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WO2023000036A1 - Récepteur antigénique chimérique utile dans le traitement du cancer - Google Patents

Récepteur antigénique chimérique utile dans le traitement du cancer Download PDF

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Publication number
WO2023000036A1
WO2023000036A1 PCT/AU2022/050776 AU2022050776W WO2023000036A1 WO 2023000036 A1 WO2023000036 A1 WO 2023000036A1 AU 2022050776 W AU2022050776 W AU 2022050776W WO 2023000036 A1 WO2023000036 A1 WO 2023000036A1
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Prior art keywords
seq
functional
cell
acid sequence
sequence
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PCT/AU2022/050776
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English (en)
Inventor
Kenneth MICKLETHWAITE
Kavitha GOWRISHANKAR
Koon Lee
Emily BLYTH
David Gottlieb
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Westmead Institute for Medical Research
University of Sydney
Western Sydney Local Health District
Original Assignee
Westmead Institute for Medical Research
University of Sydney
Western Sydney Local Health District
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Priority claimed from AU2021902252A external-priority patent/AU2021902252A0/en
Application filed by Westmead Institute for Medical Research, University of Sydney, Western Sydney Local Health District filed Critical Westmead Institute for Medical Research
Publication of WO2023000036A1 publication Critical patent/WO2023000036A1/fr
Anticipated expiration legal-status Critical
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    • 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
    • A61K40/10—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
    • A61K40/35—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4214—Receptors for cytokines
    • A61K40/4217—Receptors for interleukins [IL]
    • A—HUMAN NECESSITIES
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    • C—CHEMISTRY; METALLURGY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07K14/5403—IL-3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • 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
    • C07K14/70517—CD8
    • 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
    • C07K14/70521—CD28, CD152
    • 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
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • 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
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • 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
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • 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
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • 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
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • 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
    • 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
    • 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
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the present disclosure relates to compositions and methods for treating diseases or conditions associated with expression of CD123.
  • the disclosure provides chimeric antigen receptors (CAR) specific to CD123, and isolated nucleic acids, isolated polypeptides, and isolated vectors encoding the same, as well as recombinant cells comprising the CD123-specific CAR.
  • CAR chimeric antigen receptors
  • CAR chimeric antigen receptor
  • CARs typically comprise a T-cell activating domain, and an extracellular antibody-derived heavy and light chains sequences (otherwise known as a single chain variable fragment or 'scFv') to direct binding specificity.
  • a single chain variable fragment or 'scFv' extracellular antibody-derived heavy and light chains sequences
  • scFvs targeting known cancers can be relatively quick and inexpensive to construct.
  • Constructing and/or modifying scFvs to target previously unknown tumour antigens can also be rapid given well- studied methods of scFv engineering.
  • a major disadvantage of scFvs is the requirement for correct pairing between heavy (VH) and light (VL) chains to achieve binding specificity.
  • a VH can also pair with the VL of a neighbouring molecule and vice versa. This can result in dimerisation (i.e. formation of a diabody) or possibly oligomerisation (i.e. formation of triabodies or tetrabodies, for example) 23 .
  • dimerisation i.e. formation of a diabody
  • oligomerisation i.e. formation of triabodies or tetrabodies, for example
  • CAR chimeric antigen receptor
  • the IL-3 protein may comprise:
  • SEQ ID NO: 1 or a functional portion thereof, a functional variant of SEQ ID NO: 1 comprising at least one but not more than 20 amino acid modifications, or a functional portion thereof, or a functional variant having at least 60% identity to SEQ ID NO: 1 , or a functional portion thereof.
  • the isolated nucleic acid may encode a functional portion of a mature IL-3 protein, and wherein the functional portion comprises at least 100 amino acids, preferably at least 105 amino acids, further preferably at least 110 amino acids, most preferably not more than 115 amino acids.
  • the functional portion of the IL-3 protein comprises:
  • SEQ ID NO: 4 a functional variant of one of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 comprising at least one but not more than 20 amino acid modifications, or a functional variant having at least 60% identity to one of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  • the transmembrane domain comprises: an amino acid sequence comprising the alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a functional variant or portion thereof, SEQ ID NO: 5, or a functional variant or functional portion thereof, a functional variant of SEQ ID NO: 5 comprising at least one but not more than 20 amino acid modifications, or a functional portion thereof, or a functional variant having at least 60% identity to one of SEQ ID NO: 5, or a functional portion thereof.
  • intracellular signaling domain comprises: a functional signaling domain selected from the group consisting of: 4-1 BB, CD3 zeta, and CD27, or a combination thereof, or a functional variant or functional portion thereof,
  • SEQ ID NO: 6, or a functional variant or functional portion thereof a functional variant of SEQ ID NO: 6 comprising at least one but not more than 20 amino acid modifications, or a functional portion thereof, or a functional variant having at least 60% identity to one of SEQ ID NO: 6, or a functional portion thereof.
  • the isolated nucleic sequence may further comprise a costimulatory domain, wherein the costimulatory domain comprises: a functional costimulatory domain of a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA 1 (CDI1a/CD18), ICOS (CD278) and 4-1 BB (CD137), or a combination thereof, or a functional variant or functional portion thereof,
  • the isolated nucleic sequence may further comprise a spacer region connecting the CD123 binding domain to the transmembrane domain, wherein the spacer region comprises: a sequence derived from the stalk region of the alpha or beta chain of CD8, or a functional variant or functional portion thereof the hinge region of lgG1, lgG2, lgG3 or lgG4, or a functional variant or functional portion thereof,
  • SEQ ID NO: 8 or a functional variant or functional portion thereof, a functional variant of SEQ ID NO: 8 comprising at least one but not more than 20 amino acid modifications, or a functional portion thereof, or a functional variant having at least 60% identity to one of SEQ ID NO: 8, or a functional portion thereof.
  • the isolated nucleic sequence encodes a CAR which may comprise: a) a fusion of sequences SEQ ID NO: 1 , SEQ ID NO: 8, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 6 in that order, or a functional variant or functional portion thereof, b) a fusion of sequences SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 6 in that order, or a functional variant or functional portion thereof, c) a fusion of sequences SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 6 in that order, or a functional variant or functional portion thereof, d) a fusion of sequences SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 6 in that order, or a functional variant or functional portion thereof e) a functional variant of a CAR which may comprise:
  • isolated nucleic sequence of any one of the previous claims may comprise: a nucleic acid sequence of SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, a nucleic acid sequence having at least one modification but not more than 20 modifications to a nucleic acid sequence of SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, or a nucleic acid sequence with at least 60% identity to a nucleic acid sequence of SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14.
  • an isolated polypeptide molecule encoded by a nucleic acid of the present disclosure may comprise: an amino acid sequence comprising SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15, an amino acid sequence having at least one modification but not more than 20 modifications to an amino acid sequence of S SEQ ID NO: 11 , SEQ ID NO: 13 or SEQ ID NO: 15, or an amino acid sequence with at least 60% identity to an amino acid sequence of SEQ ID NO: 11 , SEQ ID NO: 13 or SEQ ID NO: 15.
  • a vector which may encode: an isolated nucleic acid sequence of the present disclosure, or an isolated polypeptide of the present disclosure, preferably wherein the vector: is selected from the group consisting of: a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, and a retrovirus vector, and/or further comprises at least one a promoter, a poly(A) tail, a 3'UTR, or a combination thereof.
  • an isolated cell which may comprise an isolated nucleic acid sequence of the present disclosure, an isolated polypeptide molecule of the present disclosure, or a vector of the present disclosure.
  • the cell is a T cell, further preferably a CD8+ T cell.
  • cells capable of producing a human being are excluded from the scope of protection.
  • a pharmaceutical composition which may comprise: an isolated nucleic acid, isolated polypeptide molecule, a vector, or a cell, according to the present disclosure.
  • the pharmaceutical composition may optionally comprise a pharmaceutically acceptable excipient
  • a method of manufacturing a CAR-expressing cell comprising introducing into a cell the isolated nucleic acid of any one of claims 1-10 or the vector of claim 13.
  • the cell is a T cell, further preferably a CD8+ T cell.
  • a method of treating a disease or condition associated with expression of CD123 comprising administering to a subject in need an effective amount of an isolated cell or a pharmaceutical composition of the present disclosure.
  • an isolated nucleic acid sequence an isolated polypeptide molecule, a vector, or an isolated cell, according to the present disclosure in the manufacture of a pharmaceutical composition for treating a disease or condition associated with expression of CD123.
  • the disease or condition is cancer, preferably a CD123-expressing cancer selected from the group consisting of: acute myeloid leukaemia (AML), myelodysplastic syndrome (MDS, low or high risk), acute lymphocytic leukaemia (ALL, all subtypes), diffuse large cell B-cell lymphoma (DLBCL) (including subtypes), chronic myeloid leukaemia (CML), and blastic plasmacytoid dendritic cell neoplasm (DPDCN).
  • AML acute myeloid leukaemia
  • MDS myelodysplastic syndrome
  • ALL acute lymphocytic leukaemia
  • DLBCL diffuse large cell B-cell lymphoma
  • CML chronic myeloid leukaemia
  • DPDCN blastic plasmacytoid dendritic cell neoplasm
  • FIG 1 is a schematic diagram of the CD123-specific chimeric antigen receptor constructs according to the present disclosure. All 3 constructs were identical except for the human IL-3 sequence which is either (a) a truncated human IL-3 wild type sequence (CARIL3WT; SEQ ID NO: 2); (b) a truncated human IL-3 sequence with a single K116W substitution (CARIL3S; SEQ ID NO: 3) substitution or (c) a truncated human IL-3 sequence with K116W and E22R substitutions (CARIL3D; SEQ ID NO: 4).
  • CARIL3WT truncated human IL-3 wild type sequence
  • CARIL3S truncated human IL-3 sequence with a single K116W substitution
  • CARIL3D truncated human IL-3 sequence with K116W and E22R substitutions
  • FIG. 2 is a schematic diagram of the cloning strategy for the CARIL3 constructs according to the present disclosure.
  • Panel (a) shows the strategy for creating the CARIL3WT construct; whereas panel (b) shows the strategy for creating the CARIL3S and CARIL3D constructs.
  • Figure 3 shows the enrichment of CARIL3 T-cells with differing stimulation conditions.
  • Panel (a) shows the expression of CD123 in (i) Nalm6; (ii) TF-1 and (iii )THP-1 cell lines that were used as irradiated feeders for stimulation, and as targets for intracellular cytokine flow cytometry (shown in red) overlayed with isotype control (shown in cyan).
  • Panel (b)(i) shows the gating strategy to determine the CD123 expression of cryopreserved PBMCs.
  • FIG. 4 shows the expansion of CARIL3 T-cells with differing stimulation conditions.
  • Figure 5 shows expansion of CARIL3 T-cells by weekly exposure to 4:1 irradiated autologous PBMCs.
  • Panel (b) shows CD3 proportion assessed weekly by flow cytometry.
  • Panel (d) shows proportions of CD4 + and CD8 + CAR T-cells at day 22 of culture.
  • Panel (e) shows end of culture memory phenotype, na ' ive/central memory n/scm (CD45RA+/CD62L+); cm: central memory (CD45RA-/CD62L+); em: effector memory (CD45RA-/CD62L-); emra: terminal effector (CD45RA+/CD62L-).
  • Panel (f) shows expression of immune-inhibitory receptors PD-1 , TIM3 and LAG3 on CAR T-cells at the end of culture. Pooled data represented as mean +/- SD. n.s.: P > 0.05; * : P ⁇ 0.05; ** : P ⁇ 0.01.
  • Figure 6 shows in vitro functional assessment of CARIL3 T-cells.
  • Panel (a) shows CD123 expression assessed by flow cytometry of the target cell lines (in red) overlayed with isotype control (in cyan).
  • the term "Chimeric Antigen Receptor” or alternatively a “CAR” refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "an intracellular signaling domain”).
  • the cytoplasmic signaling domain may comprise a functional signaling domain derived from a stimulatory molecule.
  • the stimulatory molecule may be the zeta chain associated with the T cell receptor complex.
  • the cytoplasmic signaling domain may further comprise one or more functional signaling domains derived from at least one co stimulatory molecule preferably chosen from 4-1 BB (i.e., CD137) and/or CD28.
  • the CAR may therefore comprise a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule.
  • the CAR may comprise a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule.
  • the CAR may comprise a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule.
  • the CAR may also comprise an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein.
  • the leader sequence may be optionally cleaved from the antigen recognition domain during cellular processing and localisation of the CAR to the cellular membrane.
  • the CAR may also comprise an optional fluorescence marker sequence at the carboxy-terminus (C-ter) of the CAR fusion protein.
  • the fluorescence marker sequence is optionally eGFP.
  • signaling domain refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.
  • stimulation refers to a primary response induced by binding of a stimulatory molecule (e.g., a T cell receptor (TCR)/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex.
  • a stimulatory molecule e.g., a T cell receptor (TCR)/CD3 complex
  • TCR T cell receptor
  • Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-b, and/or reorganisation of cytoskeletal structures, and the like.
  • the term "stimulatory molecule” refers to a molecule expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway.
  • a primary signal may be initiated by, for instance, binding of a TCR/CD3 complex with an MFIC molecule loaded with peptide, which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • a primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine based activation motif or ITAM.
  • ITAM immunoreceptor tyrosine based activation motif
  • Examples of an ITAM containing primary cytoplasmic signaling sequence that is of particular use includes, but is not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS”) and CD66d.
  • the intracellular signaling domain comprises an intracellular signaling sequence, e.g.
  • the primary signaling sequence of CD3-zeta is human derived or an equivalent from a non-human species, e.g., mouse, rodent, monkey, ape and the like.
  • PBMC peripheral blood mononuclear cells
  • PBMC sample may have been subjected to a selection step to contain non adherent PBMC (which contain T cells, B cells, natural killer (NK) cells, NK T cells and DC precursors).
  • a PBMC sample preferably contains lymphocytes (B cells, T cells, NK cells, NKT cells).
  • these cells can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates layers of blood, with the PBMC forming a cell ring under a layer of plasma.
  • PBMC can be extracted from whole blood using a hypotonic lysis buffer, which will preferentially lyse red blood cells. Such procedures are known in the art.
  • the term "antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC's) on its surface.
  • T-cells may recognise these complexes using their T-cell receptors (TCRs).
  • APCs process antigens and present them to T-cells.
  • intracellular signaling domain refers to an intracellular portion of a molecule.
  • the intracellular signaling domain generates a signal that promotes an immune effector function of the CAR containing cell, e.g. a CAR-T cell.
  • immune effector function e.g. in a CAR-T cell
  • cytolytic activity and helper activity including the secretion of cytokines.
  • Exemplary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen-dependent simulation.
  • the intracellular signaling domain may comprise a costimulatory intracellular domain.
  • Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen- independent stimulation.
  • a primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule.
  • a primary intracellular signaling domain can comprise a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP12.
  • zeta or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta”, “CD3-zeta stimulatory domain” or a “TCR-zeta stimulatory domain” is defined as the amino acid residues from the cytoplasmic domain of the CD3 zeta chain that are sufficient to functionally transmit an initial signal necessary for T cell activation.
  • the zeta chain may be human derived or comprise equivalent residues from a non human species, e.g., mouse, rodent, monkey, ape and the like, that are functional orthologues thereof.
  • costimulatory molecule refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response.
  • Costimulatory molecules include, but are not limited to, an MFIC class I molecule, BTLA and a Toll ligand receptor, as well as 0X40, CD2, CD27, CD28, CDS, ICAM-1 , LFA-1 (CD11 a/CD18) and 4-1 BB (CD137).
  • a costimulatory intracellular signaling domain can be derived from the intracellular portion of a costimulatory molecule.
  • a costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors.
  • the intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.
  • peptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogues, fusion proteins, among others.
  • a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolised in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologues, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • nucleic acid sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or a RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
  • promoter refers to a DNA sequence recognised by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
  • promoter/regulatory sequence refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence.
  • this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • constitutive promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • inducible promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • tissue-specific promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • a 5' cap (also termed an RNA cap, an RNA 7- methylguanosine cap or an RNA m 7G cap) is a modified guanine nucleotide that has been added to the "front" or 5' end of a eukaryotic messenger RNA shortly after the start of transcription.
  • the 5' cap can comprise a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition can be coupled to transcription, and occurs co-transcriptionally, such that each influences the other.
  • RNA polymerase Shortly after the start of transcription, the 5' end of the mRNA being synthesised is bound by a cap synthesising complex associated with RNA polymerase. This enzymatic complex catalyses the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction.
  • the capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.
  • a "poly(A)" is a series of adenosines attached by polyadenylation to the imRNA.
  • a polyA sequence can be between 50 and 5000 adenosines in length, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localisation, stability or efficiency of translation.
  • polyadenylation refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule.
  • mRNA messenger RNA
  • the 3'poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase.
  • poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal.
  • Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm.
  • the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase.
  • the cleavage site is usually characterised by the presence of the base sequence AAUAAA near the cleavage site.
  • adenosine residues are added to the free 3' end at the cleavage site.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • the term "expression” refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
  • the term "transfer vector” refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “transfer vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like.
  • viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • transfected or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into a host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • antibody refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen.
  • Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.
  • antibody heavy chain refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.
  • antibody light chain refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations.
  • Kappa (K) and lambda (l) light chains refer to the two major antibody light chain isotypes.
  • recombinant antibody refers to an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.
  • anti-tumour effect refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumour volume, a decrease in the number of tumour cells, a decrease in the number of metastases, an increase in life expectancy, decrease in tumour cell proliferation, decrease in tumour cell survival, or amelioration of various physiological symptoms associated with the cancerous condition.
  • An "anti-tumour effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies in prevention of the occurrence of tumour in the first place.
  • autologous refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • allogeneic refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically
  • xenogeneic refers to a graft derived from an animal of a different species.
  • cancer refers to a disease characterised by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include, but are not limited to, haematological malignancies (also known as 'blood cancers') such as various types of leukemia (acute lymphocytic (ALL), chronic lymphocytic (CLL), acute myeloid (AML), chronic myeloid (CML), acute monocytic leukemia (AMoL), for example), myeloma, and lymphoma (Hodgkin's and non-Hodgkin's (NHL)) and the like.
  • ALL acute lymphocytic
  • CLL chronic lymphocytic
  • AML acute myeloid
  • CML chronic myeloid
  • AoL acute monocytic leukemia
  • myeloma myeloma
  • NHL lymphoma
  • NHL lymphoma
  • CD123 refers to the interleukin 3 receptor, alpha (IL-3Ra) a membrane receptor polypeptide that is preferentially expressed on certain types of pluripotent stem cells and cancer cells, such as leukaemia cancer cells (e.g., acute myeloid leukaemia cells).
  • IL-3Ra alpha
  • CD123 includes any CD123 variant, isoform, and interspecific homologue unless otherwise noted.
  • CD123-expressing cancer is a cancer that is associated with CD123, either directly or indirectly, which is treatable with an anti-CD123 therapy as disclosed herein.
  • a CD123-expressing cancer may be a blood cancer, such as acute myeloid leukaemia (AML), myelodysplastic syndrome (MDS, low or high risk), acute lymphocytic leukaemia (ALL, all subtypes), diffuse large cell B-cell lymphoma (DLBCL) (including subtypes), chronic myeloid leukaemia (CML), or blastic plasmacytoid dendritic cell neoplasm (DPDCN).
  • AML acute myeloid leukaemia
  • MDS myelodysplastic syndrome
  • ALL acute lymphocytic leukaemia
  • DLBCL diffuse large cell B-cell lymphoma
  • CML chronic myeloid leukaemia
  • DPDCN blastic plasmacytoid dendritic cell neoplasm
  • composition means a product manufactured to comprise one or more active ingredients, and may include one or more inert ingredients, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the compositions of the present disclosure encompass any composition made by admixing a compound, substance or ingredient of the present disclosure and optionally a pharmaceutically or nutraceutically acceptable excipient (pharmaceutically acceptable carrier).
  • pharmaceutically acceptable derivatives includes, but is not limited to, pharmaceutically o acceptable salts, esters, salts of such esters, ethers, or any other derivative including prodrugs and metabolites, which upon administration to a subject (e.g. patient, human or animal) in need is capable of providing, directly or indirectly, a bioactive substance as otherwise described herein.
  • the term "pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animal without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • amino acid analogue (or “non-standard amino acid”) is meant to include all amino acid-like compounds that are similar in structure and/or overall shape to one or more of the twenty L-amino acids commonly found in naturally occurring proteins (Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, lie or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gin or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y, as defined and listed in WIPO Standard ST.25 (2009), Annex C, Appendix 2, Table 3).
  • An amino acid analogue can include a natural amino acid that comprises a modified side chain or backbone.
  • a modification may include, without limitation, substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as methyl, or hydroxyl group, for example) or an atom (such as Cl or Br, for example), deletion of a group, substitution of a covalent bond (single bond for double bond, for example), or combinations thereof (see also WIPO Standard ST.25 (2009), Annex C, Appendix 2, Table 4, for example).
  • amino acid with respect to compositions and methods for combating oxidative stress includes amino acid analogues.
  • an "analogue” or “derivative” of a substance in other contexts includes a functionally equivalent substance. Functional equivalence may be achieved by modifying the structure of the original substance using chemical or recombinant techniques, or by identifying a substance having a different structure having the same or similar function as the original substance.
  • nucleic acid in the context of a sequence modification is intended to refer to a nucleic acid or amino acid modification which may or may not affect or alter the functional characteristics encoded protein. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • nucleic acid is used inter-changeably herein with “gene”, “cDNA, “mRNA”, “oligonucleotide,” and “polynucleotide”.
  • Nucleotide analogues include nucleotides having modifications in the chemical structure of the base, sugar and/or phosphate, including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitution of 5-bromo- uracil, and the like; and 2'-position sugar modifications, including but not limited to, sugar-modified ribonucleotides in which the 2'-OH is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN.
  • shRNAs also can comprise non-natural elements such as non-natural bases, e.g., ionosin and xanthine, non natural sugars, e.g., 2'-methoxy ribose, or non-natural phosphodiester linkages, e.g., methylphosphonates, phosphorothioates and peptides. Modifications may be introduced into an antibody or antibody fragment of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR- mediated mutagenesis.
  • non-natural bases e.g., ionosin and xanthine
  • non natural sugars e.g., 2'-methoxy ribose
  • non-natural phosphodiester linkages e.g., methylphosphonates, phosphorothioates and peptides. Modifications may be introduced into an antibody or antibody fragment of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and
  • modification may also be used in reference to an amino acid substitutions, additions and deletions.
  • Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta- branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • one or more amino acid residues within a CAR of the present disclosure can be replaced with other amino acid residues from the same side chain family and the altered CAR, which may be referred to generally as a 'variant', can be tested using a functional assay.
  • the term “functional variant” or “functional portion” in connection with a modified nucleic acid or amino acid sequence means that the encoded protein variant or portion has equivalent or better function than the unmodified (wild type) protein.
  • isolated or “purified” refers to a material that is removed from its original environment (e.g. the natural environment, if it is naturally occurring). For example, the material is said to be “purified” when it is present in a particular composition or mixture in a higher concentration than exists in a naturally occurring or wild type organism or in combination with components not normally present upon expression from a naturally occurring or wild type organism.
  • nucleic acid or protein/polypeptide present in a living organism is not isolated, but the same nucleic acid or protein/polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • nucleic acids or proteins/polypeptides could, for example, be part of a composition, and still be isolated in that such a composition is not part of the natural environment of the nucleic acids or proteins/polypeptides.
  • the term “sufficiently identical” is intended to encompass an amino acid or nucleic acid sequence that has at least about 60% or 65% sequence identity, about 70% or 75% sequence identity, about 80% or 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to a reference sequence.
  • sequences are aligned for optimal comparison purposes.
  • the two sequences may be of the same length or of different lengths. Further, the comparison may be across the entirety of the reference sequence or a portion.
  • the percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • a non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) 7 , modified as in Karlin and Altschul (1993) 8 .
  • Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) 9 .
  • Gapped BLAST in BLAST 2.0
  • PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (see Altschul et al. (1997) 10 ).
  • the default parameters of the respective programs e.g., BLASTX and BLASTN
  • Alignment may also be performed manually by inspection.
  • ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence.
  • the ClustalW algorithm may be used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, Calif.). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed.
  • Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) 12 .
  • ALIGN program version 2.0
  • GCG Wisconsin Genetics Software Package Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA).
  • a PAM120 weight residue table a gap length penalty of 12, and a gap penalty of 4 can be used.
  • GAP Version 10 which uses the algorithm of Needleman and Wunsch (1970) 13 J. Mol. Biol. 48(3):443-453, is preferred to determine sequence identity or similarity using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity or % similarity for an amino acid sequence using GAP weight of 8 and length weight of 2, and the BLOSUM62 scoring program. Equivalent programs may also be used.
  • Equivalent program is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
  • subject or “patient” are intended to include living organisms (e.g. mammals, humans and or non-human animals).
  • administration concurrently or “co-administering” and the like refer to the administration of a single composition containing two or more actives, or the administration of each active as separate compositions and/or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such actives are administered as a single composition.
  • simultaneous is meant that the active agents are administered at substantially the same time, and preferably together in the same formulation.
  • treat means accomplishing one or more of the following: (a) reducing the severity and/or duration of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in subjects that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s).
  • prevent means preventing that a disease or disorder occurs in a subject.
  • the terms "effective amount” or “therapeutic amount” are intended to mean that amount of a substance that will elicit the biological or medical response of a tissue, a system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.
  • the term “prophylactically effective amount” is intended to mean that amount of a substance that will prevent or reduce the risk of occurrence of the biological or medical event that is sought to be prevented in a tissue, a system, animal or human by a researcher, veterinarian, medical doctor or other clinician.
  • the expression "is for administration” and “is to be administered” have the same meaning as “is prepared to be administered”.
  • the statement that an active compound "is for administration” has to be understood in that said active compound has been formulated and made up into doses so that said active compound is in a state capable of exerting its therapeutic activity.
  • the present disclosure is directed to a CAR which avoids CAR clustering that is associated with scFv-mediated dimerisation/oligomerisation.
  • the cluster of differentiation (CD) marker CD123 (also known as interleukin-3 receptor-a or 'IL-3R a') is cell surface marker that is widely overexpressed in various haematological malignancies, including acute myeloid leukaemia (AML), B-cell acute lymphoblastic leukaemia, hairy cell leukaemia, Hodgkin lymphoma and blastic plasmacytoid dendritic neoplasm (BPDCN).
  • AML acute myeloid leukaemia
  • BPDCN blastic plasmacytoid dendritic neoplasm
  • CD123 is expressed in over 90% of cases of AML and on leukaemic stem cells while exhibiting relatively low expression on normal haematopoietic stem cells and normal tissue. Elimination of the self-regenerating leukaemic stem cell population is crucial in the elimination of AML.
  • CD123 being the target of multiple therapeutic agents including CAR T-cells. While clinical outcomes for a small number of patients treated with CD123-specifc CAR T-cells in relapsed/refractory AML showed promising results 14 , the anti-CD123 scFv moiety upon which this CAR is based is problematic for the reasons alluded to above.
  • the present disclosure is predicated on the surprising discovery that a CD123-specific CAR could be constructed which avoids the use of an anti-CD123 scFv-based binding moiety.
  • the present inventors have found it possible to construct a CD123-specific CAR that comprises the IL-3 protein, the natural ligand for CD123 (IL-3Ra).
  • the CARs of the present disclosure do not include a scFv-based anti-CD123 binding moiety.
  • the present disclosure provides an isolated nucleic acid sequence encoding a CD123-specific chimeric antigen receptor (CAR), wherein the binding specificity of the CAR is mediated by the IL-3 protein, or a functional variant or functional portion thereof.
  • CAR CD123-specific chimeric antigen receptor
  • the word 'functional' in this context means that the variant or portion of IL-3 retains the ability to interact with CD123, whereby IL-3-CD123 interaction results in in the initiation of downstream signalling.
  • the isolated nucleic acid sequence encodes the mature human IL-3 protein which comprises 133 amino acids.
  • the mature, unmodified human IL-3 is represented by SEQ ID NO: 1.
  • the isolated nucleic acid encodes non-human IL-3.
  • the isolated nucleic acid sequence encodes a functional variant or functional portion of an IL-3 protein which is of at least 100 amino acids, preferably at least 105 amino acids, most preferably at least 110 amino acids.
  • the isolated nucleic acid encodes a functional variant or functional portion of an IL-3 protein that represented by SEQ ID NO: 2 (which is amino acids 15-125 of SEQ ID NO. 1 ) or a functional variant or functional portion of an IL-3 protein having an amino acid sequence at least 60% identical to SEQ ID NO: 2, preferably 95-99% identity.
  • the isolated nucleic acid sequence encodes a functional derivative or functional portion of an IL-3 protein represented by SEQ ID NO: 2 but further comprising at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions).
  • the modifications are point mutations selected from the group consisting of K116W and E22R, or the combination thereof.
  • the isolated nucleic acid encodes a functional derivative or functional portion of an IL- 3 protein comprising a sequence of SEQ ID NO: 3 (encoding K116W single mutation) or SEQ ID NO: 4 (encoding K116W and E22R dual mutation), or a sequence with at least 60% identity, preferably 95-99% identity thereto.
  • the isolated nucleic acid sequence further comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
  • the transmembrane domain is derived from CD28.
  • the transmembrane domain comprises a sequence of SEQ ID NO: 5.
  • the transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 5, or a sequence with at least 60% identity, preferably 95-99%, identity to an amino acid sequence of SEQ ID NO: 5.
  • the isolated nucleic acid sequence further comprises a sequence encoding an intracellular signaling domain.
  • the encoded intracellular signaling domain comprises a functional signaling domain selected from the group consisting of: 4-1 BB, CD3 zeta, and CD27, or a combination thereof or a functional variant or portion thereof.
  • the intracellular signaling domain of the CAR can comprise the signaling domain by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a CAR.
  • the intracellular signaling sequences within the cytoplasmic portion of a CAR may be linked to each other in a random or specified order.
  • a short oligo- or polypeptide linker for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequence.
  • a glycine-serine doublet can be used as a suitable linker.
  • a single amino acid e.g., an alanine, a glycine, can be used as a suitable linker.
  • the encoded intracellular signaling domain comprises a sequence of SEQ ID NO: 6.
  • the intracellular signaling domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 6, or a sequence with at least 60% identity, preferably 95-99% identity to an amino acid sequence of SEQ ID NO: 6.
  • the isolated nucleic acid sequence further comprises a sequence encoding a costimulatory domain.
  • the encoded costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1 , LFA 1 (CDI1 a/CD18), ICOS (CD278) and 4-1 BB (CD137), or a combination thereof, or a functional variant or portion thereof.
  • the costimulatory domain comprises a sequence of SEQ ID NO: 7.
  • the costimulatory domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 7, or a sequence with at least 60% identity, preferably 95-99% identity, to an amino acid sequence of SEQ ID NO: 7.
  • the isolated nucleic acid sequence further comprises a sequence encoding a spacer region which connects the CD123 binding domain to the transmembrane domain of the CAR.
  • the spacer region comprises a sequence derived from CD8 (e.g.
  • the spacer may further comprise other amino acid residues - for example, four glycine residues and a serine residue repeated three times (i.e. (G4S)3) .
  • the spacer region comprises an amino acid sequence of SEQ ID NO: 8.
  • the spacer region comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 8, or a sequence with at least 60% identity, preferably 95-99% identity, to an amino acid sequence of SEQ ID NO: 8.
  • the spacer region comprises an amino acid sequence of SEQ ID NO: 9.
  • the spacer region comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 9, or a sequence with at least 60% identity, preferably 95-99% identity, to an amino acid sequence of SEQ ID NO: 9.
  • the isolated nucleic acid sequence encodes a fusion of sequences SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 6 in that order, or a functional variant or functional portion thereof (i.e. a functional variant or functional portion of the fused sequence).
  • the fusion comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) compared to a fusion of combining sequences SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 6 in that order, or a sequence with at least 60% identity, preferably 95-99% identity to a fusion combining sequences SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 6 in that order.
  • the isolated nucleic acid sequence comprises SEQ ID NO: 10, a sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) to nucleic acid sequence of SEQ ID NO: 10, or a nucleic acid sequence with at least 60% identity, preferably 95-99% identity, to a nucleic acid sequence of SEQ ID NO: 10.
  • the isolated nucleic acid sequence encodes a fusion protein comprising SEQ ID NO: 11 , a sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) to amino acid sequence of SEQ ID NO: 11 , or an amino acid sequence with at least 60% identity, preferably 95-99% identity, to the amino acid sequence of SEQ ID NO: 11.
  • the isolated nucleic acid sequence encodes a fusion of sequences SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 6 in that order, or a functional variant or functional portion thereof.
  • the fusion comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) to a fusion combining sequences SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 6 in that order, or a sequence with at least 60% identity, preferably 95-99% identity, to a fusion combining sequences SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 6 in that order.
  • the isolated nucleic acid sequence comprises SEQ ID NO: 12, a sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) to nucleic acid sequence of SEQ ID NO: 12, or a nucleic acid sequence with at least 60% identity, preferably 95-99% identity, to a nucleic acid sequence of SEQ ID NO: 12.
  • the isolated nucleic acid sequence encodes a fusion protein comprising an amino acid sequence of SEQ ID NO: 13, an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) to the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence with at least 60% identity, preferably 95-99% identity, to the an amino acid sequence of SEQ ID NO: 13.
  • the isolated nucleic acid sequence encodes a fusion of sequences SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 6 in that order, or a functional variant or functional portion thereof.
  • the fusion comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) to a fusion combining SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 6 in that order, or a sequence with at least 60% identity, preferably 95-99% identity to a fusion combining SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 6 in that order.
  • the isolated nucleic acid sequence comprises SEQ ID NO: 14, a sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) to nucleic acid sequence of SEQ ID NO: 14, or a nucleic acid sequence with at least 60% identity, preferably 95-99% identity, to a nucleic acid sequence of SEQ ID NO: 14.
  • the isolated nucleic acid sequence encodes a fusion protein comprising an amino acid sequence of SEQ ID NO: 15, a sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) to an amino acid sequence of SEQ ID NO: 15, or an amino acid sequence with at least 60% identity, preferably 95-99% identity, to the sequence of SEQ ID NO: 15.
  • the expression of nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector.
  • the vectors can be suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • the expression constructs of the present disclosure may also be used for nucleic acid immunisation and gene therapy, using standard gene delivery protocols.
  • the present disclosure provides a gene therapy vector.
  • the nucleic acid can be cloned into a number of types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation.
  • these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either cooperatively or independently to activate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • CMV immediate early cytomegalovirus
  • EF-1 alpha Elongation Growth Factor-1 alpha
  • constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukaemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • the constructs of the present disclosure should not be limited to the use of constitutive promoters.
  • inducible promoters are also contemplated.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g. enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene. Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter driven transcription.
  • the vector can be readily introduced into a host cell, e.g. mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al 15 . A preferred method for the introduction of a polynucleotide into a host cell is electroporation.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. 15 , and in other virology and molecular biology manuals.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • an exemplary delivery vehicle is a nanoparticle, e.g., a liposome or other suitable sub-micron sized delivery system.
  • lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • assays include, for example, "molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g. by immunological means (ELISAs and Western blots).
  • the present disclosure provides an isolated polypeptide molecule encoded by the above isolated nucleic sequence.
  • the isolated polypeptide comprises the sequence of SEQ ID NO: 11 , 13 or 15, or a sequence having at least 60% identity thereto, or a sequence having 95-99% identity thereto.
  • the present disclosure provides an isolated cell which expresses the CD123-specific CAR described herein.
  • the cell is a T cell.
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumours.
  • any commercially available T cell line available in the art may be used.
  • T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FicollTM separation.
  • T cells are isolated from PBMCs by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient or by counterflow centrifugal elutriation.
  • a specific subpopulation of T cells such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+T cells, can be further isolated by positive or negative selection techniques known in the art.
  • Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDIIb, CD16, HLA-DR, and CD8.
  • T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.
  • a T cell population can be selected that expresses one or more of IFN-y, TNFa, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other appropriate molecules, e.g. other cytokines.
  • Methods for screening for cell expression are known in the art.
  • T cells can be obtained from a patient directly following treatment that leaves the subject with functional T cells.
  • the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo.
  • these cells may be in a preferred state for enhanced engraftment and in vivo expansion.
  • mobilisation for example, mobilization with GM-CSF
  • conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favoured, especially during a defined window of time following therapy.
  • Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.
  • the CD123-specific CAR-expressing T cells of the present disclosure may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells.
  • T cell populations may be stimulated by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilised on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.
  • a protein kinase C activator e.g., bryostatin
  • a ligand that binds the accessory molecule is used for co-stimulation of an accessory molecule on the surface of the T cells.
  • a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
  • an anti-CD3 antibody and an anti-CD28 antibody are examples of an anti- CD28 antibody.
  • an anti- CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besangon, France) can be used as can other methods commonly known in the art.
  • the primary stimulatory signal and the costimulatory signal for the T cell may be provided by different protocols.
  • the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in "cis” formation) or to separate surfaces (i.e., in "trans” formation).
  • one agent may be coupled to a surface and the other agent in solution.
  • the agent providing the costimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface.
  • both agents can be in solution.
  • the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents.
  • compositions comprising isolated nucleic acids sequences, polypeptides, vectors, and/or cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented).
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
  • Compositions may be formulated aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
  • the compositions described herein may be formulated for administration to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • Compositions of the present disclosure are preferably formulated for intravenous administration.
  • the present disclosure provides a method of treating a disease or condition associated with expression of CD123, the method comprising administering to a subject in need an effective amount of a nucleic acid sequence encoding a CD123-specific CAR, a vector encoding said sequence, a CD123-specific CAR polypeptide, or a cell expressing the CD123-specific CAR.
  • the method comprises administering to a subject in need an effective amount of a T cell expressing the CD123-specific CAR.
  • T cells can be activated from blood draws of from 10cc to 400cc.
  • T cells are activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 10Occ.
  • the cells expressing a CAR molecule according to the present disclosure are administered in combination (simultaneously or concurrently) with an agent that increases the efficacy of a cell expressing a CAR molecule, e.g. a chemotherapeutic agent (such as an antimetabolites, alkylating agent, anthracyclines, or DNA methyltransferase, or combinations thereof) or an inhibitory molecule (such as an antibody or inhibitory nucleic acid that inhibits one or more of PD1 , PD-L1 CTLA4, TIM3, LAG 3, VISTA, BTLA, TIGIT, LAIR1 , CD160, 2B4 and TGFR beta).
  • a chemotherapeutic agent such as an antimetabolites, alkylating agent, anthracyclines, or DNA methyltransferase, or combinations thereof
  • an inhibitory molecule such as an antibody or inhibitory nucleic acid that inhibits one or more of PD1 , PD-L1 CTLA
  • the cells expressing a CAR molecule according to the present disclosure are administered in combination (simultaneously or concurrently) with an agent that ameliorates one or more side effect associated with administration of a cell expressing a CAR molecule.
  • Side effects associated with the administration of a CAR-expressing cell include, but are not limited to cytokine release syndrome (CRS), or haemophagocytic lymphohistiocytosis (HLH), also termed Macrophage Activation Syndrome (MAS).
  • CRS cytokine release syndrome
  • HHLH haemophagocytic lymphohistiocytosis
  • MAS Macrophage Activation Syndrome
  • Symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like.
  • an agent that ameliorates one or more side effect associated with administration of a cell expressing a CAR molecule may include steroid, an inhibitor of TNFa, or an inhibitor of IL-6.
  • FIG. 1 Three truncated human IL-3 (amino acid residues 15-125) constructs were prepared as shown in Figure 1 - one with the wild type sequence (CARIL3WT), one with a single K116W substitution (CARIL3S) and one with dual K116W and E22R substitutions (CARIL3D).
  • Each construct comprised a CD8a stalk-derived spacer (CD8a), CD28 transmembrane (CD28TM), 4-1 BB intracellular co-stimulatory and O ⁇ 3z domains. Also included was an eGFP tag.
  • the pVAX1 super piggyBac transposase plasmid was constructed as previously described (Bishop, D et al (2016) 16 ). Briefly, The kanamycin-resistant plasmid pVAX1 (Life Technologies, Carlsbad, CA, Catalogue V260-20; SEQ ID NO: 16) was used as a backbone for transposase and transposon plasmids used to generate CAR-T cells.
  • transposase plasmid pVAXISPBase (SEQ ID NO: 17) was created by sub-cloning the Super piggyBac transposase sequence from the Super PiggyBac Transposase Expression Vector (System Biosciences, Palo Alto, CA, Catalogue PB200PA-1) into the multiple cloning site of pVAX1.
  • Generation of transposon plasmids for CAR expression required formation of an intermediate plasmid, pVAXI PB (SEQ ID NO: 18).
  • the piggyBac transposon sequences including inverted repeats, EF1a promoter, and SV40 polyadenylation sequence, were isolated from the GFP-containing PiggyBac single-expression vector (System Biosciences, Catalogue PB530A-2) by digestion with BssHII.
  • the pVAX1 backbone sequence including the kanamycin resistance gene and the plasmid origin of replication, was amplified by PCR using primers, including 15-bp extensions overlapping with 5' and 3' sequences of the linearized PB530A-2 transposon.
  • the PB530A-2 transposon and pVAX1 backbone were then joined using the Cold Fusion cloning kit (System Biosciences) creating pVAX1 PB.
  • DNA encoding the amino acid sequence of the truncated human IL-3 (SEQ ID NO: 2) comprising amino acid residues 15-125 of mature form human IL- 3 (hereafter referred to as IL-3(15-125)) linked with DNA sequences encoding (G4S)3, CD8a stalk and CD28 transmembrane domain, 4-1 BB costimulatory intracellular domains, and CD3z domain, was synthesised after codon optimisation (Genscript, NJ, USA) and cloned into pVAXI PB between EcoRI and BamFII sites, which provided the pVAX1 backbone, piggyBac recognition inverted repeats, EF1a promoter, eGFP and SV40 polyadenylation sequence to produce pVAX1 PB CARIL3WT ( Figure 2a).
  • DNA encoding amino acid sequence of the truncated human IL-3(15-125) with amino acid substitutions K116W alone (CARIL3S) and in combination with substitution E22R (CARIL3D) linked with CD8a stalk were synthesised and cloned into the pVAX1 PB CARIL3WT plasmid between the EcoRI and SgrAI sites, to generate the pVAX1 PB CARIL3S and pVAX1 PB CARIL3D plasmids, respectively ( Figure 2b).
  • PBMCs donor periphera! blood mononuclear cel!
  • Healthy donor PBMCs were used as starting material, isolated and cryopreserved as previously described (Ramanayake, S. et al (2015) 17 ). Briefly, ethics approval was obtained from the Sydney West Local Health District Human Research Ethics Committee for the collection of PBMCs from healthy donors that had provided informed consent in accordance with the Declaration of Helsinki. For each donor, separate CAR-T cell cultures were generated using each CARIL3 construct.
  • Viable cells were enumerated manually by trypan blue (Sigma-Aldrich) exclusion with light microscopy using a haemocytometer (Neubauer improved, Camlab, Cambridge UK) at magnification of 100x.
  • PBMCs Peripheral blood mononuclear cells
  • the cell lines were maintained in 75cm 2 Falcon rectangular canted neck cell culture flasks (Corning, Corning, NY, USA) at 37°C in a 5% CO2 humidified incubator at cell concentrations recommended by ATCC or DSMZ and passaged 2-3 times per week by performing 1 :5 to 1 :10 media exchange as required.
  • the cell lines were passaged for a maximum of 25 times or 3 months in culture to reduce phenotypic or genotypic drift.
  • Cryopreserved PBMCs were thawed and rested for 18-24 hours at cellular concentration of 4-5 x 10 6 /ml_ in AIM-V (Gibco-BRL, CA, USA) supplemented with 10% FBS.
  • Electroporation of PBMCs was performed using the Neon Transfection System (Life Technologies) with settings of single pulse, 20 ms, and 2,400 V. On the day after electroporation, and every 7 days thereafter, cells were enumerated by trypan blue (Sigma-Aldrich) exclusion, analysed by flow cytometry, and stimulated by the addition of twice as many autologous irradiated PBMCs to the culture. Specifically, on the day after electroporation and every 7 days thereafter, irradiated autologous PBMCs were added at a ratio of between 2-4 irradiated PBMCs:1 cultured cell. When additional irradiated cell lines (e.g.,
  • Nalm6, TF-1, THP-1) were used, these were added at a ratio of 1 irradiated cell line cell: 5 cultured cells, with irradiated autologous PBMCs also added at a ratio of 2 irradiated PBMCs:1 cultured cell.
  • CARIL3 T-cells were kept in culture for 22 days in total prior to functional assessment and cryopreservation.
  • Cultures were harvested 22 days post electroporation, and they were cryopreserved in freezing medium containing 70% PBS (Lonza), 20% heat-inactivated FBS, and 10% dimethyl sulfoxide (Sigma- Aldrich) for later infusion.
  • CARIL3 T cells were phenotyped at weekly intervals, and memory phenotype and immuno-inhibitory markers were assessed on the final product.
  • the following fluorochrome-conjugated anti-human mAbs were used:
  • lnterferon-g and TNF-a production from CARIL3 T-cells were assessed by co-culturing CAR T-cells at 1 :1 ratio with target cells negative for CD123 expression (Raji and HEK293) and target cells with varying CD123 expression (Nalm6, TF-1 and KG-1). Positive and negative controls were as previously described (Ramanayake S. et al (2015) 17 ). Detection of intracellular IFNy production by CARIL3 or control NT T cells (effectors) in response to 4 hr of exposure to Jurkat or Nalm-6 cell lines, or control stimuli, was performed as previously described (Ramanayake, S. et al (2015) 17 ). Antibodies used for detection were CD3- Pacific Blue, CD4-PerCP, CD8-FITC, and IFNy-PE (BD Biosciences).
  • Calcein release assays were performed to assess the ability of CARIL3 T cells to lyse targets as previously described by Lichtenfels, R. et al (1994) 18 . Briefly, cultured CAR T-cells were incubated with K562 cells at a 1:1 ratio in RPMI + 10% FBS for 1 hour to adsorb any natural killer (NK) cells. Target cells were then labelled with 25 mM calcein-AM (Sigma Aldrich), and 1.5 x 10 4 targets were co cultured in triplicate with decreasing numbers of effector cells in complete RPMI without phenol red (Lonza) to give E:T ratios ranging from 40:1 to 1.25:1. The spontaneous (spont) and the maximal (max) calcein release were determined by incubating target cells with complete RPMI alone or 1% Triton X-100 (Sigma Aldrich), respectively.
  • Target cells were Raji (CD123 ne a), HEK293 (CD123 ne a), KG-1 (CD123 + ) and TF-1 (CD123 + ). After incubating for 4 hours at 37°C, the fluorescence (F) due to calcein was measured with a PerkinElmer Victor X3 plate reader (PerkinElmer, Waltham, MA), using excitation and emission spectra of 485 and 538 nm, respectively. The percentage of specific target cell lysis at each E:T ratio (test) was calculated with the formula:
  • Example 8 Expansion of CARIL3 T-cells in vitro
  • Each culture contained both CD4+ and CD8+ CAR T- cells with no significant difference in the mean proportions between constructs (ranges: CD4 CAR: 40.0%% - 53.0%; CD8 CAR: 43.1% - 57.8%) ( Figure 5(d)).
  • CAR T-cells numerically expanded with 4:1 irradiated PBMCs alone showed a large proportion of terminal effector (CD45RA+/CD62L-) T-cells (mean range: 50.6% - 68.9%) but had significant preservation of na ' ive/stem cell memory (CD45RA+/CD62L+) and central memory (CD45RA-/CD62L+) compartments which comprises a combined average 25.9% - 47.1% of CAR T-cells across the different constructs.
  • the choice of construct had no significant effect on memory phenotype (Figure 5(e)).
  • CAR T-cells with the CARIL3WT construct only produced TNF-a following co-culture with the CD123+ TF-1 cell line (mean: 10.3%), with no significant increase following co-culture with all other cell lines tested (mean range: 0.2% - 2.8%) or no target control (mean: 0.3%) (Figure 6(c)).
  • Calcein-AM cytotoxicity assay showed CAR T-cell mediated dose dependent elimination of cell lines expressing CD123 (TF-1 and KG-1) but not of cell lines without CD123 expression (Raji and FIEK293) ( Figure 6(d)).
  • autologous PBMCs as feeder cells provides several advantages. In particular, it circumvents the requirement for GMP compliant stimulator cell line when used in production of therapeutic products. In addition, the cultures expanded with autologous PBMCs may reduce the risk of expanding allo-reactive T-cells due to exposure to various allo-antigens on cell lines. The lack of outgrowth of CD3 negative NK cells is also advantageous as the relatively pure CAR T-cells in final culture reduces the risk of efficacy being mediated through effectors (like NK cells) other than CAR T-cells.
  • the in vitro findings showed surprising robust expansion, enrichment, specific cytokine production and cytotoxicity with the CARIL3S construct having the IL-3 K116W mutant extracellular domain.
  • the double mutant CARIL3D construct showed similar in vitro activity although a statistically significant increase in TNF-a production was not achieved with TF-1 cell line. Cytotoxicity that achieved statistical significance against both KG-1 and TF-1 cell lines was also not achieved due to variability between the individual cultures.
  • the CARIL3WT expressing cultures showed lowest cell number expansion and CAR enrichment. The cytotoxicity of CARIL3WT expressing CAR T-cells were not significantly different from non -transfected PBMCs and cytokine response was seen only with TF-1 cell line.
  • CAR T-cells expressing the CARIL3WT construct demonstrated a cytokine response when co-cultured with the TF-1 but not the KG-1 cell line which expresses CD123 at similar levels.
  • One possible explanation is the cross-reactivity of IL-3 which can act as ligand of the closely related GM-CSF receptor due to the pre-formed heterodimeric conformation in the absence of GM-CSF.
  • the TF-1 leukaemic cell line has been reported to have high GM-CSF receptor (CD116) expression.
  • the results demonstrate the surprising finding that it is possible to generate CAR T-cells against CD123 using human IL-3 based extracellular antigen binding domain.
  • T-cells expressing these CARs could be expanded and enriched in culture, resulting in products with specific activity in vitro against leukaemic cell lines expressing CD123.
  • the single mutant IL3 expressing CAR T-cells showed robust activity against target receptor expressing cells. Flowever, while the single mutant may show a better response in vitro, the double mutant may be a safer option for in vivo targeting.
  • SEQ ID NO: 1 mature, unmodified human IL-3:
  • SEQ ID NO: 2 wild type Fluman IL-3 amino acids 15-125 (IL-3):
  • SEQ ID NO: 7 4-1 BB co-stimulatory domain (4-1 BB IC): KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL [0194]
  • SEQ ID NO: 8 CD8-alpha Stalk (CD8a Stalk): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD [0195]
  • SEQ ID NO: 10 CARIL3WT nucleic acid sequence:
  • SEQ ID NO: 12 CARIL3K116W nucleic acid sequence:
  • SEQ ID NO: 16 plasmid pVAX1 nucleic acid sequence: G ACT CTT CGCG AT GT ACGGGCCAG AT AT ACGCGTT G ACATT GATT ATT G ACT AGTT ATT AAT AGT AAT CAATT ACGGGGT CATT AGTT CAT AGCCCAT AT ATGG A GTT CCGCGTT ACAT AACTT ACGGT AAAT GGCCCGCCTGGCT G ACCGCCCAA CG ACCCCCGCCCATT G ACGT CAAT AAT G ACGT AT GTT CCCAT AGT AACGCCA AT AGGG ACTTT CCATT G ACGT CAATGGGT GG AGT ATTT ACGGT AAACTGCCC ACTT GGCAGT ACAT CAAGT GT AT CAT ATGCCAAGT ACGCCCCCT ATT GACGT CAAT GACGGT AAATGGCCCGCCTGGCATT ATGCCCAGT ACAT G ACCTT AT G GG ACTTT CCT ACTT GGCAGT ACAT CT ACGT ATT AGT

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Abstract

La présente divulgation concerne des compositions et des méthodes de traitement de maladies ou d'états associés à l'expression de CD123. Plus particulièrement, l'invention concerne des récepteurs antigéniques chimériques (CAR) spécifiques de CD123, et des acides nucléiques isolés, des polypeptides isolés, et des vecteurs isolés codant pour ceux-ci, ainsi que des cellules de recombinaison comprenant le CAR spécifique de CD123.
PCT/AU2022/050776 2021-07-22 2022-07-21 Récepteur antigénique chimérique utile dans le traitement du cancer Ceased WO2023000036A1 (fr)

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AU2021902252A AU2021902252A0 (en) 2021-07-22 Chimeric antigen receptor useful in treating cancer
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010025177A1 (fr) * 2008-08-26 2010-03-04 City Of Hope Procédé et compositions pour fonctionnement amélioré d’effecteur antitumoral de lymphocytes t
WO2015193406A1 (fr) * 2014-06-17 2015-12-23 Cellectis Récepteur d'antigène chimère multichaînes spécifique de cd123
EP3508504A1 (fr) * 2016-08-31 2019-07-10 Nanjing Legend Biotech Co., Ltd. Ligand de récepteur chimérique ciblant le cd123 humain et son application

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010025177A1 (fr) * 2008-08-26 2010-03-04 City Of Hope Procédé et compositions pour fonctionnement amélioré d’effecteur antitumoral de lymphocytes t
WO2015193406A1 (fr) * 2014-06-17 2015-12-23 Cellectis Récepteur d'antigène chimère multichaînes spécifique de cd123
EP3508504A1 (fr) * 2016-08-31 2019-07-10 Nanjing Legend Biotech Co., Ltd. Ligand de récepteur chimérique ciblant le cd123 humain et son application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KLEIN BARBARA K., FENG YIQING, MCWHERTER CHARLES A., HOOD WILLIAM F., PAIK KUMNAN, MCKEARN JOHN P.: "The Receptor Binding Site of Human Interleukin-3 Defined by Mutagenesis and Molecular Modeling", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 272, no. 36, 1 September 1997 (1997-09-01), US , pages 22630 - 22641, XP093027522, ISSN: 0021-9258, DOI: 10.1074/jbc.272.36.22630 *
MURAD, J. M. ET AL.: "Advances in the use of natural receptor- or ligand-based chimeric antigen receptors (CARs) in haematologic malignancies", BEST PRACT RES CLIN HAEMATOL., vol. 31, no. 2, 2018, pages 176 - 183, XP055868131, DOI: 10.1016/j.beha. 2018.03.00 3 *

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