US20190048085A1 - Modified cells for immunotherapy - Google Patents

Modified cells for immunotherapy Download PDF

Info

Publication number
US20190048085A1
US20190048085A1 US16/079,202 US201716079202A US2019048085A1 US 20190048085 A1 US20190048085 A1 US 20190048085A1 US 201716079202 A US201716079202 A US 201716079202A US 2019048085 A1 US2019048085 A1 US 2019048085A1
Authority
US
United States
Prior art keywords
cell
antibody
immune cell
engineered immune
cancer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US16/079,202
Other languages
English (en)
Inventor
Gianpietro Dotti
Elisa Landoni
Abdijapar Shamshiev
Titus Kretzschmar
Miriam Droste
Douglas Phillips
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of North Carolina at Chapel Hill
Kuur Therapeutics Inc
Original Assignee
Cell Medica Switzerland AG
University of North Carolina at Chapel Hill
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cell Medica Switzerland AG, University of North Carolina at Chapel Hill filed Critical Cell Medica Switzerland AG
Publication of US20190048085A1 publication Critical patent/US20190048085A1/en
Assigned to CELL MEDICA SWITZERLAND AG reassignment CELL MEDICA SWITZERLAND AG ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KRETZSCHMAR, TITUS, SHANSHIEV, ABDIJAPAR, DROSTE, Miriam, PHILLIPS, DOUGLAS
Assigned to THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL reassignment THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DOTTI, GIANPIETRO, LANDONI, Elisa
Assigned to CELL MEDICA INC. reassignment CELL MEDICA INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CELL MEDICA SWITZERLAND AG
Assigned to OAKTREE FUND ADMINISTRATION, LLC reassignment OAKTREE FUND ADMINISTRATION, LLC SECURITY INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CELL MEDICA, INC.
Assigned to CELL MEDICA, INC. reassignment CELL MEDICA, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CELL MEDICA INC.
Assigned to CELL MEDICA, INC. reassignment CELL MEDICA, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CELL MEDICA INC.
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • 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
    • A—HUMAN NECESSITIES
    • 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
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • 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
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • 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
    • 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
    • A61K2239/55—Lung
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/20—Vector systems having a special element relevant for transcription transcription of more than one cistron
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70532—B7 molecules, e.g. CD80, CD86

Definitions

  • the present invention relates generally to engineered cells expressing an antigen receptor and an antibody blocking PD-L1 as well as methods of using the same for the treatment cancer and other disorders.
  • Immunotherapy with antigen-specific immune cells offers a promising approach for the treatment of different types of diseases, e.g., cancers.
  • One therapeutic strategy is by engineering immune cells to express chimeric antigen receptors (CARs) which specifically target tumor cells and typically comprise an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain derived from, for example, the T-cell receptor CD3-zeta chain.
  • CARs chimeric antigen receptors
  • the signaling domain may be linked to one or more costimulatory molecule endodomains.
  • TCRs antigen-specific T cell receptors
  • Cell-based immunotherapy has a major advantage over the currently available immunotherapies based on monoclonal antibodies, due to the potential of such genetically modified immune cells to traffic to sites of disease, to expand and to persist even after a single dose of administration.
  • Cancer cells utilize numerous pathways to escape the immune system.
  • PD-L 1 is often expressed on tumor cells and protects tumor cells from T cell-mediated destruction by binding PD-1. Up-regulated levels of PD-L 1 correlate with increased tumor aggressiveness and an increased risk of death.
  • a therapeutic approach using antigen-specific immune cells in combination with anti-PD-L1 antibodies is promising. Particularly attractive is the generation of immune cells expressing both, an antigen receptor such as a TCR and/or a CAR and an antibody against PD-L1, as secretion of the antibody at the site of action of the immune cell would protect it from inactivation. Moreover, such localized antibody delivery treatment approach has advantages over a systemic approach as to amount of drug to be administered as well as potential side-effects.
  • WO2014134165 describes the co-expression of a chimeric antigen receptor (CAR) and an anti-mouse PD-1 scFv in mouse T cells.
  • CAR chimeric antigen receptor
  • the scFv was derived from the Armenian hamster anti-PD-1 antibody clone J43 as described in U.S. Pat. No. 7,858,746.
  • the scFv construct was cloned into a retroviral backbone expressing a CAR targeting human CD19 or human MUC-CD.
  • Primary murine T cells were transduced with the respective constructs.
  • the invention provides for an engineered cell expressing:
  • Such antibody may be a humanized antibody or a fully human antibody.
  • the antibody may comprise at least one CDR sequence as set forth in SEQ ID NOs.: 3, 4, 5, 6, 7 and 8 or variants thereof.
  • Said antibody preferably binds to an epitope on PD-L1 such that PD-L1 interaction with both CD80 and PD-1 is inhibited.
  • PD-L1 binds to CD80, a membrane receptor expressed on T cells and B cells, although the binding affinity of PD-L1 for CD80 is much lower than for PD-1.
  • PD-L1 binding to either PD-1 or CD80 transmits inhibitory signals to T-lymphocytes, suppressing T-cell migration, proliferation and secretion of cytotoxic mediators, and reducing tumor cell killing.
  • PD-1/PD-L1 interaction drives T cell exhaustion
  • PD-L1/CD80 interaction drives T cell anergy.
  • the cell is preferably a therapeutic cell.
  • Suitable cells may e.g. be a T cell, a Natural Killer T (NKT) cell, a natural killer (NK) cell, a human embryonic stem cell, or a hematopoietic stem cell (HSC) or induced pluripotent stem cells (iPS).
  • T cell a Natural Killer T (NKT) cell
  • NK natural killer
  • HSC hematopoietic stem cell
  • iPS induced pluripotent stem cells
  • engineered immune cells expressing an antigen receptor and an antibody.
  • the cell is T cell.
  • T cell may be a cytotoxic T lymphocyte (CTL), a regulatory T lymphocyte, an inflammatory T- lymphocytes, a helper T-lymphocyte, or a gamma-delta T cell.
  • CTL cytotoxic T lymphocyte
  • regulatory T lymphocyte a regulatory T lymphocyte
  • inflammatory T- lymphocytes a helper T-lymphocyte
  • helper T-lymphocyte a gamma-delta T cell.
  • the cell is a NKT cell.
  • the cell is of human in origin. In one embodiment, the cell is autologous; in one embodiment, the cell is allogenic.
  • the antigen receptor is specific to a cancer antigen, for example a antigen which is only expressed on a cancer cell or is upregulated on a cancer cell.
  • the antigen receptor is T cell receptor, for example a native T cell receptor (for example stimulated to be specific to antigen or selected for its specificity to antigen) or an engineered T cell receptor.
  • the antigen receptor is a chimeric antigen receptor (CAR).
  • the antigen receptor is membrane anchored.
  • an immune cell according to the present disclosure may comprise a TCR and a CAR, for example a native TCR and a CAR (in particular specific to cancer antigen).
  • the immune cell of the present disclosure (such as an NK cell) may comprise an engineered TCR and a CAR.
  • Exemplary engineered immune cells described herein show enhanced tumor-killing activity compared to engineered immune cells expressing the antigen receptor only.
  • immune cells encoding both an antigen receptor and an anti-PD-L1 antibody can be effective in killing cancer cells for longer periods of time than corresponding cells encoding only an antigen receptor.
  • cells according to the present disclosure are less susceptible to exhaustion, especially in the tumor microenvironment.
  • nucleic acid encoding the antigen receptor and the antibody as described herein, as well as vectors comprising such nucleic acid.
  • compositions comprising
  • engineered immune cells comprising:
  • the present invention provides substantial benefits over alternatives such as the combined use of an engineered immune cell and a purified checkpoint inhibitor antibody.
  • a checkpoint inhibitor antibody In attempting to use an engineered immune cell in combination with a checkpoint inhibitor antibody, the skilled practitioner will recognize that the timing of antibody delivery is a crucial factor. Furthermore, the local concentration of the antibody is also important.
  • the antibody offering protective function should be present when cell contact is established between the antigen receptor expressing cells (effector cells) and their target cells, e.g., at the tumor site. For the medical practitioner, it is practically impossible to determine the precise timing of systemic antibody administration in a traditional antibody formulation, for example by infusion, to provide the antibody at the requisite cancer location. Furthermore, full-length antibodies are generally not able to cross the blood brain barrier.
  • the therapy or method of treatment of the invention may be in combination with one or more therapies selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.
  • kits for treatment of cancer, pathogen infection, and/or an autoimmune disorder comprising the engineered immune cell or the expression vector as described herein, and written instructions for use.
  • FIG. 1 shows the design of the scFv anti-PD-L1 retroviral construct SFG.scFv.anti-PD-L1(I)eGFP having 8510 bp.
  • the Ncol restriction site at 5′ end, the Sphl restriction site at 3′ end and the TGA stop codon were added to the scFv anti-PD-L1 DNA by PCR.
  • PCR product was cloned into the retroviral vector SFG(I)eGFP to obtain the final vector SFG.scFv.anti-PD-L1(I)eGFP.
  • the reporter gene eGFP expressed upon IRES is used to assess the transduction efficiency.
  • LTR long terminal repeat
  • SD splicing donor
  • PS packaging signal
  • TGG truncated gagpol
  • SA splicing acceptor
  • SP signal peptide
  • VL variable light chain of the scFv
  • L linker
  • VH variable heavy chain of the scFv
  • IRES internal ribosomal entry site
  • eGFP enhanced green fluorescent protein.
  • FIG. 2 shows that CD4 and CD8 T cells could be transduced with the scFv anti-PD-L1 retroviral construct without alterations of the CD4/CD8 ratio.
  • Transduction efficiency of the SFG.scFv.anti-PD-L1(I)eGFP vector is shown as percentage of CD4 ( FIG. 2A ) or CD8 ( FIG. 2B ) T cells expressing eGFP.
  • Non Transduced T cells-PD-L1 T cells transduced only with SFG.scFv.anti-PD-L1(I)eGFP vector
  • CAR.28 PD-L1 T cells co-transduced with SFG.scFv.anti-PD-L1(I)eGFP vector and GD2.CAR encoding the CD28 endodomain
  • CAR.BB PD-L1 T cells co-transduced with SFG.scFv.anti-PD-L1(I)eGFP vector and GD2.CAR encoding the 4-IBB endodomain.
  • FIG. 3 shows that T cells co-expressed GD2.CAR and eGFP anti-PD-L1 upon double retroviral transduction.
  • FIG. 3A Transduction efficiency of the SFG.scFv.anti-PD-L1(I)eGFP vector. Shown is the percentage of eGFP positive T cells.
  • FIG. 3B Transduction efficiency of GD2.CAR encoding either the CD28 or the 4-1BB endodomains.
  • FIG. 3C Expression of GD2.CAR shown as RFI (Relative Fluorescence Intensity).
  • FIG. 3D Percentage of T cells co-transduced with the GD2.CAR and the scFv anti-PD-L1 retroviral vectors.
  • NT Non Transduced T cells
  • CAR.28 T cells transduced only with GD2.CAR encoding the CD28 endodomain
  • CAR.BB T cells transduced only with GD2.CAR encoding the 4-1BB endodomain
  • -PD-L1 T cells transduced only with SFG.scFv.anti-PD-L1(I)eGFP vector
  • CAR.28 PD-L1 T cells co-transduced with SFG.scFv.anti-PD-L1(I)eGFP vector and GD2.CAR encoding the CD28 endodomain
  • CAR.BB PD-L1 T cells co-transduced with SFG.scFv.anti-PD-L1(I)eGFP vector and GD2.CAR encoding the 4-1BB endodomain
  • CAR.BB PD-L1 T cells co-transduced with SFG.scFv.anti-PD-L1(I)eGFP vector and GD2.
  • T cells can be co-transduced with SFG.scFv.anti-PD-L1(I)eGFP and GD2.CAR vectors and that the transduction with SFG.scFv.anti-PD-L1(I)eGFP vector does not alter the expression level of GD2.CAR.
  • FIG. 4 shows that transduction with the scFv anti-PD-L1 did not affect T cell proliferation.
  • NT Non Transduced T cells
  • CAR.28 T cells transduced only with GD2.CAR encoding the CD28 endodomain
  • CAR.BB T cells transduced only with GD2.CAR encoding the 4-1BB endodomain
  • -PD-L1 T cells transduced only with SFG.scFv.anti-PD-L1(I)eGFP vector
  • CAR.28 PD-L1 T cells co-transduced with SFG.scFv.anti-PD-L1(I)eGFP vector and GD2.CAR encoding the CD28 endodomain
  • CAR.BB PD-L1 T cells co-transduced with SFG.scFv.anti-PD-L1(I)eGFP vector and GD2.CAR encoding the 4-1BB endodomain
  • CAR.BB PD-L1 T cells co-transduced with SFG.scFv.anti-PD-L1(I)eGFP vector and GD2.
  • FIG. 5 shows that expression of the anti-PD-L1 scFv did not affect T cell subset compositions.
  • FIG. 5A Scheme of T lymphocyte subpopulations determined by the expression of CD62L, CD45RA and CD95 [Na ⁇ ve (TN) CD62L+CD45RA+CD95-, Stem Cell Memory (TSCM) CD62L+CD45RA+CD95+, Central Memory (TCM) CD62L+CD45RA-, Effector Memory (TEM) CD62L-CD45RA-, Effector T cell (TEFF) CD62L-CD45RA+].
  • T cell subset compositions FIG. 5B
  • percentage of CD27 FIG. 5C
  • CD28 FIG. 5D
  • PD-1 FIG. 5E
  • FIG. 6 shows that the anti-PD-L1 scFv is released by transduced T cells.
  • FIG. 6A Quantification of anti-PD-L1 scFv released from Non-transduced (NT) and anti-PD-L1 scFv transduced T cells (scFv PD-L1) in T cell medium with 10% FBS. T cells were seeded and activated with immobilized anti-CD3/CD28 antibodies. The supernatant was collected after 18 hours and anti-PD-L1 scFv quantified by a specific sandwich ELISA.
  • FIG. 6B The anti-PD-L1 scFv secreted by transduced T cells binds PD-L.
  • NT non-transduced
  • scFv PD-L1 anti-PD-L1 transduced T cells
  • FIG. 7 shows that GD2.CAR T cells with the 4-1BB endodomain transduced with the anti-PD-L1 scFv show better killing of tumor cells in the second cycle of co-culture compared to GD2.CAR T cells with the 4-1BB endodomain that are not secreting the scFV anti-PD-L1.
  • FIG. 7A shows that in the first cycle (7 days) of antigen stimulation GD2.CAR-Ts encoding 4-1BB efficiently eliminated tumor cells and the presence of the secreted anti-PD-L1 scFv does not show any impairment of the GD2.CAR-T cytotoxic function.
  • GD2.CAR T cells with the 4-1BB endodomain transduced with the anti-PD-L1 scFv showed enhanced killing of tumor cells than the GD2.CAR T cells without the anti-PD-L1 scFv.
  • Representative plots of T cells at the end of the first (7 days of culture; FIG. 7B ) and second cycle of co-culture (14 days of culture; FIG. 7C ) T cells identifies as CD3+ and tumor cells CHLA-255 identified as GD2+ cells, respectively). E:T ratio 1:5.
  • CHLA tumor cells
  • T cells T cells alone with no tumor cells
  • Tc NT Non Transduced T cells
  • Tc PD-L1 T cells transduced only with SFG.scFv.anti-PD-L1(I)eGFP vector
  • Tc CAR-41BB T cells transduced only with GD2.CAR encoding the 4-1BB endodomain
  • Tc CAR-41BB PD-L1 T cells co-transduced with SFG.scFv.anti-PD-L1(I)eGFP vector and GD2.CAR encoding the 4-1BB endodomain.
  • FIG. 8 shows that in the first cycle (7 days) of antigen stimulation GD2.CAR-Ts encoding 4-1BB efficiently release IFNgamma and the presence of the secreted anti-PD-L1 scFv does not show any impairment of the GD2. CAR-T function (IFNgamma release).
  • GD2.CAR T cells with the 4-1BB endodomain transduced with the anti-PD-L1 scFv showed enhanced release of IFNgamma compared to GD2.CAR T cells without the anti-PD-L1 scFv IFNgamma ELISA assay to quantify the IFNgamma produced in the first 24 hours after the first (7 days of culture; FIG. 8A ) and second (14 days of culture; FIG. 8B ) tumor specific stimulation.
  • CAR.BB refers to “CAR.41BB” as well as “CAR.4-1BB”.
  • engineered immune cell refers to an immune cell which was genetically modified to express the proteins described herein.
  • antigen receptor refers to a receptor that is capable of activating an immune cell in response to antigen binding.
  • exemplary antigen receptors may be endogenous (i.e., native) or recombinant T cell receptors (TCRs) or chimeric antigen receptors (CARs).
  • TCRs are membrane-anchored heterodimeric proteins expressed on immune cells. Upon binding to antigenic molecules presented by antigen presenting cells, the immune cell is activated. Whereas some TCRs comprise variable alpha and beta chains, others comprise gamma and delta chains, the chains being expressed as part of a complex with the invariant CD3 chain molecules.
  • Each of the alpha and beta chain may comprise a variable region and a constant region, both being located extracellulary, wherein each variable domain has three complementarity determining regions (CDRs) which enable binding of the TCR to the peptide/MHC complex.
  • the variable region of the beta chain has an additional hypervariable region HV4 which typically does not contact antigen and is therefore not considered a CDR (see. e.g. Richman, S. A. et al, Mol Immunol. 2009; 46(5): 902-916).
  • CARs typically comprise an extracellular domain (ectodomain), a transmembrane domain and a cytoplasmic domain (endodomain).
  • the ectodomain provides antigen recognition and is most commonly a scFv but other antibody formats may also be used.
  • the scFv is connected via a spacer to the transmembrane domain, which is then connected to an endodomain.
  • First generation CARs had a simple structured endodomain comprising CD3-zeta. Upon antigen binding, receptors cluster and an activation signal is transmitted to the cell.
  • second generation CARs further include a co-stimulatory domain, such as CD28, OX40 and/or 4-1BB
  • third generation CARs include two or more co-stimulatory domains (Maus MV et al (2014) Blood, 123: 2625-2635).
  • CD3-zeta other ITAM-containing domains have been explored including the Fc receptor for IgE- ⁇ domain.
  • binding of the antigen to the antigen receptor activates the immune cell through induction of signal transduction or changes in protein expression in the immune cell which results in initiation of an immune response.
  • endogenous refers to a nucleic acid or a polypeptide that is normally expressed in a cell or tissue, absent recombinant engineering.
  • PD-L1 refers to the protein also known as “programmed cell death ligand 1,” “cluster of differentiation 274 (i.e., CD274)” or “B7 homolog 1 (i.e., B7-H1)”.
  • the native protein comprises two extracellular domains, a transmembrane domain, and a cytoplasmic domain.
  • the term encompasses full-length and/or unprocessed PD-L1 as well as any intermediate resulting from processing in the cell.
  • PD-L1 can exist as a transmembrane protein or as a soluble protein; thus, the term as used herein may refer to the full length or the extracellular domain of the protein.
  • the term also encompasses naturally occurring variants of PD-L1, e.g., splice variants or allelic variants.
  • the protein may additionally contain a tag, such as a his tag or Fc tag.
  • the amino acid sequence of exemplary human full-length PD-L1 protein can e.g. be found under NCBI protein database accession number NP_054862.
  • hPD-L1 refers to human PD-L1 and comprises natural hPD-L1 and recombinant human rhPD-L1.
  • rPD-L1 refers to recombinant PD-L1.
  • Recombinant PD-L1 may or may not have an amino terminal methionine residue, depending upon the method by which it is prepared.
  • rhPD-L1 refers to recombinant human PD-L1.
  • PD-L1 may also be obtained by isolation from biological samples of human or non-human origin rhPD-L1 may, e.g., be obtained from RnD Systems, USA, cat. no. 156-B7, or from Peprotech, USA, cat. no. 310-35.
  • Monkey PD-L1 refers to PD-L1 of Rhesus macacque (Macaca mulatta).
  • the amino acid sequence of exemplary monkey PD-L1 protein can e.g. be found under NCBI protein database accession number NP 001077358.
  • Monkey PD-L1 may, e.g., be obtained from Sino Biological, China, cat. no. 90251-CO2H.
  • Rat PD-L1 refers to PD-L1 of Rattus norvegicus (Norway rat).
  • the amino acid sequence of exemplary rat PD-L1 protein can e.g. be found under NCBI protein database accession number NP_001178883
  • Rat PD-L1 may, e.g., be obtained from Sino Biological, China, cat. no. 80450-RO2H.
  • “Mouse PD-L1” refers to PD-L1 of Mus musculus.
  • the amino acid sequence of exemplary mouse PD-L1 protein can e.g.
  • PD-1 is the programmed cell death protein 1, also known as CD279 is a cell surface receptor for PD-L1.
  • CD279 is a cell surface receptor for PD-L1.
  • PD-1 binds two ligands, PD-L1 and PD-L2.
  • PD-1 is a transmembrane protein including an extracellular domain followed by a transmembrane region and an intracellular domain. The term encompasses full-length and/or unprocessed PD-1 as well as any intermediate resulting from processing in the cell.
  • PD-1 can exist as a transmembrane protein or as a soluble protein; thus, the term as used herein may refer to the full length or the extracellular domain of the protein.
  • the term also encompasses naturally occurring variants of PD-1, e.g., splice variants or allelic variants.
  • the protein may additionally contain a tag, such as a his tag or Fc tag.
  • the amino acid sequence of exemplary human PD-1 protein can e.g. be found under NCBI protein database accession number NP_005009
  • the term “hPD-1” refers to human PD-1 and comprises its natural form (hPD-1) as well as the recombinant human form (rhPD-1).
  • rPD-1 refers to recombinant PD-1.
  • CD80 refers to the cluster of differentiation 80, also known as B7-1, B7.1, BB1, CD28LG, CD28LG1, LAB7. It is a membrane receptor for CD28 and CTLA-4 as well as PD-L1 and comprises extracellular domain followed by a transmembrane region and an intracellular domain. The term encompasses full-length and/or unprocessed CD80 as well as any intermediate resulting from processing in the cell. CD80 can exist as a transmembrane protein or as a soluble protein; thus, the term as used herein may refer to the full length or the extracellular domain of the protein. The term also encompasses naturally occurring variants of CD80, e.g., splice variants or allelic variants.
  • the protein may additionally contain a tag, such as a his tag or Fc tag.
  • a tag such as a his tag or Fc tag.
  • the amino acid sequence of exemplary human CD80 protein can e.g. be found under NCBI protein database accession number NP 005182, CD80 may, e.g., be obtained from RnD Systems, USA, cat. no. 9050-B1-100.
  • hCD80 refers to human CD80 and comprises its natural form (hCD80) as well as the recombinant human form (rhCD80).
  • rCD80 refers to recombinant CD80.
  • PD-L2 refers to the protein also known as “Programmed cell death 1 ligand 2”, “B7-DC”, or “CD273” (cluster of differentiation 273).
  • the term as used herein encompasses full-length and/or unprocessed PD-L2 as well as any intermediate resulting from processing in the cell.
  • PD-L2 can exist as a transmembrane protein or as a soluble protein; thus, the term as used herein may refer to the full length or the extracellular domain of the protein.
  • the term also encompasses naturally occurring variants of PD-L2, e.g., splice variants or allelic variants.
  • the protein may additionally contain a tag, such as a his tag or Fc tag.
  • exemplary human full-length PD-L2 protein can e.g. be found under NCBI protein database accession number NP 079515.
  • PD-L2 may, e.g., be obtained from RnD Systems, USA, cat. no. 1224-PL.
  • the term “rhPD-L2” refers to recombinant human PD-L2.
  • B7-H3 refers to the protein also known as CD276 (Cluster of Differentiation 276).
  • CD276 Cluster of Differentiation 276).
  • the term as used herein encompasses full-length and/or unprocessed B7-H3 as well as any intermediate resulting from processing in the cell.
  • B7-H3 can exist as a transmembrane protein or as a soluble protein; thus, the term as used herein may refer to the full length or the extracellular domain of the protein.
  • the term also encompasses naturally occurring variants of B7-H3, e.g., splice variants or allelic variants.
  • the protein may additionally contain a tag, such as a his tag or Fc tag.
  • the amino acid sequence of exemplary human full-length B7-H3 protein can e.g. be found under NCBI protein database accession number NP_079516.
  • B7-H3 may, e.g., be obtained from RnD Systems, USA, cat. no. 1027-B3.
  • the term “rhB7-H3” refers to recombinant human B7-H3.
  • T cell exhaustion as employed herein is a state of T cell dysfunction that arises during many chronic viral infections, autoimmunity and cancer. It is characterized by poor effector function, sustained expression of inhibitory receptors and a transcriptional state which is distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infectious conditions and tumors, i.e., in particular in chronic environment.
  • anti-tumor T cells are persistently exposed to antigen in the tumor microenvironment, they are particularly susceptible to exhaustion. Exhaustion is a likely mechanism contributing to T cell dysfunction in cancer patients. Accordingly, exhausted T cells have been reported for melanoma patients as well as patients with ovarian cancer and hepatocellular carcinoma. Exhausted T cells express multiple inhibitory receptors including PD-1 and LAG-3, and progressively lose cytotoxic and proliferative potential. Ultimately, they may be driven to apoptosis.
  • PD-1 programmed cell death protein 1
  • LAG-3 lymphocyte activation gene 3 protein
  • TIM-3 T-cell immunoglobulin domain and mucin domain protein 3
  • CTLA-4 cytotoxic T lymphocyte antigen-4
  • BTLA band T lymphocyte attenuator
  • TAGIT T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain
  • interferon-gamma IFN-gamma
  • GzmB granzyme B
  • T cell anergy typically refers to a hyporesponsive state which is induced by triggering the TCR either (i) without adequate concomitant co-stimulation through CD28 or (ii) in the presence of high co-inhibitory molecule signaling.
  • IL-2 is not effectively transcribed, but anergy-associated genes such as GRAIL are expressed instead which contribute to impaired TCR signaling via negative feedback.
  • isolated indicates that matter such as a peptide, a nucleic acid molecule or a cell has been removed from its normal physiological environment, e.g. a natural source, or that a peptide or nucleic acid is synthesized.
  • Use of the term “isolated” indicates that a naturally occurring sequence has been removed from its normal cellular (e.g., the chromosomal or cellular) environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment.
  • isolated in reference to a polypeptide or nucleic acid molecule means a polymer of amino acids (2 or more amino acids) or nucleotides coupled to each other, including a polypeptide or nucleic acid molecule that is isolated from a natural source or that is synthesized.
  • isolated does not imply that the sequence is the only amino acid chain or nucleotide chain present, but that it is essentially free of e.g. non-amino acid material and/or non-nucleic acid material, respectively, naturally associated with it.
  • An “isolated cell” refers to a cell that is separated from the molecular and/or cellular components that naturally accompany the cell.
  • a “variant” refers to an amino acid or nucleic acid sequence which differs from the parental sequence by virtue of addition (including insertions), deletion, modification and/or substitution of one or more amino acid residues or nucleobases while retaining at least one desired activity of the parent sequence disclosed herein. In the case of antibodies such desired activity may include specific antigen binding. Similarly, a variant nucleic acid sequence may be modified when compared to the parent sequence by virtue of addition, deletion and/or substitution of one or more nucleobases, but the encoded antibody retains the desired activity as described above. Variants may be naturally occurring, such as allelic or splice variants, or may be artificially constructed. The term “identity” as used herein refers to the sequence match between two proteins or nucleic acids.
  • the protein or nucleic acid sequences to be compared are aligned for maximum correspondence over a comparison window, for example using bioinformatics tools such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk) or by manual alignment and visual inspection.
  • EMBOSS Needle air wise alignment; available at www.ebi.ac.uk
  • the respective molecules are identical at that very position.
  • the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100%. For instance, if 6 out of 10 sequence positions are identical, then the identity is 60%. Aligning sequences for maximum correspondence may require introducing gaps.
  • the percent identity between two protein sequences can, e.g., be determined using the Needleman and Wunsch algorithm (Needlemann S. B. and Wunsch C. D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 1970, vol. 48, p.443) which has been incorporated into EMBOSS Needle, using a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5, or a method of aligning sequences manually introducing gaps in a manner which maximises identity can be used.
  • Two molecules having the same primary amino acid or nucleic acid sequence are identical irrespective of any chemical and/or biological modification.
  • two antibodies having the same primary amino acid sequence but different glycosylation patterns are identical by this definition.
  • nucleic acids for example, two molecules having the same sequence but different linkage components such as thiophosphate instead of phosphate are identical by this definition.
  • nucleobases that differ only because of exocyclic modifications for example cytosine and 5-methyl-cytosine, are identical by this definition.
  • a comparison window as used herein includes the entire sequence as claimed.
  • CDR refers to the hypervariable regions of the antibody which mainly contribute to antigen binding.
  • an antigen binding site typically includes six CDRs, embedded into a framework scaffold.
  • CDRs of the VL are referred to as CDR-L1, CDR-L2 and CDR-L3
  • CDR-H1 CDR-H2 and CDR-H3.
  • KABAT E. A., et al. Sequences of Proteins of Immunological Interest. 5th edition. Edited by U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES. NIH Publications, 1991. p. 91-3242.
  • CDR-H1 as used herein differs from the Kabat definition in that it starts with position 27 and ends prior to position 36 (AHo positions 28 to 42, inclusive).
  • the numbering system to identify amino acid residue positions in the VH and VL of the antibody corresponds to the “AHo”-system described by Honegger A. and Plückthun A. Yet another numbering scheme for immunoglobulin variable domains: An automatic modelling and analysis tool. J. Mol. Biol. 2001, vol. 309, p.657.
  • the publication further provides conversion tables between the AHo and the Kabat system (Kabat E. A. et al., Sequences of Proteins of Immunological Interest. 5th edition. Edited by U.S. Department of Health and Human Services. NIH Publications, 1991. No. 91-3242).
  • FR refers to the scaffold of the variable antibody domain, either the variable light chain (VL) or variable heavy chain (VH), embedding the respective CDRs.
  • VL and/or VH framework typically includes four framework sections, FR1, FR2, FR3 and FR4, flanking the CDR regions.
  • a VL has the general structure: (FR-L1)-(CDR-L1)-(FR-L2)-(CDR-L2)-(FR-L3)-(CDR-L3)-(FR-L4)
  • a VH has the general structure: (FR-H1)-(CDR-H1)-(FR-H2)-(CDR-H2)-(FR-H3)-(CDR-H3)-(FR-H4).
  • the invention provides an engineered immune cell expressing:
  • Such immune cell may e.g. be a T cell, a Natural Killer T (NKT) cell, a natural killer (NK) cell, a human embryonic stem cell, a hematopoietic stem cell (HSC) or a induced pluripotent stem cell (iPS).
  • T cell e.g. be a T cell, a Natural Killer T (NKT) cell, a natural killer (NK) cell, a human embryonic stem cell, a hematopoietic stem cell (HSC) or a induced pluripotent stem cell (iPS).
  • NKT Natural Killer T
  • NK natural killer
  • HSC hematopoietic stem cell
  • iPS induced pluripotent stem cell
  • Said T cell may be a cytotoxic T lymphocyte (CTL), a regulatory T lymphocyte, an inflammatory T-lymphocytes, or a helper T-lymphocyte or a gamma-delta T cell. Additionally or alternatively, said T cell is a CD4+ or CD8+ or a mixed population of CD4+ and CD8+ cells.
  • CTL cytotoxic T lymphocyte
  • regulatory T lymphocyte an inflammatory T-lymphocytes
  • helper T-lymphocyte or a gamma-delta T cell.
  • said T cell is a CD4+ or CD8+ or a mixed population of CD4+ and CD8+ cells.
  • the antigen receptor is a chimeric antigen receptor (CAR).
  • CARs comprise a cytoplasmic domain acting as intracellular signaling domain, a transmembrane domain and an extracellular domain serving antigen recognition.
  • the extracellular domain may be connected to a signal peptide, to direct the transport of the domain to the cell surface.
  • Said signal peptide may be cleavable.
  • a spacer or hinge region is present between the transmembrane domain and the extracellular domain.
  • Such hinge region may e.g. be selected from the group consisting of a CD8a hinge, an IgG1 hinge or a FcyRll hinge.
  • the CAR comprises a CD3 zeta, a CD4, a CD28, a CD8 alpha or a 4-1BB transmembrane domain.
  • the CAR may comprise one or more costimulatory domains, e.g., selected from the group consisting of CD28, 4-1BB (CD137), ICOS, or OX40 (CD134) costimulatory domains, or functional fragments thereof, respectively.
  • the CAR comprises 4-1BB costimulatory domain or a functional fragment thereof.
  • Exemplary sequences of CD28 and 4-1BB co-stimulatory domains are provided in SEQ ID NOs: 49 and 47, respectively.
  • the cytoplasmic domain comprises a CD3 zeta signaling domain.
  • An exemplary sequence of a CD3 zeta signaling domain is given in SEQ ID NO: 48.
  • the CAR architecture is as shown in FIG. 1 of Heczey A. et al, Blood. 2014 Oct. 30; 124(18):2824-33, incorporated herein by reference.
  • the CAR comprises an extracellular domain targeting GD2, such as the 14g2a scFv or an antibody comprising the 14g2a variable domains or an antibody comprising the CDRs of the 14g2a scFv, or variants thereof respectively, a CD3 zeta signaling domain and a CD28 costimulatory domain.
  • the CAR comprises an extracellular domain targeting GD2, such as the 14g2a scFv, an antibody comprising the 14g2a variable domains or an antibody comprising the CDRs of the 14g2a scFv, or variants thereof, respectively, a CD3 zeta signaling domain and a 4-1BB costimulatory domain.
  • the VH and VL sequences of the 14g2a scFv can e.g. be found in the PDB database under accession number 4TUO_A and 4TUO_B, respectively (see also SEQ ID NOs: 13 and 14).
  • variants of the 14g2a derived sequences in particular variants having framework mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations in the variable light and/or heavy chain.
  • an anti-GD2-CAR comprising at least one, preferably all of the CDR sequences as shown in SEQ ID NOs: 13 and 14, i.e. at least one, preferably all CDR sequences of SEQ ID NOs. 16 to 21.
  • Exemplary GD2-specific CAR constructs are described in Heczey A. et al, Blood. 2014 Oct. 30; 124(18):2824-33, in Pule, M. A. et al, Mol Ther, 12(5), November 2005, 933-941 (see FIG. 1 for the amino acid sequence of transmembrane and endodomains of different receptors), as well as in WO2012033885, all three incorporated herein by reference.
  • the CAR comprises an extracellular domain targeting CSPG4.
  • such CAR comprises at least one, preferably all CDRs of SEQ ID NOs.: 22 to 27.
  • such CAR comprises a VL sequence of SEQ ID NO.: 28 and/or a VH sequence of SEQ ID NO.: 29.
  • Exemplary CSPG4-specific CAR constructs are described in WO2015/080981, incorporated herein by reference.
  • the CAR comprises an extracellular domain targeting GPC3.
  • such CAR comprises at least one, preferably all CDRs of SEQ ID NOs.: 30 to 35.
  • such CAR comprises a VL sequence of SEQ ID NO.: 36 and/or a VH sequence selected from the group consisting of SEQ ID NO.: 37 and SEQ ID NO.: 38.
  • Exemplary GPC3-specific CAR constructs are described in WO2016/049459, incorporated herein by reference.
  • the CAR comprises an extracellular domain targeting 5T4.
  • such CAR comprises at least one, preferably all CDRs of SEQ ID NOs.: 39 to 44.
  • such CAR comprises a VL sequence of SEQ ID NO.: 45 and/or a VH sequence of SEQ ID NO.: 46.
  • Exemplary 5T4-specific CAR constructs are described in WO2016/034666, incorporated herein by reference.
  • the antigen receptor is a T cell receptor (TCR).
  • the TCR may be an endogenous (or native) TCR or an engineered TCR. The endogenous TCR may for example be selected for its specificity to antigen.
  • the engineered TCR is a native TCR the sequence of which is recombinantly expressed in the immune cell.
  • the engineered TCR is derived from a native TCR, but comprises point mutations.
  • the engineered TCR comprises a disulfide bond in the constant region, for example as disclosed in WO2006/000830, incorporated herein by reference. In particular the specific locations of the disulfide bonds in the constant regions are incorporated.
  • the engineered TCR is modified to increase the surface expression as described in WO2016/170320, incorporated herein by reference.
  • the TCR comprises at least one of the following amino acid residues:
  • the engineered TCR comprises non-human constant regions, for example murine constant regions.
  • the TCR such as an endogenous TCR of a NKT cell, is capable of binding tumor associated macrophages.
  • the TCR is specific for survivin.
  • Exemplary TCRs that are specific for the survivin tumor antigen but do not have “on-target off tumor” toxicity are disclosed in WO2016/070119.
  • Such survivin specific TCR preferably comprises the CDRs of SEQ ID NOs: 50 and 51.
  • the TCR comprises a beta chain of SEQ ID NOs: 50 and/or an alpha chain of 51.
  • the TCR is specific for WT-1.
  • Exemplary TCRs specific for WT-1 are disclosed in WO2005056595.
  • WT-1 specific TCRs preferably comprise at least one CDR from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59.
  • the alpha chain comprises SEQ ID NOs: 52, 53 and 54.
  • the alpha chain comprises SEQ ID NOs: 52, 53 and 55.
  • the beta chain comprises SEQ ID NOs: 56, 57 and 58.
  • the beta chain comprises SEQ ID NOs: 56, 57 and 59.
  • Such TCR may comprise the alpha chain of SEQ ID NOs: 60 or 62.
  • such TCR may comprise the beta chain of SEQ ID NOs: 61 or 63.
  • the TCR comprises SEQ ID NOs: 60 and 61.
  • the TCR comprises SEQ ID NOs: 60 and 63.
  • the TCR comprises SEQ ID NOs: 62 and 61.
  • the TCR comprises SEQ ID NOs: 62 and 63.
  • said antigen receptor is recombinantly expressed. Accordingly, the immune cell is transduced or transfected with a vector encoding said antigen receptor.
  • the antigen to which the antigen receptor binds is preferably expressed by or derived from a tumor or a pathogen.
  • the antigen receptor may bind more than one target.
  • immune cells expressing two or more, such as three, four or five, different recombinant antigen receptors.
  • Exemplary antigens to which the antigen receptor binds may include, without being limited to, GD2, WT-1, 5T4, GPC3, CSPG4, MUC16, MUC1, CA1X, CEA, CDS, CD7, CD 10, CD19, CD20, CD22, CD23, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD70, CD74, CD133, CD138, CD123, cytomegalovirus (CMV) proteins such as pp65 or IE-1, human papillomavirus (HPV) proteins such as E6 or E7, Epstein-Barr virus (EBV) proteins such as EBNA-1, LMP-1, LMP-2, or BARF-1, ADV proteins such as hexon, EGP-2, EGP-40, EpCAM, erb-B2, erb-B3, erb-B4, FBP, Fetal acetylcholine receptor, folate receptor-a, GD3, Her-1, HER-2
  • the antibody may be a full-length immunoglobulin or an antibody derivative.
  • full-length immunoglobulins have been dissected and the modules have been used to create monovalent, bivalent or multivalent derivatives as well as monospecific, bispecific or multispecific derivatives.
  • smaller antigen binding fragments were produced by proteolysis and later, artificial constructs have been generated by genetic engineering.
  • Antibody derivatives are thus recombinant molecules including functional parts or the entirety of a full-length immunoglobulin, possibly in multiple copies.
  • Exemplary antibody derivatives include, without being limited to, Fab, Fab', scFab, scFv, Fv fragment, nanobody, VHH, minimal recognition unit, diabody, single-chain diabody (scDb), tandem scDb (Tandab), a linear dimeric scDb (LD-scDb), circular dimeric scDb (CD-scDb), BiTE (or tandem di-scFv or tandem scFv), DART, tandem tri-scFv, tri(a)body, bispecific Fab2, di-miniantibody, tetrabody, scFv-Fc-scFv fusion, scFv-Fc fusion, di-diabody, DVD-Ig, CrossMab, Duobody, scFab-Fc, sc
  • antibody fragments refers to (i) monovalent and monospecific antibody derivatives which comprise the variable heavy and/or light chains or functional fragments of an antibody and lack an Fc part; and (ii) BiTE (tandem scFv), DARTs, diabodies and single-chain diabodies (scDB).
  • an antibody fragment is e.g. selected from the group consisting of: Fab, Fab', scFab, scFv, Fv fragment, nanobody, VHH, dAb, minimal recognition unit, single-chain diabody (scDb), BiTE and DART.
  • the recited antibody fragments have a molecular weight below 60 kDa.
  • the antibody derivative is an antibody fragment, preferably a humanized antibody fragment.
  • the antibody comprises an Fc domain which is capable of mediating cytotoxic immune responses.
  • Fc domain which is capable of mediating cytotoxic immune responses.
  • antibodies including an Fc domain are full-length immunoglobulins, DVD-Ig, scFv-Fc and scFv-Fc.scFv fusions, IgG-scFab, scFab-Fc, scFab-Fc-scFab,Fv2-Fc, IgG-scFv fusions (such as e.g., bsAb, Bs1Ab, Bs2Ab, Bs3Ab, Ts1Ab, Ts2Ab), DuoBody and CrossMab.
  • the antibody comprises an Fc domain which is modified such that it does not induce cytotoxic immune responses and/or or does not activate complement.
  • the antibody derivative lacks an Fc domain.
  • Exemplary antibody derivatives lacking an Fc domain are Fab, Fab', scFab, scFv, Fv fragment, nanobody, VHH, minimal recognition unit, diabody, single-chain diabody (scDb), tandem scDb (Tandab), a linear dimeric scDb (LD-scDb), circular dimeric scDb (CD-scDb), BiTE (also called tandem di-scFv or tandem scFv), tandem tri-scFv, tri(a)body, bispecific Fab2, di-miniantibody, di-diabody, scFab-dsscFv or DART.
  • the antibody comprises a Fc domain engineered using Knob into Holes (KiHs) technology.
  • the Fc part mediates cytotoxic immune responses such as ADCC, ADCP and/or CDC; however, such Fc mediated effects are not required or are even undesired when targeting the PD-1: PD-L1 axis as both proteins are expressed on the surface of antitumor cytotoxic T cells. Hence, administering full-length monoclonal antibodies with functional Fc parts may result in the depletion of the very lymphocytes they are intended to activate. Treatment with anti-PD-1 antibodies was found to correlate with lower circulating T-cell numbers in patients. PD-L1 is expressed on non-tumor cells as well and it is not desirable to target these cells and mediate ADCC, ADCP and/or CDC.
  • the antibody derivative has a molecular weight of about 60 kDa or lower, such as about 55 kDa, 50 kDa, 45 kDa, 40 kDa, 35 kDa, 30 kDa or 27 kDa or lower.
  • Solid tumors have substantial physical barriers that often prevent full-length immunoglobulins to penetrate to the center which results in reduced therapeutic effects (Christiansen, J., and Rajasekaran, A. K. (2004), Mol. Cancer Ther. 3, 1493-1501). Smaller antibody derivatives may in contrast penetrate deeper into the tumor.
  • Exemplary antibody derivates having a molecular weight of about 60 kDa or lower are antibody fragments, including, without being limited to, Fab, Fab', scFab, scFv, Fv fragment, nanobody, VHH, dAb, minimal recognition unit, single-chain diabody (scDb), or DART.
  • the size and/or architecture of the antibody has implications on its half-life. To decrease side-effects in a therapeutic setting, it may be advantageous to use antibodies with a short half-life. This may e.g. be achieved by using an antibody derivative lacking an Fc part, more preferably an antibody derivative having a low molecular weight, such as about 60 kDa or lower, such as about 55 kDa, 50 kDa, 45 kDa, 40 kDa, 35 kDa, 30 kDa or 27 kDa or lower.
  • an antibody derivative lacking an Fc part more preferably an antibody derivative having a low molecular weight, such as about 60 kDa or lower, such as about 55 kDa, 50 kDa, 45 kDa, 40 kDa, 35 kDa, 30 kDa or 27 kDa or lower.
  • Preferred antibody derivatives having these characteristics are e.g. Fab, Fab', scFab, scFv, Fv fragment, nanobody, VHH, dAb, minimal recognition unit, single-chain diabody (scDb), BiTE or DART.
  • the antibody can thus be monovalent or multivalent, i.e. having one or more antigen binding sites.
  • monovalent antibodies in particular antibody derivatives, include, without being limited to, scFv, Fv fragments, Fab, scFab, dAb, VHH, nanobody or minimal recognition unit.
  • a multivalent antibody can have two, three, four or more antigen binding sites.
  • the antibody in particular the antibody derivative, is bispecific, i.e. the antibody derivative is directed against two different targets or two different epitopes on one target molecule.
  • the antibody derivative is multivalent and comprises more than two, e.g., three or four different binding sites for three or four, respectively, different antigens. Such antibody is multivalent and multispecific, in particular tri- or tetra-specific, respectively.
  • the antibody derivative above is an scFv (a “single chain variable fragment” or a “single chain antibody”).
  • An scFv is a fusion protein that includes the VH and VL domains of an antibody connected by a linker. It thus lacks the constant Fc region which is present in a full-length antibody.
  • the VH and VL domains can be connected in either orientation, VL-linker-VH or VH-linker-VL, by a flexible linker.
  • the orientation is VL-linker-VH, i.e. the light chain variable region being at the N-terminal end and the heavy chain variable region being at the C-terminal end of the polypeptide.
  • the linker may have the sequence of SEQ ID NO: 10, however, shorter or longer linkers or variants of SEQ ID NO: 10 may also be used.
  • the cell provided herein is a T cell expressing a scFv blocking PD-L1 and at least one CAR according to the present disclosure. In one embodiment, the cell provided herein is a T cell expressing a scFv blocking PD-L1 and at least one TCR according to the present disclosure. In one embodiment, the cell provided herein is a T cell expressing a scFv blocking PD-L1 and at least one CAR as well as at least one TCR according to the present disclosure.
  • the cell provided herein is a Natural Killer T (NKT) cell, expressing a scFv blocking PD-L1 and at least one CAR according to the present disclosure.
  • the cell provided herein is a NKT cell expressing a scFv blocking PD-L1 and at least one TCR according to the present disclosure.
  • the cell provided herein is a NKT cell expressing a scFv blocking PD-L1 and at least one CAR as well as at least one TCR according to the present disclosure.
  • the cell provided herein is a Natural Killer (NK) cell expressing a scFv blocking PD-L1 and at least one CAR according to the present disclosure.
  • the cell provided herein is a NK cell expressing a scFv blocking PD-L1 and at least one TCR according to the present disclosure.
  • the cell provided herein is a NK cell expressing a scFv blocking PD-L1 and at least one CAR as well as at least one TCR according to the present disclosure.
  • the cell provided herein is a human embryonic stem cell, expressing a scFv blocking PD-L1 and at least one CAR according to the present disclosure.
  • the cell provided herein is a human embryonic stem cell expressing a scFv blocking PD-L1 and at least one TCR according to the present disclosure.
  • the cell provided herein is a human embryonic stem cell expressing a scFv blocking PD-L1 and at least one CAR as well as at least one TCR according to the present disclosure.
  • the cell provided herein is a hematopoietic stem cell (HSC) expressing a scFv blocking PD-L1 and at least one CAR according to the present disclosure.
  • the cell provided herein is a hematopoietic stem cell expressing a scFv blocking PD-L1 and at least one TCR according to the present disclosure.
  • the cell provided herein is a hematopoietic stem cell expressing a scFv blocking PD-L1 and at least one CAR as well as at least one TCR according to the present disclosure.
  • the cell provided herein is an induced pluripotent stem cell (iPS) expressing a scFv blocking PD-L1 and at least one CAR according to the present disclosure.
  • iPS induced pluripotent stem cell
  • the cell provided herein is an induced pluripotent stem cell expressing a scFv blocking PD-L1 and at least one TCR according to the present disclosure. In one embodiment, the cell provided herein is an induced pluripotent stem cell expressing a scFv blocking PD-L1 and at least one CAR as well as at least one TCR according to the present disclosure.
  • Antibodies having the frameworks as used herein have been described as being surprisingly stable in the scFv format (see, e.g., WO/2009/155726 or Borras et al., JBC, Vol. 285, no. 12, 9 Mar. 2010, pages 9054-9066).
  • the antibody preferably comprises the framework sequences as comprised in SEQ ID Nos: 1 and/or 2 or variants thereof.
  • Variants may e.g. include modifications as described in WO2014/206561, in particular including VL framework sequences SEQ ID NOs. 15 to 22 of WO2014/206561.
  • Humanized antibodies refer to antibodies that include one or more, typically all six CDR regions of a non-human parent antibody or variants thereof or synthetic CDRs, and of which the framework is, e.g., (i) a human framework, potentially including one or more framework residues of the non-human parent antibody, or (ii) a framework from a non-human antibody modified to increase similarity to naturally produced human frameworks.
  • Methods of humanizing antibodies are known in the art, see e.g. Leger O., and Saldanha J. Antibody Drug Discovery. Edited by Wood C. London: Imperial College Press, 2011. ISBN 1848166281. p.1-23.
  • the antibody is fully human.
  • the antibody binds to an epitope on PD-L1 such that PD-L1 interaction with both CD80 and PD-1 is blocked.
  • PD-L1 binding to PD-1 drives T cell exhaustion
  • PD-L1 binding to CD80 drives T cell anergy
  • simultaneously blocking the binding of PD-L1 to CD80 and PD-1 prevents anergy and reverts exhaustion.
  • the antibody comprises
  • VH variable heavy chain
  • VL variable light chain
  • the antibody comprises
  • said antibody is a scFv comprising SEQ ID NO.: 9 or a variant thereof.
  • a variant may in some embodiments be an antibody that differs from a given antibody, in one, two, three, four, five or more positions of its amino acid sequence. Such difference may e.g., be a substitution, addition, modification or deletion.
  • the variant has at least 85%, more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and most preferably 100% sequence identity to the sequences disclosed herein, in particular SEQ ID NO: 1, 2 or 9.
  • Variants of the antibodies provided herein may be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the antibody. Any combination(s) of deletions, substitutions, additions, modifications and insertions can be made to the framework or to the CDRs, provided that the generated antibody possesses the desired characteristics for which it can be screened using appropriate methods. Of particular interest are substitutions, preferably conservative substitutions.
  • conservative substitution refers to a modification and a substitution, that maintains physically, biologically, chemically and/or functionally the properties with regard to the corresponding reference.
  • a molecule that includes a sequence with conservative substitution for instance may have a similar size, shape, electric charge, chemical properties, including a comparable ability to form covalent or hydrogen bonds, and/or comparable polarity.
  • conservative modifications include, but are not limited to, one or more nucleobases and amino acid substitutions, additions and deletions.
  • conservative amino acid substitutions include those in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • amino acid residues being non-essential with regard to binding to an antigen can be replaced with another amino acid residue from the same side chain family, e.g. serine may be substituted for threonine.
  • Amino acid residues are usually divided into families based on common, similar side-chain properties, such as:
  • nonpolar side chains e.g., glycine, alanine, valine, leucine, isoleucine, methionine
  • uncharged polar side chains e.g., asparagine, glutamine, serine, threonine, tyrosine, proline, cysteine, tryptophan
  • acidic side chains e.g., aspartic acid, glutamic acid
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • a conservative substitution can be taken to be a substitution of a first amino acid within one of the six groups above by a further amino acid within the same group of the six groups.
  • Preferred conservative substitutions include:
  • valine (V) by leucine (L) and vice versa.
  • Other substitutions such as substituting proline (P) by alanine (A) are also permissible and can be determined empirically or in accord with other known conservative or non-conservative substitutions.
  • a conservative substitution may also involve the use of a non-natural amino acid.
  • the antibody described herein may comprise one or more, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more of such conservative substitutions.
  • non-conservative substitutions are introduced into any sequence disclosed herein to produce a variant.
  • the antibody comprises one or more, such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more of such non-conservative substitutions.
  • a particularly preferred type of variant is one where one or more entire CDRs are replaced.
  • the CDR-H3 and CDR-L3 contribute most significantly to antigen binding.
  • the entire CDR-L1, CDR-L2, CDR-H1 and/or CDR-H2 may be replaced by a different CDR of natural or artificial origin.
  • one or more CDRs are replaced by an alanine-cassette.
  • the variant does not show any improvement over the parent antibody.
  • (ii) has a KD to human PD-L1 of lower than 500 pM, preferably lower than 250 pM, 100 pM, 75 pM, 50 pM, 40 pM, 30 pM, 20 pM, more preferably of lower than 10 pM as measured by KinExA®; and/or
  • (v) has at least 80%, preferably at least 85%, 90%, 95% or 97% sequence identity to the sequences disclosed herein.
  • the antibody has high affinity for PD-L1 and binds hPD-L1 with a KD of lower than 100 pM, such as lower than about 75 pM, 50 pM, 25 pM or 10 pM.
  • the antibody is bivalent and binds PD-L1 with a KD of lower than 10 pM as measured by KinExA®, preferably lower than 5 pM, more preferably about 3 pM, e.g., 2.9 pM, 2.8 pM or 2.7 pM.
  • such bivalent antibody is a full-length immunoglobulin.
  • the antibody provided herein is monovalent and binds human PD-L1 with a KD of lower than 50 pM as measured by KinExA®. Said KD is preferably lower than about 10 pM, such as about 9 pM, e.g., 9.0 pM, 8.9 pM, 8.8 pM or 8.7 pM. In some embodiments, said monovalent antibody is a scFv.
  • the antibody provided herein is monovalent and binds monkey PD-L1 with a KD of lower than 50 pM as measured by KinExA®.
  • Said KD is preferably lower than about 10 pM, more preferably lower than about 5 pM, such as e.g., about 3.4 pM, 3.3 pM or 3.2 pM as measured by KinExA®.
  • Such KinExA®.measurements are preferably done at room temperature, more preferably under the conditions as described in example 1.
  • High affinity antibodies may be advantageous to provide a protective effect even if a small amount of engineered immune cells is present at its target site, accordingly expressing a limited amount of antibody.
  • engineered immune cells as described above expressing antibodies that bind to human PD-L1 as well as to monkey PD-L1.
  • the affinity to monkey PD-L1 is at least twice as tight as the affinity to human PD-L1.
  • the affinity KD of a monovalent antibody, preferably a scFv, to monkey PD-L1 is about 3.3 pM as measured by KinExA®, for example as measured at room temperature, preferably under the conditions indicated in Example 1.
  • antibodies that have no cross-reactivity to other member of the B7 family such as PD-L2 and B7-H3.
  • engineered immune cells as described above expressing antibodies that compete with the antibodies disclosed herein for binding to PD-L1.
  • the engineered immune cell described herein may secreted the antibody and/or express the antibody on its surface.
  • the antibody is secreted.
  • the cell may additionally recombinantly express at least one further protein compound such as a second antibody or a cytokine.
  • Cytokines are e.g. selected from the group consisting of IL-2, IL-4, IL-7, IL-12, IL-15, IL-21 or MIP-lalpha and are preferably of human origin, i.e. hIL-2, hIL-4, hIL-7, hIL-15, hIL-21, or hMIP-1 alpha.
  • such cell expresses hIL-15.
  • IL-15 has been described to improve in vivo persistence and anti-tumor activity of CAR NKT cells (see WO2013/040371).
  • Such second antibody may e.g., target an immune inhibitory molecule, such as transforming growth factor-beta (TGF- ⁇ ), IL-10, Fas, CD47, CTLA-4, Tim-3, LAG-3, or ligands thereof.
  • TGF- ⁇ transforming growth factor-beta
  • the engineered immune cell expresses an antigen receptor being a CAR comprising a 4-1BB costimulatory domain, an antibody as described herein and further IL-15. In some embodiments, the engineered immune cell expresses an antigen receptor being a CAR comprising a CD28 costimulatory domain, an antibody as described herein and further IL-15.
  • the NKT cell further encodes a cytokine, such as IL-15.
  • nucleic acid encoding the antigen receptor and/or the antibody described herein.
  • the proteins may be encoded by a plurality of nucleic acid sequences. In some embodiments, the proteins are encoded by a single nucleic acid sequence. Typically, the nucleic acid is an isolated nucleic acid.
  • cDNAs encoding the respective polypeptide sequence can be generated by methods well known in the art, e.g. by gene synthesis. These cDNAs can be cloned by standard cloning and mutagenesis techniques into a suitable vector such as an expression vector or a cloning vector. Thus, further contemplated is cDNA encoding the antigen receptor and/or the antibody as described herein.
  • genetic constructs may generate an antibody and/or an antigen receptor having one or more additional residues at the N-terminal or C-terminal end. It is therefore to be understood that the antibodies disclosed herein include the disclosed sequences rather than consist of them.
  • nucleic acids hybridizing with the nucleic acids described herein under stringent conditions.
  • vectors comprising the nucleic acid provided herein, such as an expression vector or a cloning vector.
  • One, two or more nucleic acids encoding the antigen receptor and the antibody described herein may be comprised in a vector, which may be the same vector (bicistronic or multicistronic) or separate vectors.
  • the expression vector may be a multicistronic vector, such as a bicistronic vector, e.g., using internal ribosome entry sites (IRES), 2A-like sequences or dual promoters.
  • IRS internal ribosome entry sites
  • the expression vector may e.g. be a lentiviral, a retroviral, an adenoviral or an Adeno-Associated Virus (AAV) vector.
  • the expression vector may also be a non-viral vector, including a plasmid, a transposon, an inserting sequence, or an artificial chromosome.
  • a nucleic acid molecule may in some embodiments define an expression cassette.
  • An expression cassette is a nucleic acid molecule capable of directing expression of a particular nucleotide sequence in an appropriate host cell.
  • An expression cassette includes a promoter operatively linked to the nucleotide sequence of interest, which is operatively linked to one or more termination signals. It may also include sequences required for proper translation of the nucleotide sequence.
  • the coding region can encode a polypeptide of interest.
  • the expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus.
  • the antibody is under the control of the 5′′ end LTR of the retrovirus.
  • the nucleic acid encoding the antigen receptor and/or the nucleic acid encoding the antibody may each be operably linked to a promoter which may be the same or different promoters.
  • the nucleic acid and/or vectors may further comprise a signal peptide.
  • a signal peptide is a 5-30 amino acid peptide attached to the N-terminus of the protein to be secreted and is attached to increase protein secretion.
  • the signal peptide is a human signal peptide.
  • the signal peptide is hIgGl.
  • the signal peptide comprises SEQ ID NO: 15.
  • the antibody is membrane anchored.
  • Such membrane anchored antibody may comprise a transmembrane domain.
  • the membrane anchored antibody does not comprise no signaling domain.
  • the antibody is secreted and in also provided as membrane anchored form.
  • a genetically engineered immune cell may comprise a safety switch, such as a suicide switch.
  • a safety switch such as a suicide switch.
  • Such switches suppress the cell's activity if serious side effects emerge, or make the cells self-destruct if they attack healthy tissue.
  • Such switches are controllable and therefore require an additional receptor or other target on the cell.
  • Such safety switches are controlled by administering a second medication to the subject.
  • the vector comprises a nucleic acid sequence encoding a safety switch, preferably a suicide switch.
  • the invention also provides a method of generating an immune cell as described herein, comprising the steps of:
  • step (b) comprises introducing the expression vector as described above into said cell.
  • the method may comprise the additional step of:
  • step (i) Introducing into said cell at least one other antigen receptor having a different antigen specificity than the antigen receptor of step (b); and/or introducing into said cell at least one other antibody having a different antigen specificity than the antibody of step (b).
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising
  • Suitable “excipients” include, without being limited to: (i) buffers such as phosphate, citrate, or other, organic acids; (ii) antioxidants such as ascorbic acid and tocopherol; (iii) preservatives such as 3-pentanol, hexamethonium chloride, benzalkonium chloride, benzyl alcohol, alkyl paraben, catechol, or cyclohexanol; (iv) amino acids, such as e.g.
  • peptides preferably up to 10 residues such as polylysine;
  • proteins such as bovine or human serum albumin;
  • hydrophilic polymers such as polyvinylpyrrolidone;
  • chelating agents e.g. EDTA;
  • salt-forming ions such as sodium, potassium, and/or chloride;
  • metal complexes e.g.
  • Zn-protein complexes Zn-protein complexes); (xii) ionic and non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG), (xiii) cryopreservatives such as dimethyl sulfoxide (DMSO).
  • ionic and non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG)
  • cryopreservatives such as dimethyl sulfoxide (DMSO).
  • the engineered immune cell described herein, the expression vector described herein and/or the pharmaceutical composition described herein are useful for therapy.
  • the engineered immune cell described herein, the expression vector described herein and/or the pharmaceutical composition described herein for use in therapy are useful for therapy.
  • the method of treatment involves the provision and administration of the expression vector described herein and/or the pharmaceutical composition described herein.
  • treatment include a prophylactic or preventative measure having a therapeutic effect and/or preventing, slowing down (lessening), or at least partially alleviating or abrogating an abnormal, including pathologic, condition in the organism of a subject.
  • Treatment according to the present disclosure involves the administration of a pharmaceutically effective amount of a molecule, nucleic acid, vector, pharmaceutical composition, and/or an engineered immune cell as described herein, i.e. inter alia, the cell, the vector or the composition disclosed herein, to a subject in need thereof to prevent, cure, delay the onset and/or progression, reduce the severity of, stabilize, modulate, cure or ameliorate one or more symptoms of the condition to be treated.
  • a treatment reduces, stabilizes, or inhibits progression of a symptom that is associated with the presence and/or progression of a disease or pathological condition.
  • the subject in need of a treatment can be a human or a non-human animal.
  • the subject is a mammal, e.g., a mouse, a rat, rabbit, a hamster, a dog, a cat, a monkey, an ape, a goat, a sheep, a horse, a chicken, a guinea pig or a pig.
  • the subject is diagnosed with a cancer and/or a PD-L1-related disorder or may acquire such a disorder.
  • the animal might be genetically engineered to develop such disorder.
  • an effective amount of the cell, the vector or the composition disclosed herein is administered to the subject.
  • An “effective amount” is an amount—either as a single dose or as part of a series of doses—which at the dosage regimen applied yields the desired therapeutic effect, i.e., to reach a certain treatment goal.
  • a therapeutically effective amount is generally an amount sufficient to provide a therapeutic benefit in the treatment or management of the relevant pathological condition, or to delay or minimize one or more symptoms associated with the presence of the condition.
  • the dosage will depend on various factors including patient and clinical factors (e.g., age, weight, gender, clinical history of the patient, severity of the disorder and/or response to the treatment), the nature of the disorder being treated, the particular composition to be administered, the route of administration, and other factors.
  • administering refers to any mode of transferring, delivering, introducing, or transporting matter such as the cell, the vector or the composition described herein, to a subject. Administration may be administered locally or systemically. Preferred modes of administration include, without being limited to, parenteral, e.g., intravenous, or systemic, administration. Administration “in combination with” further matter such as one or more therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
  • the cells, the vector or the composition are administered one or more times to said subject.
  • the cells, the vector or the composition may be administered in combination with one or more therapies selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.
  • the therapies of the present invention may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
  • the cells may have originated from the subject or from another individual of the same species, i.e. they are autologous or allogeneic.
  • Autologous adoptive transfer requires extraction of the patient's cells, their genetic modification, e.g. as described above and culturing said cells in vitro before returning them to the same patient.
  • Such individual preparation for each new patient limits application of cellular immunotherapies in treating cancer.
  • allogeneic T cells derived from healthy donors carry the risk of recognizing the patient's body as foreign, which can cause a serious side effect called graft versus host disease (GvHD).
  • GvHD graft versus host disease
  • Off-the-shelf therapies based on CAR-modified NKT cells generated in large volumes from healthy donors offer great promise. While endowed with powerful cancer-killing properties like conventional T cells, invariant NKT cells express special T cell receptors that are not associated with GvHD. Hence allogeneic NKT cells can be used to treat multiple cancer patients with minimal risk of GvHD.
  • the subject in need of treatment may have a condition, without being limited to, a pre-malignant or malignant cancer condition, such as NSCLC (non small cell lung carcinoma), urothelial cancer, melanoma, renal cell carcinoma, Hodgkin's lymphoma, head and neck squamous cell carcinoma, ovarian cancer, gastrointestinal cancer, hepatocellular cancer, glioma, breast cancer, lymphoma, small cell lung carcinoma, myelodysplastic syndromes, prostate cancer, bladder cancer, cervical cancer, non clear cell kidney cancer, colorectal cancer, sarcomas, colon cancer, kidney cancer, lung cancer, pancreatic cancer or gastric cancer, skin cancer, uterine cancer, glioblastoma, neuroblastoma, sarcoma, head and neck cancer, leukemia, carcinoma, Merkel cell carcinoma or renal cell carcinoma (RCC), multiple myeloma, lymphoblastic leukemia (ALL), B cell leukemia, chronic lymphocytic leukemia
  • the invention further provides a kit for treatment of cancer, pathogen infection, an autoimmune disorder comprising the engineered immune cell described herein, the expression vector described herein or the pharmaceutical composition described herein, and written instructions for use.
  • the kit may further comprise an inducer of a safety switch.
  • CDR1 alpha SEQ ID NO: 52 SSYSPS -WT-1 specific TCR.
  • CDR2 alpha SEQ ID NO: 53 YTSAATL -WT-1 specific TCR.
  • CDR3 alpha SEQ ID NO: 54 WSPFSGGGADGLT -WT-1 specific TCR.
  • CDR3 alpha SEQ ID NO: 55 SPFSGGGADGLT -WT-1 specific TCR.
  • CDR3 beta SEQ ID NO: 58 SARDGGEG -WT-1 specific TCR.
  • the 293T cells were obtained from ATCC while neuroblastoma tumor cell line CHLA-255 was kindly provided by Dr Leonid Metelitsa Baylor College of Medicine (Houston, Tex.). Cells were maintained in culture with IMDM (Gibco) for 293T or RPMI 1640 (Gibco) for CHLA-255, containing 10% FBS (Corning), 1% GlutaMAX and 1% penicillin/streptavidin (Gibco) in a humidified atmosphere containing 5% CO 2 at 37° C.
  • the anti-PD-L1 scFv (see SEQ ID NO: 9) is a humanized protein comprising rabbit CDRs. Its ability to inhibit the binding of PD-L1 to PD-1 was tested by competition ELISA. Briefly, rhPD-L1 Fc fusion was coated onto 96-well microplates. After blocking, serial dilutions of scFv were added to plates and incubated for one hour at room temperature. Half of the scFv dilution was replaced with biotinylated PD-1 Fc fusion and bound PD-1 was detected with streptavidin-HRP.
  • scFvs Two different processes can be observed that may affect the stability of scFvs. Firstly, scFvs could be prone to dimerization, often followed by oligomerization and further aggregation and precipitation. Secondly, scFv degradation, leading to smaller fragments, can occur over time.
  • the stability of the scFv formulated in PBS pH 7.2 at 10 mg/mL concentration upon storage at different temperature conditions (4° C., 22° C., 37° C. and ⁇ 20° C.) was investigated. Only a minor amount of dimerization of the scFv or formation of high molecule weight molecules was observed upon storage for 2 weeks at 4° C., 22° C. and 37° C. The scFv formed up to 1.8% and 2.7% of dimers after 1 or 2 weeks of storage at 37° C., respectively.
  • the thermal stability was assessed by differential scanning fluorimetry (DSF) in a real-time PCR device (Corbett, Rotor-Gene).
  • DFS differential scanning fluorimetry
  • the midpoint melting temperatures (Tm) of the scFv calculated using Rotor-Gene 6000 Series Software 1.7. was 81.5° C.
  • Proteinaceous biologics may become exposed to freeze/thaw stress during manufacturing, storing and shipping which may cause aggregation and degradation.
  • anti-PD-L1 scFv In order to assess stability of the anti-PD-L1 scFv during freeze/thaw cycles, it was formulated in PBS pH 7.2 at 10 mg/mL in 1.5 mL polypropylene tubes. The vials were submerged into liquid nitrogen, followed by incubation in a water bath at room temperature. After centrifugation, supernatants were analyzed by SE-HPLC. Virtually 100% of the scFv remained monomeric after 10 freeze/thaw cycles and no protein loss or precipitation was observed.
  • the stability of the anti-PD-L1 scFv in human serum was assessed by ELISA. There was no loss of binding activity of the scFv after up to 20 hours of incubation with human serum at 37° C.
  • the affinity of a monovalent and a bivalent antibody to PD-L1 was determined by Kinetic Exclusion Assay (KinExA®) with a KinExA 3200 (Sapidyne Instruments, USA, cat. no. 5001).
  • KinExA® measures the equilibrium binding affinity and kinetics between unmodified molecules in solution. The measurement requires the immobilization of one interaction partner on a solid phase solely to act as a probe to determine the concentration of the corresponding binding partner in solution.
  • the affinity of the scFv to PD-L1-Fc fusion was determined at room temperature in PBS with 0.02% sodium azide, pH7.4. Two curves were measured, one using 20 pM scFv1 with an incubation time of 5 hours and the other at 10 pM scFv1 with an incubation time of 9 hours.
  • the KD value for the scFv was 8.8 pM, calculated using the “n-curve analysis” of the KinExA® Pro software version 4.1.9 or 4.2.10.
  • the scFv was reformatted into IgG format and expressed in suspension-adapted CHO K1 cells originally received from ATCC and adapted to serum-free growth in suspension culture.
  • IgG antibodies were purified by Protein A chromatography followed by size exclusion chromatography.
  • the KD for binding to PD-L1-His was calculated at room temperature in PBS with 0.02% sodium azide, pH7.4, using two curves. One curve used 100 pM of IgG with an incubation time of 5 hours, and the other used 10 pM of IgG with an incubation time of 10 hours.
  • the KD value calculated for the binding of the IgG to human PD-L1 was 2.77 pM.
  • the results demonstrate that the IgG binds PD-L1 with an affinity around three times tighter than the affinity of the scFv to PD-L1.
  • Cross-reactivity of the scFv to PD-L1 from other species was determined by ELISA, using PD-L1 Fc fusions from human (RnD Systems, USA, cat. no. 156-B7), rat (Sino Biological, China, cat. no. 80450-RO2H) or monkey (Sino Biological, China, cat. no. 90251-C02H).
  • Crossreactivity of the anti-PD-L1 scFv to monkey PD-L1 was further investigated using KinExA®.
  • the KD value was calculated at room temperature in PBS with 0.02% sodium azide, pH7.4 using two curves. One curve used 50 pM of scFv with an incubation time of 6 hours, and the other used 10 pM of scFv with an incubation time of 16 hours. The KD value calculated for the scFv was 3.3 pM. The results demonstrate that the scFv binds to monkey PD-L1 with an affinity around 2.7 times tighter than binding to human PD-L1.
  • Cross-reactivity of the scFv to recombinant human proteins sharing sequence similarity to PD-L1 was determined by ELISA using rhPD-L1 Fc fusion (RnD Systems, USA, cat. no. 156-B7), rhPD-L2 Fc fusion (RnD Systems, USA, cat. no. 1224-PL) or rhB7-H3 Fc fusion (RnD Systems, USA, cat. no. 1027-B3).
  • the ability of the scFv to bind to PD-L1 on the surface of cells was determined by extracellular FACS staining of ES-2 cells (ATCC, USA, cat. no. CRL-1978). The results demonstrate that the scFv is able to specifically recognize the natural form of PD-L1 expressed on the surface of cells.
  • the binding of the scFv to cell surface PD-L1 was further investigated at room temperature in PBS with 0.02% sodium azide, pH7.4 using KinExA®.
  • One curve was constructed, using 50 pM scFv and an incubation time of 5 hours.
  • the calculated KD value for the scFv binding to cell surface expressed PD-L1 on ES-2 cells is 12.8 pM.
  • Example 2 Generation of the retroviral construct encoding the anti-PD-L1 scFv and retrovirus production
  • DNA encoding the anti-PD-L1 scFv was amplified by PCR with the following primers: Forward: 5′-TAACCATGG AGTTTGGGCTGAG-3′ (SEQ ID NO: 11) and Reverse: 5′-GACGCATGCTCAGCTCGACACGGTGACC-3′ (SEQ ID NO: 12) in order to add the NcoI restriction site at 5′ end (forward primer) and the stop codon TGA and the restriction site Sphl at the 3′ end (reverse primer).
  • the PCR product and the retroviral backbone SFG(I)eGFP were digested with Ncol and Sphl and ligated. The insert was sequenced to confirm that no mutations occurred during the cloning.
  • the final vector was named SFG.scFv.anti-PD-L1(I)eGFP (see FIG. 1 ).
  • the reporter gene GFP expressed upon IRES is used to assess the transduction efficiency.
  • Transient retroviral supernatant was prepared by transfection of 293T cells with the retroviral vector and two plasmids encoding gag-pol and RDF envelop, respectively. The supernatant collected at 48 hrs was used to transduce activated T cells isolated from healthy donors.
  • the retroviral vectors encoding the CAR that targets the GD2 antigen (GD2.CAR) including either the CD28 (CAR.CD28 or CAR.28) or the 4-1BB (CAR.41BB or CAR.BB) endodomains were previously described (Heczey A. et al, Blood. 2014 Oct. 30; 124(18):2824-33).
  • Said CAR comprises the CDR sequences of SEQ ID NOs: 16 to 21.
  • PBMCs Peripheral blood mononuclear cells
  • Lymphoprep Fresenius density gradient centrifugation.
  • Primary T cells were cultured in complete T cell media containing 44% Click's medium (Irvine Scientific), 44% RPMI 1640 (Hyclone), 10% FBS (Hyclone), 1% Glutamax and 1% penicillin/streptavidin in the presence of IL-7 (10 ng/mL) and IL-15 (5 ng/mL) (from PeproTech).
  • T cells were activated with immobilized anti-CD3 (1 mcg/mL) (Miltenyi, Catalog Number: 130-093-387) and anti-CD28 (1 mcg/mL) (BD Biociences, Catalog Number: 555725) in 24-well plate at the concentration of 0.5 ⁇ 10 6 cell/mL in T cells media without cytokines. Twenty four hours after the stimulation, IL-7 and IL-15 were added to the medium. By day 2, T cells were transduced with the retroviral supernatant SFG.scFv.anti-PD-L1(I)eGFP and/or for the GD2.CAR (1 mL/well of retroviral supernatant in a retronectin-coated 24-well plate).
  • Non-Transduced (NT) T cells were plated at the same concentration (0.25 ⁇ 10 6 cells/mL) in a non-tissue culture plate coated with retronectin. Seventy two hours after the transduction T cells were washed, counted and suspended in complete T cell media with IL-7/IL-15 at 1 ⁇ 10 6 cells/mL. T cells were expanded in vitro for 5 days and then analyzed by flow cytometry to assess transduction efficiency and T cell composition. Eleven to twelve days after initiation, T cells were tested in functional assays.
  • Example 4-T cells co-transduced with the GD2.CAR and SFG.scFv.anti-PD-L1 vectors express both GD2.CAR and anti-PD-L1 scFvs
  • the phenotype of T cells was assessed using mAbs for CD3, CD4, CD8, CD60L, CD45RA, CD95, CD27, CD2, PD-L1 (BD Bioscience or Biolegend).
  • GD2.CAR expression was detected using the anti-idiotype 1A7 mAb followed by staining with a secondary rat anti-mouse-IgG1-PE mAb (BD Bioscience).
  • the transduction efficiency of the SFG.scFv.anti-PD-L1(I)eGFP vector was assessed by measuring GFP expression.
  • GD2.CAR Relative Fluorescence Intensity (RFI) was calculated as the Mean Fluorescence Intensity (MFI) of CAR T cells divided by the MFI of non-transduced T cells.
  • CD4 and CD8 T cells were successfully transduced with the anti-PD-L1 scFv retroviral construct of Example 2 without alteration of the CD4/CD8 ratio.
  • T cells co-expressed GD2.CAR and eGFP anti-PD-L1 upon double retroviral transduction ( FIG. 3 ).
  • Transduction with the anti-PD-L1 scFv did not affect T cell proliferation ( FIG. 4 ).
  • expression of the anti-PD-L1 scFv did not affect T cell subset compositions ( FIG. 5 ).
  • This example demonstrates the generation of CART cells co-expressing anti-GD2 CARs and anti-PD-L1 scFvs.
  • Non-transduced T cells or anti-PD-L1 scFv transduced T cells were plated in tissue culture treated 24-well plates uncoated or coated with immobilized anti-CD3 (1 ⁇ g/ml, Miltenyi) and anti-CD28 (1 ⁇ g/ml, BD Bioscences) anti-CD3/CD28 anti-CD3/CD28 antibodies at the concentration of 0.5 ⁇ 10 6 cells/mL in 2 mL/well of T cells medium with 10% FBS.
  • the quantitative ELISA showed large amounts of secreted anti-PD-L1 scFv ( FIG. 6A ).
  • Non-transduced cells did not secrete scFvs.
  • the anti-PD-L1 scFv is under the control of the constitutively active 5′ LTR of a retroviral vector, transduced T cells plated on uncoated wells released basal levels of anti-PD-L1 scFvs which however significantly increased upon T cell activation.
  • the anti-PD-L1 scFv produced by activated T cells is capable of binding to immobilized PD-L1 ( FIG. 6 B).
  • recombinant human PD-L1-Fc R&D Systems
  • FIG. 6B shows that T cell-produced anti-PD-L1 scFv binds to PD-L1 equally well as a reference control, the anti-PD-L1 scFv produced in E. coli.
  • transduced T cells release functionally active anti-PD-L1 scFvs in large amounts upon activation through the endogenous TCR. These data also suggest that transduced T cells will readily secrete anti-PD-L1 scFvs when they are activated through genetically modified TCR and through CARs.
  • Example 6-T cells co-transduced with the GD2.CAR-4-1BB and SFG.scFv.anti-PD-L1 vectors have enhanced tumor-killing activity
  • Transduced and non-transduced T cells were co-cultured with the tumor cell line CHLA-255 (2.5 ⁇ 10 5 cells/well) at an effector:target (E:T) ratio of 1:5 in 24-well plates, in the absence of exogenous cytokines. After 7 days of co-culture, T cells were harvested and counted. If the percentage of residual tumor cells was ⁇ 5% (as assessed by flow cytometry), T cells were re-plated with fresh tumor cells at the same 1:5 E:T ratio for a second cycle of co-culture. After an additional 7-8 days, cells were then collected and analyzed by flow cytometry to enumerate T cells and residual tumor cells.
  • E:T effector:target
  • CD3 and GD2 antibodies were used to stain T cells and tumor cells, respectively.
  • CountBright beads (Invitrogen) were used for cell counting by flow cytometry. Supernatant was also collected after 24 hours of culture to measure IFNgamma release by ELISA (R&D System) according to the manufacturer's instruction.
  • GD2.CAR T cells with the 4-1BB endodomain transduced with the anti-PD-L1 scFv showed better killing of tumor cells in the second cycle of co-culture (14 days of culture) ( FIG. 7 ) and produced higher levels of IFNgamma in the second cycle of co-culture (14 days of culture) with tumor cells ( FIG. 8 ) than the GD2.CAR T cells without the anti-PD-L1 svFv.
  • exhaustion of GD2.CAR T cells engaging repetitively tumor cells occurs with 4-1BB costimulation, but the presence of the anti PD-L1 scFv protect CART cells from exhaustion and may provide prolonged anti tumor activity in vivo and prevent tumor recurrence.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Wood Science & Technology (AREA)
  • Hematology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Mycology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
US16/079,202 2016-02-25 2017-02-24 Modified cells for immunotherapy Abandoned US20190048085A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP16020057.2 2016-02-25
EP16020057 2016-02-25
PCT/US2017/019301 WO2017147383A1 (en) 2016-02-25 2017-02-24 Modified cells for immunotherapy

Publications (1)

Publication Number Publication Date
US20190048085A1 true US20190048085A1 (en) 2019-02-14

Family

ID=55446573

Family Applications (3)

Application Number Title Priority Date Filing Date
US16/079,210 Active 2038-04-05 US11434294B2 (en) 2016-02-25 2017-02-24 Binding members to PD-L1
US16/079,202 Abandoned US20190048085A1 (en) 2016-02-25 2017-02-24 Modified cells for immunotherapy
US17/823,355 Abandoned US20230235061A1 (en) 2016-02-25 2022-08-30 Binding Members to PD-L1

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US16/079,210 Active 2038-04-05 US11434294B2 (en) 2016-02-25 2017-02-24 Binding members to PD-L1

Family Applications After (1)

Application Number Title Priority Date Filing Date
US17/823,355 Abandoned US20230235061A1 (en) 2016-02-25 2022-08-30 Binding Members to PD-L1

Country Status (12)

Country Link
US (3) US11434294B2 (OSRAM)
EP (3) EP3420000A1 (OSRAM)
JP (3) JP7080819B2 (OSRAM)
KR (2) KR20190003938A (OSRAM)
CN (3) CN108699153B (OSRAM)
AU (2) AU2017224843A1 (OSRAM)
BR (2) BR112018067696A2 (OSRAM)
CA (2) CA3014067A1 (OSRAM)
ES (1) ES2903408T3 (OSRAM)
RU (2) RU2018128464A (OSRAM)
SG (2) SG11201807187XA (OSRAM)
WO (2) WO2017144681A1 (OSRAM)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2733430C1 (ru) * 2019-11-19 2020-10-01 Общество с ограниченной ответственностью «Реал Таргет» Создание конъюгатов gd2-специфичных антител и фрагментов gd2-специфичных антител с препаратами
WO2021113307A3 (en) * 2019-12-02 2021-07-22 Dana-Farber Cancer Institute, Inc. Antibodies against pd-l1 and methods of use thereof
WO2021252654A1 (en) * 2020-06-11 2021-12-16 Innovative Cellular Therapeutics Holdings, Ltd. Cell/gene therapies targeting mage-a4 peptide
US11535669B2 (en) 2018-07-09 2022-12-27 Precigen, Inc. Fusion constructs and methods of using thereof
US11643451B2 (en) 2016-10-19 2023-05-09 Ucl Business Ltd T cell receptor
WO2023147564A3 (en) * 2022-01-28 2023-10-12 The University Of North Carolina At Chapel Hill Natural killer t-cells and methods of using the same
US11998607B2 (en) 2016-12-08 2024-06-04 Immatics Biotechnologies Gmbh T cell receptors and immune therapy using the same
US12194094B2 (en) 2020-07-10 2025-01-14 Precigen, Inc. Fusion constructs and methods of using thereof
US12350318B2 (en) 2021-08-13 2025-07-08 Inovio Pharmaceuticals, Inc. Combination therapy to treat brain cancer
US12378298B2 (en) 2015-04-20 2025-08-05 Ucl Buseness Ltd T cell receptor

Families Citing this family (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI680138B (zh) * 2014-01-23 2019-12-21 美商再生元醫藥公司 抗ｐｄ-ｌ１之人類抗體
BR112018008865A8 (pt) 2015-11-02 2019-02-26 Five Prime Therapeutics Inc polipeptídeos do domínio extracelular cd80 e seu uso no tratamento do câncer
BR112018067696A2 (pt) * 2016-02-25 2019-01-08 Cell Medica Switzerland Ag membros de ligação à pd-l1
TWI822521B (zh) 2016-05-13 2023-11-11 美商再生元醫藥公司 藉由投予pd-1抑制劑治療皮膚癌之方法
JP7275043B2 (ja) 2016-12-16 2023-05-17 ビー－モーゲン・バイオテクノロジーズ，インコーポレーテッド 増大したｈＡＴファミリートランスポゾン媒介遺伝子導入ならびに関連する組成物、システムおよび方法
US11278570B2 (en) 2016-12-16 2022-03-22 B-Mogen Biotechnologies, Inc. Enhanced hAT family transposon-mediated gene transfer and associated compositions, systems, and methods
EP3585812A1 (en) 2017-02-21 2020-01-01 Regeneron Pharmaceuticals, Inc. Anti-pd-1 antibodies for treatment of lung cancer
SG10202111394XA (en) 2017-04-13 2021-12-30 Senti Biosciences Inc Combinatorial cancer immunotherapy
MX2019012849A (es) 2017-04-28 2019-11-28 Five Prime Therapeutics Inc Metodos de tratamiento con polipeptidos del dominio extracelular del cd80.
EP3728305A1 (en) 2017-12-20 2020-10-28 Poseida Therapeutics, Inc. Vcar compositions and methods for use
ES2960686T3 (es) * 2018-01-19 2024-03-06 Miltenyi Biotec Bv & Co Kg Células T reguladoras que expresan un receptor de antígeno quimérico
AU2019228128A1 (en) 2018-03-02 2020-09-03 Cdr-Life Ag Trispecific antigen binding proteins
WO2019185035A1 (en) * 2018-03-29 2019-10-03 Adagene Inc. Anti-pd-l1 antibodies and use thereof
US12304963B2 (en) * 2018-06-04 2025-05-20 Diatheva S.R.L. Antibody complex and uses thereof
WO2019246486A2 (en) 2018-06-21 2019-12-26 B-Mogen Biotechnologies, Inc. ENHANCED hAT FAMILY TRANSPOSON-MEDIATED GENE TRANSFER AND ASSOCIATED COMPOSITIONS, SYSTEMS, AND METHODS
KR102250234B1 (ko) * 2018-06-29 2021-05-10 주식회사 와이바이오로직스 Lag-3에 특이적으로 결합하는 단클론항체 및 이의 용도
KR20210087458A (ko) * 2018-10-01 2021-07-12 아디셋 바이오, 인크. 고형 종양 치료를 위한 조작된 및 비-조작된 γδ-T 세포에 관한 조성물 및 방법
JP2022512766A (ja) 2018-10-17 2022-02-07 センティ バイオサイエンシズ インコーポレイテッド 組み合わせがん免疫療法
US11419898B2 (en) 2018-10-17 2022-08-23 Senti Biosciences, Inc. Combinatorial cancer immunotherapy
SG11202105975SA (en) * 2018-12-06 2021-07-29 Guangdong Tcrcure Biopharma Technology Co Ltd Combinational tcr-t cell therapy targeting tumor antigens, tgf-beta, and immune checkpoints
CN113195542B (zh) 2018-12-26 2024-05-24 南京传奇生物科技有限公司 Cd30-结合部分、嵌合抗原受体及其用途
CN109652379B (zh) * 2018-12-29 2022-08-16 博生吉医药科技(苏州)有限公司 Cd7嵌合抗原受体修饰的nk-92mi细胞及其应用
CN111454358A (zh) * 2019-01-18 2020-07-28 四川科伦博泰生物医药股份有限公司 一种嵌合抗原受体及其应用
JP7641223B2 (ja) * 2019-01-28 2025-03-06 サイフォス、バイオサイエンシズ、インコーポレイテッド Ｃａｒ細胞上の非天然ｎｋｇ２ｄ受容体に、結合した異種分子を選択的に送達する改変非天然ｎｋｇ２ｄリガンド
CN113543792A (zh) * 2019-03-08 2021-10-22 奥托路斯有限公司 包含工程化嵌合抗原受体和car调节剂的组合物和方法
JP2022527081A (ja) * 2019-03-26 2022-05-30 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 血液がんおよび固形がんの治療のためのキメラ抗原受容体改変ｔ細胞（ｃａｒ－ｔ）
CN110402892A (zh) * 2019-04-30 2019-11-05 梁廷波 选择性敲除胰腺上皮细胞程序性死亡配体1分子的自发胰腺癌小鼠模型的建立方法
CN112079924B (zh) * 2019-06-12 2024-02-06 苏州工业园区唯可达生物科技有限公司 Pd-l1靶向结合剂及其用途
AU2020299024A1 (en) * 2019-07-02 2022-01-20 Telix Pharmaceuticals (Innovations) Pty Ltd Antibodies against CAIX with reduced affinity for the neonatal Fc receptor
WO2021057906A1 (zh) * 2019-09-25 2021-04-01 科济生物医药（上海）有限公司 表达il-15的免疫效应细胞
AU2020380288B2 (en) * 2019-11-04 2024-02-01 Inovio Pharmaceuticals, Inc. Combination therapy to treat brain cancer
CN110760007B (zh) * 2019-11-21 2022-08-26 博生吉医药科技(苏州)有限公司 Cd7-car-t细胞及其制备和应用
JP2023515747A (ja) * 2019-12-28 2023-04-14 シャンハイ セル セラピー グループ カンパニー カンパニー リミテッド 免疫調節分子を発現する細胞および免疫調節分子を発現するための系
EP3878858A1 (en) * 2020-03-11 2021-09-15 Affilogic Variants of sac7d and their use in cancer therapy
US20230127169A1 (en) * 2020-03-23 2023-04-27 Curigin Co.,Ltd. Structure of oncolytic virus comprising bispecific nucleic acid molecule
JP2023526605A (ja) * 2020-05-14 2023-06-22 ヴィルトゥオーソ ビンコ，インク． ヒト化ｃｄ３８およびｉｃａｍ１抗体、ならびにそれらの使用
WO2021262499A1 (en) * 2020-06-25 2021-12-30 Promab Biotechnologies, Inc. Pd-l1-specific antibody and anti-pd-l1-car-t cells
KR20230117107A (ko) * 2020-11-04 2023-08-07 센티 바이오사이언시스, 인코포레이티드 단백질 페이로드 방출
CN112375743A (zh) * 2020-11-20 2021-02-19 山东省医学科学院附属医院 一种分泌型靶向her2抗原的嵌合抗原受体t细胞及其应用
EP4251190A4 (en) * 2020-11-30 2024-10-23 Fred Hutchinson Cancer Center PD-L1-BINDING PEPTIDES, PEPTIDE COMPLEXES AND METHODS OF USE THEREOF
CN113234152B (zh) * 2021-06-03 2023-05-02 天津科技大学 程序性死亡受体-配体1(pd-l1)特异性结合多肽及应用
WO2023274387A1 (zh) * 2021-07-01 2023-01-05 宁波茂行生物医药科技有限公司 靶向gd2的通用型car-t细胞及其制备方法和应用
EP4376887A4 (en) * 2021-07-30 2025-09-24 Janssen Biotech Inc MATERIALS AND METHODS FOR MAKING OR USING IL-23 R BINDING PROTEINS
WO2023072043A1 (zh) * 2021-10-26 2023-05-04 正大天晴药业集团股份有限公司 治疗肿瘤的联用药物
CN116410331B (zh) * 2021-12-31 2024-01-30 合源生物科技(天津)有限公司 靶向cs1的嵌合抗原受体、靶向bcma/cs1的双特异性嵌合抗原受体及其应用
CN118005806A (zh) * 2024-02-08 2024-05-10 北京市眼科研究所 嵌合抗原受体、人多能干细胞分化的表达嵌合抗原受体的小胶质细胞及其应用
CN120518751B (zh) * 2025-07-23 2025-09-19 中国人民解放军军事科学院军事医学研究院 抗人4型腺病毒特异性纳米抗体lum44及在抗病毒治疗中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11434294B2 (en) * 2016-02-25 2022-09-06 Cell Medica, Inc. Binding members to PD-L1

Family Cites Families (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4237113B4 (de) 1992-11-03 2006-10-12 "Iba Gmbh" Peptide und deren Fusionsproteine, Expressionsvektor und Verfahren zur Herstellung eines Fusionsproteins
DE19641876B4 (de) 1996-10-10 2011-09-29 Iba Gmbh Streptavidinmuteine
DE10113776B4 (de) 2001-03-21 2012-08-09 "Iba Gmbh" Isoliertes streptavidinbindendes, kompetitiv eluierbares Peptid, dieses umfassendes Fusionspeptid, dafür codierende Nukleinsäure, Expressionsvektor, Verfahren zur Herstellung eines rekombinanten Fusionsproteins und Verfahren zum Nachweis und/oder zur Gewinnung des Fusionsproteins
JP4249013B2 (ja) 2001-07-31 2009-04-02 佑 本庶 Ｐｄ−１に対し特異性を有する物質
CN105175535A (zh) 2002-05-22 2015-12-23 艾斯巴技术-诺华有限责任公司 在胞内环境中表现出增强的稳定性的免疫球蛋白构架及其鉴定方法
GB0328363D0 (en) 2003-12-06 2004-01-14 Imp College Innovations Ltd Therapeutically useful molecules
DE602005022595D1 (de) 2004-06-29 2010-09-09 Immunocore Ltd Einen modifizierten t-zellen-rezeptor exprimierende zellen
NZ600281A (en) 2006-12-27 2013-03-28 Harvard College Compositions and methods for the treatment of infections and tumors
CA2689941C (en) 2007-06-25 2019-10-29 Esbatech Ag Methods of modifying antibodies, and modified antibodies with improved functional properties
MX2011000074A (es) 2008-06-25 2011-02-24 Esbatech Alcon Biomed Res Unit Optimizacion de solubilidad de inmuno-aglutinantes.
MX345395B (es) 2008-06-25 2017-01-30 Esbatech Alcon Biomed Res Unit Humanizacion de anticuerpos de conejos usando una estructura de anticuerpos universal.
HRP20170908T1 (hr) 2008-12-09 2017-09-22 F. Hoffmann - La Roche Ag Protutijela anti-pd-l1 i njihova uporaba za poboljšanje funkcije t-stanice
PT2504364T (pt) 2009-11-24 2017-11-14 Medimmune Ltd Agentes de ligação direcionados contra b7-h1
EP3467101A3 (en) 2010-09-08 2019-06-19 Baylor College of Medicine Immunotherapy of brain tumor using geneticially engineered gd2-specific t cells
WO2013040371A2 (en) 2011-09-16 2013-03-21 Baylor College Of Medicine Targeting the tumor microenvironment using manipulated nkt cells
RS61033B1 (sr) 2011-11-28 2020-12-31 Merck Patent Gmbh Antitela na pd-l1 i njihova upotreba
KR102410078B1 (ko) * 2012-05-31 2022-06-22 소렌토 쎄라퓨틱스, 인코포레이티드 Pd-l1에 결합하는 항원 결합 단백질
EP3467124A1 (en) 2012-06-06 2019-04-10 Fundació Institut de Recerca Biomèdica IRB (Barcelona) Method for the diagnosis, prognosis and treatment of lung cancer metastasis
KR102363191B1 (ko) * 2013-02-26 2022-02-17 메모리얼 슬로안 케터링 캔서 센터 면역치료용 조성물 및 방법
KR20220029763A (ko) 2013-06-26 2022-03-08 누맙 세러퓨틱스 아게 신규한 항체 기본구조
WO2015038538A1 (en) 2013-09-10 2015-03-19 Medimmune, Llc Compositions and methods for treating sepsis
WO2015080981A1 (en) 2013-11-27 2015-06-04 Baylor College Of Medicine Csgp4-specific chimeric antigen receptor for cancer
TWI680138B (zh) 2014-01-23 2019-12-21 美商再生元醫藥公司 抗ｐｄ-ｌ１之人類抗體
EP3593812A3 (en) * 2014-03-15 2020-05-27 Novartis AG Treatment of cancer using chimeric antigen receptor
JP2017518053A (ja) * 2014-06-06 2017-07-06 メモリアル スローン−ケタリング キャンサー センター メソセリン標的化キメラ抗原受容体およびその使用
KR102360693B1 (ko) * 2014-07-11 2022-02-08 벤타나 메디컬 시스템즈, 인코포레이티드 항-pd-l1 항체 및 이의 진단 용도
DK3189073T4 (da) 2014-09-04 2025-08-18 Cellectis Trofoblastglykoprotein (5t4, tpbg)-specifikke kimære antigenreceptorer til cancerimmunterapi
DE20196219T1 (de) 2014-09-26 2021-10-28 Baylor College Of Medicine Glypican-3-Spezifische Chimäre Antigenrezeptoren für adoptive Immuntherapie
SG11201703309PA (en) 2014-10-31 2017-05-30 Baylor College Medicine Survivin specific t-cell receptor targeting tumor but not t cells
RU2017125531A (ru) 2014-12-19 2019-01-21 Дана-Фарбер Кэнсер Инститьют, Инк. Химерные антигенные рецепторы и способы их применения
GB201506642D0 (en) 2015-04-20 2015-06-03 Ucl Business Plc T cell receptor
CN105331585A (zh) * 2015-11-13 2016-02-17 科济生物医药（上海）有限公司 携带pd-l1阻断剂的嵌合抗原受体修饰的免疫效应细胞

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11434294B2 (en) * 2016-02-25 2022-09-06 Cell Medica, Inc. Binding members to PD-L1

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Kontermann. Strategies to Extend Plasma Half-Lives of Recombinant Antibodies. Biodrugs 2009; 23 (2): 93-109 (Year: 2009) *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12378298B2 (en) 2015-04-20 2025-08-05 Ucl Buseness Ltd T cell receptor
US11643451B2 (en) 2016-10-19 2023-05-09 Ucl Business Ltd T cell receptor
EP3529267B1 (en) * 2016-10-19 2023-07-26 UCL Business Ltd T cell receptor
US11998607B2 (en) 2016-12-08 2024-06-04 Immatics Biotechnologies Gmbh T cell receptors and immune therapy using the same
US11535669B2 (en) 2018-07-09 2022-12-27 Precigen, Inc. Fusion constructs and methods of using thereof
RU2733430C1 (ru) * 2019-11-19 2020-10-01 Общество с ограниченной ответственностью «Реал Таргет» Создание конъюгатов gd2-специфичных антител и фрагментов gd2-специфичных антител с препаратами
WO2021113307A3 (en) * 2019-12-02 2021-07-22 Dana-Farber Cancer Institute, Inc. Antibodies against pd-l1 and methods of use thereof
JP2023504271A (ja) * 2019-12-02 2023-02-02 デイナ ファーバー キャンサー インスティチュート，インコーポレイテッド Ｐｄ－ｌ１に対する抗体およびその使用方法
WO2021252654A1 (en) * 2020-06-11 2021-12-16 Innovative Cellular Therapeutics Holdings, Ltd. Cell/gene therapies targeting mage-a4 peptide
US12194094B2 (en) 2020-07-10 2025-01-14 Precigen, Inc. Fusion constructs and methods of using thereof
US12350318B2 (en) 2021-08-13 2025-07-08 Inovio Pharmaceuticals, Inc. Combination therapy to treat brain cancer
WO2023147564A3 (en) * 2022-01-28 2023-10-12 The University Of North Carolina At Chapel Hill Natural killer t-cells and methods of using the same

Also Published As

Publication number Publication date
AU2017224843A1 (en) 2018-08-23
RU2018128464A (ru) 2020-03-25
CN116082505A (zh) 2023-05-09
US20190055312A1 (en) 2019-02-21
ES2903408T3 (es) 2022-04-01
US11434294B2 (en) 2022-09-06
AU2017223846A1 (en) 2018-08-23
CN108699153B (zh) 2022-09-23
EP4086285A1 (en) 2022-11-09
SG11201807188VA (en) 2018-09-27
BR112018067698A2 (pt) 2019-01-08
CN108699153A (zh) 2018-10-23
EP3420001B1 (en) 2021-12-01
US20230235061A1 (en) 2023-07-27
CA3014001A1 (en) 2017-08-31
WO2017147383A1 (en) 2017-08-31
JP2019513009A (ja) 2019-05-23
KR20190003938A (ko) 2019-01-10
CN108884164B (zh) 2022-12-27
WO2017147383A8 (en) 2018-11-15
SG11201807187XA (en) 2018-09-27
JP7080819B2 (ja) 2022-06-06
BR112018067696A2 (pt) 2019-01-08
RU2018128462A (ru) 2020-03-25
EP3420001A1 (en) 2019-01-02
JP7082574B2 (ja) 2022-06-08
JP2019515646A (ja) 2019-06-13
EP3420000A1 (en) 2019-01-02
CN108884164A (zh) 2018-11-23
JP2022116230A (ja) 2022-08-09
WO2017144681A1 (en) 2017-08-31
KR20180111870A (ko) 2018-10-11
CA3014067A1 (en) 2017-08-31

Similar Documents

Publication Publication Date Title
US20190048085A1 (en) Modified cells for immunotherapy
KR102898514B1 (ko) 다양한 다중 항원의 표적화를 위한 역방향 범용 키메라 항원 수용체 발현 면역 세포, 이의 제조 방법 및 암, 감염증 및 자가면역 장애의 치료를 위한 이의 용도
US10766943B2 (en) Universal chimeric antigen expressing immune cells for targeting of diverse multiple antigens and method of manufacturing the same and use of the same for treatment of cancer, infections and autoimmune disorders
ES2966099T3 (es) Receptores de antígeno quiméricos dirigidos al antígeno de maduración de células B y usos de los mismos
JP2024069500A (ja) 抗ｇｐｃ３一本鎖抗体を含むｃａｒ
IL297916A (en) Compositions and methods for tcr reprogramming using cd70 specific fusion proteins
US20220267420A1 (en) Foxp3 targeting agent compositions and methods of use for adoptive cell therapy
EP4382119A1 (en) A kit for use in the treatment of hematological cancer
EP4566619A1 (en) Targeting modules against cd19 and cd20 for use in a method for stimulating a reversed chimeric antigen receptor-mediated immune response in a mammal
RU2824391C2 (ru) Иммунные клетки, экспрессирующие обратный универсальный химерный антигенный рецептор, для нацеливания на различные многочисленные антигены и способ их получения и применения для лечения рака, инфекций и аутоиммунных заболеваний
US20240148867A1 (en) Methods of treating cancer with a combination of adoptive cell therapy and a targeted immunocytokine
HK40068149A (en) Universal chimeric antigen receptor expressing immune cells and method of manufacturing the same and the therapeutic use of the same
HK40125822A (en) Targeting modules against cd19 and cd20 for use in a method for stimulating a reversed chimeric antigen receptor-mediated immune response in a mammal
AU2023389158A1 (en) A kit for use in the treatment of hematological cancer
WO2025120563A1 (en) Switchable universal chimeric antigen receptors and their use
AU2023289687A1 (en) Targeting modules against cd123 for use in a method for stimulating a chimeric antigen receptor-mediated immune response in a mammal
CN119213128A (zh) 与Eva1蛋白质结合的人源化抗体或其功能性片段、抗体药物缀合物和嵌合抗原受体

Legal Events

Date Code Title Description
STPP Information on status: patent application and granting procedure in general

Free format text: APPLICATION UNDERGOING PREEXAM PROCESSING

STPP Information on status: patent application and granting procedure in general

Free format text: APPLICATION DISPATCHED FROM PREEXAM, NOT YET DOCKETED

AS Assignment

Owner name: THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL, N

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DOTTI, GIANPIETRO;LANDONI, ELISA;REEL/FRAME:049318/0766

Effective date: 20170405

Owner name: CELL MEDICA SWITZERLAND AG, SWITZERLAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SHANSHIEV, ABDIJAPAR;KRETZSCHMAR, TITUS;DROSTE, MIRIAM;AND OTHERS;SIGNING DATES FROM 20190211 TO 20190220;REEL/FRAME:049318/0782

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

AS Assignment

Owner name: CELL MEDICA INC., TEXAS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:CELL MEDICA SWITZERLAND AG;REEL/FRAME:054988/0114

Effective date: 20201222

Owner name: CELL MEDICA INC., TEXAS

Free format text: ASSIGNMENT OF ASSIGNOR'S INTEREST;ASSIGNOR:CELL MEDICA SWITZERLAND AG;REEL/FRAME:054988/0114

Effective date: 20201222

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

AS Assignment

Owner name: OAKTREE FUND ADMINISTRATION, LLC, CALIFORNIA

Free format text: SECURITY INTEREST;ASSIGNOR:CELL MEDICA, INC.;REEL/FRAME:056782/0954

Effective date: 20210702

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

AS Assignment

Owner name: CELL MEDICA, INC., TEXAS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:CELL MEDICA INC.;REEL/FRAME:060529/0195

Effective date: 20220609

Owner name: CELL MEDICA, INC., TEXAS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:CELL MEDICA INC.;REEL/FRAME:060529/0066

Effective date: 20220609

Owner name: CELL MEDICA, INC., TEXAS

Free format text: ASSIGNMENT OF ASSIGNOR'S INTEREST;ASSIGNOR:CELL MEDICA INC.;REEL/FRAME:060529/0195

Effective date: 20220609

Owner name: CELL MEDICA, INC., TEXAS

Free format text: ASSIGNMENT OF ASSIGNOR'S INTEREST;ASSIGNOR:CELL MEDICA INC.;REEL/FRAME:060529/0066

Effective date: 20220609

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION