatment. "therapeutically effective amount" is intended to define the amount required to achieve a therapeutic effect. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the "therapeutically effective amount" (e.g., the ED 50) of the desired pharmaceutical composition.
Provided herein are engineered myeloid cells (including but not limited to neutrophils, monocytes, myeloid dendritic cells (mdcs), mast cells, and macrophages) designed to specifically bind target cells. The engineered myeloid cells can attack and kill target cells directly (e.g., by phagocytosis) and/or indirectly (e.g., by activating T cells). In some embodiments, the target cell is a cancer cell.
Although cancer is one exemplary embodiment described in detail in the present disclosure, the methods and techniques described herein are contemplated to be useful for targeting infected or other diseased cells in vivo. Similarly, therapeutic and vaccine compositions using engineered cells are described herein.
Provided herein are compositions and methods for treating a disease or disorder (e.g., cancer). The compositions and methods provided herein utilize human myeloid cells (including but not limited to neutrophils, monocytes, myeloid dendritic cells (mdcs), mast cells, and macrophages) to target diseased cells, such as cancer cells. The compositions and methods provided herein can be used to eliminate diseased cells, such as cancer cells and/or diseased tissue, by a variety of mechanisms including T cell activation and recruitment, effector immune cell activation (e.g., CD 8T cell and NK cell activation), antigen cross-presentation, enhanced inflammatory responses, modulation of T cell reduction, and phagocytosis. For example, myeloid cells can be used to maintain an immune response against cancer cells.
Provided herein are 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 recruitment receptor (caspase-CAR) fusion proteins. CFPs encoded by the recombinant nucleic acids may comprise an extracellular domain (ECD) comprising an antigen binding domain that binds to a target cell antigen. The extracellular domain may be fused to a hinge domain or an extracellular domain derived from a receptor, such as CD2, CD8, CD28, CD68, phagocytic receptor, scavenger receptor or integrin receptor. CFPs encoded by the recombinant nucleic acids may further comprise a transmembrane domain, e.g., a transmembrane domain derived from CD2, CD8, CD28, CD68, phagocytic receptor, scavenger receptor or integrin receptor. In some embodiments, the CFP encoded by the recombinant nucleic acid further comprises an intracellular domain comprising an intracellular signaling domain, e.g., an intracellular signaling domain derived from a phagocytic receptor, a scavenger receptor, or an integrin receptor. For example, an intracellular domain may comprise one or more intracellular signaling domains derived from phagocytic receptors, scavenger receptors or integrin receptors. For example, an intracellular domain may comprise one or more intracellular signaling domains that promote phagocytic activity, inflammatory response, nitric oxide production, integrin activation, enhanced effector cell migration (e.g., via chemokine receptor expression), antigen presentation, and/or enhanced cross presentation. In some embodiments, the CFP is a phagocytic receptor fusion protein (PFP). In some embodiments, the CFP is a phagocytic scavenger receptor fusion protein (PFP). In some embodiments, the CFP is an integrin receptor fusion protein (IFP). In some embodiments, the CFP is an inflammatory receptor fusion protein. In some embodiments, the CFP encoded by the recombinant nucleic acid further comprises an intracellular domain comprising a recruitment domain. For example, the intracellular domain may comprise one or more PI3K recruitment domains, caspase recruitment domains, or Caspase Activation and Recruitment Domains (CARDs).
Provided herein is a composition comprising a recombinant nucleic acid encoding a CFP comprising a phagocytic or binding receptor (PR) subunit (e.g., a phagocytic receptor fusion protein (PFP)), the subunit comprising: (i) A transmembrane domain, and (ii) an intracellular domain comprising a phagocytic receptor intracellular signaling domain; and an extracellular antigen-binding domain specific for an antigen (e.g., an antigen of a target cell or an antigen presented on a target cell); wherein the transmembrane domain and the extracellular antigen-binding domain are operably linked such that antigen binding to the target via the extracellular antigen-binding domain of the fusion receptor is activated in the intracellular signaling domain of the phagocytic receptor.
In some embodiments, the extracellular domain of CFP comprises an Ig-binding domain. In some embodiments, the extracellular domain comprises a IgA, igD, igE, igG, igM, fcR gamma I, fcR gamma IIA, fcrgamma IIB, fcrgamma IIC, fcrgamma IIIA, fcrgamma IIIB, fcRn, TRIM, fcRL5 binding domain. In some embodiments, the extracellular domain of CFP comprises an FcR extracellular domain. In some embodiments, the extracellular domain of CFP comprises an fcrα, fcrβ, fcrepsilon, or fcrγ extracellular domain. In some embodiments, the extracellular domain comprises an Fcrα (FCAR) extracellular domain. In some embodiments, the extracellular domain comprises an fcrβ extracellular domain. In some embodiments, the extracellular domain comprises an FCER1A extracellular domain. In some embodiments, the extracellular domain comprises an FDGR1A, FCGR2A, FCGR2B, FCGR2C, FCGR a or FCGR3B extracellular domain. In some embodiments, the extracellular domain comprises an integrin domain or an integrin receptor domain. In some embodiments, the extracellular domain comprises one or more integrin α1, α2, αiib, α3, α4, α5, α6, α7, α8, α9, α10, α11, αd, αe, αl, αm, αv, αx, β1, β2, β3, β4, β5, β6, β7, or β8 domains.
In some embodiments, the CFP further comprises an extracellular domain operably linked to the transmembrane domain and the extracellular antigen-binding domain. In some embodiments, the extracellular domain further comprises an extracellular domain of a receptor, a hinge, an compartmentSpacers and/or linkers. In some embodiments, the extracellular domain comprises an extracellular portion of a phagocytic receptor. In some embodiments, the extracellular portion of the CFP is derived from the same receptor as the receptor from which the intracellular signaling domain is derived. In some embodiments, the extracellular domain comprises an extracellular domain of a scavenger receptor. In some embodiments, the extracellular domain comprises an immunoglobulin domain. In some embodiments, the immunoglobulin domain comprises an extracellular domain of an immunoglobulin or an immunoglobulin hinge region. In some embodiments, the extracellular domain comprises a phagocytic engulfing domain. In some embodiments, the extracellular domain comprises a structure capable of multimeric assembly. In some embodiments, the extracellular domain comprises a scaffold for multimerization. In some embodiments, the extracellular domain is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 amino acids in length. In some embodiments, the extracellular domain is up to 500, 400, 300, 200, or 100 amino acids in length. In some embodiments, the extracellular antigen-binding domain specifically binds to an antigen of a target cell. In some embodiments, the extracellular antigen-binding domain comprises an antibody domain. In some embodiments, the extracellular antigen-binding domain comprises a receptor domain, an antibody domain, wherein the antibody domain comprises a functional antibody fragment, a single chain variable fragment (scFv), a Fab, a single domain antibody (sdAb), a nanobody, a V H Domain, V L Domain, VNAR domain, V HH A domain, a bispecific antibody, a diabody, or a functional fragment or combination thereof. In some embodiments, the extracellular antigen-binding domain comprises a ligand, an extracellular domain of a receptor, or an adapter. In some embodiments, the extracellular antigen-binding domain comprises a single extracellular antigen-binding domain specific for a single antigen. In some embodiments, the extracellular antigen-binding domain comprises at least two extracellular antigen-binding domains, wherein each of the at least two extracellular antigen-binding domains is specific for a different antigen.
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 of the present invention, in some embodiments, the antigen is selected from the group consisting of thymidine kinase (TK 1), hypoxanthine-guanine phosphoribosyl transferase (HPRT), receptor tyrosine kinase-like orphan receptor 1 (ROR 1), mucin-1, mucin-16 (MUC 16), MUC1, EGF receptor vIII (EGFRvIII), mesothelin, HER2 (HER 2), EBNA-1, LEMD1, phosphatidylserine, carcinoembryonic antigen (CEA), B Cell Maturation Antigen (BCMA), phosphatidylinositol proteoglycan 3 (GPC 3), follicle stimulating hormone receptor, fibroblast Activating Protein (FAP), erythropoietin-producing hepatocellular carcinoma A2 (EphA 2), ephB2, natural killer group 2D (NKG 2D) ligand antigens of bissialoganglioside 2 (GD 2), CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD45, CD56, CD79B, CD97, CD117, CD123, CD133, CD138, CD171, CD179a, CD213A2, CD248, CD276, PSCA, CS-1, CLECL1, GD3, PSMA, FLT3, TAG72, EPCAM, IL-1, integrin receptor, PRSS21, VEGFR2, PDGFR beta, SSEA-4, EGFR, NCAM, prostase, PAP, ELF2M, GM3, TEM7R, CLDN6, TSHR, rc5D, ALK, dsg1, dsg3, IGLL1, and combinations thereof. In some embodiments, the antigen is an antigen of a protein selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CCR4, CD8, CD30, CD45, and CD 56. In some embodiments, the antigen is an ovarian cancer antigen or a T-lymphoma antigen. In some embodiments, the antigen is an antigen of an integrin receptor. In some embodiments, the antigen is an integrin receptor or an 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 may bind to two or more different antigens.
In some embodiments, the antigen binding domain comprises an autoantigen or fragment thereof, e.g., dsg1 or Dsg3. In some embodiments, the extracellular antigen-binding domain comprises a receptor domain or an antibody domain, wherein the antibody domain binds to an autoantigen, such as Dsg1 or Dsg3.
In some embodiments, the transmembrane domain and the extracellular antigen binding domain are operably linked by a linker. In some embodiments, the transmembrane domain and extracellular antigen binding domain are operably linked by a linker (e.g., a hinge region of CD8 a, igG1, or IgG 4).
In some embodiments, the extracellular domain comprises a multimerization scaffold.
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 FcR epsilon transmembrane domain. In some embodiments, the transmembrane domain comprises a transmembrane domain from a synaptic fusion protein (e.g., synaptic fusion protein 3 or synaptic fusion protein 4 or synaptic fusion protein 5). In some embodiments, when CFP is expressed in a cell, the transmembrane domain oligomerizes with the transmembrane domain of an endogenous receptor. In some embodiments, when CFP is expressed in a cell, the transmembrane domain oligomerizes with the transmembrane domain of an exogenous receptor. In some embodiments, when CFP is expressed in a cell, the transmembrane domain dimerizes with the transmembrane domain of an endogenous receptor. In some embodiments, when CFP is expressed in a cell, the transmembrane domain dimerizes with the transmembrane domain of an exogenous receptor. In some embodiments, the transmembrane domain is derived from a protein that is different from the protein from which the intracellular signaling domain is derived. In some embodiments, the transmembrane domain is derived from a different protein than the protein from which the extracellular domain is derived. In some embodiments, the transmembrane domain comprises a 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 DAP 12.
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. In some embodiments, the transmembrane domain is up to 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.
In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocytic receptor. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocytic receptor other than a phagocytic receptor selected from the group consisting of Megf10, merTk, fcrα, or Bai 1. In some embodiments, the intracellular signaling domain comprises a phagocytic signaling domain derived from a receptor selected from the group consisting of TNFR1, MDA5, CD40, lectin, dectin 1, CD206, scavenger receptor A1 (SRA 1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF2, CXCL16, STAB1, STAB2, SRCC RB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4/80, CCR2, CX3CR1, CSF1R, tie, huCRG (L), CD64, CD32a, CD16a, CD89, fc- α receptor I, CR1, CD35, CD3, CR4, tim-1, tim-4, and CD 169. In some embodiments, the intracellular signaling domain comprises a PI3 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, cdc42, or gtpase inhibitory domain inhibits Rac, cdc42, or gtpase at the phagolycytics cup (phagolycytics cup) of the PFP expressing cell. In some embodiments, the intracellular domain comprises an F-actin-decomposing activation domain, an ARHGAP12 activation domain, an ARHGAP25 activation domain, or an SH3BP1 activation domain. In some embodiments, the intracellular domain comprises a phosphatase inhibiting domain. In some embodiments, the intracellular domain comprises an ARP2/3 inhibition domain. In some embodiments, the intracellular domain comprises at least one ITAM domain. In some embodiments, the intracellular domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more ITAM domains. In some embodiments, the intracellular domain comprises at least one ITAM domain selected from the group consisting of a cd3ζ, cd3ε, cd3γ, cd3δ, fcε receptor 1 chain, fcε receptor 2 chain, fcγ receptor 1 chain, fcγ receptor 2a chain, fcγ receptor 2b 1 chain, fcγ receptor 2b2 chain, fcγ receptor 3a chain, fcγ receptor 3b chain, fcβ receptor 1 chain, TYROBP (DAP 12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and ITAM domains having at least one but no more than 20 modified amino acid sequences thereof. In some embodiments, at least one ITAM domain comprises a Src family kinase phosphorylation site. In some embodiments, at least one ITAM domain comprises a Syk recruitment domain. In some embodiments, the intracellular domain comprises an F-actin depolymerizing activation domain. In some embodiments, the intracellular domain lacks enzymatic activity.
In some embodiments, the intracellular domain does not comprise a domain derived from a cd3ζ intracellular domain. In some embodiments, the intracellular domain does not comprise a domain derived from a MerTK intracellular domain. In some embodiments, the intracellular domain does not comprise a domain derived from a TLR4 intracellular domain. In some embodiments, the intracellular domain comprises a CD47 inhibitory domain. In some embodiments, the intracellular signaling domain comprises a domain that activates an integrin, such as the intracellular region of PSGL-1.
In some embodiments, the intracellular signaling domain comprises a domain that activates Rap1 gtpase, such as domains from EPAC and C3G. In some embodiments, the intracellular signaling domain is derived from a pilin. 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.
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 member, NLRP1-14, NOD1, NOD2, thermal protein, AIM2, NLRC4, FCGR3A, FCERIG, CD40, tank 1-binding kinase (TBK), caspase domain, pro-caspase binding domain, or any combination thereof.
In some embodiments, the intracellular domain comprises a signaling domain derived from a connexin (Cx), such as an intracellular signaling domain. For example, the intracellular domain may comprise a signaling domain derived from Cx43, cx46, cx37, cx40, cx33, cx50, cx59, cx62, cx32, cx26, cx31, cx30.3, cx31.1, cx30, cx25, cx45, cx47, cx31.3, cx36, cx31.9, cx39, cx40.1, or Cx23, such as an intracellular signaling domain. For example, the intracellular domain can comprise a signaling domain derived from Cx43, such as an intracellular signaling domain.
In some embodiments, the intracellular domain comprises a signaling domain derived from a SIGLEC protein, e.g., an intracellular signaling domain. For example, the intracellular domain may comprise a signaling domain derived from Siglec-1 (sialoadhesin), siglec-2 (CD 22), siglec-3 (CD 33), siglec-4 (MAG), siglec-5, siglec-6, siglec-7, siglec-8, siglec-9, siglec-10, siglec-11, siglec-12, siglec-13, siglec-14, siglec-15, siglec-16, or Siglec-17, such as an intracellular signaling domain.
In some embodiments, the intracellular domain comprises a signaling domain derived from a C-type lectin protein, e.g., an intracellular signaling domain. For example, the intracellular domain may comprise a signaling domain derived from a mannose receptor protein, such as an intracellular signaling domain. For example, the intracellular domain may comprise a signaling domain derived from an asialoglycoprotein receptor protein, such as an intracellular signaling domain. For example, the number of the cells to be processed, the intracellular domain may comprise a protein derived from macrophage galactose type lectin (MGL), DC-SIGN (CLEC 4L), langerin (CLEC 4K), myeloid DAP12 associated lectin (MDL) -1 (CLEC 5A), DC associated C type lectin 1 (Dectin 1) subfamily proteins, dectin 1/CLEC7A, DNGR1/CLEC9A, myeloid C type lectin-like receptor (MICL) (CLEC 12A), CLEC2 (CLEC 1B), CLEC12B, DC immunoreceptor (DCIR) subfamily proteins, DCIR/CLEC4A, dectin 2/CLEC6A, blood DC antigen 2 (BDCA 2) (CLEC 4C), mineral (macrophage-induced C type lectin) (CLEC 4E), NOD-like receptor proteins, NOD-like receptor class II transactivators (CIITA), IPAF, BIRC1, RIG-I receptor (RLR) proteins, RIG-I, MDA, NAIP 2, biRP 5/LGRP 1, RP2, NLRP1, NLR 5, NLR 6, NLR 14, NLR 4 or any combination thereof, such as intracellular signaling domains.
In some embodiments, the intracellular domain comprises a signaling domain derived from a cell adhesion molecule, e.g., an intracellular signaling domain. For example, the intracellular domain may comprise a signaling domain derived from an IgCAM, cadherin, integrin, C-type lectin-like domain protein (CTLD), and/or proteoglycan molecule, such as an intracellular signaling domain. For example, the intracellular domain may comprise a signaling domain derived from E-cadherin, P-cadherin, N-cadherin, R-cadherin, B-cadherin, T-cadherin, or M-cadherin, e.g., an intracellular signaling domain. For example, the intracellular domain may comprise a signaling domain derived from a selectin (e.g., E-selectin, L-selectin, or P-selectin), such as an intracellular signaling domain.
In some embodiments, the CFP does not comprise a full-length intracellular signaling domain. In some embodiments, the intracellular domain is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 amino acids in length. In some embodiments, the intracellular domain is up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 amino acids in length.
In some embodiments, the recombinant nucleic acid encodes an FcR a chain extracellular domain, an FcR a chain transmembrane domain, and/or an FcR a 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 a chain or fcrβ chain forms a complex with fcrγ when expressed in a cell. In some embodiments, the FcR a chain or fcrβ chain forms a complex with endogenous fcrγ when expressed in a cell. In some embodiments, the FcR a chain or fcrβ chain is not incorporated into the cell membrane of cells that do not express fcrγ. In some embodiments, the CFP does not comprise an FcR alpha 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, TREM is TREM1, TREM2, or TREM3.
Provided herein are methods and compositions for generating therapeutic myeloid cells, e.g., engineered to target and kill diseased cells, e.g., cancer cells. While the present invention fully exemplifies compositions and methods for killing cancer or tumor cells, those of skill in the art will be able to design such macrophages to target another diseased cell, such as an infected cell, without undue experimentation.
In some embodiments, macrophages are isolated from a subject in need thereof, engineered to express a protein or more than one protein of interest, and then administered to the subject. In some embodiments, the subject has cancer or tumor. Macrophages isolated from a subject are engineered to express a protein or more than one protein of interest, and then administered to the subject are therapeutic macrophages that can target and kill cancer cells or tumor cells of the subject.
In one aspect, provided herein is a composition comprising one or more recombinant nucleic acid sequences comprising: (a) a first nucleic acid sequence encoding an exogenous polypeptide; (B) A second nucleic acid sequence encoding a myeloid cell chimeric antigen receptor fusion protein (CFP), wherein the CFP comprises: (a) An intracellular signaling subunit comprising an intracellular signaling domain having a tyrosine residue that is phosphorylated upon receptor binding to an antigen; (b) A transmembrane domain, and (c) an extracellular binding domain having binding specificity for a component on the surface of a target cell, wherein the extracellular binding domain is operably linked to the transmembrane domain and an intracellular signaling subunit; and (d) a transcriptional activator domain operably linked to the intracellular signaling subunit by a protease cleavage sequence, wherein the transcriptional activator domain facilitates transcription of the first nucleic acid sequence encoding the exogenous polypeptide; and (C) a third nucleic acid sequence encoding (a) a protease that cleaves a protease cleavage sequence operably linked to the transcriptional activator domain to the intracellular signaling subunit; (b) A domain that binds to tyrosine residues that are phosphorylated upon CFP activation; wherein the protease that cleaves the protease cleavage sequence and the domain that binds to tyrosine residues are operably linked.
In some embodiments, the third nucleic acid sequence further encodes (c) a stimulus responsive element. In some embodiments, the stimulus-responsive element (c) is fused to a domain that binds a phosphorylated tyrosine residue. In some embodiments, the stimulus-responsive element is responsive to the microenvironment of the cell expressing the nucleic acid sequence. In some embodiments, (c) is a degradation determinant operably linked to (b). In some embodiments, the degradation determinant is a HIF-1a degradation solution stator.
In some embodiments, one or more recombinant nucleic acids are expressed in myeloid cells. In some embodiments, the myeloid cell is a macrophage. In some embodiments, the target cell is a cancer cell.
In some embodiments, the first nucleic acid encodes a pro-phagocytic or pro-inflammatory polypeptide, or both. In some embodiments, the CFP is a phagocytic receptor fusion protein having one or more phagocytic receptor domains or fragments thereof. In some embodiments, the one or more phagocytic receptor domains or fragments thereof are selected from lectin, dectin 1, CD206, scavenger receptor A1 (SRA 1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF2, CXCL16, starb 1, starb 2, srrb 4D, SSC5D, CD, CD207, CD209, RAGE, CD14, CD64, F4/80, CCR2, CX3CR1, CSF1R, tie2, huCRIg (L), CD64, CD32a, CD16a, CD89, fc-alpha receptor I, CR1, CD35, CR3, CR4, tim-1, tim-4, and CD169.
In some embodiments, the intracellular domain of CFP is derived from a phagocytic receptor.
In some embodiments, the intracellular signaling domain is derived from a receptor other than a phagocytic receptor selected from the group consisting of Megf10, merTk, fcR-a, or Bai 1. In some embodiments, an intracellular signaling subunit comprising an intracellular signaling domain having a tyrosine residue comprises at least one ITAM domain. In some embodiments, the intracellular signaling subunit comprises more than one ITAM domain. In some embodiments, the at least one ITAM domain is selected from the group consisting of a CD3 ζtcr subunit, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a TCR ζ chain, an fcepsilon receptor 1 chain, an fcepsilon receptor 2 chain, an fcgamma receptor 1 chain, an fcgamma receptor 2a chain, an fcgamma receptor 2b 1 chain, an fcgamma receptor 2b2 chain, an fcgamma receptor 3a chain, an fcgamma receptor 3b chain, an fcbeta receptor 1 chain, TYROBP (DAP 12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications. In some embodiments, at least one ITAM domain comprises a Src family kinase phosphorylation site. In some embodiments, at least one ITAM domain comprises a Syk recruitment domain.
In some embodiments of the various aspects described herein, the intracellular signaling subunit further comprises a DAP12 recruitment domain. In some embodiments, the intracellular domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ITAM domains.
In some embodiments, the intracellular signaling subunit further comprises a pro-inflammatory signaling domain comprising 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, NLRP family member, NLRP1-14, NOD1, NOD2, thermal protein, AIM2, NLRC4, FCGR3A, FCERIG, CD40, caspase domain or pro-caspase binding domain, or any combination thereof.
In some embodiments, the intracellular signaling domain further comprises a domain that activates an integrin, such as the intracellular region of PSGL-1.
In some embodiments, the intracellular signaling domain further comprises a domain that activates Rap1 gtpase, such as a domain from EPAC and C3G. In some embodiments, the intracellular signaling domain further comprises a domain from a pilin.
In some embodiments, the intracellular signaling domain activates focal adhesion kinase.
In some embodiments, the extracellular binding domain having binding specificity for a component on the surface of the target cell comprises an antibody or a functional fragment thereof. In some embodiments, the extracellular binding domain comprises a single chain variable fragment (scFv).
In some embodiments of the present invention, in some embodiments, the component on the surface of the target cell is selected from thymidine kinase (TK 1), hypoxanthine-guanine phosphoribosyl transferase (HPRT), receptor tyrosine kinase-like orphan receptor 1 (ROR 1), mucin-1, mucin-16 (MUC 16), MUC1, EGFR vIII (EGFRvIII), mesothelin, EGFR2 (HER 2), mesothelin, EBNA-1, LEMD1, phosphatidylserine, carcinoembryonic antigen (CEA), B Cell Maturation Antigen (BCMA), phosphatidylinositol proteoglycan 3 (GPC 3), follicle stimulating hormone receptor, fibroblast Activating Protein (FAP), erythropoietin-producing hepatocellular carcinoma A2 (EphA 2), ephB2 antigens of natural killer group 2D (NKG 2D) ligands, bissialoganglioside 2 (GD 2), CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD45, CD56, CD79B, CD97, CD117, CD123, CD133, CD138, CD171, CD179a, CD213A2, CD248, CD276, PSCA, CS-1, CLECL1, GD3, PSMA, FLT3, TAG72, EPCAM, IL-1, integrin receptors, PRSS21, VEGFR2, PDGFR- β, SSEA-4, EGFR, NCAM, prostases, PAP, ELF2M, GM, TEM7R, CLDN, TSHR, GPRC5D, ALK, IGLL1, and combinations thereof.
In some embodiments, the component on the surface of the target cell is an antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CCR4, CD8, CD30, CD45, CD 56.
In some embodiments, the component on the surface of the target cell is an ovarian cancer antigen or a T-lymphoma antigen.
In some embodiments, the component on the surface of the target cell is an integrin receptor.
In some embodiments, the component on the surface of the target cell is an integrin receptor 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 component on the surface of the target cell comprises 2 or more different antigens.
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. In some embodiments, the transmembrane domain is up to 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. In some embodiments, the transmembrane domain comprises a transmembrane domain that oligomerizes with DAP 12. In some embodiments, the transmembrane domain and the extracellular antigen binding domain are operably linked by a linker. In some embodiments, the linker comprises a peptide. In some embodiments, the linker comprises a hinge region of CD8 a, igG1, or IgG 4. In some embodiments, the linker is a synthetic linker. In some embodiments, the transmembrane domain comprises an FcR transmembrane domain.
The composition of any of the above embodiments, wherein the transcriptional activator domain further comprises a DNA binding domain. In some embodiments, the DNA binding domain is selected from the group consisting of the DNA binding Domain (DB) of Gal4, ZFHD1, or tet-R. In some embodiments, the transcriptional activator domain comprises a VP64 transactivation domain.
In some embodiments, the protease that cleaves a protease cleavage sequence that operably links the transcriptional activator domain to an intracellular signaling subunit is a Hepatitis C Virus (HCV) NS3 protease. In some embodiments, the domain that binds to a tyrosine residue that is phosphorylated upon CFP activation is a phosphotyrosine binding (PTB) domain. In some embodiments, the PTB is Shc PTB.
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 circRNA. In some embodiments, the recombinant nucleic acid is associated with a replicon RNA.
In one aspect, provided herein is a vector encoding one or more recombinant nucleic acids of any one of the above embodiments.
In one aspect, provided herein is a myeloid cell that expresses the vector of the above embodiments.
In one aspect, provided herein is a pharmaceutical composition comprising the myeloid cells of the above embodiments.
In one aspect, provided herein is a cell comprising a recombinant nucleic acid encoding a chimeric protein comprising: (a) a cytotoxic polypeptide; (b) a protease cleavage sequence; and (c) an inhibitory polypeptide domain, wherein the inhibitory polypeptide domain inhibits a cytotoxic polypeptide; wherein the cytotoxic polypeptide, protease cleavage sequence and inhibitory polypeptide domain are operably linked.
In some embodiments, the cell is a myeloid cell.
In some embodiments, the cytotoxic polypeptide is a human eosinophil major basic protein cytotoxicity domain. In some embodiments, the cytotoxic polypeptide is a human eosinophil major basic protein acidic domain.
In some embodiments, the protease cleavage sequence is an MMP recognition sequence.
In some embodiments, the protease cleavage sequence is cleaved by MMP.
In one aspect, provided herein is a pharmaceutical composition comprising any of the cells of the embodiments described herein.
In one aspect, provided herein is a recombinant nucleic acid of any one of the embodiments described herein.
In one aspect, provided herein is a vector encoding a recombinant nucleic acid of the embodiments described herein.
In one aspect, provided herein is a method for preparing a myeloid cell therapeutic agent for cancer, the method comprising expressing in a myeloid cell a recombinant nucleic acid comprising: (a) a first nucleic acid sequence encoding an exogenous polypeptide; (B) A second nucleic acid sequence encoding a myeloid cell chimeric antigen receptor (CFP), wherein the CFP comprises: (a) An intracellular signaling subunit comprising an intracellular signaling domain having a tyrosine residue that is phosphorylated upon CFP activation; (b) A transmembrane domain, and (c) an extracellular binding domain having binding specificity for a component on the surface of a target cell, wherein the extracellular binding domain is operably linked to the transmembrane domain and an intracellular signaling subunit; and (d) a transcriptional activator domain operably linked to the intracellular signaling subunit by a protease cleavage sequence, wherein the transcriptional activator domain facilitates transcription of the first nucleic acid sequence encoding the exogenous polypeptide; and (C) a third nucleic acid sequence encoding (a) a protease that cleaves a protease cleavage sequence operably linked to the transcriptional activator domain to the intracellular signaling subunit; (b) A domain that binds to a tyrosine residue that is phosphorylated upon CAR activation; wherein the protease that cleaves the protease cleavage sequence and the domain that binds to tyrosine residues are operably linked.
In one aspect, provided herein is a method for preparing a myeloid cell therapeutic agent for cancer, the method comprising expressing in a myeloid cell a recombinant nucleic acid encoding a chimeric protein comprising: (a) Human eosinophils are the primary basic protein acidic domain; (b) an MMP recognition sequence; and (c) a human eosinophil major basic protein cytotoxicity domain.
In one aspect, provided herein is a method for treating a subject having cancer, the method comprising administering to a subject in need thereof a pharmaceutical composition of any of the embodiments described herein.
In one aspect, provided herein is a method of inducing tumor regression in a subject in need thereof, the method comprising intravenously administering to the subject a pharmaceutical composition comprising myeloid cells, wherein the myeloid cells express one or more recombinant nucleic acids encoding one or more polypeptides, and wherein at least one of the one or more polypeptides has functional activity in a tumor microenvironment and no functional activity in a non-tumor environment. In some embodiments of the several aspects described herein, the pharmaceutical composition is a pharmaceutical composition of an embodiment described herein. In some embodiments of the several aspects described herein, the pharmaceutical composition is a pharmaceutical composition of the claims of the embodiments described herein.
In some embodiments of several aspects described herein, the target cell is a cancer cell, which is a glioblastoma cell.
Provided herein is a method for treating a subject having cancer, the method comprising administering to a subject in need thereof the above pharmaceutical composition.
In some embodiments, the recombinant nucleic acid comprises a sequence encoding a pro-inflammatory polypeptide. In some embodiments, the composition further comprises a proinflammatory nucleotide or a nucleotide in a recombinant nucleic acid, e.g., ATP, ADP, UTP, UDP and/or UDP-glucose. In some embodiments, the transcriptional activator domain comprises a VP64 transactivation domain. In some embodiments, the protease that cleaves a protease cleavage sequence that operably links the transcriptional activator domain to an intracellular signaling subunit is a Hepatitis C Virus (HCV) NS3 protease. In some embodiments, the domain that binds to a tyrosine residue that is phosphorylated upon CFP activation is a phosphotyrosine binding (PTB) domain. In some embodiments, the PTB is Shc PTB. In some embodiments, the third nucleic acid encodes a down-solving stator that may be operably linked to the CFP. In some embodiments, the degradation determinant is a HIF-1a degradation solution stator. 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 circRNA.
In some embodiments, the recombinant nucleic acid is associated with a replicon RNA. Provided herein is a method for preparing a myeloid cell therapeutic agent for cancer, the method comprising expressing in a myeloid cell a recombinant nucleic acid comprising: (a) a first nucleic acid sequence encoding an exogenous polypeptide; (B) A second nucleic acid sequence encoding a myeloid cell chimeric antigen receptor (CFP), wherein the CFP comprises: (a) An intracellular signaling subunit comprising an intracellular signaling domain having a tyrosine residue that is phosphorylated upon CFP activation; (b) A transmembrane domain, and (c) an extracellular binding domain having binding specificity for a component on the surface of a target cell, wherein the extracellular binding domain is operably linked to the transmembrane domain and an intracellular signaling subunit; and (d) a transcriptional activator domain operably linked to the intracellular signaling subunit by a protease cleavage sequence, wherein the transcriptional activator domain facilitates transcription of the first nucleic acid sequence encoding the exogenous polypeptide; and (C) a third nucleic acid sequence encoding (a) a protease that cleaves a protease cleavage sequence operably linked to the transcriptional activator domain to the intracellular signaling subunit; (b) A domain that binds to tyrosine residues that are phosphorylated upon CFP activation; wherein the protease that cleaves the protease cleavage sequence and the domain that binds to tyrosine residues are operably linked. Provided herein is a method for preparing a myeloid cell therapeutic agent for cancer, the method comprising expressing in a myeloid cell a recombinant nucleic acid encoding a chimeric protein comprising: (a) Human eosinophils are the primary basic protein acidic domain; (b) an MMP recognition sequence; and (c) a human eosinophil major basic protein cytotoxicity domain.
Provided herein is a method for treating a subject having cancer, the method comprising administering to a subject in need thereof the above pharmaceutical composition. Provided herein is a method for treating a subject having cancer, the method comprising administering to a subject in need thereof a pharmaceutical composition described herein. Provided herein is a method of inducing tumor regression in a subject in need thereof, the method comprising intravenously administering to the subject a pharmaceutical composition comprising myeloid cells, wherein the myeloid cells express one or more recombinant nucleic acids encoding one or more polypeptides, and wherein at least one of the one or more polypeptides has functional activity in a tumor microenvironment and no functional activity in a non-tumor environment.
In some embodiments, the composition further comprises a pro-inflammatory polypeptide. In some embodiments, the proinflammatory polypeptide is a chemokine, a 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, IL, IP-10, RANTES, and interferon.
In some embodiments, the myeloid cells are specifically targeted for delivery. Specialized biodegradable polymers such as PLGA (poly (lactic-co-glycolic acid)) and/or polyvinyl alcohol (PVA) can be used to target myeloid cells. In some embodiments, one or more compounds may be selectively incorporated into such polymer structures to affect myeloid cell function. In some embodiments, the targeting structure is multilayered, e.g., one or more PLGA and one or more PVA layers. In some embodiments, the targeting moieties are assembled in order of layered activity. In some embodiments, the targeting polymer structure is organized into a specifically shaped component, such as an unstable structure that can adhere to the surface of the myeloid cells and deliver one or more components (e.g., growth factors and cytokines), such as to maintain the myeloid cells in a microenvironment that imparts a specific polarization. In some embodiments, the polymer structures are such that they are not phagocytosed by myeloid cells, but they may remain adhered to the surface. In some embodiments, the one or more growth factors may be M1 polarization factors, such as cytokines. In some embodiments, the one or more growth factors may be M2 polarization factors, such as cytokines. In some embodiments, the one or more growth factors may be macrophage activating cytokines, such as ifnγ. In some embodiments, the polymer structure is capable of sustained release of one or more growth factors in an in vivo environment (e.g., in a solid tumor).
In some embodiments, the recombinant nucleic acid comprises a sequence encoding a homeostatic modulator of inflammation. In some embodiments, the modulator of homeostasis of inflammation is a sequence in the untranslated region (UTR) of mRNA. In some embodiments, the sequence in UTR is a sequence that binds an RNA binding protein. In some embodiments, translation is inhibited or prevented when the RNA binding protein binds to a sequence in the untranslated region (UTR). In some embodiments, the sequence in UTR comprises the consensus sequence WWWU (AUUUA) UUUW, wherein W is a or U. In some embodiments, the recombinant nucleic acid is expressed on a bicistronic vector.
In some embodiments, the target cell is a mammalian cell. In some embodiments, the target cell is a human cell. In some embodiments, the target cell comprises a pathogen-infected cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell is a cancer cell, which is a lymphocyte. In some embodiments, the target cell is a cancer cell, which is an ovarian cancer cell. In some embodiments, the target cell is a cancer cell, which is a breast cell. In some embodiments, the target cell is a cancer cell, which is a pancreatic cell. In some embodiments, the target cell is a cancer cell, which is a glioblastoma cell.
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 an unmodified mRNA. In some embodiments, the recombinant nucleic acid is a modified mRNA. In some embodiments, the recombinant nucleic acid is circRNA. In some embodiments, the recombinant nucleic acid is a tRNA. In some embodiments, the recombinant nucleic acid is microRNA. In some embodiments, the recombinant nucleic acid is a self-replicating RNA.
Also provided herein is a vector comprising a recombinant nucleic acid sequence encoding a CFP as described herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector or a lentiviral vector. In some embodiments, the vector further comprises a promoter operably linked to at least one nucleic acid sequence encoding one or more polypeptides. In some embodiments, the vector is polycistronic. In some embodiments, each of the at least one nucleic acid sequences is operably linked to a separate promoter. In some embodiments, the vector further comprises one or more Internal Ribosome Entry Sites (IRES). In some embodiments, the vector further comprises a 5'utr and/or a 3' utr flanking the at least one nucleic acid sequence encoding the one or more polypeptides. In some embodiments, the vector further comprises one or more regulatory regions.
Also provided herein is a polypeptide encoded by a recombinant nucleic acid of a composition described herein.
Provided herein is a composition comprising a recombinant nucleic acid sequence encoding a CFP comprising a phagocytic or binding receptor (PR) subunit (e.g., a phagocytic receptor fusion protein (PFP)), the subunit comprising: a PR subunit comprising: a transmembrane domain and an intracellular domain comprising an intracellular signaling domain; and an extracellular domain comprising an antigen binding domain specific for a target cell antigen; wherein the transmembrane domain and extracellular domain are operably linked; and wherein upon binding of the CFP to an antigen of a target cell, the killing or phagocytosis activity of a myeloid cell (e.g., neutrophil, monocyte, myeloid dendritic cell (mDC), mast cell, or macrophage) expressing the CFP is increased by at least more than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% compared to a cell not expressing the CFP.
Table 1 shows exemplary sequences of chimeric fusion protein domains and/or fragments thereof, which are non-limiting for the present invention.
TABLE 1 exemplary chimeric fusion proteins and receptor domains
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Provided herein is a composition comprising a recombinant nucleic acid sequence encoding a CFP comprising a phagocytic or binding receptor (PR) subunit (e.g., a phagocytic receptor fusion protein (PFP)), the subunit comprising: a PR subunit comprising: a transmembrane domain and an intracellular domain comprising an intracellular signaling domain; and an extracellular domain comprising an antigen binding domain specific for a target cell antigen; wherein the transmembrane domain and extracellular domain are operably linked; and wherein upon binding of the CFP to an antigen of the target cell, the killing or phagocytosis activity of a myeloid cell (e.g., neutrophil, monocyte, myeloid dendritic cell (mDC), mast cell, or macrophage) 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 a cell not expressing the CFP.
CD137 binding proteins and domains
In one aspect, provided herein are recombinant proteins that can bind CD137 on a target cell. As used herein, such a recombinant protein capable of binding CD137 on a target cell is referred to as a CD137 conjugate. In some embodiments, the CD137 binding comprises an extracellular domain of a Chimeric Fusion Protein (CFP). In some embodiments, provided herein are recombinant nucleic acid sequences encoding chimeric CD 137-binder receptor fusion proteins (CFPs), referred to herein as CD 137-CFPs, as used herein. In some embodiments, a recombinant nucleic acid encoding, for example, a chimeric receptor fusion protein (e.g., a CD 137-binder receptor fusion protein) can be expressed in a suitable cell (e.g., a myeloid cell). In some embodiments, when CD137-CFP is expressed on a suitable cell (e.g., a myeloid cell), the CD137 conjugate can bind to a target cell that expresses CD137, and the myeloid cell that expresses the conjugate is activated. In some embodiments, the CD137-CFP, when engaged with CD137, activates the intracellular signaling domain of the CD137-CFP, which activates myeloid cells expressing the recombinant nucleic acid encoding the CD 137-CFP. In some embodiments, the myeloid cell is a phagocyte. In some embodiments, when the CD137 conjugate binds to CD137 expressed on another cell (target cell), a myeloid cell that is a phagocyte and expresses the CD137 conjugate may be activated and swallow the target cell. In some embodiments, the target cell may be any cell that expresses CD 137. In some embodiments, the target cell is a lymphocyte. In some embodiments, the target cell is a T lymphocyte (Y cell). In some embodiments, the target cell may be a malignant cell. In some embodiments, the target cell may be a malignant T cell. In some embodiments, myeloid cells expressing the CD 137-binder CFP can be used to generate therapeutic compositions against T-cell lymphomas.
Provided herein is a recombinant polynucleic acid comprising a sequence encoding a Chimeric Fusion Protein (CFP), wherein the CFP protein can bind CD137 on T lymphocytes. In some embodiments, provided herein is a CFP that can bind to a target on a T cell, the target being CD137. In some embodiments, the target T cell is an activated T cell. In some embodiments, CFP targets activated T cells expressed in T cell lymphoma. Provided herein is a recombinant nucleic acid encoding a CD 137-targeted CFP, wherein the CFP comprises: (i) An extracellular domain comprising an anti-CD 137 binding domain, and (ii) a transmembrane domain operably linked to the extracellular domain; and (b) a pharmaceutically acceptable carrier; wherein the myeloid cells express CFP and exhibit an increase in phagocytosis of CD137 expressing target cells by at least a factor of 1.1 compared to myeloid cells not expressing CFP. In some embodiments, the CD 137-binding domain is a CD 137-binding protein comprising an antigen-binding fragment, fab fragment, scFv domain, or sdAb domain of an antibody. In some embodiments, the CD137 binding domain comprises SEQ ID NO. 31 or a CD137 ligand sequence having at least 90% sequence identity to SEQ ID NO. 31.
Provided herein is a recombinant protein that is a chimeric fusion protein that can bind CD 137. CD137 is an activated T cell surface marker. Thus, a chimeric fusion protein that binds CD137 can specifically bind to an activated T cell, wherein the CFP comprises: (i) An extracellular domain comprising an anti-CD 137 binding domain, and (ii) a transmembrane domain operably linked to the extracellular domain. In some embodiments, the CD 137-binding domain is a CD 137-binding protein comprising an antigen-binding fragment, fab fragment, scFv domain, or sdAb domain of an antibody. In some embodiments, the CD137 binding domain comprises an scFv. Provided herein is a myeloid cell that expresses a recombinant protein that is a chimeric fusion protein that can bind CD137 expressed on T cells, wherein the CFP comprises: (i) An extracellular domain comprising an anti-CD 137 binding domain, and (ii) a transmembrane domain operably linked to the extracellular domain; myeloid cells are able to specifically target T cells expressing CD137 and phagocytose, lyse and thus lyse T cells expressing CD 137. In some embodiments, the myeloid cells expressing the recombinant protein (which is a chimeric fusion protein that can bind CD 137) are therapeutic agents for cd137+ve cancer (e.g., T cell lymphoma).
Typically, CD137 is expressed on activated immune cells. CD137 is expressed in innate and adaptive cells of the immune cascade. In healthy individuals, T cells may be abundant in lymph nodes (e.g., tonsils where T cells are found in an activated state). Cd137+ve T cells are also abundant in tumors.
Notably, cd137+ve T cells may be responsible for graft versus host and host versus graft rejection. In the case of allogeneic cell therapy, CD137 may additionally be targeted destroyed to reduce host versus graft rejection. In some embodiments, the methods and compositions described herein include (i) using allogeneic myeloid cells that express a chimeric receptor that binds to a cancer antigen and lyses cancer cells that express the cancer antigen; and (ii) administering myeloid cells that express a chimeric antigen that can bind to CD137 and lyse cd137+ve T cells, thereby reducing the killing of allogeneic myeloid cells by host cd137+ve T lymphocytes.
In one aspect, provided herein is a recombinant protein that is a chimeric fusion protein that can bind CD137 and further comprises a second, third, or additional binding domain that binds a second, third, or additional extracellular portion, wherein the extracellular portion can be a cell surface molecule. In some embodiments, any of the second or third or additional binding domains may comprise a binding domain for an additional cell surface molecule on the target cell, e.g., a cell surface molecule other than CD137, e.g., a cell surface molecule on a cancer cell or an activated T cell. The second or third or additional binding domain may comprise the binding domain of any additional cell surface molecule on the target cell, such as CD5, or any other antigen on the target cell described anywhere in this specification.
In some embodiments, any of the second or third or additional binding domains may comprise a binding domain of a cell surface molecule on a myeloid cell. In some embodiments, any of the second or third or additional binding domains comprising a myeloid cell binding domain can activate a myeloid cell upon binding.
In some embodiments, the CD 137-binding protein is a chimeric fusion protein comprising an extracellular domain capable of binding CD137, and further comprising a hinge domain derived from CD8, wherein the hinge domain is operably linked to a transmembrane domain on the cytoplasmic side and a CD 137-binding domain on the extracellular side. In some embodiments, the transmembrane domain comprises any one of the sequences set forth in SEQ ID NO 15, 16, 17, 18 or 19. In some embodiments, the transmembrane domain comprises a sequence derived from a CD8a (CD 8 a) transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 15 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 15. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 16 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 16. In some embodiments, the transmembrane domain comprises a sequence derived from a CD2 transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 17 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 17. In some embodiments, the transmembrane domain comprises a sequence derived from a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO:18 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 18. In some embodiments, the transmembrane domain comprises a sequence derived from a CD68 transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 19 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 19. In some embodiments, the ECD of a CFP comprises the amino acid sequence set forth in SEQ ID NO. 31 or a sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 31 fused to a transmembrane domain selected from any of the sequences set forth in SEQ ID NO. 15, 16, 17, 18 or 19 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NO. 15, 16, 17, 18 or 19.
In some embodiments, the extracellular hinge domain and the transmembrane domain comprise SEQ ID NO. 20 or SEQ ID NO. 21 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 20 or SEQ ID NO. 21, respectively. In some embodiments, a CFP comprises an extracellular domain fused to SEQ ID NO. 20 or to a transmembrane domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 20. In some embodiments, a CFP comprises an extracellular domain fused to SEQ ID NO. 21 or to a transmembrane domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 21. In some embodiments, a CFP comprises an ECD conjugate domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 31 fused to a hinge and transmembrane domain having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 20 or 21.
In some embodiments, the CFP comprises an extracellular domain comprising the sequence shown in SEQ ID NO. 31 or a sequence having at least 80% identity to SEQ ID NO. 31; which is operably linked to a TM domain comprising a CD16 transmembrane domain sequence (e.g., comprising a short hinge domain) as shown in table 4. In some embodiments, the CFP comprises an extracellular domain comprising the sequence shown in SEQ ID NO. 31 or a sequence having at least 80% identity to SEQ ID NO. 31; which is operably linked to a TM domain comprising the CD64 transmembrane domain sequence. In some embodiments, the CFP comprises an extracellular domain comprising the sequence shown in SEQ ID NO. 31; which is operably linked to a TM domain comprising a CD89 transmembrane domain sequence (e.g., comprising a short hinge domain) as shown in table 4. In some embodiments, the CFP comprises an ECD having the sequence of SEQ ID NO. 31 or a sequence having at least 80% identity to SEQ ID NO. 31; a TM domain comprising a sequence from the TM domain of the CD16 protein, or the TMD of the CD64 protein, or the TMD of the CD89 protein, and a corresponding intracellular domain (ICD) or portion thereof, e.g., from the CD16 protein, the CD64 protein, or the CD89 protein, respectively, optionally in addition to other different ICDs described herein. Exemplary CD16 and CD89 ICD sequences are provided in table 4.
In some embodiments, the CFP comprises one or more intracellular signaling domains comprising an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a phagocytic signaling domain. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Megf10, merTk, fcrα, and Bai 1. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Megf10, merTk, fcR, and Bai 1. In some embodiments, the intracellular signaling domain comprises a domain derived from a receptor other than cd3ζ. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from fcrγ, fcrα, or fcrepsilon.
In some embodiments, the CFP comprises an intracellular signaling domain derived from an FcR gamma protein (FcR gamma chain) comprising the amino acid sequence of any one of SEQ ID NOs 22, 23, 24 or 25 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs 22, 23, 24 or 25. In some embodiments, the one or more intracellular signaling domains further comprise a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain comprises a PI3 kinase (PI 3K) recruitment domain. In some embodiments, the pro-inflammatory signaling domain comprises SEQ ID NO. 26 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 26. In some embodiments, the pro-inflammatory signaling domain is derived from the intracellular signaling domain of CD 40. In some embodiments, the pro-inflammatory signaling domain comprises SEQ ID NO 27 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO 27. In some embodiments, the CFP comprises SEQ ID NO 28 or an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO 28. In some embodiments, the CFP comprises SEQ ID NO. 29 or an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 29. In some embodiments, the CFP comprises SEQ ID NO. 30 or an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 30.
In some embodiments, the CFP comprises: (a) an extracellular domain comprising: (i) An scFv that specifically binds CD137, and (ii) a hinge domain derived from CD 8; at least a portion of a hinge domain derived from CD28 or an extracellular domain derived from CD 68; (b) A CD8 transmembrane domain, a CD28 transmembrane domain, a CD2 transmembrane domain, or a CD68 transmembrane domain; and (c) an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise: (i) A first intracellular signaling domain derived from fcrα, fcrγ, or fcrepsilon, and (ii) a second intracellular signaling domain: (A) Comprising a PI3K recruitment domain, or (B) derived from CD40. In some embodiments, as an alternative to (c) above, the CFP comprises: an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise: (i) A first intracellular signaling domain derived from a phagocytic receptor intracellular domain, and (ii) a second intracellular signaling domain derived from a scavenger receptor phagocytic receptor intracellular domain, comprising: (A) Comprising a PI3K recruitment domain, or (B) derived from CD40. In some embodiments, the CFP comprises an intracellular signaling domain derived from an intracellular signaling domain of an innate immune receptor.
In some embodiments, the chimeric fusion protein may have more than one extracellular antigen-binding domain.
In some embodiments, the chimeric fusion protein may comprise a CD137 binding domain and a second binding domain of a second cell surface molecule, such as an extracellular protein or glycoprotein, a membrane protein or glycoprotein present on the surface of a cancer cell or target cell. In some embodiments, the second binding domain binds to any target described herein. In some embodiments, the second binding domain is designed such that the second binding domain binds to a protein on the extracellular matrix of the target cell or an adjacent cell. In some embodiments, the extracellular domain may comprise a second binding domain that can bind to a soluble target, e.g., a molecule that is released in the extracellular space of an adjacent cell or target cell.
In one aspect, provided herein is a chimeric fusion protein that can bind CD137 (also referred to as 4-1 BB) on a target cell. In some embodiments, a recombinant nucleic acid encoding a CFP that can bind CD137 can be incorporated into a myeloid cell, such that the myeloid cell expresses CD137 binds CFP. In some embodiments, the myeloid cells can be cd14+ cells. In some embodiments, the myeloid cells may be CD14+/CD16-. In some embodiments, the myeloid cells can be CD14+/CD16low. Myeloid cells expressing CD137 in combination with CFP can be used alone in various ways; or in combination with other CFP expressing myeloid cells in a therapeutic composition; or a combination therapeutic regimen alone; and may be used alone or in combination with other therapeutic agents, including other nucleic acid, peptide, protein, or small molecule therapeutic agents.
CD137 is a member of the tumor necrosis factor receptor superfamily, member 9 (TNFRSF 9), and is expressed on the surface of certain immune cells. CD137 is a cell surface protein expressed on activated T cells. The interaction of CD137 with its ligand on activated Antigen Presenting Cells (APC) (CD 137L, also known as TNFSF9 or 4-1 BBL) can lead to bi-directional activation, which promotes immunity against cancer. For example, signaling through the CD137 receptor has been shown to result in T cell activation and survival. In contrast, the conjugated CD137L may affect cells expressing it, e.g., myeloid cells, dendritic cells, resulting in their activation and maturation. The use of agonistic CD137 antibodies (e.g., wu Ruilu mab and Wu Tuolu mab) is considered a promising immunotherapeutic approach to treat various types of tumors. In some embodiments, CFPs comprising CD137L may be expressed in myeloid cells and used in anti-tumor therapy, and the signaling of CD137L to monocytes induces their differentiation into CD 137L-DCs. CD137L-DC preferentially induces type 1T helper (Th 1) cell polarization and strong type 1 CD8+ T cell (Tc 1) responses against tumors
CFP can be designed using CD137 agonistic antibodies or CD137 binding regions thereof to activate an immune response in vivo. Alternatively, CFP may be designed using CD137L or a fragment thereof to activate an immune response in vivo. In certain embodiments, CD137 binding CFP comprising a CD137 agonist antibody or fragment thereof or CD137L as an extracellular domain may be used to activate an immune response or as an anti-tumor agent. Provided herein is a chimeric fusion protein comprising a CD 137-binding extracellular domain, the CD 137-binding extracellular domain comprising a CD 137-agonistic antibody or fragment thereof, wherein the antibody can be an scFv, a bispecific antibody, a Fab fragment, a F (ab') 2 fragment, a monovalent antibody, a scFv fragment, a scr-Fc fragment, a IgNAR, hcIgG, V HH Antibodies, nanobodies, and alphabodies. In some embodiments, the CD 137-agonistic antibody is a human or humanized antibody or fragment thereof. In some embodiments, the CD 137-agonistic antibody is nivolumab or a fragment thereof. Provided herein is a nucleic acid construct encoding a chimeric fusion protein comprising a CD137 binding extracellular domain, the CD137 binding extracellular domain comprising a CD137 agonist antibody or fragment thereof, wherein the antibody may be an scFv, a bispecific antibody, a Fab fragment, a F (ab') 2 fragment, a monovalent antibody, an scFv fragment, a scr v-Fc fragmentSegment IgNAR, hcIgG, V HH Antibodies, nanobodies, and alphabodies. Provided herein is a chimeric fusion protein comprising a CD 137-binding extracellular domain, the CD 137-binding extracellular domain comprising CD137L or a fragment thereof. In some embodiments, the therapeutic cells may be generated by expressing a full length CD137L protein, which CD137L protein may be expressed in the cells (e.g., myeloid cells) such that the myeloid cells bind and activate lymphocytes. In some embodiments, the therapeutic cells may be generated by designing and expressing CFPs that express at least the extracellular fragment of CD 137L. In some embodiments, a CFP comprising extracellular CD137L as a CD137 binding domain may further comprise an intracellular domain of CD 137L. When administered to a subject as a therapeutic cell expressing a CFP or a nucleic acid construct encoding a CFP, the CFP described in this paragraph can be used for any therapeutic purpose to stimulate an immune response, wherein the nucleic acid construct is administered such that it is taken up by the cell in vivo and the cell expresses the CFP. In some embodiments, the administered nucleic acid construct may comprise a targeting moiety that targets and is taken up by a particular cell type or tissue. In some embodiments, the nucleic acid construct may encode an extracellular protein, e.g., a soluble extracellular protein, comprising a CD137 binding domain. In some embodiments, CFPs comprising a CD137 binding domain may be soluble extracellular proteins, and may be used as immunostimulatory CD137 agonistic proteins, thereby activating lymphocytes upon administration and expression in a subject. In some embodiments, the chimeric fusion protein is a receptor protein. Thus, a CFP receptor comprising a CD 137-binding extracellular domain comprises a transmembrane domain, and may optionally comprise an intracellular domain. Cells expressing fusion proteins with CD137 agonist binding domains and/or transmembrane domains can activate T lymphocytes and can be used to activate an anti-tumor response in vivo.
In some embodiments, the CD 137-binding fusion protein can be designed and expressed in a myeloid cell (e.g., a DC cell) to generate a DC vaccine, e.g., an anti-cancer DC vaccine. Provided herein is a myeloid cell (e.g., DC) that expresses a CFP comprising an extracellular domain comprising a CD137 binding ligand (e.g., human CD137L or binding fragment thereof).
However, CD137 can target cd137+ve cancers, such as T cell lymphomas. CD 137-targeted myeloid cells designed to express a CD 137-binding chimeric fusion protein as described herein can be used as an effective, safe and efficient tumoricidal agent in cd137+ tumors, and can be used for adoptive cell therapy of T cell lymphomas and other cancers (e.g., lung cancer, pancreatic cancer, white blood cell cancer, and other cancers).
In some embodiments, the chimeric fusion proteins described herein target binding to CD137 receptors on lymphocytes and NK cells. When expressed in suitable myeloid cells, killer myeloid cells can be generated, further by culturing the cells under suitable culture conditions for a limited period of time and developing the ex vivo cells as therapeutic agents. The killer myeloid cells expressing CD 137-binding CFP described herein can be used to treat a subject with the potential for CD137 cell malignancy or a disease or disorder caused by CD137 cell function.
In some embodiments, the chimeric fusion proteins described herein are therapeutic agents that can be administered in the form of a nucleic acid construct in a therapeutic composition, such that when a suitable cell ingests a nucleic acid in vivo, the chimeric fusion protein is expressed by the cell in vivo, and the cell can bind to cells expressing CD137 in vivo. Binding of CD137 to an additional domain (e.g., an intracellular signaling domain) in a CFP fusion protein can render cells in which the protein is expressed into potent cytotoxic cells, e.g., cells with enhanced phagocytic activity, thus rendering cells expressing the protein into "aggressor cells" or killer cells, e.g., lymphocytes or NK cells, targeted to destroy cells expressing CD 137.
In some embodiments, the CD 137-binding chimeric fusion protein may comprise a heterologous transmembrane domain and/or one or more heterologous intracellular domains, and may be expressed in a suitable cell (e.g., a myeloid cell) to generate a potent immunoreactive cell. In some embodiments, binding of CFP with a CD137 conjugate activates a chimeric fusion protein (receptor) intracellular domain that activates a phagocytic response, an inflammatory response, and/or a cytotoxic response in CFP expressing cells such that CFP expressing cells destroy CD137 expressing target cells. Examples of target cells expressing CD137 may be lymphocytes, such as T lymphocytes or NK cells, such as in cd137+ve cell cancers, e.g., lung cancer, leukemia, pancreatic cancer, colorectal cancer and lymphomas.
In some embodiments, CD 137-binding CFPs may be expressed in suitable cells (e.g., myeloid cells) such that cells expressing CD137 may act as modulators of immune responses by binding to and killing activated cd137+ lymphocytes. In this embodiment, CD 137-targeted CFP is used therapeutically to reduce an activated immune response, such as an alloimmune response.
In some embodiments, killer cells, such as active phagocytes engineered to express an anti-CD 137 extracellular moiety, can be used as a contiguous therapy for several non-autologous cell-based immunotherapy. Such combination therapies may be used for a variety of cancers, including but not limited to melanoma, glioblastoma, sarcoma, renal cell carcinoma, ovarian cancer, lung cancer, pancreatic cancer, breast cancer, and many others.
In some embodiments, a CD 137-targeted CFP nucleic acid construct can be incorporated into a myeloid cell to express CFP, and the CD 137-binding CFP-expressing myeloid cell can be used as an adjunct cell in CAR-P (e.g., CAR or CFP-expressing phagocyte) therapy or CAR-T (CAR-expressing T cell) therapy, wherein the CAR-expressing cell targets a cancer antigen other than CD137, such as CD5, CD19, CD40, etc., such that the CD137 CFP-expressing cell reduces or eliminates CD 137-expressing tumor-inhibiting lymphopenia, thereby enhancing the tumoricidal function of the cancer-targeted CAR-expressing cell (CAR-P or CAR-T). Thus, the helper cell may be developed using CD 137-binding CFP for co-administration in any adoptive cell therapy, wherein the helper cell is a cytotoxic cell into which the CD 137-binding CFP encoding nucleic acid construct is incorporated and the cell expresses the CD 137-binding CFP extracellular domain. The CD 137-conjugate CFP expressing cell may be a myeloid cell. The CD137 conjugate CFP expressing cell may be a phagocytic myeloid cell. The CD 137-conjugate CFP expressing cell may be a lymphocyte. The CD137 conjugate CFP expressing cell may be a cytotoxic cell.
In some embodiments, CD 137-binding CFPs may be expressed in suitable cells (e.g., myeloid cells), such that CD 137-expressing cells may be used as modulators of immune responses, for example, in allogeneic cell therapy responses, host versus graft diseases, or autoimmune diseases or disorders. For example, CD 137-binding CFP can be used in combination with CAR-P or CAR-T therapies, where CAR-P or CAR-T is an allogeneic cell that targets a disease cell, e.g., a cancer cell that expresses a cancer antigen other than CD137 (e.g., CD5, CD19, CD 40). In some embodiments, CD 137-binding CFP may be used to develop helper cells that are co-administered in any adoptive cell therapy, wherein the helper cells are cytotoxic cells comprising CD 137-binding CFP and expressing the extracellular domain of the CD 137-binder CFP. The CD 137-conjugate CFP expressing cell may be a myeloid cell. The CD137 conjugate CFP expressing cell may be a phagocytic myeloid cell. The CD 137-conjugate CFP expressing cell may be a lymphocyte. The CD137 conjugate CFP expressing cell may be a cytotoxic cell.
In some embodiments, CD 137-targeted CFPs may be expressed in myeloid cells and used in combination with CART therapies, wherein CART cells may be allogeneic or autologous. In some embodiments, CD 137-targeted CFPs may be expressed in myeloid cells and used in combination with CART therapies targeting cancer cells other than cd137+ cells, wherein CART cells may be allogeneic or autologous. In some embodiments, CD 137-targeted CFPs may be expressed in myeloid cells and used in combination with CART therapy, wherein CART cells and/or myeloid cells may be allogeneic or autologous. In some embodiments, the CD 137-targeted CFP expressing cells may be allogeneic or autologous relative to the subject to which the cells are administered as therapy.
In some embodiments, a CD 137-targeted CFP may comprise an extracellular CD137 binding domain consisting of a CD137 agonist antibody or fragment thereof. In some embodiments, the extracellular CD137 binding domain consisting of a CD137 agonist antibody or fragment thereof may comprise an scFv, a VHH, a diabody, a bispecific antibody, or a CD137 ligand or fragment thereof. In some embodiments, the CD 137-targeted CFP may further comprise a transmembrane domain (TMD) derived from cd3ζ, CD2, CD8 a, CD28, CD68, fcgR, fcG, fcR y, fcra transmembrane domain, or any other suitable domain. In some embodiments, the CD 137-targeted CFP described above may further comprise an intracellular domain comprising one or more signal transduction domains. In some embodiments, the CD 137-targeted CFP may comprise an intracellular domain derived from a PI3K recruitment domain, an intracellular signaling domain of a scavenger receptor, a CD40 intracellular domain, an FcR-derived signaling domain, a TLR intracellular domain, an NLRP3 intracellular domain, a CD 3-derived intracellular domain, and the like. The present invention shows in tables 1 and 4 the sequence information for each domain described herein in various embodiments, and it is contemplated that within the scope of the present invention, the sequence information in tables 1 and 4 can be used and various combinations thereof incorporated into a single biomolecule using molecular biology techniques known in the art to develop chimeric fusion proteins.
In some embodiments, a Chimeric Fusion Protein (CFP) comprises an extracellular domain (ECD) targeted to bind CD137, which may comprise the amino acid sequence of SEQ ID NO:31, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 31.
MEFGLSWLFLVAILKGVQCGLLDLRQGMFAQLVAQNVLLIDGPLS
WYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRV
VAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAF
GFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE(SEQ ID NO:31)。
In one aspect, the chimeric fusion protein may comprise a soluble protein having a binding domain that binds CD 137. In one embodiment, the chimeric fusion protein may comprise a soluble protein having at least one binding domain that binds CD137 and one or more additional binding domains, wherein at least one binding domain binds a cell surface molecule on a myeloid cell. In some embodiments, the chimeric fusion protein comprises a binding domain that can bind CD137 and can bind to myeloid cellsBinding domains of cell surface molecules. In some embodiments, the chimeric fusion protein comprises a binding domain that can bind CD137 and a binding domain that can bind a cell surface molecule on a myeloid cell. In some embodiments, the CFP further comprises a third or any number of additional binding domains that bind to a third or additional extracellular portion, wherein the extracellular portion may be a cell surface molecule or a soluble component in an extracellular environment. In some embodiments, the chimeric fusion proteins described herein can be referred to as bispecific conjugates (e.g., bispecific myeloid cell conjugates, bimes), wherein the bispecific conjugates comprise two "conjugate" moieties, wherein the conjugate moieties are binding domains of two different targets, wherein a first binding domain binds to CD137, e.g., on any cd137+ cell, e.g., CD137 on an activated T cell, an activated NK cell, or a cancer cell, and wherein a second binding domain binds to a surface molecule on a myeloid cell. In some aspects, the chimeric fusion proteins described herein are referred to as trispecific conjugates (trispecific myeloid-like cell conjugates, tri mes), wherein the trispecific conjugates comprise three conjugate moieties that are binding domains of three different targets, wherein a first binding domain binds to CD137 on, for example, any CD137+ cell, such as an activated T cell, an activated NK cell, or a CD 137-expressing cancer cell, and wherein a second binding domain binds to a cell surface molecule on a myeloid cell, and a third binding domain binds to a third element, wherein the third element may be a different cell surface molecule other than CD137 or a cell surface molecule on a myeloid cell to which the second binding domain binds. In some embodiments, the third binding domain binds to a cell surface molecule other than CD137 on a cd137+ cell. In some embodiments, the third binding domain binds to a cell surface molecule on a myeloid cell and binds to a molecule other than the molecule to which the second binding domain binds. In some embodiments, the BiME or TriME juxtapose the target cells with the myeloid cells such that the myeloid cells can attack, phagocytose, and lyse the target cells. In some embodiments, the engagement of the BiME or TriME with the myeloid cells not only brings the myeloid cells and the target cells into close proximity, But also activates myeloid cells to effectively bind and kill target cells. In some embodiments, the second binding domain and/or the third binding domain described herein activates myeloid cells upon binding. In some embodiments, the BiME or TriME is a secreted fusion protein. In some embodiments, the conjugate or any binding domain described herein may be an antigen binding fragment (Fab), single chain variable fragment (scFv), nanobody, V H Domain, V L Domain, single domain antibody (sdAb), VNAR domain and V HH A domain, a bispecific antibody, a diabody, or any functional fragment thereof. In some embodiments, the conjugate may be a ligand, such as a CD137 ligand (SEQ ID NO: 31) that binds CD137, or a fragment thereof.
In some embodiments, CFPs comprising a binding domain capable of binding CD137 on a tumor cell are designed for anti-tumor activity. A recombinant nucleic acid encoding a fusion protein comprising a binding domain capable of binding CD137 is expressed in a myeloid cell, wherein the chimeric fusion protein is a transmembrane protein and the myeloid cell is a phagocyte; upon engagement of CFP with target CD137 expressed on cells (e.g., tumor cells), the myeloid cells are activated, phagocytosed and lyse the target cells, e.g., tumor cells, thereby slowing or stopping tumor growth, reducing tumor size and/or eliminating tumors.
In some embodiments, CFPs comprising a binding domain capable of binding to CD137 on activated CD 137-expressing cells are designed for immunosuppression, e.g., to treat Host Versus Graft Disease (HVGD) or a disorder associated with an allogeneic response, or acute or persistent severe inflammation. In some embodiments, the CD 137-expressing cells are activated T cells or NK cells. In some embodiments, a CD137 ligand or fragment thereof on the extracellular domain of CFP expressed in a myeloid cell activates the myeloid cell when bound to CD137 on an activated T cell or NK cell, activates phagocytosis and lyses the activated T cell or NK cell and reduces inflammation.
In some embodiments, described herein are therapeutic agents, such as pharmaceutical compositions, wherein the pharmaceutical compositions may comprise: (i) a CFP comprising a binding domain capable of binding CD 137; (ii) A recombinant nucleic acid encoding a CFP comprising a binding domain capable of binding CD 137; or (iii) a cell expressing a CFP comprising a binding domain capable of binding CD 137.
In some embodiments, a CFP comprising a binding domain capable of binding CD137 is included; a recombinant nucleic acid encoding a CFP comprising a binding domain capable of binding CD 137; or a therapeutic agent (e.g., a pharmaceutical composition as described herein) expressing a cell comprising a CFP capable of binding to the binding domain of CD137 may be associated with (i) one or more additional CFPs, respectively; or (ii) a recombinant nucleic acid encoding one or more additional CFPs; or (iii) cells expressing one or more additional CFPs, or any combination thereof. As an example of the foregoing, a pharmaceutical composition may be described herein that comprises a myeloid cell expressing a CFP comprising a binding domain capable of binding to CD137, and may be used in combination with a myeloid cell expressing a CFP comprising a binding domain capable of binding to CD5 or any other binding domain described elsewhere in this specification. Similarly, any combination of the compositions described herein is contemplated as being combined or mixed in a therapeutic agent and will be within the scope of the present invention.
CD70 binding proteins and domains
CD70 may be highly expressed in certain cancers including, but not limited to, colorectal cancer (CRC), lung cancer, T-cell lymphoma, glioma, thyroid cancer, head and neck cancer, gastric cancer, liver cancer, pancreatic cancer, urothelial cancer, ovarian cancer, and melanoma. Provided herein are chimeric fusion proteins that can bind CD70 on cancer cells and are designed for therapeutic use against diseases involving CD70 overexpression (e.g., cancer).
In one aspect, provided herein is a chimeric fusion protein having an extracellular binding domain (CD 70 conjugate CFP) that can bind CD 70. In some embodiments, a CD70 conjugate CFP may comprise an extracellular binding domain having the amino acid sequence of SEQ ID NO. 32 or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO. 32. The CD70 binding domain may comprise an antigen binding fragment (Fab), a single chain variable fragment (scFv), a nanobody, V H Domain, V L Domain, single domain antibody (sdAb), VNAR domain and V HH A domain, a bispecific antibody, a diabody, or any functional fragment thereof.
In some embodiments, the extracellular domain further comprises a hinge domain derived from CD8, wherein the hinge domain is operably linked to a cytoplasmic side transmembrane domain and an extracellular CD70 binding domain. In some embodiments, the transmembrane domain comprises any one of the sequences set forth in SEQ ID NO 15, 16, 17, 18 or 19. In some embodiments, the transmembrane domain comprises a sequence derived from a CD8a (CD 8 a) transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 15 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 15. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 16 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 16. In some embodiments, the transmembrane domain comprises a sequence derived from a CD2 transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 17 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 17. In some embodiments, the transmembrane domain comprises a sequence derived from a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO:18 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 18. In some embodiments, the transmembrane domain comprises a sequence derived from a CD68 transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 19 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 19. In some embodiments, the ECD of a CFP comprises the amino acid sequence set forth in SEQ ID NO:32 or a sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:32 fused to a transmembrane domain selected from any of the sequences set forth in SEQ ID NO:15, 16, 17, 18 or 19 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NO:15, 16, 17, 18 or 19.
In some embodiments, the extracellular hinge domain and the transmembrane domain comprise SEQ ID NO. 20 or SEQ ID NO. 21 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 20 or SEQ ID NO. 21, respectively. In some embodiments, a CFP comprises an extracellular domain fused to SEQ ID NO. 20 or to a transmembrane domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 20. In some embodiments, a CFP comprises an extracellular domain fused to SEQ ID NO. 21 or to a transmembrane domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 21. In some embodiments, a CFP comprises an ECD conjugate domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 32 fused to a hinge and transmembrane domain having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 20 or 21.
In some embodiments, the CFP comprises an extracellular domain comprising the sequence shown in SEQ ID NO. 32 or a sequence having at least 80% identity to SEQ ID NO. 32; which is operably linked to a TM domain comprising a CD16 transmembrane domain sequence (e.g., comprising a short hinge domain) as shown in table 4. In some embodiments, the CFP comprises an extracellular domain comprising the sequence shown in SEQ ID NO. 32 or a sequence having at least 80% identity to SEQ ID NO. 32; which is operably linked to a TM domain comprising the CD64 transmembrane domain sequence. In some embodiments, the CFP comprises an extracellular domain comprising the sequence shown in SEQ ID NO. 32; which is operably linked to a TM domain comprising a CD89 transmembrane domain sequence (e.g., comprising a short hinge domain) as shown in table 4. In some embodiments, the CFP comprises an ECD having the sequence of SEQ ID NO. 32 or a sequence having at least 80% identity to SEQ ID NO. 32; a TM domain comprising a sequence from the TM domain of the CD16 protein, or the TMD of the CD64 protein, or the TMD of the CD89 protein, and a corresponding intracellular domain (ICD) or portion thereof, e.g., from the CD16 protein, the CD64 protein, or the CD89 protein, respectively, optionally in addition to other different ICDs described herein. Exemplary CD16 and CD89 ICD sequences are provided in table 4.
In some embodiments, the CFP comprises one or more intracellular signaling domains comprising an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a phagocytic signaling domain. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Megf10, merTk, fcrα, and Bai 1. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Megf10, merTk, fcR, and Bai 1. In some embodiments, the intracellular signaling domain comprises a domain derived from a receptor other than cd3ζ. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from fcrγ, fcrα, or fcrepsilon.
In some embodiments, the CFP comprises an intracellular signaling domain derived from an FcR gamma protein (FcR gamma chain) comprising the amino acid sequence of any one of SEQ ID NOs 22, 23, 24 or 25 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs 22, 23, 24 or 25. In some embodiments, the one or more intracellular signaling domains further comprise a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain comprises a PI3 kinase (PI 3K) recruitment domain. In some embodiments, the pro-inflammatory signaling domain comprises SEQ ID NO. 26 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 26. In some embodiments, the pro-inflammatory signaling domain is derived from the intracellular signaling domain of CD 40. In some embodiments, the pro-inflammatory signaling domain comprises SEQ ID NO 27 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO 27. In some embodiments, the CFP comprises SEQ ID NO 28 or an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO 28. In some embodiments, the CFP comprises SEQ ID NO. 29 or an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 29. In some embodiments, the CFP comprises SEQ ID NO. 30 or an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 30.
Provided herein is a recombinant nucleic acid encoding a CFP comprising a CD70 binding domain (CD 70 conjugate) as described above, which CFP can be expressed in a suitable cell (e.g., a myeloid cell). The myeloid cells may be phagocytes. In some embodiments, the CD70 conjugate activates myeloid cells expressing CD 70-binding CFP when bound to CD70 on the cells. Provided herein are recombinant nucleic acid sequences encoding chimeric CD 70-binder receptor fusion proteins (CFPs), referred to as CD 70-CFPs, as used herein. In some embodiments, a recombinant nucleic acid encoding, for example, a chimeric receptor fusion protein (e.g., a CD 70-binder receptor fusion protein) can be expressed in a suitable cell (e.g., a myeloid cell). In some embodiments, when CD70-CFP is expressed on a suitable cell (e.g., a myeloid cell), the CD70 conjugate can bind to the target cell expressing CD70 and the myeloid cell expressing the conjugate is activated. In some embodiments, the CD70-CFP, when engaged with CD70, activates the intracellular signaling domain of the CD70-CFP, which activates myeloid cells expressing the recombinant nucleic acid encoding the CD 70-CFP. In some embodiments, the myeloid cell is a phagocyte. In some embodiments, when the CD70 conjugate binds to CD70 expressed on another cell (target cell), a myeloid cell that is a phagocyte and expresses the CD70 conjugate may be activated and swallow the target cell. In some embodiments, the target cell may be any cell that expresses CD 70. In some embodiments, the target cell is a lymphocyte. In some embodiments, the target cell is a T lymphocyte (Y cell). In some embodiments, the target cell may be a malignant cell. In some embodiments, the target cell may be a malignant T cell. In some embodiments, myeloid cells expressing CD70 binding CFP can be used to generate therapeutic compositions against T cell lymphomas.
In another embodiment, provided herein is a recombinant protein, such as an extracellular protein, e.g., a soluble protein that can bind to the extracellular domain of CD70. In some embodiments, a chimeric fusion protein, such as the soluble proteins described herein, can be referred to as a bispecific conjugate (e.g., a bispecific myeloid cell conjugate, biME), wherein the bispecific conjugate comprises two "conjugate" moieties, wherein the conjugate moieties are binding domains of two different targets, wherein a first binding domain binds to CD70 on, for example, any cd70+ cell, such as activated T cell, activated NK cell, or CD70 on a cancer cell, and wherein a second binding domain binds to a surface molecule on a myeloid cell. In some aspects, the chimeric fusion proteins described herein are referred to as trispecific conjugates (trispecific myeloid cell conjugates, tri mes), wherein the trispecific conjugates comprise three conjugate moieties that are binding domains of three different targets, wherein the first binding domain binds CD70. In some embodiments, the recombinant extracellular protein (BiME or treme) having a domain that can bind CD70 further comprises a second domain that can bind a surface protein of a myeloid cell (e.g., a phagocyte). The surface protein of the myeloid cell may be a protein expressed on the myeloid cell membrane, e.g. a phagocytic receptor, a pattern recognition molecule or a scavenger receptor. Provided herein is a recombinant nucleic acid encoding an extracellular protein, e.g., a soluble protein that can bind to a CD70 extracellular domain as described herein.
Provided herein is a therapeutic agent for treating a disease in a subject, wherein the disease is associated with overexpression of a CD70 protein, the therapeutic agent comprising a recombinant protein having at least an extracellular binding domain that can bind CD70, such as any of the CD 70-binding proteins described above. In some embodiments, provided herein is a cell that expresses a CD70 binding protein. In some embodiments, provided herein is a therapeutic agent for treating a disease in a subject, wherein the disease is associated with overexpression of CD70, the therapeutic agent comprising a recombinant nucleic acid encoding at least a CD70 conjugate as described herein. In some embodiments, provided herein is a therapeutic agent for treating a disease in a subject, wherein the disease is associated with overexpression of a CD70 protein, the therapeutic agent comprising a cell comprising a recombinant nucleic acid encoding a CD70 conjugate as described herein. In some embodiments, the cell is a myeloid cell. In some embodiments, the disease is cancer, such as T cell lymphoma or colorectal cancer (CRC).
An exemplary CD70 conjugate sequence is SEQ ID NO. 32:
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADAFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDYGDYGMDYWGQGTTVTVSSGSTSGSGKPGSSEGSTKGDIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSREVPWTFGQGTKVEIK(SEQ ID NO:32)。
in some embodiments, the Chimeric Fusion Protein (CFP) comprises an extracellular domain (ECD) targeted to bind CD70, which comprises the amino acid sequence of SEQ ID NO:32, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 32.
Claudin 18.2 and Claudin 3 binding proteins and domains
The Claudin family of proteins are closely cell linked components that establish a paracellular barrier that controls the flow of molecules between cells. The transmembrane domain of Claudin includes an N-terminus and a C-terminus in the cytoplasm. Claudin proteins exhibit tissue-specific expression, and their functional alterations are often associated with cancer of specific tissues. Claudin-1 expression has been shown to have prognostic value in colon cancer. Claudin-1 is a strong prognostic indicator of stage II colon cancer and Claudin-18 is a strong prognostic indicator of gastric cancer. Claudin-18 gene down-regulation has been demonstrated by serial analysis of gene expression data analysis in gastric cancer with intestinal phenotype, and Claudin-10 down-regulation has been demonstrated in hepatocellular carcinoma. The surface protein Claudin represents a useful target for various therapeutic strategies.
Claudin 18 isoform 2 (Claudin 18.2) is a highly selective cell lineage marker. Claudin-18 splice variant 2 is a pan-cancerous target suitable for therapeutic antibody development and exhibits a highly restricted expression pattern in normal tissues with frequent ectopic activation in a variety of human cancers including gastric, ovarian and pancreatic cancers.
Claudin-3 is expressed in tight junctions and appears to be regulated during tumorigenesis in many organs and tissues (e.g., breast, ovary, uterus, prostate, and esophagus). The Caudin 3 overexpression increased epithelial resistance as measured by paracellular resistance without significant changes in transcellular resistance. Claudin-3 overexpression results in reduced permeability to sodium and chlorine, and expression of Claudin 3 molecules is elevated in many cancer tissues (e.g., ovarian, prostate, breast, uterine, liver, lung, pancreatic, gastric, bladder and colon tissues). Claudin 3 and Claudin 4 expression has been shown to be particularly high in chemotherapy-resistant uterine cancers. Claudin 3 is a protein having four transmembrane regions, and has a structure in which two peptide loops are exposed to the outside of the cell.
In one aspect, provided herein is a chimeric fusion protein having an extracellular binding domain that can bind to Claudin18.2 (Claudin 18.2 conjugate CFP).
In another aspect, provided herein is a chimeric fusion protein having an extracellular binding domain that can bind to Claudin 3.0.
In one embodiment, the extracellular binding domain comprises an scFv or fragment thereof specific for human Claudin 18.2. In some embodiments, the ECD of the chimeric fusion receptor protein comprises a binding domain that binds to the extracellular loop of Claudin18.2 on the target cell.
In some embodiments, an exemplary Claudin18.2 conjugate domain may comprise the sequence of SEQ ID NO. 33; or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO. 33.
QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGYNWHWIRQPPGKGL
EWIGYIHYTGSTNYNPALRSRVTISVDTSKNQFSLKLSSVTAADTA
VYYCARIYNGNSFPYWGQGTTVTVSSGGGGSGGGGSGGGGSDIV
MTQSPDSLAYSLGERATINCKSSQSLFNSGNQKNYLTWYQQKPGQ
PPKLLIYWASTRESGVPDRFSGSGSGTDIFITISSLQAEDVAVYYCQNAYSFPYTFGGGTKLEIKR(SEQ ID NO:33)
In some embodiments, the Chimeric Fusion Protein (CFP) comprises an extracellular domain (ECD) that targets binding Claudin18.2, comprising the amino acid sequence of SEQ ID NO. 33 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO. 33.
In some embodiments, the CFP comprises an extracellular domain comprising the sequence shown in SEQ ID NO. 33 or a sequence having at least 80% identity to SEQ ID NO. 33; which is operably linked to a TM domain comprising a CD16 transmembrane domain sequence (e.g., comprising a short hinge domain) as shown in table 4. In some embodiments, the CFP comprises an extracellular domain comprising the sequence shown in SEQ ID NO. 33 or a sequence having at least 80% identity to SEQ ID NO. 33; which is operably linked to a TM domain comprising the CD64 transmembrane domain sequence. In some embodiments, the CFP comprises an extracellular domain comprising the sequence shown in SEQ ID NO. 33; which is operably linked to a TM domain comprising a CD89 transmembrane domain sequence (e.g., comprising a short hinge domain) as shown in table 4. In some embodiments, the CFP comprises an ECD having the sequence of SEQ ID NO. 33 or a sequence having at least 80% identity to SEQ ID NO. 33; a TM domain comprising a sequence from the TM domain of the CD16 protein, or the TMD of the CD64 protein, or the TMD of the CD89 protein, and a corresponding intracellular domain (ICD) or portion thereof, e.g., from the CD16 protein, the CD64 protein, or the CD89 protein, respectively, optionally in addition to other different ICDs described herein. Exemplary CD16 and CD89 ICD sequences are provided in table 4.
In one embodiment, the extracellular binding domain comprises an scFv specific for human Claudin 3.0. In some embodiments, the extracellular binding domain comprises a Claudin 3 binding domain comprising a sequence shown in table 2. Table 2 shows exemplary sequences of human Claudin 3.0 binding domains and/or fragments thereof, which are non-limiting for the present invention.
TABLE 2
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The Claudin 3 binding domain may comprise one or more of the sequences from table 2. In some embodiments, the Claudin 3 binding domain comprises an ScFv comprising the sequence of table 2. In some embodiments, the binding domain comprises an antigen binding fragment (Fab), a single chain variable fragment (scFv), a nanobody, a VH domain, a VL domain, a single domain antibody (sdAb), a VNAR domain, and a VHH domain, a bispecific antibody, a diabody, or any functional fragment thereof, comprising a sequence of table 2. In some embodiments, an antigen binding fragment (Fab), single chain variable fragment (scFv), nanobody, VH domain, VL domain, single domain antibody (sdAb), VNAR domain and VHH domain, bispecific antibody, diabody, or any functional fragment thereof specifically binds to one or more antigens.
In some embodiments, the chimeric fusion protein is a receptor comprising an extracellular binding domain, such as an antigen binding domain; wherein the extracellular binding domain may comprise a Claudin 18.2 protein as described herein; or may bind to the sequence of a Claudin 3 protein as described herein; and may further comprise a transmembrane domain with or without a hinge domain between the extracellular antigen binding domain and the intracellular domain. In some embodiments, the extracellular domain further comprises a hinge domain derived from CD8, wherein the hinge domain is operably linked to a cytoplasmic side transmembrane domain and an extracellular Claudin 18.2 binding domain. In some embodiments, the transmembrane domain comprises any one of the sequences set forth in SEQ ID NO 15, 16, 17, 18 or 19. In some embodiments, the transmembrane domain comprises a sequence derived from a CD8a (CD 8 a) transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 15 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 15. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 16 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 16. In some embodiments, the transmembrane domain comprises a sequence derived from a CD2 transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 17 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 17. In some embodiments, the transmembrane domain comprises a sequence derived from a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO:18 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 18. In some embodiments, the transmembrane domain comprises a sequence derived from a CD68 transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 19 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 19. In some embodiments, the ECD of the CFP comprises an amino acid sequence set forth in table 2 or a sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the sequences set forth in table 2 fused to a transmembrane domain selected from any of SEQ ID NOs 15, 16, 17, 18 or 19 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs 15, 16, 17, 18 or 19.
In some embodiments, the extracellular hinge domain and the transmembrane domain comprise SEQ ID NO. 20 or SEQ ID NO. 21 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 20 or SEQ ID NO. 21, respectively. In some embodiments, a CFP comprises an extracellular domain fused to SEQ ID NO. 20 or to a transmembrane domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 20. In some embodiments, a CFP comprises an extracellular domain fused to SEQ ID NO. 21 or to a transmembrane domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 21. In some embodiments, a CFP comprises an ECD conjugate domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences in table 2 fused to a hinge and transmembrane domain having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 20 or 21.
In some embodiments, the CFP comprises one or more intracellular signaling domains comprising an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a phagocytic signaling domain. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Megf10, merTk, fcrα, and Bai 1. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Megf10, merTk, fcR, and Bai 1. In some embodiments, the intracellular signaling domain comprises a domain derived from a receptor other than cd3ζ. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from fcrγ, fcrα, or fcrepsilon.
In some embodiments, the CFP comprises an intracellular signaling domain derived from an FcR gamma protein (FcR gamma chain) comprising the amino acid sequence of any one of SEQ ID NOs 22, 23, 24 or 25 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs 22, 23, 24 or 25. In some embodiments, the one or more intracellular signaling domains further comprise a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain comprises a PI3 kinase (PI 3K) recruitment domain. In some embodiments, the pro-inflammatory signaling domain comprises SEQ ID NO. 26 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 26. In some embodiments, the pro-inflammatory signaling domain is derived from the intracellular signaling domain of CD 40. In some embodiments, the pro-inflammatory signaling domain comprises SEQ ID NO 27 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO 27. In some embodiments, the CFP comprises SEQ ID NO 28 or an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO 28. In some embodiments, the CFP comprises SEQ ID NO. 29 or an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 29. In some embodiments, the CFP comprises SEQ ID NO. 30 or an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 30.
Provided herein is a recombinant nucleic acid encoding a CFP comprising a Claudin18.2 binding domain (Claudin 18.2 conjugate) as described above, which CFP can be expressed in a suitable cell (e.g., a myeloid cell). The myeloid cells may be phagocytes. In some embodiments, the Claudin18.2 conjugate activates myeloid cells expressing Claudin18.2 binding CFP when binding to Claudin18.2 on the cells. Provided herein are recombinant nucleic acid sequences encoding chimeric Claudin 18.2-binder receptor fusion proteins (CFPs). In some embodiments, the recombinant nucleic acid encoding, for example, a chimeric receptor fusion protein (e.g., claudin 18.2-conjugate receptor fusion protein) can be expressed in a suitable cell (e.g., a myeloid cell). In some embodiments, when Claudin18.2-CFP is expressed on a suitable cell (e.g., a myeloid cell), the Claudin18.2 conjugate can bind to a target cell expressing Claudin18.2, and the myeloid cell expressing the conjugate is activated. In some embodiments, the Claudin18.2-CFP activates the intracellular signaling domain of Claudin18.2-CFP when engaged with Claudin18.2, which activates myeloid cells expressing recombinant nucleic acid encoding Claudin 18.2-CFP. In some embodiments, the myeloid cell is a phagocyte. In some embodiments, when the Claudin18.2 conjugate binds Claudin18.2 expressed on another cell (target cell), a myeloid cell that is a phagocytic cell and expresses the CD137 conjugate can be activated and swallow the target cell. In some embodiments, the target cell may be any cell that expresses Claudin 18.2. In some embodiments, the target cell is a lymphocyte. In some embodiments, the target cell is a T lymphocyte (Y cell). In some embodiments, the target cell may be a malignant cell. In some embodiments, the target cell may be a malignant T cell. In some embodiments, myeloid cells expressing Claudin18.2 binding CFP can be used to generate therapeutic compositions against T cell lymphomas.
In some embodiments, the Claudin 18.2 conjugate chimeric fusion protein is an extracellular protein comprising an antigen binding domain that binds Claudin 18.2. Provided herein is a recombinant protein, such as an extracellular protein, e.g., a soluble protein that can bind Claudin 18.2. In some embodiments, the extracellular protein comprises an antibody or fragment thereof that can bind Claudin 18.2 as described above. In some embodiments, the extracellular or soluble protein further comprises a second domain of a surface protein that can bind to a myeloid cell (e.g., a phagocyte). The surface protein of the myeloid cell may be a protein expressed on the myeloid cell membrane, e.g. a phagocytic receptor, a pattern recognition molecule or a scavenger receptor.
Provided herein is a recombinant nucleic acid encoding an extracellular protein, e.g., a soluble protein that can bind to the Claudin 18.2 extracellular domain as described herein.
In some embodiments, the Claudin 3 conjugate is an extracellular protein. Provided herein is a recombinant protein, such as an extracellular protein, e.g., a soluble protein that can bind Claudin 3. In some embodiments, the extracellular protein comprises an antibody or fragment thereof that can bind Claudin 3, e.g., any one or more of the sequences set forth in table 2. In some embodiments, the extracellular Claudin 3 conjugate protein further comprises a second domain of a surface protein that can bind to a myeloid cell (e.g., a phagocyte). The surface protein of the myeloid cell may be a protein expressed on the myeloid cell membrane, e.g. a phagocytic receptor, a pattern recognition molecule or a scavenger receptor. Provided herein is a recombinant nucleic acid encoding an extracellular protein, e.g., a soluble protein that can bind to a Claudin 3 extracellular domain as described herein.
Provided herein is a therapeutic agent for treating a disease in a subject, wherein the disease is associated with overexpression of a Claudin protein (e.g., claudin 18.2 protein or Claudin 3 protein), the therapeutic agent comprising a recombinant protein having at least an extracellular binding domain that can bind Claudin 18.2 or Claudin 3, respectively, e.g., any of the Claudin 18.2 or Claudin 3 conjugate proteins described above. In some embodiments, provided herein is a cell that expresses a Claudin 18.2 conjugate or a Claudin 3 conjugate. In some embodiments, provided herein is a therapeutic agent for treating a disease in a subject, wherein the disease is associated with overexpression of a Claudin protein (e.g., claudin 18.2 protein or Claudin 3 protein), the therapeutic agent comprising a recombinant nucleic acid encoding at least a Claudin 18.2 conjugate or a Claudin 3 conjugate as described herein. In some embodiments, provided herein is a therapeutic agent for treating a disease in a subject, wherein the disease is associated with overexpression of a Claudin protein (e.g., claudin 18.2 protein or Claudin 3 protein), the therapeutic agent comprising cells comprising a recombinant nucleic acid encoding a Claudin 18.2 conjugate or a Claudin 3 conjugate as described herein. In some embodiments, the cell is a myeloid cell.
TROP 2-binding proteins and domains
TROP2 (also known as epithelial glycoprotein-1, gastrointestinal antigen 733-1, membrane fraction surface marker-1 and tumor associated calcium signaling protein-2) is a protein product of the TACSTD2 gene. It is a transmembrane glycoprotein that plays a role in a variety of cell signaling pathways, and was first elucidated as a transduction protein of intracellular calcium signaling. TROP2 expression has been shown to depend on a variety of transcription factors. TROP2 regulates proliferation and self-renewal through b-catenin signaling, and TROP2 signaling enhances stem cell-like properties of cancer cells. Considering the involvement of several molecular pathways traditionally associated with cancer progression, TROP2 may play a role in tumor progression. TROP2 high expression is associated with poor prognosis of pancreatic cancer, hepatobiliary cancer, cervical cancer, gastric cancer and other cancers (Fong D et al, br J cancer.2008;99 (8): 1290-1295; ning S et al, J gastointest surg.2013;17 (2): 360-368; liu T et al, PLoS one.2013;8 (9): e75864; zhao W et al, oncostarget.2016; 7 (5): 6136-6145). TROP2 is overexpressed on a variety of tumors, which can target the development of novel therapeutic agents relative to upregulation of normal cellular expression.
Provided herein is a chimeric protein that can bind to a cancer cell that expresses TROP 2. Provided herein is a chimeric protein that binds to an extracellular domain of TROP2 (TROP 2 conjugate). In some embodiments, the chimeric protein is a chimeric receptor protein having an extracellular protein, a transmembrane domain, and/or a cytoplasmic domain capable of binding an extracellular domain of TROP 2. In some embodiments, the chimeric protein is an extracellular soluble protein that can bind to an extracellular domain of TROP 2. In some embodiments, an extracellular soluble chimeric protein capable of binding to an extracellular domain of TROP2 can comprise another domain capable of binding to an extracellular domain of a protein expressed on the surface of a myeloid cell (e.g., a phagocyte). A chimeric protein (TROP 2 conjugate) as described herein can be used to prepare a therapeutic agent comprising a pharmaceutical composition comprising a TROP2 conjugate for use in treating a disease associated with overexpression of a TROP2 protein (e.g., cancer). Such cancers include, but are not limited to, pancreatic cancer, hepatobiliary cancer, cervical cancer, and gastric cancer.
In one aspect, provided herein is a chimeric fusion receptor (CFP) protein having an extracellular TROP2 binding domain (TROP 2 conjugate CFP). In some embodiments, the extracellular binding domain is an antigen binding domain of an antibody capable of binding to TROP2, or a fragment, single chain variable fragment (scFv), nanobody, VH domain, VL domain, single domain antibody (sdAb), VNAR domain and VHH domain, bispecific antibody, diabody, or any functional fragment thereof. In some embodiments, an antigen binding fragment (Fab), single chain variable fragment (scFv), nanobody, VH domain, VL domain, single domain antibody (sdAb), VNAR domain and VHH domain, bispecific antibody, diabody, or any functional fragment thereof specifically binds to one or more antigens. In some embodiments, the conjugate is a natural ligand for the TROP2 receptor.
In some embodiments, the extracellular domain further comprises a hinge domain derived from CD8, wherein the hinge domain is operably linked to a cytoplasmic side transmembrane domain and an extracellular TROP2 binding domain. In some embodiments, the transmembrane domain comprises any one of the sequences set forth in SEQ ID NO 15, 16, 17, 18 or 19. In some embodiments, the transmembrane domain comprises a sequence derived from a CD8a (CD 8 a) transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 15 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 15. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 16 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 16. In some embodiments, the transmembrane domain comprises a sequence derived from a CD2 transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 17 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 17. In some embodiments, the transmembrane domain comprises a sequence derived from a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO:18 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 18. In some embodiments, the transmembrane domain comprises a sequence derived from a CD68 transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 19 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 19. In some embodiments, the ECD of the CFP comprises an amino acid sequence set forth in table 2 or a sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the sequences set forth in table 2 fused to a transmembrane domain selected from any of SEQ ID NOs 15, 16, 17, 18 or 19 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs 15, 16, 17, 18 or 19.
In some embodiments, the extracellular hinge domain and the transmembrane domain comprise SEQ ID NO. 20 or SEQ ID NO. 21 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 20 or SEQ ID NO. 21, respectively. In some embodiments, a CFP comprises an extracellular domain fused to SEQ ID NO. 20 or to a transmembrane domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 20. In some embodiments, a CFP comprises an extracellular domain fused to SEQ ID NO. 21 or to a transmembrane domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 21. In some embodiments, a CFP comprises an ECD conjugate domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences in table 2 fused to a hinge and transmembrane domain having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 20 or 21.
In some embodiments, the CFP comprises an extracellular domain comprising the sequence of SEQ ID NO. 34 or SEQ ID NO. 35 or both, or a sequence having at least 80% identity to SEQ ID NO. 34 or SEQ ID NO. 35; which is operably linked to a TM domain comprising a CD16 transmembrane domain sequence (e.g., comprising a short hinge domain) as shown in table 4. In some embodiments, the CFP comprises an extracellular domain comprising the sequence of SEQ ID NO 34 or 35, or a sequence having at least 80% identity to SEQ ID NO 34 or 35; which is operably linked to a TM domain comprising the CD64 transmembrane domain sequence. In some embodiments, the CFP comprises an extracellular domain comprising a sequence set forth in SEQ ID NO 34 or 35; which is operably linked to a TM domain comprising a CD89 transmembrane domain sequence (e.g., comprising a short hinge domain) as shown in table 4. In some embodiments, the CFP comprises an ECD having the sequence of SEQ ID NO 34 or 35 or a sequence having at least 80% identity to SEQ ID NO 34 or 35; a TM domain comprising a sequence from the TM domain of the CD16 protein, or the TMD of the CD64 protein, or the TMD of the CD89 protein, and a corresponding intracellular domain (ICD) or portion thereof, e.g., from the CD16 protein, the CD64 protein, or the CD89 protein, respectively, optionally in addition to other different ICDs described herein. Exemplary CD16 and CD89ICD sequences are provided in table 4.
In some embodiments, the CFP comprises one or more intracellular signaling domains comprising an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a phagocytic signaling domain. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Megf10, merTk, fcrα, and Bai 1. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Megf10, merTk, fcR, and Bai 1. In some embodiments, the intracellular signaling domain comprises a domain derived from a receptor other than cd3ζ. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from fcrγ, fcrα, or fcrepsilon.
In some embodiments, the CFP comprises an intracellular signaling domain derived from an FcR gamma protein (FcR gamma chain) comprising the amino acid sequence of any one of SEQ ID NOs 22, 23, 24 or 25 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs 22, 23, 24 or 25. In some embodiments, the one or more intracellular signaling domains further comprise a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain comprises a PI3 kinase (PI 3K) recruitment domain. In some embodiments, the pro-inflammatory signaling domain comprises SEQ ID NO. 26 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 26. In some embodiments, the pro-inflammatory signaling domain is derived from the intracellular signaling domain of CD 40. In some embodiments, the pro-inflammatory signaling domain comprises SEQ ID NO 27 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO 27. In some embodiments, the CFP comprises SEQ ID NO 28 or an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO 28. In some embodiments, the CFP comprises SEQ ID NO. 29 or an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 29. In some embodiments, the CFP comprises SEQ ID NO. 30 or an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 30.
Provided herein is a recombinant nucleic acid encoding a CFP comprising a TROP2 binding domain (TROP 2 conjugate) as described above, which CFP can be expressed in a suitable cell (e.g., a myeloid cell). The myeloid cells may be phagocytes. In some embodiments, the recombinant nucleic acid encoding a CFP comprises a TROP2 binding domain as described above, comprising an intracellular domain from one or more of the sequences of SEQ ID NOs 21-30; and a transmembrane domain comprising a sequence having a sequence selected from any one of SEQ ID NOS.15-21.
In another embodiment, provided herein is a recombinant protein, e.g., an extracellular protein, e.g., a soluble protein that can bind to an extracellular domain of TROP 2. In some embodiments, the recombinant extracellular protein having a domain that can bind to TROP2 (e.g., a domain that can bind to an extracellular domain of TROP 2) further comprises a second domain that can bind to a surface protein of a myeloid cell (e.g., a phagocyte). The surface protein of the myeloid cell may be a protein expressed on the myeloid cell membrane, e.g. a phagocytic receptor, a pattern recognition molecule or a scavenger receptor. Provided herein is a recombinant nucleic acid encoding an extracellular protein, e.g., a soluble protein that can bind to a TROP2 extracellular domain as described herein.
Provided herein is a therapeutic agent for treating a disease in a subject, wherein the disease is associated with overexpression of a TROP2 protein, the therapeutic agent comprising a recombinant polynucleic acid encoding at least a protein having an extracellular binding domain that can bind to TROP2, such as any of the above TROP2 binding proteins. In some embodiments, the recombinant polynucleic acid encoding a protein is an mRNA. In some embodiments, the recombinant polynucleic acid encoding the protein is DNA. In some embodiments, the recombinant polynucleic acid encodes a TROP2 binding protein, which is a chimeric fusion receptor protein (CFP) comprising an extracellular TROP2 binding domain, optionally a transmembrane domain, and one or more intracellular domains. In some embodiments, one or more intracellular domains comprise one or more cell signaling domains. In some embodiments, as described in the specification, the one or more intracellular signaling domains comprise a PI3 kinase recruitment domain, a CD40 intracellular domain, an FcR domain. In some embodiments, the recombinant polynucleotide is contained in a cell. In some embodiments, recombinant mRNA comprising a sequence encoding a TROP2 conjugate CFP is expressed in myeloid cells. In some embodiments, provided herein is a cell that expresses a TROP2 binding protein. In some embodiments, provided herein is a therapeutic agent for treating a disease in a subject, wherein the disease is associated with overexpression of TROP2, the therapeutic agent comprising a recombinant nucleic acid encoding at least a TROP2 conjugate as described herein. In some embodiments, provided herein is a therapeutic agent for treating a disease in a subject, wherein the disease is associated with overexpression of a TROP2 protein, the therapeutic agent comprising cells comprising a recombinant nucleic acid encoding a TROP2 conjugate as described herein. In some embodiments, the cell is a myeloid cell. In some embodiments, the disease is cancer, including but not limited to pancreatic cancer, hepatobiliary tract cancer, cervical cancer, gastric cancer, lung cancer. In some embodiments, the disease is non-small cell lung cancer.
In some embodiments, the therapeutic agents described herein comprise at least a recombinant Chimeric Fusion Protein (CFP) or polynucleotide encoding a recombinant CFP having an extracellular binding domain that can bind TROP2, optionally a CD8 hinge domain, and a transmembrane domain that can bind endogenous proteins in myeloid cells for proper expression and localization on myeloid cell membranes. For example, in some embodiments, the TROP2 conjugate comprises a transmembrane region that dimerizes or oligomerizes with another transmembrane protein of a myeloid cell. In some embodiments, the TROP2 conjugate comprises a transmembrane protein that dimerizes with an Fc-gamma receptor on a myeloid cell membrane, and thus can only be expressed in myeloid cells. In some embodiments, the TROP2 conjugate is designed such that the protein only functions when it dimerizes (or oligomerizes) with endogenous proteins that are expressed only in myeloid cells. In some embodiments, the TROP2 conjugate comprises an Fc-a transmembrane domain. In some embodiments, the TROP2 conjugate comprises a transmembrane domain selected from the group consisting of a CD64 (fcγr1) protein transmembrane domain, a CD89 (fcαr1) protein transmembrane domain, and a CD16a (fcγriiia) transmembrane domain. In some embodiments, the TROP2 conjugate comprises an Fc-epsilon transmembrane domain. In some embodiments, provided herein is a TROP2 binding CFP (TROP 2 conjugate) comprising an extracellular TROP2 binding domain, optionally a short hinge region (e.g., comprising a CD8 hinge domain), a CD89 transmembrane domain, optionally a CD89 intracellular region, fused to one or more intracellular signaling domains, such as a CD40 intracellular domain and a PI3 kinase recruitment domain. In some embodiments, provided herein is a TROP2 binding CFP (TROP 2 conjugate) comprising an extracellular TROP2 binding domain, optionally a short hinge region (e.g., comprising a CD8 hinge domain), a CD64 transmembrane domain, optionally a CD64 intracellular region, fused to one or more intracellular signaling domains, such as a CD40 intracellular domain and a PI3 kinase recruitment domain. The TROP2 CFP may comprise one or more human or humanized domains. Provided herein is a polynucleotide, e.g., mRNA or DNA, comprising a sequence encoding a TROP2 CFP construct comprising an anti-TROP 2 ScFv fused to a CD8 hinge domain (TROP 2 antigen-binding) operably linked to a CD89 transmembrane domain and an intracellular domain.
In some embodiments, a TROP2 CFP described herein exhibits myeloid cell-specific expression. In some embodiments, a TROP2 CFP described herein exhibits Fc-gamma-dependent expression. In some embodiments, a TROP2 CFP described herein exhibits undetectable expression in a T cell, B cell, epithelial cell, muscle cell, neuronal cell, or any non-myeloid cell when the polynucleotide is administered in vivo. In one embodiment, when the polynucleotide is administered in vivo, a TROP2 conjugate encoded by a polynucleotide described herein having a CD16, CD89 or CD64 transmembrane domain as described herein is expressed predominantly in cd14+ cells. In one embodiment, a TROP2 conjugate having a CD16, CD89 or CD64 transmembrane domain encoded by a polynucleotide described herein is expressed only in cd14+ cells when the polynucleotide is administered in vivo.
Provided herein is a pharmaceutical composition comprising cd14+ cells expressing a CFP having a TROP2 binding domain. In some embodiments, the pharmaceutical composition comprises a population of cells, wherein at least 50% of the cells are cd14+ cells that express a TROP2 conjugate CFP. In some embodiments, the pharmaceutical composition comprises at least 50% cd14+ and CD 16-cells. In some embodiments, the pharmaceutical composition comprises at least 50% of cells that express cd14+ cells, the cd14+ cells being CD 16-and expressing TROP2-CFP (CFP with TROP2 binding extracellular domain).
Provided herein is a pharmaceutical composition comprising a recombinant polynucleotide (polynucleic acid) encoding a transmembrane polypeptide comprising CD64, or CD89 or CD16a transmembrane domain, and wherein the recombinant polynucleotide is encapsulated within an LNP. In some embodiments, the transmembrane polypeptide is a CFP having an extracellular binding domain that specifically binds to an antigen on a cancer cell. In some embodiments, the CFP comprises an extracellular binding domain that specifically binds TROP 2.
Provided herein is a pharmaceutical composition for treating cancer in a human subject comprising cells expressing a TROP2 conjugate CFP as described herein, e.g., cd14+ myeloid cells, wherein the cancer is lung cancer. In some embodiments, the lung cancer is NSCLC.
Provided herein is a pharmaceutical composition for treating cancer in a human subject comprising a recombinant polynucleic acid encoding a CFP comprising a TROP2 binding extracellular domain capable of expression in vivo myeloid cells; wherein the CFP comprising a TROP2 binding extracellular domain comprises a CD64, CD16a or CD89 transmembrane domain as described herein, wherein the recombinant polynucleic acid is encapsulated in an LNP, wherein the cancer is lung cancer. In some embodiments, the lung cancer is NSCLC.
An exemplary TROP2 binding domain sequence can be the amino acid sequence of SEQ ID NO. 34, or a sequence sharing at least 80% identity with SEQ ID NO. 34.
DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKL
LIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYI
TPLTFGAGTKVEIKRGGGGSGGGGSGGGGSQVQLQQSGSELKKPG
ASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS(SEQ ID NO:34)。
In some embodiments, the Chimeric Fusion Protein (CFP) comprises an extracellular domain (ECD) targeted to bind TROP2, which comprises the amino acid sequence of SEQ ID NO:34, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 34.
An exemplary TROP2 binding domain can be the sequence set forth in SEQ ID NO. 35.
QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKR(SEQ ID NO:35)。
In some embodiments, the Chimeric Fusion Protein (CFP) comprises an extracellular domain (ECD) targeted to bind TROP2, which comprises the amino acid sequence of SEQ ID NO:35, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 35.
TMPRSS3 binding proteins and domains
Near 50% of prostate cancers have gene fusions that link androgen regulated gene transmembrane proteases, serine 2 (TMPRSS 2), to transcription factors of the erythropoiesis virus E26 conversion sequence (ETS) family, typically erythropoiesis-specific related genes (ERGs). It has recently been demonstrated that TMPRSS2: ERG fusion generally occurs early in tumor progression and is generally homogeneously distributed throughout the cancer mass. In recent studies, inconsistent ERG findings were observed in 30% of the lymph nodes of 84 prostate cancers. TMPRSS 2. ERG fusion protein can be the best target for novel therapies because it is highly specific for prostate cancer cells. In one aspect, provided herein is a chimeric fusion receptor (CFP) protein having an extracellular TMPRSS binding domain (TMPRSS conjugate CFP).
In some embodiments, the extracellular binding domain is an antigen binding domain of an antibody capable of binding TMPRSS, or a fragment, single chain variable fragment (scFv), nanobody, VH domain, VL domain, single domain antibody (sdAb), VNAR domain and VHH domain, bispecific antibody, diabody, or any functional fragment thereof. In some embodiments, an antigen binding fragment (Fab), single chain variable fragment (scFv), nanobody, VH domain, VL domain, single domain antibody (sdAb), VNAR domain and VHH domain, bispecific antibody, diabody, or any functional fragment thereof specifically binds to one or more antigens.
In some embodiments, the extracellular domain further comprises a hinge domain derived from CD8, wherein the hinge domain is operably linked to a cytoplasmic side transmembrane domain and an extracellular TMPRSS binding domain. In some embodiments, the transmembrane domain comprises any one of the sequences set forth in SEQ ID NO 15, 16, 17, 18 or 19. In some embodiments, the transmembrane domain comprises a sequence derived from a CD8a (CD 8 a) transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 15 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 15. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 16 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 16. In some embodiments, the transmembrane domain comprises a sequence derived from a CD2 transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 17 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 17. In some embodiments, the transmembrane domain comprises a sequence derived from a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO:18 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 18. In some embodiments, the transmembrane domain comprises a sequence derived from a CD68 transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO. 19 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 19. In some embodiments, the ECD of the CFP comprises an amino acid sequence set forth in table 2 or a sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the sequences set forth in table 2 fused to a transmembrane domain selected from any of SEQ ID NOs 15, 16, 17, 18 or 19 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs 15, 16, 17, 18 or 19.
In some embodiments, the extracellular hinge domain and the transmembrane domain comprise SEQ ID NO. 20 or SEQ ID NO. 21 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 20 or SEQ ID NO. 21, respectively. In some embodiments, a CFP comprises an extracellular domain fused to SEQ ID NO. 20 or to a transmembrane domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 20. In some embodiments, a CFP comprises an extracellular domain fused to SEQ ID NO. 21 or to a transmembrane domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 21. In some embodiments, a CFP comprises an ECD conjugate domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences in table 2 fused to a hinge and transmembrane domain having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 20 or 21.
In some embodiments, CFP comprising an ECD domain that can bind TMPRSS protein or a portion thereof comprises TMD and a hinge domain, optionally a short cytoplasmic domain of CD16, CD64, or CD89 protein, optionally one or more other ICDs described herein.
In some embodiments, the CFP comprises one or more intracellular signaling domains comprising an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a phagocytic signaling domain. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Megf10, merTk, fcrα, and Bai 1. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Megf10, merTk, fcR, and Bai 1. In some embodiments, the intracellular signaling domain comprises a domain derived from a receptor other than cd3ζ. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from fcrγ, fcrα, or fcrepsilon.
In some embodiments, the CFP comprises an intracellular signaling domain derived from an FcR gamma protein (FcR gamma chain) comprising the amino acid sequence of any one of SEQ ID NOs 22, 23, 24 or 25 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs 22, 23, 24 or 25. In some embodiments, the one or more intracellular signaling domains further comprise a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain comprises a PI3 kinase (PI 3K) recruitment domain. In some embodiments, the pro-inflammatory signaling domain comprises SEQ ID NO. 26 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 26. In some embodiments, the pro-inflammatory signaling domain is derived from the intracellular signaling domain of CD 40. In some embodiments, the pro-inflammatory signaling domain comprises SEQ ID NO 27 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO 27. In some embodiments, the CFP comprises SEQ ID NO 28 or an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO 28. In some embodiments, the CFP comprises SEQ ID NO. 29 or an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 29. In some embodiments, the CFP comprises SEQ ID NO. 30 or an intracellular signaling domain having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 30.
Provided herein is a recombinant nucleic acid encoding a CFP comprising a TMPRSS binding domain (TMPRSS conjugate) as described above, which CFP can be expressed in a suitable cell (e.g., a myeloid cell). The myeloid cells may be phagocytes. In some embodiments, the recombinant nucleic acid encoding a CFP comprises a TMPRSS binding domain as described above, comprising an intracellular domain from one or more of the sequences of SEQ ID NOs 21-30; and a transmembrane domain comprising a sequence having a sequence selected from any one of SEQ ID NOS.15-21.
In another embodiment, provided herein is a recombinant protein, e.g., an extracellular protein, e.g., a soluble protein that can bind to the extracellular domain of TMPRSS. In some embodiments, the recombinant extracellular protein having a domain that can bind to TMPRSS (e.g., a domain that can bind to an extracellular domain of TMPRSS) comprises a second domain that is capable of binding to a surface protein of a myeloid cell (e.g., a phagocyte). The surface protein of the myeloid cell may be a protein expressed on the myeloid cell membrane, e.g. a phagocytic receptor, a pattern recognition molecule or a scavenger receptor. Provided herein is a recombinant nucleic acid encoding an extracellular protein, e.g., a soluble protein that can bind to a TMPRSS extracellular domain as described herein.
Provided herein is a therapeutic agent for treating a disease in a subject, wherein the disease is associated with overexpression of TMPRSS protein, the therapeutic agent comprising a recombinant protein having at least an extracellular binding domain that can bind TMPRSS, such as any of the TMPRSS conjugate proteins described above. In some embodiments, provided herein is a cell that expresses a TMPRSS conjugate protein. In some embodiments, provided herein is a therapeutic agent for treating a disease in a subject, wherein the disease is associated with overexpression of TMPRSS, the therapeutic agent comprising a recombinant nucleic acid encoding at least a TMPRSS conjugate as described herein. In some embodiments, provided herein is a therapeutic agent for treating a disease in a subject, wherein the disease is associated with overexpression of TMPRSS protein, the therapeutic agent comprising a cell comprising a recombinant nucleic acid encoding a TMPRSS conjugate as described herein. In some embodiments, the cell is a myeloid cell. In some embodiments, the disease is cancer, such as prostate cancer.
BIME and TRiME structures
Provided herein are bispecific conjugates (bimes) or trispecific conjugates (tri mes) comprising any of the binding domains of the therapeutic agents described above, wherein the binding domain comprises a binding domain of an antibody, a functional fragment of an antibody, a variable domain thereof, V H Domain, V L Domain, VNAR domain, V HH A domain, a single chain variable fragment (scFv), a Fab, a single domain antibody (sdAb), a nanobody, a bispecific antibody, a diabody, or a functional fragment or combination thereof. In some embodiments, the antigen on the target cell to which the first binding domain binds is a cancer antigen or a pathogenic antigen or self on the target cellA systemic immune antigen. In some embodiments, the first therapeutic agent comprises a polypeptide less than 1000 amino acids in length or 1000 nm. In some embodiments, the first therapeutic agent comprises a polypeptide less than 500 amino acids in length or 500nm in length. In some embodiments, the first therapeutic agent comprises a polypeptide that is 200-1000 amino acids in length or 200-1000nm in length.
In some embodiments, the binding domain of the first therapeutic agent is conjugated to a cancer cell. In some embodiments, the second binding domain specifically interacts with a myeloid cell and promotes phagocytic activity of the myeloid cell. In some embodiments, the second binding domain specifically interacts with and promotes inflammatory signaling by myeloid cells. In some embodiments, the second binding domain specifically interacts with a myeloid cell or an adhesion molecule and promotes adhesion of the myeloid cell to the target cell. In some embodiments, the second binding domain specifically interacts with a myeloid cell and inhibits the antiphagic activity of the myeloid cell mediated by the target cell. In some embodiments, the second binding domain specifically interacts with a myeloid cell and inhibits anti-inflammatory activity of the myeloid cell mediated by the target cell. In some embodiments, the second binding domain and/or the third binding domain promotes phagocytic activity of the myeloid cells. In some embodiments, the second binding domain and/or the third binding domain promotes inflammatory signaling of myeloid cells. In some embodiments, the second binding domain and/or the third binding domain specifically interact with a myeloid cell or an adhesion molecule and promote adhesion of the myeloid cell to the target cell. In some embodiments, the second binding domain and/or the third binding domain inhibits antiphagic activity of the myeloid cells mediated by the target cells. In some embodiments, the second binding domain and/or the third binding domain inhibits anti-inflammatory activity of the myeloid cells mediated by the target cells.
In some embodiments, the third binding domain or additional therapeutic agent comprises a CD47 antagonist, a CD47 blocker, an antibody, a chimeric CD47 receptor, a sialidase, a cytokine, a pro-inflammatory gene, a pro-caspase, or an anti-cancer agent. In some embodiments, the first binding domain, the second binding domain, and the third binding domain bind different, non-identical target antigens. In some embodiments, the first binding domain, the second binding domain, or the third binding domain is a ligand binding domain.
In some embodiments, the first binding domain, the second binding domain, or the third binding domain is operably linked by one or more linkers. In some embodiments, the linker is a polypeptide. In some embodiments, the linker is a functional peptide. In some embodiments, the linker is a ligand for the receptor. In some embodiments, the linker is a ligand for a monocyte or macrophage receptor. In some embodiments, the linker activates the receptor. In some embodiments, the linker inhibits the receptor. In some embodiments, the linker is a ligand for M2 macrophage receptor. In some embodiments, the linker is a ligand for a TLR receptor (e.g., TLR 4). In some embodiments, the linker activates TLR receptors. In some embodiments, the first, second, and/or third binding domains are associated with a mask that binds the binding domains. In some embodiments, the mask is an inhibitor that inhibits the binding domain from interacting with its target while the mask remains associated with the corresponding binding domain. In some embodiments, the mask is associated with the binding domain via a peptide linker. In some embodiments, the peptide linker comprises a cleavable moiety. In some embodiments, the cleavable moiety is cleaved by a protein or enzyme that is selectively abundant in the cancer or tumor site.
Therapeutic compositions
In one aspect, provided herein is a myeloid cell, e.g., a CD14+ cell, a CD14+/CD 16-cell, a CD14+/CD16+ cell, a CD14-/CD 16-cell, a dendritic cell, an M0 macrophage, an M2 macrophage, an M1 macrophage, or a chimeric myeloid cell/macrophage/dendritic cell. In some embodiments, provided herein is a therapeutic composition 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+/CD 16-cells. In some embodiments, provided herein is a therapeutic composition comprising less than 20%, less than 15%, less than 10%, or less than 5% dendritic cells. Myeloid cells for use in therapeutic compositions as described herein comprise recombinant nucleic acids encoding chimeric fusion proteins encoding CFP receptor proteins or conjugate proteins as described herein. The myeloid cells used in the therapeutic compositions as described herein express CFP encoded by the recombinant nucleic acid or express the adaptor protein encoded by the recombinant nucleic acid as described herein.
In some embodiments, provided herein is a therapeutic composition comprising a chimeric fusion protein, such as a chimeric fusion receptor protein (CFP), comprising: (a) an extracellular domain comprising: (i) An scFv that specifically binds any one of the targets described herein, and (ii) a hinge domain derived from CD 8; at least a portion of a hinge domain derived from CD28 or an extracellular domain derived from CD 68; (b) A CD8 transmembrane domain, a CD28 transmembrane domain, a CD2 transmembrane domain, or a CD68 transmembrane domain; and (c) an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise: (i) A first intracellular signaling domain derived from fcrγ or fcrε, and (ii) a second intracellular signaling domain that: (A) Comprising a PI3K recruitment domain, or (B) derived from CD40.
In some embodiments, provided herein are therapeutic compositions comprising bispecific or trispecific conjugates as disclosed herein.
In one aspect, provided herein are one or more recombinant polynucleic acids encoding one or more recombinant proteins, which may be chimeric fusion proteins, such as receptors or conjugates as described herein. In some embodiments, the recombinant polynucleic acid is mRNA. In some embodiments, the recombinant polynucleotide comprises a circRNA. In some embodiments, the recombinant polynucleotide is contained in a viral vector. In some embodiments, the recombinant polynucleotide is delivered via a viral vector.
In some embodiments, provided herein is a therapeutic composition comprising a recombinant nucleic acid encoding a chimeric fusion protein (e.g., a chimeric fusion receptor protein (CFP)), the CFP comprising: (a) an extracellular domain comprising: (i) An scFv that specifically binds any one of the targets described herein, and (ii) a hinge domain derived from CD 8; at least a portion of a hinge domain derived from CD28 or an extracellular domain derived from CD 68; (b) A CD8 transmembrane domain, a CD28 transmembrane domain, a CD2 transmembrane domain, or a CD68 transmembrane domain; and (c) an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise: (i) A first intracellular signaling domain derived from fcrγ or fcrε, and (ii) a second intracellular signaling domain that: (A) Comprising a PI3K recruitment domain, or (B) derived from CD40.
In some embodiments, provided herein is a therapeutic composition comprising a recombinant nucleic acid encoding a bispecific or trispecific conjugate as disclosed herein.
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 breakdown, an agent that promotes PI3K recruitment to PFP, an agent that promotes PI3K activity, an agent that promotes phosphatidylinositol 3,4, 5-triphosphate production, an agent that promotes ARHGAP12 activity, an agent that promotes ARHGAP25 activity, an agent that promotes SH3BP1 activity, an agent that promotes lymphocyte isolation in primary and/or secondary lymphoid organs, an agent that increases primary T cell and central memory T cell concentration in secondary lymphoid organs, and any combination thereof.
In some embodiments, the myeloid cell further comprises: (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 which interacts with CFP; wherein dimerization or interaction enhances phagocytosis of the CFP-expressing myeloid cells compared to the myeloid cells that do not express CFP.
In some embodiments, the myeloid cells exhibit an increase in (i) effector activity, cross presentation, respiratory burst, ROS production, iNOS production, inflammatory mediators, extracellular vesicle production, phosphatidylinositol 3,4, 5-triphosphate production, cytostatic effect with target cells expressing an antigen, resistance to inhibition of CD 47-mediated phagocytosis, resistance to inhibition of LILRB 1-mediated phagocytosis, or any combination thereof; and/or (II) an increase in expression of IL-1, IL3, IL-6, IL-10, IL-12, IL-13, IL-23, TNF alpha, cytokine TNF family, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL-17, IP-10, RANTES, interferon, MHC class I protein, MHC class II protein, CD40, CD48, CD58, CD80, CD86, CD112, CD155, TRAIL/TNF family death receptor, TGF beta, B7-DC, B7-H2, LIGHT, HVEM, TL A, 41BBL, OX40L, GITRL, CD30L, TIM1, TIM4, SLAM, PDL1, MMP (e.g., MMP2, MMP7, and MMP 9), or any combination thereof.
In some embodiments, the intracellular signaling domain is derived from a phagocytic or tethered receptor, or wherein the intracellular signaling domain comprises a phagocytosis activating domain. In some embodiments, the intracellular signaling domain is derived from a receptor other than a phagocytic receptor selected from the group consisting of Megf10, merTk, fcR-a, or Bai 1. In some embodiments, the intracellular signaling domain is derived from a protein, e.g., a receptor (e.g., phagocytic receptor) selected from the group consisting of TNFR1, MDA5, CD40, lectin, dectin 1, CD206, scavenger receptor A1 (SRA 1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF2, CXCL16, starb 1, starb 2, srrb 4D, SSC5D, CD, CD207, CD209, RAGE, CD14, CD64, F4/80, CCR2, CX3CR1, CSF1R, tie2, huCRIg (L), CD64, CD32a, CD16a, CD89, fcα receptor I, CR1, CD35, CD3 ζ, complement receptor, CR3, CR4, tim-1, tim-4, and CD169. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain.
In some embodiments, the intracellular signaling domain is derived from a receptor comprising an ITAM domain. In some embodiments, the recombinant intracellular signaling domain comprises a first portion derived from a phagocytic receptor and a second portion derived from a non-phagocytic receptor, wherein the second portion derived from the non-phagocytic receptor comprises a phosphorylation site. In some embodiments, the phosphorylation site comprises an amino acid sequence suitable for an autophosphorylation site. In some embodiments, the amino acid residue that is phosphorylated is tyrosine. In some embodiments, the phosphorylation site comprises an amino acid sequence suitable for phosphorylation by Src family kinases. In some embodiments, the phosphorylation site comprises an amino acid sequence that, when phosphorylated, is capable of binding to an SH2 domain in a kinase. In some embodiments, the receptor tyrosine kinase domain is fused at the cytoplasmic end of the chimeric receptor, except for the first cytoplasmic portion.
In some embodiments, the phosphorylation site is a tyrosine phosphorylation site.
In some embodiments, the second intracellular domain is an immune receptor tyrosine activation motif (ITAM). Exemplary ITAM motifs are present in mammalian α and β immunoglobulins, TCR gamma receptor, fcγ receptor subunits, CD3 chain receptors and NFAT activating molecules.
In some embodiments, the chimeric receptor intracellular domain comprises one ITAM motif. In some embodiments, the chimeric receptor intracellular domain comprises more than one ITAM motif. In some embodiments, the chimeric receptor intracellular domain comprises two or more ITAM motifs. In some embodiments, the chimeric receptor intracellular domain comprises three or more ITAM motifs. In some embodiments, the chimeric receptor intracellular domain comprises four or more ITAM motifs. In some embodiments, the chimeric receptor intracellular domain comprises five or more ITAM motifs. In some embodiments, the chimeric receptor intracellular domain comprises six or more ITAM motifs. In some embodiments, the chimeric receptor intracellular domain comprises seven or more ITAM motifs. In some embodiments, the chimeric receptor intracellular domain comprises eight or more ITAM motifs. In some embodiments, the chimeric receptor intracellular domain comprises nine or more ITAM motifs. In some embodiments, the chimeric receptor intracellular domain comprises ten or more ITAM motifs.
Provided herein is a composition comprising a recombinant nucleic acid encoding a CFP, such as a phagocytosis or binding receptor (PR) fusion protein (PFP), comprising: a PR subunit comprising: a transmembrane domain and an intracellular domain comprising an intracellular signaling domain; and an extracellular domain comprising an antigen binding domain specific for a target cell antigen; wherein the transmembrane domain and extracellular domain are operably linked; and wherein the intracellular signaling domain is derived from a phagocytic receptor other than a phagocytic receptor selected from the group consisting of Megf10, merTk, fcrα, or Bai 1.
In some embodiments, upon binding of a CFP to an antigen of a target cell, the killing activity of the cell expressing the CFP is increased by at least greater than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950% or 1000% compared to a cell not expressing the CFP. In some embodiments, when CFP is expressed in a cell, CFP is functionally incorporated into the cell membrane of the cell. In some embodiments, upon binding of CFP to an antigen of a target cell, the killing activity of the cell expressing CFP is increased by at least 1.1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 75-fold, or 100-fold as compared to the cell not expressing CFP.
In some embodiments, the intracellular signaling domain is derived from a receptor, e.g., a phagocytic receptor, selected from the group consisting of TNFR1, MDA5, CD40, lectin, dectin1, CD206, scavenger receptor A1 (SRA 1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF2, CXCL16, starb 1, starb 2, srrb 4D, SSC5D, CD, CD207, CD209, RAGE, CD14, CD64, F4/80, CCR2, CX3CR1, CSF1R, tie2, huCRIg (L), CD64, CD32a, CD16a, CD89, fcα receptor I, CR1, CD35, CD3 zeta, CR3, CR4, tim-1, tim-4, and CD169. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain.
Provided herein is a composition comprising a recombinant nucleic acid encoding a CFP, such as a phagocytosis or binding receptor (PR) fusion protein (PFP), comprising: a PR subunit comprising: a transmembrane domain and an intracellular domain comprising an intracellular signaling domain; and an extracellular domain comprising an antigen binding domain specific for a target cell antigen; wherein the transmembrane domain and extracellular domain are operably linked; and wherein the intracellular signaling domain is derived from a receptor, such as a phagocytic receptor, selected from the group consisting of TNFR1, MDA5, CD40, lectin, dectin1, CD206, scavenger receptor A1 (SRA 1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD, CD207, CD209, RAGE, CD14, CD64, F4/80, CCR2, CX3CR1, CSF1R, tie, huCRG (L), CD64, CD32a, CD16a, CD89, fc alpha receptor I, CR1, CD35, CD3 zeta, CR3, CR4, tim-1, tim-4, and CD169.
In some embodiments, upon binding of a CFP to an antigen of a target cell, the killing activity of the cell expressing the CFP is increased by at least greater than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950% or 1000% compared to a cell not expressing the CFP. In some embodiments, the intracellular signaling domain is derived from a phagocytic receptor other than a phagocytic receptor selected from the group consisting of Megf10, merTk, fcR-a, or Bai 1. 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, e.g., a PI3K recruitment domain derived from CD 19. In some embodiments, the intracellular signaling domain comprises a pro-inflammatory signaling domain that is not a PI3K recruitment domain.
In some embodiments, the cells expressing CFP exhibit increased phagocytosis of target cells expressing the antigen as compared to cells not expressing CFP. In some embodiments, the cells expressing CFP exhibit at least a 1.1-fold increase in phagocytosis of target cells expressing the antigen as compared to cells not expressing CFP. In some embodiments, the cell expressing the CFP exhibits 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 the target cell expressing the antigen as compared to a cell not expressing the CFP. In some embodiments, the cell expressing CFP exhibits increased cytokine production as compared to a cell not expressing 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 as compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased cross-presentation as compared to cells not expressing CFP. In some embodiments, the CFP expressing cells exhibit increased expression of MHC class II proteins as compared to cells that do not express CFP. In some embodiments, the cell expressing CFP exhibits an increase in CD80 expression as compared to a cell not expressing CFP. In some embodiments, the cell expressing CFP exhibits an increase in CD86 expression as compared to a cell not expressing CFP. In some embodiments, the cell expressing CFP exhibits increased MHC class I protein expression compared to a cell not expressing CFP. In some embodiments, the CFP expressing cells exhibit increased TRAIL/TNF family death receptor expression compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit increased B7-H2 expression as compared to cells not expressing CFP. In some embodiments, the CFP expressing cells exhibit increased LIGHT expression as compared to cells that do not express CFP. In some embodiments, the cell expressing CFP exhibits an increase in HVEM expression as compared to a cell not expressing CFP. In some embodiments, the cell expressing CFP exhibits increased expression of CD40 as compared to a cell not expressing CFP. In some embodiments, the CFP expressing cells exhibit increased TL1A expression compared to cells that do not express CFP. In some embodiments, the cell expressing CFP exhibits an increase in 41BBL expression as compared to a cell not expressing CFP. In some embodiments, cells expressing CFP exhibit increased OX40L expression compared to cells not expressing CFP. In some embodiments, the CFP expressing cell exhibits an increase in GITRL death receptor expression as compared to a cell that does not express the CFP. In some embodiments, the cell expressing CFP exhibits an increase in CD30L expression as compared to a cell not expressing CFP. In some embodiments, the CFP expressing cells exhibit increased TIM4 expression as compared to cells that do not express CFP. In some embodiments, the CFP expressing cells exhibit increased expression of TIM1 ligand as compared to cells that do not express CFP. In some embodiments, the CFP expressing cells exhibit increased SLAM expression compared to cells that do not express CFP. In some embodiments, the CFP expressing cells exhibit increased CD48 expression as compared to cells that do not express CFP. In some embodiments, the cell expressing CFP exhibits an increase in CD58 expression as compared to a cell not expressing CFP. In some embodiments, the cell expressing CFP exhibits an increase in CD155 expression as compared to a cell not expressing CFP. In some embodiments, the cell expressing CFP exhibits an increase in CD112 expression as compared to a cell not expressing CFP. In some embodiments, the CFP expressing cells exhibit an increase in PDL1 expression compared to cells that do not express CFP. In some embodiments, cells expressing CFP exhibit an increase in B7-DC expression as compared to cells not expressing CFP. In some embodiments, the cell expressing CFP exhibits an increase in respiratory burst as compared to a cell not expressing CFP. In some embodiments, the CFP expressing cells exhibit increased ROS production compared to cells that do not express CFP. In some embodiments, the cell expressing the CFP exhibits an increase in iNOS production as compared to a cell not expressing the CFP. In some embodiments, the cell expressing the CFP exhibits an increase in iNOS production as compared to a cell not expressing the CFP. In some embodiments, the cell expressing the CFP exhibits an increase in extracellular vesicle production as compared to a cell not expressing the CFP. In some embodiments, the cell expressing the CFP exhibits an increase in the cytoplasmic effect with the target cell expressing the antigen as compared to a cell not expressing the CFP. In some embodiments, the CFP expressing cells exhibit increased resistance to CD 47-mediated inhibition of phagocytosis as compared to cells that do not express CFP. In some embodiments, the CFP expressing cells exhibit increased resistance to LILRB 1-mediated inhibition of phagocytosis as compared to cells that do not express CFP. In some embodiments, the CFP expressing cells exhibit increased production of phosphatidylinositol 3,4, 5-triphosphate.
Also provided herein is a pharmaceutical composition comprising a composition described herein, e.g., a recombinant nucleic acid described herein, a vector described herein, a polypeptide described herein, or a cell described herein; and a pharmaceutically acceptable excipient.
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 breakdown, an agent that promotes PI3K recruitment to PFP, an agent that promotes PI3K activity, an agent that promotes phosphatidylinositol 3,4, 5-triphosphate 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, lipid, or nanoparticle.
In some embodiments, the recombinant nucleic acid is mRNA or circRNA.
In some embodiments, provided herein is a therapeutic composition comprising a cell comprising a recombinant nucleic acid as described anywhere within the specification. In some embodiments, the therapeutic composition comprises a recombinant nucleic acid that expresses a chimeric protein as described anywhere herein. In some embodiments, the myeloid cell is a CD14+ cell, a CD14+/CD 16-cell, a CD14+/CD16+ cell, a CD14-/CD 16-cell, a dendritic cell, an M0 macrophage, an M2 macrophage, an M1 macrophage, or a chimeric myeloid cell/macrophage/dendritic cell.
Methods for generating novel chimeric receptor fusion protein (CFP) constructs
In one aspect, provided herein is a method for generating a novel chimeric receptor protein comprising, for example, identifying novel domains that can be used to enhance the function of a myeloid cell, such that when the fusion receptor is expressed in the myeloid cell, it functions as an effector myeloid cell of the specifications described herein. The generation of fusion proteins as described herein can be performed using well known molecular cloning techniques, and the sequence can be verified after the recombinant nucleic acid is generated.
Preparation of recombinant nucleic acid encoding CAR:recombinant nucleic acid constructs encoding Chimeric Antigen Receptors (CARs) are prepared and incorporated into plasmid vectors for amplification and/or testing expression in eukaryotic cells. Recombinant CARs were constructed using molecular cloning techniques known in the art. The recombinant CAR protein comprises an intracellular domain, a transmembrane domain, and an extracellular domain. Each domain or sub-portion of a domain may be encoded by a nucleic acid sequence generated by PCR from a heterologous source sequence and spliced together by cloning separately into a vector or ligated into a longer nucleic acid and then inserted into a suitable plasmid or multiple cloning site of a vector with suitable promoter and 3' -regulatory elements for amplification. Briefly, by incorporating a coding for one or more signaling domains (e.g For example, PI3 kinase recruitment domain), a nucleic acid sequence encoding a CD8 hinge and transmembrane domain, a nucleic acid sequence encoding an extracellular domain having a sequence encoding a target antigen binding scFv at the extracellular end. Some constructs include a FLAG peptide sequence at the extracellular end, which is designed so as not to hinder the binding of scFv to its target antigen. Together, these components are linked into a sequence encoding a fully functional transmembrane CAR. Nucleic acid subunits encoding individual domains of recombinant proteins are designed to include short flexible linker sequences interposed between the two domains. The construct is ligated in a plasmid with a promoter and 3' stabilizing building blocks. In one variant, the construct is placed within an Alu retrotransposon element encoding ORF2p and has the corresponding 5 '-and 3' -UTR sequences, i.e. the CMV promoter. Plasmids were amplified in E.coli and verified by sequencing or stored at (-) 80 ℃.
mRNA preparation: mRNA can be prepared by in vitro transcription using the digested plasmid as a template and purified to remove contaminating DNA and polyadenylation. The RNA product was purified, resuspended to 1mg/ml with RNase-free water and stored in a freezer tube.
Useful CFP ECD, TM, ICD and antigen binding domains for generating novel CFPs can be identified using the methods described herein. Briefly, a large number of potential candidate proteins can be screened to enhance phagocytic properties and their corresponding phagocytosis-related intracellular signaling. The useful domains can then be used to generate novel CFPs. Screening can be divided into two parts: A. screening of Phagocytic Receptor (PR) domains; B. screening of extracellular antigen binding domains.
Screening of PR domains:
in one embodiment, about 5,800 plasma membrane proteins are screened for phagocytic potential according to the general methods described herein. J774 macrophages can be transiently transfected with a library of 5800 plasma proteins. High throughput multiplex assays (ranging from 6-well plate assays setup to up to 384-well plate assays, robotically operated) can be established to assess various potential functions of plasma membranes. Exemplary assays include, but are not limited to, phagocytosis assays, cytokine production assays, inflammatory body activation assays, and iNOS activation assays. An exemplary simplified method may be described in the following paragraphs. Variations of each method may also be used and will be understood by the skilled person. Variations of each method may also be used and will be understood by the skilled person. Exemplary intracellular signaling domains tested include, but are not limited to, CD 40-FcRgamma; fcR gamma-CD 40; NLRP3; fcR gamma-SH 2-caspase zymogen; fcRgamma-Myd 88; fcR gamma-IFN receptor; fcR-TNFR1; fcRgamma-TNFR 2; fcR-AIM2; fcrγ -tifn; fcR gamma-caspase zymogen; TRIFC; RIG1; MDA5; TBK; CD64; CD16a; CD89; fcR epsilon; sirpβ; (two consecutive intracellular domains may be expressed as a hyphenated term, e.g., fcRgamma-Myd 88 refers to an intracellular domain comprising FcRgamma intracellular signaling domain as signaling domain 1; and Myd88 intracellular signaling domain as signaling domain 2). The extracellular linker domains selected include, but are not limited to, CD64, CD16A, CD89, sirpa, fcrepsilon, CD8 hinge. The transmembrane domains tested include, but are not limited to, CD8, CD64, CD16A, CD89, fcrepsilon, sirpa, TNFR1, and CD40. MDA5 domains were also screened.
Phagocytosis assay:
antigen-linked silica or polystyrene beads ranging in diameter from 1nm, 5nm or 10nm were used to screen macrophages. Inert beads may be coated in the supported lipid bilayer and the antigen may be linked to the lipid bilayer. J774 macrophage cell lines can be prepared, each expressing cloned recombinant plasma membrane proteins. The recombinant plasma membrane proteins may also express fluorescent tags. Cell lines can be maintained and propagated in complete RPMI medium containing heat-inactivated serum and antibiotics (penicillin/streptomycin). On the day of the assay, cells may be plated in 6 well plates at a density of 1X 10≡6 cells/ml/well or in 12 or 24 well plates in relative proportion and incubated for 2-6 hours. Cells are then washed once in phosphate buffered saline and beads can be added to serum-depleted or complement-depleted nutrient media. The cells were observed by light microscopy 30 minutes and 2 hours after the addition of the beads. Immunofluorescence reactions can be performed using labeled antibodies and fluorescence confocal microscopy can be used to detect interactions and co-localization of cellular proteins upon engulfment. Confidence levels can be determined by the Kruskal-Wallis test with Dunn multiple comparison correction.
In some examples, dye-loaded tumor cells can be fed to a macrophage cell line and phagocytosis assessed by microscopy.
Cytokine production:
macrophage cell lines can be cultured as described above. In one assay, each J774 cell line expressing plasma membrane proteins was plated in a multi-well plate and challenged with antigen-linked beads and cytokine production was determined by collecting supernatants at 4 and 24 hours. Cytokines in the supernatant can be determined by ELISA. In another fraction, cells can be collected 4 and 24 hours after incubation with beads and flow cytometry performed to detect cytokines. In each case, a plurality of cytokines may be assayed in a multiplex format, which may 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. Macrophage inflammatory cytokine array kit (R & D Systems) was used.
Intracellular signaling pathways activated by inflammatory genes and cytokines can be identified by western blot analysis of the MAP kinase, JNK, akt signaling pathways, including STAT-1 phosphorylation and phosphorylation of activated interferon activation pathways.
Functional assay
Inflammatory body activation assay:
activation of NLRP3 inflammasome was determined by ELISA to detect increased IL-1 production and by Western blotting to detect caspase-1 activation and cleavage of pro-caspase to shorter caspase. caspase-Glo (Promega Corporation) is used to read caspase 1 activation more quickly in microplate multiplex settings.
iNOS activation assay:
activation of oxidative burst potential can be measured by iNOS activation and NO production using a fluorometric NOS activity assay kit (AbCAM).
Cancer cell killing assay:
raji B cells can be used as cancer antigen presenting cells. Raji cells can be incubated with whole cell crude extracts of cancer cells and with J774 macrophage cell line. Macrophages can destroy cells 1 hour after infection, which can be detected by microscopy or by cell death assays.
Screening for high affinity antigen binding domains:
the antibody light and heavy chain variable domains of the cancer ligand can be screened to generate the extracellular binding domains of CFP. Human full length antibodies or scFv libraries can be screened. Potential ligands can also be used to immunize llamas to develop novel immunoglobulin binding domains and to produce single domain antibodies in llamas.
The specific useful domains identified from the screen can then be reverse transcribed and cloned into lentiviral expression vectors to generate a CFP construct. Recombinant nucleic acids encoding CFP can be generated using one or more domains from the extracellular, TM, and cytoplasmic regions of the highly phagocytic receptors generated from the screen. Briefly, plasma membrane receptors that exhibit high activators of pro-inflammatory cytokine production and inflammatory body activation can be identified. Bioinformatic studies can be performed to identify functional domains, including extracellular activation domains, transmembrane domains, and intracellular signaling domains, e.g., specific kinase activation sites, SH2 recruitment sites. These screened functional domains can then be cloned in modular structures to generate novel CFPs. These may be candidate CFPs, each of which are tested for enhanced phagocytosis, cytokine and chemokine production, and/or tumor cell killing in vitro and/or in vivo. A particle-based phagocytosis assay was used to examine the change in phagocytosis. Briefly, streptavidin-coupled fluorescent polystyrene microparticles (6 μm diameter) can be conjugated with biotinylated recombinant expressed and purified cancer ligands. Myeloid cells expressing the novel CFP can be incubated with ligand coated microparticles for 1-4h and analyzed and quantified for phagocytosis using flow cytometry. A CFP-engineered plasmid or lentiviral construct can then be prepared and tested for cancer cell lysis in macrophages.
Specific design of cell-targeted CFP
One aspect of developing recombinant nucleic acids encoding chimeric antigen receptors for in vivo delivery is to allow CFP to be specifically taken up, expressed and functional by macrophages in monocytes and avoid expression in non-myeloid cells that do not have phagocytic capacity. Thus, in one aspect, CFP is designed such that expression of CFP is dependent on bone marrow-specific proteins. For example, the concept of expressing CFP using bone marrow specific promoters is contemplated herein. The bone marrow-specific promoter may be encoded in a vector or in a recombinant nucleic acid, located at a position upstream of and operably linked to the CFP-encoding sequence. Certain myeloid cell-specific promoters are disclosed in the art. For example, the CD68S promoter is a bone marrow specific promoter (Scharenberg et al, nat Commun 11,3327 (2020)).
In one aspect, the recombinant nucleic acid is mRNA. Methods for bone marrow specific expression are contemplated herein, which can be used whether the nucleic acid is delivered as mRNA or DNA. Thus, in one aspect, CFP is designed such that its own expression and function is achieved by the presence of bone marrow endogenous proteins. In one aspect, bone marrow-specific expression of CFP is achieved by design, wherein CFP is expressed only in the membrane when dimerized (or multimerized) with a monocyte-specific protein. In some embodiments, bone marrow-specific function of CFP is achieved by design, where CFP is expressed only on the membrane when dimerized (or multimerized) with monocyte-specific proteins. FcR alpha receptor oligomerizes with FcR gamma receptor for membrane expression and/or function on myeloid cells. Several Fc receptors (FcR) are expressed endogenously in monocytes and myeloid cells. After they are cross-linked by immune complexes, fcrs play a variety of roles, such as modulating immune responses or modulating phagocytosis by released cytokines. FcRα receptors and FcRγ receptors are oligomerized via transmembrane domains.
In some embodiments, the CFP comprises a transmembrane domain and/or an intracellular domain from an Fc receptor that oligomerizes with an endogenous FcR.
Fcrs and their cellular expression patterns are shown in table 3 below:
table 3.
Exemplary FcR domain sequences for use in constructing the chimeric fusion protein receptors disclosed herein are provided in table 4.
TABLE 4 Table 4
In some embodiments, CFP is designed to include a transmembrane domain derived from a transmembrane domain of a protein selected from fcγr1 (CD 64). In some embodiments, the CFP is designed to comprise a transmembrane domain derived from a transmembrane domain of a protein selected from fcyriiia (CD 16). In some embodiments, the CFP is designed to comprise a transmembrane domain derived from a transmembrane domain of a protein selected from fcyriia (CD 32 a). In some embodiments, the CFP is designed to include a transmembrane domain derived from a transmembrane domain of a protein selected from fcαr1 (CD 89). Any of the domains in the CFPs described herein may be from a suitable mammalian source, depending on or irrespective of the source of any other contiguous domains. Non-human mammalian transmembrane domains are contemplated as being within the scope of the invention.
In some embodiments, a CFP may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues in the extracellular domain from a protein from which TMD is derived. In some embodiments, a CFP may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more amino acid residues from a protein from which TMD is derived in the intracellular domain.
In some embodiments, the CFP comprises an intracellular domain from a TMD-derived protein fcγr1 (CD 64), fcrγiiia (CD 16), or fcγriia (CD 32 a). In some embodiments, the TMD has a sequence identical to the TMD sequence of the proteins fcγr1 (CD 64), fcγriiia (CD 16) or fcγriia (CD 32 a).
Myeloid cell-specific CFP constructs are designed to comprise a transmembrane domain that dimerizes with endogenous Fc-gamma receptors specifically expressed in myeloid cells, thereby becoming functionally active due to dimerization.
In some embodiments, the myeloid cell-specific CFP construct comprises an fcαr1 (CD 89) transmembrane domain. In some embodiments, the myeloid cell-specific CFP construct comprises a human fcαr1 (CD 89) transmembrane domain. In some embodiments, the myeloid cell-specific CFP construct comprises a mouse fcαr1 (CD 89) transmembrane domain. In some embodiments, the myeloid cell-specific CFP construct comprises a rodent fcαr1 (CD 89) transmembrane domain. In some embodiments, the myeloid cell-specific CFP construct comprises a equine fcαr1 (CD 89) transmembrane domain. In some embodiments, the myeloid cell-specific CFP construct comprises a porcine fcαr1 (CD 89) transmembrane domain. In some embodiments, the myeloid cell-specific CFP construct comprises a sheep fcαr1 (CD 89) transmembrane domain. In some embodiments, the CD89TMD has at least 80% homology with human or mouse CD89 TMD. In some embodiments, the CD89TMD has at least 85% homology with human or mouse CD89 TMD. In some embodiments, the CD89TMD has at least 90% homology with human or mouse CD89 TMD. In some embodiments, the CD89TMD has at least 95% homology with human or mouse CD89 TMD.
In some embodiments, the myeloid cell-specific CFP construct comprises an fcyriii (CD 16) transmembrane domain. In some embodiments, the myeloid cell-specific CFP construct comprises a human fcyriii (CD 16) transmembrane domain. In some embodiments, the myeloid cell-specific CFP construct comprises a mouse fcyriii (CD 16) transmembrane domain. In some embodiments, the myeloid cell-specific CFP construct comprises a rodent fcyriii (CD 16) transmembrane domain. In some embodiments, the myeloid cell-specific CFP construct comprises a equine fcyriii (CD 16) transmembrane domain. In some embodiments, the myeloid cell-specific CFP construct comprises a porcine fcyriii (CD 16) transmembrane domain. In some embodiments, the myeloid cell-specific CFP construct comprises a sheep fcyriii (CD 16) transmembrane domain. In some embodiments, the CD16TMD has at least 80% homology with human or mouse CD16 TMD. In some embodiments, the CD16TMD has at least 85% homology with human or mouse CD16 TMD. In some embodiments, the CD16TMD has at least 90% homology with human or mouse CD16 TMD. In some embodiments, the CD16TMD has at least 95% homology with human or mouse CD16 TMD. In one embodiment, fcyriii is fcyriiia (CD 16 a).
It is contemplated herein that any myeloid-like cell-specific CFP can be designed or generated using TMD from CD89 molecule, CD16 molecule, CD64 molecule, or CD32a molecule, and that a myeloid-like cell-specific CFP having the desired antigen binding domain can be generated based on the compositions and methods described herein. In some embodiments, CFP exhibits myeloid cell-specific expression. In some embodiments, the myeloid cell-specific expression is Fc-gamma dependent expression, i.e., if a cell comprising a nucleic acid comprising a CFP construct endogenously expresses Fc-gamma, then CFP will be co-expressed in the cell. In some embodiments, CFP is only transiently expressed, and may not be detectable, even if it is expressed in cells other than myeloid cells. In some embodiments, only myeloid cells express functional CFPs in an Fc-gamma dependent manner, i.e., CFPs do not function in cells that do not endogenously express Fc-gamma. In some embodiments, the myeloid-like cell-specific CFP comprises an antigen binding domain that binds to a cancer cell-specific target antigen and an intracellular region comprising 1, 2, 3, or more domains, e.g., an intracellular cell signaling domain, e.g., a PI3 kinase recruitment domain. For example, a myeloid cell-specific CFP comprises an antigen binding domain that binds to a CD5 target antigen. The corresponding CFP is designed herein to include: an extracellular domain comprising an anti-CD 5 antibody or a portion thereof (e.g., an anti-CD 5 ScFv), a hinge domain (e.g., a CD8 hinge domain), a transmembrane domain capable of dimerizing with Fc-gamma receptors endogenously expressed by myeloid cells, and an intracellular region comprising 1, 2, 3 or more domains, e.g., an intracellular cell signaling domain comprising, e.g., a CD40 intracellular signaling domain, and/or a PI3 kinase recruitment domain.
In some embodiments, provided herein is a TROP2 binding CFP (TROP 2 conjugate) comprising an extracellular TROP2 binding domain, optionally a short hinge region (e.g., comprising a CD8 hinge domain), a CD89 transmembrane domain, optionally a CD89 intracellular region, fused to one or more intracellular signaling domains, such as a CD40 intracellular domain and a PI3 kinase recruitment domain. In some embodiments, provided herein is a TROP2 binding CFP (TROP 2 conjugate) comprising an extracellular TROP2 binding domain, optionally a short hinge region (e.g., comprising a CD8 hinge domain), a CD16 transmembrane domain, optionally a CD16 intracellular region, fused to one or more intracellular signaling domains, such as a CD40 intracellular domain and a PI3 kinase recruitment domain. The TROP2 CFP may comprise one or more human or humanized domains. Provided herein is a polynucleotide, e.g., mRNA or DNA, comprising a sequence encoding a TROP2 CFP construct comprising an anti-TROP 2 ScFv (TROP 2 antigen-binding) fused to a CD8 hinge domain operably linked to a CD16 or CD89 transmembrane domain and an intracellular domain. In some embodiments, a TROP2 CFP described herein exhibits myeloid cell-specific expression. In some embodiments, a TROP2 CFP described herein exhibits Fc-gamma dependent expression. In some embodiments, a TROP2 CFP described herein exhibits undetectable expression in a T cell, B cell, epithelial cell, muscle cell, neuronal cell, or any non-myeloid cell when the polynucleotide is administered in vivo. In one embodiment, when the polynucleotide is administered in vivo, a TROP2 conjugate encoded by a polynucleotide described herein having a CD16, CD89 or CD64 transmembrane domain as described herein is expressed predominantly in cd14+ cells. In one embodiment, a TROP2 conjugate having a CD16, CD89 or CD64 transmembrane domain encoded by a polynucleotide described herein is expressed only in cd14+ cells when the polynucleotide is administered in vivo.
In some embodiments, provided herein is a GPC3 binding CFP (GPC 3 conjugate) comprising an extracellular GPC3 binding domain, optionally a short hinge region (e.g., comprising a CD8 hinge domain), a CD89 transmembrane domain, optionally a CD89 intracellular region, fused to one or more intracellular signaling domains, such as a CD40 intracellular domain and PI3 kinase recruitment domain. In some embodiments, provided herein is a GPC3 binding CFP (GPC 3 conjugate) comprising an extracellular GPC3 binding domain, optionally a short hinge region (e.g., comprising a CD8 hinge domain), a CD16 transmembrane domain, optionally a CD16 intracellular region, fused to one or more intracellular signaling domains, such as a CD40 intracellular domain and PI3 kinase recruitment domain. The GPC3CFP may comprise one or more human or humanized domains. In some embodiments, the GPC3-CFP comprises an intracellular domain further comprising an interferon-inducing.
Provided herein is a polynucleotide, e.g., mRNA or DNA, comprising a sequence encoding a GPC3CFP construct comprising an anti-GPC 3 ScFv (GPC 3 antigen-conjugate) fused to a CD8 hinge domain operably linked to a CD16 or CD89 transmembrane domain and an intracellular domain. In some embodiments, a GPC3CFP described herein exhibits myeloid cell-specific expression. In some embodiments, a GPC3CFP described herein exhibits Fc-gamma dependent expression. In some embodiments, the GPC3 CFPs described herein exhibit undetectable expression in T cells, B cells, epithelial cells, muscle cells, neuronal cells, or any non-myeloid cells when the polynucleotide is administered in vivo. In one embodiment, when the polynucleotide is administered in vivo, GPC3 conjugates encoded by the polynucleotides described herein having a CD16, CD89, or CD64 transmembrane domain as described herein are expressed predominantly in cd14+ cells. In one embodiment, GPC3 conjugates with CD16, CD89, or CD64 transmembrane domains encoded by the polynucleotides described herein are expressed only in cd14+ cells when the polynucleotides are administered in vivo. In some embodiments, the GPC3CFP further comprises an interferon-inducing intracellular domain.
Provided herein is a pharmaceutical composition comprising, in some embodiments, GPC3 conjugates of the specifications described herein for use in treating cancer in a human subject. In some embodiments, the GPC3 conjugate is used to treat hepatocellular carcinoma (HCC).
In some embodiments, provided herein is a GP75 binding CFP (GP 75 conjugate) comprising an extracellular GP75 binding domain, optionally a short hinge region (e.g., comprising a CD8 hinge domain), a CD89 transmembrane domain, optionally a CD89 intracellular region, fused to one or more intracellular signaling domains, such as a CD40 intracellular domain and a PI3 kinase recruitment domain. In some embodiments, provided herein is a GP75 binding CFP (GP 75 conjugate) comprising an extracellular GP75 binding domain, optionally a short hinge region (e.g., comprising a CD8 hinge domain), a CD16 transmembrane domain, optionally a CD16 intracellular region, fused to one or more intracellular signaling domains, such as a CD40 intracellular domain and a PI3 kinase recruitment domain. The GP75 CFP may comprise one or more human or humanized domains. Provided herein is a polynucleotide, e.g., mRNA or DNA, comprising a sequence encoding a GP75 CFP construct comprising an anti-GP 75 ScFv (GP 75 antigen conjugate) fused to a CD8 hinge domain operably linked to a CD16 or CD89 transmembrane domain and an intracellular domain. In some embodiments, the GP75 CFPs described herein exhibit myeloid cell-specific expression. In some embodiments, the GP75 CFP described herein exhibits Fc-gamma dependent expression. In some embodiments, the GP75 CFPs described herein exhibit undetectable expression in T cells, B cells, epithelial cells, muscle cells, neuronal cells, or any non-myeloid cells when the polynucleotide is administered in vivo. In one embodiment, the GP75 conjugate encoded by a polynucleotide described herein having a CD16, CD89 or CD64 transmembrane domain as described herein is expressed predominantly in cd14+ cells when the polynucleotide is administered in vivo. In one embodiment, GP75 conjugate with a CD16, CD89 or CD64 transmembrane domain encoded by a polynucleotide described herein is expressed only in cd14+ cells when the polynucleotide is administered in vivo.
In some embodiments, any of the CFP constructs described in this section comprises at least a CD40 intracellular signaling domain. In some embodiments, any of the CFP constructs described in this section comprises at least PI3 kinase recruitment and signaling domains. In some embodiments, any of the CFP constructs described in this section comprises at least a CD40 intracellular signaling domain and a PI3 kinase recruitment domain. In some embodiments, the myeloid-like cell-specific CFP construct described in this section comprises at least three intracellular domains comprising, for example, a CD40 intracellular signaling domain and a PI3 kinase recruitment domain and a third intracellular signaling domain. In some embodiments, any of the CFP constructs described in this section comprises at least one intracellular domain that induces NF- κb activation upon receptor activation. In some embodiments, any of the CFP constructs described in this section comprises at least one intracellular domain that stimulates interferon production upon receptor activation. In some embodiments, any of the CFP constructs described in this section comprises at least one intracellular domain that induces NF- κb activation and comprises at least one intracellular domain that stimulates interferon production upon receptor activation.
Method for producing myeloid cells from a subject
Isolation of myeloid cells from PBMCs:
peripheral blood mononuclear cells can be isolated from normal donor white membranes by density centrifugation using Histopaque 1077 (Sigma). After washing, cd14+ monocytes were isolated from the monocyte fraction using a clinic GMP grade CD14 microbead and LS separation magnetic separation column (Miltenyi Biotec). Briefly, cells can be buffered with PEA buffer (phosphate buffered saline [ PBS]2.5mmol/L ethylenediamine tetraacetic acid [ EDTA ] was added]And human serum albumin [0.5% final volume Alburex 20%, octopharma ]]) Is resuspended to the appropriate concentration, incubated with CliniMACS CD14 beads according to the manufacturer's instructions, then washed and magnetizedIs a LS column of (C). After washing, the purified monocytes can be eluted from the degaussing column, washed and resuspended in the relevant medium for cultivation. Cd14+ cells were isolated from leukapheresis: PBMCs may be collected by leukapheresis from liver cirrhosis donors given informed consent for participation in the study. White blood cell apheresis of peripheral blood mononuclear cells (MNCs) was performed by sterile collection using an Optia apheresis system. Using standard collection procedures for MNC, 2.5 blood volumes were processed. Isolation of CD14 cells was performed using GMP compliant functional closure system (clinimacs Prodigy system, miltenyi Biotec). Briefly, leukocyte apheresis products were counted and aliquots were pre-isolated flow cytometry. The percentage of monocytes (CD14+) and the absolute cell number can be determined and, if desired, the volume adjusted to meet the desired selection criteria (.ltoreq.20X10) 9 Total white blood cells;<400×10 6 white blood cells/mL; is less than or equal to 3.5 multiplied by 10 9 Cd14 cells, 50-300mL in volume). CD14 cell isolation was performed using CliniMACS Prodigy (medical device class III), TS510 tube set and LP-14 program with CliniMACS CD14 microbeads. At the end of the procedure, selected cd14+ positive monocytes can be washed in PBS/EDTA buffer (clinic macs buffer, miltenyi) containing pharmaceutical grade 0.5% human albumin (Alburex) and then resuspended in TexMACS (or control) medium for culture.
Cell count and purity:
cell counts of total MNC and isolated monocyte fractions can be performed using a Sysmex XP-300 Autoanalyzer (Sysmex). The assessment of macrophage numbers was performed by flow cytometry using a TruCount tube (Becton Dickinson) to determine absolute cell numbers, as Sysmex consistently underestimated the number of monocytes. Isolated purity was assessed using flow cytometry (FACSCanto II, BD Biosciences) with a panel of antibodies to human leukocytes (CD 45-VioBlue, CD15-FITC, CD14-PE, CD 16-APC) and product quality was assessed by determining the amount of neutrophil contamination (CD 45int, CD15 pos). Cell culture-development of healthy donor sample cultures
Optimal medium for macrophage differentiation was studied and three candidates could be tested using cell products. In addition, the effect of monocyte cryopreservation on derived myeloid cells and macrophages for therapeutic use was examined. Functional assays can be performed to quantify the phagocytic capacity of myeloid cells and macrophages and their ability to further polarize, as well as phagocytic potential as described elsewhere in this invention.
Whole process validation of object samples
Monocytes cultured from the white blood cell apheresis of Prodigy isolates can be cultured in a culture bag containing GMP grade TexMACS (Miltenyi) and 100ng/mL M-CSF (MACS GMP differentiation bag, miltenyi) at 2X 10 6 Individual monocytes/cm 2 and/mL culture. Monocytes may be treated with 100ng/mL of GMP-compliant recombinant human M-CSF (R&DSystems) culture. The cells can be cultured at 37deg.C and 5% CO 2 Is cultured in a humid atmosphere for 7 days. Two 50% volume medium supplements were made during the culture period (day 2 and day 4), 50% medium removed, and then fed with fresh medium supplemented with 200ng/mL M-CSF (to restore the final concentration of 100 ng/mL).
Cell harvesting:
for normal donor-derived macrophages, cells can be removed from the wells on day 7 using cell dissociation buffer (Gibco, thermo Fisher) and paste. Cells can be resuspended in PEA buffer and counted, and then approximately 1×10 per test can be performed 6 Individual cells were stained for flow cytometry. On day 7, leukocyte apheresis-derived macrophages can be removed from the culture bag using PBS/EDTA buffer (CliniMACS buffer, miltenyi) containing pharmaceutical grade 0.5% human serum albumin (HAS; alburex). The harvested cells can be resuspended in excipients consisting of two permissive products: saline (Baxter) for 0.9% infusion containing 0.5% human albumin (Alburex).
Flow cytometry characterization:
monocyte and macrophage surface marker expression can be analyzed using FACSCanto II (BD Biosciences) or macquant 10 (Miltenyi) flow cytometry. Typically, approximately 20,000 events can be obtained for each sample. Cell surface expression of leukocyte markers was performed in freshly isolated and day 7 mature cells by incubating the cells with specific antibodies (final dilution 1:100). Cells were incubated with FcR blocker (Miltenyi) for 5min, then with the antibody mixture for 20min at 4 ℃. Cells can be washed in PEA and dead cell exclusion dye DRAQ7 (BioLegend) added at 1:100. The following series of surface markers of cells can be stained: CD45-VioBlue, CD14-PE or CD14-PerCP-Vio700, CD163-FITC, CD169-PE and CD16-APC (all from Miltenyi), CCR2-BV421, CD206-FITC, CXCR4-PE and CD115-APC (all from BioLegend), and 25F9-APC and CD115-APC (eBioscience). Monocytes and macrophages can be gated using forward and side scatter and a DRAQ7 dead cell discriminator (BioLegend) to exclude fragments, doublets and dead cells and analyzed using FlowJo softwcan be (Tree Star). Based on the initial detailed phenotypic analysis, a panel was developed as a release standard (CD 45-VB/CD206-FITC/CD14-PE/25F9 APC/DRAQ 7) defining the development of functional macrophages from monocytes. It was determined that the Mean Fluorescence Intensity (MFI) of macrophages was five times higher than the level of monocytes at day 0 of 25F9 and CD 206. A second set was developed which evaluated other markers as part of the expanded set, consisting of CCR2-BV421/CD163-FITC/CD169-PE/CD14-PerCP-Vio700/CD16-AP C/DRAQ7, but not as part of the release criteria for cell products.
Monocytes and macrophages may be isolated from the white film layer formed in sucrose gradient centrifugation samples of the isolated peripheral blood cells. Phagocytic uptake of CD14 cells can be tested using pHRodo beads that fluoresce only when taken up into acidic endosomes. Briefly, monocytes or macrophages can be incubated with 1-2uL of pHRodo escherichia coli bioparticles (Life Technologies, thermo Fisher) for 1h, then the medium removed and the cells washed to remove non-phagocytic particles. Phagocytosis was assessed using an EVOS microscope (Thermo Fisher), images were captured, and cellular uptake of the beads was quantified using ImageJ software (NIH). The ability to polarize towards defined differentiated macrophages was examined by treating macrophages with IFNγ (50 ng/mL) or IL-4 (20 ng/mL) for 48h on day 7 to induce polarization to the M1 or M2 phenotype (or M [ IFNγ ] and M [ IL-4], respectively). After 48h, the cells can be observed by EVOS bright field microscopy, and then harvested and phenotyped as described previously. The cytokine and growth factor secretion profile of macrophages after production and in response to inflammatory stimuli is further analyzed. Macrophages can be generated from healthy donor buffy coats as previously described and stimulated either with TNFα (50 ng/mL, peprotech) and polyinosinic acid, polycytidylic acid (poly I: C, a viral homolog that binds TLR3, 1g/mL, sigma) to mimic the conditions present in inflamed liver, or lipopolysaccharide (LPS, 100ng/mL, sigma) plus IFNγ (50 IU/mL, peprotech) to produce maximum macrophage activation. Macrophages can be incubated overnight on day 7, and supernatants collected and centrifuged to remove debris before storage at-80 ℃ until tested. The secretome analysis was performed on a Magpix multiplex enzyme-linked immunoassay microplate reader (BioRad) using a 27-plex human cytokine kit and a 9-plex matrix metalloproteinase kit.
Product stability:
various excipients, including PBS/EDTA buffer, can be tested during process patency; PBS/EDTA buffer (Alburex) with 0.5% HAS, 0.9% saline alone or 0.5% HAS. 0.9% saline (Baxter) with 0.5% has excipient was found to maintain optimal cell viability and phenotype (data not shown). Post-harvest stability of liver cirrhosis donor macrophages was studied in three process optimization runs, and a more limited time point range (n=3) was assessed in a process validation run. After harvesting and re-suspension in vehicle (0.9% saline for infusion, 0.5% human serum albumin), the bags can be stored at ambient temperature (21-22 ℃) and sampled at 0, 2, 4, 6, 8, 12, 24, 30 and 48 hours post-harvest. The release standard antibody set was run on each sample and the change in viability and mean fold from day 0 was measured according to the geometric MFI of 25F9 and CD 206.
Statistical analysis:
the results can be expressed as mean ± SD. The statistical significance of the differences was assessed with a unpaired two-tailed t-test using GraphPad Prism 6, where possible. When the P value is <0.05, the result can be considered statistically significant.
Also provided herein is a cell 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 myeloid cell, a macrophage, a dendritic cell, a lymphocyte, a mast cell, a monocyte, a neutrophil, a microglial cell, or an astrocyte. In some embodiments, the cell is an autologous cell. In some embodiments, the cell is an allogeneic cell. In some embodiments, the cell is an M1 cell. In some embodiments, the cell is an M2 cell. In some embodiments, the cell is an M1 phagocyte. In some embodiments, the cell is an M2 phagocyte. In some embodiments, the cell is an M1 myeloid cell. In some embodiments, the cell is an M2 myeloid cell.
Also provided herein is a method of treating a disease in a subject in need thereof, 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 selected from ovarian cancer, suitable cancers include ovarian cancer, renal cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, lung cancer. In some embodiments, the cancer is 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. In some embodiments, the cancer is NSCLC. In some embodiments, the cancer is HCC.
In some embodiments, the method further comprises administering an additional therapeutic agent to the subject. 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 breakdown, an agent that promotes PI3K recruitment to PFP, an agent that promotes PI3K activity, an agent that promotes phosphatidylinositol 3,4, 5-triphosphate 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, administering comprises infusion or injection. In some embodiments, the administering comprises directly administering to the solid cancer. In some embodiments, administering comprises a circRNA-based delivery program, a non-particle encapsulated mRNA-based delivery program, an mRNA-based delivery program, a virus-based delivery program, a particle-based delivery program, a liposome-based delivery program, or an exosome-based delivery program. In some embodiments, a cd4+ T cell response or a cd8+ T cell response is elicited in the subject.
Also provided herein is a method of making a cell, the method comprising contacting the cell with a composition described herein, a vector described herein, or a polypeptide described herein. In some embodiments, contacting comprises transduction. In some embodiments, contacting comprises chemical transfection, electroporation, nuclear transfection, or viral infection or transduction.
Provided herein is a method for administering a therapeutic agent comprising any of the above compositions. In some embodiments, the therapeutic agent is administered via a parenteral route of administration.
In some embodiments, the therapeutic agent is administered via an intramuscular route of administration. In some embodiments, the therapeutic agent is administered via an intravenous route of administration. In some embodiments, the therapeutic agent is administered via a subcutaneous route of administration.
Also provided herein is a method of preparing a pharmaceutical composition 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 carrier as described herein. In some embodiments, the lipid comprises forming a lipid nanoparticle.
In vitro and in vivo delivery of recombinant nucleic acids
In one aspect, the recombinant nucleic acid encoding the chimeric antigen receptor is encapsulated in a suitable lipid nanoparticle for in vivo delivery in a therapeutic composition. The recombinant nucleic acid may be DNA, circRNA or mRNA. In some embodiments, the nucleic acid may be introduced into the cell using naked DNA or messenger RNA (mRNA). In some embodiments, DNA or mRNA encoding the chimeric fusion protein is introduced into phagocytes by Lipid Nanoparticle (LNP) encapsulation. The mRNA may be codon optimized. In some embodiments, the mRNA may comprise one or more modified or unnatural bases, e.g., 5' -methylcytosine or pseudouridine. mRNA may be 50-10,000 bases long. In one aspect, the transgene is delivered in the form of mRNA. mRNA can comprise greater than about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 bases. In some embodiments, the mRNA may be more than 10,000 bases long. In some embodiments, the mRNA may be about 11,000 bases long. In some embodiments, the mRNA may be about 12,000 bases long.
In some embodiments, the pharmaceutical composition for local or systemic delivery in a subject comprises a messenger RNA encoding a chimeric fusion protein described herein associated with one or more lipid components. In some embodiments, one or more lipid components may comprise a cationic lipid. In some embodiments, one or more lipid components may comprise a non-cationic lipid. In some embodiments, the one or more lipid components may comprise polar lipids and/or non-polar lipids. In some embodiments, the one or more lipid components may comprise neutral lipids. In some embodiments, one or more lipid components may comprise conjugated lipids. In some embodiments, the one or more lipid components may comprise liposomes surrounding the mRNA. In some embodiments, the one or more lipid components may comprise lipid nanoparticles encapsulating mRNA.
LNP-encapsulated DNA or RNA can be used to transfect myeloid cells, such as monocytes or macrophages, or can be administered to a subject. LNP can be designed for targeted delivery to be taken up by myeloid cells upon local or systemic delivery to a subject. In some embodiments, the LNP may comprise one or more targeting moieties, such as antibodies or ligands, or biomolecules or portions thereof, that bind to surface elements of the myeloid cells and promote uptake of mRNA encapsulated LNP by the myeloid cells. In some embodiments, the LNP may be targeted to the tissue by one or more antibodies, ligands, conjugates, aptamers that may associate or be incorporated into the LNP.
In one embodiment, the LNP comprises one or more polymers.
In one embodiment, the LNP comprises a synthetic polymer.
In some embodiments, the LNP comprises a cationic lipid. In some embodiments, the LNP comprises a non-cationic lipid. In some embodiments, the LNP comprises neutral lipids. In some embodiments, the LNP comprises one or more pegylated lipids. In some embodiments, the LNP has a diameter of about 100nm to about 200nm. In some embodiments, the LNP has a diameter of about 150nm, e.g., 80-120nm, 100-140nm, 100-130nm, 70-140nm, 80-150nm, or 90-180nm. In some embodiments, the LNP has a diameter of less than 100nm, such as less than 90nm, or less than 80nm.
In some embodiments, the delivery vehicle is any of the lipid vehicles described above, e.g., a lipid component that is freely associated with a recombinant nucleic acid, an encapsulated liposome, or LNP, and wherein mRNA is designed for preferential expression in myeloid cells, as described elsewhere within this specification.
In some embodiments, the composition is formulated for in vitro delivery of mRNA encoding CFP in a myeloid cell (e.g., a monocyte in a cell population comprising monocytes) using the delivery vehicle-containing mRNA described anywhere in this specification, wherein the myeloid cell comprising mRNA encoding CFP is formulated as a pharmaceutical composition for delivery into a mammalian subject (e.g., a human subject). In some embodiments, the mRNA comprises a CFP comprising an extracellular antigen-binding domain that binds TROP2 as described herein. In some embodiments, the delivery vehicle-containing mRNA described anywhere in this specification is used to formulate a pharmaceutical composition for delivery into a mammalian subject (e.g., a human subject). In some embodiments, the mRNA comprises a sequence encoding a CFP comprising an extracellular antigen-binding domain that binds TROP2 as described herein. In some embodiments, the mRNA comprising a sequence encoding TROP2 binding CFP and the delivery vehicle are specifically taken up by myeloid cells in the heterologous cell population. In some embodiments, mRNA comprising a sequence encoding TROP2 binding CFP is specifically taken up by both myeloid cells and non-myeloid cells in the heterologous cell population, but is expressed and/or functions only in myeloid cells in the heterologous cell population.
In some embodiments, the mRNA is electroporated into a cell. In some embodiments, the composition comprising mRNA is electroporated into the cell. In some embodiments, as described above, the mRNA is associated with one or more lipid components, e.g., in an LNP. In some embodiments, a composition comprising mRNA (e.g., comprising one or more lipid components) is electroporated into a myeloid cell within a heterogeneous population of cells, designed and optimized for delivery into the myeloid cell.
Modification in CFP design for functional enhancement
In one aspect, provided herein is a composition comprising one or more recombinant nucleic acid sequences comprising: (a) a first nucleic acid sequence encoding an exogenous polypeptide; (B) A second nucleic acid sequence encoding a chimeric antigen receptor fusion protein (CFP), wherein the CFP comprises: (a) An intracellular signaling subunit comprising an intracellular signaling domain having one or more tyrosine residues that are phosphorylated upon receptor binding to an antigen; (b) A transmembrane domain, and (c) an extracellular binding domain having binding specificity for a component on the surface of a target cell, wherein the extracellular binding domain is operably linked to the transmembrane domain and an intracellular signaling subunit; and (d) a transcriptional activator domain operably linked to the intracellular signaling subunit by a protease cleavage sequence, wherein the transcriptional activator domain facilitates transcription of the first nucleic acid sequence encoding the exogenous polypeptide; and (C) a third nucleic acid sequence encoding (i) a protease that cleaves a protease cleavage sequence operably linked to the transcriptional activator domain to the intracellular signaling subunit; (ii) A domain that binds to tyrosine residues that are phosphorylated upon CFP activation; wherein the protease that cleaves the protease cleavage sequence and the domain that binds to tyrosine residues are operably linked. In some embodiments, the third nucleic acid sequence further encodes (iii) a stimulus responsive element. In some embodiments, the stimulus-responsive element (iii) is fused to a domain that binds a phosphorylated tyrosine residue. In some embodiments, the stimulus-responsive element (iii) is responsive to the microenvironment of the cell expressing the nucleic acid sequence. In some embodiments, one or more recombinant nucleic acids are expressed in myeloid cells.
In some embodiments, the stimulus-responsive element (iii) is fused to a domain that binds a phosphorylated tyrosine residue. In some embodiments, the stimulus-responsive element is responsive to the microenvironment of the cell expressing the nucleic acid sequence. In some embodiments, (iii) is a degradation determinant operably linked to (ii). In some embodiments, the degradation determinant is a HIF-1a degradation solution stator.
In some embodiments, the transcriptional activator domain comprises a VP64 transactivation domain. In some embodiments, the protease that cleaves a protease cleavage sequence that operably links the transcriptional activator domain to an intracellular signaling subunit is a Hepatitis C Virus (HCV) NS3 protease. In some embodiments, the domain that binds to a tyrosine residue that is phosphorylated upon CFP activation is a phosphotyrosine binding (PTB) domain. In some embodiments, the PTB is Shc PTB.
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 circRNA.
In some embodiments, the recombinant nucleic acid is associated with a replicon RNA. Provided herein is a method for preparing a myeloid cell therapeutic agent for cancer, the method comprising expressing in a myeloid cell a recombinant nucleic acid comprising: (a) a first nucleic acid sequence encoding an exogenous polypeptide; (B) A second nucleic acid sequence encoding a myeloid cell chimeric antigen receptor (CFP), wherein the CFP comprises: (a) An intracellular signaling subunit comprising an intracellular signaling domain having a tyrosine residue that is phosphorylated upon CFP activation; (b) A transmembrane domain, and (c) an extracellular binding domain having binding specificity for a component on the surface of a target cell, wherein the extracellular binding domain is operably linked to the transmembrane domain and an intracellular signaling subunit; and (d) a transcriptional activator domain operably linked to the intracellular signaling subunit by a protease cleavage sequence, wherein the transcriptional activator domain facilitates transcription of the first nucleic acid sequence encoding the exogenous polypeptide; and (C) a third nucleic acid sequence encoding (i) a protease that cleaves a protease cleavage sequence operably linked to the transcriptional activator domain to the intracellular signaling subunit; (ii) A domain that binds to tyrosine residues that are phosphorylated upon CFP activation; wherein the protease that cleaves the protease cleavage sequence and the domain that binds to tyrosine residues are operably linked.
Provided herein is a method for preparing a myeloid cell therapeutic agent for cancer, the method comprising expressing in a myeloid cell a recombinant nucleic acid encoding a chimeric protein comprising: (a) Human eosinophils are the primary basic protein acidic domain; (b) an MMP recognition sequence; and (c) a human eosinophil major basic protein cytotoxicity domain.
Provided herein is a method for treating a subject having cancer, the method comprising administering to a subject in need thereof the above pharmaceutical composition. Provided herein is a method for treating a subject having cancer, the method comprising administering to a subject in need thereof a pharmaceutical composition described herein. Provided herein is a method of inducing tumor regression in a subject in need thereof, the method comprising intravenously administering to the subject a pharmaceutical composition comprising myeloid cells, wherein the myeloid cells express one or more recombinant nucleic acids encoding one or more polypeptides, and wherein at least one of the one or more polypeptides has functional activity in a tumor microenvironment and no functional activity in a non-tumor environment.
Chimeric antigen receptor with modular sensing and internal programmable switching
Major objectives in designing immunotherapeutic agents include (1) disease specificity, which includes that the therapeutic agent will be active at the disease site and will act specifically on diseased cells; (2) Programmability, which includes that the therapeutic agent can be programmed or intended to perform its desired function at the desired time and location.
In one aspect, the invention includes the development of chimeric antigen receptors that can sense and utilize disease microenvironment and transform into output functions that generate resistance to disease. In one embodiment, this is referred to as a conversion function. In principle, the recombinant proteins of interest disclosed herein utilize signal transduction within the disease system, for example, to switch in the Tumor Microenvironment (TME). For example, TMEs are rich in Matrix Metalloproteinases (MMPs) that act as triggers for cleaving peptides that activate and make functional chimeric protein components. This ensures that the function of the immunotherapeutic cells is specific and not applicable to all cells in the body, thus reducing the chance of toxicity associated with immunotherapy.
Generation of target-specific programmable chimeric constructs for myeloid cell therapy
Provided herein is a method of making a myeloid cell for use in immunotherapy, wherein the myeloid cell expresses an exogenous recombinant nucleic acid encoding a chimeric fusion protein that is inducible in a tumor microenvironment. For example, myeloid cells for immunotherapy comprise recombinant nucleic acids that express at least a portion of chimeric receptors activated in the Tumor Microenvironment (TME). In some embodiments, the myeloid cells for immunotherapy comprise a recombinant nucleic acid encoding a pro-inflammatory protein or a pro-apoptotic or lytic protein, the expression of which is under the control of an inducible transcriptional activator. Under resting conditions, the transcriptional activator remains fused to the non-nuclear protein. Upon reaching the tumor environment, the transcriptional activator is released for nuclear localization and transactivates the nucleic acid encoding the pro-inflammatory or pro-apoptotic or lytic protein via cleavage from the fusion protein. This functional result can be achieved by modular design of recombinant proteins and their expression in myeloid cells.
In one embodiment, cleavable transcriptional activators may be designed by inserting a cleavable sequence that is not cleaved by an endogenous protease. In some embodiments, the transcriptional activator is fused to a transmembrane protein, such as a chimeric receptor (CFP). In some embodiments, the CFP comprises an ICD having one or more ITAM domains comprising one or more tyrosine residues that are activated upon receptor activation. Receptor activation is understood to occur when the receptor is engaged with a cancer cell via an extracellular binding domain. In some embodiments, an exemplary modular design of the inducible transcriptional activator may be achieved by: the transcriptional activator domain is operably linked to an intracellular signaling subunit by a non-endogenous protease cleavage sequence; wherein the non-endogenous protease is encoded by a recombinant nucleic acid expressed in the same cell. The non-endogenous protease may be designed such that it is fused to and operably linked to another protein domain that binds to tyrosine residues that are phosphorylated upon CFP activation for activation. The domain that binds to phosphorylated tyrosine residues is the phosphotyrosine binding domain (PTB). Upon activation of CFP, ITAM is phosphorylated, the PTB domain may in turn be activated by the ITAM domain, and the activated PTB domain may in turn activate a protease that can cleave the transcriptional activator domain from the cytoplasmic end of CFP, releasing the transcriptional activator for nuclear localization.
In addition, protease-PTB fusion proteins can be fused to degradation determinants that trigger degradation of PTB and proteases in an inactive state. In one embodiment, the degradation determinant is a HIF 1-alpha (HIF 1-a) degradation determinant sequence that naturally degrades under normoxic conditions. Thus, PTB-proteases will degrade in cells under normoxic conditions, i.e. in the circulation or in normal (non-tumour) tissues. On the other hand, when the cells expressing the protein were in a hypoxic tissue environment, the PTB-protease was no longer degraded, and homologous phosphorylated residues were found in the activated CFP intracellular sequence. Furthermore, upon binding of the degradation determinant-PTB complex to intracellular phosphotyrosine residues in CFP-ICD, the degradation determinant is inactivated by contact with a portion of ICD (see fig. 2B). Thus, the recombinant protein can be designed as follows: the recombinant protein comprises (i) a CFP comprising an extracellular domain that binds to a component on the surface of a cancer cell or tumor cell and that binds to an activated receptor (CFP), wherein the CFP comprises a transmembrane and intracellular domain (ICD) comprising an ITAM domain that is activated and phosphorylated upon activation of the receptor; (ii) Degrading the stator-PTB-protease complex, which may optionally be encoded by the same vector encoding the recombinant nucleic acid, and generating a preprotein having a degradation determinant-PTB-protease flanked by T2A self-cleavable sequences; (iii) A transcriptional activator operably linked to a CFP intracellular domain by a cleavable sequence is a substrate for a protease. In addition, the myeloid cells co-express the nucleic acid under the influence of a promoter or transcriptional activator that is responsive to the transcriptional activator operably linked to the CFP intracellular domain by a cleavable sequence. Upon activation of CFP via conjugation to cancer cells, ITAM in ICD is phosphorylated, and the activated ITAM domain promotes binding of PTB and its activation, while the binding inactivates degradation determinants that otherwise degrade free (non-binding to phosphotyrosine residues in ITAM, inactivated) degradation solves stator-PTB-proteases. The activated PTB activates proteases and stabilizes (in the absence of degradation determinant activity), which in turn cleaves transcriptional activators for nuclear localization.
Exemplary proteases that may be used for the first fusion protein include hepatitis C virus protease (e.g., NS3 and NS 2-3); a signal peptidase; proprotein convertases of the subtilisin/kexin family (furin, PC1, PC2, PC4, PACE4, PCs, PC); a proprotein convertase that cleaves at a hydrophobic residue (e.g., leu, phe, val or Met); a proprotein convertase cleaving at a small amino acid residue (e.g., ala or Thr); pro-melanoidin converting enzyme (PCE); chromogranin aspartic protease (CGAP); a prohormone thiol protease; carboxypeptidase (e.g., carboxypeptidase E/H, carboxypeptidase D and carboxypeptidase Z); aminopeptidases (e.g., arginine aminopeptidase, lysine aminopeptidase, aminopeptidase B); prolyl endopeptidase; aminopeptidase N; insulin degrading enzymes; calpain; a high molecular weight protease; and caspases 1, 2, 3, 4, 5, 6, 7, 8 and 9. Other proteases include, but are not limited to, aminopeptidase N; puromycin sensitive aminopeptidase; angiotensin converting enzyme; pyroglutamyl peptidase II; dipeptidyl peptidase IV; n-arginine dibasic invertase; endopeptidase 24.15; endopeptidase 24.16; amyloid precursor protein secretase α, β and γ; angiotensin converting enzyme secretase; tgfα secretase; tnfα secretase; FAS ligand secretase; TNF receptors-I and-II secretase; CD30 secretase; KL1 and KL2 secretase; IL6 receptor secretase; CD43, CD44 secretase; CD16-I and CD16-II secretase; an L-selectin secretase; folate receptor secretase; MMP 1, 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, and 15; urokinase plasminogen activator; tissue plasminogen activator; plasmin; thrombin; BMP-1 (procollagen C-peptidase); ADAM 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11; and granzymes A, B, C, D, E, F, G and H.
In some embodiments, the protease is Hepatitis C Virus (HCV) nonstructural protein 3 protease NS3. In some embodiments, the NS3 cleavable sequence is EDVVCC. In some embodiments, the NS3 cleavable sequence is a DEMEEC.
In some embodiments, the transcriptional activator comprises a DNA binding domain. In some embodiments, the DNA binding domain is a GAL4 domain. In some embodiments, the DNA binding domain is a DNA binding domain of ZFHD1 or tetR. In some embodiments, the transcriptional activator comprises a tetramer repeat of the VP64 transactivation domain (the minimal activation domain of herpes simplex virus protein VP16 (amino acids 437-447)).
In some embodiments, the degradation determinant is a HIF-down solving stator.
In some embodiments, provided herein are one or more recombinant nucleic acids comprising: (a) a first nucleic acid sequence encoding an exogenous polypeptide; (b) A second nucleic acid sequence encoding a chimeric antigen receptor (CFP), wherein the CFP comprises: (a) An intracellular signaling subunit comprising an intracellular signaling domain having a tyrosine residue that is phosphorylated upon CFP activation; (b) A transmembrane domain, and (c) an extracellular binding domain having binding specificity for a component on the surface of a target cell, wherein the extracellular binding domain is operably linked to the transmembrane domain and an intracellular signaling subunit; and (d) a transcriptional activator domain operably linked to the intracellular signaling subunit by a protease cleavage sequence, wherein the transcriptional activator domain facilitates transcription of the first nucleic acid sequence encoding the exogenous polypeptide; and (C) a third nucleic acid sequence encoding (i) a protease that cleaves a protease cleavage sequence operably linked to the transcriptional activator domain to the intracellular signaling subunit; (ii) A domain that binds to tyrosine residues that are phosphorylated upon CFP activation; wherein the protease that cleaves the protease cleavage sequence and the domain that binds to tyrosine residues are operably linked.
In some embodiments, provided herein is a recombinant nucleic acid for a modular polypeptide that can be expressed in myeloid cells for use in immunotherapy. A recombinant nucleic acid encodes a chimeric protein comprising: (a) a cytotoxic polypeptide; (b) a protease cleavage sequence; and (c) an inhibitory polypeptide domain, wherein the inhibitory polypeptide domain inhibits a cytotoxic polypeptide; wherein the cytotoxic polypeptide, protease cleavage sequence and inhibitory polypeptide domain are operably linked. In some embodiments, the cytotoxic polypeptide is a human eosinophil major basic protein cytotoxicity domain. In some embodiments, the cytotoxic polypeptide is a human eosinophil major basic protein acidic domain. In some embodiments, the protease cleavage sequence is an MMP recognition sequence. In some embodiments, the protease cleavage sequence is cleaved by MMP.
In some embodiments, one or more domains in the first phagocytic ICD comprises a mutation.
In some embodiments, one or more domains in the second ICD comprise mutations to enhance the kinase binding domain, create a phosphorylation site, create an SH2 docking site, or a combination thereof.
Co-expression of inflammatory genes
In one aspect, the recombinant nucleic acid comprises a coding sequence for a pro-inflammatory gene that is co-expressed with a chimeric receptor in an engineered cell. In some embodiments, the proinflammatory gene is a cytokine. Examples include, but are not limited to, TNF- α, IL-1α, IL-1β, IL-6, CSF, GMCSF, or IL-12 or an interferon.
The recombinant nucleic acid encoding a pro-inflammatory gene may be monocistronic, wherein both coding sequences of (a) CFP and (b) the pro-inflammatory gene are posttranscriptionally or posttranslationally cleaved for independent expression.
In some embodiments, the two coding sequences comprise a self-cleaving domain, e.g., a coding P2A sequence.
In some embodiments, the two coding regions are separated by an IRES site.
In some embodiments, the two coding sequences are encoded by a bicistronic genetic element. (a) The coding regions of CFP and (b) the pro-inflammatory gene may be unidirectional, each of which is under separate regulatory control. In some embodiments, the coding regions of both are bi-directional and drive in opposite directions. Each coding sequence is under separate regulatory control.
Co-expression of pro-inflammatory genes is designed to confer strong inflammatory stimuli to macrophages and to activate surrounding tissues for inflammation.
Chimeric antigen receptor for enhanced intracellular signaling and inflammatory activation
In one aspect, the recombinant nucleic acid encodes a chimeric intracellular domain other than an extracellular binding domain, a transmembrane domain, and in some cases, a portion or the entire intracellular domain of a receptor. Intracellular domains are designed to have potent pro-inflammatory immune activating capacity, for example, when macrophages are involved in combating infection. The chimeric intracellular domain (or the second ICD, as the case may be) is fused to the cytoplasmic end of the chimeric receptor such that the ICD is operably linked to the extracellular domain and activation of the extracellular domain can activate the fused ICD. The ICD provides a second signal necessary to trigger a pro-inflammatory signal. In one embodiment, the pro-inflammatory signal is a signal for activating an inflammatory body. Nod-like receptors (NLR) are a subset of receptors that form components of the inflammatory body pathway. These receptors are activated in the innate immune response and oligomerize to form a multiprotein complex that serves as a platform to recruit and induce cleavage and activation of the pro-inflammatory caspase. This results in direct activation of ROS and often in severe cell death known as cell death. There are four types of inflammasome complexes: NLRP1m, NLRP3, IPAF and AIM2.
Tumor Microenvironment (TME) constitutes an immunosuppressive environment. The effects of IL-10, glucocorticoids, apoptotic cells and immune complexes can interfere with innate immune cell function. Immune cells (including phagocytes) settle to a tolerogenic phenotype. In macrophages, this phenotype (commonly referred to as the M2 phenotype) is different from the M1 phenotype, in that macrophages are effective and capable of killing pathogens. Macrophages exposed to LPS or IFN-gamma may, for example, be polarized towards the M1 phenotype, whereas macrophages exposed to IL-4 or IL-13 will be polarized towards the M2 phenotype. LPS or IFN-gamma can interact with Toll-like receptor 4 (TLR 4) on the surface of macrophages, induce the Trif and MyD88 pathways, induce activation of transcription factors IRF3, AP-1 and NFKB, and thus activate TNF genes, interferon genes, CXCL10, NOS2, IL-12, etc., necessary for pro-inflammatory M1 macrophage responses. Similarly, IL-4 and IL-13 bind to IL-4R, activating the Jak/Stat6 pathway, which regulates the expression of CCL17, ARG1, IRF4, IL-10, SOCS3, etc., which are genes associated with anti-inflammatory responses (M2 responses). Expression of CD14, CD80, D206 and low expression of CD163 indicate that macrophages are polarized to the M1 phenotype.
In some embodiments, the recombinant nucleic acid encodes one or more additional intracellular domains comprising a cytoplasmic domain for the inflammatory response. In some embodiments, expression of a recombinant nucleic acid encoding a chimeric receptor fusion protein comprising a cytoplasmic domain for engineering an inflammatory response in macrophages confers an effective pro-inflammatory response similar to the M1 phenotype.
In some embodiments, the cytoplasmic domain for the inflammatory response may be a signaling domain or region of TLR3, TLR4, TLR9, MYD88, tif, RIG-1, MDA5, CD40, IFN receptor, NLRP-1-14, NOD1, NOD2, thermal protein, AIM2, NLRC4, CD 40.
In some embodiments, expression of a recombinant nucleic acid encoding a chimeric receptor fusion protein comprises a pro-inflammatory cytoplasmic domain for activating an IL-1 signaling cascade.
In some embodiments, the cytoplasmic portion of the chimeric receptor comprises a cytoplasmic domain from a toll-like receptor, such as an intracellular signaling domain of toll-like receptor 3 (TLR 3), toll-like receptor 4 (TLR 4), toll-like receptor 7 (TLR 7), toll-like receptor 8 (TLR 8), toll-like receptor 9 (TLR 9).
In some embodiments, the cytoplasmic portion of the chimeric receptor comprises a suitable region from interleukin-1 receptor associated kinase 1 (IRAK 1).
In some embodiments, the cytoplasmic portion of the chimeric receptor comprises a suitable region from a differentiation primary response protein (MYD 88).
In some embodiments, the cytoplasmic portion of the chimeric receptor comprises suitable regions from myelin and lymphocyte protein (MAL).
In some embodiments, the cytoplasmic portion of the chimeric receptor comprises a suitable region from a retinoic acid inducible gene (RIG-1).
In some embodiments, the transmembrane domain of the chimeric receptor comprises the transmembrane domain of any one of MYD88, TLR3, TLR4, TLR7, TLR8, TLR9, MAL, IRAK1 protein.
In some embodiments, the recombinant intracellular signaling domain comprises a first portion derived from a phagocytic receptor and a second portion derived from a non-phagocytic receptor, wherein the second portion derived from the non-phagocytic receptor comprises a phosphorylation site. In some embodiments, the phosphorylation site comprises an amino acid sequence suitable for an autophosphorylation site. In some embodiments, the amino acid residue that is phosphorylated is tyrosine. In some embodiments, the phosphorylation site comprises an amino acid sequence suitable for phosphorylation by Src family kinases. In some embodiments, the phosphorylation site comprises an amino acid sequence that, when phosphorylated, is capable of binding to an SH2 domain in a kinase. In some embodiments, the receptor tyrosine kinase domain is fused at the cytoplasmic end of the chimeric receptor, except for the first cytoplasmic portion.
In some embodiments, the phosphorylation site is a tyrosine phosphorylation site.
In some embodiments, the second intracellular domain is an immune receptor tyrosine activation motif (ITAM). Exemplary ITAM motifs are present in mammalian α and β immunoglobulins, TCR gamma receptor, fcγ receptor subunits, CD3 chain receptors and NFAT activating molecules.
In some embodiments, the chimeric receptor intracellular domain comprises one ITAM motif. In some embodiments, the chimeric receptor intracellular domain comprises more than one ITAM motif. In some embodiments, the chimeric receptor intracellular domain comprises two or more ITAM motifs. In some embodiments, the chimeric receptor intracellular domain comprises three or more ITAM motifs. In some embodiments, the chimeric receptor intracellular domain comprises four or more ITAM motifs. In some embodiments, the chimeric receptor intracellular domain comprises five or more ITAM motifs. In some embodiments, the chimeric receptor intracellular domain comprises six or more ITAM motifs. In some embodiments, the chimeric receptor intracellular domain comprises seven or more ITAM motifs. In some embodiments, the chimeric receptor intracellular domain comprises eight or more ITAM motifs. In some embodiments, the chimeric receptor intracellular domain comprises nine or more ITAM motifs. In some embodiments, the chimeric receptor intracellular domain comprises ten or more ITAM motifs.
In some embodiments, one or more domains in the first phagocytic ICD comprises a mutation.
In some embodiments, one or more domains in the second ICD comprise mutations to enhance the kinase binding domain, create a phosphorylation site, create an SH2 docking site, or a combination thereof.
Myeloid cell-specific chimeric antigen receptor encoding polynucleic acids as "ready" therapeutic compositions
Provided herein for the first time are therapeutically effective compositions that can be prepared and stored at any point in time for use by a subject in need thereof. The therapeutically effective compositions comprise nucleic acid compositions having sequences encoding Chimeric Fusion Proteins (CFPs). The polynucleotide encoding a CFP comprises (i) a sequence encoding an extracellular antigen binding domain (e.g., scFv), (ii) a sequence encoding a transmembrane domain capable of dimerizing with an Fc-gamma receptor transmembrane domain when expressed in a cell, and (iii) a sequence encoding an intracellular domain. In some embodiments, the therapeutically effective composition comprises a delivery vehicle in addition to the foregoing. In some embodiments, the delivery vehicle comprises a lipid nanoparticle. In some embodiments, the nucleic acid (i.e., polynucleotide) is mRNA.
Provided herein are myeloid-like cell-specific expression constructs encoding CFPs, wherein the CFPs comprise (i) a sequence encoding an extracellular antigen binding domain that binds to a target antigen, wherein the binding domain comprises a target-specific antibody or fragment thereof, e.g., an scFv that binds to a target antigen expressed on a cancer cell, (ii) a sequence encoding a transmembrane domain capable of dimerizing with an Fc-gamma receptor transmembrane domain when expressed in a cell, and (iii) a sequence encoding one or more intracellular domains comprising a signaling domain that can activate intracellular signaling for phagocytosis activation, inflammatory cytokine secretion, and/or immune activation in a cell expressing the construct; wherein the expression construct is a polynucleotide encapsulated in a lipid nanoparticle for delivery. In some embodiments, the polynucleotide is mRNA.
Provided herein are myeloid cell-specific expression constructs encoding CFPs, wherein the CFP comprises (i) a sequence encoding an extracellular antigen binding domain that binds to a target antigen (e.g., a target antigen expressed on a cancer cell), e.g., CD5, HER2, TROP2, GPC3, GP75, CD19, CD7, CD22, or any other conceivable target antigen, (ii) a sequence encoding a transmembrane domain capable of dimerizing with an Fc-gamma receptor transmembrane domain when expressed in a cell, e.g., CD89 TMD, CD16 TMD, CD64 TMD, or CD32a TMD, and (iii) a sequence encoding one or more intracellular domains comprising a signaling domain that can activate intracellular signaling for phagocytosis activation, inflammatory cytokine secretion, and/or immune activation in cells expressing the construct; wherein the expression construct is a polynucleotide, wherein the expression construct is encapsulated in a lipid nanoparticle for delivery. In some embodiments, the polynucleotide is mRNA.
The therapeutically effective composition is a pharmaceutical composition. The pharmaceutical composition is suitable for in vivo delivery, e.g., suitable for delivery to a human in need thereof.
In some embodiments, it is desirable to store a pharmaceutical composition comprising a myeloid cell-specific expression construct encoding CFP at a suitable temperature and under conditions to preserve the composition therein.
The therapeutically effective compositions described herein may be delivered intravenously, intramuscularly, subcutaneously, intraorbitally, intracranially, intrathecally, intranasally, or by any suitable route of administration.
Also contemplated herein are myeloid cells comprising a myeloid cell-specific expression construct encoding CFP, which as a therapeutic composition can be formulated as an "off-the-shelf product for a subject in need thereof. In some embodiments, the myeloid cells are electroporated with a construct encoding a CFP, wherein the CFP comprises (i) a sequence encoding an extracellular antigen binding domain that binds to a target antigen (e.g., a target antigen expressed on a cancer cell), e.g., CD5, HER2, TROP2, GPC3, GP75, CD19, CD7, CD22, or any other conceivable target antigen, (ii) a sequence encoding a transmembrane domain capable of dimerizing with an Fc-gamma receptor transmembrane domain when expressed in a cell, e.g., CD89 TMD, CD16 TMD, CD64 TMD, or CD32a TMD, and (iii) a sequence encoding one or more intracellular domains comprising a signaling domain that can activate intracellular signaling for phagocytosis activation, inflammatory cytokine secretion, and/or immune activation. Myeloid cells are formulated in compositions for in vivo delivery. In some embodiments, such compositions are suitable for in vivo delivery to a human in need thereof.
Detailed description of the illustrated embodiments
1. A composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP), the CFP comprising:
(a) Phagocytosis or binding receptor (PR) subunits comprising:
(i) Transmembrane domain, or
(ii) An intracellular domain comprising an intracellular signaling domain; and
(b) An extracellular domain comprising a CD137 antigen binding domain that can specifically bind CD137 on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
2. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first CD137 antigen binding domain that specifically binds to a CD137 antigen on a target cell, and (b) a second binding domain that specifically binds to a surfactant on a myeloid cell; wherein the first antigen binding domain binds to CD137 antigen on a target cell and the second binding domain binds to a surfactant on a myeloid cell.
3. A composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP), the CFP comprising: (a) Phagocytosis or binding receptor (PR) subunits comprising: (i) A transmembrane domain, or (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising a Claudin 18.2 antigen binding domain that can specifically bind Claudin 18.2 on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
4. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first Claudin18.2 antigen binding domain that specifically binds to a Claudin18.2 antigen on a target cell, and (b) a second binding domain that specifically binds to a surfactant on a myeloid cell; wherein the first antigen binding domain binds to Claudin18.2 antigen on the target cell and the second binding domain binds to a surfactant on the myeloid cell.
5. A composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP), the CFP comprising: (a) Phagocytosis or binding receptor (PR) subunits comprising: (i) A transmembrane domain, or (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising a Claudin 3 antigen binding domain that can specifically bind to Claudin 3 on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
6. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first Claudin 3 antigen binding domain that specifically binds to a Claudin 3 antigen on a target cell, and (b) a second binding domain that specifically binds to a surfactant on a myeloid cell; wherein the first antigen binding domain binds to claudin18.2 antigen on a target cell and the second binding domain binds to a surfactant on a myeloid cell.
7. A composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP), the CFP comprising: (a) Phagocytosis or binding receptor (PR) subunits comprising: (i) A transmembrane domain, or (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising a CD70 antigen binding domain that can specifically bind CD70 on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
8. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first CD70 antigen binding domain that specifically binds to a CD70 antigen on a target cell, and (b) a second binding domain that specifically binds to a surfactant on a myeloid cell; wherein the first antigen binding domain binds to a CD70 antigen on a target cell and the second binding domain binds to a surfactant on a myeloid cell.
9. A composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP), the CFP comprising: (a) Phagocytosis or binding receptor (PR) subunits comprising: (i) A transmembrane domain, or (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising a TROP2 antigen binding domain that can specifically bind to TROP2 on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
10. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first TROP2 antigen binding domain that specifically binds a TROP2 antigen on a target cell, and (b) a second binding domain that specifically binds a surfactant on a myeloid cell; wherein the first antigen binding domain binds to a TROP2 antigen on a target cell and the second binding domain binds to a surfactant on a myeloid cell.
11. A composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP), the CFP comprising: (a) Phagocytosis or binding receptor (PR) subunits comprising: (i) A transmembrane domain, or (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising a TMPRSS antigen binding domain that can specifically bind TMPRSS on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
12. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first TMPRSS antigen binding domain that specifically binds to a TMPRSS antigen on a target cell, and (b) a second binding domain that specifically binds to a surfactant on a myeloid cell; wherein the first antigen binding domain binds to TMPRSS antigen on a target cell and the second binding domain binds to a surfactant on a myeloid cell.
13. A composition comprising a recombinant nucleic acid encoding a phagocytic or binding receptor (PR) fusion protein (PFP), the fusion protein comprising: (a) a PR subunit comprising: (i) A transmembrane domain, and (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising the antigen binding domain of any one of embodiments 1, 3, 5, 7, 9 or 11, having a strong binding affinity for an antigen of a target cell; wherein the transmembrane domain and extracellular domain are operably linked; and wherein upon binding of the PFP to the antigen of the target cell, the killing or phagocytosis activity of the PFP-expressing cell is increased by at least more than 20% as compared to a cell not expressing the PFP.
14. The composition according to any of embodiments 1, 3, 5, 7, 9, 11 or 13, wherein the intracellular signaling domain is derived from a phagocytosis or binding receptor, or wherein the intracellular signaling domain comprises a phagocytosis activating domain.
15. The composition according to any one of embodiments 1, 3, 5, 7, 9, 11, 13 or 14, wherein the intracellular signaling domain comprises a pro-inflammatory signaling domain.
16. The composition according to any of embodiments 1, 3, 5, 7, 9, 11, or 13-15, wherein the pro-inflammatory signaling domain comprises a kinase activation domain or a kinase binding domain.
17. The composition according to any one of embodiments 1, 3, 5, 7, 9, 11, or 13-16, wherein the intracellular signaling domain comprises a PI3 kinase recruitment domain.
18. The composition according to any one of embodiments 1, 3, 5, 7, 9, 11, or 13-17, wherein the pro-inflammatory signaling domain comprises an IL-1 signaling cascade activation domain.
19. The composition of any of embodiments 1, 3, 5, 7, 9, 11, or 13-18, wherein 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 member, NLRP1-14, NOD1, NOD2, a thermal protein, AIM2, NLRC4, FCGR3A, FCERIG, CD, caspase domain, or pro-caspase binding domain, or any combination thereof.
20. The composition according to any one of embodiments 1, 3, 5, 7, 9, 11 and 13-19, further comprising a transmembrane domain derived from a CD2, CD8, CD28 or CD68 protein TM domain.
21. The composition according to any one of embodiments 1, 3, 5, 7, 9, 11 and 13-20, further comprising a hinge domain.
22. The composition of any one of embodiments 1, 3, 5, 7, 9, 11, or 13-22, wherein upon binding of the PFP to an antigen of a target cell, the killing activity of the PFP-expressing cell is increased by at least 20% as compared to a cell that does not express the PFP.
23. The composition according to any one of embodiments 1, 3, 5, 7, 9, 11 and 13-22, wherein upon binding of PFP to an antigen of a target cell, the killing activity of PFP-expressing cells is increased by at least a factor of 1.1 compared to cells not expressing PFP.
24. The composition of any of embodiments 2, 4, 6, 8, 10 or 12 comprising a first therapeutic agent, wherein the therapeutic agent comprises:
a. a first binding domain, wherein the first binding domain is a first antibody or functional fragment thereof that specifically interacts with an antigen on a target cell, and
b. a second binding domain, wherein the second binding domain is a second antibody or functional fragment thereof that specifically interacts with myeloid cells;
wherein, the liquid crystal display device comprises a liquid crystal display device,
(i) The first therapeutic agent is coupled to a first component, wherein the first component is an additional therapeutic agent or a third binding domain, or
(ii) The composition comprises an additional therapeutic agent.
25. The composition according to any one of embodiments 2, 4, 6, 8, 10, 12 or 24, wherein the therapeutic agent comprises: (a) a first binding domain that specifically interacts with an antigen of a target cell, (b) a second binding domain that specifically interacts with a myeloid cell, and (c) a third binding domain that specifically interacts with a myeloid cell.
26. The composition according to any of embodiments 1-25, wherein any of the binding domains of the therapeutic agent comprises the binding domain of an antibody, a functional fragment of an antibody, a variable domain thereof, V H Domain, V L Domain, VNAR domain, V HH A domain, a single chain variable fragment (scFv), a Fab, a single domain antibody (sdAb), a nanobody, a bispecific antibody, a diabody, or a functional fragment or combination thereof.
27. The composition according to any of embodiments 1-26, wherein the antigen on the target cell to which the first binding domain binds is a cancer antigen or a pathogenic antigen or an autoimmune antigen on the target cell.
28. The composition according to any one of embodiments 2, 4, 6, 8, 10, 12, or 24, wherein the first therapeutic agent comprises a polypeptide less than 1000 amino acids in length or 1000 nm.
29. The composition of any of embodiments 2, 4, 6, 8, 10, 12, 24, or 28, wherein the first therapeutic agent comprises a polypeptide less than 500 amino acids in length or 500 nm.
30. The composition according to any one of embodiments 2, 4, 6, 8, 10, 12, 24, 28, or 29, wherein the first therapeutic agent comprises a polypeptide having a length of 200-1000 amino acids or 200-1000 nm.
31. The composition according to any one of embodiments 2, 4, 6, 8, 10, 12, 24, or 28-30, wherein the binding of the binding domain of the first therapeutic agent contacts the cancer cell with the myeloid cell.
32. The composition according to any of embodiments 2, 4, 6, 8, 10, 12, 24 or 28-31, wherein the second binding domain specifically interacts with a myeloid cell and promotes phagocytic activity of the myeloid cell.
33. The composition according to any one of embodiments 2, 4, 6, 8, 10, 12, 24, or 28-32, wherein the second binding domain specifically interacts with and promotes inflammatory signaling by myeloid cells.
34. The composition according to any of embodiments 2, 4, 6, 8, 10, 12, 24 or 28-33, wherein the second binding domain specifically interacts with a myeloid cell or an adhesion molecule and promotes adhesion of the myeloid cell to a target cell.
35. The composition according to any one of embodiments 2, 4, 6, 8, 10, 12, 24, or 28-34, wherein the second binding domain specifically interacts with a myeloid cell and inhibits the antiphagic activity of the myeloid cell mediated by the target cell.
36. The composition according to any one of embodiments 2, 4, 6, 8, 10, 12, 24, or 28-35, wherein the second binding domain specifically interacts with a myeloid cell and inhibits the anti-inflammatory activity of the myeloid cell mediated by the target cell.
37. The composition according to any of embodiments 2, 4, 6, 8, 10, 12, 24 or 28-36, wherein the second binding domain and/or the third binding domain promotes phagocytic activity of myeloid cells.
38. The composition according to any of embodiments 2, 4, 6, 8, 10, 12, 24 or 28-37, wherein the second binding domain and/or the third binding domain promotes inflammatory signaling in myeloid cells.
39. The composition according to any of embodiments 2, 4, 6, 8, 10, 12, 24 or 28-38, wherein the second binding domain and/or the third binding domain specifically interacts with a myeloid cell or an adhesion molecule and promotes adhesion of the myeloid cell to a target cell.
40. The composition according to any of embodiments 2, 4, 6, 8, 10, 12, 24 or 28-39, wherein the second binding domain and/or the third binding domain inhibits phagocytic activity of the myeloid cells mediated by the target cells.
41. The composition according to any one of embodiments 2, 4, 6, 8, 10, 12, 24 or 28-40, wherein the second binding domain and/or the third binding domain inhibits anti-inflammatory activity of the myeloid cells mediated by the target cells.
42. The composition according to any of embodiments 2, 4, 6, 8, 10, 12, 24, or 28-41, wherein the third binding domain or additional therapeutic agent comprises a CD47 antagonist, a CD47 blocker, an antibody, a chimeric CD47 receptor, a sialidase, a cytokine, a pro-inflammatory gene, a pro-caspase, or an anti-cancer agent.
43. The composition according to any of the preceding embodiments, wherein the first binding domain, the second binding domain and the third binding domain bind different, non-identical target antigens.
44. The composition according to any of embodiments 2, 4, 6, 8, 10, 12, 24, or 28-43, wherein the first binding domain, the second binding domain, or the third binding domain is a ligand binding domain.
45. The composition according to any of the preceding embodiments, wherein the first binding domain, the second binding domain, or the third binding domain is operably linked by one or more linkers.
46. The composition according to embodiment 45, wherein the linker is a polypeptide.
47. The composition according to embodiment 46, wherein the linker is a functional peptide.
48. The composition according to any of embodiments 45-47, wherein the linker is a ligand for the receptor.
49. The composition according to embodiment 45, wherein the linker is a ligand for a monocyte or macrophage receptor.
50. The composition according to any of embodiments 45-49, wherein the linker activates the receptor.
51. The composition according to any of embodiments 45-50, wherein the linker inhibits the receptor.
52. The composition according to embodiment 51, wherein the linker is a ligand for M2 macrophage receptor.
53. The composition according to embodiments 48 or 49, wherein the linker is a ligand for a TLR receptor (e.g., TLR 4).
54. The composition according to any of embodiments 48, 49 or 50, wherein the linker activates a TLR receptor.
55. The composition according to any of embodiments 45-54, wherein the first, second, and/or third binding domains are associated with a mask that binds the binding domains.
56. The composition according to embodiment 55, wherein the mask is an inhibitor that inhibits the binding domain from interacting with its target while the mask remains associated with the corresponding binding domain.
57. The composition according to embodiment 56, wherein the mask is associated with the binding domain via a peptide linker.
58. The composition of embodiment 57, wherein the peptide linker comprises a cleavable moiety.
59. The composition according to embodiment 57, wherein the cleavable moiety is cleaved by a protein or enzyme that is selectively abundant in the cancer or tumor site.
60. The composition according to any one of embodiments 1-59, wherein the recombinant nucleic acid is RNA.
61. The composition according to any one of embodiments 1-60, wherein the recombinant nucleic acid is mRNA.
62. The composition according to any of embodiments 1-61, wherein the recombinant nucleic acid is associated with one or more lipids.
63. The composition according to any of embodiments 1-61, wherein the recombinant nucleic acid is encapsulated in a liposome.
64. The composition according to embodiment 63, wherein the liposome is a nanoparticle.
65. The composition according to any one of embodiments 1-64, wherein the recombinant nucleic acid is comprised in a vector.
66. A pharmaceutical composition comprising any one of the recombinant nucleic acids of the compositions of embodiments 1-65, and an acceptable excipient.
67. A pharmaceutical composition comprising a polypeptide encoded by the recombinant nucleic acid of any one of embodiments 2, 4, 6, 8, 10, 12, 24, or 28-59.
68. A cell comprising the recombinant nucleic acid of any one of embodiments 1-67.
69. The cell according to embodiment 68, wherein the cell is a myeloid cell, e.g., a cd14+ cell.
70. The cell according to embodiment 69, wherein the cell is CD14+, CD16-.
71. A pharmaceutical composition comprising a population of cells comprising the recombinant nucleic acid of any one of embodiments 1-66, wherein at least 50% of the cells are cd14+cd16-.
72. The pharmaceutical composition according to embodiment 71, wherein less than 10% of the cells are dendritic cells.
73. The pharmaceutical composition according to embodiment 71 or 72, further comprising a suitable excipient.
74. A method of preparing any of the compositions of embodiments 1-73.
75. A method of treating cancer in a subject comprising administering to the subject the pharmaceutical composition of any one of embodiments 71-73.
76. A method of treating cancer in a subject comprising administering to the subject a pharmaceutical composition of embodiment 66; or the pharmaceutical composition of embodiment 67.
77. The method according to embodiment 75 or 76, wherein the cancer is selected from the group consisting of gastric cancer, ovarian cancer, renal cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, glioblastoma, and lung cancer.
78. A composition comprising a recombinant polynucleic acid comprising a sequence encoding a Chimeric Fusion Protein (CFP), the CFP comprising: (a) An extracellular domain comprising an anti-tumor associated antigen binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is a transmembrane domain from a protein that dimerizes with an endogenous FcR-gamma receptor in a myeloid cell, and wherein the recombinant polynucleic acid comprising a sequence encoding a Chimeric Fusion Protein (CFP) is functional in a myeloid cell when expressed in the cell and non-functional in a non-myeloid cell.
79. The composition according to embodiment 78, wherein expression of the CFP is detectable in myeloid cells 24h, 36h, 48h, or 72h after transfection with a recombinant nucleic acid comprising a sequence encoding the CFP, and is undetectable in non-myeloid cells 24h, 36h, 48h, or 72h after transfection therewith.
80. A composition comprising a recombinant polynucleic acid comprising a sequence encoding a Chimeric Fusion Protein (CFP), the CFP comprising:
(a) An extracellular domain comprising an anti-tumor associated antigen binding domain, and
(b) A transmembrane domain operably linked to an extracellular domain;
wherein the recombinant polynucleic acid is encapsulated by a nanoparticle delivery vehicle; and wherein CFP is expressed on the surface of myeloid cells of the human subject after administration of the composition to the human subject.
81. A composition comprising a recombinant polynucleic acid comprising a sequence encoding a Chimeric Fusion Protein (CFP), the CFP comprising:
(a) An extracellular domain comprising an anti-tumor associated calcium signaling protein-2 (anti-TROP 2) binding domain, and
(b) A transmembrane domain operably linked to an extracellular domain;
wherein the transmembrane domain is a transmembrane domain from a protein that dimerizes with an endogenous FcR-gamma receptor in a myeloid cell; wherein the recombinant polynucleic acid is encapsulated by a nanoparticle delivery vehicle; and wherein CFP is expressed on the surface of myeloid cells of the human subject after administration of the composition to the human subject.
82. The composition according to embodiment 78, wherein the anti-TROP 2 binding domain comprises a Fab fragment, an scFv domain, or an sdAb domain.
83. The composition according to embodiment 1, wherein the extracellular domain or transmembrane domain is an extracellular domain or transmembrane domain from CD8, CD16a, CD64, CD68 or CD 89.
84. The composition according to embodiment 78, wherein the extracellular domain further comprises a hinge domain derived from CD8, wherein the hinge domain is operably linked to the transmembrane domain and the anti-TROP 2 binding domain.
85. The composition according to embodiment 78, wherein the transmembrane domain is a transmembrane domain from a protein that dimerizes with an endogenous FcR-gamma receptor in a myeloid cell, monocyte or macrophage; wherein CFP is specifically expressed in myeloid cells, monocytes or macrophages of the human subject after administration of the pharmaceutical composition to the human subject.
86. The composition of embodiment 85, wherein the transmembrane domain is a transmembrane domain from CD16a, CD64, CD68 or CD 89.
87. The composition according to embodiment 78, wherein the CFP further comprises an intracellular domain.
88. The composition according to embodiment 87, wherein the intracellular domain comprises one or more intracellular signaling domains, and wherein the one or more intracellular signaling domains comprise an intracellular signaling domain from fcγ R, fc α R, fc epsilon R, CD40 or cd3ζ.
89. The composition according to embodiment 87, wherein the one or more intracellular signaling domains further comprise a phosphoinositide 3 kinase (PI 3K) recruitment domain.
90. The composition according to embodiment 89, wherein the PI3K recruitment domain comprises a sequence having at least 90% sequence identity to SEQ ID No. 26.
91. The composition according to embodiment 87, wherein the intracellular domain comprises an intracellular domain from CD16a, CD64, CD68, or CD 89.
92. The composition according to embodiment 78, wherein the recombinant polynucleic acid is an mRNA.
93. The composition according to embodiment 78, wherein the nanoparticle delivery vehicle comprises a lipid nanoparticle.
94. The composition of embodiment 93, wherein the lipid nanoparticle comprises a polar lipid.
95. The composition of embodiment 93, wherein the lipid nanoparticle comprises a nonpolar lipid.
96. The composition of embodiment 93, wherein the lipid nanoparticle has a diameter of 100 to 300nm.
97. A pharmaceutical composition comprising the composition of embodiment 78, and a pharmaceutically acceptable excipient.
98. The pharmaceutical composition according to embodiment 97, wherein the pharmaceutical composition comprises an effective amount of the composition of embodiment 78 to inhibit the growth of cancer when administered to a human subject having cancer.
99. A method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of embodiment 97 or 98.
100. A method of introducing the composition of embodiment 78 into a myeloid cell, comprising:
electroporating a myeloid cell in the presence of a recombinant polynucleic acid comprising a sequence encoding a Chimeric Fusion Protein (CFP), the CFP comprising:
(a) An extracellular domain comprising an anti-TROP 2 binding domain, and
(b) A transmembrane domain operably linked to an extracellular domain;
wherein the recombinant polynucleic acid
(i) In myeloid cells, or
(ii) Encapsulation by nanoparticle delivery vehicle;
wherein the recombinant polynucleic acid is configured for expression of the recombinant polynucleic acid in myeloid cells of a human subject.
101. The composition according to embodiments 81-84, further comprising an interferon-inducing intracellular domain.
Examples
Example 1.cd137-FcR-PI3K CFP construct:
in this section, design and functional analysis of exemplary Chimeric Fusion Protein (CFP) receptors having an extracellular binding domain for binding antigen (e.g., a cancer antigen, e.g., CD 37) is provided, in brief, CD 137-binding substance CFP receptor protein is an exemplary case of the conjugates described herein (illustrated in fig. 1, 2A, 2B, and 3). The procedure and method may be modified as needed to generate any of the other constructs described herein. CD 137-targeted CFP was constructed using known molecular biology techniques. The nucleic acid construct comprises a sequence encoding a signal peptide fused upstream of a sequence encoding an extracellular CD137 binding domain derived from a human CD137L ligand binding fragment. The ligand binding domain of human CD137L was PCR amplified from a human complementary DNA library and fused to a backbone containing a transmembrane region, attached via a short linker to the CD8 a chain hinge and CD8 a chain TM domain. The TM domain is fused at the cytoplasmic end to the FcR gamma cytoplasmic portion and PI3K recruitment domain. An illustration is provided in fig. 4A. Constructs were prepared in vectors with fluorescent markers and drug (ampicillin) resistance and amplified by transfection of bacterial hosts.
The construct is delivered to the non-dividing mammalian cell in the form of an mRNA, accompanied by a delivery vehicle, e.g., a lipid, liposome, lipid nanoparticle, or synthetic compound, e.g., a polymer, etc. Through this group of findings and previously reported, several cell types (e.g., myeloid cells) are affected by the introduction of foreign DNA by viral transduction or by plasmid delivery. Myeloid cells rarely express foreign DNA and typically undergo cell transformation, e.g., maturation, differentiation, when the cells are handled as simply as foreign DNA. (left in FIG. 4B). In some cases, the viability of the cells is affected (upper right). On the other hand, as shown in the lower right of fig. 4B, delivery via mRNA does not lead to cell progression, aging or transformation, as cells do not release cytokines, such as TNF and IL6, as an indicator of cell activation.
Example 2 efficacy of monocytes expressing CD137 targeting chimeric antigen receptor
In this example, chimeric fusion proteins (CARs) having the exemplary designed extracellular CD137 antigen binding domains described in the present invention were analyzed for their functional efficacy as potential anti-cancer agents. The first generation lentiviral vectors can be used to generate lentiviruses for transduction of myeloid THP1 cell lines. For CFP constructs, transduction efficiency in PMA-treated THP1 cells ranged from 67% -90%, similar to CD137 binders with other binding domains. The experimental setup is depicted in the schematic of the upper graph of fig. 5, and the expected results of phagocytosis are depicted in the lower graph of fig. 5. Target cell death can be calculated by the following formula: [ (# SKOV3 alone- # SKOV3 with effector)/# SKOV3 alone ]. Times.100.
Cd14+ cells isolated from healthy donors were transduced with lentiviral CD 137-targeted CFP constructs encoding fcrγ+pi3k intracellular domains, and analyzed for phagocytosis and killing of CSFE-labeled SCOV3 tumor cells (fig. 6A).
The ATAK construct confers target specificity to cells expressing the construct (fig. 6B). For example, cells expressing a CD19 conjugate do not phagocytose targets that present CD22 but not CD19 on the surface. Figure 6C shows that incorporation of the construct into the myeloid cells does not affect migration of the cells to the tumor site (e.g., left panel, cells expressing the CAR construct are gfp+ in the tumor, cells have migrated after mRNA was introduced into the mouse). As shown in the data (right), CAR expressing cells respond in vitro to chemokines.
To test whether these cells expressing the CD137 construct are capable of differentiating into the M0, M1, M2 phenotype in a tumor environment, CAR-expressing myeloid cells are subjected to an M0, M1 or M2 polarization signal and incubated in culture for 18 hours in the presence of tumor cells or non-tumor control cells. M0 (100 ng/ml MCSF); m1 (5 ng/ml LPS+100ng/ml IFNgamma); m2 (100 ng/ml MCSF+20ng/ml IL-10+20ng/ml TGF beta); DC (100 ng/ml GMCSF+20ng/ml IL-4); and controls. Fig. 7A shows the CD80 and CD206 expression profiles expected under the different conditions, indicating the differential potential of these cells. For some of these experiments, a sequence encoding a FLAG peptide was incorporated between the scFv and the transmembrane domain in the extracellular region of the chimeric CD137 construct. Cells can be harvested and tested for cell viability, which was found to be greater than 80%. The phenotype of the cells can be checked by flow cytometry at 24 hours. Expression of several cell markers can be determined for 24, 48 and 72 hours. CAR expression alone did not increase CD16 expression.
In vivo models of CD 137-expressing tumors can be used to study tumor penetration and activation of CD137-CFP expressing cells. A schematic of the experimental design is shown in fig. 7B. Migration and penetration of CD 137-targeted CFP expressing cells can be determined 24 hours after a single infusion of CFP expressing cells that have been labeled with the cytoplasmic dye CSFE. Tumors can be removed and histologically treated. As shown in the predictive data in fig. 7C, myeloid cells expressing CD137-CFP can migrate into the tumor and accumulate around tumor cells. Twenty-four hours after administration of CFSE labeled CD 137-targeted CFP expressing cells in MSTO tumor-bearing NSG mice, the spleen can be removed and histologically treated.
EXAMPLE 3 chimeric antigen receptor for bone marrow specific expression
This example shows chimeric antigen receptor design for bone marrow specific expression. The chimeric fusion proteins of the present design have a transmembrane domain consisting of FcR TDM, whose expression in the cell membrane is dependent on its multimerization with the fcγ receptor expressed endogenously in monocytes. In this example, CFP constructs for monocyte-specific expression were designed with the transmembrane domain (TMD) of CD16 or CD 89. The specific CFP may be designed based on the general schematic outlined in fig. 8. Exemplary designs include designs having an extracellular cancer antigen binding domain operably linked to a CD16 (FcRIIIA) TMD and CD16 intracellular domain; or a design having an extracellular cancer antigen binding domain operably linked to a CD89 (fcriα) TMD and CD89 intracellular domain. There may be a hinge (H) between the extracellular antigen binding domains. In certain constructs, CD16 or CD89 TMD extends about 10 amino acids in the extracellular region to be flexible, and may or may not include a separate hinge domain. The ECD cancer antigen binding domain can be an scFv, or an antibody or fragment thereof that binds any of the various antigens contemplated herein.
EXAMPLE 4 TROP2 antigen-specific CFP construct
This example shows the design of CFP that targets TROP2 on cancer cells and is expressed on myeloid cells. Figure 9 shows an exemplary version with an extracellular FLAG tag for in vitro testing purposes. Briefly, one construct consisted of an extracellular anti-TROP 2 scFv, which in this case was fused to a FLAG tag, followed by the transmembrane domain of the FcRIIIA (CD 16) protein and the cytoplasmic domain from the same protein. Another construct consists of an extracellular anti-TROP 2 scFv, which in this case is fused to a FLAG tag, followed by the transmembrane domain of the Fc- αr (CD 89) protein and the CD89 cytoplasmic domain.
Also shown are simplified protocols for electroporation and in vitro testing of recombinant mRNA encoding each construct in PBMC for phagocytosis and cytokine production. Fig. 16 shows a generic LNP formulation for in vivo delivery.
EXAMPLE 5 preferential expression of TROP2-CD16 and TROP2-CD89 constructs in myeloid cells/monocytes
Constructs with CD16 and CD89TMD and TROP2 conjugates were tested for expression in different cell types using FLAG tags for flow cytometry assays. As also disclosed elsewhere, the TROP2 conjugate is anti-TROP 2 scFV. B cells, T cells, NK cells and monocyte-derived PBMC were electroporated with TROP2-FLAG-CD16 and TROP2-FLAG-CD89 mRNA and flow cytometry was performed at 24 hours. The data are shown in fig. 10, which shows that the constructs were successfully preferentially expressed in monocytes (rightmost), and no expression was detected in cd19+ (B cells), cd3+ (lymphocytes) or cd56+ (NK cells). The CD 89-based construct showed higher expression than the CD 16-based construct, with more than 60% of the gated cells showing the TROP2-FLAG-CD89 construct.
EXAMPLE 6 TROP2-CD16 and TROP2-CD89 cells exhibit target phagocytosis
PBMCs electroporated with TROP2 constructs were tested in vitro for effect on tumor cell phagocytosis. The target tumor cells used were SKOV3 cell lines expressing luciferase. Mock transfected and untransfected monocytes showed basal levels of phagocytosis as determined by a decrease in luciferase activity. Cells expressing both TROP2 CFPs showed a statistically significant decrease in luciferase activity compared to these controls, which showed that expression of the constructs improved phagocytic capacity of the cells (fig. 11).
Cytokine expression was examined in the presence or absence of target SKOV3 cells in the results shown in TROP2-FLAG-CD16 and TROP2-FLAG-CD89 expressing cells. The data presented in FIGS. 12A and 12B show that the expression of TROP2-CD16 and TROP2-CD89 constructs does not exhibit a strong signaling in monocytes. This is demonstrated by the fact that: cytokine or chemokine expression (IL-1 b, IL-18 or TNF- α or chemokine CCL 2) is induced in the presence of target cells (SKOV 3), i.e., when ECDs of CFPs are bound to their targets. In the absence of target cells, cytokine induction is not significant or negligible. These results indicate that specific bone marrow-targeted CFPs have been generated that can bind TROP2 on cancer cells and activate myeloid cells to target destruction of the targeted cancer cells, and that therapeutic compositions can be further developed with these constructs.
It was then tested whether the orientation of VH and VL of the scFv that bound TROP2 produced a difference in expression levels. FIG. 13A shows constructs with VL-VH (left) and VH-VL (right) orientations. An exemplary VL-VH scFv has the sequence of SEQ ID NO: 34. An exemplary VH-VL scFv has the sequence of SEQ ID NO: 35. Expression of the VH-VL construct showed higher expression in both THP-1 cells (fig. 13B) and primary monocytes (data not shown). In addition, the phagocytic capacity of THP-1 cells expressing the VH-VL construct was shown to be slightly higher than that of the VL-VH construct (FIG. 13C). TROP2 binding CFP constructs comprising a combination of transmembrane and intracellular domains were constructed. TROP2-CD8 hinge-CD 8TM-FcR gamma-PI 3K constructs were generated such that the transmembrane domain (TMD) was a TM domain comprising a CD8 hinge and a TM domain, and was an intracellular domain comprising an Fc receptor gamma-chain and a PI3 kinase recruitment domain. Likewise, the TROP2-FcR-41BB construct was generated with a CD8 hinge and TM domain, and a CD137 (4-1 BB) intracellular domain; the TROP2-41BB-FcR construct was generated with a CD8 hinge and TM domain, and an FcRgamma intracellular domain CD137 (4-1 BB) intracellular domain. The TROP2-CD40-FcR was constructed using the CD8 hinge and TM domains, the CD40 intracellular domain and the FcR intracellular domain. CD8 hinge and TM domain, fcRgamma intracellular domain, MDA5 tandem CARD domain constructs TROP2-FcR-MDA5.TROP2-CD64 and TROP2-CD89 constructs were generated with CD64 or CD89 TM domains and intracellular domains and required to dimerize or multimerize with endogenous fcrγ -chains to transmit intracellular signaling. THP-1-565 cell lines stably expressing HER2-CD8 hinge-CD 8 TM-FcRgamma-PI 3K and other constructs were used to test in vitro phagocytosis potential.
Fig. 13D shows flow cytometry data indicative of construct expression. Each of the TROP 2-binding constructs has a lower expression level compared to the HER 2-binding CFP construct. Figure 13E shows a direct comparison of the expression levels of each TROP2 conjugate and HER2 conjugate construct detected by flow cytometry.
EXAMPLE 7 GP75 targeted CAR-P constructs and expression
GP75 is a melanosome glycoprotein expressed in melanoma and normal melanocytes. GP75 can be expressed on the cell surface and in human and mouse melanoma cells. Chimeric fusion proteins with GP75 binding domains were constructed for testing in cell and animal models. As shown in fig. 14A, the experimental constructs were designed for testing expression with an extracellular FLAG domain that did not interfere with the anti-GP 75 binding extracellular domain of the chimeric receptor. In one construct, the CD8 hinge and transmembrane domain are fused to an extracellular anti-GP 75-ScFv-FLAG sequence. Fcγ domains were included as ICDs. The PI3 kinase recruitment domain was incorporated into ICD. As shown in fig. 14A, a set of exemplary CFP constructs were generated with anti-GP 75 extracellular binding domains with CD16 or CD89 transmembrane regions. In some constructs, the CD16 and CD89 domains have their cytoplasmic portions intact. The GFP coding sequence is tagged at the cytoplasmic end separated from the cytoplasmic domain of the Chimeric Fusion Protein (CFP) by a sequence encoding a self-cleavable peptide T2A. For mouse-specific expression and in vivo experiments in mice, sequences encoding the mouse CD16 or CD89 transmembrane domain were incorporated instead of human sequences. Constructs comprising the corresponding human CD89 were also designed and prepared.
As shown in particular in the lower panel of fig. 14A, some exemplary CD 16-based anti-GP 75-FLAG constructs with different hinges/TM and ICD were generated for experiments to test the effect of hinge domains on expression levels. Constructs range from lacking the hinge domain (MYL 157) to having the CD8 hinge domain (MYL 184), to having the CD28 hinge domain (MYL 185). Other constructs not shown here include anti-GP 75-FLAG-hCD89TM-hCD89 ICD (MYL 158).
Monocytes were isolated from mouse femur by negative selection and electroporated with mRNA constructs, cultured overnight, and incubated with B16 tumor cells expressing GP 75. Successful expression of the construct was observed in murine cells (fig. 14B). The incorporation of the CD8 hinge domain showed a slight advantage of the construct at the expression level (not shown). As shown in fig. 15A-15C, strong tumor cell specific phagocytosis was observed. As shown in particular in fig. 15B, construct MYL158 with anti-GP 75 ECD and CD89TM domains showed increased phagocytosis. Similarly, as shown in fig. 15C, both the MYL158 and MYL186 constructs with GP75 extracellular antigen binding domain and CD89TM domain showed increased phagocytosis. The positive control in the figure is a first generation myeloid cell CFP construct from the inventors with FcR-PI3K recruiting ICD and showing a high phagocytic index. Figure 15D shows cytokine release data for monocytes expressing the indicated constructs.
Example 8 isogenic mouse tumor model and CAR-P therapy
In this example, a HER2 isogenic mouse model was used to test the effect of CFP on endogenous tumors. Human Erb B2 expression of human HER2 in syngeneic mouse brain and breast using a whey protein promoter construct allows establishment of a non-repulsive hHER2 tumor. HER2+ tumors were established in a fully immunocompetent host. The effect of hHER2 expressing anti-HER 2 CFP was then examined in a mouse model. In this model, HER2 tumor was treated with CRISPR edited HER2-CFP expressing monocytes. Tumor growth, transport of CAR cells, persistence and infiltration of CAR cells into tumors, and immunoassays are performed.
EXAMPLE 9 in vivo delivery and bone marrow-specific expression of CFP
Figure 16 shows a graphical representation of recombinant mRNA encoding engineered CFPs with GFP-tags and/or FLAG-tags, encapsulated in LNP, introduced by injection into a mouse melanoma model (figure 17A) for testing myeloid cell-specific expression and in vivo antitumor activity.
Figure 17B shows PEI formulations for in vivo delivery of mRNA encoding CFP in vivo.
CFP has a CD89 transmembrane domain. Organs were harvested periodically as shown in fig. 18A and organ-specific expression of mRNA encoding CFP was determined. Non-myeloid cells (e.g., lymphocytes) do not express CFP. Positive verification of expression in myeloid cells of lung, liver and spleen was obtained. FIG. 18B further analyzes cell type specific expression. Cells isolated after the first or second CFP injection showed statistically significant increases in the expression of the tag in ly6c+/f4/80+ macrophages and ly6c+ly6g-monocytes in both organs. Neutrophils also showed expression of the construct. Non-myeloid cells (e.g., T cells) do not show expression. FIGS. 18C-18J further illustrate cell-specific and tissue-specific expression of constructs with specific transmembrane domains as shown, which allow for myeloid cell-specific expression in vitro and in vivo.
Example 10 immunological Effect of CAR-expressing macrophage treatment in mice
Tumor histopathological results showed that 2 tumors were significantly reduced and occluded in the 4 treated mice, while not in the untreated group. Representative results are shown in fig. 19. In addition, figures 20A and 20B show cytokine and chemokine characteristics in tissues, which indicate high myeloid cell activity necessary for tumor killing. Figure 20C shows recruitment of T cells by engineered monocytes in an in vitro assay. Figures 21 and 41 and 42 show increased inflammatory cytokine secretion from cells isolated from tumor bearing mice administered CFP constructs. This inflammatory cytokine in turn potentially converts cold tumors into hot TMEs.
Figure 22 shows data demonstrating tumor reduction in mice receiving LNP injections containing CFP. Isogenic mouse models were generated with myeloid cells expressing GP75 in combination with CFP. The generation of mouse anti-GP 75 (Trp-1) ATAK cells is shown in figure 29. As shown in the flow cytometry plot at the lower left panel, the ATAK-GP75 receptor was efficiently expressed in mouse monocytes via electroporation of mRNA. These cells were able to effectively phagocytose tumor cells expressing GP75 (bottom right panel of fig. 29). Similar data are confirmed by the data in figures 30, 36 and 37, which depict tumor reduction and survival advantage of mice expressing the corresponding CFP. Figure 30 shows a first in vivo graphical representation of the ability of ATAK myeloid cells to contain tumor growth in a tumor model that is refractory to CAR T and checkpoint inhibitors. 8 infusions 2X 10 relative to vehicle 6 And (3) ATAK cells. Infusions were 4 times per day, resting for 3 days, 4 times per day +/-SD (Mann-Whitney test). When GP-75ATAK monocytes are injected intravenously into immunocompetent C57Bl/6 mice with subcutaneously implanted gp75+b16 melanoma tumors (these mice are resistant to B16 syngeneic tumors), the mice are able to control tumor growth and survival is significantly improved compared to mice administered vehicle or control monocytes. Furthermore, animals treated with GP75-ATAK monocytes showed significantly higher levels of inflammatory monocyte/macrophage and neutrophil infiltration of tumors and an increase in dendritic cells relative to MDSC when tumors were removed from animals and examined for immune cell content. This demonstrates that ATAK monocytes transport to tumor sites, inhibit tumor growth, and recruit other immune cell populations to TME.
Figure 21 specifically shows that treatment of mice with CFP expressing myeloid cells correlates with broad T cell activity in spleen restimulation cultures, including the spread of CD4 epitopes. In this experimentIn (2) performing a spleen cell recall culture in which the spleen cells are cultured with OVA 323*329 SINFECKEL or PMA/ionomycin for 6 hours. Cytokines were detected by flow cytometry. GP75-ATAK cell therapy is associated with increased non-antigen specific activity, which is indicative of cross-presentation.
Positive CFP expression was identified in myeloid cells and some cd3+ cells (fig. 23 and 24). Increased phagocytosis and cytokine secretion are also evident in the data shown in figures 31, 32, 33 and 35. As shown in fig. 31, monocytes further became DC and mature monocytes within the tumor and spleen. Serum cytokine levels shown in fig. 32 show extensive bone marrow activity in responders. The therapeutic response is related to the anti-tumor serum cytokine profile. Other significant observations were cross-presentation of ATAK monocytes to tumor antigens (fig. 33). The ATAK myeloid cells can phagocytose, release and present new antigens to T cells, resulting in an adaptive T cell immune response. ATAK-GP75 (also known as TRP-1 ATAK) monocytes are able to phagocytose, process and present surrogate tumor neoantigens (in this case derived from OVA proteins) to T cells carrying a cognate TCR derived from OVA class I restriction peptide (SIINFEKL), whereas monocytes from the same mice that do not express the ATAK receptor are unable. This demonstrates that ATAK monocytes can process and cross-present antigens to adaptive immune cells, such as CD 8T cells. This further suggests that potential novel T cell subsets can be stimulated via the function of these myeloid cells in vivo, demonstrating broader immune coverage in contrast to the treatment of other immune cell types (e.g., T cells).
In vivo preferential expression of the construct in myeloid cells was demonstrated in the data set forth in figure 38. LNP-FcR chain receptor is expressed by inflammatory monocytes within TME, and greater than 15% of tumor myeloid cells express GP-75-FcaR construct.
EXAMPLE 11 microenvironment modification by in vivo CFP expression modification
In this example, inflammatory gene expression is depicted from in vivo tumor samples, indicating that CFP constructs convert tumor microenvironment into active inflammatory sites, expressing pro-inflammatory cytokines and chemokines (fig. 26), (heated TME), and fig. 34 (upper right). Figures 27 and 34 show data indicating that in vivo CFP injection is capable of inducing T cells. Figure 34 shows that human ATAK monocytes as disclosed herein are able to utilize innate immunity and stimulate an adaptive anti-tumor immune response. The following list shows the key total nodes that the engineered human cell products were demonstrated to have key functional mechanisms:
tumor penetration and identification
Penetration of
Accumulation of
Tumor identification
Signal transduction dynamics
Myeloid cell activation
Cytokine production
Chemokine production
Inflammatory polarization
Direct killing of tumors
Phagocytosis (phagocytosis)
Cytokine(s)
Death receptor (CD 95L)
Long term tumor control
Antigen presentation
Epitope diffusion
T cell engagement
From the data shown, it is apparent that myeloid cells have the ability to alter tumor microenvironment with the increase of inflammatory monocytes, and that DCs are shown in the data of fig. 39. The data in fig. 39 shows up-regulation of inflammatory monocytes (left) and dendritic cells (right). In addition, these cells affect T cell function. Taken together, to the applicant's knowledge, this significant effect of therapeutic potential and correlation has never been achieved and/or demonstrated before.
Example 12 characterization of T cells in vivo after mouse treatment.
The data shown in fig. 40-60 relate to the use of LNP compositions for delivery. Figure 40 shows up-regulation of cd8+ T cells in mice via tail vein injection of CFP treatment. A study of T cell subpopulation phenotypes is shown in figure 41 with cytokine profiles. A study of T cell subpopulation phenotypes is shown in figure 42 with chemokine profiles. In the study discussed herein, syngeneic mice bearing GP75 tumors were dosed once every four days or once every other day, and tumor progression was determined by measuring tumor size. As shown in fig. 43, both treatment groups showed effective inhibition of tumor progression compared to the control. FIG. 44 shows a gating strategy for studying T cell subtypes.
Analysis of the T cell subtype revealed high induction of cd8+ cells and reduction of Treg in tumors (fig. 45). Elevated T cell activation markers and reduced T cell depletion markers (PD 1) were noted in T cells (fig. 46A and 46B, fig. 47A and 47B, fig. 48A and 48B).
EXAMPLE 13 characterization of in vivo myeloid cells after mouse treatment
In addition, using the gating strategy shown in fig. 49, all cell types including lymphocytes, DCs, etc. were delineated after isolation from treated and untreated GP75 mice. Overall, immune cell frequencies did not show significant changes (fig. 50). Myeloid cells and dendritic cells show high expression of the receptor construct. As shown in fig. 51-53, expression was characterized in various cell types, indicating that myeloid cells exhibited significant expression of constructs delivered via LNP formulations. FIG. 54 shows that the bone marrow pattern (Ly 6C) was preserved at greater than 50% lo ) Receptor expression in cells. FIG. 55 depicts a graph demonstrating that treatment in mice significantly increases Ly6C hi And CCR2 hi Data on CD40 and CD206 levels in inflammatory monocytes.
As shown in FIG. 56, CD40 and CD206 levels are at Ly6C lo And CCR2 lo A significant increase in resident myeloid cells; and PDL1 levels were slightly reduced in tumor-resident macrophages (fig. 57). Figure 58 shows that treatment significantly increases the DC activation markers, including mhc ii, CD86 and CD40, but also increases PD-L1 levels in the cd103+cd11b-dendritic cell phenotype in the spleen. FIG. 59 shows that treatment significantly increases the DC activation markers, including MHCII, CD86, and CD40, but also increases the PD-L1 levels in the CD103-CD11b+ dendritic cell phenotype in the spleen.
Example 14.Protease and protease-inducible transcriptional activators with recruiting tyrosine phosphorylation domains Generation of chimeric fusion protein libraries
In this example, a target-specific programmable expression vector was designed that, when expressed in macrophages or myeloid cells, allowed for specific activation of the myeloid cell target cells, which then performed their intended function, resulting in an immunogenic response against the target cells and destruction of the target cells. The vector may be a lentiviral or adenoviral expression vector. The target cell is a cancer cell. In this example, compositions and methods are shown for preparing an exemplary myeloid cell expression vector encoding a membrane protein having a cancer-specific extracellular binding domain, a transmembrane domain, and an intracellular domain having a tyrosine phosphorylation and activation moiety (ITAM domain) that recruits phosphotyrosine binding Protein (PTB), and the intracellular domain linked via a phosphotyrosine binding protease cleavable sequence to a transcriptional activator domain and a protease fused to a HIF-degradation determinant sequence (e.g., T2A cleaving protease) (HIF-degradation stator-protease). The expression vector encodes a target gene sequence that can be activated by a transcriptional activator. The target gene or fragment thereof is a pro-inflammatory or pro-phagocytic protein or protein fragment. The vector may be designed to maintain flexibility in the use of any target gene selected for transcriptional activation. Exemplary macrophage expression vector designs are shown in FIG. 60.
The vector comprises from 5 'to 3': a nucleic acid sequence encoding each of: (a) an extracellular cancer cell binding domain having an N-terminal signal sequence that allows transmembrane localization, (b) a transmembrane domain, (c) an intracellular domain comprising a phosphotyrosine activation/recruitment domain (e.g., ITAM); (d) A DNA binding domain and a transcriptional activator comprising a GAL4DNA binding sequence and a VP64 transcriptional activator linked to an intracellular domain coding sequence by a cleavable sequence that is a substrate for a phosphotyrosine binding Protein (PTB) -dependent protease, (e) a sequence encoding a phosphotyrosine PTB-dependent protease, which in an exemplary construct is an HCV nonstructural protease (NS 3) fused to a PTB-HIF degradation determinant; the PTB-HIF-NS3 sequence is T2A cleavable. The cleavable sequence as a phosphotyrosine binding Protein (PTB) dependent protease substrate is EDVVCC or DEMEEC. A second vector construct is used to co-express the sequence encoding the target gene, the transcription of which is activated by an upstream encoded transcriptional activator. The target gene is driven by a CMV mini-promoter, with an upstream GAL4 binding and activation domain. The nucleic acid sequence encoding the extracellular cancer cell-binding portion encodes a scfv antibody, which in an exemplary construct is a CD 19-binding scfv, but may be a scfv specific for another protein specifically expressed on a cancer cell or target cell. The transcription binding and activating factor comprises a 5x GAL4 sequence fused to VP64 sequence, which can bind via the GAL-4DNA binding region upstream of the CMV promoter, thereby activating transcription of the target gene. PTB-HIF and adjacent protease sequences are flanked by T2A cleavable sequences and are self-cleavable by a T2A protease, thereby releasing the protease post-translationally.
The carrier designs encoding the various portions shown in the embodiments may be variously designed in other ways. For example, polycistronic designs or multiple vector designs are also contemplated herein.
The modular vector design with co-expression of the above components enables flexible incorporation of the target gene downstream of the CMV promoter, which can lead to macrophage-mediated activation and enhancement of phagocytosis and killing of target cells. Exemplary target genes are inflammatory genes, inflammatory body activating genes, cytokines, chemokines, REDOX genes.
The basic structural layout of the extracellular and intracellular domains and the cleavable units is shown in fig. 61A. Constructs as described above are expressed in macrophages. The expected mode of action after translation and release of the mature protein after expression is shown in figure 61B. When binding to the phosphotyrosine binding element on the intracellular domain via the interaction of PTB and ITAM, the HIF-down-resolution stator is inactivated and allows proper function of the protease, allowing cleavage and release of transcription factors. As shown in fig. 2B, in the absence of association with ITAM, the degradation determinant is responsible for degradation of HIF-protease elements. Thus, this function allows cancer cell-specific activation of transcription factors, which in turn allows transcription of specific target genes driven by the CMV promoter, as ITAM is activated and recruits PTB only when it receives a signal from the extracellular domain (e.g., scfv binding to CD19 target on cancer cells).
Example 15 production of tumor-activated eosinophil-lytic proteins
In this example, eosinophil-lysing proteins are used to prepare engineered macrophages or myeloid cells that are capable of lysing target cells (e.g., cancer cells). As shown in the previous examples, exemplary expression vectors were constructed and expressed in macrophages; while expression vectors allow target specificity and are programmable to perform cell lysis functions.
The structural design is shown in fig. 62A. Basically, the vector encodes a recombinant chimeric protein comprising a sequence encoding the acidic domain (amino acid residues 17-105) of human eosinophil-binding protein 1; a sequence encoding the cytotoxic domain of human eosinophil-binding protein 1 (amino acid residues 106-222); interspersed in the framework between the two domains of eosinophil primary binding protein are sequences encoding Matrix Metalloproteinase Protein (MMP) recognition sequences that can be cleaved by homologous MMPs. The chimeric protein coding sequence is preceded by a sequence encoding a signal peptide that allows secretion of the chimeric protein out of macrophages. Mature chimeric proteins secreted from macrophages are shown in exemplary figure 62B. The acidic domain and the cytotoxic domain form a tightly paired configuration held together by MMPs, while the acidic domain binding form holds the cytotoxic domain inactive.
Because the tumor microenvironment is rich in MMPs, secreted chimeric proteins with MMP recognition cleavable domains are easily cleaved, releasing the acidic domain from the cytotoxic domain and thus the cytotoxic domain to perform lytic activity. Tumor cells are abundant in the environment and are therefore attacked, lysed and damaged by the released eosinophil primary basic protein cytotoxic domain protein, while nearby macrophages are more likely to phagocytose and clear the lysed and/or damaged cells. On the other hand, macrophages may further express additional proteins, chimeric receptors and/or target cell-binding moieties, thereby potentiating the effect of secreted eosinophil major basic proteins on cancer cells.

Claims (129)

1. A composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP), the CFP comprising:
(a) Phagocytosis or binding receptor (PR) subunits comprising:
(i) Transmembrane domain, or
(ii) An intracellular domain comprising an intracellular signaling domain; and
(b) An extracellular domain comprising a CD137 antigen binding domain capable of specifically binding CD137 on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
2. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first CD137 antigen binding domain that specifically binds to a CD137 antigen on a target cell, and (b) a second binding domain that specifically binds to a surfactant on a myeloid cell; wherein the first antigen binding domain binds to CD137 antigen on a target cell and the second binding domain binds to a surfactant on a myeloid cell.
3. A composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP), the CFP comprising: (a) Phagocytosis or binding receptor (PR) subunits comprising: (i) A transmembrane domain, or (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising a Claudin 18.2 antigen binding domain capable of specifically binding to Claudin 18.2 on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
4. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first Claudin 18.2 antigen binding domain that specifically binds to a Claudin 18.2 antigen on a target cell, and (b) a second binding domain that specifically binds to a surfactant on a myeloid cell; wherein the first antigen binding domain binds to Claudin 18.2 antigen on a target cell and the second binding domain binds to a surfactant on a myeloid cell.
5. A composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP), the CFP comprising: (a) Phagocytosis or binding receptor (PR) subunits comprising: (i) A transmembrane domain, or (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising a Claudin 3 antigen binding domain capable of specifically binding to Claudin 3 on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
6. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first Claudin 3 antigen binding domain that specifically binds to a Claudin 3 antigen on a target cell, and (b) a second binding domain that specifically binds to a surfactant on a myeloid cell; wherein the first antigen binding domain binds to Claudin 18.2 antigen on a target cell and the second binding domain binds to a surfactant on a myeloid cell.
7. A composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP), the CFP comprising: (a) Phagocytosis or binding receptor (PR) subunits comprising: (i) A transmembrane domain, or (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising a CD70 antigen binding domain capable of specifically binding CD70 on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
8. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first CD70 antigen binding domain that specifically binds to a CD70 antigen on a target cell, and (b) a second binding domain that specifically binds to a surfactant on a myeloid cell; wherein the first antigen binding domain binds to a CD70 antigen on a target cell and the second binding domain binds to a surfactant on a myeloid cell.
9. A composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP), the CFP comprising: (a) Phagocytosis or binding receptor (PR) subunits comprising: (i) A transmembrane domain, or (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising a TROP2 antigen binding domain capable of specifically binding to TROP2 on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
10. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first TROP2 antigen binding domain that specifically binds a TROP2 antigen on a target cell, and (b) a second binding domain that specifically binds a surfactant on a myeloid cell; wherein the first antigen binding domain binds to a TROP2 antigen on a target cell and the second binding domain binds to a surfactant on a myeloid cell.
11. A composition comprising a recombinant nucleic acid encoding a Chimeric Fusion Protein (CFP), the CFP comprising: (a) Phagocytosis or binding receptor (PR) subunits comprising: (i) A transmembrane domain, or (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising a TMPRSS antigen binding domain capable of specifically binding TMPRSS on a target cell; wherein the extracellular domain and the transmembrane domain are operably linked.
12. A composition comprising a recombinant nucleic acid encoding a chimeric fusion protein comprising: (a) A first TMPRSS antigen binding domain that specifically binds to a TMPRSS antigen on a target cell, and (b) a second binding domain that specifically binds to a surfactant on a myeloid cell; wherein the first antigen binding domain binds to TMPRSS antigen on a target cell and the second binding domain binds to a surfactant on a myeloid cell.
13. A composition comprising a recombinant nucleic acid encoding a phagocytic or binding receptor (PR) fusion protein (PFP), said fusion protein comprising: (a) a PR subunit comprising: (i) A transmembrane domain, and (ii) an intracellular domain comprising an intracellular signaling domain; and (b) an extracellular domain comprising the antigen binding domain of any one of claims 1, 3, 5, 7, 9 or 11, having a strong binding affinity for an antigen of a target cell; wherein the transmembrane domain and the extracellular domain are operably linked; and wherein upon binding of said PFP to said antigen of said target cell, the killing or phagocytosis activity of cells expressing said PFP is increased by at least 20% as compared to cells not expressing said PFP.
14. The composition of any one of claims 1, 3, 5, 7, 9, 11, or 13, wherein the intracellular signaling domain is derived from a phagocytosis or binding receptor, or wherein the intracellular signaling domain comprises a phagocytosis activating domain.
15. The composition of any one of claims 1, 3, 5, 7, 9, 11, 13, or 14, wherein the intracellular signaling domain comprises a pro-inflammatory signaling domain.
16. The composition of any one of claims 1, 3, 5, 7, 9, 11, or 13-15, wherein the pro-inflammatory signaling domain comprises a kinase activation domain or a kinase binding domain.
17. The composition of any one of claims 11, 3, 5, 7, 9, 11, or 13-16, wherein the intracellular signaling domain comprises a PI3 kinase recruitment domain.
18. The composition of any one of claims 1, 3, 5, 7, 9, 11, or 13-17, wherein the pro-inflammatory signaling domain comprises an IL-1 signaling cascade activation domain.
19. The composition of any one of claims 1, 3, 5, 7, 9, 11, or 13-18, wherein the pro-inflammatory signaling domain comprises an intracellular signaling domain derived from TLR3, TLR4, TLR7, TLR9, tri, RIG-1, MYD88, MAL, IRAK1, MDA-5, IFN receptor, NLRP family member, NLRP1-14, NOD1, NOD2, thermal protein, AIM2, NLRC4, FCGR3A, FCERIG, CD, caspase domain, or caspase zymogen binding domain, or any combination thereof.
20. The composition of any one of claims 1, 3, 5, 7, 9, 11, and 13-19, further comprising a transmembrane domain derived from a CD2, CD8, CD28, or CD68 protein TM domain.
21. The composition of any one of claims 1, 3, 5, 7, 9, 11, and 13-20, further comprising a hinge domain.
22. The composition of any one of claims 1, 3, 5, 7, 9, 11, or 13-21, wherein upon binding of the PFP to the antigen of the target cell, the killing activity of a cell expressing the PFP is increased by at least greater than 20% as compared to a cell not expressing the PFP.
23. The composition of any one of claims 1, 3, 5, 7, 9, 11, or 13-22, wherein upon binding of the PFP to the antigen of the target cell, the killing activity of cells expressing the PFP is increased by at least a factor of 1.1 compared to cells not expressing the PFP.
24. The composition of any one of claims 2, 4, 6, 8, 10, or 12, comprising a first therapeutic agent, wherein the therapeutic agent comprises:
a first binding domain, wherein the first binding domain is a first antibody or functional fragment thereof that specifically interacts with an antigen on a target cell, and
A second binding domain, wherein the second binding domain is a second antibody or functional fragment thereof that specifically interacts with myeloid cells; wherein, the liquid crystal display device comprises a liquid crystal display device,
(i) The first therapeutic agent is coupled to a first component, wherein the first component is an additional therapeutic agent or a third binding domain, or
(ii) The composition comprises an additional therapeutic agent.
25. The composition of any one of claims 2, 4, 6, 8, 10, 12, or 24, wherein the therapeutic agent comprises: (a) a first binding domain that specifically interacts with an antigen of a target cell, (b) a second binding domain that specifically interacts with a myeloid cell, and (c) a third binding domain that specifically interacts with the myeloid cell.
26. The composition of any one of claims 1-25, wherein any one of the binding domains of the therapeutic agent comprises an antibody binding domain, a functional fragment of an antibody, a variable domain thereof, a VH domain, a VL domain, a VNAR domain, a VHH domain, a single chain variable fragment (scFv), a Fab, a single domain antibody (sdAb), a nanobody, a bispecific antibody, a diabody, or a functional fragment or combination thereof.
27. The composition of any one of claims 1-26, wherein the antigen on the target cell to which the first binding domain binds is a cancer antigen or a pathogenic antigen or an autoimmune antigen on the target cell.
28. The composition of any one of claims 2, 4, 6, 8, 10, 12, or 24, wherein the first therapeutic agent comprises a polypeptide less than 1000 amino acids in length or 1000 nm.
29. The composition of any one of claims 2, 4, 6, 8, 10, 12, 24, or 28, wherein the first therapeutic agent comprises a polypeptide less than 500 amino acids in length or 500 nm.
30. The composition of any one of claims 2, 4, 6, 8, 10, 12, 24, 28, or 29, wherein the first therapeutic agent comprises a polypeptide of 200-1000 amino acids in length or 200-1000 nm.
31. The composition of any one of claims 2, 4, 6, 8, 10, 12, 24, or 28-30, wherein engagement of the binding domain of the first therapeutic agent brings the cancer cell into contact with the myeloid cell.
32. The composition of any one of claims 2, 4, 6, 8, 10, 12, 24, or 28-31, wherein the second binding domain specifically interacts with a myeloid cell and promotes phagocytic activity of the myeloid cell.
33. The composition of any one of claims 2, 4, 6, 8, 10, 12, 24, or 28-32, wherein the second binding domain specifically interacts with a myeloid cell and promotes inflammatory signaling by the myeloid cell.
34. The composition of any one of claims 2, 4, 6, 8, 10, 12, 24, or 28-33, wherein the second binding domain specifically interacts with a myeloid cell or adhesion molecule and promotes adhesion of the myeloid cell to the target cell.
35. The composition of any one of claims 2, 4, 6, 8, 10, 12, 24, or 28-34, wherein the second binding domain specifically interacts with a myeloid cell and inhibits the antiphagic activity of the myeloid cell mediated by the target cell.
36. The composition of any one of claims 2, 4, 6, 8, 10, 12, 24, or 28-35, wherein the second binding domain specifically interacts with a myeloid cell and inhibits the anti-inflammatory activity of the myeloid cell mediated by the target cell.
37. The composition of any one of claims 2, 4, 6, 8, 10, 12, 24, or 28-36, wherein the second binding domain and/or the third binding domain promotes phagocytic activity of the myeloid cells.
38. The composition of any one of claims 2, 4, 6, 8, 10, 12, 24, or 28-37, wherein the second binding domain and/or the third binding domain promotes inflammatory signaling of the myeloid cells.
39. The composition of any one of claims 2, 4, 6, 8, 10, 12, 24, or 28-38, wherein the second binding domain and/or the third binding domain specifically interacts with a myeloid cell or an adhesion molecule and promotes adhesion of the myeloid cell to the target cell.
40. The composition of any one of claims 2, 4, 6, 8, 10, 12, 24, or 28-39, wherein the second binding domain and/or the third binding domain inhibits the antiphagic activity of the myeloid cells mediated by the target cells.
41. The composition of any one of claims 2, 4, 6, 8, 10, 12, 24, or 28-40, wherein the second binding domain and/or the third binding domain inhibits anti-inflammatory activity of the myeloid cells mediated by the target cells.
42. The composition of any one of claims 2, 4, 6, 8, 10, 12, 24, or 28-41, wherein the third binding domain or the additional therapeutic agent comprises a CD47 antagonist, a CD47 blocker, an antibody, a chimeric CD47 receptor, a sialidase, a cytokine, a pro-inflammatory gene, a pro-caspase, or an anticancer agent.
43. The composition of any one of the preceding claims, wherein the first binding domain, the second binding domain, and the third binding domain bind different, non-identical target antigens.
44. The composition of any one of claims 2, 4, 6, 8, 10, 12, 24, or 28-43, wherein the first binding domain, the second binding domain, or the third binding domain is a ligand binding domain.
45. The composition of any one of the preceding claims, wherein the first binding domain, the second binding domain, or the third binding domain are operably linked by one or more linkers.
46. The composition of claim 45, wherein the linker is a polypeptide.
47. The composition of claim 46, wherein the linker is a functional peptide.
48. The composition of any one of claims 45-47, wherein the linker is a ligand for a receptor.
49. The composition of claim 45, wherein the linker is a ligand for a monocyte or macrophage receptor.
50. The composition of any one of claims 45-49, wherein the linker activates the receptor.
51. The composition of any one of claims 45-50, wherein the linker inhibits the receptor.
52. The composition of claim 51, wherein the linker is a ligand for M2 macrophage receptor.
53. The composition of claim 48 or 49, wherein the linker is a ligand for a TLR receptor such as TLR 4.
54. The composition of any one of claims 48, 49 or 50, wherein the linker activates a TLR receptor.
55. The composition of any one of claims 45-54, wherein the first binding domain, the second binding domain, and/or the third binding domain is associated with a mask that binds the binding domain.
56. The composition of claim 55, wherein the mask is an inhibitor of binding domain interaction with its target when the mask remains associated with the corresponding binding domain.
57. The composition of claim 56, wherein said mask is associated with said binding domain via a peptide linker.
58. The composition of claim 57, wherein the peptide linker comprises a cleavable moiety.
59. The composition of claim 58, wherein the cleavable moiety is cleaved by a protein or enzyme that is selectively abundant in the site of the cancer or tumor.
60. The composition of any one of claims 1-59, wherein the recombinant nucleic acid is RNA.
61. The composition of any one of claims 1-60, wherein the recombinant nucleic acid is mRNA.
62. The composition of any one of claims 1-61, wherein the recombinant nucleic acid is associated with one or more lipids.
63. The composition of any one of claims 1-62, wherein the recombinant nucleic acid is encapsulated in a liposome.
64. The composition of claim 63, wherein the liposome is a nanoparticle.
65. The composition of any one of claims 1-64, wherein the recombinant nucleic acid is contained in a vector.
66. A pharmaceutical composition comprising any one of the recombinant nucleic acids of the composition of any one of claims 1-65, and an excipient.
67. A pharmaceutical composition comprising a polypeptide encoded by the recombinant nucleic acid of any one of claims 1-65.
68. A cell comprising the recombinant nucleic acid of any one of claims 1-65.
69. The cell of claim 68, wherein the cell is a myeloid cell.
70. The cell of claim 69, wherein the cell is cd14+, CD16-.
71. A pharmaceutical composition comprising a population of cells comprising the recombinant nucleic acid of any one of claims 1-65, wherein at least 50% of the cells are cd14+cd16-.
72. The pharmaceutical composition of claim 71, wherein less than 10% of the cells are dendritic cells.
73. The pharmaceutical composition of claim 71 or 72, further comprising a suitable excipient.
74. A method of preparing any of the compositions of claims 1-73.
75. A method of treating cancer in a subject comprising administering to the subject the pharmaceutical composition of any one of claims 71-73.
76. A method of treating cancer in a subject comprising administering to the subject the pharmaceutical composition of any one of claims 66, 67, and 71-73.
77. The method of claim 75 or 76, wherein the cancer is selected from the group consisting of gastric cancer, ovarian cancer, renal cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, glioblastoma, and lung cancer.
78. A composition comprising a recombinant polynucleic acid comprising a sequence encoding a Chimeric Fusion Protein (CFP), the CFP comprising:
(a) An extracellular domain comprising an antigen binding domain, and
(b) A transmembrane domain operably linked to the extracellular domain;
wherein the transmembrane domain is a transmembrane domain from a protein that dimerizes with an endogenous FcR-gamma receptor in a myeloid cell; wherein the recombinant polynucleic acid is encapsulated by a nanoparticle delivery vehicle; and wherein the CFP is expressed on the surface of myeloid cells of the human subject after administration of the composition to the human subject.
79. The composition of claim 78, wherein the antigen binding domain comprises a Fab fragment, an scFv domain, or an sdAb domain.
80. The composition of claim 78, wherein the transmembrane domain is a transmembrane domain from CD8, CD16a, CD64, CD68 or CD 89.
81. The composition of claim 78, wherein said extracellular domain further comprises a hinge domain derived from CD8, wherein said hinge domain is operably linked to said transmembrane domain and said antigen binding domain.
82. The composition of claim 78, wherein the transmembrane domain is a transmembrane domain from a protein that dimerizes with an endogenous FcR-gamma receptor in a myeloid cell, monocyte or macrophage; wherein the CFP is specifically expressed in myeloid cells, monocytes or macrophages of a human subject after administration of the pharmaceutical composition to said human subject.
83. The composition of claim 78, wherein the transmembrane domain is a transmembrane domain from CD16a, CD64, CD68 or CD 89.
84. The composition of claim 78, wherein said CFP further comprises an intracellular domain.
85. The composition of claim 84, wherein the intracellular domain comprises one or more intracellular signaling domains, and wherein the one or more intracellular signaling domains comprise an intracellular signaling domain from fcγ R, fc a R, fc epsilon R, CD40 or cd3ζ.
86. The composition of claim 84, wherein said one or more intracellular signaling domains further comprises a phosphoinositide 3 kinase (PI 3K) recruitment domain.
87. The composition of claim 86, wherein the PI3K recruitment domain comprises a sequence having at least 90% sequence identity to SEQ ID No. 26.
88. The composition of claim 84, wherein the intracellular domain comprises an intracellular domain from CD16a, CD64, CD68, or CD 89.
89. The composition of claim 78, wherein the recombinant polynucleic acid is an mRNA.
90. The composition of claim 78, wherein the nanoparticle delivery vehicle comprises lipid nanoparticles.
91. The composition of claim 90, wherein the lipid nanoparticle comprises a polar lipid.
92. The composition of claim 90, wherein the lipid nanoparticle comprises a non-polar lipid.
93. The composition of claim 90, wherein the lipid nanoparticle has a diameter of 100 to 300nm.
94. The composition of any one of claims 78-93, wherein the antigen binding domain binds an antigen selected from the group consisting of TROP2, GPC3, CD5, HER2, CD137, CD70, claudin 3, claudin 18.2, TMPRSS, CD19, CD22, CD7, and GP 75.
95. A pharmaceutical composition comprising the composition of any one of claims 78-94, and a pharmaceutically acceptable excipient.
96. The pharmaceutical composition of claim 95, wherein the pharmaceutical composition comprises an effective amount of the composition of any one of claims 78-94, wherein the growth of cancer is inhibited when administered to a human subject having the cancer.
97. A method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 95 or 96.
98. A method of introducing the composition of any one of claims 78-94 into a myeloid cell, comprising: electroporating a myeloid cell in the presence of a recombinant polynucleic acid comprising a sequence encoding a Chimeric Fusion Protein (CFP), said CFP comprising: (a) An extracellular domain comprising an anti-TROP 2 binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the recombinant polynucleic acid is (i) present in a myeloid cell, or (ii) encapsulated by a nanoparticle delivery vehicle; wherein the recombinant polynucleic acid is configured for expression of the recombinant polynucleic acid in myeloid cells of a human subject.
99. A composition comprising a recombinant polynucleic acid comprising a sequence encoding a Chimeric Fusion Protein (CFP), the CFP comprising:
(a) An extracellular domain comprising an anti-TROP 2 binding domain, said extracellular domain comprising at least one of the sequences shown in SEQ ID No. 34 and SEQ ID No. 35, or a sequence at least 80% identical to SEQ ID No. 34 or SEQ ID No. 35;
(b) A transmembrane domain operably linked to the extracellular domain, the transmembrane domain comprising a sequence from a transmembrane domain of an fcγr1 molecule (CD 64), an fcγriiia molecule (CD 16) or an fcαr1 molecule (CD 89); and
(c) An optional hinge domain operably linked to the extracellular domain and the transmembrane domain, wherein the hinge domain comprises an amino acid sequence from a CD16 protein, a CD64 protein or a CD89 protein or a CD8 a hinge domain.
100. The composition of claim 99, further comprising an intracellular domain comprising an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NOs 26, 27, or 28; or a sequence which is at least 80% identical to the amino acid sequence selected from the group consisting of the sequences shown in SEQ ID NO. 26, 27 or 28, and an optional intracellular domain from a CD16 protein, a CD64 protein or a CD89 protein or a fragment thereof.
101. The composition of claim 99 or 100, wherein the recombinant polynucleic acid is mRNA.
102. A cell comprising the composition of claims 99-101, wherein the cell is a cd14+ cell.
103. Use of the composition of any one of claims 1-65, 78-94 or 99-101 or the pharmaceutical composition of any one of claims 66-67, 71-73 or 95-96 or the cell of claim 68-70 or 102 in the treatment of a disease or disorder.
104. Use of the pharmaceutical composition of claims 66-67, 71-73 or 95-96 or the cell of claim 68-70 or 102 in treating cancer in a subject.
105. The pharmaceutical composition for use according to claim 103 or 104, wherein the cancer is selected from the group consisting of gastric cancer, ovarian cancer, renal cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, glioblastoma, and lung cancer.
106. Use of the composition of any one of claims 1-65, 78-94 or 99-101 or the pharmaceutical composition of any one of claims 66-67, 71-73 or 95-96 or the cell of claim 68-70 or 102 in the manufacture of a medicament for treating cancer in a subject, wherein the cancer is selected from the group consisting of gastric cancer, ovarian cancer, renal cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, skin cancer, pancreatic cancer, colorectal cancer, glioblastoma and lung cancer.
107. The composition of any one of claims 1, 3, 5, 7, 9, 11, or 13-22, wherein the intracellular signaling domain comprises an intracellular signaling domain having a tyrosine residue comprising at least one immune receptor tyrosine-based activation motif (ITAM) domain.
108. The composition of claim 107, wherein the at least one ITAM domain is from an intracellular domain of a protein or polypeptide selected from the group consisting of: the CD3 ζtcr subunit, CD3 εtcr subunit, CD3 γtcr subunit, CD3 δ TCR subunit, TCR ζ chain, fcε receptor 1 chain, fcε receptor 2 chain, fcγreceptor 1 chain, fcγreceptor 2a chain, fcγreceptor 2b 1 chain, fcγreceptor 2b2 chain, fcγreceptor 3a chain, fcγreceptor 3b chain, fcβreceptor 1 chain, TYROBP (DAP 12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and having at least one but no more than 20 modified amino acid sequences.
109. The composition of claim 107, wherein the at least one ITAM domain comprises a Src family kinase phosphorylation site.
110. The composition of claim 107, wherein the at least one ITAM domain comprises a Syk recruitment domain.
111. The composition of any one of claims 107-110, wherein the intracellular signaling subunit further comprises a DAP12 recruitment domain.
112. The composition of any one of claims 107-111, wherein the intracellular domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ITAM domains.
113. A composition comprising one or more recombinant nucleic acid sequences comprising:
(A) A first nucleic acid sequence encoding an exogenous polypeptide;
(B) A second nucleic acid sequence encoding a chimeric antigen receptor fusion protein (CFP),
wherein the CFP comprises:
(a) An intracellular signaling subunit comprising an intracellular signaling domain having one or more tyrosine residues that are phosphorylated upon binding of the receptor to an antigen;
(b) A transmembrane domain comprising a transmembrane domain,
(c) An extracellular binding domain having binding specificity for a component on the surface of a target cell, wherein the extracellular binding domain is operably linked to the transmembrane domain and the intracellular signaling subunit; and
(d) A transcriptional activator domain operably linked to the intracellular signaling subunit by a protease cleavage sequence, wherein the transcriptional activator domain facilitates transcription of the first nucleic acid sequence encoding the exogenous polypeptide; and
(C) A third nucleic acid sequence encoding (i) a protease that cleaves the protease cleavage sequence operably linking the transcriptional activator domain to the intracellular signaling subunit; (ii) A domain that binds to the tyrosine residue that is phosphorylated upon activation of the CFP; wherein said protease that cleaves said protease cleavage sequence and said domain that binds said tyrosine residue are operably linked.
114. The composition of claim 113, wherein the third nucleic acid sequence further encodes (iii) a stimulus responsive element.
115. The composition of claim 114, wherein the stimulus-responsive element (iii) is fused to the domain that binds the phosphorylated tyrosine residue.
116. The composition of claim 114 or 115, wherein the stimulus-responsive element (iii) is responsive to the microenvironment of the cell expressing the nucleic acid sequence.
117. The composition of claim 116, wherein the one or more recombinant nucleic acids are expressed in myeloid cells.
118. The composition of claim 113, wherein the transcriptional activator domain further comprises a DNA binding domain.
119. The composition of claim 118, wherein the DNA binding domain is selected from the group consisting of the DNA binding Domain (DB) of Gal4, ZFHD1, or tet-R.
120. The composition of claim 113, wherein the transcriptional activator domain comprises a VP64 transactivation domain.
121. The composition of any one of claims 113-120, wherein the protease that cleaves the protease cleavage sequence operably linking the transcriptional activator domain to the intracellular signaling subunit is a Hepatitis C Virus (HCV) NS3 protease.
122. The composition of any one of claims 113-121, wherein the domain that binds to the tyrosine residue that is phosphorylated upon activation of the CFP is a phosphotyrosine binding (PTB) domain.
123. The composition of claim 122, wherein the PTB is Shc PTB.
124. The composition of any of claims 113-117, wherein (c) is a degradation determinant operably linked to (b).
125. The composition of claim 124, wherein the down-solving stator is a HIF-1a down-solving stator.
126. A pharmaceutical composition comprising the composition of any one of claims 113-125, and a pharmaceutically acceptable excipient.
127. A cell comprising the composition of any one of claims 113-125.
128. The cell of claim 128, wherein the cell is cd14+.
129. A method of treating a disease in a subject, comprising administering to the subject any one of: (i) the pharmaceutical composition of claim 126; or (b)
(ii) The cell of claim 127 or claim 128.
CN202180089305.2A 2020-11-04 2021-11-04 Engineered chimeric fusion protein compositions and methods of use thereof Pending CN116916940A (en)

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