Classifications

• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/44—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
  • G01N33/5759—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds localised on the membrane of tumour or cancer cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/53—Hinge
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • 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
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• compositions and methods of making and using these compositions relate to engineered chimeric antigen receptors (CARs) and compositions thereof having specificity and affinity for fluorescein containing ligands presented on the surface of tumor cells. Accordingly, provided herein are compositions and methods of making and using these compositions.
• CARs engineered chimeric antigen receptors
• chimeric antigen receptors are synthetic receptors that include an extracellular ligand binding domain, most commonly a single chain variable fragment of a monoclonal antibody (scFv) linked to intracellular signaling components, most commonly O ⁇ 3z alone or combined with one or more costimulatory domains.
• scFv monoclonal antibody
• Much of the research in the design of chimeric antigen receptors has focused on defining scFvs and other ligand binding elements that target malignant cells without causing serious toxicity to essential normal tissues, and on defining the optimal composition of intracellular signaling modules to activate T cell effector functions.
• CAR T cell-mediated therapy that is selective for specific targets and which minimizes adverse side effects.
• a complex comprising a chimeric antigen receptor (CAR) or a T cell receptor (TCR), wherein the CAR or TCR is joined to a lipid, wherein the lipid comprises a target moiety and the CAR is joined to said lipid through an interaction with said target moiety, and wherein the CAR or TCR comprises a spacer domain having a spacer length of 1-22 amino acids, 23-50 amino acids, 51-100 amino acids, 100 to 150 amino acids or 151-250 amino acids or a spacer length that is within a range defined by any two of the aforementioned lengths.
• the CAR or TCR comprises a sequence as set forth in any one of SEQ ID NOs: 1-6.
• the spacer domain is a IgG4 hinge connected to a CH2 domain to a CH3 domain. In some such embodiments, the spacer domain comprises the amino acid sequence SEQ ID NO: 7. In some alternatives, the spacer domain is an IgG4 hinge connected to a CH3 domain. In some such embodiments, the spacer domain comprises the amino acid sequence SEQ ID NO: 8. In some alternatives, the spacer domain is an IgG4 hinge only. In some such embodiments, the spacer domain comprises the amino acid sequence SEQ ID NO: 9. Some embodiments include nucleic acids that encode any one of SEQ ID NO: 1-9, which may be present on a vector and/or introduced to a cell (e.g., a T cell).
• the spacer comprises a length of 229 amino acids.
• the lipid comprises a polar head group and a hydrophobic group.
• the polar head comprises a choline, a phosphatidylcholine, sphingomyelin, phosphoethanolamine group, an oligosaccharide residue, a sugar residue, phosphatidyl serine or phosphatidyl inositol.
• the polar-head group comprises phosphocholine, a piperidine moiety or a trimethylarseno-ethyl-phosphate moiety.
• the hydrophobic group is fatty acid, such as an aliphatic chain.
• the fatty acid is saturated or unsaturated.
• the hydrophobic group comprises an alkyl, alkenyl or alkynyl group.
• the hydrophobic group comprises a terpenoid lipid, such as a steroid or cholesterol.
• the hydrophobic group comprises an ether linkage, wherein the ether linkage is between the polar head group and the aliphatic chain.
• the sugar residue is a glycerol.
• the hydrophobic group comprises a carbon alkyl chain, wherein the carbon alkyl chain comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbons or any number that is within a range defined by any two aforementioned values.
• the carbon alkyl chain comprises 18 carbons.
• the lipid is a phospholipid ether.
• the target moiety is biotin, digoxigenin, dinitrophenol or fluorescein.
• the spacer comprises a PEG spacer, a Hapten (2x) spacer, or an alkane chain.
• the PEG spacer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 PEG molecules, or any amount of PEG molecules that is within a range defined by any two aforementioned values.
• the CAR or TCR is expressed by a cell or a T cell. In some alternatives, the CAR or TCR is on the surface of a cell or a T cell.
• the cell is a precursor T cell. In some alternatives, the precursor T cell is a hematopoietic stem cell. In some alternatives, the cell is a CD8+ T cytotoxic lymphocyte cell selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. In some alternatives, the cell is a CD4+ T helper lymphocyte cell that is selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some alternatives, the lipid is intercalated in a lipid bilayer of a target cell. In some alternatives, the target cell is a tumor cell. In some alternatives, the target cell is an immune cell. In some alternatives, the immune cell is a T cell or a B cell. In some alternatives, the target cell is present in a tumor microenvironment.
• a cell comprising a complex of any one of the alternatives herein, the cell comprising a chimeric antigen receptor (CAR) or T cell receptor (TCR), wherein the CAR or TCR is bound to or is configured to bind to a lipid, wherein the lipid comprises a target moiety and the cell comprising the CAR is bound to the target moiety of the lipid, and wherein the CAR or TCR comprises a spacer domain.
• CAR chimeric antigen receptor
• TCR T cell receptor
• the complex comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR), wherein the CAR or TCR is joined to or is configured to be joined to a lipid, wherein the lipid comprises a target moiety and the CAR is joined to or configured to be joined to said lipid through an interaction with said target moiety, and wherein the CAR or TCR comprises a spacer domain having a spacer length of 1-22 amino acids, 23-50 amino acids, 51-100 amino acids, 100 to 150 amino acids or 151-250 amino acids or a spacer length that is within a range defined by any two of the aforementioned lengths.
• the CAR or TCR comprises a sequence as set forth in any one of SEQ ID NOs: 1-6.
• the spacer domain is an IgG4 hinge connected to a CH2 domain to a CH3 domain. In some such embodiments, the spacer domain comprises the amino acid sequence SEQ ID NO: 7. In some alternatives, the spacer domain is an IgG4 hinge connected to a CH3 domain. In some such embodiments, the spacer domain comprises the amino acid sequence SEQ ID NO: 8. In some alternatives, the spacer domain is an IgG4 hinge only. In some such embodiments, the spacer domain comprises the amino acid sequence SEQ ID NO: 9. In some alternatives, the cell comprises a nucleic acid encoding any one of SEQ ID. NO: 1-9). In some alternatives, the spacer comprises a length of 229 amino acids.
• the lipid comprises a polar head group and a hydrophobic group.
• the polar head comprises a choline, a phosphatidylcholine, sphingomyelin, phosphoethanolamine group, an oligosaccharide residue, a sugar residue, phosphatidyl serine or phosphatidyl inositol.
• the polar-head group comprises phosphocholine, a piperidine moiety or a trimethylarseno-ethyl- phosphate moiety.
• the hydrophobic group is fatty acid, such as an aliphatic chain. In some alternatives, the fatty acid is saturated or unsaturated.
• the hydrophobic group comprises an alkyl, alkenyl or alkynyl group. In some alternatives, the hydrophobic group comprises a terpenoid lipid, such as a steroid or cholesterol. In some alternatives, the hydrophobic group comprises an ether linkage, wherein the ether linkage is between the polar head group and the aliphatic chain. In some alternatives, the sugar residue is a glycerol or sugar alcohol. In some alternatives, the hydrophobic group comprises a carbon alkyl chain, wherein the carbon alkyl chain comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbons or any number that is within a range defined by any two aforementioned values. In some alternatives, the carbon alkyl chain comprises 18 carbons.
• the lipid is a phospholipid ether.
• the target moiety is biotin, digoxigenin, dinitrophenol or fluorescein.
• the spacer comprises a PEG spacer, a Hapten (2X) spacer, or an alkane chain.
• the PEG spacer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 PEG molecules, or any amount of PEG molecules that is within a range defined by any two aforementioned values.
• the CAR or TCR is expressed by a cell or a T cell.
• the CAR or TCR is on the surface of a cell or a T cell.
• the cell is a precursor T cell.
• the precursor T cell is a hematopoietic stem cell.
• the cell is a CD8+ T cytotoxic lymphocyte cell selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells.
• the cell is a CD4+ T helper lymphocyte cell that is selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells.
• the lipid is intercalated in a lipid bilayer of a target cell.
• the target cell is a tumor cell.
• the target cell is an immune cell.
• the immune cell is a T cell or a B cell.
• the target cell is present in a tumor microenvironment.
• the cell is a precursor T cell.
• the precursor T cell is a hematopoietic stem cell.
• the cell is a CD8+ T cytotoxic lymphocyte cell selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells.
• the cell is a CD4+ T helper lymphocyte cell that is selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells.
• the lipid is intercalated in a lipid bilayer of a target cell.
• the target cell is a tumor cell.
• the target cell is an immune cell.
• the immune cell is a T cell or a B cell.
• the target cell is present in a tumor microenvironment.
• a method of treating, ameliorating, or inhibiting a cancer in a subject comprising a) introducing, providing, or administering to a subject a composition that comprises a lipid, which comprises a target moiety that is bound to a masking moiety, b) introducing, providing, or administering to said subject a cell comprising a chimeric antigen receptor (CAR) or T cell receptor (TCR), which is specific for the target moiety once the masking moiety is removed from the target moiety, and wherein the CAR or TCR comprises a spacer domain, c) removing the masking moiety from the target moiety thereby allowing the target moiety to bind to the CAR present on the cell, and, d) optionally, measuring or evaluating the binding of the cell comprising the CAR to the lipid, after steps a-c and/or e) optionally, measuring or evaluating the treatment, amelioration, or inhibition of said cancer after steps a-d,
• CAR chimeric antigen receptor
• TCR
• the complex or the cell is provided to the subject at the same time as the composition or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 24, 36 or 48 hours before or after administration of the composition, or any time within a range defined by any two aforementioned values.
• the cell is provided to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 24, 36 or 48 hours before administration of the composition, or any time within a range defined by any two aforementioned values.
• the cell is provided to the subject within seconds or minutes, such as less than an hour, of providing the composition to the subject.
• a boost of the cell and/or the composition is provided to the subject.
• an additional cancer therapy is provided, such as a small molecule, e.g., a chemical compound, an antibody therapy, e.g., a humanized monoclonal antibody with or without conjugation to a radionuclide, toxin, or drug, surgery, and/or radiation.
• the cancer is a solid tumor, such as colon, breast, ovarian, lung, pancreatic, prostate, melanoma, renal, pancreatic, glioblastoma, neuroblastoma, medulloblastoma, sarcoma or liver cancer; or a non-solid tumor, such as a leukemia, or a multiple myeloma.
• the spacer domain is an IgG4 hinge connected to a CH2 domain to a CH3 domain. In some such embodiments, the spacer domain comprises the amino acid sequence SEQ ID NO: 7. In some alternatives, the spacer domain is an IgG4 hinge connected to a CH3 domain. In some such embodiments, the spacer domain comprises the amino acid sequence SEQ ID NO: 8. In some alternatives, the spacer domain is a IgG4 hinge only. In some such embodiments, the spacer domain comprises the amino acid sequence SEQ ID NO: 9. In some alternatives, the spacer comprises a sequence set forth in SEQ ID NO: 7. In some alternatives, binding of the target moiety to the CAR present on the cell induces production of at least one cytokine. In some alternatives, the at least one cytokine comprises IL-2, TNF-a and/or INF-a.
• kits comprising a pharmaceutical grade FL-PLE.
• the pharmaceutical grade FL-PLE comprises a hapten.
• the pharmaceutical grade FL-PLE comprises fluorescein (e.g., FITC).
• FIG. 1A and FIG. 1B schematically depict different spacers for CAR T cells.
• FIG. 1A shows an example of: (i) a second-generation CAR. The antigen recognition moiety (uppermost portion of molecule), which is presented at a desired distance by a short spacer domain, from the cell surface. This spacer is connected to a transmembrane domain, which is connected to two signaling domains; (ii) a medium spacer CAR, where another domain is added to the short spacer; and (iii) a long spacer CAR, where another domain is added to the medium spacer.
• FIG. 1B shows an example of different spacers exposed on CAR T cells.
• long, medium, and short spacers can comprise a sequence as set forth in SEQ ID NO: 7, 8, and 9, respectively.
• FIG. 2 schematically depicts an example of spacer length and orientation on a surface of a tumor cell.
• a tumor cell whose surface is labeled with CAR T cell tumor targeting agent (CTCT).
• CTCT CAR T cell tumor targeting agent
• the tumor surface has proteins and other cellular debris protruding from the surface, which can impact a CTCT specific CAR’s ability to recognize the CAR T cell tumor targeting agent (CTCT).
• Panels A-C depict example embodiments of a PLE-CTCT imbedded into the lipid membrane
• panels D-E depict example embodiments of a CAR T cell tumor targeting agent (CTCT) conjugated to a surface protein.
• CTCT CAR T cell tumor targeting agent
• a short CAR is unable to reach the CAR T cell tumor targeting agent (CTCT) and will not be able to activate.
• C CAR T cell tumor targeting agent
• CCT CAR T cell tumor targeting agent
• D and E the long CAR from shown in panel C and the short CAR depicted in panel B are both able to recognize and activate through a CAR T cell tumor targeting agent (CTCT) that is conjugated to a surface protein.
• FIG. 3 schematically depicts an example of a CTCT CAR T cell recognizing only unmasked PLE-CTCT and being activated.
• a tumor loaded with a prodrug version of PLE-CTCT.
• the prodrug provides steric hindrance to the CTCT specific CAR T cell making the Pro-PLE-CTCT unrecognizable to the CTCT specific CAR T cell.
• FIG. 4A depicts an example embodiment of a phospholipid conjugated with a hapten, fluorescein (PLE-CTCT).
• PLE phospholipid conjugated with a hapten, fluorescein
• FL-PLE Structure of phospholipid ether (PLE) tethered to the hapten fluorescein (FL-PLE).
• FL fluorescein
• PEG Polyethylene glycol
• iii & iv) PLE is the polar head group
• iv) is the hydrophobic tail for incorporation or tethering into the cell plasma membrane.
• FIG. 4B depicts an example prodrug version of FL- PLE in which a Pro moiety is conjugated to the hapten (fluorescein) via a cleavable bond indicated by the arrow.
• the masked FL-PLE is substantially non-florescent.
• the bond can be cleaved by the presence of reactive oxygen species (ROS), such as in a tumor microenvironment.
• ROS reactive oxygen species
• FIG. 5 depicts examples of different antiFL scFvs that were selected to create an antiFL CAR library (upper portion).
• Lower portion depicts structures of example CARs with a long spacer (L), a medium spacer (M), or a short spacer (S).
• the structures include a leader sequence, an scFv, a hinge/spacer, a CD28tm domain, a 41BB domain, a O ⁇ 3z, a T2A sequence, and an EGFTt domain.
• L long spacer
• M medium spacer
• S short spacer
• the structures include a leader sequence, an scFv, a hinge/spacer, a CD28tm domain, a 41BB domain, a O ⁇ 3z, a T2A sequence, and an EGFTt domain.
• L long spacer
• M medium spacer
• S short spacer
• FIG. 6 depicts FACS analysis of CD8+ T cells containing antiFL CARs.
• Panel (a) and (b) depict T cells staining for the selection marker EGFRt containing either an antiFL(FITC-E2) CAR, which comprises SEQ ID NO: 5 [96% positive], or an antiFL(4M5.3) CAR, which comprises SEQ ID NO: 2 [80% positive], respectively.
• Panels (c) and (d) depict T cells incubated with a mouse CD19-FITC antibody, then stained with an anti-mouse-Fc- Alexa647 antibody for the bound FITC antibody, for T cells containing either the antiFL(FITC- E2) CAR [97% positive], or the antiFL(4M5.3) CAR [79% positive], respectively.
• FIG. 7A depicts a FACS analysis of cells treated with FL-PLE.
• FIG. 7B depicts specific lysis of K562 cells in the presence or absence of
• FIG. 7C depicts cytokine production from K562 cells in the presence or absence of FL-PLE, and in the presence or absence of CD8+ T cells containing an antiFL CAR.
• FIG. 8A depicts FACS of various cell lines treated with FL-PLE.
• FIG. 8B depicts specific lysis of cells treated with FL-PLE and contacted with CD8+ T cells containing an antiFL(FITC-E2) CAR containing a long spacer.
• FIG. 9 depicts a FACS analysis of CD8+ T cells contain various antiFL CARs, and staining for a surface marker, EGFRt.
• FIG. 10 depicts a FACS analysis of CD4+ T cells contain various antiFL CARs, and staining for a surface marker, EGFRt.
• FIG. 11 depicts a FACS analysis of cells labeled with FL-PLE.
• FIG. 12 depicts specific lysis of target cells incubated with FL-PLE and contacted with CD8+ T cells containing antiFL CARs.
• FIG. 13 depicts cytokine production of cells incubated with FL-PLE and contacted with CD4+ T cells containing antiFL CARs.
• FIG. 14A depicts specific lysis of target cells incubated with FL-PLE and contacted with CD8+ T cells containing antiFL CARs.
• FIG. 14B depicts cytokine production of cells incubated with FL-PLE and contacted with CD4+ T cells containing antiFL CARs.
• FIG. 15 A depicts a FACS analysis of cells containing various antiFL CARs.
• FIG. 15B depicts a FACS analysis of cells treated with FL-PLE.
• FIG. 15C depicts a series of graphs for specific lysis of various tumor cells in the presence or absence of FL-PLE, and contacted with various antiFL CARs.
• FIG. 15D depicts a series of graphs for cytokine production from T cells containing various antiFL CARs and contacted with various tumor cells in the presence or absence of FL-PLE.
• FIG. 15E depicts a series of graphs for cytokine production from T cells containing various antiFL CARs and contacted with various tumor cells in the presence or absence of FL-PLE.
• FIG. 16 depicts an image of an orthotopic glioma xenograft in a subject which had been dosed intravenously with FL-PLE, and evaluated 48 hr later.
• Left lower panel is an image of the control contralateral hemisphere of the subject. Both the orthotopic glioma xenograft and the control contralateral hemisphere of the subject were evaluated by staining with an anti-fluorescein antibody. Levels of retained FL-PLE were also quantified in both the orthotopic glioma xenograft and the control contralateral hemisphere, over time (right panel).
• FIG. 17 depicts graphs demonstrating FL-PLE retention in different tumors in vivo.
• FIG. 18A depicts a FACS analysis of cells treated with FL-PLE, or ProFL-
• FIG. 18B depicts specific lysis of cells treated with FL-PLE or ProFL-PLE, and contacted with CD8+ T cells containing an antiFL CAR
• FIG. 18C depicts a series of graphs for cytokine product for cells in the presence or absence of masked or unmasked ProFL-PLE and FL-PLE, in the presence or absence of CD4+ T cells containing an antiFL(FITCE2) CAR with a long spacer.
• FIG. 19A depicts a FACS analysis of FL-PLE or ProFL-PLE binding to cells.
• FIG. 19B depicts a series of graphs for cytokine product for cells in the presence or absence of ProFL-PLE or FL-PLE, in the presence or absence of CD4+ T cells containing an antiFL(FITCE2) CAR with a long spacer.
• FIG. 19C depicts a FACS analysis of cells treated with FL-PLE or ProFL-
• FIG. 20 depicts a graph of relative life span of mice having a neuroblastoma (Be2) tumor and treated with ProFL-PLE, FL-PLE or control, and administered either T cells containing an antiFL(FITCE2) long spacer CAR which comprised SEQ ID NO: 5, or a control.
• FIG. 21 A depicts graphs for average tumor progression (top panel), and percent survival (bottom panel) in mice injected intra-tum orally with ProFL-PLE, and administered T cells containing an antiFL(FITCE2) CAR with a long spacer.
• FIG. 21B depicts graphs for average tumor progression (top panel), and percent survival (bottom panel) in mice injected intravenously with ProFL-PLE, and administered T cells containing an antiFL(FITCE2) CAR with a long spacer.
• chimeric antigen receptors with specificity and a selected or designed affinity for the molecule fluorescein when presented on the surface of tumor cells loaded with exogenous FL-PLE, which results in redirected anti tumor T cell reactivity.
• FL-PLE is a synthetic molecular structure that is designed to integrate into the plasma membrane of tumors such that the molecule’s fluorescein is present adjacent to the outer leaflet of the plasma membrane in the extracellular space.
• the FL-PLE structure includes therapeutically important attributes.
• a CAR that targets the fluorescein (FL) moiety of the FL- PLE is provided, and methods of making and using the same are also contemplated. These CARs can either be constitutively expressed or placed under regulated control.
• the present disclosure addresses the identification, creation, and engineering of suitable CAR T cells for binding to the FL moiety of FL-PLE.
• the FL moiety may be masked and unmasked such that the FL moiety can be activated to receive the CAR T cell.
• a FL specific CAR may recognize only unmasked CAR T cell tumor targeting agents (CTCTs) and not masked FLs.
• Some embodiments of the methods and compositions provided herein can include aspects disclosed in WO 2018/148224 entitled“PHOSPHOLIPID ETHER (PLE) CAR T CELL TUMOR TARGETING (CTCT) AGENTS” which is hereby expressly incorporated by reference in its entirety.
• “a” or“an” may mean one or more than one.
• the term“about” indicates that a value includes the inherent variation of error for the method being employed to determine a value, or the variation that exists among experiments.
• Chimeric receptor refers to a synthetically designed receptor comprising a ligand binding domain of an antibody or other protein sequence that binds to a molecule associated with the disease or disorder and is linked via a spacer domain to one or more intracellular signaling domains of a T cell or other receptors, such as a costimulatory domain.
• Chimeric receptor can also be referred to as artificial T cell receptors, chimeric T cell receptors, chimeric immunoreceptors, and chimeric antigen receptors (CARs).
• CARs are genetically engineered T- cell receptors designed to redirect T-cells to target cells that express specific cell-surface antigens.
• T-cells can be removed from a subj ect and modified so that they can express receptors that can be specific for an antigen by a process called adoptive cell transfer. The T-cells are reintroduced into the patient where they can then recognize and target an antigen.
• CARs are engineered receptors that can graft a selected specificity onto an immune receptor cell.
• the term chimeric antigen receptors or“CARs” are also considered by some investigators to include the antibody or antibody fragment, the spacer, signaling domain, and transmembrane region. Due to the surprising effects of modifying the different components or domains of the CAR described herein, such as the epitope binding region (for example, antibody fragment, scFv, or portion thereof), spacer, transmembrane domain, and/ or signaling domain), the components of the CAR are frequently distinguished throughout this disclosure in terms of independent elements.
• CAR T cell targeting agent is given its plain and ordinary meaning in view of the specification and can be described, for example as a composition that that can integrate into the membrane of a target cell.
• the CTCT comprises a lipid, wherein the lipid comprises a target moiety and a masking moiety.
• the masking moiety may be unmasked in the presence of low pH, ROS species and within a tumor microenvironment, for example.
• the masking moiety inhibits specific binding of a CAR to the target moiety.
• the target moiety may be recognized and bound by a chimeric antigen receptor that is specific for the target moiety.
• the masking moiety is removed at a pH of 4, 5, 6, or 6.5 or any pH in between a range defined by any two aforementioned values.
• A“T cell receptor” or“TCR” is a molecule that is found on the surface of T lymphocytes or T cells that is responsible for the recognition of fragments of antigen bound to a major histocompatibility complex molecule.
• Target moiety refers to a specific group or site on a molecule or chemical that is a binding target for another chemical or protein of interest.
• a complex is provided, wherein the complex comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR) joined to a lipid, wherein the lipid comprises a target moiety and the CAR is joined to said lipid through an interaction with said target moiety.
• the target moiety is biotin, digoxigenin, dinitrophenol or fluorescein.
• A“single-chain variable fragment,” (scFv) is a fusion protein that can have variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to 25 amino acids.
• the short linker peptide can comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids or any number of amino acids in between a range defined by any two aforementioned values.
• the linker is usually rich in glycine for flexibility, as well as, serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa.
• the scFv can be specific for an antigen.
• “Antigen” or“ Ag” as used herein refers to a molecule that provokes an immune response. This immune response can involve either antibody production, or the activation of specific immunologically-competent cells, or both.
• An antigen can be generated, synthesized, produced recombinantly or can be derived from a biological sample.
• a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid such, for example, blood, plasma or ascites fluid.
• a composition comprising cells manufactured by any one of the alternative methods herein.
• the cells comprise a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a scFv that is specific for an antigen.
• a ScFv described herein as antiFL(FITCE2 TyrHl33 Ala) also referred to as antiFL(FITC-E2 Mut2), or as antiFL(Mut2)
• SEQ ID NO: 1 amino acid sequence of
• Some embodiments provided herein relate to a scFv described herein as antiFL(4M5.3), which can be incorporated into a CAR in accordance with this disclosure (SEQ ID NO: 2), having an amino acid sequence of
• Some embodiments provided herein relate to a ScFv described herein as antiFL(4420), which can be incorporated into a CAR in accordance with this disclosure (SEQ ID NO: 3), having an amino acid sequence of
• Some embodiments provided herein relate to a ScFv described herein as antiFL(4D5Flu), which can be incorporated into a CAR in accordance with this disclosure (SEQ ID NO: 4), having an amino acid sequence of
• Some embodiments provided herein relate to a ScFv described herein as antiFL(FITCE2), which can be incorporated into a CAR in accordance with this disclosure (SEQ ID NO: 5), having an amino acid sequence of
• Some embodiments provided herein relate to a ScFv described herein as antiFL(FITCE2 HisHl3 lAla), which can be incorporated into a CAR in accordance with this disclosure (SEQ ID NO: 6), having an amino acid sequence of S VLT QP S S V S A APGQK VTI SC S GS T SNIGNN YV S W Y Q QHPGK APKLMIYDV SKRP S G VPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFLFGTGTKLTVLGGGGG SGGGGSGGGGSQVQLVESGGNLVQPGGSLRLSCAASGFTFGSFSMSWVRQAPGGGL EWVAGLSARSSLTHYADSVKGRFTISRDNAKNSVYLQMNSLRVEDTAVYYCARRS YD S SGYWGAFYS YMD VWGQGTL VT VS .
• Antigen specific binding domains can include protein or protein domains that can specifically bind to an epitope on a protein at a low or high binding affinity (fM to mM binding capacity).
• the fusion protein comprises a protein or portion thereof that can modulate an immune response.
• the protein comprises an antigen specific binding domain.
• T-cells or“T lymphocytes” as used herein, can be from any mammalian species, preferably primate, including monkeys, dogs, and humans.
• the T- cells are allogeneic (from the same species but different donor) as the recipient subject; in some alternatives the T-cells are autologous (the donor and the recipient are the same); in some alternatives the T-cells are syngeneic (the donor and the recipients are different but are identical twins).
• Combination therapy refers to a therapy that uses more than one medication or modality for a treatment.
• Combination therapy for example, can also refer to multiple therapies for a single disease, and often all the therapies are pharmaceutical product combinations.
• Combination therapy can also involve prescribing and administering separate drugs in which the dosage can also have more than one active ingredient.
• a combination therapy is provided, wherein the combination therapy comprises administering a genetically modified immune cell for modifying a tumor microenvironment.
• the combination therapy comprises administering a genetically modified immune cell for modulating the suppression of the immune response in a tumor microenvironment.
• the combination therapy comprises administering a genetically modified immune cell for minimizing the proliferation of tumor and suppressive cells in a subject in need thereof e.g. a human.
• the combination therapy comprises administering a genetically modified immune cell for increasing the efficiency of an anti-cancer therapy, anti -infection therapy, antibacterial therapy, anti-viral therapy, or anti-tumoral therapy in a subject in need thereof e.g., a human.
• the combination therapy further comprises administration of an inhibitor.
• the inhibitor is not an enzymatic inhibitor.
• the inhibitor is an enzymatic inhibitor.
• the combination therapy comprises administering a therapeutic dose of an inhibitor or an antibody or a binding fragment thereof. These antibodies or binding fragments thereof can be humanized in some alternatives.
• the combination therapy can further comprise administering a CAR bearing T-cell to a subject in need e.g., a human.
• “Chemotherapeutic drugs” are a category of anti-cancer medicaments that can use, for example, chemical substances, such as anti-cancer drugs (chemotherapeutic agents), which can be administered as part of a standardized chemotherapy regimen. Chemotherapeutic drugs can be administered with a curative intent or to prolong life or to reduce symptoms (palliative chemotherapy). Additional chemotherapies can also include hormonal therapy and targeted therapy, as it is one of the major categories of medical oncology (pharmacotherapy for cancer). These modalities are often used in conjunction with other cancer therapies, such as radiation therapy, surgery, and/or hyperthermia therapy. In a few cases, cancer has been known to spread due to surgery.
• a genetically modified immune cell is administered to the tumor site prior to or after a surgical procedure.
• Chemotherapy in which chemotherapeutic drugs are administered, can use one drug at a time (single-agent chemotherapy) or several drugs at once (combination chemotherapy or poly chemotherapy). The combination of chemotherapy and radiotherapy is chemoradiotherapy. Chemotherapy using drugs that convert to cytotoxic activity only upon light exposure is called photochemotherapy or photodynamic therapy.
• the method can further comprise administering to a subject having cancer, photochemotherapy or photodynamic therapy after receiving the genetically modified immune cells or genetically engineered macrophages (GEMs).
• GEMs genetically engineered macrophages
• Chemotherapuetic drugs can include but are not limited to antibody-drug conjugates (for example, an antibody attached to a drug by a linker), nanoparticles (for example a nanoparticle can be 1-1000 nanometer sized particle for promoting tumor selectivity and aid in delivering low-solubility drugs), electochemotherapy, alkylating agents, antimetabolites (for example, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), Capecitabine (Xeloda®), Cladribine, Clofarabine, Cytarabine (Ara-C®), Floxuridine, Fludarabine, Gemcitabine (Gemzar®), Hydroxyurea, Methotrexate, Pemetrexed (Alimta®), Pentostatin, or Thioguanine), anti-tumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, DNA intercalating agents, or checkpoint inhibitors (for example checkpoint kinases
• the genetically modified immune cells or compositions comprising genetically modified immune cells are administered in combination with one or more anti-cancer agents, such as any one or more of the foregoing compounds or therapies.
• the one or more anti-cancer agent that is co-administered or administered in conjunction with the genetically modified immune cells comprises antibody-drug conjugates, nanoparticles, electrochemotherapy, alkylating agents, antimetabolites, anti-tumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, DNA intercalating agents, or checkpoint inhibitors.
• the antimetabolites comprise 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), Capecitabine (Xeloda®), Cladribine, Clofarabine, Cytarabine (Ara-C®), Floxuridine, Fludarabine, Gemcitabine (Gemzar®), Hydroxyurea, Methotrexate, Pemetrexed (Alimta®), Pentostatin, or Thioguanine.
• Subjects or“patient,” as described herein refers to any organism upon which the alternatives described herein may be used or administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes.
• Subjects or patients include, for example, animals.
• the subject is mice, rats, rabbits, non-human primates, and humans.
• the subject is a cow, sheep, pig, horse, dog, cat, primate or a human.
• CCT CAR T cell tumor targeting
• FL-PLE hapten fluorescein
• fluorescein is a synthetic organic compound that is soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications.
• fluorescein is a target moiety on a lipid that is specifically recognized by a chimeric antigen receptor designed and/or selected for its ability to bind or interact with the fluorescein.
• the lipid is a phospholipid ether.
• lipid as described herein, is a class of organic compounds that comprise carbon chains, fatty acids or a fatty acid derivative that is typically insoluble in water but can integrate into or mix with hydrophobic or organic solvents.
• lipids can include fats, waxes, fat soluble vitamins, monoglycerides, diglycerides, triglycerides, sphingolipids, cerebrosides, ceramides, or phospholipids. Described herein are amphiphilic lipids that can have a polar head group and a hydrophobic moiety or hydrophobic group.
• “Hydrophobic group” or hydrophobic moiety is a molecule or a part of a molecule that is repelled from a mass of water and tends to be non-polar. This can include alkanes, oils or fats.
• lipids can be glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids or polyketides.
• a complex is provided, wherein the complex comprises a lipid.
• the lipid comprises a polar head group and a hydrophobic moiety.
• the hydrophobic moiety is a hydrophobic carbon tail.
• the hydrophobic carbon tail is saturated or unsaturated. In some alternatives, the hydrophobic carbon tail comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 carbons or any number of carbons in between a range set forth in any aforementioned value. In some alternatives, the hydrophobic moiety is a steroid or a cholesterol. In some alternatives, the lipid comprises a glycerolipid, glycerophospholipid, sphingolipid, sterol lipid, prenol lipid, saccharolipid or polyketide. In some alternatives, the lipid is a phospholipid ether. In some alternatives, the lipid contains branched alkyl tails.
• the lipid can be a sphingolipid.
• the sphingolipid can contain a backbone of sphingoid bases, such as a set of aliphatic amino alcohols that includes sphingosine.
• a sphingolipid with an R group consisting of a hydrogen atom only is a ceramide.
• Other common R groups include phosphocholine, yielding a sphingomyelin, and various sugar monomers or dimers, yielding cerebrosides and globosides, respectively. Cerebrosides and globosides are collectively known as glycosphingolipids.
• the lipid is a glycosphingolipid.
• the lipid comprises a polar head group and a hydrophobic group.
• the hydrophobic group comprises a fatty acid such as an aliphatic chain. The fatty acid can be saturated or unsaturated.
• the hydrophobic group comprises an alkyl, alkenyl or alkynyl group.
• the hydrophobic group comprises a terpenoid lipid, such as a steroid or cholesterol.
• the hydrophobic group comprises an ether linkage, wherein the ether linkage is between the polar head group and the aliphatic chain.
• the lipid is a phospholipid ether.
• the polar head comprises a choline, a phosphatidylcholine, sphingomyelin, phosphoethanolamine group, an oligosaccharide residue, a sugar residue, phosphatidyl serine or phosphatidyl inositol.
• the sugar is a glycerol or sugar alcohol.
• the lipid is a single chain alkylphospholipid.
• the lipid comprises a structure of synthetic alkylphospholipids, such as edelfosine, perifosine or erucylphosphocholine.
• the lipid is a lysophosphatidylcholine, edlfosine, erucylphosphocholine, D-21805 or perfisone.
• Such lipids are described for example, in van der Lui et al (“A new class of anticancer alkylphospholipids uses lipid rafts as membrane gateways to induce apoptosis in lymphoma cells” Mol Cancer Ther 2007; 6(8), 2007; hereby expressly incorporated by reference in its entirety).
• a choline within the polar head group can be substituted with a piperidine moiety.
• the lipid is an anticancer alkylphospholipid.
• Anticancer phospholipids are described by vander Lui et al. (“A new class of anticancer alkylphospholipids uses lipid rafts as membrane gateways to induce apoptosis in lymphoma cells” Mol Cancer Ther 2007; 6(8), 2007; hereby expressly incorporated by reference in its entirety).
• the lipids provided herein are synthetic and structurally related antitumor agents that interact with a cell membrane. These types of synthetic lipids are alkylphospholipids and are described by e.g., van Blitterswijk et al. (“Anticancer mechanisms and clinical application of alkylphopholipids” Biochimica et Biophysica Acta 1831 (2013)663-674; hereby incorporated by reference in its entirety herein).
• the synthetic alkylphospholipids can include edelfosine, miltefosine, perifosine, erucylphosphocholine or Erufosine.
• the lipid is edelfosine, miltefosine, perifosine, erucylphosphocholine or Erufosine.
• the lipid is a stable analog of lysophosphatidylcholine.
• the lipid is a thio-ether variant of edelfosine, or l-hexadecylthio- 2-methoxymethyl-rac-glycero-3-phosphocholine.
• the lipid is LysoPC, edelfosine, Ilmofosine, Miltefosine, Perifosine, Erucylphophocholine, or Erufosine.
• “Polar-head group” as described herein is the hydrophilic group of a lipid, such as a phospholipid.“Phospholipids” as described herein are a specific class of lipids that can form lipid bilayers due to their amphiphilic characteristic.
• the phospholipid molecule comprises at least one hydrophobic fatty acid“tail” and a hydrophilic“head” or“polar-head group.”
• the phospholipid or phospholipid ether comprises a polar- head group.
• the polar-head group comprises phosphocholine, a piperidine moiety or a trimethylarseno-ethyl-phosphate moiety.
• the lipid comprises a target moiety and the CAR is joined to or is configured to join to said lipid through an interaction with said target moiety.
• the lipid comprises a polar-head group (e.g., comprising an aromatic ring) and a carbon alkyl chain.
• a complex is provided, wherein the complex comprises one or more of said lipids.
• the lipid comprises a polar head group.
• the lipid is a phospholipid ether.
• the phospholipid ether comprises a target moiety and the CAR is joined to or is configured to join to said phospholipid ether through an interaction and/or binding with said target moiety.
• the phospholipid ether comprises a polar-head group and a carbon alkyl chain.
• the polar head group comprises a choline, a phosphatidylcholine, sphingomyelin, phosphoethanolamine group, an oligosaccharide residue, a sugar residue, phosphatidyl serine or phosphatidyl inositol.
• the polar head group comprises phosphocholine, a piperidine moiety or a trimethylarseno-ethyl-phosphate moiety.
• the lipid is a phospholipid ether.
• the sugar is a glycerol or sugar alcohol.
• the polar head group comprises a sugar group.
• the lipid comprises a mannose- containing head group.
• the polar head group comprises sphingosine.
• the polar head group comprises a glucose.
• the polar head group comprises a di-, tri- or tetra-saccharide.
• the lipid is a glucosylcerebroside.
• the lipid is a lactosylceramide.
• the lipid is a glycolipid.
• the glycolipid comprises sugar units, such as n- glucose, n-galactose or N-actyl-n-glactosamine.
• the lipid comprises a hydrocarbon ring such as a sterol.
• the polar head group of the lipid comprises glycerol or a sugar alcohol. In some alternatives, the polar head group of the lipid comprises a phosphate group. In some alternatives, the polar head group of the lipid comprises choline. In some alternatives, the lipid is a phosphatidylethanolomine. In some alternatives, the lipid is a phosphatidylinositol. In some alternatives, the lipid comprises a sphingoid base backbone. In some alternatives, the lipid comprises a sterol lipid, such as cholesterol or its derivatives. In some alternatives, the lipid comprises saccharolipids. In some alternatives, the polar head group comprises choline, phosphate and/or glycerol.
• the lipid is a glycolipid. In some alternatives, the lipid comprises a sugar. In some alternatives, the lipid is derived from sphingosine. In some alternatives, the lipid is a glycerol-glycolipid or a sphingo-glycolipid.
• the lipid is an ether lipid with branched hydrophobic chains.
• Saturated as described herein is a fatty acid molecule, in which there are no double bonds within the carbon molecules. Unsaturated as described herein indicates that there are one or more double bonds in a fatty acid chain.
• a complex comprising a lipid is provided.
• the lipid comprises a fatty acid chain, in which the fatty acid is saturated or unsaturated.
• Alkyl as described herein, is an alkyl substituent that has a missing hydrogen.
• An“alkenyl” group is an unsaturated hydrocarbon that contains at least one carbon-carbon double bond.
• An“alkynyl” group is an unsaturated hydrocarbon containing at least one carbon-carbon triple bond.
• Tepenoid as described herein, is a molecule that is derived from five carbon isoprene units. Steroids and sterols can be produced from terpenoid precursors. For example, steroids and cholesterol can be biosynthesized by terpenoid precursors.
• Phospholipid ether as described herein is a lipid in which one or more of the carbon atoms on a polar head group are bonded to an alkyl chain via an ether linkage, as opposed to the more common ester linkage.
• the polar head group is a glycerol.
• the spacer for a chimeric antigen receptor refers to a polypeptide spacer, which spacer length is configured to or is selected for its ability to promote an increase in binding or interaction with a chimeric antigen receptor or to reduce or minimizes an adverse side effect associated with CAR T cell therapy.
• a short spacer domain of a CAR has about 12 amino acids or less but greater than 1 amino acid and comprises all or a portion of a IgG4 hinge region sequence or variant thereof.
• an intermediate spacer domain of a CAR has about 119 amino acids or less but greater than 1 amino acid and comprises all or a portion of a IgG4 hinge region sequence and a CH3 region or variant thereof.
• a long spacer domain of a CAR has about 229 amino acids or less but greater than 1 amino acid and comprises all or a portion of a IgG4 hinge region sequence, a CH2 region, and a CH3 region or variant thereof.
• the lipid can also comprise a spacer that separates the target moiety from the lipid and is bound to the polar-head group of the lipid.
• the spacer of the lipid can comprise a PEG spacer, a Hapten spacer, a small peptide or an alkane chain.
• the hapten spacer comprises two haptens (hapten (2X) spacer).
• the lipid comprises a hydrophobic group, such as an alkane chain.
• the alkane chain can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbons, or any number of carbons between a range defined by any two aforementioned values.
• the PEG spacer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 PEG molecules, or any amount of PEG molecules that is within a range defined by any two aforementioned values.
• “Hapten” as described herein is a small molecule that elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one that also does not elicit an immune response by itself.
• the small-molecule hapten may also be able to bind to the antibody, but it will usually not initiate an immune response; usually only the hapten-carrier adduct can do this.
• a hapten is a small molecule binding moiety, which can be bound by or have specificity towards a scFv or antibody.
• the cells provided are cytotoxic T lymphocytes.
• “Cytotoxic T lymphocyte” (CTL) as used herein refers to a T lymphocyte that expresses CD8 on the surface thereof (e.g., a CD8 + T cell).
• such cells are preferably “memory” T cells (TM cells) that are antigen-experienced.
• the cell is a precursor T cell.
• the precursor T cell is a hematopoietic stem cell.
• the cell is a CD8+ T cytotoxic lymphocyte cell selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells.
• the cell is a CD4+ T helper lymphocyte cell that is selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells.
• “Masking moiety” as used herein refers to a moiety on a lipid ether that is bound to the target moiety.
• the masking moiety functions as a protective group to prevent recognition of the lipid’s target moiety by blocking binding and recognition of a chimeric antigen receptor that is specific for the target moiety.
• the masking moiety can be self-cleaved, thus allowing binding, interaction and/or recognition of the target moiety by the chimeric antigen receptor.
• the lipid is a phospholipid ether.
• the masking moiety comprises a phenolic hydroxyl group or PEG.
• the phenolic hydroxyl group is bound to a hydroxyl on a xanthene moiety of fluorescein.
• the masking moiety is bound to the target moiety by a cleavable moiety, which is optionally configured to be specifically cleavable in a tumor microenvironment.
• the cleavable moiety which is configured to be cleavable in a tumor microenvironment, is cleaved by a reactive oxygen species reaction, an acidic pH, hypoxia, or nitrosylation.
• the phospholipid ether comprises a target moiety and the CAR is joined to said phospholipid ether through an interaction with said target moiety.
• the phospholipid ether comprises a polar-head group and a carbon alkyl chain.
• Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body.
• Subjects that can be addressed using the methods described herein include subjects identified or selected as having cancer, including but not limited to colon, lung, liver, breast, renal, prostate, ovarian, skin (including melanoma), bone, leukemia, multiple myeloma, or brain cancer, etc. Such identification and/or selection can be made by clinical or diagnostic evaluation.
• the tumor associated antigens or molecules are known, such as melanoma, breast cancer, brain cancer, squamous cell carcinoma, colon cancer, leukemia, myeloma, or prostate cancer.
• Examples include but are not limited to B cell lymphoma, breast cancer, brain cancer, prostate cancer, and/or leukemia.
• one or more oncogenic polypeptides are associated with kidney, uterine, colon, lung, liver, breast, renal, prostate, ovarian, skin (including melanoma), bone, brain cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leukemia or leukemia.
• a method of treating, ameliorating, or inhibiting a cancer in a subject is provided.
• the cancer is breast, ovarian, lung, pancreatic, prostate, melanoma, renal, pancreatic, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, liver, colon, skin (including melanoma), bone or brain cancer.
• the subject that receives one of the therapies described herein is also selected to receive an additional cancer therapy, which can include a cancer therapeutic, radiation, chemotherapy, or a cancer therapy drug.
• the cancer therapy drug provided comprises Abiraterone, Alemtuzumab, Anastrozole, Aprepitant, Arsenic trioxide, Atezolizumab, Azacitidine, Bevacizumab, Bleomycin, Bortezomib, Cabazitaxel, Capecitabine, Carboplatin, Cetuximab, Chemotherapy drug combinations, Cisplatin, Crizotinib, Cyclophosphamide, Cytarabine,Denosumab, Docetaxel, Doxorubicin, Eribulin, Erlotinib, Etoposide, Everolimus, Exemestane, Filgrastim, Fluorouracil, Fulvestrant, Gemcitabine, Imatinib, Imiquimod, Ipilimumab, Ixabepilone, Lapatinib, Lenalidomide, Letrozole, Leuprolide, Mesna, Methotrexate, Nivolumab
• Tumor microenvironment as described herein is a cellular environment, wherein a tumor exists.
• the tumor microenvironment can include surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules or the extracellular matrix (ECM).
• ECM extracellular matrix
• the method includes human tumor therapy in which patients receive infusions of FL-PLE or derivatives thereof in combination with infusions of FL-specific CAR T cells.
• this system represents a universal target antigen used in combination with a universal CAR and/or universal CAR expressing anti-tumor effector cells.
• a PLE (18C alkyl chain) having a FL appended CAR recognition element, which is appended to the polar head group’s choline via three repeat PEG spacers is provided.
• An antiFL CAR library may be generated with different single chain variable fragments (scFvs), such as 6 or more different scFvs, with dissociation constants ranging from 200 fM to 10 nM, such as 200 fM, 210 fM, 220 fM, 230 fM, 240 fM, 250 fM, 260 fM, 270 fM, 280 fM, 290 fM, 300 fM, 400 fM, 500 fM, 600 fM, 700 fM, 800 fM, 900 fM, 1 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, or 10 nm, or
• Each scFv binds FL in a slightly different manner.
• the binding of scFv to FL is partially due to the difference in binding affinities through mutations in the binding pocket and due to the fact that some scFvs were derived from phage display compared to yeast display.
• three different spacers may be used, including, for example: long, IgG4 hinge connected to a CH2 domain to a CH3 domain; medium, IgG4 hinge connected to a CH3 domain; and short, IgG4 hinge only.
• FL-specific scFv CAR T cells with the correct orientation and spacer length exhibit redirected anti-tumor function in both in vitro and in vivo tumors loaded with FL PLE-CTCTs.
• shape and size of the scFv’s binding pocket can play a role in how well the CAR can activate.
• Some embodiments provided herein relate to a FL-specific CAR T cell comprising a targeting and recognition domain presented in a correct orientation with spacing and a binding affinity selected to create a desired T cell response.
• the spacing is selected for generating a T cell response in the specific cells of interest.
• the binding affinity is selected for generating a T cell response in the specific cells of interest.
• different tumors or cells benefit from different orientation, different spacer lengths, and affinities of CAR T cells to allow for desired CAR T cell responses. Therefore, some embodiments provided herein relate to generating a desired or selected combination of orientation, spacer length, and binding affinity for a selected therapy.
• an indication or measure of successful CAR T cells in vitro include cell lysis and the production of cytokines.
• short spacer CARs typically do not produce cytokines; whereas the long spacer CARs produce a large quantity of cytokines.
• an antiFL(4D5Flu) CAR which comprises SEQ ID NO: 4, and which is not able to produce cytokines with FL-PLE, is provided; whereas when cells are labeled with FL by another approach the antiFL(4D5Flu) CAR produces cytokines highlighting that the orientation of scFv to FL moiety of FL-PLE is an important and previously unrecognized variable in T cell activity.
• lowering the dissociation constant such as improving the binding affinity, does not necessarily improve the activation of the CAR.
• asymmetric T cell antitumor reactivity does not correlate with scFv KD.
• asymmetric T cell antitumor reactivity may be spacer length dependent.
• an antiFL(FITC-E2 Mut2) CAR which comprises SEQ ID NO: 1
• is a robust CAR which could not have been predicted from any prior knowledge of CAR structure function or scFv studies. In contrast, the field teaches that the highest scFv affinity results in the best CAR.
• FL-PLE administered to tumor-bearing mammals serves as a generic tumor-targeting agent that is retained on the tumor cell plasma membrane for recognition by FL-specific CAR T cells.
• Identification of a FL- specific CAR with unexpected T cell signaling robustness upon engagement of tumor cells loaded with FL-PLE is provided herein.
• This disclosure relates, in some embodiments, to the full development of a pharmaceutical-grade FL-PLE to be used in conjunction with a CAR T cell product, including in a kit. Other haptens could replace FL with CAR designed to react with these PLEs.
• Some embodiments of the methods and compositions provided herein include methods of treating or ameliorating or inhibiting a cancer in a subj ect. Some such embodiments include administering an effective amount to the subj ect a composition comprising a lipid conjugated to a target moiety, wherein the target moiety comprises a masking moiety; and administering a cell, such as a population of the cells, to the subj ect, wherein the cell comprises a chimeric antigen receptor (CAR) or T cell receptor (TCR), which specifically binds to the target moiety in the absence of the masking moiety, wherein the CAR or TCR comprises: an amino acid sequence having at least 95% identity with a sequence selected from SEQ ID NO:0 l -06; and/or a spacer domain comprising, consisting essentially of, or consisting of: a IgG4 hinge connected to a CH2 domain to a CH3 domain, such as a long spacer having an amino acid sequence with at least
• the CAR or TCR comprises an amino acid sequence selected from SEQ ID NO: 1-6.
• the spacer comprises a length of 229 amino acids.
• the CAR or TCR comprises an scFv domain having the amino acid sequence of SEQ ID NO: 1 (FITC-E2 Mut2); and a spacer domain having the amino acid sequence of SEQ ID NO: 07 (an exemplary long spacer).
• the CAR or TCR comprises an scFv domain having the amino acid sequence of SEQ ID NO:2 (4M5.3); and a spacer domain having the amino acid sequence of SEQ ID NO: 07 (an exemplary long spacer).
• the CAR or TCR comprises: an scFv domain having the amino acid sequence of SEQ ID NO:5 (FITC-E2); and a spacer domain having the amino acid sequence of SEQ ID NO: 07 (an exemplary long spacer).
• Some embodiments of the methods and compositions provided herein include methods method of treating, ameliorating, or inhibiting a cancer in a subject comprising (a) introducing, providing, or administering to a subject a composition that comprises a lipid, which comprises a target moiety that is bound to a masking moiety; (b) introducing, providing, or administering to said subject a cell comprising a chimeric antigen receptor (CAR) or T cell receptor (TCR), which is specific for the target moiety once the masking moiety is removed from the target moiety, wherein the CAR or TCR comprises a spacer domain having a spacer length of 1-22 amino acids, 23-50 amino acids, 51-100 amino acids, 100 to 150 amino acids or 151-250 amino acids, wherein: the CAR or TCR comprises a sequence selected from SEQ ID NO :01-06; and/or the spacer domain comprises, consists essentially of, or consists of: an IgG4 hinge connected to a CH2 domain to a CH3 domain,
• the cell is provided to the subject at the same time or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 24, 36 or 48 hours before or after administration of the composition, or any time within a range defined by any two aforementioned values. In some embodiments, the cell is provided to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 24, 36 or 48 hours before administration of the composition, or any time within a range defined by any two aforementioned values. In some embodiments, the cell is provided to the subject within seconds or minutes, such as less than an hour, of providing the composition to the subject.
• a boost of the cell and/or the composition is provided to the subject.
• an additional cancer therapy is provided to said subject, such as a small molecule, e.g., a chemical compound, an antibody therapy, e.g., a humanized monoclonal antibody with or without conjugation to a radionuclide, toxin, or drug, surgery, and/or radiation.
• a small molecule e.g., a chemical compound
• an antibody therapy e.g., a humanized monoclonal antibody with or without conjugation to a radionuclide, toxin, or drug, surgery, and/or radiation.
• the cancer is a solid tumor.
• the cancer is a colon cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, renal cancer, pancreatic cancer, brain cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, bone cancer, bladder cancer, head and neck cancer, or liver cancer.
• the cancer is a non-solid tumor, such as a leukemia or multiple myeloma. Examples of solid tumors include sarcomas, carcinomas, and lymphomas.
• cancers such as solid and non-solid tumors are listed in Amin, M.B., et al.,. (Eds.). AJCC Cancer Staging Manual (8th edition). Springer International Publishing: American Joint Commission on Cancer; 2017 which is incorporated herein by reference in its entirety.
• binding of the target moiety to the CAR present on the cell induces production of at least one cytokine.
• the at least one cytokine comprises IL-2, TNF-a and/or INF-a.
• the lipid comprises a polar head group and a hydrophobic group.
• the polar head comprises a choline, a phosphatidylcholine, sphingomyelin, phosphoethanolamine group, an oligosaccharide residue, a sugar residue, phosphatidyl serine or phosphatidyl inositol.
• the polar-head group comprises phosphocholine, a piperidine moiety or a trimethylarseno-ethyl-phosphate moiety.
• the hydrophobic group is fatty acid, such as an aliphatic chain. In some embodiments, the fatty acid is saturated or unsaturated.
• the hydrophobic group comprises an alkyl, alkenyl or alkynyl group. In some embodiments, the hydrophobic group comprises a terpenoid lipid, such as a steroid or cholesterol. In some embodiments, the hydrophobic group comprises an ether linkage, wherein the ether linkage is between the polar head group and the aliphatic chain. In some embodiments, the sugar residue is a glycerol or a sugar alcohol. In some embodiments, the hydrophobic group comprises a carbon alkyl chain, wherein the carbon alkyl chain comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbons or any number that is within a range defined by any two aforementioned values. In some embodiments, the carbon alkyl chain comprises 18 carbons. In some embodiments, the lipid is a phospholipid ether.
• the target moiety is biotin, digoxigenin, dinitrophenol, or fluorescein, or a derivative thereof. In some embodiments, the target moiety is fluorescein, or a derivative thereof.
• the spacer comprises a polyethylene glycol (PEG) spacer, a Hapten (2x) spacer, or an alkane chain.
• PEG spacer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 PEG molecules, or any amount of PEG molecules that is within a range defined by any two aforementioned values.
• the cell is a precursor T cell.
• the precursor T cell is a hematopoietic stem cell.
• naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells are selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells.
• kits can include a pharmaceutical grade lipid conjugated with a targeting moiety.
• the lipid comprises a polar head group and a hydrophobic group.
• the polar head comprises a choline, a phosphatidylcholine, sphingomyelin, phosphoethanolamine group, an oligosaccharide residue, a sugar residue, phosphatidyl serine or phosphatidyl inositol.
• the polar- head group comprises phosphocholine, a piperidine moiety or a trimethylarseno-ethyl- phosphate moiety.
• the hydrophobic group is fatty acid, such as an aliphatic chain. In some embodiments, the fatty acid is saturated or unsaturated.
• the hydrophobic group comprises an alkyl, alkenyl or alkynyl group. In some embodiments, the hydrophobic group comprises a terpenoid lipid, such as a steroid or cholesterol. In some embodiments, the hydrophobic group comprises an ether linkage, wherein the ether linkage is between the polar head group and the aliphatic chain. In some embodiments, the sugar residue is a glycerol or a sugar alcohol. In some embodiments, the hydrophobic group comprises a carbon alkyl chain, wherein the carbon alkyl chain comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbons or any number that is within a range defined by any two aforementioned values. In some embodiments, the carbon alkyl chain comprises 18 carbons. In some embodiments, the lipid is a phospholipid ether.
• the target moiety is biotin, digoxigenin, dinitrophenol, or fluorescein, or a derivative thereof. In some embodiments, the target moiety is fluorescein, or a derivative thereof.
• the spacer comprises a polyethylene glycol (PEG) spacer, a Hapten (2x) spacer, or an alkane chain.
• PEG spacer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 PEG molecules, or any amount of PEG molecules that is within a range defined by any two aforementioned values.
• T cells were isolated from leukocyte reduction“cones” that are normally disposed of after plateletpheresis. Specifically, Ficoll density centrifugation was used to generate the T cell-containing peripheral blood mononuclear cell (PBMC) fraction and CD8+ and CD4+ T cells were sequentially purified using the appropriate magnetic enrichment kits. T cells were either immediately placed into CAR production the same day as isolation. T cells, ranging from 0.5-5 million cells, were stimulated with anti-human CD3/CD28 activator beads at a 1 : 1 ratio on Day 0. In some alternatives, the concentrations of the cells can range between 0.5, 1, 2, 3, 4 or 5 million cells that are produced for use with the anti-human CD3/CD28 activator beads.
• PBMC peripheral blood mononuclear cell
• T cells were transduced with CAR-containing lentivirus at a multiplicity of infection (MOI) ranging from 1 to 6 in the presence of protamine sulfate via spinoculation.
• MOI multiplicity of infection
• Half or full-media exchanges were conducted every 2-3 days to maintain the cell cultures at appropriate cell densities and expanded to larger culture vessels as needed. In general, cells were moved to larger culture vessels when cell concentrations reach 1.5-2 million cells/mL or when cultures appear visibly dense and media is yellow.
• CD4+ T cells were reconstituted with fresh rhIL-7 and rhlL- 15 at a final concentration of 50 ng/mL and 0.5 ng/mL, respectively, and CD8+ T cells were reconstituted with fresh rhIL-2 and rhU.- l 5 at a final concentration of 50 U/mL and 0.5 ng/mL, respectively.
• Activator beads were magnetically removed on Days 9-11 of stimulation.
• CAR T cells that did not contain DHFRdm for methotrexate-mediated selection of CAR expression such as those used in examples depicted in FIG. 8B, FIG. 15 A, FIG. 15C, FIG. 15D, FIG. 15D, FIG. 15E, FIG. 18C, FIG. l9b, FIG. 19C, FIG. 21A and FIG. 21B
• cells were magnetically sorted based on reporter EGFRt expression using biotinylated antibody and anti -biotin microbeads during Days 10-21 of culture.
• CAR T cells that did contain DHFRdm for methotrexate-mediated enrichment of CAR expression such as those used in examples depicted in FIG. 9, FIG. 10, FIG. 12, FIG.
• cells were first treated on Days 7-14 of culture with 50 nM methotrexate, then ramped up to 100 nM methotrexate on Days 14-19 of culture, and finally brought back down to 50 nM for Days 19- 21 of culture. To prevent cultures from crashing due to poor cell viability from methotrexate selection, cells were separated on Ficol on Day 12 of culture to remove dead cells and improve culture viability.
• T cells for each CAR were placed into a rapid expansion protocol (REP) with irradiated feeder PBMCs and TM-LCL cells. If fresh, unfrozen PBMCs were used, 25 million PBMCs and 5 million TM-LCL cells were used in each REP culture. If frozen PBMCs were used, these feeder cell numbers were doubled. PBMCs and TM-LCL cells were irradiated at 3500 and 8000 rads, respectively, using a Cesium source irradiator. In addition to the normal cytokines mentioned above, cells were also supplemented with OKT3 antibody at 30 ng/pL for Days 0-5 of REP to provide acute TCR stimulation.
• OKT3 antibody OKT3 antibody at 30 ng/pL for Days 0-5 of REP to provide acute TCR stimulation.
• Irradiated feeder cells disintegrated by Day 5 of REP, and CAR T cells were maintained similarly to as described herein.
• CAR T cells that did contain DHFRdm for methotrexate-mediated enrichment of CAR expression such as those used in examples depicted in FIG. 9, FIG. 10, FIG. 12, FIG. 13, FIG. 14 A, and FIG. 14B
• 100 nM methotrexate was not introduced until Day 5 of REP and was maintained until Day 12 of REP to further enrich CAR-positive cells.
• all CAR T cells described herein were introduced into functional chromium release cytotoxicity assays (CD8+ T cells only) as well as functional 3-plex cytokine release assays (CD4+ and CD8+ T cells).
• Target cells were incubated with 51 Cr overnight.
• the CTCT-PLE was also present in the media overnight with the 51 Cr.
• the following day the target cells were washed and seeded in a 96 well plate at a concentration 5000 cell per well.
• CD8+ antiFL and mock T cell effectors (usually in day 8-16 of a rapid expansion protocol) were washed, seeded with the target cells in triplicate at various E:T ratios (30: 1, 10: 1, 3 : 1, 1 : 1), and allowed to co-incubate for 4 hours at 37°C.
• each target cell line was seeded with media only and for maximum 51 Cr release each target cell line was seeded and lysed with 2% SDS. Control groups were done in sextuplicate. After co-incubation, the supernatant was harvested, dispensed on LUMA plates, and allowed to dry overnight. The next day samples were run on the Top Count instrument. Percent-specific lysis was calculated by the following formula: (experimental 51 Cr release) - (control 51 Cr release)
• the CTCT-PLE was incubated overnight in media. The next day all target cells were harvested, washed, and seeded in a 96 well plate at a concentration of 5xl0 4 cells per well. CD8+ antiFL and mock T cell effectors (usually in day 8-16 of a rapid expansion protocol) were washed and seeded (lxlO 5 cells/well) with the target cells allowed to co-incubate for 24 hours at 37°C. After 24hr the supernatant was harvested and IFN-gamma, TNF-alpha, and IL-2 concentration in the supernatant were measured by using a Bio-Plex ® 200 system (Bio-Rad).
• FIG. 6 depicts cell positivity of CD8+ antiFL CARs T cell via staining for the selection marker EGFRt.
• a mouse CD19-FITC antibody was incubated with the T cells and washed out.
• FIG. 7A, FIG. 7B, and FIG. 7C show CAR T cell recognition and activation through FL-PLE in vitro.
• K562 leukemia
• FIG. 7A shows CAR T cell recognition and activation through FL-PLE in vitro.
• K562 leukemia
• FIG. 7B shows CAR T cell recognition and activation through FL-PLE in vitro.
• K562 leukemia
• FIG. 7A shows CAR T cell recognition and activation through FL-PLE in vitro.
• K562 leukemia
• the K562 OKT3+ cells (a cell line created to test the endogenous activation of T cells through the TCR) matched the K562 parental exactly. These cells were used in a chromium release assay (FIG. 7B) and a cytokine release assay (FIG. 7C) to test the activation of CD8+ antiFL(FITC-E2) CAR T cells compared with a CD8+ mock T cells from FIG. 6. From these experiments, the antiFL (FITC-E2) CAR T cells recognized the FL moiety of the FL-PLE integrated into the plasma membrane and that the cells were activated. The amount of the activation was associated with the amount FL exposed on the surface of the cell.
• FIG. 8A and FIG. 8B depict the universality of FL-PLE loading in tumor cells and recognition of the fluorescein moiety by antiFL CAR T cells.
• K562 leukemia
• U87 glioblastoma
• 251T glioma
• Be2 neuroblastoma
• MDA-MB-231 adenocarcinoma
• NIH OVCAR adenocarcinoma
• 143B osteosarcoma
• CAR T cells were selected for with methotrexate.
• FIG. 9 shows CD8+ antiFL CAR T cells selected by methotrexate, a chemotherapeutic in which antiFL CARs include CARs with FITC-E2 scFv domain and either a long, medium or short spacer, and other CARs having an antiFL scFv domain as labeled and a long spacer. CAR positivity of CD8+ antiFL CAR T cells staining for the surface marker EGFRt.
• antiFL CARs also harbored a gene for a double mutant dihydrofolate reductase (DHFRdm) that confered methotrexate resistance for methotrexate-mediated enrichment of CAR-positive cells. Every cell line except minimum EGFRt+ cells were diluted with mock T cells to make equivalent stocks to use in functional assays. Target: 18.1% EGFRt+. Actual: about 13-20% EGFRt+.
• DHFRdm dihydrofolate reductase
• FIG. 10 shows another example in which CD4+ antiFL CAR T cells selected by methotrexate, a chemotherapeutic. CAR positivity of CD4+ antiFL CAR T cells via staining for the surface marker EGFRt.
• These antiFL CARs also harbor a gene for a double mutant dihydrofolate reductase (DHFRdm) that confers methotrexate resistance and, thus allows for methotrexate-mediated enrichment of CAR-positive cells. Every cell line except minimum EGFRt+ cells were diluted with mock T cells to make equivalent stocks to use in functional assays. Target: 18.1% EGFRt+. Actual: about 15-24% EGFRt+.
• DHFRdm dihydrofolate reductase
• Target cells were labelled with FL-PLE.
• FIG. 11 shows loading of cells with FL-PLE.
• Target cells for functional assays were made using K562 cells and MDA-MB-231 cells. Each cell group was labeled with FL-PLE.
• Cell integration of FL-PLE to the plasma membrane was analyzed by flow cytometry.
• FIG. 12 shows CD8+ antiFL CAR T cell cytotoxicity assays.
• MDA-MB-231 cells were incubated with FL-PLE. Cell integration of FL-PLE was analyzed by flow cytometry. These cells and the CAR T cells were used in a chromium release assay.
• the negative controls K562 Parental and MDA-MB-231) both showed no killing and the positive control (K562 OKT3+ cells) showed cell lysis as expected.
• the long CAR barely outperformed the medium; whereas only slight killing was seen with the short CAR.
• FIG. 14A shows CD8+ antiFL CAR T cell assays with different antiFL scFv.
• MDA-MB-231 cells were incubated with FL-PLE. Cell integration of FL-PLE was analyzed by flow cytometry. These cells and the CAR T cells were used in a chromium release assay.
• the negative controls K562 Parental and MDA-MB-231) both showed no killing and the positive control (K562 OKT3+ cells) showed cell lysis as expected (FIG. 14A).
• This chromium assay tested four different antiFL scFv’s in the context of a long CAR. For MDA- MB-23 !
• antiFL(4D5Flu) which comprised SEQ ID NO: 4 exhibited almost no killing.
• AntiFL (4D5Flu) in other experiments has shown the ability to lysis cells labeled with FL. Accordingly, antiFL(4D5Flu) was not orientated in the correct relationship to recognize the FL moiety on the FL-PLE when integrated into a cell membrane.
• FIG. 13 depicts CD4+ antiFL CAR T cell cytokine release assays.
• K562 and MDA-MB-231 cells were incubated with FL-PLE. Cell integration of FL-PLE was analyzed by flow cytometry. These cells and the antiFL CAR T cells were used in a cytokine release assay.
• the negative controls K562 Parental and MDA-MB-231) both showed no cytokine production and the positive control (K562 OKT3+ cells) showed production of all three cytokines for all cell lines.
• This assay was designed to study the relationship of the spacer length of CAR and FL-PLE. This data shows that only the long spacer CAR in relationship with FL-PLE was able to produce all three cytokines.
• FIG. 14B depicts CD4+ antiFL CAR T cell cytokine release assays with different antiFL scFv.
• K562 and MDA-MB-231 cells were incubated with FL-PLE. Cell integration of FL-PLE was analyzed by flow cytometry. These cells and the CAR T cells were used in a cytokine release assay.
• the negative controls K562 Parental and MDA-MB-231) both showed no cytokine production and the positive control (K562 OKT3+ cells) showed production of all three cytokines for all cell lines.
• This cytokine release assay tested five different antiFL scFv’s in the context of a long CAR.
• antiFL(4M5.3) which comprised SEQ ID NO: 2
• antiFL(FITC- E2 Mut2) which comprised SEQ ID NO: 1
• antiFL(4M5.3) and antiFL(FITC-E2 Mut2) produced the most cytokine with MDA-MB-231 cells labeled with 5 mM FL-PLE.
• AntiFL(4M5.3) and antiFL(FITC-E2 Mut2) showed the best activation with FL-PLE and had vastly different disassociation constants, 270 fm and 3.1 nM respectively, showing that just lowering a CAR’s disassociation constant did not necessarily make for the best CAR.
• the CAR that did not work in conjunction with FL-PLE was antiFL(4D5Flu), which comprised SEQ ID NO: 4.
• AntiFL(4D5Flu) exhibited no cytokine production in either cell line labeled with 5 mM FL- PLE.
• AntiFL(4D5Flu) in other experiments has shown the ability to lysis cells labeled with FL. Accordingly, antiFL(4D5Flu) was not orientated in the correct relationship to recognize the FL moiety on FL-PLE when integrated into a cell membrane.
• FIG. 15A shows cell pure population of CD4+ and CD8+ antiFL CARs T cell via staining for the selection marker EGFRt.
• K562 leukemia
• MDA- MB-231 adenocarcinoma
• FIG. 16 shows in vivo targeting and integration of FL-PLE with FL moiety available for binding.
• FIG. 16 at top left shows 10 X fluorescent image, tumor was very bright compared to the normal healthy tissue, labeled as N. This showed the selective integration of FL-PLE into tumor membranes with FL moiety available for binding. Bottom left: bright signals were be seen when looking at the contralateral image of the brain. Right: To quantify this the MFI of the tumor and the contralateral side were individually calculated. This analysis was then repeated at multiple time points. These values were then plotted, generating a multiday retention time plot for FL-PLE upon this dose of FL-PLE.
• Results depicted in FIG. 17 demonstrate the universality of in vivo targeting and integration of FL-PLE.
• adenocarcinoma MDA-MB-231
• osteosarcoma 143B
• glioblastoma U87
• the adenocarcinoma and osteosarcoma each received two flank tumors in their respective groups and the glioblastoma only received one flank tumor.
• the fourth group had an adenocarcinoma tumor on one flank and an osteosarcoma on the opposite flank. After tumors were established in the groups of mice by subcutaneous injection, the mice received an intravenous injection of FL-PLE.
• FIG. 18A and FIG. 18B show ProFL-PLE ability to block CAR T cell recognition until unmasked.
• K562 (leukemia) cells were incubated with FL-PLE (high and low doses) or ProFL-PLE overnight.
• Cell integration of FL-PLE and ProFL- PLE was analyzed by flow cytometry (FIG. 18 A). There was a clear shift from the control K562 parental with the K562 parental incubated with high dose FL-PLE whereas there was a smaller shift with K562 parental incubated with low dose FL-PLE.
• FIG. 18B shows cells used in a chromium release assay to test the activation of CD8+ antiFL CAR T cells compared with a CD8+ mock T cells.
• antiFL CAR T cells recognized the FL moiety of the FL-PLE integrated into the plasma membrane.
• the ProFL-PLE completely blocked the recognition of the antiFL CARs; whereas once the Pro protection was unmasked the FL moiety was available for recognition of antiFL CARs.
• the lysis of the unmasked ProFL-PLE was about the same as the Low Dose FL-PLE, which correlated with the amount of FL exposed on the surface of the k562 cells.
• FIG. 18A and 18C shows cells used in a cytokine release assay to demonstrate the activation of CD4+ antiFL CAR T cells compared with a CD4+ mock T cells.
• Cell integration of FL-PLE and ProFL-PLE was analyzed by flow cytometry (FIG. 18 A).
• negative controls K562 Parental
• K562 OKT3+ cells showed production of all three cytokines for all cell lines.
• FL-PLE labeled cells were able to generate all three cytokines and was dependent on the amount of FL exposed on the surface.
• ProFL-PLE cells labeled with the pro moiety still intact produced no cytokine meaning the antiFL CAR T cells were not activated. After unmasking, ProFL-PLE labeled cells produced all 3 cytokines showing that upon removal of the Pro moiety the antiFL CAR T cell activated through ProFL-PLE integrated into the surface of a cancer cell.
• labelling tumor cells with a masked hapten conjugated with a lipid, such as ProFL-PLE unmasking the hapten to obtain a unmasked hapten conjugated to a lipid integrated into a tumor cell membrane, and contacting the unmasked hapten with an anti hapten CAR T cell activates the T cell in at least an in vitro environment.
• FIG. 19 A, FIG. 19B, and FIG. 19C depict ProFL-PLE blocking CAR T cell recognition until unmasked, after unmasking the antiFL CAR T cell recognized the newly exposed fluorescein on the surface was then activated.
• K562 (leukemia) cells were incubated with FL-PLE or ProFL-PLE overnight.
• Cell integration of FL-PLE and ProFL-PLE was analyzed by flow cytometry (FIG. 19A). There was a clear shift from the control K562 parental with the K562 parental incubated with FL-PLE corresponding to the amount of FL exposed on the surface of the cell for CAR T cell recognition.
• ProFL-PLE was not fluorescent due to the presence of the masking agent, a phenolic hydroxy group.
• FIG. 19B shows cells used in a cytokine release assay to continue to prove the activation of CD4+ and CD8+ antiFL CAR T cells compared with a CD4+ and CD8+ mock T cells.
• the negative controls showed no cytokine production and the positive control (K562 OKT3+ cells) showed production of all three cytokines for all cell lines.
• FL-PLE labeled cells were able to generate all three cytokines.
• ProFL-PLE cells labeled with the pro moiety still intact produced no cytokine meaning the antiFL CAR T cells were not activating as designed.
• After unmasking ProFL-PLE labeled cells produced all 3 cytokines showing that upon removal of the Pro moiety the antiFL CAR T cell activated through the unmasked ProFL-PLE integrated into the surface of the cancer cell. This shows the design of the ProFL-PLE works in an in vitro environment.
• 19C depicts results of staining cells from the co-culture of the cytokine release assay to investigate the up regulation of activation markers (LAG3, 41BB, and PD-l) on the CAR T cells after 24hrs.
• the live CD8+ antiFL(FITC-E2) long spacer CAR T cells are shown.
• the negative control co- culture with k562 Parental showed no up regulation of activation markers and the positive control (K562 OKT3+ cells) showed up regulation of all three activation markers.
• the K562 cells with ProFL-PLE showed only slightly elevated amounts of the activation markers meaning the intact Pro moiety was blocking antiFL CAR T cell recognition and activation.
• K562 cells with FL-PLE or unmasked ProFL-PLE had similar levels of activation as the positive control showing that the antiFL CAR T cells were activating similarly through FL- PLE and unmasked ProFL-PLE.
• Example 12 In vivo activity of antiFL CAR T cells
• FIG. 20 shows initial FL-PLE and ProFL- PLE in vivo therapy.
• a neuroblastoma (Be2) tumor was established in 3 groups of mice by intracranial injection, the mice received an intracranial injection of T cells comprising an antiFL(FITCE2) long spacer CAR, which comprised SEQ ID NO: 5.
• the control group only received the antiFL(FITCE2) long spacer CAR T cells and the tumor progressed as normal (black bar).
• the second group receive a single intravenous injection of FL-PLE prior to T cell injection. This group lived -20% longer than the control group.
• the third group received 3 scheduled doses of ProFL-PLE via intravenous injection (one before T cell injection and two post T cell injection), which led to -20% longer life span for the mice. These results additionally confirmed that ProFL-PLE was safe for re-dosing.
• Example 13 In vivo activity of antiFL CAR T cells with masked FL-PLE
• FIG. 21A and FIG. 21B show ProFL-PLE in vivo therapy for a flank tumor model.
• adenocarcinoma MDA- MB-231 eGFPTfLuc IL2+
• tumors (2 tumors per mouse) were established in three groups of mice by subcutaneous injection, two groups of mice received injection of ProFL-PLE either intratumorally (FIG. 21 A) or intravenously (IV) (FIG. 21B) and the control group received no injection of drug. Following the first injection of drug all three groups received IV injection of cells containing the antiFL(FITC-E2) long spacer CAR.
• the grey vertical dotted lines on the graphs represent the days of injection.
• the control group died after 16 days from tumor burden.
• FIG. 21 A The ProFL-PLE intratum orally injected group received 12 doses of ProFL-PLE over 45 days and the tumor regressed to baseline levels by day 40 for all 3 mice. The mice lived tumor free till the end of the study on day 90.
• FIG. 21B The ProFL-PLE IV injected group received 10 doses of ProFL-PLE over 34 days and the tumor regressed to baseline levels by day ⁇ 40 for 2 mice. These mice lived tumor free till the end of the study on day 90. One mouse from this group died on day 16 with the control mice.
• BALB/c mice comparative characterization of several monoclonal antiFLuorescyl antibodies. Mol Immunol, 18(10), 889-898.

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