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  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
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  • A61K40/41—Vertebrate antigens
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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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  • 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
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  • 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
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  • 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
  • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
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  • 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)
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  • 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
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
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  • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
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  • C12N2510/00—Genetically modified cells

Definitions

• HCT Allogeneic hematopoietic cell transplantation
• HCT is an effective therapy for hematological malignancies but it is limited by acute graft-versus-host disease (GVHD).
• GVHD arises when donor T cells respond to genetically defined proteins on host cells, and is a key contributor to the high mortality associated with HCT.
• DC Dendritic cells
• Chimeric antigen receptor (CAR) polypeptides are disclosed that can be used with adoptive cell transfer to suppress alloreactive cells, such as donor T cells.
• the disclosed CAR polypeptides contain in an ectodomain an anti-CD83 binding agent that can bind CD83-expressing cells.
• an immune effector cell genetically modified to express the disclosed CAR polypeptide.
• the anti-CD83 binding agent is in some embodiments an antibody fragment that specifically binds CD83.
• the antigen binding domain can be a Fab or a single-chain variable fragment (scFv) of an antibody that specifically binds CD83.
• the anti-CD83 binding agent is in some embodiments an aptamerthat specifically binds CD83.
• the anti-CD83 binding agent can be a peptide aptamer selected from a random sequence pool based on its ability to bind CD83.
• the anti-CD83 binding agent can also be a natural ligand of CD83, or a variant and/or fragment thereof capable of binding CD83.
• the anti-CD83 scFv can comprise a variable heavy (V H ) domain having CDR1 , CDR2 and CDR3 sequences and a variable light (VL) domain having CDR1 , CDR2 and CDR3 sequences.
• V H variable heavy
• VL variable light
• the CDR1 sequence of the V H domain comprises the amino acid sequence GFSITTGGYWWT (SEQ ID NO:1), SDGIS (SEQ ID NO:7), or SNAMI (SEQ ID NO:13);
• CDR2 sequence of the V H domain comprises the amino acid sequence GYIFSSGNTNYNPSIKS (SEQ ID NO:2), IISSGGNTYYASWAKG (SEQ ID NO:8), or AMDSNSRTYYATWAKG (SEQ ID NO:14);
• CDR3 sequence of the V H domain comprises the amino acid sequence CARAYGKLGFDY (SEQ ID NO:3), WGGTYSI (SEQ ID NO:9), or GDGGSSDYTEM (SEQ ID NO:15);
• CDR1 sequence of the VL comprises the amino acid sequence TLSSQHSTYTIG (SEQ ID NO:4), QSSQSVYNNDFLS (SEQ ID NO:10).
• CDR2 sequence of the V L domain comprises the amino acid sequence VNSDGSHSKGD (SEQ ID NO:5), YASTLAS (SEQ ID NO:11), or QASSLAS (SEQ ID NO:17); and CDR3 sequence of the V L domain comprises the amino acid sequence GSSDSSGYV (SEQ ID NO:6),
• TGTYGNSAWYEDA SEQ ID NO:12
• LGEYSISADNH SEQ ID NO:18
• the CDR1 sequence of the V H domain comprises the amino acid sequence GFSITTGGYWWT (SEQ ID NO:1)
• CDR2 sequence of the V H domain comprises the amino acid sequence
• CDR3 sequence of the V H domain comprises the amino acid sequence CARAYGKLGFDY (SEQ ID NO:3)
• CDR1 sequence of the VL comprises the amino acid sequence TLSSQHSTYTIG (SEQ ID NO:4)
• CDR2 sequence of the VL domain comprises the amino acid sequence VNSDGSHSKGD (SEQ ID NO:S)
• CDR3 sequence of the V L domain comprises the amino acid sequence GSSDSSGYV (SEQ ID NO:6).
• the CDR1 sequence of the VH domain comprises the amino acid sequence SDGIS (SEQ ID NO:7)
• CDR2 sequence of the V H domain comprises the amino acid sequence IISSGGNTYYASWAKG (SEQ ID NO:8)
• CDR3 sequence of the VH domain comprises the amino acid sequence WGGTYSI (SEQ ID NO:9)
• CDR1 sequence of the V L comprises the amino acid sequence QSSQS VYNNDFLS (SEQ ID NO:10)
• CDR2 sequence of the V L domain comprises the amino acid sequence YASTLAS (SEQ ID NO:11)
• CDR3 sequence of the VL domain comprises the amino acid sequence TGTYGNSAWYEDA (SEQ ID NO:12).
• the CDR1 sequence of the V H domain comprises the amino acid sequence SNAMI (SEQ ID NO:13)
• CDR2 sequence of the V H domain comprises the amino acid sequence AMDSNSRTYYATWAKG (SEQ ID NO:14)
• CDR3 sequence of the V H domain comprises the amino acid sequence GDGGSSDYTEM (SEQ ID NO: 15)
• CDR1 sequence of the V L comprises the amino acid sequence QSSQSVYGNNELS (SEQ ID NO:16)
• CDR2 sequence of the V L domain comprises the amino acid sequence QASSLAS (SEQ ID NO: 17)
• CDR3 sequence of the V L domain comprises the amino acid sequence LGEYSISADNH (SEQ ID NO: 18).
• the anti-CD83 scFv V H domain comprises the amino acid sequence:
• the anti-CD83 scFv V L domain comprises the amino acid sequence:
• the anti-CD83 scFv V H domain comprises the amino acid sequence:
• the anti-CD83 scFv V L domain comprises the amino acid sequence:
• the anti-CD83 scFv V H domain comprises the amino acid sequence:
• the anti-CD83 scFv VL domain comprises the amino acid sequence:
• the anti-CD83 scFv V H domain comprises the amino acid sequence:
• the anti-CD83 scFv VL domain comprises the amino acid sequence:
• the anti-CD83 scFv V H domain comprises the amino acid sequence:
• the anti-CD83 scFv VL domain comprises the amino acid sequence:
• the anti-CD83 scFv V H domain comprises the amino acid sequence:
• the anti-CD83 scFv VL domain comprises the amino acid sequence:
• the anti-CD83 scFv V H domain comprises the amino acid sequence:
• the anti-CD83 scFv VL domain comprises the amino acid sequence:
• the anti-CD83 scFv V H domain comprises the amino acid sequence:
• the anti-CD83 scFv VL domain comprises the amino acid sequence:
• the anti-CD83 scFv V H domain comprises the amino acid sequence:
• the anti-CD83 scFv V L domain comprises the amino acid sequence:
• the anti-CD83 scFv V L domain comprises the amino acid sequence:
• the anti-CD83 scFv VL domain comprises the amino acid sequence:
• the anti-CD83 scFv VL domain comprises the amino acid sequence:
• the anti-CD83 scFv VL domain comprises the amino acid sequence:
• the anti-CD83 scFv VL domain comprises the amino acid sequence: LTQSPLSLPVTLGQPASISCKSSQSLVDSDGNTYLNWFQQRPGQSPRRLIYKVSNR DSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPRTFGQGTKVEIKR
• the anti-CD83 scFv V L domain comprises the amino acid sequence:
• the anti-CD83 scFv V_, domain comprises the amino acid sequence:
• the anti-CD83 scFv V L domain comprises the amino acid sequence:
• the anti-CD83 scFv V_. domain comprises the amino acid sequence:
• the anti-CD83 scFv V L domain comprises the amino acid sequence:
• the anti-CD83 scFv V L domain comprises the amino acid sequence:
• the anti-CD83 scFv V H domain has been humanized and comprises the amino acid sequence:
• the anti-CD83 scFv V H domain has been humanized and comprises the amino acid sequence:
• the anti-CD83 scFv V H domain has been humanized and comprises the amino acid sequence:
• the anti-CD83 scFv V H domain has been humanized and comprises the amino acid sequence:
• the anti-CD83 scFv V H domain has been humanized and comprises the amino acid sequence:
• the anti-CD83 scFv V H domain has been humanized and comprises the amino acid sequence:
• the anti-CD83 scFv V L domain has been humanized and comprises the amino acid sequence:
• the anti-CD83 scFv V_, domain has been humanized and comprises the amino acid sequence:
• the heavy and light chains are preferably separated by a linker.
• Suitable linkers for scFv antibodies are known in the art.
• the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:56).
• the anti-CD83 scFv comprises an amino acid sequence:
• the anti-CD83 scFv comprises an amino acid sequence:
• the anti-CD83 scFv comprises an amino acid sequence:
• the anti-CD83 scFv comprises an amino acid sequence:
• the anti-CD83 scFv comprises an amino acid sequence:
• the anti-CD83 scFv comprises an amino acid sequence:
• the anti-CD83 scFv comprises an amino acid sequence:
• the anti-CD83 scFv comprises an amino acid sequence:
• the anti-CD83 scFv comprises an amino acid sequence:
• the anti-CD83 scFv comprises an amino acid sequence:
• the anti-CD83 scFv comprises an amino acid sequence:
• the anti-CD83 scFv comprises an amino acid sequence:
• the anti-CD83 scFv comprises an amino acid sequence:
• the anti-CD83 scFv comprises an amino acid sequence:
• the anti-CD83 scFv comprises an amino acid sequence:
• the disclosed polypeptides can also contain a transmembrane domain and an endodomain capable of activating an immune effector cell.
• the endodomain can contain a signaling domain and one or more co-stimulatory signaling regions.
• the intracellular signaling domain is a CD3 zeta (CD3Q signaling domain.
• the costimulatory signaling region comprises the cytoplasmic domain of CD28, 4-1 BB, or a combination thereof. In some cases, the costimulatory signaling region contains 1 , 2, 3, or 4 cytoplasmic domains of one or more intracellular signaling and/or costimulatory molecules. In some embodiments, the co-stimulatory signaling region contains one or more mutations in the cytoplasmic domains of CD28 and/or 4-1 BB that enhance signaling.
• the CAR polypeptide contains an incomplete endodomain.
• the CAR polypeptide can contain only an intracellular signaling domain or a co-stimulatory domain, but not both.
• the immune effector cell is not activated unless it and a second CAR polypeptide (or endogenous T-cell receptor) that contains the missing domain both bind their respective antigens. Therefore, in some embodiments, the CAR polypeptide contains a CD3 zeta (CD3Q signaling domain but does not contain a costimulatory signaling region (CSR). In other embodiments, the CAR polypeptide contains the cytoplasmic domain of CD28, 4-1 BB, or a combination thereof, but does not contain a CD3 zeta (CD3Q signaling domain (SD).
• CD3 zeta CD3Q signaling domain
• the cell can be an immune effector cell selected from the group consisting of an alpha-beta T cells, a gamma-delta T cell, a Natural Killer (NK) cells, a Natural Killer T (NKT) cell, a B cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, and a regulatory T cell.
• an immune effector cell selected from the group consisting of an alpha-beta T cells, a gamma-delta T cell, a Natural Killer (NK) cells, a Natural Killer T (NKT) cell, a B cell, an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, and a regulatory T cell.
• NK Natural Kill
• the cell suppresses alloreactive donor cells, such as T cells, when the antigen binding domain of the CAR binds to CD83.
• tissue transplantation comprises a bone marrow transplantations.
• tissue transplantation comprises a solid organ transplant, including but not limited to, face transplant, abdominal wall transplant, limb transplant, upper extremity transplant, vascularized composite allograft, or whole tissue graft.
• the subject has an autoimmune diseases, sepsis, rheumatological diseases, diabetes, and/or asthma.
• FIG. 1 is a schema of a human CD83 CAR construct according to one embodiment disclosed herein.
• An anti-CD83 single chain variable fragment is followed by a CD8 hinge and transmembrane domain, as well as a 41 BB co- stimulatory domain and 003 ⁇ activation domain.
• the CAR is tagged with a fluorescent reporter at the 3' end.
• the CAR Reporter gene is cloned into a SFG retroviral vector.
• FIGs. 2A to 2E show characterization of the human CD83 CAR T cell.
• FIG. 2A is a bar graph showing the amount (mean ⁇ SEM) of T cells expressing the eGFP reporter post production among mock transduced (eGFP negative) or the CD83 CAR (eGFP positive) T cells.
• FIG. 2B is a bar graph demonstrating the relative amount (mean ⁇ SEM) of CD4 or CD8 expression among the mock transduced or the CD83 CAR T cells, Sidak's test.
• FIG. 2C shows the amount of IFNy released by mock transduced or CD83 CAR T cells after stimulation with CD83+ DCs.
• 2D shows cytotoxicity of CD83 CAR T cells or mock transduced T cells co-cultured with CD83+ DCs, measured on a real-time cell analysis system. The data are presented as the average normalized cell index over time for duplicate wells. Normalized cell index is calculated as cell index at a given time point divided by cell index at the normalized time point which is day 1 after addition of T cells. 1 representative experiment of 2 shown, Dunnett's test.
• FIG. 3 shows human CD83 chimeric antigen receptor T cells reduce alloreactivity.
• Human T cells were cultured with allogeneic, cytokine matured, monocyte-derived dendritic cells (moDC) at a DC:T cell ratio of 1 :30 (ie 100,000 T cells and 3333 moDCs).
• CD83 CAR T autologous to the cultured T cells
• T cell proliferation was measured by Ki-67 expression at day +5.
• CAR T were gated out by their expression of GFP. Controls included T cells alone (ie no proliferation), mock transduced T cells, and CD19 CAR T cells.
• FIGs. 4Ato 4D show CD83 is differentially expressed on human activated conventional CD4+ T cells (Tcon) compared to regulatory T cells (Tregs).
• Human T cells were stimulated by allogeneic moDCs (DC:T cell ration 1 :30) or CD3/CD28 beads (Bead:T cell ratio 1 :30).
• CD83 expression on activated Tcon CD4+, CD127+, CD25+
• Treg CD4+, CD127-, CD25+, Foxp3+
• FIGs. 4A and 4B are representative contour plots showing CD83 expression among Tcon (FIG. 4A) and Treg (FIG. 4B) at various time points post stimulation.
• FIGs. 4C and 4D are bar graphs showing the amount of CD83+ Tconv or Treg (mean ⁇ SEM) after allogeneic DC (FIG. 4C) or CD3/CD28 bead (FIG. 4D) stimulation.
• FIGs. 5A and 5B show human CD83 CAR T cells prevents xenogeneic GVHD.
• NSG mice received 25x10 6 human PBMCs and were inoculated with low (1x10°) or high dose (10x10 s ) CD83 CAR or mock transduced T cells. The CARs were autologous to the PBMC donor. An additional control group of mice received PBMCs alone.
• FIGs 6Ato 6D show CD83 CAR T cells significantly reduce GVHD target- organ damage by human T cells.
• NSG mice were transplanted with 25x10 6 human PBMCs plus 1x10 8 CD83 CAR or mock transduced T cells.
• Control groups consisted of mice that received no PBMCs (negative control) and mice that received PBMCs without modified T cells (secondary positive control).
• Recipient mice were humanely euthanized at day +21 and tissue GVHD severity was evaluated by an expert, blinded pathologist.
• Xenogeneic GVHD path scores (FIGs. 6A , 6C) and
• FIG 7 shows human CD83 CAR T cells reduce the expansion of donor cell expansion in vivo.
• NSG mice were transplanted with 25x10 6 human PBMCs plus 1x10 6 CD83 CAR or mock transduced T cells.
• Control groups consisted of mice that received no PBMCs (negative control) and mice that received PBMCs without modified T cells (secondary positive control).
• Recipient mice were humanely euthanized at day +21 and their spleens were removed for gross assessment and flow cytometry studies.
• a representative image shows mice that received PBMCs and CD83 CAR T cells exhibit reduced spleen size, supporting suppression of donor T cell expansion in vivo. 1 representative experiment of 2, up to 6 mice per experimental arm.
• FIGs. 8 A to 8E show human CD83 CAR T cell significantly reduces circulating mature, CD83+ DCs in vivo. NSG mice received 25x10 6 human PBMCs plus 1x10 6 CD83 CAR or mock transduced T cells.
• FIG. 8A contains representative contour plots showing the frequency of human CD83+, CD1c+ DCs in the mouse spleens at day +21.
• FIG. 8B ⁇ is a bar graph showing the absolute number (mean ⁇ SEM) of human CD83+, CD1c+ DCs in the mouse spleens at day +21 , Dunnett's test.
• FIG. 8C contains representative contour plots showing the percentage of MHC class II+, CD1c+ DCs in the recipient spleens at day +21.
• FIG. 8D is a bar graph depicting the absolute number (mean ⁇ SEM) of these cells, Dunnett's test.
• FIGs. 9 A to 9I show human CD83 CAR T cells significantly reduce pathogenic Th1 cells, and increase the Treg:Tconv ratio.
• NSG mice received 25x10 8 human PBMCs plus 1x10 8 CD83 CAR or mock transduced T cells as described. On day +21 , the mice were humanely euthanized and the amount of donor, human T cells were enumerated and characterized.
• FIG. 9A contains representative contour plots showing the frequency of human CD4+ T cells in the recipient spleens.
• FIGs. ⁇ and 9C are bar graphs showing the absolute numbers (mean ⁇ SEM) of CD4+ (FIG. 9B) and CD8+ (FIG.
• FIG. 9C T cells in the mouse spleens at day +21 , Dunnett's test.
• FIG. 9D contains contour plots depict the percentage of CD4+, CD127-, CD25+, Foxp3+ Tregs in the mouse spleens at day +21.
• FIGs. 9E and 9F are bar graphs showing the amount (mean ⁇ SEM) of Tregs (FIG. 9E) and the Treg:CD4+, CD25+ alloreactive Tconv (FIG. 9F) at day +21 in the recipient mice, Dunnett's test.
• FIG. 9E is bar graphs showing the amount (mean ⁇ SEM) of Tregs (FIG. 9E) and the Treg:CD4+, CD25+ alloreactive Tconv (FIG. 9F) at day +21 in the recipient mice, Dunnett's test.
• FIGs. 9G contains contour plots depicting the frequency of CD4+, IFNv+ Th1 cells and CD4+, IL-4+ Th2 cells in the mouse spleens at day +21.
• FIG. 10 Human CD83 CAR T cells permit CTL-mediated anti-tumor immunity.
• NSG mice received 25x10" human PBMCs plus 1x10 6 CD83 CAR or mock transduced T cells as described.
• An inoculum of irradiated K562 cells (10 7 ) was given on days 0 and +7.
• FIGs. 11 A and 11 B show CD83 expression among human CD8+ T cells after stimulation of allogeneic dendritic cells (FIG. 11 A) or CD3/CD28 beads (FIG. 11 B).
• CAR chimeric antigen receptors
• immune effector cells such as T cells or Natural Killer (NK) cells
• NK Natural Killer
• CAR T cells expressing these CARs can suppress alloreactive donor cells, such as T cells.
• CD83-specific chimeric antigen receptors CAR
• CARs generally incorporate an antigen recognition domain from the single- chain variable fragments (scFv) of a monoclonal antibody (mAb) with transmembrane signaling motifs involved in lymphocyte activation (Sadelain M, et al. Nat Rev Cancer 2003 3:35-45).
• scFv single- chain variable fragments
• mAb monoclonal antibody
• CD83-specific chimeric antigen receptor CAR that can be that can be expressed in immune effector cells to suppress alloreactive donor cells.
• the disclosed CAR is generally made up of three domains: an ectodomain, a transmembrane domain, and an endodomain.
• the ectodomain comprises the CD83- binding region and is responsible for antigen recognition. It also optionally contains a signal peptide (SP) so that the CAR can be glycosylated and anchored in the cell membrane of the immune effector cell.
• SP signal peptide
• the transmembrane domain (TD) is as its name suggests, connects the ectodomain to the endodomain and resides within the cell membrane when expressed by a cell.
• the endodomain is the business end of the CAR that transmits an activation signal to the immune effector cell after antigen recognition.
• the endodomain can contain an intracellular signaling domain (ISD) and optionally a co-stimulatory signaling region (CSR).
• ISD intracellular signaling domain
• CSR co-stimulatory signaling region
• a “signaling domain (SD) * generally contains immunoreceptortyrosine-based activation motifs (ITAMs) that activate a signaling cascade when the ITAM is phosphorylated.
• ITAMs immunoreceptortyrosine-based activation motifs
• CSR co-stimulatory signaling region
• the endodomain contains an SD or a CSR, but not both.
• an immune effector cell containing the disclosed CAR is only activated if another CAR (or a T-cell receptor) containing the missing domain also binds its respective antigen.
• the disclosed CAR is defined by the formula:
• SP represents an optional signal peptide
• CD83 represents a CD83-binding region
• HG represents an optional hinge domain
• TM represents a transmembrane domain
• CSR represents one or more co-stimulatory signaling regions
• SD represents a signaling domain
• the CAR can be a TRUCK, Universal CAR, Self-driving CAR,
• CAR T cells engineered to be resistant to immunosuppression may be genetically modified to no longer express various immune checkpoint molecules (for example, cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD1)), with an immune checkpoint switch receptor, or may be administered with a monoclonal antibody that blocks immune checkpoint signaling.
• immune checkpoint molecules for example, cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD1)
• CTL4 cytotoxic T lymphocyte-associated antigen 4
• PD1 programmed cell death protein 1
• a self-destruct CAR may be designed using RNA delivered by electroporation to encode the CAR.
• inducible apoptosis of the T cell may be achieved based on ganciclovir binding to thymidine kinase in gene-modified lymphocytes or the more recently described system of activation of human caspase 9 by a small- molecule dimerizer.
• a conditional CAR T cell is by default unresponsive, or switched 'off, until the addition of a small molecule to complete the circuit, enabling full transduction of both signal 1 and signal 2, thereby activating the CAR T cell.
• T cells may be engineered to express an adaptor-specific receptor with affinity for subsequently administered secondary antibodies directed at target antigen.
• TanCAR T cell expresses a single CAR consisting of two linked single-chain variable fragments (scFvs) that have different affinities fused to intracellular co-stimulatory domain(s) and a CD3 ⁇ 4 domain. TanCAR T cell activation is achieved only when target cells co-express both targets.
• scFvs linked single-chain variable fragments
• a dual CAR T cell expresses two separate CARs with different ligand binding targets; one CAR includes only the CD3 ⁇ 4 domain and the other CAR includes only the co-stimulatory domain(s). Dual CAR T cell activation requires co-expression of both targets.
• a safety CAR consists of an extracellular scFv fused to an intracellular inhibitory domain.
• sCAR T cells co-expressing a standard CAR become activated only when encountering target cells that possess the standard CAR target but lack the sCAR target.
• the antigen recognition domain of the disclosed CAR is usually an scFv.
• An antigen recognition domain from native T- cell receptor (TCR) alpha and beta single chains have been described, as have simple ectodomains (e.g. CD4 ectodomain to recognize HIV infected cells) and more exotic recognition components such as a linked cytokine (which leads to recognition of cells bearing the cytokine receptor). In fact almost anything that binds a given target with high affinity can be used as an antigen recognition region.
• the endodomain is the business end of the CAR that after antigen recognition transmits a signal to the immune effector cell, activating at least one of the normal effector functions of the immune effector cell.
• Effector function of a T cell may be cytolytic activity or helper activity including the secretion of cytokines. Therefore, the endodomain may comprise the "intracellular signaling domain" of a T cell receptor (TCR) and optional co-receptors. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.
• TCR T cell receptor
• Cytoplasmic signaling sequences that regulate primary activation of the TCR complex that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs (ITAMs).
• ITAMs immunoreceptortyrosine-based activation motifs
• Examples of ITAM containing cytoplasmic signaling sequences include those derived from CD8, CDZi, CD30, CD3Y, CD3E, CD32 (Fc gamma Rlla), DAP10, DAP12, CD79a, CD79b, FcyRlY, FcvRlllY, FceRIf (FCERIB), and FceRly (FCERIG).
• the intracellular signaling domain is derived from CD3 zeta (CD3Q (TCR zeta, GenBank accno. BAG36664.1).
• CD3Q TCR zeta, GenBank accno. BAG36664.1
• T-cell surface glycoprotein CD3 zeta CD3Q chain, also known as T-cell receptor T3 zeta chain or CD247 (Cluster of Differentiation 247), is a protein that in humans is encoded by the CD247 gene.
• First-generation CARs typically had the intracellular domain from the 003 ⁇ chain, which is the primary transmitter of signals from endogenous TCRs.
• Second- generation CARs add intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41 BB, ICOS) to the endodomain of the CAR to provide additional signals to the T cell.
• costimulatory protein receptors e.g., CD28, 41 BB, ICOS
• third-generation CARs combine multiple signaling domains to further augment potency.
• T cells grafted with these CARs have demonstrated improved expansion, activation, persistence, and tumor-eradicating efficiency independent of costimulatory receptor/ligand interaction (Imai C, et al. Leukemia 2004 18:676-84; Maher J, et al. Nat Biotechnol 2002 20:70-5).
• the endodomain of the CAR can be designed to comprise the CD3 ⁇ 4 signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention.
• the cytoplasmic domain of the CAR can comprise a ⁇ chain portion and a costimulatory signaling region.
• the costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.
• a costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen.
• Examples of such molecules include CD27, CD28, 4-1 BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD123, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D.
• CD28 CD28
• 4-1 BB CD137
• OX40 CD30
• CD40 CD40
• ICOS lymphocyte function-associated antigen-1
• LFA-1 lymphocyte function-associated antigen-1
• CD2 CD7
• LIGHT lymphocyte function-associated antigen-1
• NKG2C NKG2C
• B7-H3 lymphocyte function-associated antigen-1
• the CAR comprises a hinge sequence.
• a hinge sequence is a short sequence of amino acids that facilitates antibody flexibility (see, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)).
• the hinge sequence may be positioned between the antigen recognition moiety (e.g., anti-CD83 scFv) and the transmembrane domain.
• the hinge sequence can be any suitable sequence derived or obtained from any suitable molecule. In some embodiments, for example, the hinge sequence is derived from a CD8a molecule or a CD28 molecule.
• the transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from (i.e.
• CD1 comprises at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11 a, CD18) , ICOS (CD278) , 4-1 BB (CD137) , GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRF1) , CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1 , VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD
• CD29 CD29, ITGB2, CD18, LFA-1 , ITGB7, TNFR2, DNAM1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile) , CEACAM1.
• CRTAM Ly9 (CD229) , CD160 (BY55) , PSGL1 , CD 100 (SEMA4D) , SLAMF6 (NTE3-A, Ly108) , SLAM (SLAMF1 , CD150, IPO-3) , BLAME (SLAMF8) , SELPLG (CD162) , LTBR, and PAG/Cbp.
• the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
• a short oligo- or polypeptide linker such as between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the endoplasmic domain of the CAR.
• the CAR has more than one transmembrane domain, which can be a repeat of the same transmembrane domain, or can be different transmembrane domains.
• the CAR is a multi-chain CAR, as described in WO2015/039523, which is incorporated by reference for this teaching.
• a multi-chain CAR can comprise separate extracellular ligand binding and signaling domains in different transmembrane polypeptides.
• the signaling domains can be designed to assemble in juxtamembrane position, which forms flexible architecture closer to natural receptors, that confers optimal signal transduction.
• the multichain CAR can comprise a part of an FCERI alpha chain and a part of an FCERI beta chain such that the FCERI chains spontaneously dimerize together to form a CAR.
• Tables 1 , 2, and 3 below provide some example combinations of CD83- binding region, co-stimulatory signaling regions, and intracellular signaling domain that can occur in the disclosed CARs.
• CD83 b2c FCYRI-Y CD83 DAP10 CD79a
• CD83 CD286 FCYRIII-Y CD83 BTNL3 CD79b CD83 CD286 FCER ⁇ CD83 NKG2D CD8 CD83 CD286 FCERIY CD83 NKG2D 0 ⁇ 3 ⁇
• CD83 CD28 CD137/41 BB CD32
• CD83 CD28 OX40 CD32
• CD83 CD28 NKG2D CD32
• CD83 CD8 OX40 CD32
• CD83 CD8 BTNL3 CD3Y

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