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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
• • 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
  • A61K40/31—Chimeric antigen receptors [CAR]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4254—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4264—Cancer antigens from embryonic or fetal origin
  • A61K40/4266—Carcinoembryonic antigen [CEA]
• • 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
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/7051—T-cell receptor (TcR)-CD3 complex
• • 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
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
• • 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
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
  • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
• • 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
  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
  • A61K2239/53—Liver
• • 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
  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
  • A61K2239/54—Pancreas
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
• • C—CHEMISTRY; METALLURGY
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
• • C—CHEMISTRY; METALLURGY
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2800/00—Nucleic acids vectors
  • C12N2800/10—Plasmid DNA

Definitions

• the current disclosure provides systems and methods to direct cellular therapies in vivo to an organ.
• the organ can be a secondary treatment location, such as an organ to which a primary tumor has metastasized.
• the systems and methods disclosed herein can be used to facilitate localized action of cellular therapies at secondary treatment locations and to avoid action at primary treatment locations and/or healthy, non-cancerous tissues.
• the systems and methods disclosed herein also can be used to facilitate localized action of cellular therapies at a primary treatment location and to avoid action at secondary treatment locations and/or healthy, non- cancerous tissues.
• the systems and methods disclosed herein also can be used to facilitate localized action of cellular therapies at a primary treatment location and secondary treatment locations and to avoid action at healthy, non-cancerous tissues.
• cancer cells For many years, the chosen treatments for cancer have been surgery, chemotherapy, and/or radiation therapy. In recent years, more targeted therapies have emerged to specifically target cancer cells by identifying and exploiting specific molecular and/or immunophenotypic changes seen primarily in those cells. For example, many cancer cells preferentially express particular markers on their cellular surfaces and these markers have provided targets for antibodybased therapeutics.
• CAR chimeric antigen receptor
• CAR can additionally include a transmembrane domain that can link the extracellular component to the intracellular component.
• CAR T cell-based therapies can be limited in some cancers due to toxicity resulting from on-target, off-tumor killing. This toxicity can occur when a targeted antigen is expressed by cancer cells as well as cells within healthy tissues. The CAR T cells will attack and destroy all cells expressing the antigen. Thus, strategies to improve CAR T cell efficacy, while reducing off-tumor toxicities are needed.
• the current disclosure provides cells genetically modified to achieve at least two of: expression of a recombinant receptor, modulated expression of a trafficking signal, or modulated expression of a retention signal.
• Genetic constructs described herein can be used to facilitate localized action of cellular therapies, for example at a selected organ.
• Expression of a trafficking signal and/or retention signal by genetically modified cells can induce the genetically modified cells to infiltrate a treatment site (e.g., organ or tissue) and/or prevent the genetically modified cells from leaving a treatment site, respectively.
• a surface molecule e.g., trafficking signal and/or retention signal
• systems and methods disclosed herein are used to direct a treatment to an organ that represents a secondary treatment location.
• a secondary treatment location refers to a location in the body that is different from where a condition to be treated emerged.
• a secondary treatment location is an organ to which a primary tumor has metastasized.
• systems and methods disclosed herein are used to direct a treatment to a secondary treatment location and to avoid, reduce, or prevent direction of the treatment to a primary treatment location.
• a primary treatment location is a location in the body where a condition to be treated emerged.
• Particular embodiments of the systems and methods disclosed herein can be used to facilitate localized action of cellular therapies at secondary treatment locations and to avoid action at primary treatment locations and/or healthy, non-cancerous tissues.
• the systems and methods disclosed herein also can be used to facilitate localized action of cellular therapies at a primary treatment location and to avoid action at secondary treatment locations and/or healthy, non- cancerous tissues.
• the systems and methods disclosed herein also can be used to facilitate localized action of cellular therapies at a primary treatment location and secondary treatment locations and to avoid action at healthy, non-cancerous tissues.
• a recombinant receptor includes a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), or a hybrid thereof.
• the recombinant receptor includes a binding domain.
• the binding domain binds a target antigen.
• the target antigen is expressed by a cancer cell.
• the cancer includes metastases of colorectal cancer, pancreatic cancer, breast cancer, colon cancer, gastric cancer, prostate cancer, hepatocellular carcinoma, ovarian cancer, endometrial cancer, breast cancer, or uterine cancer.
• the target antigen includes a carcinoembryonic antigen (CEA), an epithelial cell adhesion molecule (EpCAM), a human epidermal growth factor receptor 2 (HER2), a prostate-specific antigen (PSA), a glypican-3 (GPC3), a mesothelin, a folate receptor alpha (FOLR1), a mucin short variant S1 (MUC1), or a carbohydrate antigen 19-9 (CA19-9).
• the binding domain is an AHB7 scFv.
• the binding domain is a G8.8 scFv.
• the recombinant receptor includes a CAR.
• the CAR includes a binding domain, a CD28 transmembrane domain, a CD28 intracellular signaling domain, and a CD3 intracellular signaling domain.
• a trafficking signal includes up- or down-regulated expression of CXCR6, integrin 02 (LFA-1), CCR1 , CCR2, CXCR4, 07 integrin, CCR4, CXCR3, or CCR3.
• a trafficking signal includes expression of CXCR6, integrin 02 (LFA-1), CCR1 , CCR2, or CXCR4 to provide a positive directional signal (referred to as a “go here” trafficking signal).
• a trafficking signal includes down-regulated expression (e.g., by gene knockout) of 07 integrin, CCR4, CXCR3, or CCR3 to provide a negative directional signal (referred to as a “don’t go here” inhibitory signal).
• a retention signal includes expression of CD69, Runx3, Hobit, or CD103.
• a retention signal includes down-regulated expression of CD69, Runx3, Hobit, or CD103.
• administering can cause toxicity at a primary treatment location (e.g., an organ where a primary tumor developed) because cancer cells and healthy tissue cells of the same organ can both express the antigen that the recombinant receptor targets (i.e., target antigen).
• a primary treatment location e.g., an organ where a primary tumor developed
• cancer cells and healthy tissue cells of the same organ can both express the antigen that the recombinant receptor targets (i.e., target antigen).
• the genetically modified cells are administered to secondary treatment locations (e.g,. sites of metastasis.
• the genetically modified cells are delivered locally to a desired site of activity.
• the genetically modified cells are delivered systemically.
• the genetically modified cells include a trafficking signal and/or retention signal which causes the genetically modified cells to go to, stay away from, and/or stay at the desired secondary treatment location.
• a sequence encoding a trafficking signal e.g., CXCR6
• a retention signal e.g., CD69
• an anti-CEA CAR includes the sequence as set forth in SEQ ID NOs: 59, 60, 61 , or 62.
• a sequence encoding a trafficking signal e.g., CXCR6
• a retention signal e.g., CD69
• an anti-EpCAM CAR includes the sequence as set forth in SEQ ID NO: 199 or encoded by the sequence as set forth in SEQ ID NO: 202.
• cells are engineered to knockout or reduce expression of a trafficking signal.
• knockout or reduction of expression includes delivering a nuclease and single guide RNA (sgRNA) targeting the trafficking signal.
• the trafficking signal includes CXCR3 or 7 integrin.
• sgRNA targeting CXCR3 includes SEQ ID NO: 242, SEQ ID NO: 243, and/or SEQ ID NO: 244.
• sgRNA targeting [37 integrin includes SEQ ID NO: 245, SEQ ID NO: 246, and/or SEQ ID NO: 247.
• the nuclease includes a Cas9 nuclease.
• cells can be engineered to express a recombinant receptor (e.g., CAR).
• a recombinant receptor e.g., CAR
• the recombinant receptor includes the sequence as set forth in SEQ ID NO: 199 or encoded by the sequence as set forth in SEQ ID NO: 202.
• the recombinant receptor includes the sequence as set forth in SEQ ID NO: 200 or encoded by the sequence as set forth in SEQ ID NO: 201.
• the current disclosure provides a system to improve cancer treatments by directing cellular therapies in vivo.
• Genetic modification to alter expression of trafficking signals and/or retention signals by a cell induces trafficking of genetically modified cells to a desired site of activity (e.g., target organ or tissue).
• a desired site of activity e.g., target organ or tissue.
• down-regulated expression of a trafficking signal and/or retention signal within genetically modified cells can instruct the genetically modified cells to avoid a tissue, restricting access to healthy tissues, thereby reducing on-target, off-tumor toxicity.
• the current disclosure differs significantly from other approaches that have been developed to direct the migration of CAR T cells.
• Previous approaches have directed the migration of CAR T cells to the sites of cancer cells regardless of the location of the cancer cells.
• cells are directed not to cancer cells, but to specific organs.
• cells are only directed to sites of metastasis and are not directed to a site of a primary tumor. This distinction provides an important advance in the ability to provide targeted therapies with reduced toxicity and side effects.
• FIGs. 1A-1 F Development and validation of carcinoembryonic antigen (CEA)-targeting chimeric antigen receptor (CAR)-T in human and mouse T cells.
• CEA carcinoembryonic antigen
• CAR chimeric antigen receptor
• 1A Modular structure of human CAR targeting human protein CEA (CEACAM5).
• SRP signal recognition particle.
• scFV single chain fragment variable.
• hCD28 human CD28.
• TMD transmembrane domain.
• ICD intracellular domain.
• FIGs. 2A-2H Targeting epithelial cell adhesion molecule (EpCAM) to model CAR-T cell targeting of a tumor associated antigen in solid cancer.
• EpCAM epithelial cell adhesion molecule
• FIGs. 3A-3F EpCAM CAR-T cells have some anti-tumor activity against subcutaneous KPC tumors, but exert lethal toxicity at higher doses.
• (3A-3C) Tumor growth curves from C57B6 mice that were subcutaneously implanted with KPC parental (3A), EpCAM KO (3B), or EpCAM OE (3C) tumor cells. 10 days after tumor implantation, mice received 5Gy total body irradiation (control) or 5Gy total body irradiation with subsequent retroorbital transfer of 1 ,5e6 EpCAM CAR- T cells.
• (3D) Change in body mice in C57B6 mice that received 4Gy total body irradiation followed by retro-orbital transfer of CD8+ T cells transduced with control or EpCAM CAR-T encoding retrovirus, n 3-6 per group.
• FIGs. 4A-4G Liver tissue residency programing induces liver tropism in OT1 transgenic T cells.
• Liver TRP transduced cells have high co-expression of CXCR6 and CD69.
• 4D Summary graphs from transwell migration assay where CD8+ T cells transduced with the control retrovirus or Liver TRP retrovirus are placed on top of a transwell filter within a well containing CXCL16 or not. CXCL16 is the specific ligand for CXCR6. Data depicts the amount of migration through the transwell filter toward the bottom of the well containing CXCL16 or not.
• FIGs. 5A-5E Prior to transfer, hosts received 5Gy radiotherapy (RT) total body irradiation. At time of transfer, hosts received T cell mix along with vaccinia virus-OVA.
• TRP Liver tissue residency programmed EpCAM CAR-T cells
• 5A Summary data from mice who received 5Gy total body irradiation and co-transfer of EpCAM CAR-T cells with or without Liver TRP. Data depict ratio of Liver TRP EpCAM CAR-T cells to control EpCAM CAR-T cells in liver, lung, and spleen.
• 5B Change in body mass of mice that received 5Gy total body irradiation followed by retroorbital transfer of 3e6 control EpCAM CAR-T cells or Liver TRP EpCAM CAR-T cells.
• mice with intrahepatic KPC-luciferase tumors 7 days after receiving 5Gy total body irradiation alone (control, left) or 5Gy RT and Liver TRP EpCAM CAR-T cells (right).
• FIG. 6 Schematic of CEA CAR construct with CXCR6 and CD69, a cell expressing the construct, and schematic of the administration of said cell and its migration to the liver.
• FIGs. 7A, 7B CEA is a tumor associated antigen present in cancers and the healthy lung and intestine.
• FIGs. 8A-8C hCEA transgenic C57BI/6 mice express CEA in a representative distribution and suffer CEA CAR-T cell off-tumor toxicity. To appropriately capture off-tumor toxicities in a mouse model, mouse embryos transgenic for human CEA were generated.
• 8A Expression of hCEA in transgenic (hCEAtg) and wildtype (WT) C57BL/6 mice in various sites.
• 8B Weight over time of hCEA CAR-T cell recipients.
• FIGs. 9A, 9B CEA CAR T cells slow growth of the mouse implantable KPC cell line engineered to express human CEA.
• FIGs. 10A, 10B Liver tissue residency programmed CEA CAR T cells preferentially traffic to liver and reduce growth of intrahepatic (IH) tumors.
• IH intrahepatic
• 10A Summary data from mice who received 5Gy total body irradiation and co-transfer of CEA CAR-T cells with or without Liver tissue residency programming (TRP). Data depict ratio of Liver TRP CEA CAR-T cells to control CEA CAR-T cells in liver, lung, spleen, and gut.
• 10B Intrahepatic tumor weights from mice 9 days after treatment with 5Gy RT, and control PBS or Liver TRP CEA CAR T Cells.
• FIGs. 11 A, 11 B Validation of Cxcr3 and Itgb7 CRISPR disruption followed by retroviral CAR transduction.
• FIGS. 12A-12C Genetic ablation of CXCR3 in EpCAM CAR T cells limits trafficking to lung and pancreas Flow cytometric evaluation of relative frequency of EpCAM CAR T cells in lung (12A) and pancreas (12B) with or without (KO) intact CXCR3; evaluated 6 days after cell transfer. (12C) Ratio of CXCR3 knockout to scramble (Scr) EpCAM CAR T cells in IH tumor, lung, pancreas, and spleen.
• FIGS. 13A, 13B Genetic ablation of Itgb7 in CEA CAR T cells limits trafficking to intestine.
• 13A Flow cytometric evaluation of relative frequency of CEA CAR T cells in gut (Colon + Small intestine) with or without (KO) intact a4[37 integrin; evaluated 6 days after cell transfer.
• 13B Ratio of a4 7 knockout to scramble (Scr) CEA CAR T cells in IH tumor, gut, lung, and spleen.
• FIG. 14 Table of cancer antigen target, desired site of activity (secondary treatment location), and the signals used to traffic and/or retain genetically modified cells to the desired site of activity. Trafficking and inhibitory signals can be combinatorial or used individually. Acronyms: Exp (expression of provided genetic construct); KO (knockout). Alternative retention signals include Runx3 and Hobit expression (Crowl et al. Nat. Immunol. 23, 1121-1131 (2022); Christo et al. Nat. Immunol. 22, 1140-1151 (2021); and Milner and Goldrath. Curr. Opin. Immunol. 51 , 162-169 (2016)).
• FIG. 15 Sequences supporting the disclosure including CXCR6 Coding Sequence (SEQ ID NO: 1), RAKR Coding Sequence (SEQ ID NO: 2), T2A Coding Sequence (SEQ ID NO: 3), CD69 Coding Sequence (SEQ ID NO: 4), 2A Coding Sequence (SEQ ID NO: 5), Myc Coding Sequence (SEQ ID NO: 6), Extracellular Domain Spacer Coding Sequence (SEQ ID NO: 7), CD28 Transmembrane Domain Coding Sequence (SEQ ID NO: 8), CD28 Intracellular Signaling Domain Coding Sequence (SEQ ID NO: 9), CD3z Intracellular Signaling Domain Coding Sequence (SEQ ID NO: 10), lgG4 hinge (SEQ ID NO: 11) and coding sequence (SEQ ID NOs: 12-14), lgG4-CH2 domain (SEQ ID NOs: 15 and 16) and coding sequence (SEQ ID NOs: 17 and 18), lgG4-CH3 domain (SEQ ID NO:
• cancer cells For many years, the chosen treatments for cancer were surgery, chemotherapy, and/or radiation therapy. In recent years, more targeted therapies have emerged to specifically target cancer cells by identifying and exploiting specific molecular and/or immunophenotypic changes seen primarily in those cells. For example, many cancer cells preferentially express particular antigens on their cellular surfaces and these antigens have provided targets for successful therapeutics.
• CAR chimeric antigen receptor
• the current disclosure provides cells genetically modified to achieve at least two of: express a recombinant receptor, modulate expression of a trafficking signal, or modulate expression of a retention signal.
• the genetic constructs described herein can be used to facilitate localized action of cellular therapies. Expression of a trafficking signal and/or retention signal by genetically modified cells can induce the genetically modified cells to infiltrate a treatment site (e.g., organ or tissue) and/or prevent the genetically modified cells from leaving a treatment site, respectively. Alternatively, down-regulated expression of a trafficking signal can keep genetically modified cells from entering a particular tissue.
• a surface molecule e.g., trafficking signal and/or retention signal
• systems and methods disclosed herein are used to direct a treatment to an organ that represents a secondary treatment location.
• a secondary treatment location refers to a location in the body that is different from where a condition to be treated emerged.
• a secondary treatment location is an organ to which a primary tumor has metastasized.
• systems and methods disclosed herein are used to direct a treatment to a secondary treatment location and to avoid, reduce, or prevent direction of the treatment to a primary treatment location.
• a primary treatment location is a location in the body where a condition to be treated emerged.
• Particular embodiments of the systems and methods disclosed herein can be used to facilitate localized action of cellular therapies at secondary treatment locations and to avoid action at primary treatment locations and/or healthy, non-cancerous tissues.
• the systems and methods disclosed herein also can be used to facilitate localized action of cellular therapies at a primary treatment location and to avoid action at secondary treatment locations and/or healthy, non- cancerous tissues.
• the systems and methods disclosed herein also can be used to facilitate localized action of cellular therapies at a primary treatment location and secondary treatment locations and to avoid action at healthy, non-cancerous tissues.
• healthy, non-cancerous tissues can be referred to as “off-target”.
• a recombinant receptor includes a CAR, an engineered T cell receptor (eTCR), or a hybrid thereof.
• the recombinant receptor includes a binding domain.
• the binding domain binds a target antigen.
• the target antigen is expressed by a cancer cell.
• the cancer includes colorectal cancer, pancreatic cancer, breast cancer, colon cancer, gastric cancer, prostate cancer, hepatocellular carcinoma, ovarian cancer, endometrial cancer, breast cancer, or uterine cancer.
• the target antigen includes a carcinoembryonic antigen (CEA), an epithelial cell adhesion molecule (EpCAM), a human epidermal growth factor receptor 2 (HER2), a prostate-specific antigen (PSA), a glypican-3 (GPC3), mesothelin, a folate receptor alpha (FOLR1), a mucin short variant S1 (MUC1), or a carbohydrate antigen 19-9 (CA19-9).
• the binding domain binds human CEA and is an AHB7 scFv.
• the binding domain binds mouse EpCAM and is a G8.8 scFv.
• the recombinant receptor includes a CAR.
• the CAR includes a binding domain, a CD28 transmembrane domain, a CD28 intracellular signaling domain, and a CD3 intracellular signaling domain.
• a trafficking signal includes up- or down regulated expression of CXCR6, integrin 02 (LFA-1), CCR1 , CCR2, CXCR4, 07 integrin, CCR4, CXCR3, or CCR3.
• a trafficking signal includes expression of CXCR6, integrin 02 (LFA-1), CCR1 , CCR2, or CXCR4 to provide a positive directional signal (referred to as a “go here” trafficking signal).
• a trafficking signal includes down regulated expression (e.g., by gene knockout) of [37 integrin, CCR4, CXCR3, or CCR3 to provide a negative directional signal (referred to as a “don’t go here” inhibitory signal).
• a retention signal includes expression of CD69, Runx3, Hobit, or CD103.
• a retention signal includes down regulated expression of CD69, Runx3, Hobit, or CD103.
• administering can cause toxicity at the organ where a primary tumor developed because cancer cells and healthy tissue cells of the same organ can both express the antigen that the recombinant receptor targets (i.e., target antigen). Furthermore, if delivered systemically, toxicity can occur at an organ not involved with the cancer but happens to also express the target antigen. Therefore, in particular embodiments, the genetically modified cells of the present disclosure are administered to tissue sites to target metastasis (e.g., at desired site of activity). In particular embodiments, the genetically modified cells are delivered locally to a desired site of activity. In particular embodiments, the genetically modified cells are delivered systemically. In particular embodiments, the genetically modified cells include a trafficking signal and/or a retention signal which causes the genetically modified cells to go to, stay away from, and/or stay at the desired site of activity.
• a recombinant receptor e.g., CAR
• the genetically modified cells of the present disclosure are administered to tissue sites to target metastasis (e.g., at desired site
• a sequence encoding a trafficking signal e.g., CXCR6
• a retention signal e.g., CD69
• an anti-CEA CAR includes the sequence as set forth in SEQ ID NOs: 59, 60, 61 , or 62.
• a sequence encoding a trafficking signal e.g., CXCR6
• a retention signal e.g., CD69
• an anti-EpCAM CAR includes the sequence as set forth in SEQ ID NO: 199 or encoded by the sequence as set forth in SEQ ID NO: 202.
• cells are engineered to knockout or reduce expression of a trafficking signal.
• knockout or reduction of expression includes delivering a nuclease and single guide RNA (sgRNA) targeting the trafficking signal.
• the trafficking signal includes CXCR3 or [37 integrin.
• sgRNA targeting CXCR3 includes SEQ ID NO: 242, SEQ ID NO: 243, and/or SEQ ID NO: 244.
• sgRNA targeting [37 integrin includes SEQ ID NO: 245, SEQ ID NO: 246, and/or SEQ ID NO: 247.
• the nuclease includes a Cas9 nuclease.
• cells can be engineered to express a recombinant receptor (e.g., CAR).
• a recombinant receptor e.g., CAR
• the recombinant receptor includes the sequence as set forth in SEQ ID NO: 199 or encoded by the sequence as set forth in SEQ ID NO: 202.
• the recombinant receptor includes the sequence as set forth in SEQ ID NO: 200 or encoded by the sequence as set forth in SEQ ID NO: 201.
• the current disclosure provides a system to improve cancer treatments by directing cellular therapies in vivo.
• Expression of a trafficking signal and a retention signal by genetically modified cells induces trafficking of genetically modified cells to a select tissue and retains the genetically modified cells within the select tissue, thereby reducing on-target, off-tumor toxicity at both the tumor site and/or other off-tumor sites.
• down regulated expression of a trafficking signal and/or retention signal within genetically modified cells can instruct the genetically modified cells to avoid a select tissue.
• the current disclosure differs significantly from other approaches that have been developed to direct the migration of CAR T cells.
• Previous approaches have directed the migration of CAR T cells to the sites of cancer cells regardless of the location of the cancer cells.
• cells are directed not to cancer cells, but to specific organs.
• cells are only directed to sites of metastasis and are not directed to a site of a primary tumor. This distinction provides an important advance in the ability to provide targeted therapies with reduced toxicity and side effects.
• Cells The present disclosure describes cells genetically modified to achieve at least two of: express a recombinant receptor, modulate expression of a trafficking signal, or modulate expression of a retention signal.
• Cells to be genetically modified can include immune cells, stem or progenitor cells, or adult cells. Immune cells can include T-cells, B cells, natural killer (NK) cells, NK-T cells, monocytes/macrophages, and lymphocytes.
• Stem or progenitor cells can include hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPC), pluripotent stem cells, mesenchymal stem cells, neural stem cells, endothelial stem cells, and/or a mixture thereof.
• genetically modified cells include T-cells.
• T-cells Several different subsets of T-cells have been discovered, each with a distinct function. For example, a majority of T-cells have a T-cell receptor (TCR) existing as a complex of several proteins. The actual T-cell receptor is composed of two separate peptide chains, which are produced from the independent T-cell receptor alpha and beta (TCRa and TCR[3) genes and are called a- and [3-TCR chains.
• y8 T-cells represent a small subset of T-cells that possess a distinct T-cell receptor (TCR) on their surface.
• TCR T-cell receptor
• the TCR is made up of one y-chain and one 8-chain. This group of T-cells is much less common (2% of total T-cells) than the a
• CD3 is expressed on all mature T cells. Activated T-cells express 4-1 BB (CD137), CD69, and CD25. CD5 and transferrin receptor are also expressed on T-cells.
• T-cells can further be classified into helper cells (CD4+ T-cells) and cytotoxic T-cells (CTLs, CD8+ T-cells), which include cytolytic T-cells.
• T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T-cells and macrophages, among other functions. These cells are also known as CD4+ T-cells because they express the CD4 protein on their surface.
• Helper T-cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.
• APCs antigen presenting cells
• Cytotoxic T-cells destroy virally infected cells and tumor cells and are also implicated in transplant rejection. These cells are also known as CD8+ T-cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body.
• Central memory T-cells refers to an antigen experienced CTL that expresses CD62L or CCR7 and CD45RO on the surface thereof and does not express or has decreased expression of CD45RA as compared to naive cells.
• central memory cells are positive for expression of CD62L, CCR7, CD25, CD127, CD45RO, and CD95, and have decreased expression of CD45RA as compared to naive cells.
• Effective memory T-cell refers to an antigen experienced T-cell that does not express or has decreased expression of CD62L on the surface thereof as compared to central memory cells and does not express or has decreased expression of CD45RA as compared to a naive cell.
• effector memory cells are negative for expression of CD62L and CCR7, compared to naive cells or central memory cells, and have variable expression of CD28 and CD45RA.
• Effector T-cells are positive for granzyme B and perforin as compared to memory or naive T-cells.
• naive T-cells refers to a non-antigen experienced T cell that expresses CD62L and CD45RA and does not express CD45RO as compared to central or effector memory cells.
• naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T-cells including CD62L, CCR7, CD28, CD127, and CD45RA.
• Natural killer cells also known as NK cells, K cells, and killer cells
• NK cells are activated in response to interferons or macrophage-derived cytokines. They serve to contain viral infections while the adaptive immune response is generating antigen-specific cytotoxic T cells that can clear the infection.
• NK cells express CD8, CD16 and CD56 but do not express CD3.
• NK cells include NK-T cells.
• NK-T cells are a specialized population of T cells that express a semi invariant T cell receptor (TCR ab) and surface antigens typically associated with natural killer cells.
• TCR ab semi invariant T cell receptor
• NK-T cells contribute to antibacterial and antiviral immune responses and promote tumor-related immunosurveillance or immunosuppression.
• NK-T cells can also induce perforin-, Fas-, and TNF-related cytotoxicity.
• Activated NK-T cells are capable of producing IFN-y and IL-4.
• NK-T cells are CD3+/CD56+.
• Macrophages (and their precursors, monocytes) reside in every tissue of the body (in certain instances as microglia, Kupffer cells and osteoclasts) where they engulf apoptotic cells, pathogens and other non-self-components.
• Monocytes/macrophages express CD11b, F4/80; CD68; CD11c; IL-4Ra; and/or CD163.
• Immature dendritic cells engulf antigens and other non-self- components in the periphery and subsequently, in activated form, migrate to T-cell areas of lymphoid tissues where they provide antigen presentation to T cells.
• Dendritic cells express CD1a, CD1 b, CD1c, CD1d, CD21 , CD35, CD39, CD40, CD86, CD101 , CD148, CD209, and DEC-205.
• Stem cells are cells capable of differentiation into other cell types, including those having a particular, specialized function (e.g., tissue specific cells, parenchymal cells and progenitors thereof).
• tissue specific cells e.g., tissue specific cells, parenchymal cells and progenitors thereof.
• progenitor cells can be either multipotent or pluripotent.
• Progenitor cells are cells that can give rise to different terminally differentiated cell types, and cells that are capable of giving rise to various progenitor cells.
• pluripotency refers to cells with the ability to give rise to progeny cells that can undergo differentiation, under the appropriate conditions, into cell types that collectively demonstrate characteristics associated with cell lineages from all of the three germinal layers (endoderm, mesoderm, and ectoderm).
• Hematopoietic Stem/Progenitor Cells or HSPC refer to a combination of hematopoietic stem cells and hematopoietic progenitor cells.
• Hematopoietic stem cells refer to undifferentiated hematopoietic cells that are capable of self-renewal either in vivo, essentially unlimited propagation in vitro, and capable of differentiation to all other hematopoietic cell types.
• a hematopoietic progenitor cell is a cell derived from hematopoietic stem cells or fetal tissue that is capable of further differentiation into mature cell types.
• hematopoietic progenitor cells are CD24 10 Lin’ CD117 + hematopoietic progenitor cells.
• HPC can differentiate into (i) myeloid progenitor cells which ultimately give rise to monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, or dendritic cells; or (ii) lymphoid progenitor cells which ultimately give rise to T-cells, B-cells, and NK-cells.
• HSPC can be positive for a specific marker expressed in increased levels on HSPC relative to other types of hematopoietic cells.
• markers include CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLA DR, or a combination thereof.
• the HSPC can be negative for an expressed marker relative to other types of hematopoietic cells.
• markers include Lin, CD38, or a combination thereof.
• the HSPC are CD34 + cells.
• Pluripotent stem cells are a type of cell that undergoes self-renewal while maintaining an ability to give rise to all three germ layer-derived tissues and germ cell lineages.
• Pluripotent stem cells include induced pluripotent stem cells and embryonic stem cells.
• Mesenchymal stem cells also known as mesenchymal stromal cells are multipotent stromal cells. Mesenchymal stem cells are more differentiated than pluripotent stem cells, but retain the ability to differentiate into a variety of cell types, including osteoblasts, chondrocytes, myocytes and adipocytes.
• Neural stem cells are stem cells found in the adult brain. Endothelial stem cells are a type of multipotent stem cell found in the bone marrow. Endothelial stem cells differentiate into endothelial cells or cells that line blood vessels as well as lymphatic vessels.
• adult cells include any cell type that is terminally differentiated.
• adult cells include fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, kidney cells, chondrocytes, or adipocytes.
• a statement that a cell or population of cells is "positive" for or expressing a particular marker refers to the detectable presence on or in the cell of the particular marker.
• the term can refer to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype- matched control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.
• a statement that a cell or population of cells is "negative" for a particular marker or lacks expression of a marker refers to the absence of substantial detectable presence on or in the cell of a particular marker.
• the term can refer to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.
• immune cells to be genetically modified can be patient-derived cells (autologous) or allogeneic.
• immune cells can be genetically modified in vivo or ex vivo.
• cells are derived from humans, for example a patient to be treated.
• Cells can be derived from cell lines.
• T cells are derived or isolated from samples such as whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom.
• PBMCs peripheral blood mononuclear cells
• the samples contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, HSC, HPC, HSPC, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets and further processing is necessary.
• T cells are derived from PBMCs.
• blood cells collected from a subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
• Isolation can include one or more of various cell preparation and separation steps, including separation based on one or more properties, such as size, density, sensitivity or resistance to particular reagents, and/or affinity, e.g., immunoaffinity, to antibodies or other binding partners.
• the isolation is carried out using the same apparatus or equipment sequentially in a single process stream and/or simultaneously.
• the isolation, culture, and/or engineering of the different populations is carried out from the same starting material, such as from the same sample.
• a sample can be enriched for T cells by using density-based cell separation methods and related methods.
• white blood cells can be separated from other cell types in the peripheral blood by lysing red blood cells and centrifuging the sample through a Percoll or Ficoll gradient.
• a bulk T cell population can be used that has not been enriched for a particular T cell type.
• a selected T cell type can be enriched for and/or isolated based on cell-marker based positive and/or negative selection.
• positive selection cells having bound cellular markers are retained for further use.
• negative selection cells not bound by a capture agent, such as an antibody to a cellular marker are retained for further use.
• both fractions can be retained for a further use.
• CD4+ and/or CD8+ T cells are enriched from PBMCs.
• CD8+ T cells are enriched from PBMCs.
• the separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker.
• positive selection of or enrichment for cells of a particular type refers to increasing the number or percentage of such cells but need not result in a complete absence of cells not expressing the marker.
• negative selection, removal, or depletion of cells of a particular type refers to decreasing the number or percentage of such cells but need not result in a complete removal of all such cells.
• multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection.
• an antibody or binding domain for a cellular marker is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection.
• affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). MACS systems are capable of high-purity selection of cells having magnetized particles attached thereto.
• MACS magnetic-activated cell sorting
• a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream.
• T cells for different T cell subpopulations are described above.
• specific subpopulations of T cells such as cells positive or expressing high levels of one or more surface markers, e.g., CCR7, CD45RO, CD8, CD27, CD28, CD62L, CD127, CD4, and/or CD45RA T cells, are isolated by positive or negative selection techniques.
• CD3+, CD28+ T cells can be positively selected for and expanded using anti-CD3/anti- CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
• anti-CD3/anti- CD28 conjugated magnetic beads e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander.
• a CD8+ or CD4+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells.
• Such CD8+ and CD4+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
• PBMC can be enriched for or depleted of CD62L, CD8 and/or CD62L+CD8+ fractions, such as by using anti-CD8 and anti-CD62L antibodies.
• the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CCR7, CD45RO, CD27, CD62L, CD28, CD3, and/or CD127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B.
• isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CCR7, CD45RO, and/or CD62L.
• enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L.
• CD34+ HSC, HSP, and HSPC can be enriched using anti-CD34 antibodies directly or indirectly conjugated to magnetic particles in connection with a magnetic cell separator, for example, the CliniMACS® Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany).
• Cell populations are genetically modified to include a genetic construct that results in two or more of: expression of a recombinant receptor, up- or down-regulated expression of a trafficking signal, or up- or down-regulated expression of a retention signal.
• (lll-A) Genetic Engineering Techniques Genetic constructs encoding a molecule of interest (e.g., CAR, eTCR, trafficking signal, retention signal) and/or resulting in down-regulated expression of a molecule of interest (e.g., trafficking signal, retention signal) can be introduced into cells by any method known in the art, including transfection, electroporation, microinjection, lipofection, calcium phosphate mediated transfection, infection with a viral or bacteriophage vector including the gene sequences, cell fusion, chromosome-mediated gene transfer, microcell- mediated gene transfer, spheroplast fusion, in vivo nanoparticle-mediated delivery, etc.
• a molecule of interest e.g., CAR, eTCR, trafficking signal, retention signal
• resulting in down-regulated expression of a molecule of interest e.g., trafficking signal, retention signal
• a viral or bacteriophage vector including the gene sequences, cell fusion, chromos
• Methods of genetic engineering described herein can be used to cause expression or down-regulate (knockdown/knockout) expression of molecules described herein.
• the term “gene” refers to a nucleic acid sequence (used interchangeably with polynucleotide or nucleotide sequence) that encodes a molecule of interest. This definition includes various sequence polymorphisms, mutations, and/or sequence variants wherein such alterations do not substantially affect the function of the encoded molecule of interest.
• the term “gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. Gene sequences encoding the molecule can be DNA or RNA that directs the expression of the molecule of interest. These nucleic acid sequences may be a DNA strand sequence that is transcribed into RNA or an RNA sequence that is translated into protein.
• sequences can also include degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specific cell type (e.g., a mammalian cell). Portions of complete gene sequences are referenced throughout the disclosure as is understood by one of ordinary skill in the art.
• Gene sequences encoding molecules of interest are provided herein and can also be readily prepared by synthetic or recombinant methods from the relevant amino acid sequences and other description provided herein.
• the gene sequence encoding any of these sequences can also have one or more restriction enzyme sites at the 5' and/or 3' ends of the coding sequence in order to provide for easy excision and replacement of the gene sequence encoding the sequence with another gene sequence encoding a different sequence.
• Encoding refers to the property of specific sequences of nucleotides in a gene, such as a cDNA, or an mRNA, to serve as templates for synthesis of other macromolecules such as a defined sequence of amino acids.
• Polynucleotide gene sequences encoding more than one portion of an expressed genetic construct can be operably linked to each other and relevant regulatory sequences. For example, there can be a functional linkage between a regulatory sequence and an exogenous nucleic acid sequence resulting in expression of the latter. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
• a "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid.
• Vectors may be, e.g., plasmids (DNA plasmids or RNA plasmids), transposon-based systems, cosmids, bacterial artificial chromosomes, viruses, or phage.
• An "expression vector” is a vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment.
• Lentivirus refers to a genus of retroviruses that are capable of infecting dividing and nondividing cells.
• HIV human immunodeficiency virus: including HIV type 1, and HIV type 2
• equine infectious anemia virus feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
• HIV human immunodeficiency virus: including HIV type 1, and HIV type 2
• equine infectious anemia virus HIV
• feline immunodeficiency virus (FIV) feline immunodeficiency virus
• BIV bovine immune deficiency virus
• SIV simian immunodeficiency virus
• a lentiviral vector is a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et ah, Mol. Ther. 17(8): 1453-1464 (2009).
• Other examples of lentivirus vectors that may be used in the clinic include: the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAXTM vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
• cells are genetically modified (or genetically engineered) to express a recombinant receptor or surface molecule using a lentivirus or lentiviral vector.
• Retroviruses are viruses having an RNA genome.
• Gammaretrovirus refers to a genus of the retroviridae family.
• Exemplary gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.
• Retroviral vectors can be used.
• the gene to be expressed is cloned into the retroviral vector for its delivery into cells.
• a retroviral vector includes all of the cis-acting sequences necessary for the packaging and integration of the viral genome, i.e. , (a) a long terminal repeat (LTR), or portions thereof, at each end of the vector; (b) primer binding sites for negative and positive strand DNA synthesis; and (c) a packaging signal, necessary for the incorporation of genomic RNA into virions.
• LTR long terminal repeat
• retroviral vectors More detail about retroviral vectors can be found in Boesen, et al., 1994, Biotherapy 6:291-302; Clowes, et al., 1994, J. Clin. Invest. 93:644-651 ; Kiem, et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141 ; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.
• Adenoviruses, adeno-associated viruses (AAV) and alphaviruses can also be used.
• Retroviral and lentiviral viral vector constructs and expression systems are also commercially available.
• CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
• Cas CRISPR-associated protein
• ZFNs zinc finger nucleases
• US 6,534,261 US 6,607,882; US 6,746,838; US 6,794,136; US 6,824,978; 6,866,997; US 6,933,113; 6,979,539; US 7,013,219; US 7,030,215; US 7,220,719; US 7,241 ,573; US 7,241 ,574; US 7,585,849; US 7,595,376; US 6,903,185; US 6,479,626; US 2003/0232410 and US 2009/0203140 as well as Gaj et al., Nat Methods, 2012, 9(8):805-7; Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim et al., Genome Res, 2012, 22(7): 13
• TALENs transcription activator like effector nucleases
• MegaTALs have a sc rare-cleaving nuclease structure in which a TALE is fused with the DNA cleavage domain of a meganuclease.
• Meganucleases also known as homing endonucleases, are single peptide chains that have both DNA recognition and nuclease function in the same domain. In contrast to the TALEN, the megaTAL only requires the delivery of a single peptide chain for functional activity.
• transposon-based systems as gene editing agents to mediate the integration of a genetic construct into cells.
• transposon/transposase systems have been adapted for genetic insertions of heterologous DNA sequences.
• transposases include sleeping beauty (“SB”, e.g., derived from the genome of salmonid fish); piggyback (e.g., derived from lepidopteran cells and/or the Myotis lucifugusy mariner (e.g., derived from Drosophila); frog prince (e.g., derived from Rana pipiens Toll ; Tol2 (e.g., derived from medaka fish); TcBuster (e.g., derived from the red flour beetle Tribolium castaneum), Helraiser, Himarl , Passport, Minos, Ac/Ds, PIF, Harbinger, Harbinger3-DR, HSmarl , and spinON.
• SB sleeping beauty
• piggyback e.g., derived from lepidopteran cells and/or the Myotis lucifugusy
• Nanoparticles that result in selective in vivo genetic modification of targeted cell types can be used within the teachings of the current disclosure.
• the nanoparticles can be those described in WO2014153114, WO2017181110, and WO201822672.
• (lll-A) Trafficking Signals refers to any molecule that impacts the location of a cell in the body via a positive directional signal or a negative directional signal. As shown in FIG. 14, a positive directional signal can provide a ‘go here’ signal and a negative directional signal can provide a ‘don’t go here’ signal by either up- or down-regulated expression of the trafficking signal.
• cells can be engineered to traffic to a select tissue by introducing a genetic construct expressing a trafficking signal to the cell.
• a cell can be engineered to avoid a non-selected tissue by down-regulating expression of an endogenous trafficking signal.
• the product of an introduced coding sequence may be expressed at low levels in non-targeted cells, for example at less than 1 % or 1%, 2%, 3%, 5%, 10%, 15% or 20% of the levels at which the product is expressed in targeted cells.
• a “select” or “target” tissue as it refers to genetically modified cells being trafficked to or residing in the select tissue means that the genetically modified cells are trafficked to or reside in the intended tissue and are not substantially trafficked to nor residing in other tissue types, as explained in additional detail below.
• not substantially trafficked to nor residing in other tissue types means that less than 50% are found in a reference tissue type as compared to a select tissue type; less than 40% found in a reference tissue type as compared to a select tissue type; less than 30% found in a reference tissue type as compared to a select tissue type; less than 20% found in a reference tissue type as compared to a select tissue type; or less than 10% found in a reference tissue type as compared to a select tissue type.
• a reference tissue type refers to non-selected tissues.
• a reference tissue type is a non-selected tissue from a different organ or location within the body or with a different function than the selected tissue.
• the select tissue is the site of desired activity.
• a “non-targeted” or “non-selected” tissue refers to any tissue or multiple tissues in which the genetically modified cells are not intentionally trafficked to nor are intended to reside in.
• genetically modified cells are not substantially trafficked to nor substantially reside in a non-selected tissue.
• a trafficking signal results in trafficking of genetically modifieds cell to a selected tissue.
• down- regulated expression of a trafficking signal results in trafficking such that genetically modified cells are not trafficked to a non-selected tissue.
• a trafficking signal can be up-regulated or down-regulated in order to direct genetically modified cells to a select tissue or not toward a non-selected tissue.
• a trafficking signal includes CXCR6, integrin [32 (LFA-1), CCR1 , CCR2, CXCR4, [37 integrin, CCR4, CXCR3, or CCR3.
• a trafficking signal includes expression of or down-regulated expression of CXCR6, integrin [32 (LFA-1), CCR1 , CCR2, CXCR4, [37 integrin, CCR4, CXCR3, or CCR3.
• a trafficking signal includes expression of CXCR6, integrin [32 (LFA-1), CCR1 , CCR2, or CXCR4 to provide a positive directional signal (referred to as a “go here” trafficking signal).
• a trafficking signal includes down-regulated expression of [37 integrin, CCR4, CXCR3, or CCR3 to provide a negative directional signal (referred to as a “don’t go here” inhibitory signal).
• expression of CXCR6 by genetically modified cells results in trafficking to the liver.
• expression of integrin [32 (LFA-1), CCR1, and/or CCR2 by genetically modified cells results in trafficking to the pancreas.
• expression of CXCR4 by genetically modified cells results in trafficking to the bone.
• down-regulated expression of [37 integrin by genetically modified cells results in trafficking away from the colon and/or small intestine.
• down-regulated expression of CCR4, CXCR3, and/or CCR3 by genetically modified cells results in trafficking away from the lung.
• down-regulated expression of CXCR3 by genetically modified cells results in trafficking away from the prostate.
• down-regulated expression of CCR4, CXCR3, CCR3, and/or [37 integrin by genetically modified cells results in trafficking away from the lung, kidney, and/or stomach.
• Down-regulated expression refers to cellular treatment that decreases expression of an endogenously expressed protein under comparable conditions to the same cell type that has not been exposed to the cellular treatment.
• down-regulated expression is achieved through gene knockout.
• down- regulated expression results from a genetic modification that disrupts expression of a gene or genetic sequence within a cell.
• Other methods to down-regulate endogenous gene expression is through interfering RNA, administration of compounds that de-stabilize mRNA or cDNA, or DNA modification.
• CXCR6, or CXC motif chemokine receptor 6 increases the migration of cells expressing CXCR6 to tissues expressing the CXCR6 ligand, CXCL16.
• CXCR6 Homo sapiens
• NCBI Reference Sequence: NP_001373364.1 includes the sequence: MAEHDYHEDYGFSSFNDSSQEEHQDFLQFSKVFLPCMYLWFVCGLVGNSLVLVISIFYHKLQS LTDVFLVNLPLADLVFVCTLPFWAYAGIHEWVFGQVMCKSLLGIYTINFYTSMLILTCITVDRFIVV VKATKAYNQQAKRMTWGKVTSLLIWVISLLVSLPQIIYGNVFNLDKLICGYHDEAISTVVLATQMT LG FFLPLLTM I VCYSVI I KTLLH AGG FQKH RSLKI I FLVM AVFLLTQM PFN LM KFI RSTH WEYYAMT SFHYTIMVTE
• Integrin is the beta chain of an integrin known to bind with CD11a, CD11 b, CD11c, or CD11d, resulting in the formation of the Lymphocyte Functions Associated Antigen 1 (LFA-1).
• LFA-1 is a protein expressed by B cells, T cells, macrophages, neutrophils, and NK cells. It is involved in the adhesion and binding to antigen presenting cells through interactions with ICAM-1.
• integrin [32 [Homo sapiens] (UniProtKB/Swiss-Prot: P05107.2) includes the sequence: MLGLRPPLLALVGLLSLGCVLSQECTKFKVSSCRECIESGPGCTWCQKLNFTGPGDPDSIRCD TRPQLLMRGCAADDIMDPTSLAETQEDHNGGQKQLSPQKVTLYLRPGQAAAFNVTFRRAKGY PIDLYYLMDLSYSMLDDLRNVKKLGGDLLRALNEITESGRIGFGSFVDKTVLPFVNTHPDKLRNP CPNKEKECQPPFAFRHVLKLTNNSNQFQTEVGKQLISGNLDAPEGGLDAMMQVAACPEEIGW RNVTRLLVFATDDGFHFAGDGKLGAILTPNDGRCHLEDNLYKRSNEFDYPSVGQLAHKLAENNI QPIFAVTSRMVKTYEKLTEIIPKSAVGELSEDSSNVVQLIKNAYNKLSSRVFLDHNALP
• CCR1 C-C motif chemokine receptor 1
• MIP-1 alpha macrophage inflammatory protein 1 alpha
• RANTES regulated on activation normal T expressed and secreted protein
• MCP-3 monocyte chemoattractant protein 3
• MPIF-1 myeloid progenitor inhibitory factor-1
• Chemokines and their receptors mediated signal transduction are critical for the recruitment of effector immune cells to the site of inflammation.
• CCR1 Homo sapiens
• NCBI Reference Sequence: NP_001286.1 includes the sequence: METPNTTEDYDTTTEFDYGDATPCQKVNERAFGAQLLPPLYSLVFVIGLVGNILVVLVLVQYKRL KNMTSIYLLNLAISDLLFLFTLPFWIDYKLKDDWVFGDAMCKILSGFYYTGLYSEIFFIILLTIDRYL AIVHAVFALRARTVTFGVITSIIIWALAILASMPGLYFSKTQWEFTHHTCSLHFPHESLREWKLFQ ALKLNLFGLVLPLLVMIICYTGIIKILLRRPNEKKSKAVRLIFVIMIIFFLFWTPYNLTILISVFQDFLFT HECEQSRHLDLAVQVTEVIAYTHCCVNPVIYAFVGERFRKYLRQLFHRRVAVHLVKWLPFLSVD RLERVSSTSPSTGEHELSAGF (SEQ ID NO: 68).
• CCR2 C-C motif chemokine receptor 2
• monocyte chemoattractant protein-1 a chemokine which specifically mediates monocyte chemotaxis.
• Monocyte chemoattractant protein-1 is involved in monocyte infiltration in inflammatory diseases such as rheumatoid arthritis as well as in the inflammatory response against tumors.
• CCR2 isoform A [Homo sapiens] (NCBI Reference Sequence: NP_001116513.2) includes the sequence: MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVL ILINCKKLKCLTDIYLLNLAISDLLFLITLPLWAHSAANEWVFGNAMCKLFTGLYHIGYFGGIFFIILL TIDRYLAIVHAVFALKARTVTFGVVTSVITWLVAVFASVPGIIFTKCQKEDSVYVCGPYFPRGWN NFHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHRAVRVIFTIMIVYFLFWTPYNIVILLNTF QEFFGLSNCESTSQLDQATQVTETLGMTHCCINPIIYAFVGEKFRSLFHIALGCRIAPLQKPVCG GPGVRPGKNVKVTTQGLLDGRGKGKSIGRAPEASLQDKEGA (SEQ ID NO:
• CXCR4 C-X-C motif chemokine receptor 4
• CXCR4 is a receptor specific for stromal cell-derived factor-1. The protein has 7 transmembrane regions and is located on the cell surface.
• CXCR4 [Homo sapiens] (GenBank: CAA12166.1) includes the sequence: MEGISSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLV ILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSS VLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFY PNDLWVWFQFQHIMVGLILPGMLSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGI SIDSFI LLEI I KQGCEFENTVH
• Integrin a4[37 is expressed on memory T cells destined to home to the gut, where the ligands CCL25 and MadCAMI are constitutively expressed (Mora et al., 2003, Nature. 424:88- 93).
• integrin a4 subunit [Homo sapiens] (GenBank: CAD53329.1) includes the sequence: RVCALEQQKSAMQTLKGIVQFLSKTDKRLLYCIKADPHCLNFLCNFGKMESGKEASVHIQLEGR PSILEMDETSALKFEIRATGFPEPNPRVIELNKDENVAHVLLEGLHHQRPKRYFTIVIISSSLLLGLI VLLLISYVMWKAGFFKRQYKSILQEENRRDSWSYINSKSNDD (SEQ ID NO: 72).
• integrin 7 [Homo sapiens] (UniProtKB/Swiss-Prot: P26010.1) includes the sequence: MVALPMVLVLLLVLSRGESELDAKIPSTGDATEWRNPHLSMLGSCQPAPSCQKCILSHPSCAW CKQLNFTASGEAEARRCARREELLARGCPLEELEEPRGQQEVLQDQPLSQGARGEGATQLAP QRVRVTLRPGEPQQLQVRFLRAEGYPVDLYYLMDLSYSMKDDLERVRQLGHALLVRLQEVTH SVRIGFGSFVDKTVLPFVSTVPSKLRHPCPTRLERCQSPFSFHHVLSLTGDAQAFEREVGRQS VSGNLDSPEGGFDAILQAALCQEQIGWRNVSRLLVFTSDDTFHTAGDGKLGGIFMPSDGHCHL DSNGLYSRSTEFDYPSVGQVAQALSAANIQPIFAVTSAALPVYQELSKLIPKSAVGELSED
• CCR4 [Homo sapiens] (NCBI Reference Sequence: NP_005499.1) includes the sequence:
• CXCR3 (C-X-C Motif Chemokine Receptor 3) is a Gai protein-coupled receptor that promotes recruitment of Th17 cells from the blood into the liver after liver injury.
• CXCR3 [Homo sapiens] (UniProtKB/Swiss-Prot: P49682.2) includes the sequence: MVLEVSDHQVLNDAEVAALLENFSSSYDYGENESDSCCTSPPCPQDFSLNFDRAFLPALYSLL FLLGLLGNGAVAAVLLSRRTALSSTDTFLLHLAVADTLLVLTLPLWAVDAAVQWVFGSGLCKVA GALFNINFYAGALLLACISFDRYLNIVHATQLYRRGPPARVTLTCLAVWGLCLLFALPDFIFLSAH HDERLNATHCQYNFPQVGRTALRVLQLVAGFLLPLLVMAYCYAHILAVLLVSRGQRRLRAMRL VVVVVVA FA LC WTPYH LVVLVD I LM
• CCR3 C-C motif chemokine receptor 3
• CCR3 is important in leukocyte chemotaxis.
• CCR3 [Homo sapiens] (GenBank: AAL85154.1) includes the sequence: MTTSLDTVETFGTTSYYDDVGLLCEKADTRALMAQFVPPLYSLVFTVGLLGNWWMILIKYRRL RIMTNIYLLNLAISDLLFLVTLPFWIHYVRGHNWVFGHGMCKLLSGFYHTGLYSEIFFIILLTIDRYL AIVHAVFALRARTVTFGVITSIVTWGLAVLAALPEFIFYETEELFEETLCSALYPEDTVYSWRHFH TLRMTIFCLVLPLLVMAICYTGIIKTLLRCPSKKKYKAIRLIFVIMAVFFIFWTPYNVAILLSSYQSILF GNDCERSKHLDLVMLVTEVIAYSHCCMNPVIYAFVGERFRKYLRHFFHRHLLMHLGRYIPFLPS EKLERTSSVSPST
• RNA knockout includes a trafficking signal gRNA.
• Example gRNA sequences are presented in Table 1.
• Table 1 gRNA Targeting Trafficking Signals.
• expression of CXCR6 results in trafficking to the liver.
• expression of integrin 02 (LFA-1), CCR1 , and/or CCR2 results in trafficking to the pancreas.
• expression of CXCR4 results in trafficking to the bone.
• down-regulating expression of 07 integrin results in trafficking away from the colon.
• down-regulating expression of CCR4, CXCR3, and/or CCR3 results in trafficking away from the lung.
• down-regulating expression of CXCR3 results in trafficking away from the prostate.
• down-regulating expression of CCR4, CXCR3, CCR3, and/or [37 integrin results in trafficking away from the lung and kidney.
• down-regulating expression of CCR4, CXCR3, CCR3, and/or (37 integrin results in trafficking away from the stomach, lung, and/or kidney.
• down-regulating expression of [37 integrin results in trafficking away from the small intestine and/or colon.
• Cells can be engineered to remain within a select tissue by introducing a genetic construct expressing a retention signal to the cell. Alternatively, a cell can be engineered to not remain within a tissue by knocking out or down-regulating an endogenous retention signal.
• a retention signal includes CD69, Runx3, Hobit, or CD103.
• CD69, Runt-related transcription factor 3 (Runx3), Hobit, or CD103 is expressed to create a retention signal.
• expression of a retention signal by genetically modified cells can induce the genetically modified cells to remain in a select tissue.
• down-regulated expression of a retention signal within genetically modified cells can prevent the genetically modified cells from remaining in a select tissue.
• CD69 expression is considered a marker of tissue residency or retention in certain tissues.
• CD69 is a membrane-bound, type II C-lectin receptor and a marker for early lymphocyte activation and T cell differentiation.
• CD69 is commonly used as a marker for tissue-resident-memory CD8+ T cells (TRM).
• TRM are an example of non-circulating T cells that persist at a specific site.
• CD69 Homo sapiens (GenBank: CAA80298.1) includes the sequence: MSSENCFVAENSSLHPESGQENDATSPHFSTRHEGSFQVPVLCAVMNVVFITILIIALIALSVGQ YNCPGQYTFSMPSDSHVSSCSEDWVGYQRKCYFISTVKRSWTSAQNACSEHGATLAVIDSEK DMNFLKRYAGREEHWVGLKKEPGHPWKWSNGKEFNNWFNVTGSDKCVFLKNTEVSSMECE KNLYWICNKPYK (SEQ ID NO: 104).
• CD69 promotes retention in a tissue.
• CD69 promotes retention in liver, pancreas, or bone.
• Runx3 expression enhances tumor-specific CD8+ T cell abundance whereas Runx3- deficiencies result in a failure of T cells to accumulate in tumors.
• Runx3 [Homo sapiens] (GenBank: AAH13362.1) includes the sequence:
• Hobit (homolog of Blimp-1 in T cells), also referred to as zinc finger protein ZNF683, is almost exclusively expressed in effector T cells and contribute to residency in tissues.
• Hobit [Homo sapiens] (NP_001294854.1) includes the sequence: MKEESAAQLGCCHRPMALGGTGGSLSPSLDFQLFRGDQVFSACRPLPDMVDAHGPSCASWL CPLPLAPGRSALLACLQDLDLNLCTPQPAPLGTDLQGLQEDALSMKHEPPGLQASSTDDKKFT VKYPQNKDKLGKQPERAGEGAPCPAFSSHNSSSPPPLQNRKSPSPLAFCPCPPVNSISKELPF LLHAFYPGYPLLLPPPHLFTYGALPSDQCPHLLMLPQDPSYPTMAMPSLLMMVNELGHPSARW ETLLPYPGAFQASGQALPSQARNPGAGAAPTDSPGLERGGMASPAKRVPLSSQTGTAALPYP LKKKNG
• CD103 is a marker for alloantigen-induced regulatory CD8+ T cells.
• CD103 is also known as integrin, alpha E.
• Szabo et al. Sc/ lmmunol.2019, 4(34):eaas9673.
• CD103 [Homo sapiens] (GenBank: AAI13437.1) includes the sequence: MWLFHTLLCIASLALLAAFNVDVARPWLTPKGGAPFVLSSLLHQDPSTNQTWLLVTSPRTKRTP GPLHRCSLVQDEILCHPVEHVPIPKGRHRGVTVVRSHHGVLICIQVLVRRPHSLSSELTGTCSLL GPDLRPQAQANFFDLENLLDPDARVDTGDCYSNKEGGGEDDVNTARQRRALEKEEEEDKEEE EDEEEEEAGTEIAIILDGSGSIDPPDFQRAKDFISNMMRNFYEKCFECNFALVQYGGVIQTEFDL RDSQDVMASLARVQNITQVGSVTKTASAMQHVLDSIFTSSHGSRRKASKVMWLTDGGIFEDP LNLTTVINSPKMQGVERFAIGVGEEFKSARTARELNLIASDPDETHAFKVTNYMALDGLLSKLRY NIISMEGTVGDALHYQLAQ
• gRNA guide RNA
• a retention signal knockout includes a retention signal gRNA.
• Example gRNA sequences are presented in Table 2.
• a recombinant receptor is or includes a binding domain that binds a target antigen, wherein the recombinant receptor is expressed by a cell following the artificial introduction of nucleic acid encoding the recombinant receptor into the cell.
• the recombinant receptor can be, e.g., a CAR, an engineered T-cell receptor (eTCR), or a CAR/TCR hybrid.
• CAR include several distinct subcomponents that allow genetically modified cells to recognize and kill unwanted cells, such as cancer cells.
• the subcomponents include at least an extracellular component and an intracellular component.
• the extracellular component includes a binding domain that specifically binds a marker that is preferentially present on the surface of unwanted cells. When the binding domain binds such markers, the intracellular component activates the cell to destroy the bound cell.
• CAR additionally include a transmembrane domain that links the extracellular component to the intracellular component, and other subcomponents that can increase the CAR’s function. For example, the inclusion of a spacer and/or one or more linker sequences can allow the CAR to have additional conformational flexibility, often increasing the binding domain’s ability to bind the target antigen.
• eTCR include a binding domain that binds a target antigen linked to the Ca and/or C
• a TCR is a heterodimeric fusion protein that typically includes an a and p chain. Each chain includes a variable region (Va and VP) and a constant region (Co and CP).
• Va and VP variable region
• Co and CP constant region
• an eTCR does not include the native TCR variable region but does include the native TCR constant region.
• the eTCR includes an scFv as the variable region of either the a or p chain.
• the eTCR includes an scFv as the variable region of both the a and p chain.
• eTCR include a Ca and/or Cp chain sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a known or identified TCR Ca or Cp.
• binding domains include any substance that binds to a cellular marker to form a complex. The choice of binding domain can depend upon the type and number of cellular markers that define the surface of a target cell. Examples of binding domains include cellular marker ligands, receptor ligands, antibodies, peptides, peptide aptamers, receptors (e.g., T cell receptors), or combinations and engineered fragments or formats thereof.
• Antibodies are one example of binding domains and include whole antibodies or binding fragments of an antibody, e.g., Fv, Fab, Fab', F(ab')2, and single chain (sc) forms and fragments thereof that bind specifically a cellular marker.
• Antibodies or antigen binding fragments can include all or a portion of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, non-human antibodies, recombinant antibodies, chimeric antibodies, bispecific antibodies, mini bodies, and linear antibodies.
• Antibodies are produced from two genes, a heavy chain gene and a light chain gene.
• an antibody includes two identical copies of a heavy chain, and two identical copies of a light chain.
• segments referred to as complementary determining regions (CDRs) dictate epitope binding.
• Each heavy chain has three CDRs (i.e., CDRH1 , CDRH2, and CDRH3) and each light chain has three CDRs (i.e., CDRL1 , CDRL2, and CDRL3).
• CDR regions are flanked by framework residues (FR).
• CDR sets can be based on, for example, Kabat numbering (Kabat et al. (1991) “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme)); Chothia (Al-Lazikani et al. (1997) JMB 273:927-948 (“Chothia” numbering scheme)); Martin (Abinandan et al. (2008) Mol Immunol. 45:3832-3839 (“Martin” numbering scheme)); Gelfand (Gelfand and Kister (1995) Proc Natl Acad Sci USA. 92:10884-10888; Gelfand et al. (1998) Protein Eng.
• scFvs based on the binding domains described herein and for use in a recombinant receptor can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
• ScFv molecules can be produced by linking VH and VL regions of an antibody together using flexible polypeptide linkers. If a short polypeptide linker is employed (e.g., between 5-10 amino acids) intrachain folding is prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site.
• linker orientations and sizes see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, US 2005/0100543, US 2005/0175606, US 2007/0014794, and W02006/020258 and W02007/024715. More particularly, linker sequences that are used to connect the VL and VH of an scFv are generally five to 35 amino acids in length. In particular embodiments, a VL-VH linker includes from five to 35, ten to 30 amino acids or from 15 to 25 amino acids. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies. scFv are commonly used as the binding domains of recombinant receptors.
• binding fragments such as Fv, Fab, Fab', F(ab')2
• Fv, Fab, Fab', F(ab')2 can also be used within the recombinant receptors disclosed herein.
• Additional examples of antibody-based binding domain formats for use in a recombinant receptor include scFv-based grababodies and soluble VH domain antibodies. These antibodies form binding regions using only heavy chain variable regions. See, for example, Jespers et al., Nat. Biotechnol. 22:1161 , 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.
• a VL region in a binding domain of the present disclosure is derived from or based on a VL of an antibody disclosed herein and contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VL of the antibody disclosed herein.
• An insertion, deletion or substitution may be anywhere in the VL region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain containing the modified VL region can still specifically bind its target with an affinity similar to the wild type binding domain.
• a binding domain VH region of the present disclosure can be derived from or based on a VH of an antibody disclosed herein and can contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VH of the antibody disclosed herein.
• one or more e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10
• amino acid substitutions e.g., conservative amino acid substitutions or non-conservative amino acid substitutions
• An insertion, deletion or substitution may be anywhere in the VH region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain containing the modified VH region can still specifically bind its target with an affinity similar to the wild type binding domain.
• the recombinant receptor includes a binding domain that binds a target antigen.
• the target antigen is a cancer antigen.
• target antigens are expressed by cancer cells or tumors.
• target antigens are preferentially expressed by target cells (e.g., cancer cells). “Preferentially expressed” means that a target antigen is found at higher levels on target cells as compared to other cell types. In some instances, a target antigen is only expressed by the targeted cell type. In other instances, the target antigen is expressed on the targeted cell type at least 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 96%, 97%, 98%, 99%, or 100% more than on non-targeted cells. In particular embodiments, the target antigen is expressed on the target cell and healthy tissue cell.
• target antigens can be significantly expressed on cancer cells and on healthy tissue cells. This means that there may be negative side effects due to on- target/off-tumor toxicities.
• Cetuximab is an anti-EGFR antibody associated with severe skin rashes thought to be caused by EGFR expression in the skin.
• Herceptin is an anti-HER2 (ERBB2) antibody. Herceptin is associated with cardiotoxicity due to target expression in the heart.
• targeting HER2 with a CAR-T cell was lethal in a patient due to on-target, off-cancer expression in the lung.
• Table 3 provides examples of cancer antigens that are more likely to be expressed in particular primary tumor types.
• target antigens can be expressed by cancer cells that have metastasized and by primary tumors.
• healthy tissues in other parts of the body may express the target antigens and methods disclosed herein describe how to avoid these on-target/off-tumor scenarios. These methods rely on trafficking cells to particular organs rather than to sites of cancer generally.
• CEA is a protein found in various malignancies including colorectal cancer, medullary thyroid cancer, breast cancer, mucinous ovarian cancer, and pancreatic adenocarcinoma.
• CEA includes the sequence referenced in GenBank: CAE75559.1 , Accession: AAB64011.1, or Accession: AAA40910.1.
• a binding domain that binds human CEA i.e., a CEA binding domain
• the AHB7 scFv includes a variable light chain including the sequence:
• variable light chain and variable heavy chain are connected by a GS linker.
• the GS linker is includes the sequence: GSGGGGSGGGGS (SEQ ID NO: 197).
• the AHB7 scFv includes a variable light chain encoded by the sequence:
• the anti-CEA binding domain includes a variable light chain including a CDRL1 including the sequence RASSSVTYIH (SEQ ID NO: 207), a CDRL2 including the sequence ATSNLAS (SEQ ID NO: 208), and CDRL3 including the sequence QHWSSKPPT (SEQ ID NO: 209), and a variable heavy chain including a CDRH1 including the sequence DYYMN (SEQ ID NO: 210), CDRH2 including the sequence FIGNKANGYTTEYSASVKG (SEQ ID NO: 211), and CDRH3 including the sequence DRGLRFYFDY (SEQ ID NO: 212), according to Kabat. CDR predictions according to other CDR definitions can also be determined.
• EpCAM is a protein found various malignancies.
• EpCAM includes the sequence referenced in Accession: P16422.2, Accession: AAH05618.1 , Accession: 055159.1 , Accession: KAF6429286.1 , or Accession: XP_016803985.1.
• a binding domain that binds EpCAM i.e. , an EpCAM binding domain
• a binding domain that binds mouse EpCAM includes G8.8.
• the anti-EpCAM antibody includes a variable light chain including the sequence: EVQLLEQSGAELVRPGTSVKISCKASGYAFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNE KFKGKATLTADKSSSTAYMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVTVSSGGGG SGGGGSGGGGSELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQP PKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLEIK GGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNGNTYLSWYLQKPGQSPQSLIYKVSN RFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIKGEGTSTGSG
• the anti-EpCAM antibody includes a variable light chain including a CDR1 including the sequence RASQSVSSNLA (SEQ ID NO: 127), CDR2 including the sequence GASTTAS (SEQ ID NO: 128), and CDR3 including the sequence QQYNNWPPAYT (SEQ ID NO: 129) and a variable heavy chain including a CDR1 including the sequence SYAIS (SEQ ID NO: 124), a CDR2 including the sequence SYAIS (SEQ ID NO: 125), and CDR3 including the sequence SYAIS (SEQ ID NO: 126).
• a variable light chain including a CDR1 including the sequence RASQSVSSNLA (SEQ ID NO: 127), CDR2 including the sequence GASTTAS (SEQ ID NO: 128), and CDR3 including the sequence QQYNNWPPAYT (SEQ ID NO: 129) and a variable heavy chain including a CDR1 including the sequence SYAIS (SEQ ID NO: 124), a CDR2 including the
• a G8.8 antibody includes a variable light chain including the sequence: DIQMTQSPASLSASLGETVSIECLASEGISNDLAWYQQKSGKSPQLLIYATSRLQDGVPSRFSG SGSGTRYSLKISGMQPEDEADYFCQQSYKYPWTFGGGTKLELK (SEQ ID NO: 195) and a variable heavy chain including the sequence:
• variable light chain and variable heavy chain are connected with a GlySer linker including the sequence: GSGGGGSGGGGS (SEQ ID NO: 197).
• the anti-EpCAM binding domain includes a variable light chain including a CDRL1 including the sequence LASEGISNDLA (SEQ ID NO: 206), a CDRL2 including the sequence ATSRLQD (SEQ ID NO: 213), and CDRL3 including the sequence QQSYKYPWT (SEQ ID NO: 214), and a variable heavy chain including a CDRH1 including the sequence NFPMA (SEQ ID NO: 215), CDRH2 including the sequence TISTSGGSTYYRDSVKG (SEQ ID NO: 216), and CDRH3 including the sequence TLYILRVFYFDY (SEQ ID NO: 217), according to Kabat. CDR predictions according to other CDR definitions can also be determined.
• HER2 is a protein found on the surface of all breast cells. It can help breast cancer cells grow more quickly.
• HER2 includes the sequence referenced in Accession: P04626.1.
• a binding domain that binds HER2 includes trastuzumab or pertuzumab.
• the trastuzumab includes a variable light chain includes the sequence: DIQMTQSPILLSASVGDRVTITCRASQDVNTAVAWYQQRTNGSPRLLIYSASFLYSGVPSRFSG SRSGTDFTLTISSLQPEDIADYYCQQHYTTPPTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 130) and a variable heavy chain includes the sequence:
• the anti-HER2 binding domain includes a variable light chain including a CDRL1 including the sequence RASQDVNTAVA (SEQ ID NO: 218), a CDRL2 including the sequence SASFLYS (SEQ ID NO: 219), and CDRL3 including the sequence QQHYTTPPT (SEQ ID NO: 220), and a variable heavy chain including a CDRH1 including the sequence DTYIH (SEQ ID NO: 221), CDRH2 including the sequence RIYPTNGYTRYADSVKG (SEQ ID NO: 222), and CDRH3 including the sequence WGGDGFYAMDY (SEQ ID NO: 223), according to Kabat. CDR predictions according to other CDR definitions can also be determined.
• the pertuzumab includes a variable light chain includes the sequence: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSG SGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 132) and a variable heavy chain includes the sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYN QRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTK GPSVFPLAPSSKST
• the anti-HER2 binding domain includes a variable light chain including a CDRL1 including the sequence KASQDVSIGVA (SEQ ID NO: 224), a CDRL2 including the sequence SASYRYT (SEQ ID NO: 225), and CDRL3 including the sequence QQYYIYPYT (SEQ ID NO: 226), and a variable heavy chain including a CDRH1 including the sequence DYTMD (SEQ ID NO: 227), CDRH2 including the sequence DVNPNSGGSIYNQRFKG (SEQ ID NO: 228), and CDRH3 including the sequence NLGPSFYFDY (SEQ ID NO: 229), according to Kabat.
• a variable light chain including a CDRL1 including the sequence KASQDVSIGVA (SEQ ID NO: 224), a CDRL2 including the sequence SASYRYT (SEQ ID NO: 225), and CDRL3 including the sequence QQYYIYPYT (SEQ ID NO: 226)
• PSA is a protein produced by normal and malignant cells of the prostate gland.
• PSA is a member of the kallikrein-related peptidase family and is secreted by the epithelial cells.
• PSA includes the sequence referenced in Accession: NP_001639.1, Accession: NP_001025218.1 , or Accession: NP_001025219.1.
• a binding domain that binds PSA includes MSVA-603R, AS10 1352, 8A9B8, or A94916.
• an anti-PSA antibody includes a variable light chain including a CDRL1 including the sequence RASQNVNTDVA (SEQ ID NO: 134), a CDRL2 including the sequence STSYLQS (SEQ ID NO: 135), and a CDRL3 including the sequence QQYSNYPLT (SEQ ID NO: 136); and a variable heavy chain including a CDRH1 including the sequence TTGMGVS (SEQ ID NO: 137), a CDRH2 including the sequence HIYWDDDKRYSTSLK (SEQ ID NO: 138), and a CDRH3 including the sequence KGYYGYFDY (SEQ ID NO: 139).
• GPC3 is a heparan sulfate proteoglycan that is highly expressed in hepatocellular carcinoma.
• GPC3 includes the sequence referenced in Accession: P51654.1 , Accession: NP_001158090.1, Accession: NP_001158089.1 , Accession: NP_004475.1 , or Accession: NP_001158091.1.
• the anti- GPC3 antibody includes a variable light chain including a CDRL1 including the sequence RASQSISSYLN (SEQ ID NO: 140), a CDRL2 including the sequence AASSLQS (SEQ ID NO: 141), and a CDRL3 including the sequence QQSYSTPLT (SEQ ID NO: 142); and a variable heavy chain including a CDRH1 including the sequence GYTFTSYFLH (SEQ ID NO: 143), a CDRH2 including the sequence IIDPPTGRTTYAQKFQG (SEQ ID NO: 144), and a CDRH3 including the sequence GNYGGRYFDY (SEQ ID NO: 145).
• a variable light chain including a CDRL1 including the sequence RASQSISSYLN (SEQ ID NO: 140), a CDRL2 including the sequence AASSLQS (SEQ ID NO: 141), and a CDRL3 including the sequence QQSYSTPLT (SEQ ID NO: 142); and a variable heavy chain including
• mesothelin is a tumor-associated antigen broadly expressed on various malignant tumor cells, while its expression is generally limited to normal mesothelial cells.
• mesothelin includes the sequence referenced in Accession: AAH09272.1 , Accession: AAH03512.1 , Accession: KAI4052676.1 , Accession: KAI4052675.1 , or Accession: AAV87530.1.
• the anti-mesothelin antibody includes a variable light chain including a CDRL1 including the sequence RASQSVSSYLA (SEQ ID NO: 146), a CDRL2 including the sequence DASNRAT (SEQ ID NO: 147), and a CDRL3 including the sequence QQRSNWPLT (SEQ ID NO: 148); and a variable heavy chain including a CDRH1 including the sequence IYGMH (SEQ ID NO: 149), a CDRH2 including the sequence VIWYDGSHEYYADSVKG (SEQ ID NO: 150), and a CDRH3 including the sequence DGDYYDSGSPLDY (SEQ ID NO: 151).
• a variable light chain including a CDRL1 including the sequence RASQSVSSYLA (SEQ ID NO: 146), a CDRL2 including the sequence DASNRAT (SEQ ID NO: 147), and a CDRL3 including the sequence QQRSNWPLT (SEQ ID NO: 148); and a variable heavy chain including
• FOLR1 expression is tumor specific and is expressed in various epithelial tumors such as ovarian, breast, endometrial, and lung cancers.
• FOLR1 includes the sequence referenced in Accession: P15328.3, Accession: NP_057937.1 , or Accession: CAG46816.1.
• a binding domain that binds FOLR1 includes Farletuzumab.
• the Farletuzumab includes a variable light chain includes the sequence: DIQLTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFS GSGSGTDYTFTISSLQPEDIATYYCQQWSSYPYMYTFGQGTKVEIK (SEQ ID NO: 152) and a variable heavy chain includes the sequence: EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGGSYTYYA DSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQGTPVTVSS (SEQ ID NO: 153).
• the FOLR1-binding domain includes the Farletuzumab antibody (MorAb-003).
• the FOLR1 -binding domain is a human or humanized binding domain including a variable heavy chain including a CDRH1 sequence including GYGLS (SEQ ID NO: 154), a CDRH2 sequence including MISSGGSYTYYADSVKG (SEQ ID NO: 155), and a CDRH3 sequence including HGDDPAWFAY (SEQ ID NO: 156), and a variable light chain including a CDRL1 sequence including SVSSSISSNNLH (SEQ ID NO: 157), a CDRL2 sequence including GTSNLAS (SEQ ID NO: 158), and a CDRL3 sequence including QQWSSYPYMYT (SEQ ID NO: 159), according to Kabat numbering scheme.
• MUC1 is an oncoprotein found in 40% of breast cancers.
• MUC1 includes the sequence referenced in Accession: CAA56734.1 or Accession: P15941.3.
• a binding domain that binds MUC1 includes GP1.4 or SM3.
• the SM3 includes a variable light chain includes the sequence: DIVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARF SGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEKSSPSVTLFPPSSEEL ETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWER HSSYSCQVTHEGHTVEKSLSRADCS (SEQ ID NO: 160) and a variable heavy chain includes the sequence:
• the MUC1 binding domain includes a variable light chain including a CDRL1 including the sequence RSSTGAVTTSNYAN (SEQ ID NO: 230), a CDRL2 including the sequence GTNNRAP (SEQ ID NO: 231), and CDRL3 including the sequence ALWYSNHWV (SEQ ID NO: 232), and a variable heavy chain including a CDRH1 including the sequence NYWMN (SEQ ID NO: 233), CDRH2 including the sequence EIRLKSNNYATHYAESVKG (SEQ ID NO: 234), and CDRH3 including the sequence VGQFAY (SEQ ID NO: 235), according to Kabat. CDR predictions according to other CDR definitions can also be determined.
• CA 19-9 expression is often a sign of pancreatic cancer or other types of cancers.
• CA 19-9 is also known as sialyl Le A , SLe A , or as the IUPAC name: (12S,14S,15R,16R,32R,33R,34S,35S,36R,52R,53S,54R,55R,56E,72S,73S,74R,75S,76S)- 15,55-Diacetamido-14,33,35,56,73,74,75-heptahydroxy-36,52-bis(hydroxymethyl)-76-methyl- 16-[(1 R,2R)-1 ,2,3-trihydroxypropyl]-2,4,6-trioxa-1,7(2),3(4,2),5(4,3)-tetraoxanaheptaphane-12- carboxylic acid.
• a binding domain that binds CA 19-9 includes 5B1.
• the 5B1 antibody includes a variable light chain including the sequence: MAGFPLLLTLLTHCAGSWAQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVYWYQQLPGTA PKLLIYRNNQRPSGVPDRFSGSRSGTSASLAISGLRSEDEADYYCAAWDDSLGGHYVFGTGTK VTVL (SEQ ID NO: 162) and a variable heavy chain includes the sequence: MEFGLSWLFLVAILKGVQCQVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHWVRQPPG KGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNSLRLEDTAFYYCAKDIRRFSTG GAEFEYWGQGTLVTVSS (SEQ ID NO: 163).
• the anti-CA 19-9 binding domain includes a variable light chain including a CDRL1 including the sequence SGSSSNIGSNFVY (SEQ ID NO: 236), a CDRL2 including the sequence RNNQRPS (SEQ ID NO: 237), and CDRL3 including the sequence AAWDDSLGGHYV (SEQ ID NO: 238), and a variable heavy chain including a CDRH1 including the sequence AYAMH (SEQ ID NO: 239), CDRH2 including the sequence SINWNSGRIAYADSVKG (SEQ ID NO: 240), and CDRH3 including the sequence DIRRFSTGGAEFEY (SEQ ID NO: 241), according to Kabat. CDR predictions according to other CDR definitions can also be determined.
• FIG. 14 presents a table of cancer antigens and the primary tumor type by which they are expressed.
• the table further displays which trafficking signals, retention signals, and inhibitory signals (i.e., down-regulation of trafficking signals) can be used to direct genetically modified cells to a select tissue and avoid a non-selected tissue for the particular cancer types and organs that they commonly affect.
• trafficking signals, retention signals, and inhibitory signals i.e., down-regulation of trafficking signals
• transmembrane Domains As indicated, a transmembrane domain within a recombinant receptor serves to connect the extracellular component and intracellular component through the cell membrane. The transmembrane domain can anchor the expressed molecule in the modified cell’s membrane.
• the transmembrane domain can be derived either from a natural and/or a synthetic source. When the source is natural, the transmembrane domain can be derived from any membrane-bound or transmembrane protein.
• Transmembrane domains can include at least the transmembrane region(s) of the a, p or £ chain of a T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137 CD154, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9.
• TLR1 Toll-like receptor 1
• a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, 0X40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1 BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rp, IL2RY, IL7R a, ITGA1 , VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI Id, ITGAE, CD103, ITGAL, CDI la, ITGAM, CDI lb, ITGAX, CDI Ic, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1 (CD226),
• a variety of human hinges can be employed as well including the human Ig (immunoglobulin) hinge (e.g., an lgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge or a CD8a hinge.
• the recombinant receptor e.g., CAR
• CAR includes a CD28 transmembrane domain. It has been shown that a CD28 transmembrane domain reduces the antigen-threshold for second-generation 4-1 BB CAR T cell activation.
• a transmembrane domain has a three-dimensional structure that is thermodynamically stable in a cell membrane, and generally ranges in length from 15 to 30 amino acids.
• the structure of a transmembrane domain can include an a helix, a p barrel, a p sheet, a p helix, or any combination thereof.
• a transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid within the extracellular region of the recombinant receptor (e.g., up to 15 amino acids of the extracellular region) and/or one or more additional amino acids within the intracellular region of the recombinant receptor (e.g., up to 15 amino acids of the intracellular components).
• the transmembrane domain is from the same protein that the signaling domain, co-stimulatory domain or the hinge domain is derived from.
• the transmembrane domain is not derived from the same protein that any other domain of the recombinant receptor is derived from.
• the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other unintended members of the receptor complex.
• the transmembrane domain is encoded by the nucleic acid sequence encoding the CD28 transmembrane domain (SEQ ID NOs: 8, 30, 31, 32, or 33).
• the transmembrane domain includes the amino acid sequence of the CD28 transmembrane domain (SEQ ID NOs: 27, 28, or 29).
• the intracellular component of a recombinant receptor includes an effector domain that is responsible for activation of the cell in which the recombinant receptor is expressed.
• effector domain is thus meant to include any portion of the intracellular domain sufficient to transduce an activation signal.
• An effector domain can directly or indirectly promote a biological or physiological response in a cell when receiving the appropriate signal.
• an effector domain is part of a protein or protein complex that receives a signal when bound, or it binds directly to a target molecule, which triggers a signal from the intracellular effector domain.
• An effector domain may directly promote a cellular response when it contains one or more signaling domains or motifs, such as an immunoreceptor tyrosinebased activation motif (ITAM).
• ITAM immunoreceptor tyrosinebased activation motif
• an effector domain will indirectly promote a cellular response by associating with one or more other proteins that directly promote a cellular response, such as co-stimulatory domains.
• Effector domains can provide for activation of at least one function of a modified cell upon binding to the cellular marker expressed by a cancer cell. Activation of the modified cell can include one or more of differentiation, proliferation and/or activation or other effector functions.
• an effector domain can include an intracellular signaling component including a T cell receptor and a co-stimulatory domain which can include the cytoplasmic sequence from co-receptor or co-stimulatory molecule.
• An effector domain can include one, two, three or more intracellular signaling components (e.g., receptor signaling domains, cytoplasmic signaling sequences), co-stimulatory domains, or combinations thereof.
• exemplary effector domains include signaling and stimulatory domains selected from: 4-1 BB (CD137), CARD11, CD3y, CD35, CD3s, CD3 , CD27, CD28, CD79A, CD79B, DAP10, FcRa, FcR (FceRIb), FcRy, Fyn, HVEM (LIGHTR), ICOS, LAG3, LAT, Lek, LRP, NKG2D, NOTCH1 , pTa, PTCH2, 0X40, ROR2, Ryk, SLAMF1 , Slp76, TCRa, TCRp, TRIM, Wnt, Zap70, or any combination thereof.
• exemplary intracellular effector domains include signaling and co-stimulatory domains selected from: CD86, FcyRlla, DAP12, CD30, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1 , GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8a, CD8p, IL2R
• Intracellular signaling domains that act in a stimulatory manner may include iTAMs.
• iTAMs including primary cytoplasmic signaling sequences include those derived from CD3y, CD35, CD3E, CD3 , CD5, CD22, CD66d, CD79a, CD79b, and common FcRy (FCER1G), FcyRlla, FcR[3 (Fee Rib), DAP10, and DAP12.
• variants of CD3 retain at least one, two, three, or all ITAM regions.
• an effector domain includes a cytoplasmic portion that associates with a cytoplasmic signaling protein, wherein the cytoplasmic signaling protein is a lymphocyte receptor or signaling domain thereof, a protein including a plurality of ITAMs, a costimulatory domain, or any combination thereof.
• intracellular signaling domains include the cytoplasmic sequences of the CD3 chain, and/or co- receptors that act in concert to initiate signal transduction following binding domain engagement.
• a co-stimulatory domain is a domain whose activation can be required for an efficient lymphocyte response to cellular marker binding. Some molecules are interchangeable as intracellular signaling domains or co-stimulatory domains. Examples of costimulatory domains include CD27, CD28, 4-1 BB (CD 137), 0X40, CD30, CD40, PD-1 , ICOS, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.
• costimulatory domains include CD27, CD28, 4-1 BB (CD 137), 0X40, CD30, CD40, PD-1 , ICOS, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.
• CD27 co-stimulation has been demonstrated to enhance expansion, effector function, and survival of human CAR T cells in vitro and augments human T cell persistence and anti-cancer activity in vivo (Song et al. Blood. 2012; 119(3):696-706).
• co-stimulatory domain molecules include CDS, ICAM-1 , GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8a, CD8 , IL2Rp, IL2Ry, IL7Ra, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIId, ITGAE, CD103, ITGAL, CDIIa, ITGAM, CDI lb, ITGAX, CDIIc, ITGBI, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), PSGL
• the nucleic acid sequences encoding the intracellular component include a CD3 encoding sequence (SEQ I D NOs: 47 or 48) and a variant of the CD28 signaling encoding sequence (SEQ ID NOs: 9 or 41).
• the amino acid sequence of the intracellular component includes a variant of CD3 (SEQ ID NOs: 44, 45, or 46) and a portion of the CD28 (SEQ ID NOs: 40) intracellular component.
• the intracellular component includes (i) all or a portion of the signaling domain of CD3 , (ii) all or a portion of the signaling domain of CD28, or (iv) all or a portion of the signaling domain of CD3 and CD28.
• Intracellular components may also include one or more of a protein of a Wnt signaling pathway (e.g., LRP, Ryk, or ROR2), NOTCH signaling pathway (e.g., NOTCH1 , NOTCH2, NOTCH3, or NOTCH4), Hedgehog signaling pathway (e.g., PTCH or SMO), receptor tyrosine kinases (RTKs) (e.g., epidermal growth factor (EGF) receptor family, fibroblast growth factor (FGF) receptor family, hepatocyte growth factor (HGF) receptor family, insulin receptor (IR) family, platelet-derived growth factor (PDGF) receptor family, vascular endothelial growth factor (VEGF) receptor family, tropomycin receptor kinase (Trk) receptor family, ephrin (Eph) receptor family, AXL receptor family, leukocyte tyrosine kinase (LTK) receptor family, tyrosine kinase with immunoglobul
• linkers are a molecule that serves to connect two subcomponents or domains (e.g., of a recombinant receptor).
• linkers can provide flexibility for different components (e.g., different components of a recombinant receptor).
• Linkers can also include spacer regions and junction amino acids.
• proline-rich linkers can be used.
• Spacers are used to create appropriate distances and/or flexibility from other subcomponents.
• the length of a spacer is customized for binding targeted cells and mediating destruction in a recombinant receptor.
• a spacer length can be selected based upon the location of a cellular marker epitope, affinity of a binding domain for the epitope, and/or the ability of the target-binding agent to mediate cell destruction following target binding.
• Spacers typically include those having 10 to 250 amino acids, 10 to 200 amino acids, 10 to 150 amino acids, 10 to 100 amino acids, 10 to 50 amino acids, or 10 to 25 amino acids.
• a spacer is 10 amino acids, 12 amino acids, 14 amino acids, 20 amino acids, 21 amino acids, 26 amino acids, 27 amino acids, 45 amino acids, 50 amino acids, 55 amino acids, 60 amino acids, 65 amino acids, 70 amino acids, or 75 amino acids. These lengths qualify as short spacers.
• a spacer is 76 amino acids, 778 amino acids, 80 amino acids, 85 amino acids, 90 amino acids, 95 amino acids, 100 amino acids, 110 amino acids, 120 amino acids, 125 amino acids, 128 amino acids, 131 amino acids, 135 amino acids, 140 amino acids, 150 amino acids, 160 amino acids, or 179 amino acids. These lengths qualify as intermediate spacers.
• a spacer is 180 amino acids, 190 amino acids, 200 amino acids, 210 amino acids, 212 amino acids, 214 amino acids, 216 amino acids, 218 amino acids, 220 amino acids, 228 amino acids, 230 amino acids, 240 amino acids, 250 amino acids, 260 amino acids, or 270 amino acids. These lengths qualify as long spacers.
• spacers include all or a portion of an immunoglobulin hinge region.
• An immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region.
• an immunoglobulin hinge region is a human immunoglobulin hinge region.
• a “wild type immunoglobulin hinge region’’ refers to a naturally occurring upper and middle hinge amino acid sequences interposed between and connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) or interposed between and connecting the CH1 and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody.
• An immunoglobulin hinge region may be an IgG, IgA, IgD, IgE, or IgM hinge region.
• An IgG hinge region may be an I gG 1 , lgG2, 1 gG3, or lgG4 hinge region. Sequences from IgG 1 , lgG2, lgG3, lgG4 or IgD can be used alone or in combination with all or a portion of a CH2 region; all or a portion of a CH3 region; or all or a portion of a CH2 region and all or a portion of a CH3 region.
• Other examples of hinge regions that can be used in include the hinge region present in the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type or variants thereof.
• a spacer includes a hinge region that includes a type II C-lectin interdomain (stalk) region or a cluster of differentiation (CD) molecule stalk region.
• a “stalk region” of a type II C-lectin or CD molecule refers to the portion of the extracellular domain (ECD) of the type II C-lectin or CD molecule that is located between the C-type lectin-like domain (CTLD; e.g., similar to CTLD of natural killer cell receptors) and the hydrophobic portion (transmembrane domain).
• CCD extracellular domain
• CCD C-type lectin-like domain
• transmembrane domain transmembrane domain
• AAC50291.1 corresponds to amino acid residues 34-179, but the CTLD corresponds to amino acid residues 61-176, so the stalk region of the human CD94 molecule includes amino acid residues 34-60, which are located between the hydrophobic portion (transmembrane domain) and CTLD (see Boyington et al., Immunity 10:15, 1999; for descriptions of other stalk regions, see also Beavil et al., Proc. Nat'l. Acad. Sci. USA 89:153, 1992; and Figdor et al., Nat. Rev. Immunol. 2:11 , 2002).
• These type II C-lectin or CD molecules may also have junction amino acids (described below) between the stalk region and the transmembrane region or the CTLD.
• the 233 amino acid human NKG2A protein (GenBank Accession No. P26715.1) has a hydrophobic portion (transmembrane domain) ranging from amino acids 71-93 and an ECD ranging from amino acids 94-233.
• the CTLD includes amino acids 119-231 and the stalk region includes amino acids 99-116, which may be flanked by additional junction amino acids.
• Other type II C- lectin or CD molecules, as well as their extracellular ligand-binding domains, stalk regions, and CTLDs are known in the art (see, e.g., GenBank Accession Nos.
• Linkers can, for example, link VL and VH of antibody derived binding domains of scFvs and serve as junction amino acids between subcomponent portions of a recombinant receptor or other protein.
• a linker includes the Whitlow linker (GSTSGSGKPGSGEGSTKG; SEQ ID NO: 248).
• Linkers can be flexible, rigid, or semi-rigid, depending on the desired function of the linker.
• Linkers can include junction amino acids.
• linkers provide flexibility and room for conformational movement between different components.
• Commonly used flexible linkers include Gly-Ser linkers.
• the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (Gly x Ser y ) n , wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10).
• Particular examples include (Gly4Ser) n (SEQ ID NO: 164), (Gly 3 Ser) n (Gly 4 Ser)n (SEQ ID NO: 165), (Gly 3 Ser) n (Gly 2 Ser)n (SEQ ID NO: 166), or (Gly 3 Ser) n (Gly 4 Ser)i (SEQ ID NO: 167).
• the linker is (Gly 4 Ser) 4 (SEQ ID NO: 168), (Gly 4 Ser) 3 (SEQ ID NO: 169), (Gly 4 Ser) 2 (SEQ ID NO: 170), (Gly 4 Ser)i (SEQ ID NO: 171), (Gly 3 Ser) 2 (SEQ ID NO: 172), (Gly 3 Ser)i (SEQ ID NO: 173), (Gly 2 Ser) 2 (SEQ ID NO: 174) or (Gly 2 Ser)i, GGSGGGSGGSG (SEQ ID NO: 175), GGSGGGSGSG (SEQ ID NO: 176), or GGSGGGSG (SEQ ID NO: 177).
• a linker region is (GGGGS) n (SEQ ID NO: 164) wherein n is an integer including, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more.
• the linker is (EAAAK) n (SEQ ID NO: 178) wherein n is an integer including 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more.
• flexible linkers may be incapable of maintaining a distance or position (e.g., of a recombinant receptor) needed for a particular use.
• rigid or semirigid linkers may be useful.
• rigid or semi-rigid linkers include proline-rich linkers.
• a proline-rich linker is a peptide sequence having more proline residues than would be expected based on chance alone.
• a proline-rich linker is one having at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51 % proline residues.
• proline-rich linkers include fragments of proline-rich salivary proteins (PRPs).
• Linkers can be susceptible to cleavage (cleavable linker), such as, acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase- induced cleavage, and disulfide bond cleavage.
• linkers can be substantially resistant to cleavage (e.g., stable linker or noncleavable linker).
• the linker is a procharged linker, a hydrophilic linker, or a dicarboxylic acid-based linker.
• junction amino acids can be a linker which can be used to connect sequences when the distance provided by a spacer is not needed and/or wanted.
• junction amino acids can be short amino acid sequences that can be used to connect co-stimulatory intracellular signaling components.
• junction amino acids are 9 amino acids or less (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 amino acids).
• a glycine-serine doublet can be used as a suitable junction amino acid linker.
• a single amino acid e.g., an alanine, a glycine, can be used as a suitable junction amino acid.
• the recombinant receptor can optionally include a multimerization domain.
• Protein biological activities depend upon their tertiary and quaternary structure. The quaternary structure requires the physical and chemical interaction of different protein subunits or polypeptides.
• a “multimerization domain” is a domain that causes two or more proteins (monomers) to interact with each other through covalent and/or non-covalent association(s). Multimerization domains present in proteins can result in protein interactions that form dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., depending on the number of units/monomers incorporated into the multimer.
• genetic constructs can include one or more tag cassettes and/or transduction markers.
• Tag cassettes and transduction markers can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and/or eliminate genetically modified cells in vitro, in vivo and/or ex vivo.
• Tag cassette refers to a unique synthetic peptide sequence affixed to, fused to, or that is part of a genetic construct, to which a cognate binding molecule (e.g., ligand, antibody, or other binding partner) is capable of specifically binding where the binding property can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and/or eliminate the tagged protein and/or cells expressing the tagged protein.
• Transduction markers can serve the same purposes but are derived from naturally occurring molecules and are often expressed using a skipping element that separates the transduction marker from the rest of the expressed molecule (e.g., CAR, trafficking signal, retention signal).
• Tag cassettes that bind cognate binding molecules include, for example, His tag (HHHHHH; SEQ ID NO: 179), Flag tag (DYKDDDDK; SEQ ID NO: 180), Xpress tag (DLYDDDDK; SEQ ID NO: 181), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 182), Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 183), Polyglutamate tag, HA tag (YPYDVPDYA; SEQ ID NO: 184), Myc tag (EQKLISEEDL; SEQ ID NO: 185), Strep tag (which refers the original STREP® tag (WRHPQFGG; SEQ ID NO: 186), STREP® tag II (WSHPQFEK SEQ ID NO: 187 (IBA Institut fur Bioanalytik, Germany); see, e.g., US 7,981 ,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO
• Conjugate binding molecules that specifically bind tag cassette sequences disclosed herein are commercially available.
• His tag antibodies are commercially available from suppliers including Life Technologies, Pierce Antibodies, and GenScript.
• Flag tag antibodies are commercially available from suppliers including Pierce Antibodies, GenScript, and Sigma- Aldrich.
• Xpress tag antibodies are commercially available from suppliers including Pierce Antibodies, Life Technologies and GenScript.
• Avi tag antibodies are commercially available from suppliers including Pierce Antibodies, IsBio, and Genecopoeia.
• Calmodulin tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Pierce Antibodies.
• HA tag antibodies are commercially available from suppliers including Pierce Antibodies, Cell Signal and Abeam.
• Myc tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Cell Signal.
• Strep tag antibodies are commercially available from suppliers including Abeam, Iba, and Qiagen.
• Transduction markers may be selected from at least one of Cluster of Differentiation 90 (CD90 or CD90.1); a truncated HER2 extracellular protein (Her2tG); a truncated CD19 (tCD19; see Budde ef a/., Blood 122: 1660, 2013); a truncated human EGFR (tEGFR or EGFRt; see Wang et al., Blood 118: 1255, 2011); an ECD of human CD34; RQR8 which combines target epitopes from CD34 (see Fehse et al, Mol.
• CD90 or CD90.1 Cluster of Differentiation 90
• Her2tG truncated HER2 extracellular protein
• CD19 tCD19
• tCD19 see Budde ef a/., Blood 122: 1660, 2013
• a truncated human EGFR tEGFR or EGFRt; see Wang et al., Blood 118: 12
• cells are genetically modified to express CD90.1 , Her2tG, EGFRt, or tCD19.
• genetic constructs can include a polynucleotide that encodes a self-cleaving polypeptide, wherein the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding an expressed molecule within the genetic construct and a polynucleotide encoding a transduction marker (e.g., CD90.1 or EGFRt).
• a transduction marker e.g., CD90.1 or EGFRt
• Exemplary self-cleaving polypeptides include 2A peptide from porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or variants thereof. Further exemplary nucleic acid and amino acid sequences of 2A peptides are set forth in, for example, Kim et al. (PLOS One 6:e18556 (2011). In particular embodiments, cells are genetically modified to include a self-cleaving polypeptide. In particular embodiments, the self-cleaving polypeptide includes T2A.
• Control features may be present in multiple copies in a genetic construct or can be expressed as distinct molecules with the use of a skipping element.
• a genetic construct can have one, two, three, four or five tag cassettes and/or one, two, three, four, or five transduction markers could also be expressed.
• embodiments can include a genetic construct having two Myc tag cassettes, or a His tag and an HA tag cassette, or a HA tag and a Softag 1 tag cassette, or a Myc tag and a SBP tag cassette. Exemplary transduction markers and cognate pairs are described in US 13/463,247.
• modified cells expressing a genetic construct may be detected or tracked in vivo by using antibodies that bind with specificity to a control feature (e.g., anti-Tag antibodies), or by other cognate binding molecules that specifically bind the control feature, which binding partners for the control feature are conjugated to a fluorescent dye, radio-tracer, ironoxide nanoparticle or other imaging agent known in the art for detection by X-ray, CT-scan, MRI- scan, PET-scan, ultrasound, flow-cytometry, near infrared imaging systems, or other imaging modalities (see, e.g., Yu, et al., Theranostics 2:3, 2012).
• a control feature e.g., anti-Tag antibodies
• binding partners for the control feature are conjugated to a fluorescent dye, radio-tracer, ironoxide nanoparticle or other imaging agent known in the art for detection by X-ray, CT-scan, MRI- scan, PET-scan, ultrasound, flow-cytometry, near infrared imaging systems, or
• One advantage of including at least one control feature in a genetic construct is that cells expressing a genetic construct administered to a subject can be increased or depleted using the cognate binding molecule to a tag cassette.
• the present disclosure provides a method for depleting a modified cell expressing a genetic construct by using an antibody specific for the tag cassette, using a cognate binding molecule specific for the control feature, or by using a second modified cell expressing a genetic construct and having specificity for the control feature. Elimination of modified cells may be accomplished using depletion agents specific for a control feature.
• an anti-EGFRt binding domain e.g., antibody, scFv
• a cell-toxic reagent such as a toxin, radiometal
• an anti-EGFRt /anti-CD3 bispecific scFv, or an anti-EGFRt CAR T cell may be used.
• a control feature can also include a suicide gene or suicide switch.
• suicide gene refers to a suicide or apoptosis-inducing gene operably linked to a promoter, which may be constitutive or inducible.
• examples of a suicide gene include, but are not limited to, a herpes simplex virus thymidine kinase (HSV-TK), the cytoplasmic domain of Fas, a caspase such as caspase-8 or caspase-9, cytosine deaminase, E1A, FHIT, and other known suicide or apoptosis-inducing genes.
• HSV-TK herpes simplex virus thymidine kinase
• caspase such as caspase-8 or caspase-9
• cytosine deaminase E1A
• FHIT FHIT
• suicide switch refers to the expression product of the suicide gene.
• a polynucleotide encoding an iCaspase9 construct iCasp9
• iCasp9 a polynucleotide encoding an iCaspase9 construct
• Modified cells expressing at least one control feature within a genetic construct can be, e.g., more readily identified, isolated, sorted, induced to proliferate, tracked, and/or eliminated as compared to a modified cell without a tag cassette.
• the modified (i.e., engineered) cells can be assessed for expression of the genetic constructs.
• at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the genetically modified cells express a detectable level of the genetic constructs.
• Surface protein expression can be determined by flow cytometry or other appropriate methods well known to those of ordinary skill in the art. By labeling a population of cells with an element that targets the desired cell surface marker (e.g., an antibody) and is tagged with a fluorescent molecule, flow cytometry can be used to quantify the portion of the population that is positive for the surface marker, as well as the level of surface marker expression.
• an element that targets the desired cell surface marker e.g., an antibody
• flow cytometry can be used to quantify the portion of the population that is positive for the surface marker, as well as the level of surface marker expression.
• Genomic incorporation of a genetic construct within genetically modified cells can be determined by digital droplet PCR (ddPCR).
• Digital PCR enables quantification of DNA concentration in a sample.
• Digital PCR is performed by fractionating a mixture of a PCR reaction (e.g., containing a sample of nucleic acid molecules and copies of a PCR probe) such that some fractions contain no PCR probe copy, while other fractions contain one or more PCR probe copies.
• a PCR amplification of the fractions is performed and the fractions are analyzed for a PCR reaction.
• a fraction containing one or more probes and one or more target DNA molecules yields a positive end-point, while a fraction containing no PCR probe yields a negative end-point.
• Digital droplet PCR is a variation of digital PCR wherein a sample of nucleic acids is fractionated into droplets using a water-oil emulsion. PCR amplification is performed on the droplets collectively, whereupon a fluidics system is used to separate the droplets and provide analysis of each individual droplet.
• ddPCR is used to provide an absolute quantification of DNA in a sample, to perform a copy number variation analysis, or to assess efficiency of genomic edits.
• Genetically modified cells can also be assessed for cytokine-independent growth. Genetically modified cells are expected to only grow in the presence of stimulatory cytokines (e.g., IL-2, IL-7). Growth in the absence of cytokines is an indicator of tumorigenic potential. In particular embodiments, genetically modified cells are grown for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days in either the presence or in the absence of one or more stimulatory cytokines (e.g., IL-2, IL- 7).
• stimulatory cytokines e.g., IL-2, IL-7
• proliferation is assessed by cell count and viability using conventional methods (e.g., flow cytometry, microscopy, optical density, metabolic activity).
• proliferation is assessed starting on day 1, day 2, day 3, day 4, day 5, day 6.
• proliferation is assessed every 1 day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, or every 8 days.
• growth in the absence of cytokines is assessed at the end of a growth period.
• genetically modified cells with no growth in the absence of cytokines is defined as lacking tumorigenic potential.
• no growth is defined as an expansion of the population that is less than 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1.1 , 1.2, 1.3, 1 .4, or 1.5 fold between the end of the growth period relative to the beginning of the growth period.
• the genetically modified cells do not proliferate in the absence of cytokine stimulation, growth factor stimulation, or antigen stimulation.
• Cell populations can be incubated in a cultureinitiating composition to expand cell populations.
• the incubation can be carried out in a culture vessel, such as a bag, cell culture plate, flask, chamber, chromatography column, cross-linked gel, cross-linked polymer, column, culture dish, hollow fiber, microtiter plate, silica-coated glass plate, tube, tubing set, well, vial, or other container for culture or cultivating cells.
• a culture vessel such as a bag, cell culture plate, flask, chamber, chromatography column, cross-linked gel, cross-linked polymer, column, culture dish, hollow fiber, microtiter plate, silica-coated glass plate, tube, tubing set, well, vial, or other container for culture or cultivating cells.
• the cell population can be incubated in the culture-initiating composition before or after genetic engineering the cell populations.
• the incubation can be carried out for 1 day to 6 days, 1 day to 5 days, 1 day to 4 days, 1 day to 3 days, 1 day to 2 days, or 1 day before genetically engineering the cell populations.
• the incubation can be carried out for 1 day to 6 days, 1 day to 5 days, 1 day to 4 days, 1 day to 3 days, 1 day to 2 days, or 1 day after genetically engineering the cell populations.
• the incubation can be carried out at the same time as genetically engineering the cell populations.
• Culture conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
• agents e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
• incubation is carried out in accordance with techniques such as those described in US 6,040,1 77, Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1 :72-82, and/or Wang et al. (2012) J Immunother. 35(9): 689-701.
• Exemplary culture media for culturing T cells include (i) RPMI supplemented with non- essential amino acids, sodium pyruvate, and penicillin/streptomycin; (ii) RPMI with HEPES, 5- 15% human serum, 1-3% L-Glutamine, 0.5-1.5% penicillin/streptomycin, and 0.25x10-4 - 0.75x10-4 M p-MercaptoEthanol; (iii) RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 10mM HEPES, 100 U/ml penicillin and 100 m/mL streptomycin; (iv) DMEM medium supplemented with 10% FBS, 2mM L-glutamine, 10mM HEPES, 100 U/ml penicillin and 100 m/mL streptomycin; and (v) X-Vivo 15 medium (Lonza, Walkersville, MD) supplemented with 5% human AB serum
• the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g., for a time sufficient to expand the numbers of T cells).
• the non-dividing feeder cells can include gamma-irradiated PBMC feeder cells.
• the PBMC are irradiated with gamma rays in the range of 3000 to 3600 rads to prevent cell division.
• the feeder cells are added to culture medium prior to the addition of the populations of T cells.
• the incubation may further include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells.
• LCL can be irradiated with gamma rays in the range of 6000 to 10,000 rads.
• the LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least 10: 1 .
• the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least 25°C, at least 30°C, or 37°C.
• the activating culture conditions for T cells include conditions whereby T cells of the culture-initiating composition proliferate or expand.
• T cell activating conditions can include one or more cytokines, for example, interleukin (IL)-2, IL-7, IL-15 and/or IL-21.
• IL-2 can be included at a range of 10 - 100 ng/ml (e.g., 40, 50, or 60 ng/ml).
• IL-7, IL-15, and/or IL-21 can be individually included at a range of 0.1 - 50 ng/ml (e.g., 5, 10, or 15 ng/ml). Particular embodiments utilize IL- 2 at 50 ng/ml. Particular embodiments utilize, IL-7, IL-15 and IL-21 individually included at 10 ng/ml.
• T cell activating culture conditions can include T cell stimulating epitopes.
• T cell stimulating epitopes include CD3, CD27, CD2, CD4, CD5, CD7, CD8, CD28, CD30, CD40, CD56, CD83, CD90, CD95, 4-1 BB (CD 137), B7-H3, CTLA-4, Frizzled-1 (FZD1), FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, HVEM, ICOS, IL-1 R, LAT, LFA- 1 , LIGHT, MHCI, MHCII, NKG2D, 0X40, ROR2, and RTK.
• a T-cell activating culture media includes a fluorescence- activated cell sorting (FACS)-sorted T cell population cultured within RPMI with HEPES, 5-15% human serum, 1-3% L-Glutamine, 0.5-1.5% Pen/strep, 0.25x1 O' 4 - 0.75x1 O' 4 M [3- MercaptoEthanol, with IL-7, IL-15 and IL-21 individually included at 5-15 (e.g., 10) ng/ml.
• the culture is carried out on a flat-bottom well plate with 0.1-0.5x10 6 plated cells/well. On Day 3 post activation cells are transferred to a tissue culture (TC)-treated plate.
• TC tissue culture
• a T-cell activating culture media includes a FACS-sorted CD8+ T population cultured within RPMI with HEPES, 10% human serum, 2% L-Glutamine, 1% Pen/strep, 0.5x1 O’ 4 M [3-MercaptoEthanol, with IL-7, IL-15 and IL-21 individually included at 5-15 (e.g., 10) ng/ml.
• the culture is carried out on a flat-bottom non-tissue culture-treated 96/48-well plate with 0.1 -0.5x10 6 plated cells/well. On Day 3 post activation cells are transferred to TC- treated plate.
• Culture conditions for HSC/HSP can include expansion with a Notch agonist (see, e.g., US 7,399,633; US 5,780,300; US 5,648,464; US 5,849,869; and US 5,856,441 and growth factors present in the culture condition as follows: 25-300 ng/ml SCF, 25-300 ng/ml Flt-3L, 25-100 ng/ml TPO, 25-100 ng/ml IL-6 and 10 ng/ml IL-3.
• a Notch agonist see, e.g., US 7,399,633; US 5,780,300; US 5,648,464; US 5,849,869; and US 5,856,441
• growth factors present in the culture condition as follows: 25-300 ng/ml SCF, 25-300 ng/ml Flt-3L, 25-100 ng/ml TPO, 25-100 ng/ml IL-6 and 10 ng/ml IL-3.
• 50, 100, or 200 ng/ml SCF; 50, 100, or 200 ng/ml of Flt-3L; 50 or 100 ng/ml TPO; 50 or 100 ng/ml IL-6; and 10 ng/ml IL-3 can be used.
• culturing immune cells and/or stem cells includes placing immune cells and/or stem cells in a medium including potassium ion at a concentration higher than 40 mM.
• the potassium ion concentration is at least 40 mM, at least 45 mM, 50 mM, at least 55 mM, at least 60 mM, at least 65 mM, at least 70 mM, at least 75 mM, at least 80 mM, at least 85 mM, at least 90 mM, or at least 100mM.
• the medium further includes one or more cytokines selected from IL-2, IL-21 , IL-15, IL-7, or any combination thereof.
• the medium includes potassium ion at a concentration higher than 40 mM and IL-2.
• the medium includes potassium ion at a concentration higher than 40 mM, IL-2, IL-7, and IL-15.
• the medium includes potassium ion at a concentration higher than 40 mM, IL-7, and IL-21.
• the medium includes potassium ion at a concentration higher than 40 mM, IL-15, and IL-21.
• the IL-2 is at a concentration of 0.1 ng/mL to 20 ng/mL.
• the IL-21 is at a concentration of 0.1 ng/mL to 20 ng/mL.
• the IL-7 is at a concentration of 0.1 ng/mL to 20 ng/mL.
• the IL-15 is at a concentration of 0.1 ng/mL to 20 ng/mL.
• the medium is hypotonic.
• the medium is isotonic.
• the medium includes a cell expansion agent.
• the cell expansion agent includes a GSK3B inhibitor, an ACLY inhibitor, a PI3K inhibitor (e.g., LY294002, pictilisib, CAL101 , IC87114), an AKT inhibitor (e.g., MK2206, A443654, AKTi-VI 11), or any combination thereof.
• a GSK3B inhibitor e.g., LY294002, pictilisib, CAL101 , IC87114
• an AKT inhibitor e.g., MK2206, A443654, AKTi-VI 11
• the medium includes a sodium ion.
• the sodium ion is at a concentration of 25 mM to 100mM.
• the medium includes glucose.
• the concentration of glucose is more than 10 mM.
• the medium includes calcium ion.
• the calcium ion concentration is more than 0.4 mM. Additional detailed methods for formulating the cell medium can be found in US Publication No. 2021/0332326.
• cell formulations refers to the formulations including cells genetically modified to include a genetic construct (e.g., a genetic construct encoding a recombinant receptor, trafficking signal, and/or retention signal or a genetic construct resulting in down- regulated expression of a trafficking signal and/or retention signal) and prepared for administration.
• a genetic construct e.g., a genetic construct encoding a recombinant receptor, trafficking signal, and/or retention signal or a genetic construct resulting in down- regulated expression of a trafficking signal and/or retention signal
• Exemplary carriers include saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Normosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Inc., Morton Grove, IL), and combinations thereof.
• carriers can be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum.
• HSA human serum albumin
• a carrier for infusion includes buffered saline with 5% HSA or dextrose.
• Additional isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.
• Carriers can include buffering agents, such as citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and/or trimethylamine salts.
• buffering agents such as citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and/or trimethylamine salts.
• Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which helps to prevent cell adherence to container walls.
• Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate
• cell formulations can include a local anesthetic such as lidocaine to ease pain at a site of injection.
• a local anesthetic such as lidocaine to ease pain at a site of injection.
• Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.
• cells are generally in a volume of a liter or less, 500 ml or less, 250 ml or less or 100 ml or less. Hence the density of administered cells is typically greater than 10 4 cells/ml, 10 7 cells/ml or 10 8 cells/ml.
• cell formulations can include one or more genetically modified cell types (e.g., modified T cells, NK cells, or stem cells).
• Cell formulations can include different types of genetically-modified cells (e.g., T cells, NK cells, and/or stem cells in combination).
• Different types of genetically-modified cells or cell subsets can be provided in different ratios e.g., a 1 :1 :1 ratio, 2:1 :1 ratio, 1 :2:1 ratio, 1 :1 :2 ratio, 5:1 :1 ratio, 1 :5:1 ratio, 1 :1:5 ratio, 10:1:1 ratio, 1 :10:1 ratio, 1 :1 :10 ratio, 2:2:1 ratio, 1 :2:2 ratio, 2:1 :2 ratio, 5:5:1 ratio, 1:5:5 ratio, 5:1 :5 ratio, 10:10:1 ratio, 1:10:10 ratio, 10:1 :10 ratio, etc.
• These ratios can also apply to numbers of cells expressing the same or different genetic construct components.
• the cell formulations disclosed herein can be prepared for administration by, e.g., injection, infusion, perfusion, instillation, or lavage.
• the cell formulations can further be formulated for bone marrow, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intratumoral, intramuscular, intravesical, and/or subcutaneous injection.
• the cell formulation can further be formulated for physical locoregional delivery such as hepatic arterial infusion pump, isolated limb perfusion, instillation of cells into the abdominal cavity, or instillation of cells into the thoracic cavity.
• Targeted Viral Vectors & Nanoparticles for In Vivo Cell Modification Targeted viral vectors and/or nanoparticles can also be used to genetically-modify immune cells in vivo or ex vivo. Modifying formulations refer to targeted viral vectors and/or nanoparticles used to genetically modify immune cells to include a genetic construct disclosed herein within a pharmaceutically acceptable carrier. Viral vectors that can be used to deliver genes to cells are described elsewhere herein, and numerous targeted (e.g., pseudotyped) viral vectors and nanoparticles are known in the art.
• Exemplary cell-targeted nanoparticles include a cell targeting ligand (e.g., that binds CD3, CD4, CD8, CD34) on the surface of the nanoparticle wherein the cell targeting ligand results in selective uptake of the nanoparticle by a selected cell type (e.g., by an immune cell).
• the nanoparticle then delivers gene modifying components that result in expression of the genetic construct or down-regulated expression of a targeted gene.
• Exemplary nanoparticles include liposomes (microscopic vesicles including at least one concentric lipid bilayer surrounding an aqueous core), liposomal nanoparticles (a liposome structure used to encapsulate another smaller nanoparticle within its core); and lipid nanoparticles (liposome-like structures that lack the continuous lipid bilayer characteristic of liposomes).
• Other polymer-based nanoparticles can also be used as well as porous nanoparticles constructed from any material capable of forming a porous network.
• Exemplary materials include metals, transition metals and metalloids (e.g., lithium, magnesium, zinc, aluminum and silica).
• Therapeutically effective amounts of vectors and/or nanoparticles within modifying formulations can range from 0.1 to 5 pg/kg or from 0.5 to 1 pg /kg.
• a dose can include 1 pg /kg, 30 pg /kg, 90 pg/kg, 150 pg/kg, 500 pg/kg, 750 pg/kg, 0.1 to 5 mg/kg or from 0.5 to 1 mg/kg.
• a dose can include 1 mg/kg, 10 mg/kg, 30 mg/kg, 50 mg/kg, 70 mg/kg, 100 mg/kg, 300 mg/kg, 500 mg/kg, 700 mg/kg, 1000 mg/kg or more.
• Methods disclosed herein include treating subjects (humans, nonhuman primates, veterinary animals (dogs, cats, reptiles, birds, etc.) livestock (horses, cattle, goats, pigs, chickens, etc.) and research animals (monkeys, rats, mice, fish, etc.)) with formulations disclosed herein (cell formulations and/or modifying formulations). Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and/or therapeutic treatments.
• an "effective amount” is the amount of a formulation necessary to result in a desired physiological change in the subject.
• an effective amount can provide an immunogenic anti-cancer effect or anti-infection effect.
• Effective amounts are often administered for research purposes.
• Effective amounts disclosed herein can cause a statistically significant effect in an animal model or in vitro assay relevant to the assessment of a cancer’s or infection’s development or progression.
• An immunogenic formulation can be provided in an effective amount, wherein the effective amount stimulates an immune response.
• a prophylactic treatment includes a treatment administered to a subject who does not display signs or symptoms of a cancer or infection or displays only early signs or symptoms of a cancer or infection such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the cancer or infection further.
• a prophylactic treatment functions as a preventative treatment against cancer or infection.
• prophylactic treatments reduce, delay, or prevent metastasis from a primary a cancer tumor site from occurring.
• a "therapeutic treatment” includes a treatment administered to a subject who displays symptoms or signs of a cancer or infection and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the cancer or infection.
• the therapeutic treatment can reduce, control, or eliminate the presence or activity of the cancer or infection and/or reduce control or eliminate side effects of the cancer or infection.
• prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.
• therapeutically effective amounts provide anti-cancer effects.
• Anti-cancer effects include a decrease in the number of cancer cells, decrease in the number of metastases, a decrease in tumor volume, an increase in life expectancy, induced chemo- or radiosensitivity in cancer cells, inhibited angiogenesis near cancer cells, inhibited cancer cell proliferation, inhibited tumor growth, reduced metastases, prolonged subject life, reduced cancer- associated pain, and/or reduced relapse or re-occurrence of cancer following treatment.
• a “tumor” is a swelling or lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells).
• a “tumor cell” is an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be benign, pre-malignant or malignant.
• therapeutically effective amounts induce an immune response.
• the immune response can be against a target cell (e.g., cancer cell or infected cell (e.g., infected with bacteria, virus, fungi, parasite, or arthropod)).
• a target cell e.g., cancer cell or infected cell (e.g., infected with bacteria, virus, fungi, parasite, or arthropod)).
• Target cell refers to a cell that expresses a target antigen on its surface.
• expression of an antigen on the cell surface is determined, for example, using antibodies to the target in a method such as immunohistochemistry, FACS, etc.
• target mRNA expression is considered to correlate to target expression on the cell surface and can be determined by, for example, in situ hybridization and/or RT-PCR (including quantitative RT-PCR).
• therapeutically effective amounts can be used to direct cells expressing a genetic construct to a select tissue and not to non-selected tissues.
• Example uses of genetic constructs and/or genetically modified cells disclosed herein include cancer treatment, tissue engineering, and genetic engineering.
• cancers that can be treated with genetic constructs disclosed herein include colorectal cancer, pancreatic cancer, breast cancer, colon cancer, gastric cancer, prostate cancer, hepatocellular carcinoma, ovarian cancer, endometrial cancer, uterine cancer, esophageal cancer, lung cancer, head and neck squamous cell cancer, cholangiocarcinoma, and bladder cancer.
• breast cancer includes triple-negative breast cancer (TNBC) and basal breast cancer.
• bladder cancer includes urothelial carcinoma, squamous cell carcinoma, and adenocarcinoma.
• genetic constructs disclosed herein are used to treat metastases of the cancers described above.
• Particular embodiments include providing localized treatment to the liver to a subject having a primary gastric cancer. Particular embodiments include providing localized treatment to the liver to a subject having a primary colon cancer. Particular embodiments include providing localized treatment to the liver to a subject having a primary colorectal cancer. Particular embodiments include providing localized treatment to the liver to a subject having a primary breast cancer. Particular embodiments include providing localized treatment to the liver to a subject having a primary pancreatic cancer. Particular embodiments include providing localized treatment to the liver to a subject having a primary ovarian cancer. Particular embodiments include providing localized treatment to the liver to a subject having a primary endometrial cancer. Particular embodiments include providing localized treatment to the liver to a subject having a primary uterine cancer. Particular embodiments include providing localized treatment to the liver to a subject having a primary hepatocellular carcinoma.
• Particular embodiments include providing localized treatment to the bone to a subject having a primary breast cancer. Particular embodiments include providing localized treatment to the bone to a subject having a primary prostate cancer.
• Particular embodiments include providing localized treatment to the pancreas to a subject having a primary pancreatic cancer.
• a treatment of a cancer in a subject includes i) assessing the type of cancer and choosing a recombinant receptor binding domain that will bind a target antigen expressed on the cancer cell; ii) determining desired site of activity (e.g., target tissue) and choosing a trafficking signal corresponding to the desired site of activity; iii) determine if there is a site of on-target/off-tumor toxicity and choose a trafficking signal to knockout in order to avoid trafficking to that site; iv) determine if a retention signal is desired; v) prepare a genetic construct; and vi) deliver a therapeutically effective amount of the treatment to the subject.
• the genetic construct is used to genetically engineer cells ex vivo and then administer the cells to the subject.
• the genetic construct is associated with a nanoparticle and administered to the subject in order to genetically engineer cells in vivo.
• genetically modified cells can be directed to a select tissue to increase the population of cells within a tissue.
• fibroblasts can be directed to skin for wound healing
• hepatocytes can be directed to the liver
• chondrocytes can be directed to cartilage.
• a stem cell can be directed to a particular tissue.
• the stem cell can include a genetic construct to direct the stem cell’s differentiation or activity.
• genetically modified cells can include a genetic construct encoding a therapeutic protein.
• genetically modified cells are directed to a select tissue where it can express the therapeutic protein.
• the therapeutic protein includes an enzyme, an antibody, a peptide, a growth factor, a transcription factor, or any other useful protein or molecule.
• beta cells can be genetically engineered to go to and reside in the pancreas where they can produce insulin.
• therapeutically effective amounts can be initially estimated based on results from in vitro assays and/or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest.
• the actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of cancer, stage of cancer, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.
• Therapeutically effective amounts of cell formulations can include 10 4 to 10 9 cells/kg body weight, or 10 3 to 10 11 cells/kg body weight.
• Therapeutically effective amounts to administer can include greater than 10 2 cells, greater than 10 3 cells, greater than 10 4 cells, greater than 10 5 cells, greater than 10 6 cells, greater than 10 7 cells, greater than 10 8 cells, greater than 10 9 cells, greater than 10 10 cells, or greater than 10 11 .
• Therapeutically effective amounts of modifying formulations can range from 0.1 to 5 pg/kg or from 0.5 to 1 pg /kg.
• a dose can include 1 pg /kg, 30 pg /kg, 90 pg/kg, 150 pg/kg, 500 pg/kg, 750 pg/kg, 0.1 to 5 mg/kg or from 0.5 to 1 mg/kg.
• a dose can include 1 mg/kg, 10 mg/kg, 30 mg/kg, 50 mg/kg, 70 mg/kg, 100 mg/kg, 300 mg/kg, 500 mg/kg, 700 mg/kg, 1000 mg/kg or more.
• Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly).
• a treatment regimen e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly.
• the treatment protocol may be dictated by a clinical trial protocol or an FDA- approved treatment protocol.
• Therapeutically effective amounts can be administered by, e.g., injection, infusion, perfusion, instillation, or lavage.
• Routes of administration can include bolus intravenous, intradermal, intraarterial, intraparenteral, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesical, limb perfusion, intrathoracic, intraabdominal, and/or subcutaneous administration.
• cell formulations and/or modifying formulations are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities.
• cells may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and irradiation.
• immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies
• immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin,
• cell formulations and/or modifying formulations may be administered in conjunction with any number of chemotherapeutic agents.
• chemotherapeutic agents include alkylating agents; alkyl sulfonates; aziridines; ethylenimines and methylamelamines; nitrogen mustards; nitrosureas; antibiotics; anti-metabolites; folic acid analogues; purine analogs; pyrimidine analogs; androgens; anti-adrenals; folic acid replenisher; platinum analogs; retinoic acid; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
• anti-hormonal agents that act to regulate or inhibit hormone action on tumors
• anti-estrogens and anti-androgens and pharmaceutically acceptable salts, acids or derivatives of any of the above.
• Combinations of chemotherapeutic agents are also administered where appropriate, including, CHOP, i.e., Cyclophosphamide (Cytoxan®), Doxorubicin (hydroxydoxorubicin), Vincristine (Oncovin®), and Prednisone.
• the chemotherapeutic agent is administered at the same time or within one week after the administration of the cell formulation and/or modifying formulation. In other embodiments, the chemotherapeutic agent is administered from 1 to 4 weeks or from 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after the administration of the cell formulation and/or modifying formulation. In other embodiments, the chemotherapeutic agent is administered at least 1 month before administering the cell formulation and/or modifying formulation. In some embodiments, the methods further include administering two or more chemotherapeutic agents.
• formulations can be administered with an anti-inflammatory agent.
• Anti-inflammatory agents or drugs include steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal antiinflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.
• steroids and glucocorticoids including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone
• NSAIDS nonsteroidal antiinflammatory drugs
• formulations described herein are administered in conjunction with a cytokine.
• Cytokine as used herein is meant to refer to proteins released by one cell population that act on another cell as intercellular mediators. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones.
• cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-1 and -II; erythropoietin (EPO); osteoinductive
• kits can include various components to practice methods disclosed herein.
• kits could include one or more of nucleic acids encoding a recombinant receptor disclosed herein; nucleic acids encoding a CAR disclosed herein; nucleic acids encoding a trafficking signal (e.g., CXCR6, integrin 2 (LFA-1), CCR1 , CCR2, CXCR4, 7 integrin, CCR4, CXCR3, CCR3) disclosed herein; nucleic acids encoding a retention signal (e.g., CD69, Runx3, Hobit, CD103) disclosed herein; nucleic acids resulting in down-regulated expression of a trafficking signal disclosed herein; nucleic acids resulting in down-regulated expression of a retention signal disclosed herein; a vector (e.g., lentiviral vector or retroviral vector) carrying a genetic construct; a nucleic acid en
• a method of treating a subject in need thereof including identifying a subject with a cancer; and administering to the subject a therapeutically effective amount of a cell genetically modified to express i) a chimeric antigen receptor including a binding domain that binds a target antigen of the cancer; and ii) a (a) trafficking signal that directs the cell to a treatment location,
• the cancer is a primary cancer that has metastasized to a second location; and the cell is genetically modified to express i) a chimeric antigen receptor including a binding domain that binds a target antigen of the primary cancer; and ii) a (a) trafficking signal that directs the cell to the second location,
• the primary cancer is colorectal cancer, the second location is liver, the target antigen is CEA, the trafficking signal includes CXC motif chemokine receptor 6 (CXCR6), and the retention signal includes CD69.
• the primary cancer is colorectal cancer, the second location is liver, the target antigen is CEA, and the trafficking signal includes CXCR6;
• the primary cancer is breast, colon, or gastric cancer;
• the second location is liver, the target antigen is HER2, and the trafficking signal CXCR6;
• the primary cancer is breast cancer, the second location is bone, the target antigen is HER2, and the trafficking signal includes CXCR4;
• the primary cancer is prostate cancer, the second location is bone, the target antigen is PSA, and the trafficking signal includes CXCR4;
• the primary cancer is pancreatic cancer, the second location is liver, the target antigen is mesothelin or CA19-9, and the trafficking signal includes CXCR6;
• the primary cancer is ova
• the retention signal includes CD69.
• the method of any of embodiments 2-5, wherein the off-target location is small intestine or colon.
• the method of any of embodiments 2-5, wherein the off-target location is prostate gland.
• the method of any of embodiments 2-5, wherein the off-target location is lung.
• the method of any of embodiments 2-5, wherein the off-target location is lung, kidney, or stomach.
• the inhibitory signal includes knockout of (37 integrin, CCR4, CXCR3, and CCR3.
• an inhibitory signal that reduces migration of the cell to an off-target location; thereby treating the subject in need thereof.
• the cancer is a primary cancer that has metastasized to a second location; and the cell is genetically modified to express i) a chimeric antigen receptor including a binding domain that binds a target antigen of the primary cancer; and ii) a (a) trafficking signal that directs the cell to the organ of the primary cancer location,
• an inhibitory signal that reduces migration of the cell to the second location and/or an off-target location; thereby treating the subject in need thereof.
• the trafficking signal includes CXCR6, integrin (32, CCR1 , CCR2, CXCR4, 37 integrin, CCR4, CXCR3, or CCR3.
• the cell of embodiments 18 or 19, wherein modulation of the trafficking signal includes up- or down-regulating expression of CXCR6, integrin (32, CCR1 , CCR2, CXCR4, (37 integrin, CCR4, CXCR3, or CCR3.
• the cell of any of embodiments 18-20 genetically modified to express CXCR6, integrin (32, CCR1 , CCR2, or CXCR4.
• the cell of any of embodiments 18-21 genetically modified to express CXCR6.
• the cell of any of embodiments 18-21 genetically modified to express integrin 02.
• the cell of any of embodiments 18-21 genetically modified to express CCR1.
• the cell of any of embodiments 18-21 genetically modified to express CCR2.
• the cell of any of embodiments 18-21 genetically modified to express CXCR4.
• the cell of any of embodiments 18-20 genetically modified to down-regulate expression of
• the cell of any of embodiments 27-31 wherein down- regulated expression is by administration of a guide RNA (gRNA) and a nuclease to the cell.
• gRNA guide RNA
• the cell of embodiment 32 wherein the gRNA includes a sequence as set forth in one of SEQ ID NOs: 77-103.
• the cell of embodiment 34, wherein the sgRNA targeting 37 integrin includes a sequence as set forth in SEQ ID Nos: 245-247.
• the cell of any of embodiments 18-37, wherein the retention signal includes CD69, Runx3, Hobit, or CD103.
• the cell of any of embodiments 18-38, wherein modulation of the retention signal includes up- or down regulating expression of CD69, Runx3, Hobit, or CD103.
• the cell of any of embodiments 18-41 genetically modified to down-regulate expression of CD69, Runx3, Hobit, or CD103.
• the cell of embodiment 42, wherein the down-regulated expression is by administration of a gRNA and a nuclease to the cell.
• the cell of embodiment 43 wherein the gRNA includes a sequence as set forth in one of SEQ ID NOs: 107-121 or 192-194.
• the cell of any of embodiments 18-45 genetically modified to express the recombinant receptor.
• CAR chimeric antigen receptor
• eTCR engineered T cell receptor
• CAR/TCR hybrid a CAR/TCR hybrid.
• the recombinant receptor includes i) an extracellular component including a binding domain that binds an antigen expressed on a surface of targeted cells; and ii) an intracellular component.
• the cell of embodiment 48, wherein the targeted cells include cancer cells or infected cells.
• the cell of embodiments 48 or 49, wherein the targeted cells include cancer cells.
• CEA carcinoembryonic antigen
• EpCAM epithelial cell adhesion molecule
• HER2 human epidermal growth factor receptor 2
• PSA prostate-specific antigen
• GPC3 glypican-3
• the cell of embodiment 53, wherein the anti-CEA antibody includes AHB7.
• CDR complementarity determining region
• the anti-CEA antibody includes a variable light chain having a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 204 and a variable heavy chain having a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 205.
• the anti-CEA antibody includes a variable light chain encoded by a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 63 and a variable heavy chain encoded by a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 64.
• the cell of embodiment 60 wherein the linker is encoded by the sequence as set forth in SEQ ID NO: 65.
• the cell of embodiment 48, wherein the antigen includes EpCAM.
• the cell of embodiment 63, wherein the anti-EpCAM antibody includes G8.8, ab71916, AUA- 1 , Ber-EP4, HEA125, EpMab-16, MOC31 , or VU-1 D9.
• the anti-EpCAM antibody includes a variable light chain including a CDRL1 as set forth in SEQ ID NO: 127, a CDRL2 as set forth in SEQ ID NO: 128, and a CDRL3 as set forth in SEQ ID NO: 129, and a variable heavy chain including a CDRH1 as set forth in SEQ ID NO: 124, a CDRH2 as set forth in SEQ ID NO: 125, and a CDRH3 as set forth in SEQ ID NO: 126.
• the cell of embodiment 68, wherein the linker is encoded by the sequence as set forth in SEQ ID NO: 197.
• the cell of any of embodiments 48-71 wherein the intracellular component is linked to the extracellular component through a transmembrane domain.
• the cell of embodiment 77, wherein the lymphocyte includes a T cell, B cell, natural killer (NK) cell, or NK-T cell.
• the stem or progenitor cell includes a hematopoietic stem cell (HSC), a hematopoietic progenitor cell (HPC), a pluripotent stem cell, a mesenchymal stem cell, a neural stem cell, or an endothelial stem cell.
• HSC hematopoietic stem cell
• HPC hematopoietic progenitor cell
• pluripotent stem cell a mesenchymal stem cell
• a neural stem cell or an endothelial stem cell.
• the adult cell includes a fibroblast, a hepatocyte, a myoblast, a neuron, an osteoblast, an osteoclast, a kidney cell, a chondrocyte, or an adipocyte.
• the cell of embodiment 84 wherein the cell expresses the anti-CEA CAR, CXCR6, and CD69 by administration of a sequence as set forth in SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 , or SEQ ID NO: 62.
• the cell of any of embodiments 18-83 wherein the cell expresses an anti-EpCAM CAR, CXCR6, and CD69.
• the cell of embodiment 90 wherein the cell expresses the anti-CEA CAR and down-regulates expression of 07 integrin by administration of an sgRNA sequence as set forth in SEQ ID NOs: 245-247 and a nuclease and administration of a sequence as set forth in SEQ ID NO:
• 201 The cell of any of embodiments 18-83, wherein the cell expresses anti-EpCAM CAR and down-regulates expression of CXCR3.
• a genetic construct including at least two of: a sequence encoding a recombinant receptor; a sequence encoding a trafficking signal or a sequence down-regulating a trafficking signal; or a sequence encoding a retention signal or a sequence down-regulating a retention signal.
• the genetic construct of embodiments 100 or 101 wherein the trafficking signal gRNA includes a trafficking signal single guide RNA (sgRNA). .
• the genetic construct of embodiment 103, wherein the sgRNA targeting 3 integrin includes a sequence as set forth in SEQ ID Nos: 245-247. .
• the genetic construct of embodiment 102, wherein the trafficking signal sgRNA includes an sgRNA targeting CXCR3. .
• the genetic construct of embodiment 105, wherein the sgRNA targeting CXCR3 includes a sequence as set forth in SEQ ID Nos: 242-244. .
• the genetic construct of any of embodiments 96-107, wherein the retention signal includes CD69, Runx3, Hobit, or CD103. .
• CAR chimeric antigen receptor
• eTCR engineered T cell receptor
• the recombinant receptor includes i) an extracellular component including a binding domain that binds a target antigen, wherein the target antigen is expressed on a surface of targeted cells; and ii) an intracellular component.
• the targeted cells include cancer cells or infected cells.
• the target antigen includes a carcinoembryonic antigen (CEA), an epithelial cell adhesion molecule (EpCAM), a human epidermal growth factor receptor 2 (HER2), a prostate-specific antigen (PSA), a glypican-3 (GPC3), mesothelin, a folate receptor alpha (FOLR1), a mucin short variant S1 (MUC1), or a carbohydrate antigen 19-9 (CA19-9).
• CEA carcinoembryonic antigen
• EpCAM epithelial cell adhesion molecule
• HER2 human epidermal growth factor receptor 2
• PSA prostate-specific antigen
• GPC3 glypican-3
• mesothelin mesothelin
• FOLR1 folate receptor alpha
• MUC1 mucin short variant S1
• CA19-9 carbohydrate antigen 19-9
• the genetic construct of embodiment 125 wherein the linker is encoded by the sequence as set forth in SEQ ID NO: 65. .
• the anti-EpCAM antibody includes a variable light chain including a CDRL1 as set forth in SEQ ID NO: 127, a CDRL2 as set forth in SEQ ID NO: 128, and a CDRL3 as set forth in SEQ ID NO: 129, and a variable heavy chain including a CDRH1 as set forth in SEQ ID NO: 124, a CDRH2 as set forth in SEQ ID NO: 125, and a CDRH3 as set forth in SEQ ID NO: 126. .
• the genetic construct of embodiment 133 wherein the linker is encoded by the sequence as set forth in SEQ ID NO: 197.
• the genetic construct of any of embodiments 113-134 wherein the intracellular component includes a CD3 signaling domain, a 4-1 BB signaling domain and/or a CD28 signaling domain.
• the genetic construct of any of embodiments 113-136 wherein the intracellular component is linked to the extracellular component through a transmembrane domain.
• the genetic construct of embodiment 137 wherein the transmembrane domain includes a CD28 transmembrane domain. .
• the genetic construct of embodiment 143 wherein the tag cassette includes a sequence as set forth in SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181 , SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, or SEQ ID NO: 190. .
• any of embodiments 96-148 including a sequence as set forth in SEQ ID NO: 201 , SEQ ID NO: 202, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 1 , and/or SEQ ID NO: 4 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 201 , SEQ ID NO: 202, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 1 , and/or SEQ ID NO: 4.
• the genetic construct of any of embodiments 96-153 wherein the genetic construct includes the sequence as set forth in SEQ ID NOs: 59, 60, 61 , or 62 or a sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NOs: 59, 60, 61 , or 62.
• the genetic construct of any of embodiments 96-154 wherein the genetic construct includes the sequence as set forth in SEQ ID NO: 203 or a sequence having at least 90% sequence identity to a sequence as set forth in SEQ ID NOs: 203.
• the vector of embodiment 156, wherein the vector is a viral vector. .
• the vector of embodiment 157 wherein the viral vector includes a retroviral vector or a lentiviral vector.
• a nanoparticle encapsulating the genetic construct of any of embodiments 96-155. A population of cells genetically modified to include the genetic construct of any of embodiments 96-155 or the vector any of embodiments 156-158. .
• a formulation including (i) the population of cells of embodiment 160 or a cell of any of embodiments 18-95 and (ii) a pharmaceutically acceptable carrier. .
• a method of treating a subject in need thereof including administering a therapeutically effective amount of the formulation of embodiment 161 or nanoparticle of embodiment 159 to the subject, thereby treating the subject in need thereof. .
• the method of embodiment 162, wherein the administering a therapeutically effective amount includes physical locoregional delivery. .
• the method of any of embodiments 162-164, wherein the administering a therapeutically effective amount includes administering intravenously, intravesically, intradermally, intraarterially, intraparenterally, intranodally, intralymphaticaly, intraperitoneally, intralesionally, intraprostaticaly, intravaginally, intrarectally, topically, intrathecally, intratumorally, intramuscularly, or subcutaneously. .
• a method of treating a cancer within a subject in need thereof including administering a therapeutically effective amount of a formulation to the subject, wherein the formulation includes cells genetically modified to i) express a recombinant receptor including a binding domain that binds a target antigen, and ii) direct location of the cell to a target tissue and/or away from a non-targeted tissue; thereby treating the subject in need thereof.
• the method of embodiment 167 wherein the subject has colorectal cancer and the target antigen is CEA. .
• the method of embodiments 167 or 171 wherein the target tissue is a liver and the cell expresses CXCR6. .
• the method of embodiments 167 or 171 wherein the target tissue is a pancreas and the cell expresses integrin
• the method of embodiment 167 wherein the subject has colon cancer or gastric cancer and the target antigen is HER2. .
• the method of embodiments 167 or 181 wherein the target tissue is a liver and the cell expresses CXCR6. .
• the method of embodiment 167, wherein the subject has prostate cancer and the target antigen is PEA. .
• any of embodiments 167-185 wherein the non-targeted tissue is a prostate and the cell has down-regulated expression of CXCR3. .
• the trafficking signal includes CXCR6, integrin P2, CCR1 , CCR2, CXCR4, (37 integrin, CCR4, CXCR3, or CCR3. .
• the kit of any of embodiments 199-201 wherein the genetic construct includes sequences encoding CXCR6, integrin (32, CCR1 , CCR2, or CXCR4. .
• the kit of embodiment 210 wherein the retention signal gRNA includes a sequence as set forth in one of SEQ ID NOs: 107-121 or 192-194. .
• the kit of any of embodiments 209-211 further including a nuclease or sequence encoding a nuclease.
• the kit of embodiment 212 wherein the nuclease includes Cas9 or Cpfl .
• CAR chimeric antigen receptor
• eTCR engineered T cell receptor
• CAR/TCR hybrid a CAR/TCR hybrid.
• control feature includes a transduction marker.
• transduction marker includes CD90.1, Her2tG, epidermal growth factor receptor (EGFRt), or tCD19.
• control feature includes a selection cassette.
• control feature includes a tag cassette. .
• the kit of embodiment 224 wherein the tag cassette includes a sequence as set forth in SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181 , SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, or SEQ ID NO: 190. .
• the kit of any of embodiments 199-225, wherein the genetic construct further includes a skip sequence. .
• the target antigen includes a carcinoembryonic antigen (CEA), an epithelial cell adhesion molecule (EpCAM), a human epidermal growth factor receptor 2 (HER2), a prostate-specific antigen (PSA), a glypican-3 (GPC3), a mesothelin, a folate receptor alpha (FOLR1), a mucin short variant S1 (MUC1), or a carbohydrate antigen 19-9 (CA19-9).
• CEA carcinoembryonic antigen
• EpCAM epithelial cell adhesion molecule
• HER2 human epidermal growth factor receptor 2
• PSA prostate-specific antigen
• GPC3 glypican-3
• mesothelin a folate receptor alpha
• MUC1 mucin short variant S1
• the anti-CEA antibody includes a variable light chain including a complementarity determining region (CDRL)1 as set forth in SEQ ID NO: 207, a CDRL2 as set forth in SEQ ID NO: 208, and a CDRL3 as set forth in SEQ ID NO: 209, and a variable heavy chain including a CDRH1 as set forth in SEQ ID NO: 210, a CDRH2 as set forth in SEQ ID NO: 211 , and a CDRH3 as set forth in SEQ ID NO: 212.
• CDRL complementarity determining region
• the kit of embodiment 234, wherein the binding domain that binds EpCAM includes a variable light chain including a CDRL1 as set forth in SEQ ID NO: 127, a CDRL2 as set forth in SEQ ID NO: 128, and a CDRL3 as set forth in SEQ ID NO: 129, and a variable heavy chain including a CDRH1 as set forth in SEQ ID NO: 124, a CDRH2 as set forth in SEQ ID NO: 125, and a CDRH3 as set forth in SEQ ID NO: 126.
• the kit of embodiment 236, wherein the binding domain that binds HER2 includes a variable light chain including a CDRL1 as set forth in SEQ ID NO: 218, a CDRL2 as set forth in SEQ ID NO: 219, and a CDRL3 as set forth in SEQ ID NO: 220, and a variable heavy chain including a CDRH1 as set forth in SEQ ID NO: 221 , a CDRH2 as set forth in SEQ ID NO: 222, and a CDRH3 as set forth in SEQ ID NO: 223.
• the kit of embodiment 238, wherein the binding domain that binds PSA includes a variable light chain including a CDRL1 including the sequence as set forth in SEQ ID NO: 134, a CDRL2 including the sequence as set forth in SEQ ID NO: 135, and a CDRL3 including the sequence as set forth in SEQ ID NO: 136; and a variable heavy chain including a CDRH1 including the sequence as set forth in SEQ ID NO: 137, a CDRH2 including the sequence as set forth in SEQ ID NO: 138, and a CDRH3 including the sequence as set forth in SEQ ID NO: 139.
• the kit of embodiment 240, wherein the binding domain that binds GPC3 includes a variable light chain including a complementarity determining region (CDRL)1 including the sequence as set forth in SEQ ID NO: 140, a CDRL2 including the sequence as set forth in SEQ ID NO: 141 , and a CDRL3 including the sequence as set forth in SEQ ID NO: 142; and a variable heavy chain including a CDRH1 including the sequence as set forth in SEQ ID NO: 143, a CDRH2 including the sequence as set forth in SEQ ID NO: 144, and a CDRH3 including the sequence as set forth in SEQ ID NO: 145. .
• CDRL complementarity determining region
• the kit of embodiment 246, wherein the binding domain that binds MUC1 includes a variable light chain including a CDRL1 as set forth in SEQ ID NO: 230, a CDRL2 as set forth in SEQ ID NO: 231 , and a CDRL3 as set forth in SEQ ID NO: 232, and a variable heavy chain including a CDRH1 as set forth in SEQ ID NO: 233, a CDRH2 as set forth in SEQ ID NO: 234, and a CDRH3 as set forth in SEQ ID NO: 235. .
• the kit of embodiment 248, wherein the binding domain that binds CA19-9 includes a variable light chain including a CDRL1 as set forth in SEQ ID NO: 236, a CDRL2 as set forth in SEQ ID NO: 241 , and a CDRL3 as set forth in SEQ ID NO: 238, and a variable heavy chain including a CDRH1 as set forth in SEQ ID NO: 239, a CDRH2 as set forth in SEQ ID NO: 240, and a CDRH3 as set forth in SEQ ID NO: 241. .
• the kit of embodiment 251 wherein the cell is within a formulation. .
• the kit of any of embodiments 251-253 wherein the cell is cultured in a culture medium..
• the kit of any of embodiments 261 wherein the cell expansion agent includes a GSK3B inhibitor, an ACLY inhibitor, a PI3K inhibitor, or an AKT inhibitor.
• the kit of any of embodiments 254-262 wherein the culture medium includes a sodium ion, glucose, or a calcium ion.
• the kit of any of embodiments 254-263 wherein the culture medium includes a sodium ion at a concentration of 25 mM to 100 mM.
• Example 1 Materials and Methods. Mice and cell lines. C57BL/6 mice (obtained from Jackson Laboratories, Bar Harbor, ME) of 6-8 weeks of age were used as recipient hosts for adoptive transfer unless otherwise indicated. OT-1 Ly5.1 transgenic mice or C57BL/6 mice were used at cell source for adoptive cell transfer experiments, as indicated. To obtain OT-1 Ly5.1 mice OT-1 (C57BL/6-Tg (TcraTcrb)1100Mjb/J) were crossed with Ly5.1 mice (B6.SJL-PtprcaPepcb/BoyJ). All mice were maintained under specific pathogen-free conditions.
• Kasumi-2 cells were maintained in RPMI 16540 (Gibco) with 10% fetal bovine serum (FBS), 1 % glutamine, and 1% penicillin-streptomycin.
• LS174T cell line was obtained from American Type Culture Collection (ATCC).
• KPC cells were transduced to express full length human carcinoembryonic antigen (CEA), mouse epithelial cell adhesion molecule (EpCAM), or firefly luciferase (pl_X313-Firefly luciferase (Addgene plasmid # 118017) to generate KPC-hCEA, KPC-EpCAM, or KPC luciferase overexpression (OE) cell lines, respectively.
• CEA human carcinoembryonic antigen
• EpCAM mouse epithelial cell adhesion molecule
• pl_X313-Firefly luciferase pl_X313-Firefly luciferase (
• KPC EpCAM knockout (KO) cells were generated via CRISPR-Cas9 mediated deletion of Epcam. Briefly, Epcam targeted single guide RNAs (sgRNAs) were purchased from Synthego. Guides were complexed with Cas9 and mixed with KPC cells; mixtures were electroporated with the Lonza 4D Nucleofector. Knockout was confirmed by flow cytometry staining from mouse EpCAM. Cell lines were used for in vitro co-culture assays and in vivo experiments, as indicated. Cell lines were maintained in complete media DMEM (Gibco) with 10% FBS, 1% glutamine and 1% penicillinstreptomycin.
• DMEM Gibco
• CD8+ T cells from OT-1 or C57BL/6 mice were isolated using a negative bead selection (StemCell) and stimulated in vitro with plate-bound anti- CD3 and anti-CD28 (5 g/ml; BioXcell) and expanded in RPMI 1640 containing 10% FBS and 100 lU/mL of recombinant human IL-2 (Peprotech).
• Human blood samples were enriched for T cells using negative bead selection (StemCell) and stimulated in vitro in media containing a 1 :100 titer of TransAct (Miltenyi Biotec), 5% FBS and 100 lU/mL of rhlL-2.
• Intracellular cytokine staining and flow cytometry T cells were stained with a fixable live/dead stain (Invitrogen) in phosphate buffered saline (PBS) followed by surface antibody staining in fluorescence-activated cell sorting (FACS) buffer (PBS with 0.5% bovine serum albumin (BSA) and 0.1% sodium azide).
• FACS fluorescence-activated cell sorting
• cells were first stained for surface markers and later stained for intracellular molecules following fixation and permeabilization following the manufacturer’s protocols (BD Cytofix/Cytoperm).
• Antibodies for surface staining and intracellular cytokine staining were purchased from BD Biosciences, BioLegend, eBiosciences and Invitrogen. All experiments were conducted on a BD (Becton Dickinson) Fortessa or CyTek Biosciences Aurora cytometer and analyzed with FlowJo software (Tree Star, Inc).
• Transduced T cells were mixed 1:1 with tumor cell lines as indicated. Cells were incubated overnight (16 hours) in the presence of protein transport inhibitor (BD Golgi Stop and BD GolgiPlug). After 16 hours, cells were washed and stained for flow cytometry as described.
• protein transport inhibitor BD Golgi Stop and BD GolgiPlug
• Retroviral transduction Platinum-E ecotropic or Platinum-A amphotropic packaging cells (Cell Biolabs) were plated one day before transfections on poly-D-lysine-coated 10-cm plates (Corning) at a concentration of 6 * 10 6 cells per plate.
• Packaging cells were transfected with retroviral plasmid DNA, pCL-Eco or pCL-Ampho plasmid DNA, and jetOPTIMUS reagent for 8 h, according to manufacturer instructions (Polyplus).
• Lenti-X packaging cells ATCC were plated one day before transfections on poly-D-lysine-coated 10-cm plates (Corning) at a concentration of 6 x 10 6 cells per plate.
• Packaging cells were transfected with lentivirus plasmid DNA (Addgene plasmid #118017), pMLDg/ pRRE plasmid DNA, pRSV/ REV plasmid DNA, pMD2.G plasmid DNA, and jetOPTIMUS reagent for8 h, according to manufacturer instructions (Polyplus). Medium was replaced 8 h after transfection and cells were incubated for a further 48 h.
• Viral supernatants were then collected and spun at 2,000g for 2 h at 32 °C onto 24-well non-tissue-culture-treated plates coated overnight in Retronectin (Takara Bio) or spinfected with T cells for 90 minutes at 32°C in 1 peg mL‘ 1 polybrene, which was then removed after an additional 4 hour incubation.
• Non-targeting control sgRNA, Cxcr3, Itgb7 targeted modified single guide RNAs (sgRNAs) were purchased from and synthesized by Synthego.
• Cxcr3 loci were targeted with sgRNAs mixed in equal parts (murine Cxcr3 sgRNAs AGGCUGAAAUCCUGUGGGCA (SEQ ID NO: 242), UUCCCCAUAAUCGUAGGGAG (SEQ ID NO: 243), ACGUCAAGUGCUAGAUGCCU (SEQ ID NO: 244)).
• Itgb7 loci were targeted with sgRNAs mixed in equal parts (murine Itgb7 sgRNAs AGAAUACACUUCUGGCAGGA (SEQ ID NO: 245), GUCAGGAUCCUCCCAUUCUG (SEQ ID NO: 246), UUUCUGCUGGUUCUGGGCGG (SEQ ID NO: 247)).
• Adoptive cell transfer C57BL/6 were sub-lethally irradiated (500cGy), randomized, and injected intravenously with transduced T cells, as described elsewhere herein. Mice received intraperitoneal injection of interleukin-2 (IL-2) in PBS (5 x 10 4 III in 0.5 mL) once daily for 2 days starting on the day of cell transfer. Mice were monitored for changes in body weight, body composition, or behavior. Jaundice was evaluated by eye with criteria being yellowing of skin. For endpoint analysis of transferred cells, organs were collected and processed to single cell suspensions prior to staining for flow cytometry.
• IL-2 interleukin-2
• Murine tumor models For subcutaneous tumor experiments, C57BL/6 mice were implanted with KPC (5 x 10 5 ), KPC EpCAM KO (5 x 10 5 ), KPC EpCAM OE (1 x 1 o 6 ), or KPC hCEA OE (5 x 10 5 ) cells. 11 days after tumor implantation, mice were enrolled in the adoptive cell transfer experiment and treated as described above. Tumors were measured using digital calipers. Tumor size was measured in a blinded fashion every two-three days after transfer and tumor area was calculated as length x width of the tumor. Mice with tumors greater than 400 mm 2 were euthanized.
• mice C57BL/6 mice were implanted with KPC-Luciferase (7.5 x 10 5 ) or KPC hCEA (5 x 10 5 ) cells via intrahepatic injection.
• KPC-Luciferase 7.5 x 10 5
• KPC hCEA 5 x 10 5
• mice with Luciferase-expressing tumors four days after implantation, mice were imaged using the IVIS Spectrum In Vivo Imaging System (Perkin Elmer) and D-Luciferin (GoldBio), according to manufacturer instructions. Images were analyzed using the automated ROI tool in Living Image software (Perkin Elmer). Mice without detectable tumors were excluded from the example.
• mice with I VIS-confirmed tumors were enrolled and treated as described with ACT. I IS imaging was repeated two-three times a week for the duration of the experiment. For mice with KPC-hCEA intrahepatic tumors, autopsy time-course studies showed 100% of mice developed tumors. At the example’s endpoint, mice were euthanized and tumors excised from the liver and weighed.
• Functional variants include one or more residue additions or substitutions that do not substantially impact the physiological effects of the protein.
• Functional fragments include one or more deletions or truncations that do not substantially impact the physiological effects of the protein. A lack of substantial impact can be confirmed by observing experimentally comparable results in an activation study or a binding study.
• Functional variants and functional fragments of intracellular domains e.g., intracellular signaling components
• Functional variants and functional fragments of binding domains bind their cognate antigen or ligand at a level comparable to a wild-type reference.
• amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids.
• a conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
• Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1 : Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gin), Asp, and Glu; Group 4: Gin and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Vai) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gin, Cys, Ser, and Thr
• the hydropathic index of amino acids may be considered.
• the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982).
• amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
• variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and/or mutations that do not affect the function of an encoded product to a statistically-significant degree.
• Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.
• % sequence identity refers to a relationship between two or more sequences, as determined by comparing the sequences.
• identity also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences.
• Identity (often referred to as “similarity") can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H.
• Variants also include nucleic acid molecules that hybridize under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence.
• Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 pg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C.
• 5XSSC 750 mM NaCI, 75 mM trisodium citrate
• 50 mM sodium phosphate pH 7.6
• 5XDenhardt's solution 10% dextran sulfate
• 20 pg/ml denatured, sheared salmon sperm DNA followed by washing the filters in 0.1XSSC at 50 °C.
• Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature.
• washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5XSSC).
• Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments.
• Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
• the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
• Specifically binds refers to an association of a binding domain (of, for example, a CAR binding domain) to its cognate binding molecule with an affinity or K a (/.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10 5 M -1 , while not significantly associating with any other molecules or components in a relevant environment sample. Binding domains may be classified as "high affinity” or "low affinity”.
• "high affinity" binding domains refer to those binding domains with a Ka of at least 10 7 M’ 1 , at least 10 8 M’ 1 , at least 10 9 M -1 , at least 10 10 M’ 1 , at least 10 11 M -1 , at least 10 12 M’ 1 , or at least 10 13 M' 1 .
• "low affinity" binding domains refer to those binding domains with a K a of up to 10 7 M -1 , up to 10 6 M’ 1 , up to 10 5 M’ 1 .
• affinity may be defined as an equilibrium dissociation constant (K d ) of a particular binding interaction with units of M (e.g., 10- 5 M to 10’ 13 M).
• a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain.
• enhanced affinity may be due to a Ka (equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a Kd (dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off- rate (K O ff) for the cognate binding molecule that is less than that of the reference binding domain.
• a variety of assays are known for detecting binding domains that specifically bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE® analysis (see also, e.g., Scatchard, et a!., 1949, Ann. N.Y. Acad. Sci. 51:660; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).
• each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component.
• the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.”
• the transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts.
• the transitional phrase “consisting of” excludes any element, step, ingredient or component not specified.
• the transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would reduce the ability of a localized therapy to treat metastases from a primary tumor site.
• the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ⁇ 20% of the stated value; ⁇ 19% of the stated value; ⁇ 18% of the stated value; ⁇ 17% of the stated value; ⁇ 16% of the stated value; ⁇ 15% of the stated value; ⁇ 14% of the stated value; ⁇ 13% of the stated value; ⁇ 12% of the stated value; ⁇ 11 % of the stated value; ⁇ 10% of the stated value; ⁇ 9% of the stated value; ⁇ 8% of the stated value; ⁇ 7% of the stated value; ⁇ 6% of the stated value; ⁇ 5% of the stated value; ⁇ 4% of the stated value; ⁇ 3% of the stated value; ⁇ 2% of the stated value; or ⁇ 1% of the stated value.

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