Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
  • A61K40/35—Cytokines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
  • A61K38/208—IL-12
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
  • A61K40/33—Antibodies; T-cell engagers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K40/00—Cellular immunotherapy
  • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
  • A61K40/36—Immune checkpoint inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5434—IL-12
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
  • C07K16/244—Interleukins [IL]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0693—Tumour cells; Cancer cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/39—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by a specific adjuvant, e.g. cytokines or CpG
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• mRNA messenger RNA
• IL-12 interleukin 12
• the IL-12 polypeptide comprises a membrane-bound IL-12 polypeptide. In some embodiments, the IL-12 polypeptide is complexed to the cell surface of the enucleated cell. In some embodiments, the enucleated cell induces an endogenous immune response at a site associated with the disease or condition of the subject. In some embodiments, the method further comprises treating the WSGR Docket No.53712-720.601 disease or condition of the subject, at least in part, by inducing an endogenous immune response in the subject. In some embodiments, the site associated with the disease or condition comprises a microenvironment. In some embodiments, the microenvironment is a tumor microenvironment.
• the IL-12 polypeptide is not delivered to the microenvironment.
• the site associated with the disease or condition comprises a lymphatic microenvironment, a hepatic microenvironment, a spleen microenvironment, a pancreatic microenvironment, an epidermis microenvironment, or a combination thereof.
• the site associated with the disease or condition comprises a cell associated with the disease or condition.
• the cell associated with the disease or condition is a cancer cell.
• the cell associated with the disease or condition is an immune cell.
• the immune cell is a leukocyte.
• the IL-12 polypeptide is not delivered to the cell associated with the disease or condition.
• the disease or condition comprises cancer.
• the IL-12 polypeptide is covalently connected to the cell surface of the enucleated cell for at least 24 hours. In some embodiments, the IL-12 polypeptide is complexed with the enucleated cell for the at least 24 hours. In some embodiments, the IL-12 polypeptide is not secreted by the enucleated cell. In some embodiments, the IL-12 polypeptide encoded by the exogenous mRNA results in a decreased toxicity in the subject compared to a toxicity caused by direct administration of an IL-12 therapy.
• the administering further comprises administering to the subject a plurality of the enucleated cell formulated in a pharmaceutical formulation, wherein the pharmaceutical formulation comprises (a) the plurality of the enucleated cell, and (b) a pharmaceutically acceptable: excipient, diluent, or a combination thereof.
• the IL-12 polypeptide comprises IL-12 or antigen-binding fragment of the IL-12.
• a method for inducing endogenous immune response in a subject comprising: administering to the subject an enucleated cell comprising (a) an exogenous mRNA encoding a cytokine polypeptide; and (b) one or more organelles for expressing the cytokine polypeptide on a cell surface of the enucleated cell, wherein the cytokine polypeptide induces the endogenous immune response in the subject.
• the cytokine polypeptide comprises a membrane-bound cytokine polypeptide.
• the cytokine polypeptide is complexed to the cell surface of the enucleated cell.
• the enucleated cell induces the endogenous immune response at a microenvironment.
• the microenvironment is a tumor microenvironment.
• the microenvironment is a lymphatic microenvironment, a hepatic microenvironment, a spleen microenvironment, a pancreatic WSGR Docket No.53712-720.601 microenvironment, an epidermis microenvironment, or a combination thereof.
• the enucleated cell induces the endogenous immune response in proximity of a cell.
• the cell is a cancer cell.
• the cell is an immune cell.
• the immune cell is a leukocyte.
• the cytokine polypeptide is expressed on a cell surface of the enucleated cell for at least 24 hours. In some embodiments, the cytokine polypeptide is complexed with the enucleated cell for the at least 24 hours. In some embodiments, the cytokine polypeptide is not secreted by the enucleated cell. In some embodiments, the cytokine polypeptide is not delivered to the microenvironment. In some embodiments, the cytokine polypeptide is not delivered to the cell. In some embodiments, the cytokine polypeptide comprises a full-length cytokine or antigen binding fragment thereof of the cytokine.
• the cytokine polypeptide encoded by the exogenous mRNA results in a decreased toxicity in the subject compared to a toxicity caused by direct delivery of the cytokine polypeptide.
• the cytokine polypeptide comprises an interleukin polypeptide.
• the interleukin polypeptide is an IL-12 polypeptide.
• the cytokine polypeptide comprises an interleukin antibody or antigen binding fragment thereof.
• the cytokine polypeptide comprises IL-12 or antigen binding fragment thereof.
• the endogenous immune response comprises an innate immune response or adaptive immune response.
• the enucleated cell further comprises an immune checkpoint inhibitor.
• the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a B7 inhibitor, a CD 137 inhibitor, a CTLA-4 inhibitor, or any combination thereof.
• the immune checkpoint inhibitor comprises the PD-1 inhibitor.
• the immune checkpoint inhibitor is encoded by a second exogenous mRNA in the enucleated cell.
• the enucleated cell is depleted of an immune recognition molecule.
• the immune recognition molecule comprises HLA antigen, proteoglycan, sugar moiety, embryonic antigen, or any combination thereof.
• the enucleated cell comprises a targeting moiety.
• targeting moiety comprises CXCR2, CCR2, PSGL-1, or a combination thereof.
• a composition comprising: an enucleated cell comprising: an exogenous mRNA that encodes a cytokine polypeptide; and organelles for expressing the cytokine polypeptide, wherein the cytokine is expressed on a cell surface of the enucleated cell.
• the enucleated cell further comprises a transmembrane domain coupled to the cytokine polypeptide.
• the transmembrane and the cytokine polypeptides are covalently connected.
• the cytokine polypeptide WSGR Docket No.53712-720.601 is expressed extracellularly on the cell surface of the enucleated cell.
• the cytokine polypeptide is complexed to a cell surface of the enucleated cell.
• the cytokine polypeptide is expressed on a cell surface of the enucleated cell for at least 24 hours, at least 48 hours, at least 72 hours, or at least 96 hours.
• the enucleated cell expresses an abundance of the cytokine polypeptide that is at least 0.1 fold, at least 0.2 fold, at least 0.5 fold, at least 1.0 fold, at least 2.0 fold, or at least 5.0 fold higher than the enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
• the abundance of the cytokine polypeptide and the abundance of the comparable naturally-occurring cytokine polypeptide is determined by FACS.
• the cytokine polypeptide is not secreted by the enucleated cell.
• the cytokine polypeptide comprises an interleukin polypeptide.
• the interleukin polypeptide is IL-12 polypeptide.
• the enucleated cell further comprises an immune checkpoint inhibitor.
• the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a B7 inhibitor, a CD 137 inhibitor, a CTLA-4 inhibitor, or any combination thereof.
• the immune checkpoint inhibitor comprises the PD-1 inhibitor.
• the immune checkpoint inhibitor is encoded by a second exogenous mRNA in the enucleated cell.
• the enucleated cell is depleted of an immune recognition molecule.
• the immune recognition molecule comprises HLA antigen, proteoglycan, sugar moiety, embryonic antigen, or any combination thereof.
• the enucleated cell comprises a targeting moiety.
• the targeting moiety comprises CXCR2, CCR2, PSGL- 1, or a combination thereof.
• the composition further comprises an immune checkpoint inhibitor.
• the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a B7 inhibitor, a CD 137 inhibitor, a CTLA-4 inhibitor, or any combination thereof.
• the immune checkpoint inhibitor comprises the PD- 1 inhibitor. In some embodiments, the immune checkpoint inhibitor is encoded by a second exogenous mRNA in the enucleated cell. In some embodiments, the immune checkpoint inhibitor is encoded by a second enucleated cell.
• Fig.1A illustrates a non-limiting example of IL-12 mRNA design.
• Fig.1B illustrates mesenchymal stem cells (MSCs), MSC-derived enucleated cells transfected with IL-12 mRNA (MSC IL-12 and Enucleated cell IL-12 respectively), or conditioned medium (CM) collected at the indicated time points.
• Fig.1C illustrates CM from cells/enucleated cells as shown in Fig.1B assessed in mouse splenocytes for the activation of the phosphorylated/activated form of Stat4 (p-Stat4) by western blot.
• Fig.1D illustrates a schematic for using the enucleated cell to treat a triple negative breast cancer (TNBC) mouse model.
• TNBC triple negative breast cancer
• Fig.1E illustrates measurements of IL-12 secreted by the enucleated cell within tumor microenvironment.
• Fig.1F illustrates serum IL-12 measurement.
• Fig.1G illustrates measurements of IL-12 mediated inflammatory biomarkers.
• Fig.1H illustrates mice bearing SQ E0771 tumors injected with a total of three doses of either PBS, MSCs secreting IL-12, or CA-IL-12.
• Fig.1I illustrates FACS analysis for detecting leukocyte activation.
• Fig.2A illustrates a schematic of enucleated cell that secreted IL-12 alone or in combination with the checkpoint inhibitor anti-PD-1 antibody for treating cancer mouse model.
• Fig.2B illustrates a survival curve for animals injected as described in Fig.2A.
• Fig.2C illustrates a graph of tumor size over time post-injection.
• Fig.2D illustrates a graph of fold change in animal weight during the treatment phase of the survival experiments shown in Fig.2B.
• Fig.2E illustrates fold change in tumor size for each side (injected and contralateral/noninjected) for animals that were bilaterally injected with E0771 cells and then were intratumorally injected unilaterally with 3 doses of CA-IL-12 or PBS.
• Fig.3A illustrates scRNA-seq datasets generated from two human TNBC primary tumors were integrated with eight normal mammary datasets. VCAM1-positive vascular endothelial cells from clusters 0 and 4 (Top panel of Fig.3A) were subsetted for differential WSGR Docket No.53712-720.601 expression analysis.
• Fig.3B illustrates Boyden Chamber migration assay showing mesenchymal stem cells or enucleated cells engineered for expressing CCR2, CXCR4, and leukocyte adhesion molecule (PSGL-1; termed CCP MSCs or CCP enucleated cells respectively) for robustly homing towards CCL2, SDF-1a, and EO771 conditioned medium relative to a no-chemoattractant negative control.
• PSGL-1 leukocyte adhesion molecule
• Fig.3C illustrates RNA-seq analysis of the EO771 and 4T1 TNBC cell lines highly expressing Ccl2 and Cxcl12 (encoding SDF-1a).
• Fig.3D illustrates RNA-seq of the 4T1 cell line revealing that the enucleated cell adhesion molecules/chemoattractants (encoding P-selectin), Ccl2, and Cxcl12 were upregulated with tumorigenesis (relative to tissue culture) and further upregulated in lung metastases.
• RNA- Seq data [0028]
• Fig.4A illustrates a schematic showing irradiation mouse model with RT and untreated bilateral tumors.
• Fig.4B illustrates homing of the enucleated cells to tumors.
• Fig.4C illustrates FACS for determining the abundance of enucleated cells homed to tumors.
• Fig.5A illustrate infiltration of the enucleated cell infiltration.
• Fig.5B illustrates optical sectioning and 3D reconstruction of the center of macrometastasis revealed that clusters of CCP enucleated cells.
• Fig.5C illustrates single channel acquisition of DAPI stained sections showing normal lung architecture and no tumor mass.
• Fig.6A illustrates compiled BLI data of E0771-LUC metastases that were untreated or treated with: empty CA + PD-1 mAB, CA-IL-12 + IgG, or CA-IL-12 + PD-1 mAB.
• Fig.6B illustrates representative images of resected lungs with metastases treated as indicated (top) and representative IHC images of CD8+ cytotoxic T cells (bottom) infiltrating EO771 lung metastases from the resected lungs shown in the top row.
• Fig.7 illustrates FACS analysis of indicated leukocyte populations in EO771 lung metastases harvested at day 21 as in Fig.6 relative to total CD45+ immune cells).
• Fig.8A illustrates an exemplary design of the enucleated cell expressing cell membrane tethered IL-12.
• Fig.8B illustrates non-limiting example of a vector map of a vector encoding IL-12.
• Fig.8C illustrates engineering and screening for the enucleated cell expressing membrane-bound scIL-12.
• WSGR Docket No.53712-720.601 illustrates an exemplary mechanism of immune cell activation by CA-scIL-12.
• Fig.9A illustrates MSCs or enucleated cells engineered with or without scIL-12 that were co-cultured with primary murine splenocytes and concanavalin A for 48 hours.
• Fig.9B illustrates enucleated cells (1 x10 6 ) engineered to secrete IL-12 (CA-IL-12).
• Fig.9D illustrates serum cytokine levels in the CA-scIL-12 or CA-IL-12 treated animals.
• Fig.10A illustrates an exemplary design of the enucleated cell expressing cell membrane tethered IL-15.
• Fig.10B illustrates non-limiting example of a vector map of a vector encoding IL-15.
• Fig.10C illustrates hTERT MSCs infected with lentivirus construct encoding membrane-bound IL-15 receptor alpha.
• Fig.11A illustrates a schematic for engineering the hTERT-MSC for expression both scIL-12 and IL-15R ⁇ and the subsequent enucleation of the hTERT-MSC.
• Fig.11B illustrates hTERT MSCs infected with two lentiviruses encoding constructs for either scIL-12 or IL-15R ⁇ . Following drug selection, cells were FACS sorted for single cell clones. Clone 4 was enucleated to generate enucleated cells expressing both scIL-12 and IL- 15R ⁇ , and receptor expression was analyzed by FACS at the indicated time points.
• Fig.11C illustrates functionality of expressing membrane-bound IL-12, IL-15, or a combination of both IL-12 and IL-15.
• Fig.12A illustrates biodistribution of the enucleated cells by bioluminescence.
• Fig.12B illustrates quantification of biodistribution of the enucleated cells of Fig.12A.
• Fig.13A illustrates increased homing of the enucleated cell described herein compared to nucleated mesenchymal stem cell (MSC).
• MSC nucleated mesenchymal stem cell
• Fig.13B illustrates quantification of homing of the enucleated cell of Fig.13A.
• Fig.14A illustrates homing of the enucleated cell based on increased chemoattractants in the TNBC tumor metastatic sites.
• Fig.14B illustrates dot plots of expression of Cxcl (Top) and Ccl (Bottom) chemokines within Vcam1-positive cells in Cluster 1 confirming Cxcl12 (encoding SDF-1 ⁇ ) and Ccl2 were the highest expressed Cxcl and Ccl chemokines, respectively.
• enucleated cells engineered to express at least one cytokine on a cell surface of the enucleated cells.
• the expressing of the at least one cytokine on the cell surface of the enucleated cells confers therapeutic effects.
• the enucleated cell comprises messenger RNA (mRNA) that encodes the cytokine.
• mRNA messenger RNA
• an enucleated cell described herein can be engineered to express a cell membrane bound cytokine for triggering or inducing endogenous immunity response in a subject administered with the enucleated cell.
• the enucleated cell expressing the membrane bound cytokine can be utilized to treat a disease or condition in the subject.
• the disease or condition can be cancer.
• the cancer can be attributed from manifestation of a cancer cell, a tumor microenvironment (TME), a metastatic cell, or a combination thereof.
• the membrane bound cytokine can be a membrane bound interleukin.
• the enucleated cell can be engineered to express at least one additional therapeutic.
• the enucleated cell can be engineered to express at least one additional therapeutic such as an immune checkpoint inhibitor or an immune checkpoint blocker (ICB).
• the active agent may be a protein.
• the active agent may be an antibody, antigen, or a fragment of said antibody or antigen.
• cells comprising mRNA to express a protein, an antibody, an antigen, or portion thereof, and compositions containing such cells.
• the cells are enucleated.
• the protein, antibody, antigen, or portion thereof is a therapeutic agent.
• the enucleated cells are capable of expressing the protein, antibody, antigen, or portion thereof (e.g., therapeutic agent) in the absence of a nucleus using one or more intracellular organelles retained by the enucleated cells from parent cells.
• the protein, antibody, antigen, or portion thereof e.g., therapeutic agent
• the enucleated cell expresses the protein, antibody, antigen, or portion thereof (e.g., therapeutic agent) at the surface of the enucleated cell.
• the composition comprises an enucleated cell described herein.
• the enucleated cell is obtained or derived from a nucleated cell (e.g., a parent cell). In some aspects, the enucleated cell comprises a transmembrane moiety. In some embodiments, the enucleated cells comprises a targeting moiety. In some embodiments, the enucleated cell comprises a therapeutic agent. In some embodiments, the enucleated cell comprises a protein. In some embodiments, the enucleated cell comprises a cytokine. In some WSGR Docket No.53712-720.601 embodiments, the enucleated cell comprises an antibody or antigen binding fragment thereof or a single-domain antibody or an antigen binding fragment thereof.
• the targeting moiety comprises an antibody or antigen binding fragment thereof or a single-domain antibody or an antigen binding fragment thereof.
• the therapeutic agent comprises an antibody or antigen binding fragment thereof.
• the enucleated cell is formulated into a pharmaceutical formulation described herein.
• Enucleated cells [0060] The enucleated cells of the present disclosure are obtained or derived from a corresponding nucleated cell (referred to herein as a “parent cell”). The parent cell may be derived from a variety of different cell types, including eukaryotic cells.
• an enucleated cell may be derived from an adult stem cell, a mesenchymal stromal cell (MSC), a natural killer (NK) cell, a macrophage, a myoblast, a neutrophil, endothelial cell, endothelial precursor cell, and/or a fibroblast.
• an enucleated cell is derived from a mesenchymal stromal cell.
• the enucleated cell is derived from an inducible pluripotent stem cell (iPSC).
• the parent cell is derived from a cell is immortalized using suitable methods.
• parent cell is immortalized by expressing human telomerase reverse transcriptase (hTERT), an oncogene, or a viral gene such as simian virus 40 (SV40).
• hTERT human telomerase reverse transcriptase
• SV40 viral gene
• the cytoplast is derived from a parent cell using suitable methods provided in United States Patent No.10,927,349, which is hereby incorporated by reference in its entirety.
• the enucleated cell retains one or more intracellular organelles for synthesis of an exogenous protein.
• the enucleated cell retains one or more intracellular organelles for synthesis of an exogenous cytokine.
• the enucleated cell does not require differentiation of the parent cell.
• the parent cell containing a nucleus is engineered to express the single-domain antibody or antigen-binding fragment thereof, therapeutic agent, transmembrane moiety, immune-evading moiety, and/or targeting moiety described herein; and subsequently, the nucleus of the parent cell is removed.
• the parent cell containing the nucleus is enucleated, and the enucleated cell is engineered to express single-domain antibody or antigen-binding fragment thereof, therapeutic agent, transmembrane moiety, immune-evading moiety, and/or targeting moiety described herein.
• the parent cell is engineered to express one or more of the biomolecules above (e.g., immune-evading moiety and/or targeting moiety), and the resulting enucleated cell (e.g., already expressing the immune- evading moiety and/or targeting moiety) is further engineered to express a second of the biomolecules above (e.g., a therapeutic agent).
• the biomolecules above e.g., immune-evading moiety and/or targeting moiety
• a second of the biomolecules above e.g., a therapeutic agent
• the enucleated cells of the present WSGR Docket No.53712-720.601 disclosure can be extensively engineered prior to enucleation, stored for long periods of time as needed (through for e.g., lyophilization, cryohibernation, cryopreservation), and quickly engineered to express a therapeutic agent closer to the time of need.
• the cell can originate from any organism having one or more cells.
• Non-limiting examples of cells include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g.
• algal cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like), seaweeds (e.g.
• a fungal cell e.g., a yeast cell, a cell from a mushroom
• an animal cell e.g. fruit fly, cnidarian, echinoderm, nematode, etc.
• a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
• a cell from a mammal e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.
• a mammal e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.
• a cell is not originating from a natural organism (e.g., a cell can be a synthetically made, sometimes termed an artificial cell).
• the cell is a somatic cell.
• the cell is a stem cell or a progenitor cell.
• the cell is a mesenchymal stem or progenitor cell.
• the cell is a hematopoietic stem or progenitor cell.
• the cell is a muscle cell, a skin cell, a blood cell, or an immune cell.
• lymphoid cells such as B cell, T cell (Cytotoxic T cell, Natural Killer T cell, Regulatory T cell, T helper cell), Natural killer cell, cytokine induced killer (CIK) cells; myeloid cells such as granulocytes (Basophil granulocyte, Eosinophil granulocyte, Neutrophil granulocyte/Hypersegmented neutrophil), Monocyte/Macrophage, Red blood cell (Reticulocyte), Mast cell, Thrombocyte/Megakaryocyte, Dendritic cell; cells from the endocrine system, including thyroid (Thyroid epithelial cell, Parafollicular cell), parathyroid (Parathyroid chief cell, Oxyphil cell), adrenal (Chromaffin cell), pineal (Pinealocyte) cells; cells of the nervous system, including glial cells (Astrocyte, Microglia), Magnocellular neurosecretory cell, Stellate cell, Boettcher cell, and pituitary (Gonadotrope
• Apocrine sweat gland cell odoriferous secretion, sex -hormone sensitive
• Gland of Moll cell in eyelid specialized sweat gland
• Sebaceous gland cell lipid-rich sebum secretion
• Bowman's gland cell in nose washes olfactory epithelium
• Brunner's gland cell in duodenum enzymes and alkaline mucus
• Seminal vesicle cell secretes seminal fluid components, including fructose for swimming sperm), Prostate gland cell (secretes seminal fluid components), Bulbourethral gland cell (mucus secretion), Bartholin's gland cell (vaginal lubricant secretion), Gland of Littre cell (mucus secretion), Uterus endometrium cell (carbohydrate secretion), Isolated goblet cell of respiratory and digestive tracts (mucus secretion), Stomach lining mucous cell (mucus secretion), Gas
• the cell is a eukaryotic cell.
• eukaryotic cells include mammalian (e.g., rodent, non-human primate, or human), non-mammalian animal (e.g., fish, bird, reptile, or amphibian), invertebrate, insect, fungal, or plant cells.
• the eukaryotic cell is a yeast cell such as Saccharomyces cerevisiae.
• the eukaryotic cell is a higher eukaryote such as mammalian, avian, plant, or insect cells.
• the nucleated cell is a primary cell.
• the nucleated cell is an immune cell (e.g., a lymphocyte (e.g., a T cell, a B cell), a macrophage, a natural killer cell, a neutrophil, a mast cell, a basophil, a dendritic cell, a monocyte, a myeloid- derived suppressor cell, an eosinophil).
• the nucleated cell is a phagocyte or a leukocyte.
• the nucleated cell is a stem cell (e.g., an adult stem cell (e.g., a hematopoietic stem cell, a mammary stem cell, an intestinal stem cell, mesenchymal stem cell, an endothelial stem cell, a neural stem cell, an olfactory adult stem cell, a neural crest stem cell, a testicular cell), an embryonic stem cell, an inducible pluripotent stem cell (iPS)).
• the nucleated cell is a progenitor cell.
• the nucleated cell is from a cell line.
• the nucleated cell is a suspension cell.
• the nucleated cell is an adherent cell.
• the nucleated cell is a cell that has been immortalized by expression of an oncogene.
• the nucleated cell is immortalized by the expression of human telomerase reverse transcriptase (hTERT) or any oncogene.
• the nucleated cell is immortalized by the expression of a viral gene such as simian virus 40 (SV40).
• the nucleated cell is a patient or subject derived cell (e.g., an autologous patient-derived cell, or an allogenic patient-derived cell).
• the enucleated cell is derived from an immune cell.
• the enucleated cell is derived from a natural killer (NK) cell, a neutrophil, a macrophage, a lymphocyte, a fibroblast, an adult stem cell (e.g., hematopoietic stem cell, a mammary stem cell, an intestinal stem cell, a mesenchymal stem cell, a mesenchymal stromal cell, an endothelial stem cell, a neural stem cell, an olfactory adult stem cell, a neural crest stem cell, a skin stem cell, or a testicular cell), a mast cell, a basophil, an eosinophil, an endothelial cell, an endothelial cell precursor cell, or an inducible pluripotent stem cell.
• NK natural killer
• neutrophil e.g., hematopoietic stem cell, a mammary stem cell, an intestinal stem cell, a mesenchy
• the parent cell is not an erythrocyte or erythroid precursor cell. In some embodiments, the parent cell is a platelet cell. In some embodiments, the parent cell is not an endothelial cell. In some embodiments, the parent cell is not an endothelial precursor cell. In some embodiments, the enucleated cell is not an erythrocyte or erythroid precursor cell. In some embodiments, the enucleated cell is not a platelet cell. In some embodiments, the enucleated cell is not an endothelial cell. In some embodiments, the enucleated cell is not an endothelial precursor cell.
• the enucleated cell does not express complement receptor one (CR1). In some embodiments, the enucleated cell does not express CD44. In some embodiments, the enucleated cell does not express VLA-4. In some embodiments, the enucleated cell does not express BCAM. In some embodiments, the enucleated cell does not express ICAM. In some embodiments, the enucleated cell does not express a receptor for collagen. In some embodiments, the enucleated cell does not express a receptor for thrombopoietin. In some embodiments, the enucleated cell does not express a receptor for collagen.
• CR1 complement receptor one
• the enucleated cell does not express CD44. In some embodiments, the enucleated cell does not express VLA-4. In some embodiments, the enucleated cell does not express BCAM. In some embodiments, the enucleated cell does not express ICAM. In some embodiments, the enucleated cell does not express
• the enucleated cell does not express a receptor for von Willebrand factor (VWF). In some embodiments, the enucleated cell does not express a receptor for fibrinogen. In some embodiments, the enucleated cell does not express GP1b-IX-V receptor. In some embodiments, the enucleated cell does not express GPIIb/IIIa receptor. In some embodiments, the enucleated cell does not express prostanoid receptor. In some embodiments, the enucleated cell does not express purinergic receptor. In some embodiments, the enucleated cell does not express thromboxane receptor.
• VWF von Willebrand factor
• the parent cell may be enucleated and engineered for therapeutic use.
• the parent cell is any one of the nucleated cells described herein.
• the parent cell is an adult stem cell.
• the parent cell is a mesenchymal stromal cell (MSC).
• the enucleated cell is derived from an inducible pluripotent stem cell (iPSC).
• iPSC inducible pluripotent stem cell
• the parent cell is not an erythrocyte or erythroid precursor cell.
• the parent cell is a platelet cell.
• the parent cell is not an endothelial cell.
• the parent cell is not an endothelial precursor cell.
• a parent cell may be treated with cytochalasin to soften the cortical actin cytoskeleton.
• the nucleus may then physically extracted from the cell body by high-speed centrifugation in gradients of Ficoll to generate an enucleated cell. Because enucleate cells and intact nucleated cells sediment to different layers in the Ficoll gradient, enucleated cells may be easily isolated and prepared for therapeutic purposes or fusion to other cells (nucleated or enucleated).
• the enucleation process is clinically scalable to process tens of millions of cells.
• enucleated cells may be used as a disease-homing vehicle to express clinically relevant cargos/payloads to treat various diseases.
• the enucleated cells described herein retain one or more intracellular organelles that are endogenous to the parent cell. In some embodiments, all of the one or more intracellular organelles are retained. In some embodiments, fewer than all of the one or more intracellular organelles are retained. In some embodiments, the Golgi apparatus and/or the endoplasmic reticulum are retained, which are involved in protein synthesis and secretion.
• Enucleated cells may be smaller than their nucleated counterparts (e.g., the nucleated parent cells), and for this reason may migrate better through small openings in the vasculature and tissue parenchyma.
• removing the large dense nucleus alleviates a major physical barrier allowing the cell to move freely through small openings in the vessels and tissue parenchyma.
• an enucleated cell has improved bio-distribution in the body and movement into target tissues.
• an enucleated cell comprises at least 1 ⁇ m in diameter. In some embodiments, an enucleated cell is greater than 1 ⁇ m in diameter.
• an enucleated cell is 1-100 ⁇ m in diameter (e.g., 1- 90 ⁇ m, 1-80 ⁇ m, 1-70 ⁇ m, 1- 60 ⁇ m, 1-50 ⁇ m, 1-40 ⁇ m, 1-30 ⁇ m, 1-20 ⁇ m, 1-10 ⁇ m, 1-5 ⁇ m, 5- 90 ⁇ m, 5-80 ⁇ m, 5-70 ⁇ m, 5- 60 ⁇ m, 5-50 ⁇ m, 5-40 ⁇ m, 5-30 ⁇ m, 5-20 ⁇ m, 5-10 ⁇ m, 10-90 ⁇ m, 10-80 ⁇ m, 10-70 ⁇ m, 10-60 ⁇ m, 10-50 ⁇ m, 10-40 ⁇ m, 10-30 ⁇ m, 10-20 ⁇ m, 10-15 ⁇ m 15-90 ⁇ m, 15-80 ⁇ m, 15-70 ⁇ m, 15- WSGR Docket No.53712-720.601 60 ⁇ m, 15-50 ⁇ m, 15-40 ⁇ m, 15-30 ⁇ m, 15-20 ⁇
• an enucleated cell is 10-30 ⁇ m in diameter. In some embodiments, the diameter of an enucleated cell is between 5-25 ⁇ m (e.g., 5-20 ⁇ m, 5-15 ⁇ m, 5-10 ⁇ m, 10-25 ⁇ m, 10-20 ⁇ m, 10-15 ⁇ m, 15- 25 ⁇ m, 15-20 ⁇ m, or 20-25 ⁇ m). In some embodiments, the enucleated cell has a diameter that is about 8 ⁇ m. In some embodiments, some enucleated cells may advantageously be small enough to allow for better homing to a target site.
• the enucleated cells described herein may pass through passages in narrow lung tissues or lung structures such as alveolar duct or microcapillary that most cells such as the parent cells may not pass through.
• enucleated cells possess significant therapeutic value, because they remain viable, do not differentiate into other cell types, secrete bioactive molecules, and may physically migrate/home for fewer than or equal to about 5 days, may be extensively enucleated ex vivo to perform specific therapeutic functions, and may be fused to the same or other cell types to transfer desirable production, natural or enucleated.
• enucleated cells have wide utility as a cellular vehicle to express therapeutically important biomolecules and disease-targeting cargos including genes, viruses, bacteria, mRNAs, shRNAs, siRNA, polypeptides (including antibodies and antigen binding fragments), plasmids, gene-editing machinery, or nanoparticles.
• the present disclosure enables the generation of safe (e.g., no unwanted DNA is transferred to the subject), and controllable (e.g., cell death occurs in 3-4 days) cell-based carrier that may be genetically enucleated to express specific disease-fighting and health promoting cargos to humans.
• the enucleated cell remains viable and retain the function to migrate or home for greater than or equal to about 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, or 5 days after being administered to the subject in need thereof.
• the enucleated cell is engineered to express at least one of an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, or an exogenous protein, gene-editing machinery or combinations thereof.
• the exogenous DNA molecule is a single-stranded DNA, a double-stranded DNA, an oligonucleotide, a plasmid, a bacterial DNA molecule, a DNA virus, or combinations thereof.
• the exogenous RNA molecule is messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), a RNA virus, or combinations thereof.
• the exogenous protein is a cytokine, a growth factor, a hormone, an antibody or the antigen-binding fragment thereof, an enzyme, or combinations thereof.
• the antibody is a single-domain antibody or antigen-binding fragment thereof.
• parental cells e.g., nucleated cells
• parental cells are WSGR Docket No.53712-720.601 genetically enucleated before enucleation (e.g., pre-enucleation).
• the parent cell is genetically enucleated after enucleation (e.g., post-enucleation).
• the enucleated cell described herein comprises a transmembrane moiety.
• the enucleated cell comprises an immune-evading moiety.
• the immune-evading comprises a “don’t eat me” signaling peptide such as CD47, PD-L1, HLA-E, HLA-G, a fragment thereof, or a combination thereof.
• the enucleated cell is depleted of an immune recognition molecule.
• immune recognition molecules include an HLA antigen, a proteoglycan, a sugar moiety, and an embryonic antigen.
• the enucleated cell or the composition comprising the enucleated cell may be cryopreserved (e.g., storing the enucleated cell or the composition comprising the enucleated cell at freezing temperature) or cryohibernated (e.g., storing the enucleated cell or the composition comprising the enucleated cell at a temperature that is between the ambient temperature and freezing temperature).
• the duration of cryopreservation or cryohibernation may be greater than or equal to about one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time.
• the enucleated cell exhibits a viability after cryopreservation or cryohibernation that is greater than or equal to about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar to a comparable cell (e.g., a parent cell or an enucleated cell described herein that has not been cryopreserved or cryo-hibernated) after same the period of time of cryopreservation or cryohibernation.
• a comparable cell e.g., a parent cell or an enucleated cell described herein that has not been cryopreserved or cryo-hibernated
• the viability is reduced by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as compared with the comparable cell.
• the viability is increased by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as compared with the comparable cell. Viability in this context may be measured by Trypan blue dye exclusion as described herein.
• the Trypan blue dye exclusion is performed by: (a) centrifuging an aliquot of a plurality of the cell without the nucleus in a suspension to create a cell pellet; (b) resuspending the cell pellet in serum-free medium to produce a serum-free cell suspension; (c) mixing 1 part Trypan blue dye and 1 part of the serum-free cell suspension; (d) counting the plurality of the cells without the nucleus within 3-5 minutes of (c), wherein at least some of the plurality of cells without the nucleus are unstained with the Trypan blue dye, which is indicative of viability.
• the viability is measured using Annexin-V cell surface staining.
• the viability is measured by expression of the exogenous polypeptide.
• the viability of the enucleated cell can be determined by the expression of the WSGR Docket No.53712-720.601 exogenous antibody or single-domain antibody expressed by the enucleated cell.
• the viability is measured by expression of cell surface markers of any one of the cell surface markers described herein such as CD105, CD90, CD45, CXCR4, PSGL-1, or CCR2.
• the viability is measured by the cell activity of the enucleated cell.
• the viability is measured by the homing capability of the enucleated cell as determined by the chemosensing or chemokine homing activity described herein.
• the enucleated cell or the composition comprising the enucleated cell may be lyophilized.
• the enucleated cell exhibits a viability after being reconstituted from lyophilization that is greater than or equal to about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar to a comparable cell (e.g., a parent cell or an enucleated cell described herein that has not been lyophilized).
• the enucleated cell or the composition comprising the enucleated cell may be dehydrated.
• the enucleated cell exhibits a viability after being rehydrated from lyophilization that is greater than or equal to about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar to a comparable cell (e.g., a parent cell or an enucleated cell described herein that has not been dehydrated).
• the enucleated cell or the composition comprising the enucleated cell is stable at 4°C for greater than or equal to about one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time.
• the composition is stable at room temperature for greater than or equal to about one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time.
• the composition is stable at 37°C for greater than or equal to about one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time.
• the enucleated cell or the composition comprising the enucleated cell may remain viable after being administered to a subject in need thereof for treating the disease or condition described herein.
• the enucleated cell or the composition comprising the enucleated cell may remain viable after being administered to the subject for greater than or equal to about one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time.
• WSGR Docket No.53712-720.601 the enucleated cell may be obtained from a parent cell that is autologous to the subject, who is in need of the treatment by the enucleated cell described herein.
• the enucleated cell may be obtained from a parent cell that is allogenic to the subject, who is in need of the treatment by the enucleated cell described herein.
• Active Agents [0077]
• An enucleated cell may comprise an active agent.
• the active agent may be encoded by an exogenous messenger RNA (mRNA).
• the active agent comprises a polypeptide, a sugar, a metabolite, a nucleic acid (e.g., RNA, DNA), a small molecule, or any combination thereof.
• the polypeptide may be a cytokine, cytokine receptor, integrin, or an adhesion protein, or a catalytically active fragment thereof.
• the adhesion protein may be PSGL-1.
• the cytokine may be a chemokine, for example, SDF-1 ⁇ , CCL2, IL-12, and IL-15.
• the cytokine may be a chemokine receptor, for example, CXCR2, CCR2, and CXCR4.
• the small molecule may be a chemosensor.
• the polypeptide may be expressed by the enucleated cell (in absence of the nucleus). In some embodiments, the polypeptide may be expressed by the nucleated parent cell prior to the enucleation to produce an enucleated cell comprising the polypeptide. In some embodiments, the active agent is expressed on the surface of the enucleated cell. In some embodiments, the active agent is or comprises a therapeutic agent of the present disclosure.
• the active agent may be a targeting moiety of the present disclosure.
• the active agent is or comprises a transmembrane moiety.
• the active agent is covalently coupled to the enucleated cell surface.
• the active agent is non-covalently coupled.
• the amount of active agent is about 0.10 to about 150.0 fold increase over endogenous amount of the active agent in an otherwise identical cell or a nucleated parent cell.
• the amount of the active agent is about 0.10 to about 0.50 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 0.10 to about 1.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 0.10 to about 2.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 3.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 4.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 5.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 10.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 15.00 fold increase over endogenous amount of the active agent.
• the amount of the WSGR Docket No.53712-720.601 active agent is about 0.10 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 40.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 0.10 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 70.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 0.10 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 100.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 0.10 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 130.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 0.10 to about 135.00 fold WSGR Docket No.53712-720.601 increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 1.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 0.50 to about 2.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 3.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 4.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 5.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 10.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 15.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 0.50 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 45.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 0.50 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 75.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 0.50 to about 80.00 fold increase over endogenous amount of the active WSGR Docket No.53712-720.601 agent. In some embodiments, the amount of the active agent is about 0.50 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 100.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 0.50 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 130.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 0.50 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.50 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 2.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 3.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 1.00 to about 4.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 5.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 10.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 15.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 25.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 1.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the WSGR Docket No.53712-720.601 active agent is about 1.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 50.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 1.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 80.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 1.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 110.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 1.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 140.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 1.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 1.00 to about 150.00 fold WSGR Docket No.53712-720.601 increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 3.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 4.00 fold increase over endogenous amount of the active agent. n some embodiments, the amount of the active agent is about 2.00 to about 5.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 2.00 to about 10.00 fold increase over endogenous amount of the active agent. n some embodiments, the amount of the active agent is about 2.00 to about 15.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 35.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 2.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 55.00 fold increase over endogenous amount of the active agent. n some embodiments, the amount of the active agent is about 2.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 65.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 2.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 95.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 2.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 105.00 fold increase over endogenous amount of the active WSGR Docket No.53712-720.601 agent. In some embodiments, the amount of the active agent is about 2.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 120.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 2.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 2.00 to about 150.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 3.00 to about 4.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 5.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 10.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 15.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 25.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 3.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 55.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 3.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the WSGR Docket No.53712-720.601 active agent is about 3.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 80.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 3.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 110.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 3.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 140.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 3.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 3.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 5.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 10.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 15.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 20.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 4.00 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 35.00 fold WSGR Docket No.53712-720.601 increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 45.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 4.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 75.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 4.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 105.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 4.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 135.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 4.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 4.00 to about 150.00 fold increase over endogenous amount of the active WSGR Docket No.53712-720.601 agent. In some embodiments, the amount of the active agent is about 5.00 to about 10.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 15.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 5.00 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 45.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 5.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 75.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 5.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 105.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 5.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the WSGR Docket No.53712-720.601 active agent is about 5.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 130.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 5.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 5.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 15.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 20.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 10.00 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 50.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 10.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 80.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 10.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 100.00 fold WSGR Docket No.53712-720.601 increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 105.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 10.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 135.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 10.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 10.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 20.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 30.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 15.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 60.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 15.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 80.00 fold increase over endogenous amount of the active WSGR Docket No.53712-720.601 agent. In some embodiments, the amount of the active agent is about 15.00 to about 85.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 15.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 115.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 15.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 15.00 to about 145.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 15.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 25.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 45.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 20.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the WSGR Docket No.53712-720.601 active agent is about 20.00 to about 70.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 20.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 100.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 20.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 130.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 20.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 20.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 30.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 35.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 25.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 60.00 fold WSGR Docket No.53712-720.601 increase over endogenous amount of the active agent.
• the amount of the active agent is about 25.00 to about 65.00 fold increase over endogenous amount of the active agent. n some embodiments, the amount of the active agent is about 25.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 90.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 25.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 120.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 25.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 25.00 to about 150.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 30.00 to about 35.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 55.00 fold increase over endogenous amount of the active WSGR Docket No.53712-720.601 agent.
• the amount of the active agent is about 30.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 85.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 30.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 95.00 fold increase over endogenous amount of the active agent. n some embodiments, the amount of the active agent is about 30.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 115.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 30.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 30.00 to about 145.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 30.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 40.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 55.00 fold increase over endogenous amount of the active agent.
• the amount of the WSGR Docket No.53712-720.601 active agent is about 35.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 80.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 35.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 110.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 35.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 140.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 35.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 35.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 45.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 60.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 40.00 to about 65.00 fold WSGR Docket No.53712-720.601 increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 85.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 40.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 115.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 40.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 40.00 to about 145.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 40.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 50.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 55.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 70.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 45.00 to about 75.00 fold increase over endogenous amount of the active WSGR Docket No.53712-720.601 agent. In some embodiments, the amount of the active agent is about 45.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 95.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 45.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 125.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 45.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 45.00 to about 150.00.In some embodiments, the amount of the active agent is about 50.00 to about 55.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 50.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 85.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 50.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 95.00 fold increase WSGR Docket No.53712-720.601 over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 110.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 50.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 140.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 50.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 50.00 to about 150.00.In some embodiments, the amount of the active agent is about 55.00 to about 60.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 70.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 75.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 55.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 105.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 55.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the WSGR Docket No.53712-720.601 active agent is about 55.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 130.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 55.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 55.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 65.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 70.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 60.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 100.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 60.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 130.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 60.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 145.00 fold WSGR Docket No.53712-720.601 increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 60.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 70.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 65.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 100.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 65.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 130.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 65.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 65.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 75.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 80.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 70.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 95.00 fold increase over endogenous amount of the active WSGR Docket No.53712-720.601 agent. In some embodiments, the amount of the active agent is about 70.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 105.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 70.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 135.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 70.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 70.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 80.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 90.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 75.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 120.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 75.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the WSGR Docket No.53712-720.601 active agent is about 75.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 75.00 to about 145.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 75.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 85.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 105.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 80.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 80.00 to about 130.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 80.00 to about 135.00. In some embodiments, the amount of the active agent is about 80.00 to about 140.00.In some embodiments, the amount of the active agent is about 80.00 to about 145.00.In some embodiments, the amount of the active agent is about 80.00 to about 150.00.In some embodiments, the amount of the active agent is about 85.00 to about 90.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 95.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 100.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 85.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 85.00 to about 120.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 85.00 to about 125.00. In some embodiments, the amount of the active agent is about 85.00 WSGR Docket No.53712-720.601 to about 130.00.In some embodiments, the amount of the active agent is about 85.00 to about 135.00.In some embodiments, the amount of the active agent is about 85.00 to about 140.00.In some embodiments, the amount of the active agent is about 85.00 to about 145.00.In some embodiments, the amount of the active agent is about 85.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 95.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 90.00 to about 100.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 125.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 90.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 90.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 100.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 95.00 to about 105.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 130.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 95.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is WSGR Docket No.53712-720.601 about 95.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 95.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 105.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 100.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 135.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 100.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 100.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 110.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 120.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 105.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 105.00 to about 150.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 110.00 to about 115.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 120.00 fold increase over WSGR Docket No.53712-720.601 endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 135.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 110.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 110.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 115.00 to about 120.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 115.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 115.00 to about 130.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 115.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 115.00 to about 140.00 fold increase over endogenous amount of the active agent. n some embodiments, the amount of the active agent is about 115.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 115.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 120.00 to about 125.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 120.00 to about 130.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 120.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 120.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 120.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 120.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 125.00 to about 130.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 125.00 to about 135.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 125.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 125.00 to about 145.00 fold increase over endogenous amount of the active agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the active agent is about 125.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 130.00 to about 135.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 130.00 to about 140.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 130.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 130.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 135.00 to about 140.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 135.00 to about 145.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 135.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 140.00 to about 145.00 fold increase over endogenous amount of the active agent.
• the amount of the active agent is about 140.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 145.00 to about 150.00 fold increase over endogenous amount of the active agent. In some embodiments, the amount of the active agent is about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 11
• the amount of the active agent is greater than or equal to about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, 150.0 fold increase over endogenous amount of the active agent in an otherwise identical cell or a nucleated parent cell.
• the amount of the active agent is less than or equal to about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, 150.0 fold increase over endogenous amount of the active agent in an otherwise identical cell or a nucleated parent cell.
• the WSGR Docket No.53712-720.601 amount of the active agent and the amount of the comparable endogenous active agent is determined by flow cytometry (FACS).
• the active agent may be a cytokine.
• the cytokine may be expressed by the mRNA in the enucleated cell.
• the cytokine may be expressed by the enucleated cell on the surface of the enucleated cell.
• the cytokine polypeptide encoded by the exogenous mRNA has decreased toxicity in the subject as compared to direct delivery of the cytokine polypeptide.
• the cytokine may comprise a transmembrane domain.
• the mRNA may encode the cytokine and the transmembrane domain.
• the cytokine may be attached to the cell surface of the enucleated cells.
• the cytokine may be attached, complexed, tethered, connected, coupled, linked, or any of the like to the cell surface of the enucleated cell.
• the cytokine may be attached to the cell surface of an enucleated cell exogenously.
• the cytokine is not delivered to a target tissue or microenvironment by an enucleated cell.
• the cytokine is not secreted to a target tissue or microenvironment by an enucleated cell.
• the mRNA encodes a cytokine or an antigen-binding fragment of a cytokine.
• the cytokine is an antigen-binding fragment of said cytokine.
• the cytokine is a full-length cytokine.
• the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.1 fold to at least 5.0 fold higher than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
• the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.1 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.2 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.3 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
• the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.4 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.5 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.6 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
• the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.7 fold than an enucleated cell expressing an abundance of a WSGR Docket No.53712-720.601 comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.8 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
• the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 0.9 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 1.0 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 1.2 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
• the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 1.4 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 1.6 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 1.8 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide.
• the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 2.0 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 2.2 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 2.4 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
• the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 2.6 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 2.8 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 3.0 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
• the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 3.2 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine WSGR Docket No.53712-720.601 polypeptide that is at least 3.4 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
• the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 3.6 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 3.8 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 4.0 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
• the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 4.2 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 4.4 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 4.6 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide.
• the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 4.8 fold than an enucleated cell expressing an abundance of a comparable naturally- occurring cytokine polypeptide. In some embodiments, the enucleated cell expresses an abundance of a cytokine polypeptide that is at least 5.0 fold than an enucleated cell expressing an abundance of a comparable naturally-occurring cytokine polypeptide. In some embodiments, the abundance of the cytokine polypeptide and the abundance of the comparable naturally- occurring cytokine polypeptide is determined by flow cytometry (FACS).
• FACS flow cytometry
• the cytokine polypeptide is coupled to the enucleated cell surface for a certain length of time. In some embodiments, the length of time is following delivery to a subject (e.g., administration) disclosed herein. In some embodiments, a cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 12 hours. In some embodiments, a cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 24 hours. In some embodiments, a cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 36 hours.
• a cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 48 hours. In some embodiments, a cytokine polypeptide is attached to the cell surface of the enucleated cell for at least 72 hours. In some embodiments, a cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 12 hours. In some embodiments, a cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 24 hours.
• a WSGR Docket No.53712-720.601 cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 36 hours. In some embodiments, a cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 48 hours. In some embodiments, a cytokine polypeptide is covalently linked or connected to the cell surface of the enucleated cell for at least 72 hours. In some embodiments, a cytokine polypeptide is expressed on a cell surface of an enucleated cell for at least 12 hours.
• a cytokine polypeptide is expressed on a cell surface of an enucleated cell for at least 24 hours. In some embodiments, a cytokine polypeptide can be expressed on a cell surface of an enucleated cell for at least 36 hours. In some embodiments, a cytokine polypeptide is expressed on a cell surface of an enucleated cell for at least 48 hours. In some embodiments, a cytokine polypeptide can be expressed on a cell surface of an enucleated cell for at least 72 hours. [0082]
• the enucleated cell disclosed herein may comprise an mRNA encoding a polypeptide.
• the polypeptide may be a cytokine.
• the cytokine may comprise an interleukin.
• the interleukin may comprise IL-12.
• the IL-12 may comprise IL-12 ⁇ .
• IL-12 ⁇ comprises SEQ ID NO: 35.
• IL-12 ⁇ comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35.
• the IL-12 may comprise IL-12 ⁇ .
• IL-12 ⁇ comprises SEQ ID NO: 36.
• IL-12 ⁇ comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 36.
• the IL-12 may comprise IL-12 ⁇ and IL-12 ⁇ .
• the IL-12 ⁇ and IL-12 ⁇ interact with a disulfide bond connecting the IL-12 ⁇ and IL-12 ⁇ .
• the IL-12 may comprise a transmembrane domain, such as a transmembrane from a B7 cell surface ligand.
• the transmembrane from the B7 cell surface ligand is from a B7-2 cell surface ligand.
• B7-2 cell surface ligand comprises SEQ ID NO: 37. In some embodiments, B7-2 cell surface ligand comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 37.
• the IL-12 comprises IL-12 ⁇ , IL-12 ⁇ , and a transmembrane B7-2 domain. In some embodiments, IL-12 ⁇ , IL-12 ⁇ , and the transmembrane B7-2 domain comprises SEQ ID NO: 33.
• IL-12 ⁇ , IL-12 ⁇ , and the transmembrane B7-2 domain comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33.
• the transmembrane domain e.g., transmembrane B7-2, can allow the IL-12 to be tethered to the cell surface.
• the interleukin may be IL-15.
• IL-15 comprises SEQ ID NO: 38.
• IL-15 comprises an amino acid WSGR Docket No.53712-720.601 sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 38.
• the IL-15 may further comprise IL-15 receptor alpha (RA).
• IL-15 RA comprises SEQ ID NO: 40.
• IL-15 RA comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 40.
• the IL-15 RA may comprise a sushi domain.
• the sushi domain comprises SEQ ID NO: 41.
• sushi domain comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 41.
• the sushi domain can allow the IL-15RA to interact with the IL-15.
• the sushi domain can allow the IL-15RA to bind to the IL-15.
• the IL-15 RA may comprise a transmembrane domain In some embodiments, the transmembrane domain comprises SEQ ID NO: 42.
• transmembrane domain comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 42.
• the IL-15 RA may comprise a sushi domain and a transmembrane domain.
• the sushi domain comprises cysteine residues that form disulfide bonds, such as cysteine residues at position 3, 29, 45, and 63 of SEQ ID NO: 41.
• the IL-15 and IL-15 RA may be connected.
• the IL-15 and IL-15 RA may be connected by a linker.
• the linker comprises SEQ ID NO: 39.
• linker comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 39.
• the polypeptide may comprise the IL-15, the IL-15 RA, and the linker.
• the IL-15, the IL-15 RA, and the linker comprises SEQ ID NO: 34.
• the IL- 15, the IL-15 RA, and the linker comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34.
• the sequences for these regions may be found in Table 9.
• Table 9 Amino acid sequences for IL-12 and IL-15 complexes WSGR Docket No.53712-720.601 [0083]
• the enucleated cell disclosed herein may comprise more than one mRNAs encoding more than one polypeptides.
• the polypeptides may comprise cytokines.
• the cytokines may comprise interleukins.
• the interleukins may comprise IL-12 and IL-15.
• IL-12 and IL-15 are expressed on the surface of the enucleated cell.
• the polypeptides comprise a cytokine and an immune checkpoint inhibitor.
• the enucleated cell disclosed herein may comprise an immune checkpoint inhibitor.
• the immune checkpoint inhibitor may comprise a PD-1 inhibitor, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a B7 inhibitor, a CD 137 inhibitor, a CTLA-4 inhibitor, or any combination thereof.
• the immune checkpoint inhibitor may be encoded by a second exogenous mRNA in the enucleated cell.
• the cells of the present disclosure comprise at least one therapeutic agent.
• the cells are enucleated, such as with the methods of enucleation disclosed herein.
• the therapeutic agent is or comprises an active agent of the present disclosure.
• an active agent comprises at least one of a DNA molecule, a RNA molecule, a protein (e.g., an enzyme, an antibody, an antigen, a toxin, cytokine, a protein hormone, a growth factor, a cell surface receptor, or a vaccine), a peptide (e.g., a peptide hormone or an antigen), a small molecule (e.g., a steroid, a polyketide, an alkaloid, a toxin, an antibiotic, an antiviral, a colchicine, a taxol, a mitomycin, or emtansine), a gene editing factor, a nanoparticle, or another active agent (e.g., bacteria, bacterial spores, bacteriophages, bacterial components, viruses (e.g., oncolytic viruses), exosomes, lipids, or ions).
• a protein e.g., an enzyme, an antibody, an antigen, a toxin
• Non-limiting examples of RNA molecules include messenger RNA (mRNA), short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA, long non-coding RNA (lncRNA) and a RNA virus.
• Non-limiting examples of DNA molecules include a single-stranded DNA, double-stranded DNA, an oligonucleotide, a plasmid, a bacterial DNA molecule and a DNA virus.
• Non-limiting examples of proteins include a cytokine, a growth factor, a hormone, an antibody or an antigen-binding fragment thereof, a single-domain antibody or antigen binding fragment thereof, a small-peptide based drug, and an enzyme.
• Non-limiting examples of oncolytic viruses include Talimogene laherparepvec, Onyx-015, GL-ONC1, CV706, Voyager- V1, and HSV-1716. Some wild-type viruses also show oncolytic behavior such as Vaccinia virus, Vesicular stomatitis virus, Poliovirus, Reovirus, Senecavirus, ECHO-7, or Semliki Forest virus.
• an enucleated cell is engineered to produce (e.g., express, and in some cases have tethered) the therapeutic agent.
• the parent cell from which the enucleated cell was obtained may be engineered to produce the therapeutic agent prior to enucleation to produce the enucleated cell.
• the enucleated cell is engineered to produce the therapeutic agent after enucleation (in absence of the nucleus).
• the therapeutic agent is exogenous to the enucleated cell or parent cell thereof.
• the therapeutic agent is endogenous to the enucleated cell or parent cell thereof.
• the enucleated cell of the present disclosure comprises at least one, two, three, four, five, six, seven, eight, nine, ten, or more therapeutic agents.
• the therapeutic agent comprises modified version of the DNA molecule, the RNA molecule, the protein, the peptide, the small molecule active agent, and/or the gene-editing factor as compared to a naturally occurring version.
• the WSGR Docket No.53712-720.601 therapeutic agent is a corrected, a truncated, or a non-mutated version and/or copy of the DNA molecule, the RNA molecule, the protein, the peptide, the small molecule active agent, and/or the gene-editing factor.
• the therapeutic agent can correct a mutated tumor protein p53 (p53) or epidermal growth factor receptor (EGFR) in the target cell or target environment as part of the treatment for lung cancer.
• the therapeutic agent may be coupled to the surface of the enucleated cell.
• the therapeutic agent may be, or include, a targeting moiety described herein.
• Non-limiting example of the targeting moieties that may be produced by or contained in an enucleated cell includes chemokine receptors, adhesion molecules, and antigen-binding polypeptides (e.g., single-domain antibodies and antigen-binding fragments thereof), or a portion thereof.
• the therapeutic agent may be, or include, a transmembrane moiety described herein.
• the therapeutic agent is covalently coupled to the enucleated cell surface. In some embodiments, the therapeutic agent is non-covalently coupled. In some embodiments, the amount of therapeutic agent is about 0.10 to about 150.0 fold increase over endogenous amount of the therapeutic agent in an otherwise identical cell or a nucleated parent cell. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 0.50 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 1.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 2.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 0.10 to about 3.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 4.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 5.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 10.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 15.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 20.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 0.10 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 0.10 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 45.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 0.10 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 75.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 0.10 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 105.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 0.10 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 135.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 0.10 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 0.10 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.10 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 1.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 2.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 0.50 to about 3.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 4.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 5.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 10.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 15.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 20.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 0.50 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 50.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 0.50 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 75.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 0.50 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 100.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 0.50 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 130.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 0.50 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 0.50 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 2.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 3.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 1.00 to about 4.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 5.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 10.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 15.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 1.00 to about 20.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 1.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 1.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 1.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 1.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 1.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 1.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 1.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 3.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 4.00 fold increase over endogenous amount of the therapeutic agent. n some embodiments, the amount of the therapeutic agent is about 2.00 to about 5.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 2.00 to about 10.00 fold increase over endogenous amount of the therapeutic agent. n some embodiments, the amount of the therapeutic agent is about 2.00 to about 15.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 20.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 2.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. n some embodiments, the amount of the therapeutic agent is about 2.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 2.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 2.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 2.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 2.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 3.00 to about 4.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 5.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 10.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 15.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 3.00 to about 20.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 3.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 3.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 3.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 3.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 3.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 3.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 3.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 5.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 10.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 15.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 4.00 to about 20.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 4.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 4.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 4.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 4.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 4.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 10.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 15.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 5.00 to about 20.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 5.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 5.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 5.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 5.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 5.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 5.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 5.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 15.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 10.00 to about 20.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 10.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 10.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 10.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 10.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 10.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 20.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 15.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 15.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 15.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 15.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 15.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 15.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 15.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 25.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 20.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 20.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 20.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 20.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 20.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 20.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 30.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 25.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 25.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. n some embodiments, the amount of the therapeutic agent is about 25.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 25.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 25.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 25.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 25.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 25.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 35.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 30.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 30.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. n some embodiments, the amount of the therapeutic agent is about 30.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 30.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 30.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 30.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 30.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 40.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 35.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 35.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 35.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 35.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 35.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 45.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 40.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 40.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 40.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 40.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 40.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 40.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 50.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 45.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 45.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 45.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 45.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 45.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 45.00 to about 150.00. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 55.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 50.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 50.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 115.00 fold increase WSGR Docket No.53712-720.601 over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 50.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 50.00 to about 150.00.In some embodiments, the amount of the therapeutic agent is about 55.00 to about 60.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 55.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 55.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 55.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 55.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 60.00 to about 65.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 60.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 60.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 60.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 60.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 60.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 70.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 65.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 65.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 65.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 70.00 to about 75.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent. In some WSGR Docket No.53712-720.601 embodiments, the amount of the therapeutic agent is about 70.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 70.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 70.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 70.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 80.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 75.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 75.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 75.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 75.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 85.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 90.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 80.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 80.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 80.00 to about 135.00.In some embodiments, the amount of the therapeutic agent is about 80.00 to about 140.00.In some embodiments, the amount of the therapeutic agent is about 80.00 to about 145.00.In some embodiments, the amount of the therapeutic agent is about 80.00 to about 150.00.In some embodiments, the amount of the therapeutic agent is about 85.00 to about 90.00 fold increase WSGR Docket No.53712-720.601 over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 85.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 85.00 to about 125.00. In some embodiments, the amount of the therapeutic agent is about 85.00 to about 130.00.In some embodiments, the amount of the therapeutic agent is about 85.00 to about 135.00.In some embodiments, the amount of the therapeutic agent is about 85.00 to about 140.00.In some embodiments, the amount of the therapeutic agent is about 85.00 to about 145.00.In some embodiments, the amount of the therapeutic agent is about 85.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 95.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 90.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 90.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 145.00 fold increase WSGR Docket No.53712-720.601 over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 90.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 95.00 to about 100.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 95.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 95.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 105.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 100.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 100.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 145.00 fold increase WSGR Docket No.53712-720.601 over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 100.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 110.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 105.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 105.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 105.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 115.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 110.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 110.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 115.00 to about 120.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 115.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 115.00 to about 130.00 fold increase WSGR Docket No.53712-720.601 over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 115.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 115.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. n some embodiments, the amount of the therapeutic agent is about 115.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 115.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 120.00 to about 125.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 120.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 120.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 120.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 120.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 120.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 125.00 to about 130.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 125.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 125.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 125.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 125.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 130.00 to about 135.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 130.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 130.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 130.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 135.00 to about 140.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 135.00 to about 145.00 fold increase WSGR Docket No.53712-720.601 over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 135.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 140.00 to about 145.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 140.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent. In some embodiments, the amount of the therapeutic agent is about 145.00 to about 150.00 fold increase over endogenous amount of the therapeutic agent.
• the amount of the therapeutic agent is about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, 150.0 fold increase over endogenous amount of the therapeutic agent in an otherwise identical cell or a nucleated parent cell.
• the amount of the therapeutic agent is greater than or equal to about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, 150.0 fold increase over endogenous amount of the therapeutic agent in an otherwise identical cell or nucleated parent cell.
• the amount of the therapeutic agent is less than or equal to about 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.0, 3.10, 3.20, 3.30, 3.40, 3.50, 3.60, 3.70, 3.80, 3.90, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0, 115.0, 120.0, 125.0, 130.0, 135.0, 140.0, 145.0, 150.0 fold increase over endogenous amount of the therapeutic agent in an otherwise identical cell or nucleated parent cell. In some embodiments, the amount of the therapeutic agent and the amount of the
• the therapeutic agent is recombinantly expressed by the enucleated cell or parent cell thereof.
• the parent cell from which the enucleated cell is derived or obtained is engineered to produce or express the therapeutic agent.
• expression of the therapeutic agent is stable (e.g., permanent).
• the expression of the therapeutic agent by the parent cell is transient (e.g., non- permanent).
• the parent cell is enucleated prior to engineering the WSGR Docket No.53712-720.601 enucleated cell to recombinantly express the therapeutic agent.
• the parent cell is engineered to recombinantly express the therapeutic agent prior to enucleation.
• the therapeutic agent is not naturally expressed (e.g., in the absence of engineering) in the cell from which the enucleated cell was derived or obtained (e.g., the therapeutic agent is exogenous to the parent cell). In some embodiments, the therapeutic agent is not naturally expressed in the subject (e.g., the therapeutic agent is exogenous to the subject).
• the therapeutic agent is not naturally expressed in the subject at the intended site of therapy (e.g., a tumor, or a particular tissue such as the brain, the intestine, the lungs, the heart, the liver, the spleen, the pancreas, muscles, eyes, and the like) (e.g., the therapeutic agent is exogenous to the intended site of therapy).
• the level of the therapeutic agent is not naturally occurring in the enucleated cell of the parent cell, such as over expression or under expression of the therapeutic agent.
• the therapeutic agent is derived from a synthetic cell and loaded into the enucleated cell.
• the therapeutic agent may be endocytosed into the cell prior to or after enucleation of the cell.
• the therapeutic agent may be synthesized by the cell.
• the enucleated cell comprises a plurality of therapeutic agents.
• the enucleated cell comprises at least 1 (e.g., at least 1, 2, 3, 4, 5, or more) different DNA molecules, RNA molecules, proteins, peptides, small molecule active agents, or gene-editing factors, in any combination.
• a therapeutic agent comprises a DNA molecule and a small molecule active agent.
• the therapeutic agent comprises two different small molecule active agents.
• the therapeutic agent comprises a chemokine receptor (e.g., for targeting) and a small molecule active agent.
• the therapeutic agent comprises a polypeptide.
• the polypeptide is exogenous.
• the polypeptide is encoded by an exogenous polynucleotide delivered into the parent cell or the enucleated cell.
• the polypeptide is synthesized by at least one intracellular organelle of the enucleated cell.
• the polypeptide is tethered to the enucleated cell.
• the polypeptide is expressed on the cell surface or the enucleated cell.
• the enucleated cell expresses the polypeptide in a target environment.
• the target environment is a microenvironment.
• the microenvironment is a tumor microenvironment.
• the enucleated cell expresses the polypeptide at a target cell.
• the target cell is a cancer cell.
• the cancer cell expresses the cancer biomarker of any cancer described WSGR Docket No.53712-720.601 herein.
• the target cell is an endothelial cell.
• the endothelial cell expresses an endothelial biomarker described herein.
• the endothelial cell is a blood vessel cell. In some embodiments, the endothelial cell is a lymphatic vessel cell.
• the exogenous polypeptide comprises a cytokine.
• An “exogenous” polypeptide provided herein may refer to a presence of the polypeptide that is exogenous to the cell or enucleated cell, or an amount or level of expression of the polypeptide that exogenous to the cell or enucleated cell.
• the cytokine comprises a membrane binding domain. In some embodiments, the exogenous polypeptide comprises a soluble cytokine.
• the exogenous polypeptide can comprise an extracellular domain or fragment of the cytokine.
• the exogenous polypeptide comprises a solubility as determined by turbidimetric solubility assay or thermodynamic solubility assay by dissolving the exogenous polypeptide in solvent such as organic solvent, including dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetonitrile, etc., or inorganic solvent, including water or phosphate-buffered saline (PBS).
• solvent such as organic solvent, including dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetonitrile, etc.
• solvent such as organic solvent, including dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetonitrile, etc., or inorganic solvent, including water or phosphate-buffered saline (PBS).
• the exogenous polypeptide comprises a solubility that is at least 0.0001 mg/ml, 0.0005 mg/ml, 0.001 mg/ml, 0.005 mg/ml, 0.01 mg/ml, 0.05 mg/ml, 0.1 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 5.0 mg/ml, 10 mg/ml, 50 mg/ml, 100 mg/ml, 500 mg/ml 1,000 mg/ml 5,000 mg/ml, 10,000 mg/ml, 50,000 mg/ml, or 100,000 mg/ml.
• the cytokine comprises an interleukin. In some embodiments, the cytokine comprises chemokines.
• the cytokine comprises interferons. In some embodiments, the cytokine comprises a lymphokine. In some embodiments, the cytokine comprises a tumor necrosis factor. In some embodiments, the cytokine comprises a monokine. In some embodiments, the cytokine comprises a colony-stimulating factor. In some embodiments, the cytokine comprises a transforming growth factor.
• Non-limiting examples of cytokines include bFGF, TNF- ⁇ , IL-10, IL-12(p70), IL-1 ⁇ , IL-2, IL-6, GM-CSF, IL-13, IFN- ⁇ , TGF- ⁇ 1, TGF-02, TGF- ⁇ 3, and IL-15.
• the exogenous polypeptide comprises an interleukin. In some embodiments, the exogenous polypeptide comprises more than one interleukin.
• interleukins include IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, or IL36.
• a cytokine of the composition is an interleukin or a mutant thereof, including, but not limited to, wild-type and mutant forms of IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8. (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, Il29, IL30, IL31, IL32, IL33, IL35, or IL36.
• the cytokine comprises IL-12.
• the IL-12 comprises an IL-12 complex.
• the IL-12 complex comprises SEQ ID NO: 33.
• the IL-12 complex comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33.
• the IL*12 complex comprises IL-12 ⁇ , IL-12 ⁇ , and a transmembrane domain.
• the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 33. In some embodiments, the exogenous polypeptide comprises IL-12 ⁇ or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 35. In some embodiments, the exogenous polypeptide comprises IL-12 ⁇ or a catalytically active fragment thereof.
• the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 36. In some embodiments, the exogenous polypeptide comprises a transmembrane region or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 37. [0096] In some embodiments, the cytokine comprises IL-15. In some embodiments, the IL-15 is an IL-15 complex. In some embodiments, the IL-15 complex comprises SEQ ID NO: 34.
• the IL-15 complex comprises an amino acid sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34.
• the IL-15 complex is formed by the noncovalent assembly of IL-15 with dimeric or monomeric sushi domain of IL-15 receptor ⁇ (SuIL-15 RA).
• the complex is a fusion protein, e.g., with IgG4 Fc.
• the IL-15 complex comprises IL-15 RA with a sushi domain and a transmembrane domain connected to IL-15 by a linker.
• the cytokine comprises IL-15 and IL-12 in the same enucleated cell.
• the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 34.
• the exogenous polypeptide comprises IL-15 or a catalytically active fragment thereof.
• the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 38.
• the exogenous polypeptide comprises a linker.
• the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 39.
• the exogenous WSGR Docket No.53712-720.601 polypeptide comprises IL-15 RA or a catalytically active fragment thereof.
• the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 40.
• the exogenous polypeptide comprises a sushi domain of IL-15 RA or a catalytically active fragment thereof.
• the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 41. In some embodiments, the exogenous polypeptide comprises a transmembrane domain of IL-15 RA or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more identical to SEQ ID NO: 42. In some embodiments, the exogenous polypeptide comprises a tumor necrosis factor (TNF) superfamily member polypeptide or a catalytically active fragment thereof.
• TNF tumor necrosis factor
• TNF superfamily member polypeptide examples include Lymphotoxin alpha (TNF ⁇ ), Tumor necrosis factor (TNF ⁇ ), Lymphotoxin beta (TNF ⁇ ), OX40 ligand (CD252, Gp34, or CD134L), CD40 ligand (CD154, TRAP, Gp39, or T-BAM), Fas ligand (CD178, APTL, or CD95L), CD27 ligand (CD70), CD30 ligand (CD153), CD137 ligand (4-1 BBL), TNF-related apoptosis-inducing ligand (CD253 or APO-2L), Receptor activator of nuclear factor kappa- ⁇ ligand (CD254, OPGL, TRANCE, or ODF), TNF-related weak inducer of apoptosis (APO-3L or DR3L), a proliferation-inducing ligand (CD256, TALL-2, or TRDL1), B-cell activating factor (CD257,
• the therapeutic agent comprises any one of the immune checkpoint molecule described herein or an immune checkpoint molecule inhibitor for inhibiting any one of the immune checkpoint molecule described herein.
• the immune checkpoint molecule include PD-1, PD-L1, CTLA-4, VISTA, PDCD1LG2 (CD273), B7-H3 (also called CD276), A2AR, CD27, LAG3, TIM-3, T cell immunoreceptor with Ig and ITIM domains (TIGIT), CD73, NKG2A, PVRIG, PVRL2, CEACAM1, CEACAM5, CEACAM6, FAK, CCR-2, CCL-2, LIF, CD47, SIRP ⁇ , M-CSF, CSF-1R, IL-3, IL-1RAP, IL-8, SEMA4D, Angiopoietin-2, CLEVER-1, Axl, phosphatidylserine or a fragment thereof.
• the therapeutic agent comprises an antibody.
• the antibody is a single-domain antibody described herein.
• the antibody or the single-domain antibody binds to an immune checkpoint molecule.
• the single-domain antibody bind to, and modulates the expression or the activity of the immune checkpoint molecule.
• the single-domain antibody is an inhibitor of the activity or expression of the immune checkpoint molecule.
• the single-domain antibody is an activator of the activity or expression of the immune checkpoint molecule.
• the antibody or single-domain antibody binds to PD-1, PD-L1, CTLA-4, VISTA, PDCD1LG2 (CD273), B7-H3 (also called CD276), A2AR, CD27, LAG3, TIM-3, T cell immunoreceptor with Ig and ITIM domains (TIGIT), CD73, NKG2A, PVRIG, PVRL2, CEACAM1, CEACAM5, CEACAM6, FAK, CCR-2, CCL-2, LIF, CD47, SIRP ⁇ , M-CSF, CSF-1R, IL-3, IL-1RAP, IL-8, SEMA4D, Angiopoietin-2, CLEVER-1, Axl, phosphatidylserine or a fragment thereof.
• the therapeutic agent comprising the antibody or the single-domain antibody binds to PD-L1. In some embodiments, the therapeutic agent comprising the antibody or the single-domain antibody binds to CTLA-4.
• Targeting moiety [0100] Described herein, in some embodiments, are cell or enucleated cells comprising a targeting moiety. In some embodiments, the cells are enucleated, such as with the methods of enucleation disclosed herein.
• the targeting moiety described herein is designed to guide the enucleated cell to a target cell or target environment (e.g., tissue) in a subject following delivery (e.g., systemic delivery) of the enucleated cell to the subject.
• the targeting moiety is expressed on the surface of the enucleated cell. In some embodiments, the targeting moiety is complexed with a transmembrane moiety described herein. In some embodiments, the targeting moiety is secreted by the enucleated cell.
• the enucleated cells comprising the targeting moiety localizes at the target cell or target environment in a subject with a 2 fold, 5 fold, 10 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1,000 fold, 5,000 fold, or 10,000 fold increase as compared to localization of a comparable enucleated cell lacking the targeting moiety.
• the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 2 fold increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 5 fold increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 10 fold increased as compared to localization of a comparable enucleated cell lacking the targeting moiety.
• the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 20 fold increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 50 fold increased as compared to WSGR Docket No.53712-720.601 localization of a comparable enucleated cell lacking the targeting moiety.
• the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 5% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 10% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 20% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety.
• the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 30% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 40% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 50% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety.
• the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 60% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 70% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 80% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety.
• the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 90% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, the enucleated cell comprising the targeting moiety localizes at the target cell or target environment in a subject with at least a 100% increased as compared to localization of a comparable enucleated cell lacking the targeting moiety. [0101] In some embodiments, the targeting moiety comprises an exogenous antibody or an exogenous antigen-binding fragment for targeting a biomarker described herein.
• the targeting moiety comprises an exogenous antibody or an exogenous antigen- WSGR Docket No.53712-720.601 binding fragment for targeting a chemokine receptor or a chemokine ligand, or portion thereof, involved in chemokine signaling.
• the exogenous antibody is an exogenous single-domain antibody or fragment thereof.
• the targeting moiety targets the biomarker expressed by, or associated with, a target cell or with a microenvironment.
• the biomarker may be released by the target cell. The biomarker may indicate the presence of the disease or the condition.
• the biomarker is expressed by immune cells responding to the target cell or the microenvironment associated with the disease or the condition.
• the biomarker may be an epitope or antigen.
• the biomarker comprising the epitope may be bound by an antibody that is different from the antibody or the antigen-binding fragment thereof that confers therapeutic property (e.g., the therapeutic agent).
• the targeting moiety targets a biomarker expressed or released by a cancer cell that has metastasized.
• the cancer cell may arise from one tissue and subsequently metastasizes to a different location.
• the targeting moiety targets a biomarker expressed or released by an endothelial cell.
• the endothelial cells are a blood vessel cell. In some embodiments, the endothelial cell is a lymphatic vessel cell. In some embodiments, the biomarker is expressed or released by a blood vessel cell. In some embodiments, the biomarker is expressed or released by a lymphatic vessel cell.
• the enucleated cells may further include (e.g., by engineering or from the cell from which they were obtained) a surface marker that aids in their evasion of the subject immune system.
• the enucleated cells may include a CD47, PD-L1, HLA-E, HLA-G, a fragment thereof, or a combination thereof.
• a CD47, PD-L1, HLA-E, HLA-G, a fragment thereof, or a combination thereof helps to prevent the enucleated cells from being phagocytosed by macrophages.
• Non-limiting examples of cell-matrix receptors and cell-cell adhesion molecules include integrins, cadherins, glycoproteins, or heparin sulfate proteoglycans.
• the cell-matrix receptors or cell-cell adhesion molecules include PD-L1, HLA-E, or HLA-G.
• therapeutic molecules include tumor antigens and immunomodulatory peptides, polyamines, and ATP.
• the therapeutic molecules can be recognized by immune cells and can induce immune response.
• the therapeutic molecules can be 4-1BB or any one of the cytokine described herein to induce immune response.
• the enucleated cell is depleted of an immune recognition molecule.
• immune recognition molecules include an HLA antigen, a proteoglycan, a sugar moiety, and an embryonic antigen.
• WSGR Docket No.53712-720.601 Transmembrane moiety [0105] Described herein, in some embodiments, are cells or compositions comprising the enucleated cell comprising at least one transmembrane moiety.
• the cells are enucleated, such as with the methods of enucleation disclosed herein.
• transmembrane moiety is coupled to a polypeptide.
• the polypeptide is a cytokine.
• the polypeptide is an interleukin.
• the polypeptide is a single-domain antibody or antigen-binding fragment thereof, a therapeutic agent disclosed here, or a combination thereof.
• the transmembrane moiety is coupled by way of a covalent bond.
• the transmembrane moiety is a fusion protein comprising the single-domain antibody or antigen-binding fragment thereof, a therapeutic agent disclosed here, or a combination thereof.
• the exogenous polypeptide is complexed to the transmembrane moiety.
• the transmembrane moiety comprises a full length protein or a variation thereof or a fragment thereof.
• the transmembrane moiety is endogenous to the parent cell that is being enucleated for obtaining the enucleated cell.
• the transmembrane moiety may be an exogenous transmembrane moiety to the parent cell or to the enucleated cell.
• the transmembrane moiety comprises a single transmembrane ⁇ -helix (bitopic membrane protein.
• the transmembrane moiety comprises a polytopic transmembrane ⁇ - helical protein.
• the transmembrane moiety comprises a polytopic transmembrane ⁇ -sheet protein.
• the transmembrane moiety comprises a Type I, II, III, or IV transmembrane protein.
• Non-limiting examples of transmembrane protein may include CD4, CD14, glycophorin a (GPA), or any combination of integrins.
• the transmembrane moiety is added to the exogenous polypeptide by way of a modification.
• a transmembrane moiety may be added to the N or C- terminus of the exogenous polypeptide to insert the exogenous polypeptide into the cell membrane of the enucleated cell described herein.
• modifications that are made to the exogenous polypeptide to add the transmembrane moiety may include adding an anchor molecule.
• Anchor molecule can be any molecule (e.g., a glycolipid) that can be inserted and remain in the cellular membrane.
• the anchor molecules comprises a glycosylphosphatidylinositol, a farnesyl, a palmitate, a myristate, or a combination thereof.
• the enucleated cell comprises a cytokine.
• the cytokine comprises an interleukin.
• the cytokine comprises an IL-12 (interleukin 12).
• the cytokine comprises an IL-15 (interleukin 15).
• the exogenous cytokine or fragment thereof is complexed with a transmembrane moiety.
• the transmembrane moiety comprises a WSGR Docket No.53712-720.601 transmembrane polypeptide.
• the exogenous cytokine or fragment thereof is complexed with N-terminus of the transmembrane polypeptide.
• the exogenous cytokine or fragment thereof is complexed with C-terminus of the transmembrane polypeptide.
• the exogenous cytokine or fragment thereof comprises a modification relative to an otherwise identical reference cytokine or fragment thereof, wherein the modification anchors the exogenous cytokine or fragment thereof to an exoplasmic or a cytosolic side of a cell membrane of the enucleated cell.
• the modification comprises complexing the exogenous single-domain antibody or fragment thereof to glycosylphosphatidylinositol, famesyl, palmitate, myristate, or a combination thereof.
• the transmembrane domain comprises IL-12.
• IL-12 is coupled to a cell surface protein ligand.
• the cell surface protein ligand is from the B7 family of cell surface ligand proteins.
• B7 family members includes but is not limited to B7.1, B7.2, B7-H4, and B7-H3.
• Il-12 is coupled to a portion of a cell surface protein ligand.
• IL-12 is coupled to only a transmembrane portion of a cell surface protein ligand.
• IL-12 is coupled to the transmembrane portion of a B7 family member.
• IL-12 is coupled to the transmembrane portion of B7.1.
• IL-12 comprises IL-12 ⁇ .
• IL-12 comprises IL-12 ⁇ and IL-12 ⁇ .
• IL-12 ⁇ comprises a signal peptide and an IL-12 subunit alpha chain.
• IL-12 ⁇ comprises an IL-12 subunit alpha chain.
• the IL-12 subunit alpha chain comprises a disulfide linkage. In some embodiments, the IL-12 subunit alpha chain comprises more than one disulfide linkages. In some embodiments, the IL-12 subunit alpha chain comprises less than five disulfide linkages. In some embodiments, the IL-12 subunit alpha chain comprises four disulfide linkages. In some embodiments, the IL-12 subunit alpha chain comprises three disulfide linkages. In some embodiments, the IL-12 subunit alpha chain comprises a glycosylation modification. In some embodiments, the IL-12 subunit alpha chain comprises more than one glycosylation modification. In some embodiments, the glycosylation modification is N-linked.
• the glycosylation modification is N-Acetylglucosamine. In some embodiments, the glycosylation modification takes place on an asparagine residue. In some embodiments, the asparagine residue is located near the center of the amino acid sequence of the IL-12 subunit alpha chain. In some embodiments, the IL-12 subunit alpha chain has a disulfide bond that WSGR Docket No.53712-720.601 connects to an IL-12 subunit beta chain. In some embodiments, IL-12 ⁇ comprises a signal peptide and an interleukin-12 subunit beta. In some embodiments, IL-12 ⁇ comprises an interleukin-12 subunit beta.
• the IL-12 subunit beta chain comprises a disulfide linkage. In some embodiments, the IL-12 subunit beta chain comprises more than one disulfide linkages. In some embodiments, the IL-12 subunit beta chain comprises less than five disulfide linkages. In some embodiments, the IL-12 subunit beta chain comprises four disulfide linkages. In some embodiments, the IL-12 subunit beta chain comprises three disulfide linkages. In some embodiments, the IL-12 subunit beta chain comprises two disulfide linkages. In some embodiments, the IL-12 subunit beta chain comprises a glycosylation modification. In some embodiments, the IL-12 subunit beta chain comprises more than one glycosylation modification.
• the glycosylation modification is N-linked. In some embodiments, the glycosylation modification is N-Acetylglucosamine. In some embodiments, the glycosylation modification takes place on an asparagine residue. In some embodiments, the glycosylation modification is C-linked. In some embodiments, the glycosylation modification is C- mannosylation. In some embodiments, the glycosylation modification takes place on a tryptophan residue. [0109] In some embodiments, the transmembrane domain comprises IL-15. In some embodiments, IL-15 may be coupled to a transmembrane domain through a linker. In some embodiments, the transmembrane domain comprises IL-15 receptor-alpha (IL-15 R ⁇ ).
• IL-15 R ⁇ IL-15 receptor-alpha
• IL-15 may be coupled to IL-15 R ⁇ to the exoplasmic side of the cell membrane of the enucleated cell. As depicted in FIG.10A, IL-15 R ⁇ spans the cell membrane of an enucleated cell. The portion of IL-15 R ⁇ on the exoplasmic surface is coupled to IL-15 by a linker. On the opposite end of IL-15 from the linker connecting to IL-15 R ⁇ is a signal peptide. In some embodiments, IL-15 R ⁇ comprises a Sushi domain and a transmembrane region.
• the IL-15 R ⁇ comprises a Sushi domain, linker, proline/threonine-rich region, a transmembrane region, and a cytoplasmic domain. In some embodiments, IL-15 R ⁇ comprises the Sushi domain, a transmembrane region and any combination of a linker, proline/threonine-rich region, or a cytoplasmic domain. In some embodiments, the Sushi domain comprises two disulfide linkages. In some embodiments, the Sushi domain may bind to IL-15. In some embodiments, IL-15 R ⁇ comprises a glycosylation modification. In some embodiments, the Sushi domain comprises a glycosylation modification. In some embodiments, the glycosylation modification is N-linked.
• the glycosylation modification is N-Acetylglucosamine. In some embodiments, the glycosylation modification takes place on an asparagine residue. In some embodiments, IL-15 comprises a disulfide linkage. In some embodiments, IL-15 comprises two disulfide linkages. In some embodiments, IL-15 comprises a WSGR Docket No.53712-720.601 glycosylation modification. In some embodiments, IL-15 comprises two glycosylation modifications. In some embodiments, IL-15 comprises three glycosylation modifications. In some embodiments, IL-15 comprises one or more glycosylation modifications. In some embodiments, the glycosylation modification is N-linked.
• the glycosylation modification is N-Acetylglucosamine. In some embodiments, the glycosylation modification takes place on an asparagine residue.
• the enucleated cells described herein may be engineered to express one or more targeting moieties that, when expressed by the enucleated cell (e.g., on its surface), guide the enucleated cell to the target cell or tissue in vivo. In order to avoid unintended clearance of the enucleated cells in vivo, the enucleated cell may also include an immune system evading moiety (a “don't eat me” signaling polypeptide) such as CD47, PD-L1, HLA-E, or HLA-G.
• an immune system evading moiety such as CD47, PD-L1, HLA-E, or HLA-G.
• the enucleated cell described herein comprises an additional active agent such as a therapeutic agent.
• the enucleated cells may be engineered before or after enucleation to express targeting moieties (e.g., homing receptors), immune-evading moieties (e.g., “don’t eat me” signaling peptides), among other biomolecules sufficient to target the enucleated cell to a target tissue without risk of clearance by the immune system before they get there.
• the enucleated cells may be cryopreserved, cryo-hibernated, or cryodesiccated, and stored for long periods of time with their biological activity slowed or stopped.
• the enucleated cells may be engineered with immune evading moieties (e.g., CD34+) to avoid an antigenic response in the host.
• Enucleated cells may also be engineered to express cell-surface receptors (e.g, adhesion molecules, chemokine receptors) used for cellular homing, chemokine sensing, and other biological functions that are essential to targeting damaged tissue.
• An immune-evading moiety may comprise a signaling peptide, or portion thereof, that reduces cellular phagocytosis through its interaction with a signal receptor protein expressed by phagocytic cells such as macrophages and dendritic cells.
• the immune-evading moiety blocks immune cell recognition or immune cell activation.
• the compositions, methods and systems of the present disclosure utilize an enucleated cell platform developed by the inventors of the present disclosure and initial described in United States Patent Application No.10,927,349, which is hereby incorporated by reference in its entirety.
• additional utility and advantages of the enucleated cells disclosed herein are discussed in U.S. Patent Application No.18/176,259, filed February 28, 2023; U.S. Patent Application No.17/885,867, filed August 11, 2022; and U.S. Patent Application No. 18/190,838, filed March 27, 2023, each of which is hereby incorporated by reference in its entirety.
• nucleic Acids WSGR Docket No.53712-720.601 Disclosed herein, in some embodiments, are nucleic acids encoding or comprising a biomolecule (e.g., the therapeutic agent, transmembrane moiety, immune-evading moiety, and/or targeting moiety described herein) of the present disclosure. In some embodiments, the nucleic acid may be introduced into the parent cell or the enucleated cell.
• a biomolecule e.g., the therapeutic agent, transmembrane moiety, immune-evading moiety, and/or targeting moiety described herein
• Non-limiting examples for introducing a biomolecule into the parent cell or the enucleated cell include: liposome mediated transfer, an adenovirus, an adeno-associated virus, a herpes virus, a retroviral based vector, a lentiviral vector, electroporation, microinjection, lipofection, transfection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalection, hydrodynamic delivery, magnetofection, nanoparticle transfection, chemicalporation, or combinations thereof.
• a therapeutic agent, a virus, an antibody, or a nanoparticle may be introduced into the enucleated cells.
• the nucleated cell is transfected with a vector (e.g., a viral vector (e.g., a retrovirus vector (e.g., a lentivirus vector), an adeno-associated virus (AAV) vector, a vesicular virus vector (e.g., vesicular stomatitis virus (VSV) vector), or a hybrid virus vector), a plasmid) before the nucleated cell is enucleated using any of the enucleation techniques described herein.
• a viral vector e.g., a retrovirus vector (e.g., a lentivirus vector), an adeno-associated virus (AAV) vector, a vesicular virus vector (e.g., vesicular stomatitis virus (VSV) vector), or a hybrid virus vector
• IL-12 is encoded in a vector.
• IL-12 and a transmembrane region are encoded in a vector.
• IL-12, a transmembrane region, and a signal peptide are encoded in a vector.
• the vector is a lentiviral vector.
• the vector is a mammalian expression vector. As depicted in FIG.8B, IL-12 and the transmembrane region of B7 (B7TM) may be inserted in a vector such as the vector depicted (vector ID: VB190513-1151unt).
• the IL-12 B7TM coding region is preceded by a human eukaryotic translation elongation factor 1 ⁇ 1 promoter (EF1A) and a signal peptide or Kozak.
• the IL-12 B7TM coding region comprises SEQ ID NO: 10.
• the IL-12 B7TM coding region comprises a nucleotide sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10.
• IL-15 is encoded by a vector.
• IL-15 and IL-15 R ⁇ are encoded by a vector.
• IL-15, IL-15 R ⁇ , and a linker are encoded by a vector.
• IL-15, IL-15 R ⁇ , a linker, and a signal peptide are encoded by a vector.
• the vector is a lentiviral vector.
• the vector is a mammalian expression vector. As depicted in FIG.10B, IL-15 and IL-15 R ⁇ may be inserted in a vector such as the vector depicted (vector ID: VB190220- WSGR Docket No.53712-720.601 1118ayw).
• the IL-15 and IL-15 R ⁇ coding region is preceded by a human eukaryotic translation elongation factor 1 ⁇ 1 promoter (EF1A) and a signal peptide or Kozak.
• the IL-15 and IL-15 R ⁇ coding region comprises SEQ ID NO: 25.
• the IL-15 and IL-15 R ⁇ coding region comprises a nucleotide sequence having greater than or equal to about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 25.
• compositions described herein comprising the enucleated cells or the compositions described herein.
• the pharmaceutical formulations further comprise a pharmaceutically acceptable: carrier, excipient, diluent, or nebulized inhalant.
• the pharmaceutical formulations include two or more active agents, or two or more therapeutic agents as disclosed herein.
• the two or more active agents are contained in a single dosage unit such as, for example, when the enucleated cell comprises two or more therapeutic agents.
• the two or more active agents are contained in separate dosage units such as when the enucleated cell is administered separately from an additional therapeutic agent or adjuvant.
• the active agents that may be, in some embodiments, the additional therapeutic agent include a chemotherapeutic agent, cytotoxic agent, cytokine, growth-inhibitory agent, anti-hormonal agent, anti-angiogenic agent, cardio protectant, and/or checkpoint inhibitor.
• Non-limiting checkpoint inhibitor includes IMP321/Eftilagimod alpha (Immutep), Relatlimab BMS-986016, Ipilimumab (Yervoy), Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), Ipilimumab (Yervoy), LAG525, MK-4280, Irinotecan, Oxaliplatin, REGN3767, TSR-033, BI754111, Sym022, FS118 (a bi-specific anti-LAG3/PD-L1 antagonistic mAb), MGD013 (a bi-specific anti-LAG3/PD-1 antagonistic mAb), TSR-022, Niraparib, Bevacizumab, MBG453, Decitabine, Spartalizumab, Sym
• Non-limiting examples of active agents that may be, in some embodiments, the additional therapeutic agent include CPI- WSGR Docket No.53712-720.601 006 (for inhibiting CD73 and allowing T cell and APC activation); Monalizumab (for inhibiting NKG2A); COM701 (for inhibiting PVRIG/PVRL2 and activating T cell); CM24 (for inhibiting CEACAM1 and allowing T and NK cells activation); NEO-201 (for inhibiting CEACAM5 and CEACAM6 which allows T cell activation while interfering with tumor cell growth); Defactinib (for inhibiting FAK and interfering with tumor growth); PF-04136309 (for inhibiting CCR-2 and CCL-2 and allowing T cell recruitment and activation); MSC-1 (for inhibiting LIF and allowing T cell and APC activation while interfering with cancer growth); Hu5F9-G4 (5F9), ALX148,
• composition described herein may include, but not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.
• the pharmaceutical formulations including a therapeutic agent may be manufactured in a conventional manner such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.
• the pharmaceutical formulations may include at least an exogenous therapeutic agent as an active ingredient in free-acid or free-base form, or in a pharmaceutically acceptable salt form.
• the methods and compositions described herein include the use of N-oxides (if appropriate), crystalline forms, amorphous phases, as well as active metabolites of these compounds having the same type of activity.
• therapeutic agents exist in WSGR Docket No.53712-720.601 unsolvated form or in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the therapeutic agents are also considered to be disclosed herein.
• pharmaceutical formulations provided herein include one or more preservatives to inhibit microbial activity.
• Suitable preservatives include mercury- containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.
• pharmaceutical formulations described herein benefit from antioxidants, metal chelating agents, thiol containing compounds and other general stabilizing agents.
• stabilizing agents include, but are not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, I about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate 80, (g) 0.001% to about 0.05% w/v.
• polysorbate 20 (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.
• aqueous oral dispersions liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.
• a therapeutic agent as discussed herein e.g., therapeutic agent is formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection.
• formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for rehydration into sterile injectable solutions or dispersions.
• suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propyleneglycol, polyethylene- glycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
• Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
• formulations suitable for subcutaneous injection also contain additives such as preserving, wetting, WSGR Docket No.53712-720.601 emulsifying, and dispensing agents.
• Prevention of the growth of microorganisms may be ensured by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and the like.
• it is desirable to include isotonic agents such as sugars, sodium chloride, and the like.
• Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption such as aluminum monostearate and gelatin.
• a pharmaceutical formulations described herein is formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer.
• physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer.
• penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• appropriate formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are known.
• Parenteral injections may involve bolus injection or continuous infusion.
• Pharmaceutical formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative.
• the composition described herein may be in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
• a therapeutic agent is formulated for use as an aerosol, a mist or a powder.
• compositions described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or nebulizers, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of such as, by way of example only, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the therapeutic agent described herein and a suitable powder base such as lactose or starch.
• Formulations that include a composition are prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
• these compositions and formulations are prepared with suitable nontoxic pharmaceutically acceptable ingredients.
• suitable carriers is dependent upon the exact nature of the nasal dosage form desired, e.g., solutions, suspensions, ointments, or gels.
• Nasal dosage forms generally contain large amounts of water in addition to the active ingredient.
• WSGR Docket No.53712-720.601 other ingredients such as pH adjusters, emulsifiers or dispersing agents, preservatives, surfactants, gelling agents, or buffering and other stabilizing and solubilizing agents are optionally present.
• the nasal dosage form should be isotonic with nasal secretions.
• Pharmaceutical preparations for oral use are obtained by mixing one or more solid excipient with one or more of the compositions described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
• Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents are added such as the cross linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol
• cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose,
• the pharmaceutical formulations of the exogenous therapeutic agents are in the form of a capsules, including push fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol.
• the push fit capsules contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active therapeutic agent is dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers are added.
• a capsule may be prepared, for example, by placing the bulk blend of the formulation of the therapeutic agent inside of a capsule.
• the formulations non-aqueous suspensions and solutions
• the formulations are placed in standard gelatin capsules or non-gelatin capsules such as capsules comprising HPMC.
• the formulation is placed in a sprinkle capsule, wherein the capsule is swallowed whole or the capsule is opened and the contents sprinkled on food prior to eating.
• compositions for oral administration are in dosages suitable for such administration.
• solid oral dosage forms are prepared by mixing a composition with one or more of the following: antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.
• the solid dosage forms disclosed herein are in the form of a tablet, (including a suspension tablet, a fast-melt WSGR Docket No.53712-720.601 tablet, a bite-disintegration tablet, a rapid-disintegration tablet, an effervescent tablet, or a caplet), a pill, a powder, a capsule, solid dispersion, solid solution, bioerodible dosage form, controlled release formulations, pulsatile release dosage forms, multiparticulate dosage forms, beads, pellets, granules.
• the composition is in the form of a powder.
• Compressed tablets are solid dosage forms prepared by compacting the bulk blend of the formulations described above.
• tablets will include one or more flavoring agents.
• the tablets will include a film surrounding the final compressed tablet.
• the film coating may provide a delayed release of a therapeutic agent from the formulation.
• the film coating aids in patient compliance. Film coatings typically range from about 1% to about 3% of the tablet weight.
• solid dosage forms e.g., tablets, effervescent tablets, and capsules, are prepared by mixing particles of a therapeutic agent with one or more pharmaceutical excipients to form a bulk blend composition. The bulk blend is readily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules.
• the individual unit dosages include film coatings.
• dosage forms include microencapsulated formulations.
• one or more other compatible materials are present in the microencapsulation material.
• materials includes pH modifiers, erosion facilitators, anti- foaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.
• Liquid formulation dosage forms for oral administration are optionally aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups.
• the liquid dosage forms optionally include additives such as: (a) disintegrating agents; (b) dispersing agents; (c) wetting agents; (d) at least one preservative, (e) viscosity enhancing agents, (f) at least one sweetening agent, and (g) at least one flavoring agent.
• the aqueous dispersions further include a crystal-forming inhibitor.
• the pharmaceutical formulations described herein are self- emulsifying drug delivery systems (SEDDS).
• SEDDS self- emulsifying drug delivery systems
• Emulsions are dispersions of one immiscible phase in another, usually in the form of droplets.
• emulsions are created by vigorous mechanical dispersion.
• SEDDS as opposed to emulsions or microemulsions, spontaneously form emulsions when added to an excess of water without any external mechanical dispersion or agitation.
• An advantage of SEDDS is that only gentle mixing is required to distribute the WSGR Docket No.53712-720.601 droplets throughout the solution. Additionally, water or the aqueous phase is optionally added just prior to administration, which ensures stability of an unstable or hydrophobic active ingredient.
• the SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients.
• SEDDS provides improvements in the bioavailability of hydrophobic active ingredients.
• Buccal formulations are administered using a variety of formulations known in the art.
• the buccal dosage forms described herein may further include a bioerodible (hydrolysable) polymeric carrier that also serves to adhere the dosage form to the buccal mucosa.
• the compositions may take the form of tablets, lozenges, or gels formulated in a conventional manner.
• a pharmaceutical formulation is optionally formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer.
• physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• appropriate formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients.
• Parenteral injections optionally involve bolus injection or continuous infusion. Formulations for injection are optionally presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative.
• a composition described herein is in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• the compositions for parenteral administration include aqueous solutions of an agent that modulates the activity of a carotid body in water soluble form. Additionally, suspensions of an agent that modulates the activity of a carotid body are optionally prepared as appropriate, e.g., oily injection suspensions.
• compositions include, e.g., one or a combination of methods: (1) dry mixing, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion.
• Other methods include, e.g., spray drying, pan coating, melt granulation, granulation, fluidized bed spray drying or coating (e.g., wurster coating), tangential coating, top spraying, tableting, extruding and the like.
• the compositions are provided that include particles of a therapeutic agent and at least one dispersing agent or suspending agent for oral administration to a subject.
• the formulations may be a powder and/or granules for suspension, and upon admixture with water, a substantially uniform suspension is obtained.
• the pharmaceutical formulations optionally include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride.
• acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.
• the pharmaceutical formulations optionally include one or more salts in an amount required to bring osmolality of the composition into an acceptable range.
• salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.
• Other the pharmaceutical formulations optionally include one or more preservatives to inhibit microbial activity.
• Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.
• the aqueous suspensions and dispersions described herein remain in a homogenous state for at least 4 hours.
• an aqueous suspension is re- suspended into a homogenous suspension by physical agitation lasting less than 1 minute.
• no agitation is necessary to maintain a homogeneous aqueous dispersion.
• An aerosol formulation for nasal administration is generally an aqueous solution designed to be administered to the nasal passages in drops or sprays.
• Nasal solutions may be similar to nasal secretions in that they are generally isotonic and slightly buffered to maintain a pH of about 5.5 to about 6.5, although pH values outside of this range may additionally be used.
• Antimicrobial agents or preservatives may also be included in the formulation.
• An aerosol formulation for inhalations and inhalants may be designed so that the agent or combination of agents is carried into the respiratory tree of the subject when administered by the nasal or oral respiratory route. Inhalation solutions may be administered, for example, by a nebulizer.
• Inhalations or insufflations comprising finely powdered or liquid drugs, may be delivered to the respiratory system as a pharmaceutical aerosol of a solution or suspension of the agent or combination of agents in a propellant, e.g., to aid in disbursement.
• Propellants may be liquefied gases, including halocarbons, for example, fluorocarbons such as fluorinated chlorinated hydrocarbons, hydrochlorofluorocarbons, and hydrochlorocarbons, as well as hydrocarbons and hydrocarbon ethers.
• Halocarbon propellants may include fluorocarbon propellants in which all hydrogens are replaced with fluorine, chlorofluorocarbon propellants in which all hydrogens are replaced with chlorine and at least one fluorine, hydrogen-containing fluorocarbon propellants, and hydrogen- containing chlorofluorocarbon propellants.
• Hydrocarbon propellants useful include, for example, propane, isobutane, n-butane, pentane, isopentane and neopentane.
• a blend of hydrocarbons may also be used as a propellant.
• Ether propellants include, for example, dimethyl ether as well as the ethers.
• An aerosol formulation may also comprise more than one propellant.
• the aerosol formulation comprises more than one propellant from the same class such as two or more fluorocarbons; or more than one, more than two, more than three propellants from different classes such as a fluorohydrocarbon and a hydrocarbon.
• the compositions of the present disclosure may also be dispensed with a compressed gas, e.g., an inert gas such as carbon dioxide, nitrous oxide or nitrogen.
• Aerosol formulations may also include other components, for example, ethanol, isopropanol, propylene glycol, as well as surfactants or other components such as oils and detergents. These components may serve to stabilize the formulation and/or lubricate valve components.
• the aerosol formulation may be packaged under pressure and may be formulated as an aerosol using solutions, suspensions, emulsions, powders and semisolid preparations.
• a solution aerosol formulation comprises a solution of an agent such as a transporter, carrier, or ion channel inhibitor in (substantially) pure propellant or as a mixture of propellant and solvent.
• the solvent may be used to dissolve the agent and/or retard the evaporation of the propellant.
• Solvents may include, for example, water, ethanol and glycols. Any combination of suitable solvents may be use, optionally combined with preservatives, antioxidants, and/or other aerosol components.
• An aerosol formulation may be a dispersion or suspension.
• a suspension aerosol formulation comprises a suspension of an agent or combination of agents, e.g., a transporter, carrier, or ion channel inhibitor, and a dispersing agent.
• Dispersing agents may include, for example, sorbitan trioleate, oleyl alcohol, oleic acid, lecithin and corn oil.
• a suspension aerosol formulation may also include lubricants, preservatives, antioxidant, and/or other aerosol components.
• An aerosol formulation may similarly be formulated as an emulsion.
• An emulsion aerosol formulation may include, for example, an alcohol such as ethanol, a surfactant, water and a propellant, as well as an agent or combination of agents, e.g., a transporter, carrier, or ion channel.
• the surfactant used may be nonionic, anionic or cationic.
• One example of an emulsion aerosol formulation comprises, for example, ethanol, surfactant, water and propellant.
• Another WSGR Docket No.53712-720.601 example of an emulsion aerosol formulation comprises, for example, vegetable oil, glyceryl monostearate and propane.
• the methods comprise treating a disease or a condition of a subject by administering the enucleated cells to the subject.
• the method comprises administering to the subject having the disease or the condition an enucleated cell, wherein the enucleated cell comprises an active agent (e.g., exogenous polypeptide or a catalytically active fragment thereof) coupled to a surface of the enucleated cell.
• the active agent is expressed by the enucleated cell in the absence of the nucleus at the surface of the enucleated cell.
• the active agent is or comprises a cytokine polypeptide, or a catalytically active fragment thereof.
• methods of producing the enucleated cell do not require differentiation of the parent cell.
• the parent cell containing a nucleus is engineered to express the single-domain antibody or antigen-binding fragment thereof, therapeutic agent, transmembrane moiety, immune-evading moiety, and/or targeting moiety described herein; and subsequently, the nucleus of the parent cell is removed.
• the parent cell containing the nucleus is enucleated, and the enucleated cell is engineered to express single-domain antibody or antigen-binding fragment thereof, therapeutic agent, transmembrane moiety, immune-evading moiety, and/or targeting moiety described herein.
• the parent cell is engineered to express one or more of the biomolecules above (e.g., immune-evading moiety and/or targeting moiety), and the resulting enucleated cell (e.g., already expressing the immune- evading moiety and/or targeting moiety) is further engineered to express a second of the biomolecules above (e.g., a therapeutic agent).
• the enucleated cells of the present disclosure can be extensively engineered prior to enucleation, stored for long periods of time as needed (through for e.g., lyophilization, cryohibernation, cryopreservation), and quickly engineered to express a therapeutic agent closer to the time of need.
• the parent cell may be engineered prior to enucleation to express adhesion molecules, chemokine or retention receptors or both, that target the lymph tissue (e.g., lymph nodes) in a subject.
• the resulting enucleated cell is engineered to express and, in some cases, secrete the therapeutic agent such as, for example, an antibody or an antigen- binding fragment thereof (e.g., single-domain antibody).
• the enucleated WSGR Docket No.53712-720.601 cell may be administered to a subject in need thereof to treat a disease or a condition in the subject.
• the enucleated cell of the present disclosure may be prepared in advance and cryopreserved for a length of time. This means, the enucleated cell of the present disclosure (e.g., engineered to express the homing receptors, immune activators, etc.) may be rapidly deployed. Such technical aspect is particular important when the enucleated cell is used to treat a disease or a condition stemmed from an outbreak of pathogen exposure or infection.
• removal of the nucleus involves mechanically removing the nucleus.
• the parent cell may be treated with cytochalasin to soften the cortical actin cytoskeleton.
• enucleated cells are then physically extracted from the cell body by high-speed centrifugation in gradients of Ficoll to generate an enucleated cell. Because enucleate cells and intact nucleated cells sediment to different layers in the Ficoll gradient, enucleated cells may be easily isolated and prepared for therapeutic purposes or fusion to other cells (nucleated or enucleated). The enucleation process is clinically scalable to process tens of millions of cells. In some embodiments, enucleated cells may be used as a disease-homing vehicle to deliver clinically relevant cargos/payloads to treat various diseases.
• a biomolecule e.g., the therapeutic agent, transmembrane moiety, immune-evading moiety, and/or targeting moiety described herein.
• Non-limiting examples of methods that may be used to introduce a biomolecule into the parent cell or the enucleated cell include: liposome mediated transfer, an adenovirus, an adeno-associated virus, a herpes virus, a retroviral based vector, a lentiviral vector, electroporation, microinjection, lipofection, transfection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalection, hydrodynamic delivery, magnetofection, nanoparticle transfection, or combinations thereof.
• a therapeutic agent, a virus, an antibody, or a nanoparticle may be introduced into the enucleated cells.
• the enucleated cell is preserved via cryopreservation, cryohibernation, or lyophilization.
• Cryopreservation comprises freezing the enucleated cell
• cryohibernation comprises storing the enucleated cell at a temperature that is below room WSGR Docket No.53712-720.601 temperature but without freezing the enucleated cell.
• the enucleated cell is lyophilized.
• the lyophilized enucleated cell can be reconstituted, and the reconstituted enucleated cell exhibits comparable viability to the enucleated cell that has not been lyophilized.
• the lyophilization comprises components: freezing the cell; subjecting the cell to drying under a very low pressure (e.g., ⁇ 3000 mTorr) using vacuum. The drying component can lead to sublimation and dehydrate the cell while maintaining cellular viability and biologic function.
• the freezing phase comprises balancing the duration and temperature of the freezing to for maintaining cell viability and stability, appropriate crystal formation, and the speed of reconstitution.
• the triple point of a substance is the temperature and the pressure at which the sublimation curve, fusion curve and vaporization curve meet. Achievement of the triple point which varies for different substances ensures that sublimation rather than melting will occur in the following drying steps.
• larger ice crystals are preferred, because they form a network within the product that promotes faster removal of water vapor during sublimation.
• the product should be frozen slowly or the temperature can be cycled up and down in a process called annealing.
• Fresh or frozen living tissue or cells do not have a single homogeneous melting point (eutectic point) and consequently the freezing stage of the material (cells or tissue) is cooled below its triple point which represents the temperature and pressure at which the solid, liquid and gas phases of the material can coexist.
• Living cells do have a critical point on a phase diagram at which both the liquid and the gas phase of an object or substance have the same density and are therefore indistinguishable.
• the product critical point temperature must be maintained to prevent melt-back or cake collapse occurring during primary and secondary drying which reflects incomplete sublimation. In the case of substances where preservation of structure is required like living cells, large ice crystals maybe detrimental and may break the cell walls which can result in increasingly poor texture and loss of nutritive content.
• the freezing should be done rapidly, in order to lower the material to below its critical point quickly, thus avoiding the formation of large ice crystals.
• the freezing temperatures for cells or tissue can vary but ranges in general between ⁇ 50 °C ( ⁇ 58 °F) and ⁇ 80 °C ( ⁇ 112 °F).
• the ambient pressure is lowered to the range of a few millibars, and then heat is supplied by conduction or radiation to the material for the ice to sublime.
• the amount of heat necessary can be calculated using the sublimating molecules’ latent heat of sublimation. In this initial drying phase, about 95% of the water in the material or substance is sublimated.
• This phase is often slow and can even last for several days depending on the substance and technology employed but if too much heat is added to quickly the material's WSGR Docket No.53712-720.601 structure could be altered.
• pressure is controlled through the application of a partial vacuum.
• the vacuum speeds up the sublimation, making it useful as a deliberate drying process.
• a cold condenser chamber and/or condenser plates are used as a surface(s) for the water vapor to re-liquify and solidify on. It is important to note that in this range of pressure, the heat cannot be provided by a convection effect because of the low air density.
• the drying phase also aims to remove remaining unfrozen water molecules since the ice induced with freezing should be removed during the primary drying phase.
• This part of the freeze-drying process is governed by the material's adsorption isotherms.
• the temperature is raised higher than in the primary drying phase and can even be above 0 °C (32 °F), to break any physico-chemical interactions that have formed between the water molecules and the frozen material.
• the pressure is also lowered in this stage to encourage desorption.
• the vacuum is usually broken with an inert gas such as nitrogen before the material is sealed.
• the residual water content in the product is extremely low and should range from ⁇ 1% to 4% of the original concentration.
• the lyophilization of the enucleated cell comprises the use of lyoprotectants for retaining cell viability and biologic function.
• Lyoprotectant comprises addition of reagents, salts, or additives that protects cell during the desiccation process.
• Common lyoprotectants include trehalose, DMSO, methylcellulose, sucrose, antioxidants, human or animal serum proteins, and cellular stress proteins. Additionally, methods for increasing the transport of lyoprotectants inside the cells in suspension can be utilized as a way of improving the viability and function of cells after lyophilization.
• Methods of Treatment include electroporation, addition of reagents that enhance intracellular transport, genetic modification of cells to upregulate the expression of pores on cell membranes, and mechanical microfluidic devices that partially disrupt cell membrane integrity and potentially promote intracellular transport of lyoprotectants.
• Methods of Treatment [0158] Provided herein are methods of treating a disease or a condition in a subject by administering a composition described herein to the subject.
• the methods comprise engaging an innate and/or adaptive immunity (anti-tumor immunity) in a subject by activating the effector Th1 response in the subject.
• the active agent is a cytokine (e.g., IL-12, IL-15) or a catalytically-active fragment thereof.
• administration is by any suitable mode of administration, including systemic administration (e.g., intravenous, inhalation, etc.).
• the subject is human.
• the disease or the condition comprises cancer.
• the WSGR Docket No.53712-720.601 composition comprises an enucleated cell disclosed herein that has been engineered to comprise or express one or more active agents coupled to the surface of the enucleated cell.
• the one or more active agents comprises a cytokine (e.g., IL-12 or IL-15) or a catalytically active fragment thereof.
• the enucleated cell induces an endogenous immune response in a subject.
• the enucleated cell comprising a therapeutic agent induces an endogenous immune response in a microenvironment of a subject.
• the disease or condition affects a microenvironment, such as tumor microenvironment (in the case of cancer).
• the microenvironment is a tumor microenvironment.
• the microenvironment is a lymphatic microenvironment.
• the microenvironment is a hepatic microenvironment.
• the microenvironment is a spleen microenvironment.
• the microenvironment is a pancreatic microenvironment.
• the microenvironment is an epidermis microenvironment.
• the disease or condition affects two or more microenvironments disclosed herein.
• the disease or condition is associated with, or caused by, a cell.
• the cell comprises a cancer cell.
• the cell comprises an immune cell.
• the immune cell may comprise a leukocyte.
• the disease or condition is associated with a tissue.
• the disease or the condition comprises an infection (e.g., human immunodeficiency virus (HIV)-infection, Chagas disease, tuberculosis), a neurological disease (e.g., Parkinson’s Disease, Huntington’s Disease, Alzheimer’s Disease) an autoimmune disease (e.g., diabetes, Crohn’s disease, multiple sclerosis, sickle cell anemia), a cardiovascular disease (e.g., acute myocardial infarction, heart failure, refractory angina), a ophthalmologic disease, a skeletal disease, a metabolic disease (e.g., phenylketonuria, glycogen storage deficiency type 1A, Gaucher disease), an inflammatory disease (e.g., cancer, inflammatory bowel disease), or a disease caused by external pathogen or toxin in a subject.
• an infection e.g., human immunodeficiency virus (HIV)-infection, Chagas disease, tuberculosis
• a neurological disease e.g
• the subject is in need of, or has been determined to be in need of such an enucleated cell treatment.
• the enucleated cell comprises a receptor that binds to an epitope expressed by a cancer cell or an epitope associated with a tumor microenvironment.
• the receptor is an antibody or antigen-binding fragment.
• the receptor is a chimeric antigen receptor (CAR).
• the enucleated cell comprises atherapeutic agent that provides a therapeutic effect to treat cancer in a subject.
• the enucleated cell comprises an active agent that recruits immune cells to activate immune response against the cancer.
• the cancer comprises a cancer in lung tissue.
• the cancer is lung cancer.
• Non-limiting examples of cancer may include Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymph
• the target cancer cell represents a subpopulation within a cancer cell population such as a cancer stem cell.
• the cancer may be lung cancer, including non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), or any other lung cancer type.
• the lung cancer may include adenocarcinoma, squamous carcinoma, large cell (undifferentiated) carcinoma, large cell neuroendocrine carcinoma, adenosquamous carcinoma, sarcomatoid carcinoma, lung carcinoid tumor, or adenoid cystic carcinoma.
• Other non-limiting examples of lung cancer includes lymphoma, sarcoma, benign lung tumor, or hamartoma.
• the cancer is metastatic cancer.
• the cancer metastasized to the lung from a different tissue or source.
• the metastatic cancer that may be found in the lung may include breast cancer, colon cancer, prostate cancer, sarcoma, bladder cancer, neuroblastoma, and Wilm’s tumor.
• the disease or condition may be caused by a pathogen.
• the enucleated cell described herein comprises a cytokine that responds to an epitope expressed by the pathogen or an epitope associated with a microenvironment associated with the pathogen. In some cases, the response of the cytokine to the epitope confers therapeutic property against the pathogen.
• the reaction of the cytokine to the epitope recruits immune cells to activate immune response to confer therapeutic property against the pathogen.
• the disease or condition may be caused by virus, bacterium, fungus, WSGR Docket No.53712-720.601 parasite, or molecule resulted from detoxification.
• the pathogens may be easily disseminated or transmitted from person to person; result in high mortality rates and have the potential for major public health impact; may cause public panic and social disruption; and require special action for public health preparedness.
• pathogens may include Anthrax (Bacillus anthracis), Botulism (Clostridium botulinum toxin), Plague (Yersinia pestis), Smallpox (variola major), Tularemia (Francisella tularensis), or Viral hemorrhagic fevers, including Filoviruses (Ebola, Marburg) and Arenaviruses (Lassa, Machupo).
• the pathogen may be moderately easy to disseminate; result in moderate morbidity rates and low mortality rates; and require specific enhancements of diagnostic capacity and enhanced disease surveillance.
• Example of these pathogens may include Brucellosis (Brucella species), Epsilon toxin of Clostridium perfringens, Food safety threats (e.g., Salmonella species, Escherichia coli O157:H7, or Shigella), Glanders (Burkholderia mallei), Melioidosis (Burkholderia pseudomallei), Psittacosis (Chlamydia psittaci), Q fever (Coxiella burnetii), Ricin toxin from Ricinus communis (castor beans), Staphylococcal enterotoxin B, Typhus fever (Rickettsia prowazekii), Viral encephalitis (alphaviruses such as eastern equine encephalitis, Venezuelan equine encephalitis, and western equine encephalitis), or Water safety threats (e.g., Vibrio cholerae and Cryptosporidium parvum).
• the pathogen may include emerging pathogen that has a high potential for mortality and morbidity, but the extend of which is not fully understood.
• Non- limiting examples of these pathogens may include Nipah virus and hantavirus.
• Methods of Delivery are methods of delivering the compositions described herein.
• the methods include treating a disease or a condition of a subject by administering a composition described herein (e.g., a pharmaceutical composition containing enucleated cells) to the subject.
• administering an enucleated cell induces an endogenous immune response in a subject.
• administering an enucleated cell induces an endogenous immune response in a microenvironment of a subject. In some embodiments, administering an enucleated cell induces an endogenous immune response in a cell of a subject. In some embodiments, administering an enucleated cell induces an endogenous immune response in a proximity of a cell of a subject. In some embodiments, the endogenous immune response comprises an innate immune response. In some embodiments, the endogenous immune response comprises an adaptive immune response.
• therapeutically effective amounts of pharmaceutical formulations comprising the enucleated cells of the present disclosure are administered to a subject having a WSGR Docket No.53712-720.601 disease, disorder, or condition to be treated, e.g., cancer.
• the subject is a mammal.
• the mammal is a human.
• a therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent used and other factors.
• the therapeutic agents, and in some cases, pharmaceutical formulations described herein may be used singly or in combination with one or more therapeutic agents as components of mixtures.
• the pharmaceutical formulations described herein may be administered to a subject by appropriate administration routes, including but not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration routes.
• administration routes including but not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration routes.
• immortalized cells e.g., Human telomerase reverse transcriptase (hTERT) cells or cancer cells
• the administration vaccinates the subject against a disease or a condition disclosed herein.
• Non-limiting examples of human immortalized cells include HeLa (human epithelial) cells, 293/293T/ HEK-293T (human embryonic kidney) cells, SH-SY5Y (human neuroblastoma, cloned from bone marrow) cells, and the like.
• Non-limiting examples of mammalian immortalized cells include 3T3 (mouse embryonic fibroblast) cells, COS (monkey kidney) cells, MDCK (dog kidney epithelial) cells, CHO (Chinese hamster ovary) cells, PC12 (Rat pheochromacytoma chromaffin) cells, and Neuro-2a/N2a (mouse neuroblastoma) cells.
• cancer cells obtained from the subject can be administered to the subject to induce immunity against a tumor without risk of metastasis.
• the enucleated cancer cell can be allogenic, thus does not trigger other harmful immune response to the subject.
• the enucleated cell may comprise one or more of a targeting moiety, immune system evading moiety, therapeutic agent (e.g., mRNA encoding a cytokine), or a combination thereof.
• the enucleated cell can activate the innate or adaptive immunity of the subject by either immunizing the subject with a pathogen or an antigen.
• the enucleated cell can activate the innate or adaptive immunity of the subject by expressing a therapeutic agent that can activate the immune response of the subject.
• a therapeutic agent that can activate the immune response of the subject.
• the composition is administered at least once during a period of time (e.g., every 2 days, twice a week, once a week, every week, three times per month, two times per month, one time per month, every 2 months, every 3 months, every 4 months, every 5 WSGR Docket No.53712-720.601 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, once a year).
• the composition is administered two or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60,70, 80, 90, 100 times) during a period of time.
• the composition is administered in a therapeutically-effective amount by various forms and routes including, for example, oral, or topical administration.
• a composition may be administered by parenteral, intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral, subarachnoid, intraocular, intrasternal, ophthalmic, endothelial, local, intranasal, intrapulmonary, rectal, intraarterial, intrathecal, inhalation, intralesional, intradermal, epidural, intracapsular, subcapsular, intracardiac, transtracheal, subcuticular, subarachnoid, or intraspinal administration, e.g., injection or infusion.
• a composition may be administered by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa administration). In some embodiments, the composition is delivered via multiple administration routes. [0175] In some embodiments, the composition is administered by intravenous infusion. In some embodiments, the composition is administered by slow continuous infusion over a long period such as more than 24 hours. In some embodiments, the composition is administered as an intravenous injection or a short infusion. [0176] A composition may be administered in a local manner, for example, via injection of the agent directly into an organ, optionally in a depot or sustained release formulation or implant.
• a composition may be provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation.
• a rapid release form may provide an immediate release.
• An extended release formulation may provide a controlled release or a sustained delayed release.
• a pump may be used for delivery of the composition.
• a pen delivery device may be used, for example, for subcutaneous delivery of a composition of the disclosure.
• the composition provided herein may be administered in conjunction with other therapies disclosed herein, including, for example, an antiviral therapy, a chemotherapy, an antibiotic, a cell therapy, a cytokine therapy, or an anti-inflammatory agent.
• a circular polyribonucleotide or the antibody or the antigen-binding fragment thereof described herein may be used singly or in combination with one or more therapeutic agents as a component of mixtures.
• a linear polyribonucleotide or the antibody or the antigen-binding fragment thereof described herein may be used singly or in combination with one or more therapeutic agents as a component of mixtures.
• WSGR Docket No.53712-720.601 The compositions (e.g., enucleated cells or pharmaceutical formulation comprising the enucleated cell described herein) may be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the composition containing a therapeutic agent may vary.
• the composition may be used as a prophylactic and may be administered continuously to subjects (e.g., the subject for immunization or the subject for treatment) with a susceptibility to a coronavirus or a propensity to a condition or disease associated with a coronavirus.
• Prophylactic administration may lessen a likelihood of the occurrence of the infection, disease or condition, or may reduce the severity of the infection, disease or condition.
• the composition may be administered to a subject before the onset of the symptoms.
• the composition may be administered to a subject (e.g., the subject for immunization or the subject for treatment) after (e.g., as soon as possible after) a test result, for example, a test result that provides a diagnosis, a test that shows the presence of a coronavirus in a subject (e.g., the subject for immunization or the subject for treatment), or a test showing progress of a condition, e.g., decreased blood oxygen levels.
• a therapeutic agent may be administered after (e.g., as soon as is practicable after) the onset of a disease or condition is detected or suspected.
• a therapeutic agent may be administered after (e.g., as soon as is practicable after) a potential exposure to a coronavirus, for example, after a subject (e.g., the subject for immunization or the subject for treatment) has contact with an infected subject, or learns they had contact with an infected subject that may be contagious.
• a subject e.g., the subject for immunization or the subject for treatment
• Actual dosage levels of an agent of the disclosure e.g., antibody or antigen-binding fragment thereof, or therapeutic agent
• an agent of the disclosure may be varied so as to obtain an amount of the agent to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject (e.g., the subject for immunization or the subject for treatment).
• the selected dosage level may depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
• Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic and/or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
• Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects (e.g., the subjects for immunization or the subjects for treatment); each unit contains a predetermined quantity of active agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the dosage unit forms of the disclosure may be determined by and directly dependent on (a) the unique characteristics of the active agent and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active agent for the treatment of sensitivity in individuals.
• a dose may be determined by reference to a plasma concentration or a local concentration of the circular polyribonucleotide or antibody or antigen-binding fragment thereof.
• a dose may be determined by reference to a plasma concentration or a local concentration of the linear polyribonucleotide or antibody or antigen-binding fragment thereof.
• a composition described herein may be in a unit dosage form suitable for a single administration of a precise dosage.
• the formulation may be divided into unit doses containing appropriate quantities of the compositions.
• the formulation may be divided into unit doses containing appropriate quantities of one or more linear polyribonucleotides, antibodies or the antigen-binding fragments thereof, and/or therapeutic agents.
• the unit dosage may be in the form of a package containing discrete quantities of the formulation.
• Non-limiting examples are packaged injectables, vials, and ampoules.
• An aqueous suspension composition disclosed herein may be packaged in a single- dose non-reclosable container. Multiple-dose reclosable containers may be used, for example, in combination with or without a preservative.
• a formulation for injection disclosed herein may be present in a unit dosage form, for example, in ampoules, or in multi dose containers with a preservative.
• a dose may be based on the amount of the agent per kilogram of body weight of a subject (e.g., the subject for immunization or the subject for treatment).
• a dose of an agent is in the range of 10-3000 mg/kg, e.g., 100-2000 mg/kg, e.g., 300-500 mg/kg/day for 1-10 or 1-5 days; e.g., 400 mg/kg/day for 3-6 days; e.g., 1 g/kg/d for 2-3 days
• a dose may be based on the number of the enucleated cells per kilogram of body weight of a subject.
• a dose may be is administered in a dosage amount of between about 1000 cells/kg body weight to about 1,000,000,000,000 cells/kg body weight. about 1,000 cells/kg body weight to about 1,000,000,000,000 cells/kg body weight.
• a dose may be is administered in a dosage amount of between about 1000 cells/kg body weight to about 1,000,000,000,000 cells/kg body weight.
• a dose may be is administered in a dosage amount of between about 1000 cells/kg body weight to about 1000000000000 cells/kg body weight. about 1,000 cells/kg body weight, about 10,000 cells/kg body weight, about 100,000 cells/kg body weight, about 1,000,000 cells/kg body weight, about 10,000,000 cells/kg body weight, about 100,000,000 cells/kg body weight, about 1,000,000,000 cells/kg body weight, about 10,000,000,000 cells/kg body weight, about 100,000,000,000 cells/kg body weight, or about 1,000,000,000,000 cells/kg body weight. In some embodiments, a dose may be is administered in a dosage amount of between about 1000 cells/kg body weight to about 1000000000000 cells/kg body weight.
• a dose may be is administered in a dosage amount of between about 1000 cells/kg body weight to about 1000000000 cells/kg body weight.
• the cell without the nucleus is administered to the subject twice within at least an hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 1 day, 2 days, a week, 2 weeks, 3 weeks, a month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, a year, 2 years, 3 years, or 4 years.
• each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and/or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.
• “or” may refer to “and”, “or,” or “and/or” and may be used both exclusively and inclusively.
• the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning.
• any systems, methods, software, and platforms described herein are modular. Accordingly, terms such as “first” and “second” do not necessarily imply priority, order of importance, or order of acts.
• the term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and the number or numerical range may vary from, for example, from 1% to 15% of the stated number or numerical range. In examples, the term “about” refers to ⁇ 10% of a stated number or value.
• the terms “increased”, “increasing”, or “increase” are used herein to generally mean an increase by a statically significant amount.
• the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, standard, or control.
• Other examples of “increase” include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.
• “decreased”, “decreasing”, or “decrease” are used herein generally to mean a decrease by a statistically significant amount.
• “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level.
• the decrease WSGR Docket No.53712-720.601 can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.
• Other examples of “decrease” include a decrease of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.
• the term “percent (%) identity”, or “percent sequence identity,” with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
• the term “percent (%) identity”, or “percent sequence identity,” with respect to a reference nucleic acid sequence is the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
• Alignment for purposes of determining percent sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
• the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
• the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code.
• the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
• the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues WSGR Docket No.53712-720.601 in B.
• Example 1 Engineered enucleated cells to secrete bioactive IL-12 for inducing antitumor immunity in a syngeneic mouse model of TNBC WSGR Docket No.53712-720.601 [0197] Enucleated cells were generated from hTERT immortalized human MSCs and engineered to express mouse IL-12 (Figs.1A-1C).
• Fig.1A illustrates an exemplary schematic of IL-12 mRNA design.5’ UTR and 3’ UTR of human alpha globin (HBA) mRNA were added to the 5’ and 3’ end of IL-12 coding regions (CDS), respectively. Artificial 5’ Cap and pseudouridine modification was implemented to increase mRNA stability.
• Fig.1B illustrates mesenchymal stem cells (MSCs), MSC-derived enucleated cells transfected with IL-12 mRNA (MSC IL-12 and Enucleated cell IL-12 respectively), or conditioned medium (CM) collected at the indicated time points. Graph shows the secreted IL-12 concentration in CM by ELISA.
• FIG. 1C illustrates CM from cells/enucleated cells as shown in Fig.1B assessed in mouse splenocytes for the activation of the phosphorylated/activated form of Stat4 (p-Stat4) by western blot.
• Complete media splenocytes treated with fresh media only.
• IL-12 protein splenocytes treated with purified recombinant IL-12 protein.
• enucleated cells that secreted IL-12 were injected intratumorally and readily secreted bioactive mouse IL-12 within the tumor microenvironment (Fig.1E), maintained minimal levels of serum IL-12 (Fig.1F), induced transcription of IL-12- mediated inflammatory biomarkers IFN-g, PD-L1, and CXCL9 (Fig.1G), and recruited key immune effector cells to the tumor site (cytotoxic CD8+ T cells, CD4+ T cells, increased M1/M2 macrophage ratio, and decreased Tregs, Fig.1H).
• Fig.1D illustrates exemplary schematic of syngeneic mouse E0771 TNBC model.
• Mice were subcutaneously (SQ) injected with E0771 cells.
• mice received intratumoral (IT) injection of either PBS, MSC transfected with IL-12 mRNA, or enucleated cells transfected with IL-12 mRNA (also known as CA-IL-12).
• Fig.1E illustrates mice treated as in Fig.1D, tumors (Fig.1E) and blood (Fig.1F) were harvested at the indicated timepoints post-IT injection to measure IL-12 by ELISA.
• Fig. 1G illustrates mice treated as in Fig.1D. Tumors were analyzed 24 hours after IT injection for indicated markers by PCR.
• Fig.1H illustrates mice bearing SQ E0771 tumors injected with a total of three doses of either PBS, MSCs secreting IL-12, or CA-IL-12. Forty-eight hours after the last dose, tumors were harvested for immunohistochemistry (Fig.1H) for indicated intratumoral leukocytes or FACS analysis (Fig.1I) of indicated leukocytes. All statistical analyses performed by one-way ANOVA with Dunnett’s correction. P values above indicated comparisons. NS, not significant; ****, p ⁇ 0.0001. In addition to low serum IL-12, no adverse health issues were noted in treated animals. In addition to low serum IL-12, no adverse health issues were noted in treated animals. Example 2.
• the same mouse model as in Example 1 was used to compare the survival of animals treated with enucleated cells that secreted IL-12 alone or in combination with the checkpoint inhibitor anti-PD-1 antibody (Fig.2A).
• Enucleated cells were transfected with IL-12 mRNA (CA-IL-12, 1 x10 6 ) and were intratumorally (IT) injected every 3 days into mice bearing subcutaneous (SQ) E0771 tumors.
• mice received IT injected PBS. Twenty-four hours after the third dose, either anti-PD-1 or control anti-IgG isotype was administered intraperitoneally (IP). The next week, a final enucleated cell expressing IL-12 or PBS dose was administered IT followed by anti-PD-1 or administered intraperitoneally (IP) anti-IgG IP the next day. Tumors were measured and animals euthanized when tumors grew > 2 cm diameter (Fig.2A). Animals treated with enucleated cells that secreted IL-12 plus anti-PD-1 had significantly improved survival compared with enucleated cells that secreted IL-12 or anti-PD-1 alone.
• Fig.2B shows a survival curve for animals injected, as described above (Fig.2A), pooled from two separate experiments.
• n 10 (5 males, 5 females)
• Mice in the enucleated cells that secreted IL-12+ anti-PD-1 group that survived greater than 175 days post-tumor initiation were re-challenged with SQ E0771 cells in the contralateral flank, and tumor size was assessed.
• Fig.2C shows a graph of tumor size over time post-injection. Complete tumor regression was again achieved, indicating a durable antitumor response. Injection of enucleated cells that secreted IL-12 did not negatively impact animal health, and no significant weight changes were noted in mice treated with enucleated cells that secrete IL-12 and PD-1.
• Fig.2D shows a graph of fold change in animal weight during the treatment phase of the survival experiments shown in Fig.2B.
• the arrows on the graph depict administration of IT CA-IL-12 or PBS.
• the downward triangle on the graph depicts administration of IP anti-PD-1 or anti-IgG.
• MSCs or enucleated cells were intravenously injected into mice with serum collected 2 and 24 hours post intravenous (i.v.) injection. The serum was analyzed for inflammation cytokines by ELISA (Table 1). The enucleated cells that secreted IL-12 that were injected intravenously (Table 1), did not have changes in acute phase immune markers detected indicating that enucleated cells that secreted IL-12 were minimally immunogenic.
• Inflammation cytokine measurements [0199] Finally, in a bilateral E0771 TNBC model, tumors were injected unilaterally with 3 doses of intertumoral enucleated cells that secreted IL-12 or PBS (control), and bilateral tumor size measured over time. Unilateral injection of intertumoral enucleated cells that secreted IL-12 reduced tumor growth in both tumors compared to controls, indicating that injection at a single site of enucleated cells that secreted IL-12 induced a systemic antitumor response to limit tumor growth at a distant site (contralateral tumor).
• Fig.2E shows fold change in tumor size for each side (injected and contralateral/noninjected) for animals that were bilaterally injected with E0771 cells and then were intratumorally injected unilaterally with 3 doses of CA-IL-12 or PBS. Therefore, enucleated cells effectively delivered complex immune-modulatory biologics to tumor sites and induced robust systemic antitumor immunity with a notable number of animals cured of TNBC.
• Example 3 Development of CA-IL-12 for intravenous (i.v.) delivery to metastatic TNBC [0200] The findings in Example 1 and Example 2 indicated that CA-IL-12 had important potential as an anti-cancer agent for IT applications. However, since many patients primary and secondary tumors are not amenable to IT injection, and i.v.
• enucleated cell therapeutics was also developed for i.v. delivery.
• Intravenous administration can be ideally suited for targeting systemic late-stage metastatic disease.
• a first in class enucleated cell vehicle for therapeutic delivery of immunomodulatory agents to metastatic tumors was developed based on cell chemotaxis and inflammatory diseases. To achieve this goal, enucleated cells were engineered with a leukocyte- like GPS system for improved homing to primary and secondary tumors. Leukocytes expressing chemoattractant receptors home to tumors in response to chemoattractant gradients produced by tumor cells and the TME.
• RNA-seq datasets generated from human TNBC primary tumors TME, and well-established mouse models of TNBC (EO771 and 4T1) were interrogated.
• Fig.3A shows scRNA-seq datasets generated from two human TNBC primary tumors were integrated with eight normal mammary datasets.
• VCAM1-positive vascular endothelial cells from clusters 0 and 4 Top panel of Fig.
• TNBC data were from NCBI SRA: SRP323816 and ERR3276428.
• Normal mammary data were from SRP301923 and SRP140533.
• Fig.3B shows Boyden Chamber migration assay showing mesenchymal stem cells or enucleated cells engineered for expressing CCR2, CXCR4, and leukocyte adhesion molecule (PSGL-1; termed CCP MSCs or CCP enucleated cells respectively) for robustly homing towards CCL2, SDF-1a, and EO771 conditioned medium relative to a no-chemoattractant negative control.
• SDF-1a (CXCL12) and CCl2 chemoattractant cytokines were appreciably upregulated in TNBC compared to normal mammary tissue controls in patient and murine tumors.
• Fig.3C shows RNA-seq analysis of the EO771 and 4T1 TNBC cell lines highly expressing Ccl2 and Cxcl12 (encoding SDF-1a).
• Fig.3D shows RNA-Seq of the 4T1 cell line revealing that the enucleated cell adhesion molecules/chemoattractants (encoding P-selectin), Ccl2, and Cxcl12 were upregulated with tumorigenesis (relative to tissue culture) and further upregulated in lung metastases.
• RNA-seq data RNA-seq replicates were from independent mice (biological replicates). RPKM denotes normalized expression. Horizontal bars denote medians.
• enucleated cells were reverse engineered with optimal levels of CXCR4 and CCR2 receptors that respond to SDF-1a and CCL2 guidance cues, respectively.
• circulating leukocytes uniquely expressed adhesion receptors that specifically recognize endothelial cell surface zip code molecules (i.e., selectins) upregulated on the luminal surface of the aberrant tumor vasculature.
• endothelial cell surface zip code molecules i.e., selectins
• CCP enucleated cells were engineered with CXCR4, CCR2, and PSGL-1 using lentiviruses, drug and/or FACS selection for high expressors.
• the cell surface expression of these molecules was monitored at various times post enucleation by FACS, shown to be stably expressed at high levels for at least 72 hours, deemed functional using established in vitro Boyden chamber cell migration assays (CCR2/CCL2, CXCR4/SDF-1a) (Fig.3B) and activated endothelial adhesion assays (PSGL-1/E-/P-selectins).
• CCP enucleated cells showed significantly improved chemotaxis migration toward a gradient of 4T1 and EO771 conditioned media relative to non-engineered hTERT MSCs (Fig.3B).
• enucleated cells could be reverse-engineered with both specific chemoattractant receptors that mediated migration to cancer cell-derived chemoattractants SDF-1a and CCL2 as well as PSGL-1 adhesion molecules that mediate attachment to activated endothelial cells.
• EO771 tumors showed significantly upregulated CCL2, SDF-1a1ct and P- /E-selectin by qPCR relative to untreated contralateral tumor that peaked on day 4 post RT (Fig. 4B).
• CCP enucleated cells (1x10 6 labeled with DiD) dye
• the animals were sacrificed 24 hours later. Both tumors were then resected, and processed for FACS quantification of enucleated cells.
• Bars the number of WSGR Docket No.53712-720.601 DiD enucleated cells relative to the total number of RFP+ EO771 tumor cells for irradiated and untreated contralateral tumors from the same animal. All statistical analyses performed by one- way ANOVA) (p ⁇ 0.01). CCP enucleated cells were labeled with DiD dye and injected i.v. on day 3 post RT. The number of CCP enucleated cells that home to untreated contralateral and RT tumors were analyzed by FACS 24 hours later relative to total extracted cells. Enucleated cells readily homed to untreated EO771 tumors which was further enhanced by 2-fold in tumors that received RT (Fig.4C).
• the lungs were resected, and thick precision sections were cut on a vibratome (400 ⁇ m) and examined for enucleated cell homing and intratumor distribution within metastatic foci by multiwave length confocal microscopy and the number of enucleated cell clusters per foci counted per field.
• Stepwise optical sections through small micrometastasis were prepared and rendered in 3D computer topography.
• Lungs from animals at this advanced stage of disease typically display numerous small micrometastasis (200 ⁇ m) and several large macrometastasis (200 ⁇ m to >1.0 mm), which is ideal for assessing enucleated cell homing and intratumoral distribution in small and large metastatic tumor nodules in the same lung sections. Strikingly, CCP enucleated cells strongly homed to micrometastasis with 97 ⁇ 11% of foci showing enucleated cell clustering and cancer cell colocalization (Fig.5A).
• Fig.5B CCP enucleated cells
• Fig.5B nonengineered enucleated cells localized deep within the dense tumor mass in most foci (53 ⁇ 6%).
• Confocal sections were acquired in a stepwise manner through the center of the tumor mass and then rendered in 3D.
• the top middle panel of Fig.5B is a DAPI stained section showing center of tumor mass.
• the top right panel has merged images showing DAPI, tumor and enucleated cells.
• the bottom panel is a single channel acquisition of enucleated cells with optical zoom to enhance clarity. Results representative of 3 animals with macrometastatic disease. The similar numbers of CCP enucleated cells (31 ⁇ 12%, clusters/field) and enucleated cell clusters (35 ⁇ 9%, cluster/field) were observed to distribute randomly within adjacent noninvolved lung tissue (Fig.5C).
• the top left panel of Fig.5C shows the single channel acquisition of enucleated cells distributed in the surrounding normal lung tissue.
• the top right panel of Fig.5C shows single channel acquisition of DAPI stained sections showing normal lung architecture and no tumor mass.
• the bottom left panel of Fig.5C shows single channel acquisition of EO771-RFP+ (Red) cells showing no tumor cells present.
• Intravenous treatment of mice with TNBC metastatic to the lung with CA-IL- 12 and PD-1 antibody robustly reduced metastatic burden and induced antitumor immunity.
• the following example looked to determine if CA-IL-12 could reduce tumor progression in mice harboring heavy TNCB metastatic burden. Mice with well-established lung metastases were injected i.v. with three doses of CA-IL-12 every 3 or 4 days alone or in combination with PD-1 antibody. Mice were treated IP with PD-1 antibody alone did not significantly impact metastatic progression in this model, which is refractory to checkpoint inhibitor therapy.
• Fig.6B shows representative images of resected lungs with metastases treated as indicated (top) and representative IHC images of CD8+ cytotoxic T cells (bottom) infiltrating EO771 lung metastases from the resected lungs shown in the top row.
• Fig. 7 shows FACS analysis of indicated leukocyte populations in EO771 lung metastases harvested at day 21 as in Fig.6 relative to total CD45+ immune cells. All statistical analyses performed by one-way ANOVA with Dunnett’s correction. P values above indicated comparisons. NS, not significant; ***, p ⁇ 0.0001. ** p ⁇ 0.00. * p ⁇ 0.05.
• CCP enucleated cells could home to and integrate within tumor tissue and deliver IL-12 (either secreted or membrane tethered) to the tumor proper and its surrounding stroma to activate a potent and localized anti-tumor immune response.
• IL-12 either secreted or membrane tethered
• the choice of a suitable transmembrane anchored sequence was important in achieving high and stable expression of chimeric proteins on the plasma membrane, the highest level of surface expression being achieved when proteins were fused to the transmembrane domain and cytoplasmic tail of B7-1 (B7TM).
• Fig.8A a schematic diagram of enucleated cell expressing soluble IL-12 (also known as CA-scIL-12), which was composed of the murine immunoglobulin ⁇ -chain signal peptide, the murine single-chain IL-12 p40 fused to the murine B7-1 transmembrane domain (B7TM).
• the single-chain IL-12 was in the p40-p35 subunit orientation, with the two subunits separated by a flexible peptide linker.
• Fig.8B illustrates a non-limiting example of a vector map of a vector encoding IL-12.
• Tables 3-5 illustrate: vector summary of the vector encoding IL-12; vector components of the vector encoding IL-12; and a nucleic acid sequence (SEQ ID NO: 1) of the vector encoding IL-12.
• Table 3. Vector summary of the vector encoding IL-12 Table 4.
• Vector components of the vector encoding IL-12 WSGR Docket No.53712-720.601
• WSGR Docket No.53712-720.601 Table 5.
• Nucleic acid sequence of the vector (SEQ ID NO: 1) encoding IL-12 WSGR Docket No.53712-720.601 961 CAATTGGAGA AGTGAATTAT ATAAATATAA AGTAGTAAAA ATTGAACCAT TAGGAGTAGC ACCCACCAAG GCAAAGAGAA 1041 GAGTGGTGCA GAGAGAAAAA AGAGCAGTGG GAATAGGAGC TTTGTTCCTT GGGTTCTTGG GAGCAGCAGG AAGCACTATG 1121 GGCGCAGCGT CAATGACGCT GACGGTACAG GCCAGACAAT TATTGTCTGG TATAGTGCAG CAGCAGAACA ATTTGCTGAG 1201 GGCTATTGAG GCGCAACAGC ATCTGTTGCA ACTCACAGTC TGGGGCATCA AGCAGCTCCA GGCAAGAATC CTGGCTGTGG 1281 AAAGATACCT AAAGGATCAA CAGCTCCTGG GGATTTGGGG TTGCTCTGGA AAACTCATTT GCACCACTGC TG
• FIG.8C illustrates immortalized mesenchymal stem cells (hTERT MSCs) infected with lentivirus vector encoding scIL-12 B7TM. Following drug selection, the engineered cells were FACS sorted to generate single cell clones, and expression of membrane-bound IL-12 was analyzed by FACS.
• Right panel of Fig.8C illustrates western blot of splenocytes activated with concanavalin A for 48 hours, then co-cultured with indicated MSCs (10M splenocytes versus 1 million MSCs, in 1 ml full media) at 37 ⁇ C for 30 minutes. Cells were pelleted and analyzed by western blot.
• Fig.8D illustrates an exemplary mechanism of immune cell activation by CA-scIL-12.
• CCP MSCs expressing cell surface IL-12 were generated using lentiviruses then WSGR Docket No.53712-720.601 FACS sorted for the top 1% highest expressors.
• CCP enucleated cells derived from these cells (CA-scIL-12) showed robust scIL-12 with stably expression for at least 72 hours post enucleation.
• Fig.8D shows that CCP MSCs were infected with lentiviruses encoding scIL-12 and a stable resistant cell line generated by drug selection, then enriched by fluorescence- activated cell sorting (FACS) using anti-IL-12 monoclonal antibody (mAb) to select the top 1% cells expressing the highest amounts of scIL-12, designated CA-scIL-12 MSC parent.
• FACS fluorescence- activated cell sorting
• mAb anti-IL-12 monoclonal antibody
• CA- scIL-12 also induced strong IFN- ⁇ secretion in a standard mixed splenocyte assay that is comparable to purified recombinant IL-12 indicating that it is highly bioactive (Fig.9A).
• Fig. 9A shows MSCs or enucleated cells engineered with or without scIL-12 that were co-cultured with primary murine splenocytes and concanavalin A for 48 hours.
• splenocytes were treated with 20 ng of purified mouse recombinant IL-12 concanavalin A for 48 hours. IFN- ⁇ protein levels were then determined by ELISA of 1:100 diluted co-culture conditioned media collected from the indicated groups.
• CA-IL-12 and CA-scIL-12 induced significant CXCL9 and IFN- ⁇ expression in the lungs with CA-IL-12 showing the highest response (Fig.9B and Fig.9C).
• CXCL9/IFN- ⁇ levels were substantially reduced in the liver compared to lungs for both CA-IL-12 and CA-scIL-12 treated groups (Fig. 9B and Fig.9C).
• Fig.9B enucleated cells (1 x10 6 ) were engineered to secrete IL-12 (CA-IL- 12), while in Fig.9C enucleated cells were engineered with cell surface IL-12 (CA-scIL-12) and were injected i.v.
• IL-12 and IFN- ⁇ levels were 10-fold and 4-fold lower in the CA-scIL-12 treated group than CA-IL-12 treated animals, respectively (Fig.9D).
• the reported maximum tolerated dose (MTD) of IL-12 was 500 ng/kg/day when administered i.v. for two weeks.
• Pilot experiments were performed to determine if CA-IL-12 could home to TNBC lung metastases using the approach described above in Fig.5 and Fig.6.
• CA-IL-12 readily homed to and integrated into micro- (93 ⁇ 11%) and macrometastasis (67% ⁇ 15%) in the lungs, which was similar to CCP enucleated cells (97 ⁇ 11% and 59 ⁇ 9%). Changes were not noted in integrated enucleated cells as in Fig.5. Strikingly, 93 ⁇ 11% and 97 ⁇ 11% of metastases observed showed infiltrated CA-IL-12 and CCP enucleated cells, respectively.
• CA-IL-12 product has significant potential to clear the lungs of micrometastatic disease, which has implications as an adjuvant therapy for TNBC as well as late-stage metastatic cancers.
• a single i.v. dose of CA-IL-12 did not impact the number of macrometastasis indicating that additional doses of CA-IL-12 are not necessary to impact large metastatic nodules.
• CA-scIL-12 with similar findings.
• Fig.10A illustrates an exemplary design of the enucleated cell expressing cell membrane tethered IL-15.
• the IL-15 is connected to the IL-15 receptor alpha by a linker.
• a signal peptide can also be connected to the IL-15.
• the fusion of IL-15 receptor alpha can increase IL-15 activity and response.
• Fig.10B illustrates a non-limiting example of a vector map for a vector encoding IL-15.
• Tables 6-8 illustrate: vector summary of the vector encoding IL-12; vector components of the vector encoding IL-15; and a nucleic acid sequence (SEQ ID NO: 2) of the vector encoding IL-15.
• Fig.10C illustrates hTERT MSCs infected with lentivirus construct encoding membrane-bound IL-15 receptor alpha. Following drug selection, cells underwent FACS sorting to generate single cell clones. Clone 1 was enucleated to generate enucleated cell WSGR Docket No.53712-720.601 expression IL-15Ra, which were analyzed by FACS for expression of IL-15Ra at the indicated time points. Table 6. Vector summary of the vector encoding IL-15 Table 7.
• Fig.11A illustrates a schematic for engineering the hTERT-MSC for expression both WSGR Docket No.53712-720.601 scIL-12 and IL-15R ⁇ and the subsequent enucleation of the hTERT-MSC.
• Fig.11B illustrates hTERT MSCs infected with two lentiviruses encoding constructs for either scIL-12 or IL-15R ⁇ . Following drug selection, cells were FACS sorted for single cell clones. Clone 4 was enucleated to generate enucleated cells expressing both scIL-12 and IL-15R ⁇ , and receptor expression was analyzed by FACS at the indicated time points.
• Fig.11C illustrates functionality of expressing membrane-bound IL-12, IL-15, or a combination of both IL-12 and IL-15.
• Pan-T cells were isolated from C57BL/6 mice.1.4x10 ⁇ 6 Pan-T cells/2 ml were co-cultured with hTERT MSCs expressing surface IL-12 a+b, hTERT MSCs expressing surface IL-15 R ⁇ , or hTERT MSCs expressing surface IL-12 a+b & IL-15 R ⁇ .
• 1.4x10 ⁇ 6 Pan-T cells/ml were co-cultured with hTERT MSCs without transfection or with IL-12 a+b transfection for 1 hour. The T cell only was taken as a negative control.
• CA-IL-12 and CA-scIL-12 Comparing the potential of CA-IL-12 and CA-scIL-12 to induce antitumor immunity against primary orthotopic TNBC tumors as well as secondary tumors in lungs and liver. Changes can be measured in a panel of key protein, gene, and immune cell biomarkers that report immune activation in the TME using established preclinical murine models of TNBC. The ability of CA-IL-12 medicines to transform the TME from relatively immunologically “cold” to “hot” tumors based on established biomarkers of antitumor immunity can be assessed. Changes in tumor regression and metastatic burden of CA-IL-12L medicine in combination with immune checkpoint blockade (ICB) therapy can be detailed.
• IRB immune checkpoint blockade
• Enucleated cell preparation can be developed as delivery vehicles engineered to decrease vascular trapping and allow for optimal systemic delivery to both primary and secondary tumors.
• MSCs can be cultured in spinner flask bioreactors to generate 3D-cultured MSCs that are smaller than traditional 2D-cultured MSCs (12 ⁇ m in diameter vs.16 ⁇ m, respectively).
• 3D-cultured MSCs are enucleated, the resulting 3D-enucleated cells are smaller (9.0 ⁇ m) and exhibit less vascular trapping improved extravasation, tumor penetration and best biodistribution kinetics following i.v. administration without major accumulation in liver, spleen, and kidney (Fig.12A and Fig.12B).
• the biodistribution of engineered MSCs and enucleated cells were monitored by bioluminescence.
• the mouse ear dermis was treated with WSGR Docket No.53712-720.601 LPS and i.v.-injected with 3D cultured CCP (CXCR4, CCR2, and PSGL-1) MSC or CCP enucleated cells each transfected with firefly luciferase mRNA.
• Mice were IP injected with D- luciferin at indicated time points after i.v. injection and mouse organs were subjected to bioluminescence imaging with IVIS Lumina Series III. Representative images were shown from two independent experiments in Fig.12A.
• significant numbers of 3D CCP enucleated cells rapidly home to SDF-1 ⁇ , CCL2, and P-selectin produced within inflamed tissues within 2 hours post injection (Fig.13A and Fig.13B).
• Fig.13A and Fig.13B show that CCP enucleated cells home better to the inflamed mouse ear than MSCs.
• the excised ears from the same mice as in Fig.12 were used and the dorsal skin of the ears was peeled away from the underlying cartilage.
• the subcutaneous surfaces of the tissue were directly exposed to D- luciferin substrate and subjected to quantitative bioluminescence imaging; representative images are shown from two independent experiments in Fig.13A.
• Primary tumors can be formed by inoculating 4T1 or E0771.LMB TNBC cells (50,000 in 50 ⁇ L) orthotopically into the inguinal mammary fat pad (mfp), and tumor growth can be monitored using digital calipers and by whole body luminescence. Under these conditions, primary tumors measuring 100 mm 3 form reproducibly (100% of mice) between 14-18 days.
• Pulmonary metastases can be formed by seeding TNBC cells by tail vein injection (200 ⁇ L).
• liver specific metastases can be established by standard hemi spleen injection. These transplantation models generate secondary tumors in nearly 100% of mice within 14 days and secondary tumor growth can be monitored using whole body luminescence BLI.
• enucleated cells can be labelled with Vybrant DiO dye (3,3'- Dioctadecyloxacarbocyanine perchlorate ( ⁇ ex 484 nm/ ⁇ em 501 nm) for direct fluorescence imaging (Figs.5-7) or PCR using human mitochondrial DNA loci.
• Vybrant DiO dye (3,3'- Dioctadecyloxacarbocyanine perchlorate ( ⁇ ex 484 nm/ ⁇ em 501 nm) for direct fluorescence imaging (Figs.5-7) or PCR using human mitochondrial DNA loci.
• Animal weight and tumor size can be recorded by BLI and analyzed semi-quantitatively by luminoscore and compared across groups by two-way ANOVA. Growth of primary tumors can be terminated when tumors reach 2,000 mm 3 or animals show signs of physical distress.
• mice euthanized and mammary fat pad (mfp), lungs, liver, heart, kidney, spleen as well as inguinal and popliteal lymph nodes excised and profiled for changes in health and immune biomarkers.
• mfp mammary fat pad
• Portions of each tissue can be prepared for fluorescent microscopy, IHC, nanostring analyses, or disaggregated into single cell suspensions for flow cytometry (FACS).
• FACS flow cytometry
• Serum and tissue samples from tumor bearing mice can be used for measurements of IL-12 and its effector IFN- ⁇ .
• tissue and serum concentrations of IL-12 and IFN- ⁇ can be measured by ELISA and by measuring changes in IL-12-induced biomarkers IFN- ⁇ , PD- 1/PD-L1, CXCL9, and CXCL10 by RT-PCR.
• Host-derived vs enucleated cell-derived IL-12 mRNA expression in breast, spleen, and liver by RT-PCR using oligonucleotide primers that distinguish innate host and engineered forms of IL-12 mRNAs can be measured.
• Tissues can also be examined for changes in 200 nCounter® Inflammation Biomarkers using a standard NanoString nCounter Analysis.
• Tissues and tumors can be collected for FACS analysis of key infiltrating leukocyte populations, including: CD8+ T cells (CD45.2+CD8+CD3+CD4-), CD4+ T cells (CD45.2+CD8- CD3+CD4+); T regulatory cells (CD45.2+CD4+CD25+FoxP3+); M1 macrophages (CD45+Ly6c-F4/80+MHC II+); M2 macrophages (CD45+Ly6c-F4/80+MHC II-); dendritic cells (CD45+ and Btla+Flt3+ or Ly-6C+TLR7+TLR9+), activated NK cells (CD45+CD3-NK1.1+ and CD107A, Granzyme B+ IFN- IFN-y+), and mature memory effector T cells (CD45ROne
• the remaining tumors can be split in half for analysis by ELISA and IHC.
• One set of halves can be analyzed by ELISA for IL-12 and IFN- ⁇ levels.
• the other half can be used for IHC staining with anti-CD8, anti-CD4, anti-FoxP3, and PD-1 and PD-L1 antibodies.
• the intratumor location and number of infiltrating leukocytes of each type can be quantified by counting immunopositive cells in 10 high-powered (400X) within the tumor and tumor/stroma interface. For FACS and IHC, the relative number of each leukocyte type can be compared between groups using two-way ANOVA.
• Organs can be WSGR Docket No.53712-720.601 weighed and undergo routine histologic processing to evaluate the presence or absence of metastatic disease and general histopathology. The number and size of visual metastatic nodules can be compared using one-way ANOVA.
• CA-IL-12/CA-scIL-12 can home to primary and secondary tumor tissues, deliver effective concentrations of mouse IL-12 within the TME, maintain minimal levels of serum IL-12/IFN- ⁇ , induce transcription of known IL-12-mediated inflammation signature biomarkers (IFN- ⁇ , PD-L1, and CXCL9, etc.), and recruit into the tumor tissue key immune effector cells (cytotoxic CD8+ T cells, CD4+ T cells, T Cell effector memory cells, increased M1/M2 macrophage ratio, and decreased Tregs), thereby igniting innate and adaptive antitumor and antimetastatic immunity.
• tumor tissue key immune effector cells cytotoxic CD8+ T cells, CD4+ T cells, T Cell effector memory cells, increased M1/M2 macrophage ratio, and decreased Tregs
• CA-IL-12 or CA-scIL-12 treatment can also increase the number of activated cytotoxic effector cells locally in the tumor containing tissues, peripherally in inguinal, and popliteal lymph nodes as measured by IFN- ⁇ and Granzyme B-secreting (or expressing) cells.
• activated cytotoxic effectors cells cannot be present in peripheral non-tumor bearing tissues.
• no adverse health, weight loss, or organ pathology can be noted in IL-12 treated animals.
• CA-IL-12 and CA-scIL-12 can induce adaptive and innate immune responses that can reprogram the immunologically “cold” TME into a “hot” TME in primary tumors and secondary tumors in lungs and liver without overt animal toxicity.
• the proposed dosing regimen of CA-IL-12 and CA-scIL-12 can slow tumor growth and metastatic progression, and this response is likely be attenuated by IL-12/ IFN- ⁇ induced immune cell exhaustion due to upregulation of PD-1/PD-L1 signaling, leading to reduction in primary and secondary tumor burden. Therefore, local delivery of IL-12 medicines in combination with anti-PD-1 therapy can result in more robust and sustained antitumor and antimetastatic activity.
• Example 1 Data discussed in Example 1 and Example 6 indicates that IT administration of CA-IL- 12 ignites a favorable shift in the composition of infiltrating immune cells in the TME without adverse health events, including: a) five-fold increase in CD8+ cells, b) two-fold increase in the M1/M2 ratio and c) two-fold decrease in CD25+ T regulatory cells, d) no or minimal changes in animal health as indicated by body weight, serum IFN-IL-12/IFN-y levels, and pathology analyses of the liver and other vital organs. Similar changes in immune cell composition in the TME and health measures in mice receiving CA-IL-12L medicines injected i.v. can be considered as criterion for success of the experiments described in this example.
• CA-IL-12 activates innate and adaptive immunity in tumor tissue better than CA-scIL- 12.
• CA-IL-12 and CA-sc-IL-12 are equally competent with regards to their ability to home to and interdigitate within secondary TNBC tumors. It is expected that CA-scIL-12 can show the most favorable toxicity profile since membrane tethered IL-12 has limited access to the circulation relative to secreted forms of extracellular IL-12; however, its efficacy may be inferior compared to CA-IL-12. If this is the case, CA-IL-12 development can be continued, but only if it shows a favorable toxicity profile based on the following criteria.
• IFN- ⁇ is the prime mediator of both the beneficial and adverse effects of high dose rIL-12 immunotherapy. This relationship is further complicated by the observations that: 1) humans are significantly more sensitive to IL-12/ IFN- ⁇ toxicity than mice, 2) direct measurements of IFN- ⁇ in tissue in humans have not been determined although tissue IFN- ⁇ has been linked to toxicity. The concentrations of serum IFN- ⁇ can be determined and examined to see if they remain lower than concentrations known to be toxic in humans. For reasons stated above, tissue IFN- ⁇ can also be measured and its association with toxicity can be determined.
• a threshold for IFN- ⁇ toxicity can be set at 5 ng/mL, and if CA-IL-12 produces serum concentrations of IFN- ⁇ ⁇ 5 ng/mL than this modality of localized IL-12 delivery can be dismissed. Leveraging the genetic versatility of the enucleated cell platform, two next generation enucleated cell products can be developed with improved benefit/risk ratios.
• CA-scIL-12 can be engineered to co-express known immune cell surface anchored co-stimulatory proteins 4- 1BBL and/or CD40L, which is expected to synergistically drive potent immune activation in combination with scIL-12 within the TME and maintain a safe profile.
• CA-IL-12 can be engineered to secrete IL-12 that is conditionally activated by the high proteolytic activity in the TME.
• IL-12 can be engineered with a peptide mask with a protease linker that prevents binding to the IL-12 receptor using a similar approach for masking cytokines and antibodies.
• the linker sequence was selected using cellular libraries of peptide substrates (CLiPS) for its responsiveness to multiple tumor-associated proteases, including urokinase-type plasminogen activator (uPA), membrane-type serine protease 1 (MT-SP1/matriptase), and legumain, all of which are strongly upregulated in the tumor microenvironment of most human tumors.
• uPA urokinase-type plasminogen activator
• MT-SP1/matriptase membrane-type serine protease 1
• legumain all of which are strongly upregulated in the tumor microenvironment of most human tumors.
• the mask contains specific protease consensus cleavage sites that upon exposure to high protease activity in the TME removes the mask and restores IL-12 function. Because proteolytic activity is largely absent in blood and normal tissues IL-12 bioactivity is retained within the TME.
• Criteria for selecting CA-IL-12 medicine can be based on detecting a favorable shift in the composition of infiltrating immune cells in the TME without adverse health events while the experiments detailed for reducing metastatic burden, improving overall animal survival, and inducing durable antitumor immunity can be conducted using; a) CA-IL-12, b) CA-scIL-12, c) WSGR Docket No.53712-720.601 CA-scIL-12 engineered to contain additional cell surface immune activators 4-1BBL/CD40L or d) CA-IL-12 secreting protease activated IL-12. The medicine developed can then be referred to as CA-IL-12L.
• CA-IL-12L can then be further evaluated for efficacy.
• Example 9 Determination of the ability of IL-12 medicine (CA-IL-12L) in combination with ICB therapy to reduce metastatic burden, improve overall animal survival, and induce durable antitumor immunity [0227] Coupling the potent adaptive and innate immune activity of IL-12 with ICB is a powerful combinatorial therapy to overcome ICB resistance and drive antitumor immunity. While IL-12 monotherapy can reduce tumor progression, it is not sufficient to drive durable antitumor responses.
• ICB therapy (Atezolizumab) have been achieved in the adjuvant setting and tumor-free and overall survival in mice whose primary tumors are resected prior to CA-IL-12L can be evaluated, and anti-PD-1 therapy as a clinical trial correlate.
• 4T1 and EO771 tumors can be used, which are refractory to ICB therapy as is the case with most human TNBCs.
• Primary tumors can be formed following orthotopic transplantation of 4T1 or E0771.LMB TNBC cells as described previously. At day 14, primary tumors can be excised, and spontaneous metastases can be monitored by bioluminescence imaging (BLI). Under these conditions, tumor weights at resection show a low coefficient of variation ( ⁇ 15% CV).
• Mice can receive enucleated cells (i.v.) as described in previously (3 doses of 10 6 enucleated cells on 3-day intervals).
• PD-1 ab or isotype matched ab can be administered i.v. on the day WSGR Docket No.53712-720.601 following the first and last enucleated cell injection.
• Overall tumor burden and treatment response can be analyzed using a semi-quantitative method.
• Local tumor recurrence rates at the resection site can be calculated as an increase in radiance (total photon flux) within a demarcated region of interest (ROI), the fourth inguinal mammary fat pad, and summed from luminescent images acquired in both the dorsal and ventral acquisition views (luminoscore).
• Animal weight and tumor size can be recorded by BLI and analyzed semi-quantitatively by luminoscore once a week and compared across groups by two-way ANOVA. Metastases can be measured by marking the whole body minus the 4th inguinal mfp as a ROI. Survival rates can be determined from Kaplan-Meier plots and the log-rank test. The durability of the antitumor response can be evaluated by rechallenging surviving mice (from group 10) with tumor cells. In these experiments, in mice with no measurable primary tumors 60 days after treatment, tumor cells can be reintroduced into the 4th mfp and tumor growth can again be monitored by BLI. These experiments can be terminated when tumors reach 2,000 mm 3 or show signs of labored respiration.
• CA-IL- 12L medicine in combination with ICB therapy can yield an 80% overall response rate with ⁇ 50% reduction in metastatic burden, and an improved overall animal survival of at least 20 days with a ⁇ 40% complete response rate based on Kaplan–Meier plots, the log rank test, and the Cox proportional hazards test.
• CA-IL-12L in combination with ICB therapy can generate durable antitumor immunity ( ⁇ 120 days disease free) and prevent tumor regrowth following tumor rechallenge in 100% of the surviving animals.
• CA-IL-12L can be present in tumor tissue but fail to induce immune cell infiltration robust enough to drive anticancer immunity. If this is the case, CA-IL-12L may be engineered to secrete potent NK and cytotoxic T cell chemoattractants (CXCL9 or CXCL10). On the other hand, if CA-IL-12L does promote immune cell infiltration, activate IL-12 biomarkers, and prevent immune exhaustion markers as expected, but fails to impact overall survival an in-depth analyses of the infiltrated immune cell phenotypes and immune cytokines can be performed to identify potential new targets and immunomodulators that could be targeted by bioengineered enucleated cells alone or in combination with ICB therapy.
• CXCL9 or CXCL10 cytotoxic T cell chemoattractants
• the PyMT mouse is ideally suited as a model for testing ICB in combination with CA-IL-12L because the primary tumors are ICB refractory and substantial work supports a role for innate and adaptive immune systems in metastasis regulation.
• MMTV-PyMT tumor vasculature overexpresses CCP enucleated cell ligands.
• CD31-positive endothelial cells within MMTV-PyMT tumors were FACS sorted and utilized for 10x Genomics scRNA-seq (Fig.14A and Fig.14B).
• Fig.14B shows dot plots of expression of Cxcl (Top) and Ccl (Bottom) chemokines within Vcam1-positive cells in Cluster 1 confirming Cxcl12 (encoding SDF-1 ⁇ ) and Ccl2 were the highest expressed Cxcl and Ccl chemokines, respectively. Undetected chemokines were removed from plots.
• NCBI SRA SRR14141127CCP enucleated cells also show greater than 3-fold increase in WSGR Docket No.53712-720.601 chemotaxis to PyMT tumor cell conditioned media compared to non-engineered enucleated cells, which is consistent with chemotaxis data obtained with 4T1 and EO771 tumor conditioned media (Fig.3B).
• a larger number of animals can be included in each treatment group (20 mice).
• Two separate sets of experiments can be performed to measure efficacy of CA-IL12L plus ICB.
• One experiment can measure antitumor and antimetastatic response, while the other experiment can measure overall survival.
• mice with similar sized primary tumors can be selected and treatment can begin at day 80 when the majority of mice show either micrometastases, contralateral tumors, or frank metastasis.
• Each experiment can consist of 4 experimental groups: a) no treatment, b) CA-empty plus control antibody, c) CA-IL-12L plus PD-1 antibody, and d) CA-IL-12L plus control antibody.
• Enucleated cells and antibody dosing can be the same as described previously (3 treatments of 10 6 enucleated cells on 3-day intervals with PD-1 or control ab on the day following the first and last enucleated cell delivery).
• mice can be euthanized on the day following the last treatment (day 90 when >90% of mice have metastatic disease).
• Animal health and biomarker analyses of blood and tissues can be performed as described previously with minor modifications.
• the presence of primary and secondary tumors in mfp, lungs, liver, and brain can be evaluated by gross inspection at the time of necropsy and by H&E and IHC using specific PyMT (Novus, NB100-2749), CK18 (Abcam, EPR17347) and EpCam (Abcam, EPR20533-63) antibodies.
• PCR can be used to measure overall metastatic burden using oligonucleotide primers specific for PyMT. Antitumor and antimetastatic activity can be measured by evaluating the composition of immune effector cells in tumor infiltrates by IHC as before. ELISA and PCR can also be used to measure IL-12 expression and downstream biomarker activity including IFN- ⁇ and PD-1/PD-L1 as before. For the experiment measuring overall survival, the endpoint can be death (or when primary tumors reach 2,000 mm 3 or mice show signs of respiratory distress or other measures of terminal morbidity) and surviving mice can be sacrificed at day 120 ( ⁇ 40 days after treatment and at a time where 100% of untreated MMTV-PyMT mice die).
• the total number of enucleated cells may be increased for the experiment measuring overall survival or the number of CA-IL-12L injections may be increased.

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