EP4568999A1 - Chimeric antigen receptor domains - Google Patents

Chimeric antigen receptor domains

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Publication number
EP4568999A1
EP4568999A1 EP23853152.9A EP23853152A EP4568999A1 EP 4568999 A1 EP4568999 A1 EP 4568999A1 EP 23853152 A EP23853152 A EP 23853152A EP 4568999 A1 EP4568999 A1 EP 4568999A1
Authority
EP
European Patent Office
Prior art keywords
amino acid
acid sequence
car
cell
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23853152.9A
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German (de)
French (fr)
Inventor
Lionel Jianrong LOW
Kar Wai TAN
Chee Hoe NG
Jin Wei TAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tikva Allocell Pte Ltd
Agency for Science Technology and Research Singapore
Original Assignee
Tikva Allocell Pte Ltd
Agency for Science Technology and Research Singapore
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Application filed by Tikva Allocell Pte Ltd, Agency for Science Technology and Research Singapore filed Critical Tikva Allocell Pte Ltd
Publication of EP4568999A1 publication Critical patent/EP4568999A1/en
Pending legal-status Critical Current

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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
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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]
    • A61K2239/17—Hinge-spacer domain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—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 the fields of molecular biology, more specifically chimeric antigen receptor (CAR) technology.
  • the present disclosure also relates to methods of medical treatment and prophylaxis.
  • CAR chimeric antigen receptor
  • MHC non-major histocompatibility complex
  • CARs are synthetic receptors that comprise of 4 main domains: the antigen specific domain, commonly a single chain variable fragment of an antibody (scFv), the spacer domain that connects the scFv to the third or transmembrane domain of the receptor, and finally the signalling domains (8).
  • scFv single chain variable fragment of an antibody
  • the spacer domain that connects the scFv to the third or transmembrane domain of the receptor
  • signalling domains (8).
  • Design and engineering of the CAR receptor is critical for the efficacy and safety of the CAR T cell strategy.
  • the spacer or hinge domain plays an important role in the function of the CAR, providing reach and flexibility for the scFv. Flexibility of the spacer allow the scFv to access otherwise sterically hindered epitopes, whilst the length of the spacer ensures that the optimal synapse distance between the CAR T cell and the target cell is maintained for efficient delivery of granzymes and perforins for potent cytotoxicity (8).
  • the most commonly used spacer domains are derived from antibody Fc domains, i.e. IgG 1 , lgG2 and lgG4-Fc, CD8a and CD28.
  • IgG-derived spacers can interact with Fc receptors on other immune cells and thus can result in the generation of immune responses mounted against T cells expressing the CARs. In addition, this interaction can also lead to nonantigen specific activation of CAR T cells, resulting in elevated cytokine release, exhaustion, activation induced cell death (11 ).
  • CARs bearing CD8a- and CD28-derived spacers have been shown to be highly-effective and have been clinically approved in CAR T cell therapies.
  • CARs with the CD28 spacer have been shown to lower the threshold of activation of CD19 CAR T cells compared to those with the CD8a spacer (12).
  • CARs with CD28 and CD8a spacers facilitate hetero-dimerization of endogenous CD28, and also with CARs CARs having CD28 transmembrane and endodomains (13), potentially lowering the threshold for CAR T activation, and producing a stronger activation signal that could increase the risk of CAR T therapy associated toxicities such as cytokine release syndrome and neurotoxicity (14).
  • the present disclosure provides a chimeric antigen receptor (CAR) comprising: (i) an antigen-binding domain which binds specifically to a target antigen, (ii) a spacer domain comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID N0s:100, 99, 101 and 102, (iii) a transmembrane domain, and (iv) a signalling domain comprising an amino acid sequence comprising an immunoreceptor tyrosine-based activation motif (ITAM).
  • the CAR comprises a spacer domain comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:100.
  • the antigen-binding domain binds specifically to a target antigen selected from CD30, CD19, CD20, CD22, ROR1 R, CD4, CD7, CD38, BCMA, Mesothelin, EGFR, GPC3, MUC1 , HER2, GD2, CEA, EpCAM, LeY and PSCA.
  • the antigen-binding domain binds specifically to CD30.
  • the antigen-binding domain comprises:
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:2
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:3
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:4;
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NQ:10
  • LC-CDR2 having the amino acid sequence of SEQ ID NO:1
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:12.
  • the antigen-binding domain comprises: a VH region having an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs:1 , 45, 52, 17, 21 , 24, 26, 28; and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs:9, 49, 54, 30, 35, 38, 41 , 43, 147, 148, 149, 150, 151 or 152.
  • the antigen-binding domain comprises:
  • VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:1
  • VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:9
  • VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:52, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:54;
  • VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:21 , and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:35;
  • the antigen-binding domain is or comprises a single chain variable fragment (scFv) comprising the VH region and the VL region.
  • scFv single chain variable fragment
  • the transmembrane domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs:103, 104 or 105.
  • the signalling domain comprises an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs:106, 107 or 108.
  • the signalling domain comprises an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:111 .
  • the CAR comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs:124, 144, 190, 210, 117, 118, 119, 120, 121 , 122, 123, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 145, 146, 183, 184, 185, 186, 187, 188, 189, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 211 or 212.
  • the present disclosure also provides a nucleic acid, or a plurality of nucleic acids, optionally isolated, encoding a CAR according to the present disclosure.
  • the present disclosure also provides an expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to the present disclosure.
  • the present disclosure also provides a cell comprising a CAR, a nucleic acid or a plurality of nucleic acids, or an expression vector or a plurality of expression vectors according to the present disclosure.
  • the present disclosure also provides a method comprising culturing a cell according to the present disclosure under conditions suitable for expression of an CAR by the cell.
  • the present disclosure also provides a composition
  • a composition comprising a CAR, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, or a cell according to the present disclosure, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • the present disclosure also provides a CAR, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the present disclosure, for use in a method of medical treatment or prophylaxis.
  • the present disclosure also provides a CAR, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the present disclosure, for use in the treatment or prevention of a cancer.
  • the cancer is selected from the group consisting of: a cancer expressing the target antigen for the CAR, a CD30-positive cancer, an EBV-associated cancer, a hematological cancer, a myeloid hematologic malignancy, a hematopoietic malignancy a lymphoblastic hematologic malignancy, myelodysplastic syndrome, leukemia, T cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, B cell nonHodgkin’s lymphoma, diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, EBV- associated lymphoma, EBV-positive B cell lymphoma, EBV-positive diffuse large B cell lymphoma, EBV- positive lymphoma associated with X-linked lymphoproliferative disorder, E
  • the present disclosure also provides a CAR, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the present disclosure, for use in the treatment or prevention of a disease or condition characterised by an alloreactive immune response.
  • the disease or condition characterised by an alloreactive immune response is selected from the group consisting of: a disease or condition associated with allotransplantation, graft versus host disease (GVHD) or graft rejection.
  • GVHD graft versus host disease
  • the present disclosure also provides the use of a CAR, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the present disclosure to deplete or increase killing of cells expressing the target antigen for the CAR.
  • the present disclosure also provides an in vitro complex, optionally isolated, comprising a CAR or a cell according to the present disclosure, bound to the target antigen for the CAR.
  • the present disclosure provides novel chimeric antigen receptor (CAR) constructs comprising novel spacer region sequences.
  • CAR chimeric antigen receptor
  • T cells expressing the novel CAR constructs are shown to expand/proliferate similarly, to have a improved safety profile, to display similar cytotoxicity to cells expressing the target antigen for the CAR, to have improved persistence in vivo, and to display improved tumor growth inhibition of cancer comprising cells expressing the target antigen for the CAR, as compared to equivalent CAR constructs instead having the known IgG 1 CH2-CH3 spacer region.
  • CARs are recombinant receptors that provide both antigen-binding and T cell activating functions.
  • CAR structure and engineering is reviewed, for example, in Dotti et al., Immunol Rev (2014) 257(1 ), hereby incorporated by reference in its entirety.
  • CARs comprise an antigen-binding domain linked via a transmembrane domain to a signalling domain.
  • An optional hinge or spacer domain may provide separation between the antigen-binding domain and transmembrane domain and may act as a flexible linker. When expressed by a cell, the antigenbinding domain is provided in the extracellular space, and the signalling domain is intracellular.
  • the antigen-binding domain mediates binding to the target antigen for which the CAR is specific.
  • the antigen-binding domain of a CAR may be based on the antigen-binding region of an antibody which is specific for the antigen to which the CAR is targeted.
  • the antigen-binding domain of a CAR may comprise amino acid sequences for the complementarity-determining regions (CDRs) of an antibody which binds specifically to the target antigen.
  • CDRs complementarity-determining regions
  • the antigen-binding domain of a CAR may comprise or consist of the light chain and heavy chain variable region amino acid sequences of an antibody which binds specifically to the target antigen.
  • the antigen-binding domain may be provided as a single chain variable fragment (scFv) comprising the sequences of the light chain and heavy chain variable region amino acid sequences of an antibody.
  • Antigen-binding domains of CARs may target antigens based on other protein protein interactions, such as ligand:receptor binding; for example an IL-13Ra2-targeted CAR has been developed using an antigen-binding domain based on IL-13 (see e.g. Kahlon et al. 2004 Cancer Res 64(24): 9160-9166).
  • a spacer domain provides separation between the antigen-binding domain and the transmembrane domain, and may act as a flexible linker. Such domains may be or comprise flexible regions allowing the binding moiety to orient in different directions. Spacer domains are sometimes derived from constant regions of immunoglobulin molecules.
  • the transmembrane domain is provided between the antigen-binding domain and the signalling domain of the CAR.
  • the transmembrane domain provides for anchoring the CAR to the cell membrane of a cell expressing a CAR, with the antigen-binding domain in the extracellular space and signalling domain inside the cell.
  • Transmembrane domains of CARs may be derived from transmembrane region sequences for cell membrane-bound proteins (e.g. CD28, CD8, CD4, CD3-£, etc.).
  • the signalling domain comprises amino acid sequences required for activation of immune cell function.
  • the CAR signalling domains may comprise the amino acid sequence of the intracellular domain of CD3- , which provides immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation and activation of the CAR-expressing cell.
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • Signalling domains comprising sequences of other ITAM- containing proteins have also been employed in CARs, such as domains comprising the ITAM containing region of FcyRI (Haynes et al., 2001 J Immunol 166(1 ):182-187).
  • CARs comprising a signalling domain derived from the intracellular domain of CD3-£ are often referred to as first generation CARs.
  • the signalling domains of CARs typically also comprise the signalling domain of a costimulatory protein (e.g. CD28, 4-1 BB etc.), for providing the costimulation signal necessary for enhancing immune cell activation and effector function.
  • CARs having a signalling domain including additional costimulatory sequences are often referred to as second generation CARs.
  • CARs are engineered to provide for costimulation of different intracellular signalling pathways. For example, CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, whereas 4-1 BB costimulation triggers signalling is through TNF receptor associated factor (TRAF) adaptor proteins.
  • TNF receptor associated factor TNF receptor associated factor
  • CARs therefore sometimes contain costimulatory sequences derived from signalling domains of more than one costimulatory molecule.
  • CARs comprising a signalling domain with multiple costimulatory sequences are often referred to as third generation CARs.
  • polypeptides, domains and amino acid sequences which are ‘derived from’ a reference polypeptide/domain/amino acid sequence have at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference polypeptide/domain/amino acid sequence.
  • Polypeptides, domains and amino acid sequences which are ‘derived from’ a reference polypeptide/domain/amino acid sequence preferably retain the functional and/or structural properties of the reference polypeptide/domain/amino acid sequence.
  • an amino acid sequence derived from the intracellular domain of CD28 may comprise an amino acid sequence having 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the intracellular domain of CD28, e.g. as shown in SEQ ID NO:106.
  • an amino acid sequence derived from the intracellular domain of CD28 preferably retains the functional properties of the amino acid sequence of SEQ ID NO:106, i.e. the ability to activate CD28-mediated signalling.
  • amino acid sequence of a given polypeptide or domain thereof can be retrieved from, or determined from a nucleic acid sequence retrieved from, databases known to the person skilled in the art.
  • databases include GenBank, EMBL and UniProt.
  • immune cells Through engineering to express a CAR specific for a particular target antigen, immune cells (typically T cells, but also other immune cells such as NK cells) can be directed to kill cells expressing the target antigen. Binding of a CAR-expressing T cell (CAR-T cell) to the target antigen for which it is specific triggers intracellular signalling, and consequently activation of the T cell. The activated CAR-T cell is stimulated to divide and produce factors resulting in killing of the cell expressing the target antigen.
  • CAR-T cell CAR-expressing T cell
  • an ‘antigen-binding domain’ refers to a domain which is capable of binding to a target antigen.
  • the target antigen may e.g. be a peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof.
  • Antigen-binding domains according to the present disclosure may be derived from antibodies (i.e. immunoglobulins (Igs)) and antigen-binding fragments thereof.
  • antibodies include monoclonal antibodies, polyclonal antibodies, monospecific and multispecific (e.g., bispecific, trispecific, etc.) antibodies, and antibody-derived antigen-binding molecules such as scFv, scFab, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH), etc.).
  • Antigen-binding fragments of antibodies include e.g. Fv, Fab, F(ab’)2 and F(ab’) fragments.
  • an antigen-binding domain according to the present disclosure may comprise or consist of an antibody or an antigen-binding fragment thereof.
  • the antigen-binding domain comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specific binding to the target antigen.
  • An antigen-binding domain formed by a VH and a VL may also be referred to herein as an Fv.
  • the domain capable of binding to a target antigen comprises or consists of an antigen-binding peptide/polypeptide, e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (/.e.
  • sdAb single-domain antibody
  • ArmRP armadillo repeat protein
  • OBody fibronectin - reviewed e.g. in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082-1101 , which is hereby incorporated by reference in its entirety (see also e.g. Boersma et al., J Biol Chem (2011 ) 286:41273-85 and Emanuel et al., Mabs (2011 ) 3:38-48).
  • the antigen-binding domains of the present disclosure generally comprise a VH and a VL of an antibody capable of specific binding to the target antigen.
  • Antibodies generally comprise six complementaritydetermining regions CDRs; three in the heavy chain variable region (VH): HC-CDR1 , HC-CDR2 and HC- CDR3, and three in the light chain variable region (VL): LC-CDR1 , LC-CDR2, and LC-CDR3.
  • the six CDRs together define the paratope of the antibody, which is the part of the antibody which binds to the target antigen.
  • the VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs.
  • VHs comprise the following structure: N term-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C term; and VLs comprise the following structure: N term-[LC-FR1 ]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]- [LC-CDR3]-[LC-FR4]-C term.
  • VH and VL sequences may be provided in any suitable format provided that the antigen-binding domain can be linked to the other domains of the CAR.
  • Formats contemplated in connection with the antigenbinding domain of the present disclosure include those described in Carter, Nat. Rev. Immunol (2006), 6: 343-357, such as scFv, dsFV, (scFv)2 diabody, triabody, tetrabody, Fab, minibody, and F(ab)2 formats.
  • the antigen-binding domain comprises the CDRs of an antibody/antibody fragment which is capable of binding to the target antigen.
  • the antigen-binding domain comprises the VH region and the VL region of an antibody/antibody fragment which is capable of binding to the target antigen.
  • a moiety comprised of the VH and a VL of an antibody may also be referred to herein as a variable fragment (Fv).
  • the VH and VL may be provided on the same polypeptide chain, and joined via a linker sequence; such moieties are referred to as single-chain variable fragments (scFvs).
  • Suitable linker sequences for the preparation of scFv are known to the skilled person, and may comprise serine and glycine residues.
  • the antigen-binding domain comprises, or consists of, Fv capable of binding to the target antigen.
  • the antigen-binding domain may be provided with any suitable format, e.g. scFv, scFab, etc.
  • the antigen-binding domain comprises, or consists of, a scFv capable of binding to the target antigen.
  • the target antigen for which the antigen-binding domain (and thus the CAR) is specific may be any target antigen.
  • the target antigen is an antigen whose expression/activity, or whose upregulated expression/activity, is positively associated with a disease or disorder (e.g. a cancer, an infectious disease or an autoimmune disease).
  • the target antigen is preferably expressed at the cell surface of a cell expressing the target antigen. It will be appreciated that the CAR directs effector activity of the cell expressing the CAR against cells/tissues expressing the target antigen for which the CAR comprises a specific antigen-binding domain.
  • a target antigen may be a cancer cell antigen.
  • a cancer cell antigen is an antigen which is expressed or over-expressed by a cancer cell.
  • a cancer cell antigen may be any peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof.
  • a cancer cell antigen’s expression may be associated with a cancer.
  • a cancer cell antigen may be abnormally expressed by a cancer cell (e.g. the cancer cell antigen may be expressed with abnormal localisation), or may be expressed with an abnormal structure by a cancer cell.
  • a cancer cell antigen may be capable of eliciting an immune response.
  • the antigen is expressed at the cell surface of the cancer cell (i.e.
  • the cancer cell antigen is a cancer cell surface antigen).
  • the part of the antigen which is bound by the antigen-binding molecule described herein is displayed on the external surface of the cancer cell (i.e. is extracellular).
  • the cancer cell antigen may be a cancer- associated antigen.
  • the cancer cell antigen is an antigen whose expression is associated with the development, progression or severity of symptoms of a cancer.
  • the cancer- associated antigen may be associated with the cause or pathology of the cancer, or may be expressed abnormally as a consequence of the cancer.
  • the cancer cell antigen is an antigen whose expression is upregulated (e.g. at the RNA and/or protein level) by cells of a cancer, e.g.
  • the cancer-associated antigen may be preferentially expressed by cancerous cells, and not expressed by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue/cell type).
  • the cancer- associated antigen may be the product of a mutated oncogene or mutated tumor suppressor gene.
  • the cancer-associated antigen may be the product of an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein.
  • Cancer cell antigens are reviewed by Zarour HM, DeLeo A, Finn OJ, et al. Categories of Tumor Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003.
  • Cancer cell antigens include oncofetal antigens: CEA, Immature laminin receptor, TAG-72; oncoviral antigens such as HPV E6 and E7; overexpressed proteins: BING-4, calcium-activated chloride channel 2, cyclin-B1 , 9D7, Ep-CAM, EphA3, HER2/neu, telomerase, mesothelin, SAP-1 , survivin; cancer-testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1 , PRAME, SSX-2; lineage restricted antigens: MARTI , Gp100, tyrosinase, TRP-1/2, MC1 R, prostate specific antigen; mutated antigens: 0-catenin, BRCA1/2, CDK4, CML66, Fibronectin, MART-2, p53, Ras, TGF-pRII; post-translationally altered antigens:
  • cancer cell antigens include heat-shock protein 70 (HSP70), heat-shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, feto-acinar pancreatic protein (FAPP), alkaline phosphatase placental-like 2 (ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1 ), protein tyrosine kinase 7 (PTK7), and cyclophilin B.
  • the cancer cell antigen is a cancer cell antigen described in Zhao and Cao, Front Immunol. (2019) 10: 2250, which is hereby incorporated by reference in its entirety.
  • a cancer cell antigen is selected from CD30, CD19, CD20, CD22, ROR1 R, CD4, CD7, CD38, BCMA, Mesothelin, EGFR, GPC3, MUC1 , HER2, GD2, CEA, EpCAM, LeY and PSCA.
  • a cancer cell antigen is an antigen expressed by cells of a hematological malignancy. In some embodiments, a cancer cell antigen is selected from CD30, CD19, CD20, CD22, ROR1 R, CD4, CD7, CD38 and BCMA. In some embodiments, a cancer cell antigen is an antigen expressed by cells of a solid tumor. In some embodiments, a cancer cell antigen is selected from Mesothelin, EGFR, GPC3, MUC1 , HER2, GD2, CEA, EpCAM, LeY and PSCA.
  • the antigen-binding domain (and thus the CAR) is multispecific.
  • multispecific it is meant that the antigen-binding domain displays specific binding to more than one target.
  • the antigen-binding domain is a bispecific antigen-binding domain.
  • the antigen-binding molecule comprises at least two different antigen-binding moieties (i.e. at least two antigen-binding moieties, e.g. comprising non-identical VHs and VLs). Individual antigen-binding moieties of multispecific antigen-binding domains may be connected, e.g. via linker sequences.
  • the antigen-binding domain binds to at least two, non-identical target antigens, and so is at least bispecific.
  • the term ‘bispecific’ means that the antigen-binding domain is able to bind specifically to at least two distinct antigenic determinants.
  • at least one of the target antigens for the multispecific antigen-binding domain/CAR is CD30.
  • Each of the target antigens may independently be a target antigen as described herein.
  • each target antigen is independently a cancer cell antigen as described herein.
  • an antigen-binding domain comprises antigen-binding moieties capable of binding to the target(s) for which the antigen-binding domain is specific.
  • an antigen-binding domain which is capable of binding to CD30 and an antigen other than CD30 may comprise: (i) an antigen-binding moiety which is capable of binding to CD30, and (ii) an antigen-binding moiety which is capable of binding to a target antigen other than CD30.
  • the target antigen is CD30. Accordingly, in some aspects and embodiments of the present disclosure the antigen-binding domain is a CD30-binding domain.
  • CD30 (also known as TNFRSF8) is the protein identified by UniProt: P28908. CD30 is a single pass, type I transmembrane glycoprotein of the tumor necrosis factor receptor superfamily. CD30 structure and function is described e.g. in van der Weyden et al., Blood Cancer Journal (2017) 7: e603 and Muta and Podack Immunol. Res. (2013) 57(1 -3) :151 -8, both of which are hereby incorporated by reference in their entirety.
  • isoform 1 (‘long’ isoform; UniProt: P28908-1 , v1 ; SEQ ID NO:87)
  • isoform 2 (‘cytoplasmic’, ‘short’ or ‘C30V’ isoform, UniProt: P28908-2; SEQ ID NO:88) in which the amino acid sequence corresponding to positions 1 to 463 of SEQ ID NO:87 are missing
  • isoform 3 (UniProt: P28908-3; SEQ ID NO:89) in which the amino acid sequence corresponding to positions 1 to 111 and position 446 of SEQ ID NO:87 are missing.
  • SEQ ID NO:87 The N-terminal 18 amino acids of SEQ ID NO:87 form a signal peptide (SEQ ID NQ:90), which is followed by a 367 amino acid extracellular domain (positions 19 to 385 of SEQ ID NO:87, shown in SEQ ID NO:91 ), a 21 amino acid transmembrane domain (positions 386 to 406 of SEQ ID NO:87, shown in SEQ ID NO:92), and a 189 amino acid cytoplasmic domain (positions 407 to 595 of SEQ ID NO:87, shown in SEQ ID NO:93).
  • CD30 refers to CD30 from any species and includes CD30 isoforms, fragments, variants or homologues from any species.
  • a ‘fragment’, ‘variant’ or ‘homologue’ of a reference protein may optionally be characterised as having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference protein (e.g. a reference isoform).
  • fragments, variants, isoforms and homologues of a reference protein may be characterised by ability to perform a function performed by the reference protein.
  • the CD30 is from a mammal (e.g. a primate (rhesus, cynomolgous, or human) and/or a rodent (e.g. rat or murine) CD30).
  • a mammal e.g. a primate (rhesus, cynomolgous, or human
  • a rodent e.g. rat or murine
  • the CD30 is a human CD30.
  • Isoforms, fragments, variants or homologues may optionally be characterised as having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature CD30 isoform from a given species, e.g. human.
  • a fragment of CD30 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 590 amino acids, and may have a maximum length of one of 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 595 amino acids.
  • the CD30 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:87, 88, 89 or 91 .
  • the CD30 comprises an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:92 or 95.
  • a fragment of CD30 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:92 or 95.
  • the CD30-binding domain of the CAR of the present disclosure preferably displays specific binding to CD30 or a fragment thereof.
  • the CD30-binding domain of the CAR of the present disclosure preferably displays specific binding to the extracellular domain of CD30.
  • the CD30-binding domain may be derived from an anti-CD30 antibody or other CD30-binding agent, e.g. a CD30-binding peptide or CD30-binding small molecule.
  • the CD30-binding domain may be derived from the antigen-binding moiety of an anti-CD30 antibody.
  • Anti-CD30 antibodies include HRS3 and HRS4 (described e.g. in Hornbach et al., Scand J Immunol (1998) 48(5):497-501 ), HRS3 derivatives described in Schlapschy et al., Protein Engineering, Design and Selection (2004) 17(12): 847-860, BerH2 (MBL International Cat# K0145-3, RRID:AB_590975), SGN-30 (also known as cAC10, described e.g. in Forero-Torres et al., Br J Haematol (2009) 146:171-9), MDX- 060 (described e.g.
  • a CD30-binding domain according to the present disclosure comprises the CDRs of an anti-CD30 antibody. In some embodiments a CD30-binding domain according to the present disclosure comprises the VH and VL regions of an anti-CD30 antibody. In some embodiments a CD30- binding domain according to the present disclosure comprises a scFv comprising the VH and VL regions of an anti-CD30 antibody.
  • the CDRs and FRs of the VH regions and VL regions of the antibody clones described herein are defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77.
  • the antigen-binding domain comprises a polypeptide or polypeptides comprising:
  • the antigen-binding domain comprises a polypeptide or polypeptides comprising a VH region comprising the heavy chain FRs, and a VL region comprising the light chain FRs, of an antibody selected from HRS3, VH1 VK1 , VH1 VK2, VH1VK3, VH1 VK4, VH1 VK5, VH2VK1 , VH2VK2, VH2VK3, VH2VK4, VH2VK5, VH3VK1 , VH3VK2, VH3VK3, VH3VK4, VH3VK5, VH4VK1 , VH4VK2, VH4VK3, VH4VK4, VH4VK5, VH5VK1 , VH5VK2, VH5VK3, VH5VK4, VH5VK5, HRS3Cys, VH1 VK1 Cys, VH1VK2Cys, VH1VK3C
  • the antigen-binding molecule comprises a polypeptide or polypeptides comprising: (i) a VH region comprising HC-FR1 , HC-FR2, HC-FR3 and HC-FR4 as indicated in column A of Table B, and (ii) a VL region comprising LC-FR1 , LC-FR2, LC-FR3, and LC-FR4 as indicated in column B of Table B, wherein the sequences of columns A and B are selected from the same row of Table B.
  • the antigen-binding domain comprises: a VH comprising, or consisting of, an amino acid sequence having at least 70% sequence identity (e.g. at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to the amino acid sequence of SEQ ID NO:1 , 45, 52, 17, 21 , 24, 26, 28; and a VL comprising, or consisting of, an amino acid sequence having at least 70% sequence identity (e.g.
  • the antigen-binding domain comprises a polypeptide or polypeptides comprising: (i) an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to an amino acid sequence indicated in column A of Table C, and (ii) an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to an amino acid sequence indicated in column B of Table C, wherein the sequences of columns A and B are selected from the same row of Table C.
  • the antigen-binding domain comprises: a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:1 , and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:9; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:45, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:49; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:52, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:54; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:17, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:30; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:21
  • the antigen-binding domain comprises a polypeptide or polypeptides comprising a VH region and a VL region of an antibody selected from HRS3, VH1 VK1 , VH1 VK2, VH1 VK3, VH1 VK4, VH1 VK5, VH2VK1 , VH2VK2, VH2VK3, VH2VK4, VH2VK5, VH3VK1 , VH3VK2, VH3VK3, VH3VK4, VH3VK5, VH4VK1 , VH4VK2, VH4VK3, VH4VK4, VH4VK5, VH5VK1 , VH5VK2, VH5VK3, VH5VK4, VH5VK5, HRS3Cys, VH1VK1 Cys, VH1VK2Cys, VH1VK3Cys, VH1VK4Cys, VH1 VK4Cys
  • the antigen-binding molecule comprises a polypeptide or polypeptides comprising: (i) an amino acid sequence indicated in column A of Table C, and (ii) an amino acid sequence indicated in column B of Table C, wherein the sequences of columns A and B are selected from the same row of Table C.
  • the antigen-binding domain comprises or consists of an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 or 182.
  • the antigen-binding domain does not comprise, or does not consist of, a polypeptide having the amino acid sequence of SEQ ID NO:59.
  • a VH region according to the present disclosure does not comprise, or does not consist of, the amino acid sequence of SEQ ID NO:1 . In some embodiments, a VL region according to the present disclosure does not comprise, or does not consist of, the amino acid sequence of SEQ ID NO:9.
  • a VH region according to the present disclosure does not comprise HC-FR1 having the amino acid sequence of SEQ ID NO:5. In some embodiments, a VH region does not comprise HC-FR2 having the amino acid sequence of SEQ ID NO:6. In some embodiments, a VH region does not comprise HC-FR3 having the amino acid sequence of SEQ ID NO:7.
  • a VL region according to the present disclosure does not comprise LC-FR1 having the amino acid sequence of SEQ ID NO:13. In some embodiments, a VL region does not comprise LC-FR2 having the amino acid sequence of SEQ ID NO:14. In some embodiments, a VL region does not comprise LC-FR3 having the amino acid sequence of SEQ ID NO:15.
  • the antigen-binding domain comprises, or consists of, a CD30-binding Fv (/.e. a VH and VL pair) as described herein.
  • the antigen-binding domain comprises, or consists of, a CD30-binding Fv, in which the VH and VL are covalently linked.
  • the VH and VL of a CD30-binding Fv sequences are linked by a flexible linker sequence, e.g. a flexible linker sequence as described herein. The flexible linker sequence may be joined to ends of the VH sequence and VL sequence, thereby linking the VH and VL sequences.
  • a CD30-binding domain may comprise or consist of a single chain variable fragment (scFv) comprising a VH sequence and a VL sequence as described herein.
  • the VH sequence and VL sequence may be covalently linked.
  • the VH and the VL sequences are linked by a flexible linker sequence, e.g. a flexible linker sequence as described herein.
  • the flexible linker sequence may be joined to ends of the VH sequence and VL sequence, thereby linking the VH and VL sequences.
  • the VH and VL are joined via a linker sequence comprising one or more copies of an amino acid sequence according to SEQ ID NO:57.
  • the linker sequence comprises at least 1 , 2, 3 or 4 copies of an amino acid sequence according to SEQ ID NO:57.
  • the VH and VL are joined via a linker sequence comprising, or consisting of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:58.
  • the antigen-binding molecule is capable of binding to the epitope of CD30 which is bound by antibody HRS3, e.g. within the region of amino acid positions 185-335 of human CD30 numbered according to SEQ ID NO:87, shown in SEQ ID NO:95 (Schlapschy et al., Protein Engineering, Design and Selection (2004) 17(12): 847-860, hereby incorporated by reference in its entirety).
  • the antigen-binding domain is capable of binding the same region of CD30, or an overlapping region of CD30, to the region of CD30 which is bound by an antibody comprising the VH and VL regions (see e.g.
  • the CAR comprises a spacer domain.
  • the spacer domain may be provided between the antigen-binding domain and the transmembrane domain.
  • the spacer domain may also be referred to as a hinge domain.
  • a spacer domain is an amino acid sequence which provides for flexible linkage of the antigen-binding and transmembrane domains of the CAR.
  • Spacer domains include those derived from: the CH2-CH3 region of human lgG1 (e.g. as shown in SEQ ID NO:96), the CH2-CH3 region of human IgG 1 (e.g. as shown in SEQ ID NO:98), the CH1 -CH2 hinge region of human IgG 1 , CD8a, e.g. as described in WO 2012/031744 A1 , and CD28, e.g.
  • Spacer domains can also be engineered to regulate synaptic cleft distances, and hence modulate signalling.
  • membrane-proximal epitopes typically require shorter spacers, while membrane-distal epitopes require longer spacers (Hudecek et al., Clin Cancer Res. (2013) 19:3153-3164).
  • Increasing epitope-paratope distance permits the interference by inhibitory phosphatases, and can result in impaired delivery of perforins and granzymes to the target cell, thereby reducing the efficiency of cytolysis.
  • Spacer lengths have been found to influence cytolytic activity and signalling by CAR-T cells.
  • Introduction of an IgG 1 -Fc spacer into a first generation anti-CEA CAR was found to reduce secretion of IFNy without comprising lytic efficiency (Guest et al., J Immunother. (2005) 28:203-21 1 ).
  • Spacer length has also been shown to affect mechano-transduction of ligand recognition. Chang et al., Nat Chem Biol. (2016)14:317— 324 found that cells expressing CARs specific for soluble homo-dimeric TGF-0 with longer lgG4-Fc spacers had a decreased activation profile compared to cells expressing CARs having a shorter lgG4 hinge only spacer.
  • T cell activation is too strong, it can impair CAR-T cell persistence and function in vivo due to activation-induced cell death (AICD) (Kuunkele et al., Cancer Immunol. Res. (2015) 3:368-379), and excessive activation of CAR-T cells can moreover contribute to undesirable side effects such as cytokine release syndrome.
  • AICD activation-induced cell death
  • Spacer domain engineering can be useful to fine-tune activation of CAR-expressing immune cells to an optimal level.
  • IgG 1 Fc, lgG4 Fc and lgG2 Fc spacer domains have been employed in CARs, and variants of IgG 1 Fc and lgG4 Fc spacers designed to reduce/eliminate binding to FcyRs have been shown to reduce depletion of cells expressing the CARs and improve persistence in vivo.
  • Non-IgG Fc-based spacers such as CD8 and CD28 spacer domains have also been used in clinically- approved CAR-T cell therapies.
  • a CD28-derived spacer has been shown to result in increased CAR activation compared to a CD8a-derived spacer, which might be a consequence of an increased propensity of the CD28-derived spacer to participate in homotypic associations (Alabanza et al., Mol. Ther. (2017) 25:2452-2465).
  • Spacer domains are also commonly used in the detection of CAR-expressing cells in vitro and in vivo.
  • Fc- specific antibodies can be employed to detect CARs comprising a spacer domain comprising the appropriate Fc region, and specific epitope tags have also been incorporated into CAR spacer domains for such purposes (see e.g. Liu et al., Nat. Biotechnol. (2016) 34:430-434).
  • a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence derived from the amino acid sequence of one of 41 BB, 0X40, CD96 CD44, lgG2 or IgG 1 . In some embodiments, a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence derived from the amino acid sequence of one of 41 BB, 0X40, CD96 or CD44. In some embodiments, a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence derived from the amino acid sequence of one of 41 BB.
  • a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:96.
  • a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:97.
  • a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:98.
  • a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:99.
  • a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NQ:100.
  • a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NQ:101 .
  • a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NQ:102.
  • the CAR of the present disclosure comprises a transmembrane domain.
  • a transmembrane domain refers to any three-dimensional structure formed by a sequence of amino acids which is thermodynamically stable in a biological membrane, e.g. a cell membrane.
  • the transmembrane domain may be an amino acid sequence which spans the cell membrane of a cell expressing the CAR.
  • the transmembrane domain may comprise or consist of a sequence of amino acids which forms a hydrophobic alpha helix or beta-barrel.
  • the amino acid sequence of the transmembrane domain of the CAR of the present disclosure may be, or may be derived from, the amino acid sequence of a transmembrane domain of a protein comprising a transmembrane domain.
  • Transmembrane domains are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as TMHMM (Krogh et al., 2001 J Mol Biol 305: 567-580).
  • the amino acid sequence of the transmembrane domain of the CAR of the present disclosure may be, or may be derived from, the amino acid sequence of the transmembrane domain of a protein expressed at the cell surface.
  • the protein expressed at the cell surface is a receptor or ligand, e.g. an immune receptor or ligand.
  • the amino acid sequence of the transmembrane domain may be, or may be derived from, the amino acid sequence of the transmembrane domain of one of ICOS, ICOSL, CD86, CTLA-4, CD28, CD80, MHC class I a, MHC class II a, MHC class II p, CD3e, CD35, CD3y, CD3 TCRa TCRp, CD4, CD8a, CD8p, CD40, CD40L, PD-1 , PD-L1 , PD-L2, 4-1 BB, 4-1 BBL, 0X40, OX40L, GITR, GITRL, TIM-3, Galectin 9, LAG3, CD27, CD70, LIGHT, HVEM, TIM-4, TIM-1 , ICAM1 , LFA-1 , LFA-3, CD2, BTLA, CD160, LILRB4, LILRB2, VTCN1 , CD2, CD48, 2B4, SLAM, CD30, CD30L,
  • the transmembrane is, or is derived from, the amino acid sequence of the transmembrane domain of CD28, CD3- , CD8a, CD80 or CD4. In some embodiments, the transmembrane is, or is derived from, the amino acid sequence of the transmembrane domain of CD28.
  • the transmembrane domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:103.
  • the transmembrane domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:104.
  • the transmembrane domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:105.
  • the chimeric antigen receptor of the present disclosure comprises a signalling domain.
  • the signalling domain provides sequences for initiating intracellular signalling in cells expressing the CAR.
  • ITAM-containinq sequence :
  • the signalling domain comprises ITAM-containing sequence.
  • An ITAM-containing sequence comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs comprise the amino acid sequence YXXL/I (SEQ ID NO:109), wherein ‘X’ denotes any amino acid.
  • SEQ ID NO:109 sequences according to SEQ ID NO:109 are often separated by 6 to 8 amino acids; YXXL/I (X)6-sYXXL/l (SEQ ID NO:110).
  • the signalling domain comprises one or more copies of an amino acid sequence according to SEQ ID NO:109 or SEQ ID NO:110. In some embodiments, the signalling domain comprises at least 1 , 2, 3, 4, 5 or 6 copies of an amino acid sequence according to SEQ ID NO:109. In some embodiments, the signalling domain comprises at least 1 , 2, or 3 copies of an amino acid sequence according to SEQ ID NO:110.
  • the signalling domain comprises an ITAM-containing sequence which is, or which is derived from, the amino acid sequence of an ITAM-containing sequence of a protein having an ITAM- containing amino acid sequence.
  • the signalling domain comprises an ITAM- containing sequence which is, or which is derived from, the amino acid sequence of the intracellular domain of one of CD3 FcyRI, CD3e, CD35, CD3y, CD79a, CD79p, FcyRIIA, FcyRIIC, FcyRIIIA, FcyRIV or DAP12.
  • the signalling domain comprises an ITAM-containing sequence which is, or which is derived from, the amino acid sequence of the intracellular domain of CD3-
  • the signalling domain comprises an ITAM-containing sequence which comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:111.
  • the signalling domain may additionally comprise one or more costimulatory sequences.
  • a costimulatory sequence is an amino acid sequence which provides for costimulation of the cell expressing the CAR of the present disclosure. Costimulation promotes proliferation and survival of a CAR-expressing cell upon binding to the target antigen, and may also promote cytokine production, differentiation, cytotoxic function and memory formation by the CAR-expressing cell. Molecular mechanisms of T cell costimulation are reviewed in Chen and Flies, (2013) Nat Rev Immunol 13(4):227-242.
  • a costimulatory sequence may be, or may be derived from, the amino acid sequence of a costimulatory protein.
  • the costimulatory sequence is an amino acid sequence which is, or which is derived from, the amino acid sequence of the intracellular domain of a costimulatory protein.
  • the costimulatory sequence Upon binding of the CAR to the target antigen, the costimulatory sequence provides costimulation to the cell expressing the CAR of the kind which would be provided by the costimulatory protein from which the costimulatory sequence is derived upon ligation by its cognate ligand.
  • the costimulatory sequence is capable of delivering the costimulation signal of the costimulatory protein from which the costimulatory sequence is derived.
  • the costimulatory protein may be a member of the B7-CD28 superfamily (e.g. CD28, ICOS), or a member of the TNF receptor superfamily (e.g. 4-1 BB, 0X40, CD27, DR3, GITR, CD30, HVEM).
  • the costimulatory sequence is, or is derived from, the intracellular domain of one of CD28, 4-1 BB, ICOS, CD27, 0X40, HVEM, CD2, SLAM, TIM-1 , CD30, GITR, DR3, CD226 and LIGHT.
  • the costimulatory sequence is, or is derived from, the intracellular domain of CD28.
  • the signalling domain comprises more than one costimulatory sequence. In some embodiments the signalling domain comprises 1 , 2, 3, 4, 5 or 6 costimulatory sequences. Plural costimulatory sequences may be provided in tandem.
  • Whether a given amino acid sequence is capable of initiating signalling mediated by a given costimulatory protein can be investigated e.g. by analysing a correlate of signalling mediated by the costimulatory protein (e.g. expression/activity of a factor whose expression/activity is upregulated or downregulated as a consequence of signalling mediated by the costimulatory protein).
  • a correlate of signalling mediated by the costimulatory protein e.g. expression/activity of a factor whose expression/activity is upregulated or downregulated as a consequence of signalling mediated by the costimulatory protein.
  • Costimulatory proteins upregulate expression of genes promoting cell growth, effector function and survival through several transduction pathways.
  • CD28 and ICOS signal through phosphatidylinositol 3 kinase (PI3K) and AKT to upregulate expression of genes promoting cell growth, effector function and survival through NF-KB, mTOR, NFAT and AP1/2.
  • PI3K phosphatidylinositol 3 kinase
  • AKT phosphatidylinositol 3 kinase
  • CD28 also activates AP1/2 via CDC42/RAC1 and ERK1 Z2 via RAS
  • ICOS activates C-MAF.
  • 4-1 BB, 0X40, and CD27 recruit TNF receptor associated factor (TRAF) and signal through MAPK pathways, as well as through PI3K.
  • TNF receptor associated factor TNF receptor associated factor
  • the signalling domain comprises a costimulatory sequence which is, or which is derived from CD28.
  • Kofler et al. Mol. Ther. (2011 ) 19: 760-767 describes a variant CD28 intracellular domain in which the lek kinase binding site is mutated in order to reduce induction of IL-2 production on CAR ligation, in order to minimise regulatory T cell-mediated suppression of CAR-T cell activity.
  • the amino acid sequence of the variant CD28 intracellular domain is shown in SEQ ID NO:107.
  • the signalling domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:106.
  • the signalling domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NQ:107.
  • the signalling domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:108.
  • the signalling domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:115.
  • the CAR comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:116.
  • the CARs of the present disclosure may additionally comprise further amino acids or sequences of amino acids.
  • amino acid sequence described herein may comprise one or more additional amino acids at one or both ends (i.e. the N- or C- terminus) of the reference amino acid sequence.
  • the peptide/polypeptide comprises e.g. 1 -5, 1 -10, 1 -20, 1 -30, 1 -40, 1 -50, 5-10, 5-20, 5-30, 5-40, 5-50, 10-20, 10-30, 10-40, 10-50, 20-30, 20-40 or 20-50 additional amino acids at one or both ends of the reference amino acid sequence.
  • the additional amino acids correspond to the amino acids provided at those positions relative to the reference amino acid sequence, in the context of the protein from which the reference amino acid sequence is derived.
  • a reference amino acid sequence corresponds to amino acid positions 20 to 30 of the amino acid sequence of the protein from which the reference amino acid sequence is derived
  • those additional 5 amino acids may correspond to positions 15 to 19 of the amino acid sequence of the protein from which the reference amino acid sequence is derived.
  • the CARs of the present disclosure may comprise one or more linker sequences between sequences of amino acids.
  • a linker sequence may be provided between domains of a CAR (e.g. between the antigen-binding domain and spacer domain, and/or between the spacer domain and the transmembrane domain, and/or between the transmembrane domain and the signalling domain).
  • a linker sequence may be provided between subsequences of the domains of a CAR (e.g. between VH and VL of an antigen-binding domain, and/or between the costimulatory and sequence and ITAM-containing sequence of a signalling domain).
  • Linker sequences are known to the skilled person, and are described, for example in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety.
  • a linker sequence may be a flexible linker sequence.
  • Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence.
  • Flexible linkers are known to the skilled person, and several are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and/or serine residues.
  • the linker sequence comprises or consists of (G4S)4 or (G4S)e. In some embodiments, the linker sequence has a length of 1 -2, 1 -3, 1 -4, 1 -5, 1 -10, 1 -15, 1 -20, 1 -25, or 1 -30 amino acids.
  • the linker sequence comprises one or more copies of an amino acid sequence according to SEQ ID NO:57. In some embodiments, the linker sequence comprises at least 1 , 2, 3 or 4 copies of an amino acid sequence according to SEQ ID NO:57.
  • the linker sequence comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:58.
  • the CARs of the present disclosure may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification or detection of the antigen-binding molecule/polypeptide.
  • antigen-binding molecules and polypeptides of the present disclosure may additionally comprise a sequence of amino acids forming a detectable moiety, e.g. as described hereinbelow.
  • the CARs of the present disclosure may additionally comprise a signal peptide (also known as a leader sequence or signal sequence).
  • Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides.
  • Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt and Ensembl, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).
  • the signal peptide may be present at the N-terminus of the CAR, and may be present in the newly synthesised CAR.
  • the signal peptide provides for efficient trafficking of the CAR to the cell surface. Signal peptides are often removed by cleavage, and thus are not comprised in the mature CAR expressed at the cell surface.
  • Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 201 1 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176).
  • SignalP Protein et al., 201 1 Nature Methods 8: 785-786
  • Signal-BLAST Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176.
  • the signal peptide comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:1 12.
  • the signal peptide comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:1 13.
  • the signal peptide comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:1 14.
  • the CAR of the present disclosure additionally comprise a detectable moiety.
  • a detectable moiety is a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label (e.g. an epitope tag), radiolabel, chemical, nucleic acid or enzymatic label.
  • the CAR may be covalently or non-covalently labelled with the detectable moiety.
  • Fluorescent labels include e.g. fluorescein, rhodamine, allophycocyanin, eosine and NDB, green fluorescent protein (GFP), chelates of rare earths such as europium (Eu), terbium (Tb) and samarium (Sm), tetramethyl rhodamine, Texas Red, 4-methyl umbelliferone, 7-amino-4-methyl coumarin, Cy3, and Cy5.
  • fluorescein e.g. fluorescein, rhodamine, allophycocyanin, eosine and NDB
  • GFP green fluorescent protein
  • Eu europium
  • Tb terbium
  • Sm samarium
  • tetramethyl rhodamine Texas Red
  • 4-methyl umbelliferone 7-amino-4-methyl coumarin
  • Cy3 Cy5
  • Radiolabels include radioisotopes such as Hydrogen 3 , Sulfur 35 , Carbon 14 , Phosphorus 32 , Iodine 123 , Iodine 125 , Iodine 126 , Iodine 131 , Iodine 133 , Bromine 77 , Technetiurn 99m , Indium 111 , Indium 113 " 1 , Gallium 67 , Gallium 68 , Ruthenium 95 , Ruthenium 97 , Ruthenium 103 , Ruthenium 105 , Mercury 207 , Mercury 203 , Rhenium 99m , Rhenium 101 , Rhenium 105 , Scandium 47 , Tellurium 121 “ 1 , Tellurium 122 “ 1 , Tellurium 125 “ 1 , Thulium 165 , Thuliuml 167 , Thulium 168 , Copper 67 , Fluorine 18 , Yttrium 90 , Palladium 100 , Bis
  • Luminescent labels include as radioluminescent, chemiluminescent (e.g. acridinium ester, luminol, isoluminol) and bioluminescent labels.
  • Immuno-detectable labels include haptens, peptides/polypeptides, antibodies, receptors and ligands such as biotin, avidin, streptavidin or digoxigenin.
  • Nucleic acid labels include aptamers.
  • the CAR comprises an epitope tag, e.g. a His, (e.g. 6XHis), FLAG, c-Myc, StrepTag, haemagglutinin, E, calmodulin-binding protein (CBP), glutathione-s-transferase (GST), maltose-binding protein (MBP), thioredoxin, S-peptide, T7 peptide, SH2 domain, avidin, streptavidin, and haptens (e.g. biotin, digoxigenin, dinitrophenol), optionally at the N- or C- terminus of the antigen-binding molecule/polypeptide/CAR.
  • an epitope tag e.g. a His, (e.g. 6XHis), FLAG, c-Myc, StrepTag, haemagglutinin, E, calmodulin-binding protein (CBP), glutathione-s-transferase (GST), mal
  • the CAR comprises a moiety having a detectable activity, e.g. an enzymatic moiety.
  • Enzymatic moieties include e.g. luciferases, glucose oxidases, galactosidases (e.g. beta- galactosidase), glucorinidases, phosphatases (e.g. alkaline phosphatase), peroxidases (e.g. horseradish peroxidase) and cholinesterases.
  • the CAR of the present disclosure is conjugated to a chemical moiety.
  • the chemical moiety may be a moiety for providing a therapeutic effect, i.e. a drug moiety.
  • a drug moiety may be a small molecule (e.g. a low molecular weight ( ⁇ 1000 daltons, typically between -300-700 daltons) organic compound).
  • Drug moieties are described e.g. in Parslow et al., Biomedicines. 2016 Sep; 4(3):14 (hereby incorporated by reference in its entirety).
  • a drug moiety may be or comprise a cytotoxic agent.
  • a drug moiety may be or comprise a chemotherapeutic agent.
  • Drug moieties include e.g. calicheamicin, DM1 , DM4, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5 and PBD.
  • the CAR according to the present disclosure comprises, or consists of:
  • An antigen-binding domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 or 182;
  • a spacer domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:100, 98, 99, 101 , or 102;
  • a transmembrane domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:103, 104 or 105; and
  • a signalling domain comprising:
  • a costimulatory sequence comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NQ:106, 107 or 108; and
  • An ITAM-containing sequence comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:1 1 1 .
  • the CAR comprises, or consists of:
  • An antigen-binding domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 or 182;
  • a spacer domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:100, 98, 99, 101 , or 102;
  • a transmembrane domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:103;
  • a signalling domain comprising:
  • a costimulatory sequence comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NQ:106; and
  • An ITAM-containing sequence comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:111.
  • the CAR comprises, or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:117, 118, 119, 120, 121 ,
  • the CAR comprises, or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:117, 118, 119, 120, 121 ,
  • the CAR comprises, or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:119, 124, 129, 134, 139,
  • the CAR comprises, or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:124, 144, 190 or 210.
  • Nucleic acids and vectors are provided.
  • the present disclosure provides a nucleic acid, or a plurality of nucleic acids, encoding a CAR according to the present disclosure.
  • the nucleic acid(s) comprise or consist of DNA and/or RNA.
  • the nucleic acid(s) may be, or may be comprised in, a vector, or a plurality of vectors. That is, the nucleotide sequence(s) of the nucleic acid(s) may be contained in vector(s).
  • the CAR according to the present disclosure may be produced within a cell by transcription from a vector encoding the antigen-binding molecule, polypeptide or CAR, and subsequent translation of the transcribed RNA.
  • the present disclosure also provides a vector, or plurality of vectors, comprising the nucleic acid or plurality of nucleic acids according to the present disclosure.
  • the vector may facilitate delivery of the nucleic acid(s) encoding an CAR according to the present disclosure.
  • the vector may be an expression vector comprising elements required for expressing nucleic acid(s) comprising/encoding an CAR according to the present disclosure.
  • Nucleic acids and vectors according to the present disclosure may be provided in purified or isolated form, i.e. from other nucleic acid, or naturally-occurring biological material.
  • the nucleotide sequence may be contained in a vector, e.g. an expression vector.
  • a ‘vector’ as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell.
  • the vector may be a vector for expression of the nucleic acid in the cell.
  • Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed.
  • a vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.
  • operably linked may include the situation where a selected nucleic acid sequence and regulatory nucleic acid sequence (e.g. promoter and/or enhancer) are covalently linked in such a way as to place the expression of nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette).
  • a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence.
  • the resulting transcript(s) may then be translated into a desired peptide(s)/polypeptide(s).
  • Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g. retroviral vectors, e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors, e.g. SFG vector), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes), e.g.
  • retroviral vectors e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors, e.g. SFG vector)
  • lentiviral vectors e.g. murine Leukemia virus (MLV)-derived vectors, e.g. SFG vector
  • lentiviral vectors e.g. murine
  • the vector may be a eukaryotic vector, e.g. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell.
  • the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.
  • CMV cytomegalovirus
  • the present disclosure provides a cell comprising a CAR according to the present disclosure.
  • the CAR according to the present disclosure may be used to generate a CAR- expressing cell, e.g. a CAR-expressing immune cells(e.cj. CAR-T or CAR-NK cell).
  • CAR-expressing cells may comprise or express nucleic acid encoding a CAR according to the present disclosure. It will be appreciated that a CAR-expressing cell comprises the CAR it expresses. It will also be appreciated that a cell expressing nucleic acid encoding a CAR also expresses and comprises the CAR encoded by the nucleic acid.
  • the cell may be a eukaryotic cell, e.g. a mammalian cell.
  • the mammal may be a primate (rhesus, cynomolgous, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate).
  • rodent including any animal in the order Rodentia
  • cat, dog, pig, sheep, goat, cattle including cows, e.g. dairy cows, or any animal in the order Bos
  • horse including any animal in the order Equidae
  • donkey and non-human primate
  • CAR-expressing cell is preferably an immune cell.
  • An immune cell may be a cell of hematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte.
  • a lymphocyte may be e.g. a T cell, B cell, NK cell, NKT cell or innate lymphoid cell (ILC), or a precursor thereof.
  • the immune cell may express e.g. CD3 polypeptides (e.g. CD3y CD3e CD3 or CD35), TCR polypeptides (TCRa or TCR0), CD27, CD28, CD4 or CD8.
  • the immune cell is a T cell, e.g.
  • the T cell is a CD3+, CD4+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a T helper cell (TH cell). In some embodiments, the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)).
  • CTL cytotoxic T lymphocyte
  • T cells comprising/expressing CD30-specific CARs according to the present disclosure.
  • the immune cell may be a virus-specific immune cell.
  • a ‘virus-specific immune cell’ as used herein refers to an immune cell which is specific for a virus.
  • a virus-specific immune cell expresses/comprises a receptor (preferably a T cell receptor) capable of recognising a peptide of an antigen of a virus (e.g. when presented by an MHC molecule).
  • the virus-specific immune cell may express/comprise such a receptor as a result of expression of endogenous nucleic acid encoding such antigen receptor, or as a result of having been engineered to express such a receptor.
  • the virus-specific immune cell preferably expresses/comprises a TCR specific for a peptide of an antigen of a virus.
  • a virusspecific T cell may display certain functional properties of a T cell in response to the viral antigen for which the T cell is specific, or in response a cell comprising/expressing the virus/antigen.
  • the properties are functional properties associated with effector T cells, e.g. cytotoxic T cells.
  • a virus-specific T cell may display one or more of the following properties: cytotoxicity to a cell comprising/expressing the virus /the viral antigen for which the T cell is specific; proliferation, IFNy expression, CD107a expression, IL-2 expression, TNFa expression, perforin expression, granzyme expression, granulysin expression, and/or FAS ligand (FASL) expression in response to stimulation with the virus/the viral antigen for which the T cell is specific, or in response to exposure to a cell comprising/expressing the virus /the viral antigen for which the T cell is specific.
  • FAS ligand FAS ligand
  • Virus-specific T cells express/comprise a TCR capable of recognising a peptide of the viral antigen for which the T cell is specific when presented by the appropriate MHC molecule.
  • Virus-specific T cells may be CD4+ T cells and/or CD8+ T cells.
  • the virus for which the virus-specific immune cell is specific may be any virus.
  • the virus may be a dsDNA virus (e.g. adenovirus, herpesvirus, poxvirus), ssRNA virus (e.g. parvovirus), dsRNA virus (e.g. reovirus), (+)ssRNA virus (e.g. picornavirus, togavirus), (-)ssRNA virus (e.g. orthomyxovirus, rhabdovirus), ssRNA-RT virus (e.g. retrovirus) or dsDNA-RT virus (e.g. hepadnavirus).
  • dsDNA virus e.g. adenovirus, herpesvirus, poxvirus
  • ssRNA virus e.g. parvovirus
  • dsRNA virus e.g. reovirus
  • (+)ssRNA virus e.g. picornavirus, togavirus
  • (-)ssRNA virus e.g. ortho
  • the present disclosure contemplates viruses of the families adenoviridae, herpesviridae, papillomaviridae, polyomaviridae, poxviridae, hepadnaviridae, parvoviridae, astroviridae, caliciviridae, picornaviridae, coronaviridae, flaviviridae, togaviridae, hepeviridae, retroviridae, orthomyxoviridae, arenaviridae, bunyaviridae, filoviridae, paramyxoviridae, rhabdoviridae and reoviridae.
  • the virus is selected from Epstein-Barr virus, adenovirus, Herpes simplex type 1 virus, Herpes simplex type 2 virus, Varicella-zoster virus, Human cytomegalovirus, Human herpesvirus type 8, Human papillomavirus, BK virus, JC virus, Smallpox, Hepatitis B virus, Parvovirus B19, Human Astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, TBE virus, Rubella virus, Hepatitis E virus, Human immunodeficiency virus, influenza virus, lassa virus, Crimean-Congo hemorrhagic fever virus, Hantaan virus, ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, picornavirus, respiratory syncytial virus, rabies virus, he
  • the virus is selected from Epstein-Barr virus (EBV), adenovirus, cytomegalovius (CMV), human papilloma virus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), or herpes simplex virus (HSV).
  • EBV Epstein-Barr virus
  • CMV cytomegalovius
  • HPV human papilloma virus
  • influenza virus measles virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • HMV human immunodeficiency virus
  • LCMV lymphocytic choriomeningitis virus
  • HSV herpes simplex virus
  • the virus-specific immune cell may be specific for a peptide/polypeptide of a virus e.g. selected from Epstein-Barr virus (EBV), adenovirus, cytomegalovius (CMV), human papilloma virus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), or herpes simplex virus (HSV).
  • a virus e.g. selected from Epstein-Barr virus (EBV), adenovirus, cytomegalovius (CMV), human papilloma virus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), or herpes
  • a T cell which is specific for an antigen of a virus may be referred to herein as a virus-specific T cell (VST).
  • VST virus-specific T cell
  • a T cell which is specific for an antigen of a particular virus may be described as being specific for the relevant virus; for example, a T cell which is specific for an antigen of EBV may be referred to as an EBV-specific T cell, or ‘EBVST’.
  • the virus-specific immune cell is an Epstein-Barr virus-specific T cell (EBVST), adenovirus-specific T cell (AdVST), cytomegalovius-specific T cell (CMVST), human papilloma virus (HPVST), influenza virus-specific T cell, measles virus-specific T cell, hepatitis B virus-specific T cell (HBVST), hepatitis C virus-specific T cell (HCVST), human immunodeficiency virus-specific T cell (HIVST), lymphocytic choriomeningitis virus-specific T cell (LCMVST), or herpes simplex virus-specific T cell (HSVST).
  • EBVST Epstein-Barr virus-specific T cell
  • AdVST adenovirus-specific T cell
  • CMVST cytomegalovius-specific T cell
  • HPVST human papilloma virus
  • influenza virus-specific T cell measles virus-specific T cell
  • HBVST hepatitis
  • the virus-specific immune cell is specific for a peptide/polypeptide of an EBV antigen.
  • the virus-specific immune cell is an Epstein-Barr virus-specific T cell (EBVST).
  • EBV infects epithelial cells via binding of viral protein BMFR2 to pi integrins, and binding of viral protein gH/gL with integrins av06 and av08.
  • EBV infects B cells through interaction of viral glycoprotein gp350 with CD21 and/or CD35, followed by interaction of viral gp42 with MHC class II. These interactions trigger fusion of the viral envelope with the cell membrane, allowing the virus to enter the cell. Once inside, the viral capsid dissolves and the viral genome is transported to the nucleus.
  • EBV has two modes of replication; latent and lytic.
  • the latent cycle does not result in production of virions, and can take place in place B cells and epithelial cells.
  • the EBV genomic circular DNA resides in the cell nucleus as an episome and is copied by the host cell’s DNA polymerase.
  • latency only a fraction of EBV's genes are expressed, in one of three different patterns known as latency programs, which produce distinct sets of viral proteins and RNAs.
  • the latent cycle is described e.g. in Amon and Farrell, Reviews in Medical Virology (2004) 15(3): 149-56, which is hereby incorporated by reference in its entirety.
  • Latency programs II and III further involve expression of EBNALP, LMP1 , LMP2A and LMP2B proteins, and latency program III further involves expression of EBNA2, EBNA3A, EBNA3B and EBNA3C.
  • EBNA1 is multifunctional, and has roles in gene regulation, extrachromosomal replication, and maintenance of the EBV episomal genome through positive and negative regulation of viral promoters (Duellman et al., J Gen Virol. (2009); 90(Pt 9): 2251-2259).
  • EBNA2 is involved in the regulation of latent viral transcription and contributes to the immortalisation of cells infected with EBV (Kempkes and Ling, Curr Top Microbiol Immunol. (2015) 391 :35-59).
  • EBNA-LP is required for transformation of native B cells, and recruits transcription factors for viral replication (Szymula et al., PLoS Pathog.
  • EBNA3A, 3B and 3C interact with RBPJ to influence gene expression, contributing to survival and growth of infected cells (Wang et al., J Virol. (2016) 90(6):2906-2919).
  • LMP1 regulates expression of genes involved in B cell activation (Chang et al., J. Biomed. Sci. (2003) 10(5): 490-504).
  • LMP2A and LMP2B inhibit normal B cell signal transduction by mimicking the activated B cell receptor (Portis and Longnecker, Oncogene (2004) 23(53): 8619-8628).
  • EBERs form ribonucleoprotein complexes with host cell proteins, and are proposed to have roles in cell transformation.
  • the latent cycle can progress according to any of latency programs I to III in B cells, and usually progresses from III to II to I.
  • EBV Upon infection of a resting naive B cell, EBV enters latency program III. Expression of latency III genes activates the B cell, which becomes a proliferating blast. EBV then typically progresses to latency II by restricting expression to a subset of genes, which cause differentiation of the blast to a memory B cell. Further restriction of gene expression causes EBV to enter latency I.
  • EBNA1 expression allows EBV to replicate when the memory B cell divides. In epithelial cells, only latency II occurs.
  • EBV In primary infection, EBV replicates in oropharyngeal epithelial cells and establishes Latency III, II, and I infections in B-lymphocytes. EBV latent infection of B-lymphocytes is necessary for virus persistence, subsequent replication in epithelial cells, and release of infectious virus into saliva. EBV Latency III and II infections of B-lymphocytes, Latency II infection of oral epithelial cells, and Latency II infection of NK- or T cell can result in malignancies, marked by uniform EBV genome presence and gene expression.
  • Latent EBV in B cells can be reactivated to switch to lytic replication.
  • the lytic cycle results in the production of infectious virions and can take place in place B cells and epithelial cells, and is reviewed e.g. by Kenney in Chapter 25 of Arvin et al., Human Herpesviruses: Biology, Therapy and Immunoprophylaxis; Cambridge University Press (2007), which is hereby incorporated by reference in its entirety.
  • Lytic replication requires the EBV genome to be linear.
  • the latent EBV genome is episomal, and so it must be linearised for lytic reactivation.
  • lytic replication normally only takes place after reactivation from latency.
  • Immediate-early lytic gene products such as BZFL1 and BRLF1 act as transactivators, enhancing their own expression, and the expression of later lytic cycle genes.
  • Early lytic gene products have roles in viral replication (e.g. EBV DNA polymerase catalytic component BALF5; DNA polymerase processivity factor BMRF1 , DNA binding protein BALF2, helicase BBLF4, primase BSLF1 , and primase-associated protein BBLF2/3) and deoxynucleotide metabolism (e.g. thymidine kinase BXLF1 , dUTPase BORF2).
  • Other early lytic gene products act transcription factors (e.g. BMRF1 , BRRF1 ), have roles in RNA stability and processing (e.g. BMLF1 ), or are involved in immune evasion (e.g. BHRF1 , which inhibits apoptosis).
  • Late lytic gene products are traditionally classed as those expressed after the onset of viral replication. They generally encode structural components of the virion such as nucleocapsid proteins, as well as glycoproteins which mediate EBV binding and fusion (e.g. gp350/220, gp85, gp42, gp25). Other late lytic gene products have roles in immune evasion; BCLF1 encodes a viral homologue of IL-10, and BALF1 encodes a protein with homology to the anti-apoptotic protein Bcl2.
  • An ‘EBV-specific immune cell’ as used herein refers to an immune cell which is specific for Epstein-Barr virus (EBV).
  • An EBV-specific immune cell expresses/comprises a receptor (preferably a T cell receptor) capable of recognising a peptide of an antigen of EBV (e.g. when presented by an MHC molecule).
  • the EBV-specific immune cell preferably expresses/comprises a TCR specific for a peptide of an EBV antigen presented by MHC class I.
  • the EBV-specific immune cell is a T cell, e.g. a CD3+ T cell.
  • the T cell is a CD3+, CD4+ T cell.
  • the T cell is a CD3+, CD8+ T cell.
  • the T cell is a T helper cell (TH cell)).
  • the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)).
  • CTL cytotoxic T lymphocyte
  • EBV-specific T cells preferably express/comprise a TCR capable of recognising a peptide of the EBV antigen for which the T cell is specific when presented by the appropriate MHC molecule.
  • EBV-specific T cells may be CD4+ T cells and/or CD8+ T cells.
  • An immune cell specific for EBV may be specific for any EBV antigen, e.g. an EBV antigen described herein.
  • a population of immune cell specific for EBV, or a composition comprising a plurality of immune cells specific for EBV, may comprise immune cells specific for one or more EBV antigens.
  • an EBV antigen is an EBV latent antigen, e.g. a type III latency antigen (e.g. EBNA1 , EBNA-LP, LMP1 , LMP2A, LMP2B, BARF1 , EBNA2, EBNA3A, EBNA3B or EBNA3C), a type II latency antigen (e.g. EBNA1 , EBNA-LP, LMP1 , LMP2A, LMP2B or BARF1 ), or a type I latency antigen, (e.g. EBNA1 or BARF1 ).
  • an EBV antigen is an EBV lytic antigen, e.g.
  • an immediate-early lytic antigen e.g. BZLF1 , BRLF1 or BMRF1
  • an early lytic antigen e.g. BMLF1 , BMRF1 , BXLF1 , BALF1 , BALF2, BARF1 , BGLF5, BHRF1 , BNLF2A, BNLF2B, BHLF1 , BLLF2, BKRF4, BMRF2, FU or EBNA1 -FUK
  • a late lytic antigen e.g. BALF4, BILF1 , BILF2, BNFR1 , BVRF2, BALF3, BALF5, BDLF3 or gp350.
  • cells may comprise/express more than one (e.g. 2, 3, 4, etc.) CAR.
  • the cells may comprise/express more than one, non-identical CAR.
  • Cells comprising/expressing more than one non-identical CAR may comprise/express CARs specific for non- identical target antigens.
  • each non-identical target antigen is independently a cancer cell antigen as described herein.
  • Cells e.g. immune cells, e.g. T cells
  • a CAR may display certain functional properties in response to the target antigen for the CAR (e.g. CD30), or in response a cell comprising/expressing the target antigen for the CAR.
  • the properties are functional properties associated with effector T cells, e.g. cytotoxic T cells.
  • Cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure may display one or more of the following properties: expression of one or more cytotoxic/effector factors (e.g. IFNy, TNFa, GM-CSF), proliferation/population expansion, and/or growth factor (e.g. IL-2) expression in response to cells expressing the target antigen for the CAR (e.g. CD30); cytotoxicity to cells expressing the target antigen for the CAR (e.g. CD30); no cytotoxicity (i.e. above baseline) to cells which do not express the target antigen for the CAR (e.g. CD30); anti-cancer activity (e.g.
  • cytotoxic/effector factors e.g. IFNy, TNFa, GM-CSF
  • proliferation/population expansion e.g. IL-2
  • growth factor e.g. IL-2
  • cytotoxicity to cancer cells, tumor growth inhibition, reduction of metastasis, etc. against cancer comprising cells expressing the target antigen for the CAR (e.g. CD30); cytotoxicity to alloreactive immune cells, e.g. alloreactive immune cells expressing the target antigen for the CAR (e.g. CD30).
  • the target antigen for the CAR e.g. CD30
  • alloreactive immune cells e.g. alloreactive immune cells expressing the target antigen for the CAR (e.g. CD30).
  • CAR-expressing T cell may display one or more of the following properties: cytotoxicity to a cell comprising/expressing the target antigen for the CAR; proliferation, IFNy expression, CD107a expression, IL-2 expression, TNFa expression, perforin expression, granzyme expression, granulysin expression, and/or FAS ligand (FASL) expression in response to stimulation with the target antigen for the CAR, or in response to exposure to a cell comprising/expressing the target antigen for the CAR; anti-cancer activity (e.g. cytotoxicity to cancer cells, tumor growth inhibition, reduction of metastasis, etc.) against cancer comprising cells expressing the target antigen for the CAR.
  • FAS ligand FAS ligand
  • Cell proliferation/population expansion can be investigated by analysing cell division or the number of cells over a period of time.
  • Cell division can be analysed, for example, by in vitro analysis of incorporation of 3 H-thymidine or by CFSE dilution assay, e.g. as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564, hereby incorporated by reference in entirety.
  • Proliferating cells can also be identified by analysis of incorporation of 5-ethynyl-2'-deoxyuridine (EdU) by an appropriate assay, as described e.g. in Buck et al., Biotechniques. 2008 Jun; 44(7):927-9, and Sali and Mitchison, PNAS USA 2008 Feb 19; 105(7): 2415-2420, both hereby incorporated by reference in their entirety.
  • EdU 5-ethynyl-2'-deoxyuridine
  • ‘expression’ may be gene or protein expression.
  • Gene expression encompasses transcription of DNA to RNA, and can be measured by various means known to those skilled in the art, for example by measuring levels of mRNA by quantitative real-time PCR (qRT-PCR), or by reporterbased methods.
  • protein expression can be measured by various methods well known in the art, e.g. by antibody-based methods, for example by western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or reporter-based methods.
  • Cytotoxicity and cell killing can be investigated, for example, using any of the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (2011 ), 9(6):601 -616, hereby incorporated by reference in its entirety.
  • Examples of in vitro assays of cytotoxicity/cell killing assays include release assays such as the 51 Cr release assay, the lactate dehydrogenase (LDH) release assay, the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) release assay, and the calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cell killing based on the detection of factors released from lysed cells.
  • LDH lactate dehydrogenase
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide
  • calcein-AM calcein-acetoxy
  • Cell killing of a given test cell type by a given effector immune cell type can be analysed e.g. by co-culturing the test cells with the effector immune cells, and measuring the number/proportion of viable/dead (e.g. lysed) test cells after a suitable period of time.
  • suitable assays include the xCELLigence real-time cytolytic in vitro potency assay described in Cerignoli et al., PLoS One. (2016) 13(3): e0193498 (hereby incorporated by reference in its entirety).
  • cell killing of cells expressing the target antigen for the CAR by CAR-expressing cells may be evaluated by xCELLigence assay as described in Example 1 .5 herein.
  • Cell killing by CAR- expressing cells can also be evaluated in vivo, e.g. by evaluating the number/proportion of cells expressing the target antigen for the CAR, and inferring their kil ling/depletion by CAR-expressing cells.
  • Cells may be evaluated for anti-cancer activity by analysis in an appropriate in vitro assays or in vivo models of the relevant cancer.
  • the cells comprising a CAR/nucleic acid encoding a CAR preferably possess novel and/or improved properties compared to cells comprising a CAR/nucleic acid encoding an equivalent CAR construct identical in all respects, with the exception that the spacer domain consists of the sequence shown in SEQ ID NO:96, 97 or 98.
  • the CAR construct of SEQ ID NO:213 is identical to that of SEQ ID NO:119, with the exception that the CAR of SEQ ID NO:213 comprises the human IgG 1 CH2-CH3 spacer domain shown in SEQ ID NO:96, whereas the CAR of SEQ ID NO:119 comprises the 41 BB spacer domain shown in SEQ ID NO:100.
  • the cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure possess novel and/or improved properties compared to cells comprising a CAR/nucleic acid encoding an equivalent CAR construct identical in all respects, with the exception that the spacer domain consists of the sequence shown in SEQ ID NO:96.
  • such reference CARs comprising the human IgG 1 CH2- CH3 spacer domain may be referred to simply as ‘an equivalent CAR comprising the hlgG 1 spacer’.
  • cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure: expand and/or proliferate to an extent which is similar to, or greater than, the extent to which cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer expand/proliferate; express cytotoxic/effector factors (e.g.
  • IFNy, TNFa, GM-CSF in response to stimulation with cells expressing the target antigen for the CAR to an extent which is similar to, or greater than, the extent to which cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer express such factors; kill cells expressing the target antigen for the CAR with potency/rate which is similar to, or greater than, the potency/rate of killing of such cells by cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG1 spacer; display off-target cytotoxicity (e.g.
  • NK cells which is similar to, or less than, the off-target cytotoxicity displayed by cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG1 spacer; induce systemic inflammation to a recipient subject to an extent which is similar to, or less than, the systemic inflammation induced by administration of cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer; inhibit tumor growth, e.g.
  • Cell proliferation and cell population expansion can be measured as described hereinabove.
  • the rate of cell proliferation/population expansion for a given cell type can be determined by analysing the number of such cells over time, e.g. at different time points.
  • Cell proliferation/population expansion may be measured in vitro, or alternatively in vivo, e.g. following administration to a subject (e.g. a subject having a cancer expressing the target antigen for the cancer).
  • the rate of cell proliferation or population expansion of cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure is > 0.5 times and ⁇ 2 times, e.g.
  • the rate of cell proliferation/population expansion of cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure is greater than 1 times, e.g.
  • the level of expression of one or more cytotoxic/effector factors e.g. IFNy, TNFa, GM-CSF
  • cytotoxic/effector factors e.g. IFNy, TNFa, GM-CSF
  • the level of expression of one or more cytotoxic/effector factors e.g. IFNy, TNFa, GM-CSF
  • cytotoxic/effector factors e.g. IFNy, TNFa, GM-CSF
  • the potency or rate of cell killing of cells expressing the target antigen for the CAR by cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure is > 0.5 times and ⁇ 2 times, e.g.
  • the potency or rate of cell killing of cells expressing the target antigen for the CAR by cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure is greater than 1 times, e.g.
  • the cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure induce cell killing of cells not expressing the target antigen for the CAR to an extent which is similar to or less than the extent to which cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer induce cell killing of cells not expressing the target antigen for the CAR.
  • the cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure induce cell killing of cells not expressing the target antigen for the CAR (e.g. in vitro or in vivo) to a level which is > 0.5 times and ⁇ 2 times, e.g.
  • the cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure induce cell killing of cells not expressing the target antigen for the CAR (e.g. in vitro or in vivo) to a level which is less than 1 times, e.g.
  • cells comprising a CAR/nucleic acid encoding a CAR induce systemic inflammation (e.g. cytokine release syndrome) in a receipt subject of such cells to an extent which is similar to, or less than, that of cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer.
  • systemic inflammation e.g. cytokine release syndrome
  • Induction of systemic inflammation by CAR-expressing cells in a recipient subject can be evaluated by measuring one or more markers of systemic inflammation in a recipient subject following administration of such cells. Markers of systemic inflammation include e.g. levels of proinflammatory cytokines (e.g. IL-6, IL-8 and TNFa and GM-CSF) in the peripheral blood.
  • cytokines e.g. IL-6, IL-8 and TNFa and GM-CSF
  • cells comprising a CAR/nucleic acid encoding a CAR induce the expression of one or more proinflammatory cytokines (e.g. selected from IL-6, IL-8 and TNFa and GM-CSF) to an extent which is similar to, or less than, that of cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer.
  • proinflammatory cytokines e.g. selected from IL-6, IL-8 and TNFa and GM-CSF
  • the cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure induce the expression of one or more proinflammatory cytokines (e.g. selected from IL-6, IL-8 and TNFa and GM-CSF) to a level which is > 0.5 times and ⁇ 2 times, e.g.
  • proinflammatory cytokines e.g. selected from IL-6, IL-8 and TNFa and GM-CSF
  • the cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure induce the expression of one or more proinflammatory cytokines (e.g. selected from IL-6, IL-8 and TNF- a and GM-CSF) to a level which is less than 1 times, e.g.
  • proinflammatory cytokines e.g. selected from IL-6, IL-8 and TNF- a and GM-CSF
  • the level of inhibition of tumor growth (e.g. of a tumor expressing the target antigen for the CAR) achieved by cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure is > 0.5 times and ⁇ 2 times, e.g.
  • administration of cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure increases survival of recipient subjects having a cancer expressing the target antigen for the CAR to > 0.5 times and ⁇ 2 times, e.g.
  • administration of cells comprising a CAR/nucleic acid encoding a CAR increases survival of recipient subjects having a cancer expressing the target antigen for the CAR to greater than 1 times, e.g. one of >1 .01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1 .1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the level of survival achieved by administration of a comparable quantity of cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer, as determined in a given assay.
  • the in vivo persistence of cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure following administration to a subject is > 0.5 times and ⁇ 2 times, e.g.
  • the in vivo persistence of cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure following administration to a subject is greater than 1 times, e.g.
  • the present disclosure provides methods for producing a cell comprising a nucleic acid(s) or vector(s) according to the present disclosure, comprising introducing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present disclosure into a cell.
  • introducing an isolated nucleic acid(s) or vector(s) according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g. retroviral transduction).
  • the present disclosure also provides methods for producing a cell expressing/comprising a CAR according to the present disclosure, comprising introducing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present disclosure in a cell.
  • the methods additionally comprise culturing the cell under conditions suitable for expression of the nucleic acid(s) or vector(s) by the cell.
  • the methods are performed in vitro.
  • Methods for producing CAR-expressing cells are well known to the skilled person. They generally involve modifying cells (e.g. immune cells, e.g. T cells or NK cells) to express/comprise a CAR, e.g. introducing nucleic acid encoding a CAR into the immune cells.
  • cells e.g. immune cells, e.g. T cells or NK cells
  • CAR nucleic acid encoding a CAR into the immune cells.
  • Immune cells may be modified to comprise/express a CAR or nucleic acid encoding a CAR described herein according to methods that are well known to the skilled person.
  • the methods generally comprise nucleic acid transfer for permanent (stable) or transient expression of the transferred nucleic acid.
  • Any suitable genetic engineering platform may be used to modify a cell according to the present disclosure.
  • Suitable methods for modifying a cell include the use of genetic engineering platforms such as gammaretroviral vectors, lentiviral vectors, adenovirus vectors, DNA transfection, transposon-based gene delivery and RNA transfection, for example as described in Maus et al., Annu Rev Immunol (2014) 32:189-225, hereby incorporated by reference in its entirety.
  • Methods also include those described e.g. in Wang and Riviere Mol Ther Oncolytics. (2016) 3:16015, which is hereby incorporated by reference in its entirety.
  • Suitable methods for introducing nucleic acid(s)/vector(s) into cells include transduction, transfection and electroporation. Methods for generating/expanding populations of CAR-expressing immune cells in vitro/ex vivo are well known to the skilled person.
  • Suitable culture conditions i.e. cell culture media, additives, stimulations, temperature, gaseous atmosphere
  • cell numbers i.e. to Hornbach et al.
  • cultures of cells according to the present disclosure may be maintained at 37°C in a humidified atmosphere containing 5% CO2.
  • the cells of cell cultures can be established and/or maintained at any suitable density, as can readily be determined by the skilled person.
  • Cultures can be performed in any vessel suitable for the volume of the culture, e.g. in wells of a cell culture plate, cell culture flasks, a bioreactor, etc.
  • cells are cultured in a bioreactor, e.g. a bioreactor described in Somerville and Dudley, Oncoimmunology (2012) 1 (8):1435-1437, which is hereby incorporated by reference in its entirety.
  • cells are cultured in a GRex cell culture vessel, e.g. a GRex flask or a GRex 100 bioreactor.
  • Immune cells may be activated prior to introduction of nucleic acid encoding the CAR.
  • T cells within a population of PBMCs may be non-specifically activated by stimulation in vitro with agonist anti-CD3 and agonist anti-CD28 antibodies, in the presence of IL-2.
  • nucleic acid(s)/vector(s) into a cell may comprise transduction, e.g. retroviral transduction. Accordingly, in some embodiments the nucleic acid(s) is/are comprised in a viral vector(s), or the vector(s) is/are a viral vector(s). Transduction of immune cells with viral vectors is described e.g. in Simmons and Alberola-lla, Methods Mol Biol. (2016) 1323:99-108, which is hereby incorporated by reference in its entirety.
  • Hexadimethrine bromide is a cationic polymer which is commonly used to improve transduction, through neutralising charge repulsion between virions and sialic acid residues expressed on the cell surface.
  • Other agents commonly used to enhance transduction include e.g. the poloxamer-based agents such as LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech) and also SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs).
  • the methods comprise centrifuging the cells into which it is desired to introduce nucleic acid encoding the CAR in the presence of cell culture medium comprising viral vector comprising the nucleic acid (referred to in the art as ‘spinfection’).
  • the methods comprise introducing a nucleic acid or vector according to the present disclosure into an immune cell by electroporation, e.g. as described in Koh et al., Molecular Therapy - Nucleic Acids (2013) 2, e114, which is hereby incorporated by reference in its entirety.
  • the methods generally comprise introducing a nucleic acid encoding a CAR into a cell, and culturing the cell under conditions suitable for expression of the nucleic acid/CAR by the cell.
  • the methods comprise culturing immune cells into which nucleic acid encoding a CAR has been introduced in order to expand their number.
  • the methods comprise culturing immune cells into which nucleic acid encoding a CAR has been introduced in the presence of IL-7 and/or IL-15 (e.g. recombinant IL-7 and/or IL-15).
  • the methods further comprise purifying/isolating CAR-expressing cells, e.g. from other cells (e.g. cells which do not express the CAR).
  • CAR-expressing cells e.g. from other cells (e.g. cells which do not express the CAR).
  • Methods for purifying/isolating immune cells from heterogeneous populations of cells are well known in the art, and may employ e.g. FACS- or MACS- based methods for sorting populations of cells based on the expression of markers of the immune cells.
  • the methods purifying/isolating cells of a particular type, e.g. CAR-expressing CD8+ T cells, CAR-expressing CTLs.
  • CAR-expressing T cells may be generated from T cells within populations of PBMCs by a process comprising: stimulating PBMCs with antagonist anti-CD3 and andti-CD28 antibodies, transducing the cells with a viral vector (e.g. a gamma-retroviral vector) encoding the CAR, and subsequently culturing the cells in the presence of IL-7 and IL-15.
  • a viral vector e.g. a gamma-retroviral vector
  • the present disclosure also provides CAR-expressing cells obtained or obtainable by the methods according to the present disclosure.
  • compositions comprising the CARs, nucleic acids, expression vectors and cells described herein.
  • the cells, CARs, nucleic acids and expression vectors described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • compositions of the present disclosure may comprise one or more pharmaceutically-acceptable carriers (e.g. liposomes, micelles, microspheres, nanoparticles), diluents/excipients (e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben), anti-oxidants (e.g.
  • pharmaceutically-acceptable carriers e.g. liposomes, micelles, microspheres, nanoparticles
  • diluents/excipients e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium stearate
  • vitamin A vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium
  • lubricants e.g. magnesium stearate, talc, silica, stearic acid, vegetable stearin
  • binders e.g. sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol
  • solubilisers e.g., surfactants (e.g., wetting agents), masking agents or colouring agents (e.g. titanium oxide).
  • pharmaceutically-acceptable refers to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation.
  • Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed. A. Adejare), 23rd Edition (2020), Academic Press.
  • compositions may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration.
  • a pharmaceutical composition/medicament may be formulated for administration by injection or infusion, or administration by ingestion.
  • Suitable formulations may comprise the relevant article in a sterile or isotonic medium.
  • Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form.
  • Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the human or animal body.
  • the composition is formulated for injection or infusion, e.g. into a blood vessel, tissue/organ of interest, or tumor.
  • the present disclosure also provides methods for the production of pharmaceutically useful compositions, such methods of production may comprise one or more steps selected from: producing a CAR or a cell comprising/expressing a CAR according to the present disclosure; isolating a CAR or a cell comprising/expressing a CAR according to the present disclosure; and/or mixing a CAR or a cell comprising/expressing a CAR according to the present disclosure with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.
  • a further aspect the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in the treatment of a disease/condition (e.g. a cancer), the method comprising formulating a pharmaceutical composition or medicament by mixing a CAR or a cell comprising/expressing a CAR according to the present disclosure with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.
  • a disease/condition e.g. a cancer
  • a pharmaceutically acceptable carrier, adjuvant, excipient or diluent e.g. a cancer
  • the CARs, nucleic acids, expression vectors, cells and compositions described herein find use in therapeutic and prophylactic methods.
  • the present disclosure provides a CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein for use in a method of medical treatment or prophylaxis. Also provided is a CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein for use in a method of treating or preventing a disease or condition described herein. Also provided is the use of a CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition described herein.
  • the methods may be effective to reduce the development or progression of a disease/condition, alleviation of the symptoms of a disease/condition or reduction in the pathology of a disease/condition.
  • the methods may be effective to prevent progression of the disease/condition, e.g. to prevent worsening of, or to slow the rate of development of, the disease/condition.
  • the methods may lead to an improvement in the disease/condition, e.g. a reduction in the symptoms of the disease/condition or reduction in some other correlate of the severity/activity of the disease/condition.
  • the methods may prevent development of the disease/condition a later stage (e.g. a chronic stage or metastasis).
  • the articles of the present disclosure may be used for the treatment/prevention of any disease/condition that would derive therapeutic or prophylactic benefit from a reduction in the level/activity of the target antigen for the CAR (e.g. CD30), or a reduction in the number or activity of cells comprising/expressing the target antigen for the CAR.
  • the target antigen for the CAR e.g. CD30
  • the number or activity of cells comprising/expressing the target antigen for the CAR may be used for the treatment/prevention of any disease/condition that would derive therapeutic or prophylactic benefit from a reduction in the level/activity of the target antigen for the CAR (e.g. CD30), or a reduction in the number or activity of cells comprising/expressing the target antigen for the CAR.
  • the disease/condition may be a disease/condition in which the target antigen for the CAR, or cells comprising/expressing the target antigen for the CAR are pathologically-implicated, e.g. a disease/condition in which an increased level/activity of the target antigen for the CAR, or an increase in the number/proportion of cells comprising/expressing the target antigen for the CAR is positively associated with the onset, development or progression of the disease/condition, and/or severity of one or more symptoms of the disease/condition.
  • an increased level/activity of the target antigen for the CAR, or an increase in the number/proportion of cells comprising/expressing the target antigen for the CAR may be a risk factor for the onset, development or progression of the disease/condition.
  • the level of expression or activity of the target antigen for the CAR may be greater than the level of expression or activity of the target antigen for the CAR in equivalent non- cancerous cells/non-tumor tissue.
  • a cancer/cell thereof may comprise one or more mutations (e.g. relative to equivalent non-cancerous cells/non-tumor tissue) causing upregulation of expression or activity of the target antigen for the CAR.
  • Treatment in accordance with the methods of the present disclosure may achieve one or more of the following in a subject (compared to an equivalent untreated subject, or subject treated with an appropriate control): a reduction in the level of the target antigen for the CAR; a reduction in the activity of the target antigen for the CAR; and/or a reduction in the number/proportion of cells comprising/expressing the target antigen for the CAR.
  • cells comprising/expressing a CAR according to the present disclosure are provided for therapeutic and prophylactic use.
  • the methods generally comprise administering a population of immune cells expressing a CAR according to the present disclosure to a subject.
  • immune cells expressing a CAR according to the present disclosure may be administered in the form of a pharmaceutical composition comprising such cells.
  • immune cells expressing a CAR in methods to treat/prevent diseases/conditions by adoptive cell transfer (ACT) is contemplated.
  • Adoptive cell transfer generally refers to a process by which cells (e.g. immune cells) are obtained from a subject, typically by drawing a blood sample from which the cells are isolated. The cells are then typically modified and/or expanded, and then administered either to the same subject (in the case of adoptive transfer of autologous/autogeneic cells) or to a different subject (in the case of adoptive transfer of allogeneic cells).
  • the treatment is typically aimed at providing a population of cells with certain desired characteristics to a subject, or increasing the frequency of such cells with such characteristics in that subject.
  • Adoptive transfer may be performed with the aim of introducing a cell or population of cells into a subject, and/or increasing the frequency of a cell or population of cells in a subject.
  • the target antigen for the CAR is CD30.
  • the biology of CD30 and intervention targeting CD30 for the treatment and prevention of disease is reviewed e.g. in van der Weyden et al., Blood Cancer Journal (2017) 7:e603 and Muta and Podack, Immunol Res (2013), 57(1 -3):151 -8, both of which are hereby incorporated by reference in their entirety.
  • the immune cells expressing a CAR according to the present disclosure may be employed in the treatment/prevention of diseases/conditions by allotransplantation or autotransplantation.
  • allotransplantation refers to the transplantation to a recipient subject of cells, tissues or organs which are genetically non-identical to the recipient subject.
  • the cells, tissues or organs may be from, or may be derived from, cells, tissues or organs of a donor subject that is genetically non-identical to the recipient subject.
  • Allotransplantation is distinct from autotransplantation, which refers to the transplantation of cells, tissues or organs which are from/derived from a donor subject genetically identical to the recipient subject (i.e. autologous material). It will be appreciated that adoptive transfer of allogeneic immune cells is a form of allotransplantation, and that adoptive transfer of autologous immune cells is a form of autotransplantation.
  • the present disclosure provides methods comprising administering immune cells comprising/expressing a CAR according to the present disclosure, or immune cells comprising/expressing nucleic acid encoding a CAR according to the present disclosure, to a subject.
  • the methods comprise modifying an immune cell to comprise/express a CAR according to the present disclosure. In some embodiments, the methods comprise modifying an immune cell specific for a virus to comprise/express nucleic acid encoding a CAR according to the present disclosure.
  • the methods comprise:
  • the methods comprise:
  • the methods comprise:
  • immune cells e.g. PBMCs
  • the methods comprise administering to a subject an EBV-specific immune cell modified to express or comprise a CD30-specific CAR according to the present disclosure, or modified to express or comprise a nucleic acid encoding a CD30-specific CAR according to the present disclosure.
  • the subject from which the immune cells (e.g. PBMCs) are isolated is the same subject to which cells are administered (i.e., adoptive transfer may be of autologous/autogeneic cells). In some embodiments, the subject from which the immune cells (e.g. PBMCs) are isolated is a different subject to the subject to which cells are administered (i.e., adoptive transfer may be of allogeneic cells).
  • the methods may comprise one or more of: obtaining a blood sample from a subject; isolating immune cells (e.g. PBMCs) from a blood sample which has been obtained from a subject; generating/expanding a population of immune cells; culturing the immune cells in in vitro or ex vivo cell culture; modifying an immune cell to express or comprise a CAR according to the present disclosure, or to express or comprise a nucleic acid encoding a CAR according to the present disclosure (e.g.
  • the methods may additionally comprise treating the cells or subject to induce/enhance expression of CAR and/or to induce/enhance proliferation or survival of virus-specific immune cells comprising/expressing the CAR.
  • the disease to be treated/prevented in accordance with the present disclosure is a cancer.
  • Cancer may refer to any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor.
  • the cancer may be benign or malignant and may be primary or secondary (metastatic).
  • a neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue.
  • the cancer may be of tissues/cells derived from e.g. the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g.
  • kidney oesophagus
  • glial cells heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, and/or white blood cells.
  • Tumors may be nervous or non-nervous system tumors.
  • Nervous system tumors may originate either in the central or peripheral nervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma.
  • Non-nervous system cancers/tumors may originate in any other non-nervous tissue, examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer , colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, NSCLC, hematologic cancer and sarcoma.
  • NHL Non-Hodgkin’s lymphoma
  • CML chronic myelogenous leukemia
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • CTCL
  • the cancer is selected from the group consisting of: a solid cancer, a hematological cancer, gastric cancer (e.g. gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma), liver cancer (hepatocellular carcinoma, cholangiocarcinoma), head and neck cancer (e.g. head and neck squamous cell carcinoma), oral cavity cancer (e.g. oropharyngeal cancer (e.g. oropharyngeal carcinoma), oral cancer, laryngeal cancer, nasopharyngeal carcinoma, oesophageal cancer), colorectal cancer (e.g.
  • gastric cancer e.g. gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma
  • liver cancer hepatocellular carcinoma, cholangiocarcinoma
  • head and neck cancer e.g. head and neck squamous cell carcinoma
  • oral cavity cancer e.g. oropharynge
  • lung cancer e.g. NSCLC, small cell lung cancer, lung adenocarcinoma, squamous lung cell carcinoma
  • bladder cancer urothelial carcinoma
  • skin cancer e.g. melanoma, advanced melanoma
  • renal cell cancer e.g. renal cell carcinoma
  • ovarian cancer e.g. ovarian carcinoma
  • mesothelioma breast cancer
  • brain cancer e.g.
  • glioblastoma glioblastoma
  • prostate cancer pancreatic cancer
  • a myeloid hematologic malignancy a lymphoblastic hematologic malignancy
  • myelodysplastic syndrome MDS
  • acute myeloid leukemia AML
  • chronic myeloid leukemia CML
  • acute lymphoblastic leukemia ALL
  • lymphoma non-Hodgkin’s lymphoma (NHL), thymoma or multiple myeloma (MM).
  • NHL non-Hodgkin’s lymphoma
  • MM multiple myeloma
  • the cancer is a cancer in which the target antigen for the CAR (e.g. CD30) is pathologically implicated. That is, in some embodiments the cancer is a cancer which is caused or exacerbated by the expression of the target antigen for the CAR, a cancer for which expression of the target antigen for the CAR is a risk factor and/or a cancer for which expression of the target antigen for the CAR is positively associated with onset, development, progression, severity or metastasis of the cancer.
  • the cancer may be characterised by expression of the target antigen for the CAR, e.g. the cancer may comprise cells expressing the target antigen for the CAR. Such cancers may be referred to as being positive for the target antigen for the CAR.
  • a cancer which is ‘positive’ for the the target antigen for the CAR may be a cancer comprising cells expressing the target antigen for the CAR (e.g. at the cell surface).
  • a cancer which is ‘positive’ for the target antigen for the CAR may overexpress the target antigen for the CAR.
  • CD30-positive cancers are described e.g. in van der Weyden eta!., Blood Cancer Journal (2017) 7:e603 and Muta and Podack, Immunol Res (2013), 57(1 -3) :151 -8.
  • CD30 is expressed on small subsets of activated T and B lymphocytes, and by various lymphoid neoplasms including classical Hodgkin’s lymphoma and anaplastic large cell lymphoma.
  • CD30 expression has also been shown for peripheral T cell lymphoma, not otherwise specified (PTCL-NOS), adult T cell leukemia/lymphoma, cutaneous T cell lymphoma (CTCL), extra-nodal NK-T cell lymphoma, various B cell non-Hodgkin’s lymphomas (including diffuse large B cell lymphoma, particularly EBV-positive diffuse large B cell lymphoma), and advanced systemic mastocytosis.
  • PTCL-NOS peripheral T cell lymphoma
  • CCL cutaneous T cell lymphoma
  • B cell non-Hodgkin’s lymphomas including diffuse large B cell lymphoma, particularly EBV-positive diffuse large B cell lymphoma
  • advanced systemic mastocytosis CD30 expression has also been observed in some non-hematopoietic malignancies, including germ cell tumors and testicular embryonal carcinomas.
  • the transmembrane glycoprotein CD30 is a member of the tumor necrosis factor receptor superfamily (Falini et al., Blood (1995) 85(1 ):1 -14).
  • TNF-R TNF/TNF-receptor
  • CD30 plays a role in regulating the function or proliferation of normal lymphoid cells.
  • CD30 was originally described as an antigen recognised by a monoclonal antibody, Ki-1 , which was raised by immunizing mice with a HL-derived cell line, L428 (Muta and Podack, Immunol Res (2013) 57: 151 -158).
  • CD30 antigen expression has been used to identify ALCL and Reed-Sternberg cells in Hodgkin's disease (Falini eta!., Blood (1995) 85(1 ):1 -14). With the wide expression in the lymphoma malignant cells, CD30 is therefore a potential target for developing both antibody-based immunotherapy and cellular therapies. Importantly, CD30 is not typically expressed on normal tissues under physiologic conditions, thus is notably absent on resting mature or precursor B or T cells (Younes and Ansell, Semin Hematol (2016) 53: 186-189).
  • Brentuximab vedotin an antibody-drug conjugate that targets CD30 was initially approved for the treatment of CD30-positive HL (Adcetris® US Package Insert 2018). Data from brentuximab vedotin trials support CD30 as a therapeutic target for the treatment of CD30-positive lymphoma.
  • HL Hodgkin lymphoma
  • the incidence of HL is bimodal with most patients diagnosed between 15 and 30 years of age, followed by another peak in adults aged 55 years or older. In 2019 it is estimated there will be 8,110 new cases (3,540 in females and 4570 in males) in the United States and 1 ,000 deaths (410 female and 590 males) from this disease (American Cancer Society 2019). Based on 2012-2016 cases in National Cancer Institute’s SEER database, the incidence rate for HL for the pediatric HL patients in US is as follows: Age 1 -4: 0.1 ; Age 5-9: 0.3; Age 10-14: 1 .3; Age 15-19: 3.3 per 100,000 (SEER Cancer Statistics Review, 1975-2016]).
  • the World Health Organization (WHO) classification divides HL into 2 main types: classical Hodgkin lymphoma (cHL) and nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL).
  • cHL classical Hodgkin lymphoma
  • NLPHL nodular lymphocyte-predominant Hodgkin lymphoma
  • cHL accounts for 95%
  • NLPHL accounts for 5% of all HL (National Comprehensive Cancer Network Guidelines 2019).
  • the cancer to be treated/prevented is an EBV-associated cancer.
  • EBV infection is implicated in several cancers, as reviewed e.g. in Jha et al., Front Microbiol. (2016) 7:1602, which is hereby incorporated by reference in its entirety.
  • the cancer is a cancer which is caused or exacerbated by infection with EBV, a cancer for which infection with EBV is a risk factor and/or a cancer for which infection with EBV is positively associated with onset, development, progression, severity or metastasis of the cancer.
  • the cancer may be characterised by EBV infection, e.g. the cancer may comprise cells infected with EBV. Such cancers may be referred to as EBV-positive cancers.
  • EBV-associated cancers which may be treated/prevented in accordance with the present disclosure include B cell-associated cancers such as Burkitt’s lymphoma, post-transplant lymphoproliferative disease (PTLD), central nervous system lymphoma (CNS lymphoma), Hodgkin’s lymphoma, nonHodgkin’s lymphoma, and EBV-associated lymphomas associated with immunodeficiency (including e.g.
  • B cell-associated cancers such as Burkitt’s lymphoma, post-transplant lymphoproliferative disease (PTLD), central nervous system lymphoma (CNS lymphoma), Hodgkin’s lymphoma, nonHodgkin’s lymphoma, and EBV-associated lymphomas associated with immunodeficiency (including e.g.
  • EBV-positive lymphoma associated with X-linked lymphoproliferative disorder EBV-positive lymphoma associated with HIV infection/AIDS, and oral hairy leukoplakia
  • epithelial cell-related cancers such as nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC).
  • the cancer is selected from lymphoma (e.g. EBV-positive lymphoma), head and neck squamous cell carcinoma (HNSCC; e.g. EBV-positive HNSCC), nasopharyngeal carcinoma (NPC; e.g. EBV-positive NPC), and gastric carcinoma (GC; e.g. EBV-positive GC).
  • lymphoma e.g. EBV-positive lymphoma
  • HNSCC head and neck squamous cell carcinoma
  • NPC nasopharyngeal carcinoma
  • GC gastric carcinoma
  • a CD30-positive cancer may be selected from: a solid cancer, a hematological cancer, a hematopoietic malignancy, Hodgkin’s lymphoma (HL), anaplastic large cell lymphoma (ALCL), ALK-positive anaplastic T cell lymphoma, ALK-negative anaplastic T cell lymphoma, peripheral T cell lymphoma (e.g. PTCL-NOS), T cell leukemia, T cell lymphoma, cutaneous T cell lymphoma (CTCL), NK- T cell lymphoma (e.g.
  • extra-nodal NK-T cell lymphoma extra-nodal NK-T cell lymphoma
  • non-Hodgkin’s lymphoma NHL
  • B cell nonHodgkin’s lymphoma diffuse large B cell lymphoma (e.g. diffuse large B cell lymphoma-NOS), primary mediastinal B cell lymphoma, EBV-positive B cell lymphoma, EBV-positive diffuse large B cell lymphoma, advanced systemic mastocytosis, a germ cell tumor and testicular embryonal carcinoma.
  • the cancer is selected from: a CD30-positive cancer, an EBV-associated cancer, a hematological cancer, a myeloid hematologic malignancy, a hematopoietic malignancy a lymphoblastic hematologic malignancy, myelodysplastic syndrome, leukemia, T cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, lymphoma, Hodgkin’s lymphoma, nonHodgkin’s lymphoma, B cell non-Hodgkin’s lymphoma, diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, EBV-associated lymphoma, EBV-positive B cell lymphoma, EBV-positive diffuse large B cell lymphoma, EBV-positive lymphoma associated with X-linked lymphoproliferative disorder, EBV- positive lymphoma associated with HIV infection/AIDS, oral hairy leu
  • the cancer may be a relapsed cancer.
  • a ‘relapsed’ cancer refers to a cancer which responded to a treatment (e.g. a first line therapy for the cancer), but which has subsequently re-emerged/progressed, e.g. after a period of remission.
  • a relapsed cancer may be a cancer whose growth/progression was inhibited by a treatment (e.g. a first line therapy for the cancer), and which has subsequently grown/progressed.
  • the cancer may be a refractory cancer.
  • a ‘refractory’ cancer refers to a cancer which has not responded to a treatment (e.g. a first line therapy for the cancer).
  • a refractory cancer may be a cancer whose growth/progression was not inhibited by a treatment (e.g. a first line therapy for the cancer).
  • a refractory cancer may be a cancer for which a subject receiving treatment for the cancer did not display a partial or complete response to the treatment.
  • the cancer may be relapsed or refractory with respect to treatment with chemotherapy, brentuximab vedotin, or crizotinib.
  • the cancer is peripheral T cell lymphoma
  • the cancer may be relapsed or refractory with respect to treatment with chemotherapy or brentuximab vedotin.
  • the cancer is extranodal NK-T cell lymphoma
  • the cancer may be relapsed or refractory with respect to treatment with chemotherapy (with or without asparaginase) or brentuximab vedotin.
  • the cancer may be relapsed or refractory with respect to treatment with chemotherapy (with or without rituximab) or CD19 CAR-T therapy.
  • chemotherapy with or without rituximab
  • CD19 CAR-T therapy In embodiments where the cancer is primary mediastinal B cell lymphoma, the cancer may be relapsed or refractory with respect to treatment with chemotherapy, immune checkpoint inhibitor (e.g. PD-1 inhibitor) or CD19 CAR-T therapy.
  • immune checkpoint inhibitor e.g. PD-1 inhibitor
  • Treatment of a cancer in accordance with the methods of the present disclosure achieves one or more of the following treatment effects: reduces the number of cancer cells in the subject, reduces the size of a cancerous tumor/lesion in the subject, inhibits (e.g. prevents or slows) growth of cancer cells in the subject, inhibits (e.g. prevents or slows) growth of a cancerous tumor/lesion in the subject, inhibits (e.g. prevents or slows) the development/progression of a cancer (e.g. to a later stage, or metastasis), reduces the severity of symptoms of a cancer in the subject, increases survival of the subject (e.g.
  • Subjects may be evaluated in accordance with the Revised Criteria for Response Assessment: The Lugano Classification (described e.g. in Cheson et al., J Clin Oncol (2014) 32: 3059-3068, incorporated by reference hereinabove) in order to determine their response to treatment.
  • treatment of a subject in accordance with the methods of the present disclosure achieves one of the following: complete response, partial response, or stable disease.
  • the CAR-expressing immune cells and compositions of the present disclosure can be used in methods involving allotransplantation, e.g. to treat/prevent a disease/condition in a subject.
  • the CAR-expressing immune cells and compositions of the present disclosure are useful in methods to reduce/prevent alloreactive immune responses (particularly T cell-mediated alloreactive immune responses) and the deleterious consequences thereof.
  • Alloreactive T cells express CD30.
  • Chan et al., J Immunol (2002) 169(4):1784-91 identify CD30- expressing T cells as a subset of activated T cells (also expressing CD25 and CD45RO) having an important role in CD30 alloimmune responses.
  • CD30 expression and the proliferation of CD30- expressing T cells increases in response to alloantigen. Chen et al., Blood (2012) 120(3):691 -6 identifies CD30 expression on CD8+ T cell subsets as a potential biomarker for GVHD, and propose CD30 as a therapeutic target for GVHD.
  • the CAR-expressing immune cells and compositions of the present disclosure are particularly useful in methods involving allotransplantation, and also in the processing/production of allotransplants.
  • the CAR-expressing immune cells and compositions are contemplated for use in the production and administration of ‘off-the-shelf’ materials for use in therapeutic and prophylactic methods comprising administration of allogeneic material.
  • CAR-expressing immune cells of the present disclosure are useful for the treatment/prevention of diseases/conditions by adoptive cell transfer.
  • CAR-expressing immune cells of the present disclosure are less susceptible to T cell-mediated alloreactive immune responses of the recipient following adoptive transfer, and thus exhibit enhanced proliferation/survival in the recipient after transfer, and superior therapeutic/prophylactic effects.
  • the CAR-expressing immune cells and compositions of the present disclosure are also useful in methods comprising allotransplantation of allogeneic cells other than the CAR-expressing immune cells of the present disclosure.
  • the CAR-expressing immune cells and compositions of the present disclosure are useful for depleting allotransplants (populations of cells, tissues and organs) and subjects of alloreactive immune cells (e.g. alloreactive T cells).
  • the CAR-expressing immune cells and compositions are useful for conditioning of donor and/or recipient subjects, and/or treatment of the allotransplant to reduce/prevent an alloreactive immune response following allotransplantation.
  • Cells, tissues and organs to be allotransplanted include e.g. immune cells (e.g. adoptive cell transfer), the heart, lung, kidney, liver, pancreas, intestine, face, cornea, skin, hematopoietic stem cells (bone marrow), blood, hands, leg, penis, bone, uterus, thymus, islets of Langerhans, heart valve and ovary.
  • immune cells e.g. adoptive cell transfer
  • allotransplants populations of cells, tissues or organs to be allotransplanted may be referred to as ‘allotransplants’.
  • the disease/condition to be treated/prevented by the allotransplantation can be any disease/condition which would derive therapeutic or prophylactic benefit from the allotransplantation.
  • the disease/condition to be treated/prevented by allotransplantation may e.g. be a T cell dysfunctional disorder, a cancer, an infectious disease or an autoimmune disease.
  • a T cell dysfunctional disorder may be a disease/condition in which normal T cell function is impaired causing downregulation of the subject’s immune response to pathogenic antigens, e.g. generated by infection by exogenous agents such as microorganisms, bacteria and viruses, or generated by the host in some disease states such as in some forms of cancer (e.g. in the form of tumor-associated antigens).
  • the T cell dysfunctional disorder may comprise T cell exhaustion or T cell anergy.
  • T cell exhaustion comprises a state in which CD8+ T cells fail to proliferate or exert T cell effector functions such as cytotoxicity and cytokine (e.g. IFNy) secretion in response to antigen stimulation.
  • Exhausted T cells may also be characterised by sustained expression of one or more markers of T cell exhaustion, e.g. PD-1 , CTLA-4, LAG-3, TIM-3.
  • the T cell dysfunctional disorder may manifest as an infection, or inability to mount an effective immune response against an infection.
  • the infection may be chronic, persistent, latent or slow, and may be the result of bacterial, viral, fungal or parasitic infection.
  • treatment may be provided to patients having a bacterial, viral or fungal infection.
  • bacterial infections include infection with Helicobacter pylori.
  • examples of viral infections include infection with HIV, hepatitis B or hepatitis C.
  • the T cell dysfunctional disorder may be associated with a cancer, such as tumor immune escape. Many human tumors express tumor-associated antigens recognised by T cells and capable of inducing an immune response.
  • An infectious disease may be e.g. bacterial, viral, fungal, or parasitic infection.
  • it may be particularly desirable to treat chronic/persistent infections, e.g. where such infections are associated with T cell dysfunction or T cell exhaustion.
  • T cell exhaustion is a state of T cell dysfunction that arises during many chronic infections (including viral, bacterial and parasitic), as well as in cancer (Wherry Nature Immunology Vol.12, No.6, p492-499, June 2011 ).
  • bacterial infections examples include infection by Bacillus spp., Bordetella pertussis, Clostridium spp., Corynebacterium spp., Vibrio chloerae, Staphylococcus spp., Streptococcus spp. Escherichia, Klebsiella, Proteus, Yersinia, Erwina, Salmonella, Listeria sp, Helicobacter pylori, mycobacteria (e.g. Mycobacterium tuberculosis) and Pseudomonas aeruginosa.
  • the bacterial infection may be sepsis or tuberculosis.
  • viral infections examples include infection by influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), Herpes simplex virus and human papilloma virus (HPV).
  • fungal infections examples include infection by Alternaria sp, Aspergillus sp, Candida sp and Histoplasma sp. The fungal infection may be fungal sepsis or histoplasmosis.
  • parasitic infections examples include infection by Plasmodium species (e.g.
  • the parasitic infection may be a disease such as malaria, leishmaniasis and toxoplasmosis.
  • the disease/condition is an autoimmune disease.
  • the treatment may be aimed at reducing the number of autoimmune effector cells.
  • the autoimmune disease is selected from: diabetes mellitus type 1 , celiac disease, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.
  • the CAR-expressing immune cells and compositions of the present disclosure are also useful for the treatment/prevention of an alloreactive immune response, and diseases/conditions characterised by an alloreactive immune response.
  • Diseases and conditions characterised by an alloreactive immune response include diseases/conditions caused or exacerbated by alloreactive immune responses associated with allotransplantation. Such diseases/conditions include graft versus host disease (GVHD) and graft rejection, and are described in detail in Perkey and Maillard Annu Rev Pathol. (2016) 13:219-245, which is hereby incorporated by reference in its entirety.
  • GVHD graft versus host disease
  • graft rejection are described in detail in Perkey and Maillard Annu Rev Pathol. (2018) 13:219-245, which is hereby incorporated by reference in its entirety.
  • graft-versus-host disease can occur following allotransplantation of large numbers of donor immune cells, and involves reactivity of donor-derived immune cells against allogeneic recipient cells/tissues/organs.
  • Graft rejection refers to the destruction of transplanted cells/tissue/organs by a recipient’s immune system following transplantation. Where graft rejection is of an allotransplant, it may be referred to as allograft rejection.
  • the CAR-expressing immune cells and compositions of the present disclosure may be used to deplete alloreactive T cells in an allotransplant, which could otherwise lead to graft versus host disease (GVHD) in a recipient upon allotransplantation.
  • GVHD graft versus host disease
  • the CAR-expressing immune cells and compositions of the present disclosure may be used to deplete alloreactive T cells in a donor for an allotransplant (e.g. prior to harvesting/collecting the allotransplant), which could otherwise lead to GVHD in a recipient upon allotransplantation.
  • the CAR-expressing immune cells and compositions of the present disclosure may be used to deplete alloreactive T cells in the recipient for an allotransplant, which could otherwise cause/promote graft rejection.
  • the present disclosure provides methods of treating/preventing graft-versus-host disease (GVHD) following allotransplantation, comprising administering a CAR-expressing immune cell or composition according to the present disclosure to a donor subject for an allotransplant.
  • the present disclosure also provides methods of treating/preventing graft-versus-host disease (GVHD) following allotransplantation, comprising contacting an allotransplant with a CAR-expressing immune cell or composition according to the present disclosure.
  • the aim for such methods is to reduce/remove the ability of alloreactive immune cells in the allograft to mount an alloreactive immune response to cells, tissue and/or organs of the recipient for the allotransplant.
  • the present disclosure provides methods of treating/preventing graft rejection following allotransplantation, comprising administering a CAR-expressing immune cell or composition according to the present disclosure to a recipient subject for an allotransplant.
  • the aim for such methods is to reduce/remove the ability of the receipt subject to mount an alloreactive immune response to the allotransplant.
  • the CAR-expressing immune cells are useful to eliminate immune cells in the recipient that would otherwise effect an alloreactive immune response against donor cells, tissue and/or organs.
  • the present disclosure provides methods comprising depleting an allotransplant of alloreactive immune cells (e.g. alloreactive T cells), comprising contacting an allotransplant (e.g. a population of cells, tissue or an organ to be transplanted) with a CAR-expressing immune cell or composition of the present disclosure.
  • the methods may comprise administering a CAR-expressing immune cell or composition of the present disclosure to a donor subject for the allotransplant.
  • the aim for such methods is to reduce/remove the ability of alloreactive immune cells in the allograft to mount an alloreactive immune response to cells, tissue and/or organs of the recipient for the allotransplant.
  • the methods comprise one or more of: obtaining/collecting a population of cells, tissue or organ from a subject; contacting a population of cells, tissue or organ with a CAR-expressing immune cell or composition according to the present disclosure; culturing a population of cells, tissue or organ in vitro or ex vivo in the presence of a CAR- expressing immune cell according to the present disclosure; harvesting/collecting a population of cells, tissue or organ depleted of alloreactive immune cells; and transplanting/administering a population of cells, tissue or organ depleted of alloreactive immune cells to a subject.
  • the present disclosure also provides methods comprising depleting a subject of alloreactive immune cells (e.g. alloreactive T cells), comprising administering a CAR-expressing immune cell or composition of the present disclosure to the subject.
  • the subject may be a donor subject for an allotransplant, or may be an intended recipient subject for an allotransplant.
  • the methods comprise one or more of: administering a CAR-expressing immune cell or composition according to the present disclosure to a subject, in order to deplete alloreactive immune cells in the subject; obtaining/collecting a population of cells, tissue or organ from a subject to which a CAR- expressing immune cell or composition according to the present disclosure has been administered; and transplanting/administering a population of cells, tissue or organ depleted of alloreactive immune cells to a subject.
  • the methods comprise one or more of: administering a CAR-expressing immune cell or composition according to the present disclosure to a subject, in order to deplete alloreactive immune cells in the subject; and transplanting/administering a population of cells, tissue or organ to a subject to which a CAR- expressing immune cell or composition according to the present disclosure have previously been administered.
  • Depletion of alloreactive immune cells may result in e.g. a 2-fold, 10-fold, 100-fold, 1000-fold, 10000-fold or greater reduction in the quantity of alloreactive immune cells in the allotransplant or subject.
  • the methods may be performed in vitro or ex vivo, or in vivo in a subject. Method steps performed in vitro or ex vivo may comprise in vitro or ex vivo cell culture.
  • the methods may further comprise method steps for the production of CAR-expressing immune cells and compositions according to the present disclosure.
  • administration of a CAR-expressing immune cell or composition according to the present disclosure to a recipient subject for an allotransplantation and allotransplantation are performed simultaneously (/.e. at the same time, or within e.g. 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 8 hrs, 12 hrs, 24 hrs, 36 hrs or 48 hrs).
  • administration of a CAR-expressing immune cell or composition according to the present disclosure to a recipient subject for an allotransplantation and allotransplantation are performed sequentially.
  • the time interval between administration of a CAR-expressing immune cell or composition and allotransplantation may be any time interval, including hours, days, weeks, months, or years.
  • the CAR-expressing immune cell or composition may be administered to the recipient subject before or after allotransplantation.
  • the CAR-expressing immune cell or composition are preferably administered to the recipient subject prior to allotransplantation.
  • administration of a CAR-expressing immune cell or composition according to the present disclosure to a donor subject for an allotransplantation and collection of the allotransplant are performed simultaneously (i.e. at the same time, or within e.g. 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 8 hrs, 12 hrs, 24 hrs, 36 hrs or 48 hrs).
  • administration of a CAR-expressing immune cell or composition according to the present disclosure to a donor subject for an allotransplantation and collection of the allotransplant i.e.
  • the time interval between administration of a CAR-expressing immune cell or composition and collection of the allotransplant may be any time interval, including hours, days, weeks, months, or years.
  • the CAR- expressing immune cell or composition may be administered to the donor subject before or after collection of the allotransplant.
  • the CAR-expressing immune cell or composition are preferably administered to the donor subject prior to collection of the allotransplant.
  • the methods comprise additional intervention to treat/prevent an alloreactive immune response, graft rejection and/or GVHD.
  • the methods to treat/prevent alloreactivity, graft rejection and/or GVHD comprise administration of immunosuppressive and/or lymphodepletive therapy such as treatment with corticosteroids (e.g. prednisolone, hydrocortisone), calcineurin inhibitors (e.g. cyclosporin, tacrolimus) anti-proliferative agents (e.g. azathioprinem, mycophenolic acid) and/or mTOR inhibitors (e.g. sirolimus, everolimus).
  • corticosteroids e.g. prednisolone, hydrocortisone
  • calcineurin inhibitors e.g. cyclosporin, tacrolimus
  • anti-proliferative agents e.g. azathioprinem, mycophenolic acid
  • mTOR inhibitors e.g. sirolimus, everolimus.
  • the methods to treat/prevent alloreactivity and/or graft rejection comprise antibody therapy, such as treatment with monoclonal anti-IL-2Ra receptor antibodies (e.g. basiliximab, daclizumab), anti-T cell antibodies (e.g. anti-thymocyte globulin, anti-lymphocyte globulin) and/or anti- CD20 antibodies (e.g. rituximab).
  • monoclonal anti-IL-2Ra receptor antibodies e.g. basiliximab, daclizumab
  • anti-T cell antibodies e.g. anti-thymocyte globulin, anti-lymphocyte globulin
  • anti- CD20 antibodies e.g. rituximab
  • the methods to treat/prevent alloreactivity and/or graft rejection comprise blood transfusion and/or bone marrow transplantation.
  • the present disclosure also provides the CAR-expressing immune cells and compositions of the present disclosure for use in such methods. Also provided is the use of the CAR-expressing immune cells or compositions of the present disclosure in the manufacture of products (e.g. medicaments) for use in such methods.
  • the methods of various aspects of the present disclosure cause less depletion and/or increased survival of non-alloreactive immune cells as compared to methods employing immunosuppressive agent(s).
  • the present methods are useful for preserving/maintaining the non-alloreactive immune cell compartment in a recipient subject for an allotransplant, or in an allotransplant.
  • the present methods are associated with an increased number/proportion of non-alloreactive immune cells in the recipient subject for the allotransplant as compared to methods involving treatment with an immunosuppressive agent. In some embodiments of the methods of the present disclosure comprising adoptive transfer of allogeneic immune cells, the present methods are associated with an increased number/proportion of non-alloreactive immune cells in the recipient subject for the allogeneic immune cells as compared to methods involving treatment with an immunosuppressive agent. In some embodiments of the methods of the present disclosure comprising allotransplantation, the present methods are associated with an increased number/proportion of non-alloreactive immune cells in the allotransplant as compared to methods involving treatment with an immunosuppressive agent.
  • the present disclosure also provides the CAR-expressing immune cell or composition of the present disclosure for use in a method of: killing a cell expressing the target antigen for which the CAR is specific (e.g. a cell expressing CD30); and/or killing an alloreactive immune cell (e.g. a T cell expressing CD30).
  • a cell expressing the target antigen for which the CAR is specific e.g. a cell expressing CD30
  • an alloreactive immune cell e.g. a T cell expressing CD30.
  • the present disclosure also provides the use of such CAR-expressing immune cells and compositions in such methods, and methods using the CAR-expressing immune cell and compositions to such ends.
  • Administration of the articles of the present disclosure is preferably in a ‘therapeutically-effective’ or ‘prophylactically-effective’ amount, this being sufficient to show therapeutic or prophylactic benefit to the subject.
  • the actual amount administered, and rate and time-course of administration will depend on the nature and severity of the disease/condition and the particular article administered.
  • Prescription of treatment e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease/disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s ‘The Science and Practice of Pharmacy’ (ed. A. Adejare), 23rd Edition (2020), Academic Press.
  • Administration of the articles of the present disclosure may be topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal or transdermal. Administration may be by injection or infusion. Administration of the articles of the present disclosure may be intratumoral.
  • the methods comprise intravenous, intra-arterial, intramuscular or subcutaneous administration and wherein the relevant article is formulated in a targeted agent delivery system.
  • Suitable targeted delivery systems include, for example, nanoparticles, liposomes, micelles, beads, polymers, metal particles, dendrimers, antibodies, aptamers, nanotubes or micro-sized silica rods.
  • Such systems may comprise a magnetic element to direct the agent to the desired organ or tissue.
  • Suitable nanocarriers and delivery systems will be apparent to one skilled in the art.
  • the articles of the present disclosure are formulated for targeted delivery to specific cells, a tissue, an organ and/or a tumor. Further intervention
  • Administration may be alone or in combination with other treatments, either simultaneously or sequentially dependent upon the disease/condition to be treated.
  • the antigen-binding molecule, CAR, cell or composition described herein and another prophylactic/therapeutic agent may be administered simultaneously or sequentially.
  • the methods comprise additional therapeutic or prophylactic intervention, e.g. for the treatment/prevention of a cancer.
  • the therapeutic or prophylactic intervention is selected from chemotherapy, immunotherapy, radiotherapy, surgery, vaccination and/or hormone therapy.
  • the therapeutic or prophylactic intervention comprises leukapheresis.
  • the therapeutic or prophylactic intervention comprises a stem cell transplant.
  • Simultaneous administration refers to administration of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition and therapeutic agent together, for example as a pharmaceutical composition containing both agents (combined preparation), or immediately after each other and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel.
  • Sequential administration refers to administration of one of the antigen-binding molecule/composition or therapeutic agent followed after a given time interval by separate administration of the other agent. It is not required that the two agents are administered by the same route, although this is the case in some embodiments.
  • the time interval may be any time interval.
  • treatment of cancer further comprises chemotherapy and/or radiotherapy.
  • Chemotherapy and radiotherapy respectively refer to treatment of a cancer with a drug or with ionising radiation (e.g. radiotherapy using X-rays or y-rays).
  • the drug may be a chemical entity, e.g. small molecule pharmaceutical, antibiotic, DNA intercalator, protein inhibitor (e.g. kinase inhibitor), or a biological agent, e.g. antibody, antibody fragment, aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide, or protein.
  • the drug may be formulated as a pharmaceutical composition or medicament.
  • the formulation may comprise one or more drugs (e.g. one or more active agents) together with one or more pharmaceutically acceptable diluents, excipients or carriers.
  • Chemotherapy may involve administration of more than one drug.
  • a drug may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • the chemotherapy may be administered by one or more routes of administration, e.g. parenteral, intravenous injection, oral, subcutaneous, intradermal or intratumoral.
  • routes of administration e.g. parenteral, intravenous injection, oral, subcutaneous, intradermal or intratumoral.
  • the chemotherapy may be administered according to a treatment regime.
  • the treatment regime may be a pre-determined timetable, plan, scheme or schedule of chemotherapy administration which may be prepared by a physician or medical practitioner and may be tailored to suit the patient requiring treatment.
  • the treatment regime may indicate one or more of: the type of chemotherapy to administer to the patient; the dose of each drug or radiation; the time interval between administrations; the length of each treatment; the number and nature of any treatment holidays, if any etc.
  • a single treatment regime may be provided which indicates how each drug is to be administered.
  • Chemotherapeutic drugs may be selected from: Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE- PC, AC, Acalabrutinib, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aliqopa (Copanlisib Hydrochloride), Alkeran for
  • the treatment may comprise administration of a corticosteroid, e.g. dexamethasone and/or prednisone.
  • a corticosteroid e.g. dexamethasone and/or prednisone.
  • a subject is administered lymphodepleting chemotherapy prior to administration of immune cells expressing/comprising a CAR described herein (or expressing/comprising nucleic acid encoding such a CAR).
  • methods of treating/preventing a disease/condition in accordance with the present disclosure comprise: (i) administering a lymphodepleting chemotherapy to a subject, and (ii) subsequently administering an immune cell expressing/comprising a CAR according to the present disclosure, or expressing/comprising a nucleic acid encoding a CAR according to the present disclosure.
  • lymphocytes e.g. T cells, B cells, NK cells, NKT cells or innate lymphoid cell (ILCs), or precursors thereof
  • a ‘lymphodepleting chemotherapeutic agent’ refers to a chemotherapeutic agent which results in depletion of lymphocytes.
  • Lymphodepleting chemotherapy and its use in methods of treatment by adoptive cell transfer are described e.g. in Klebanoff et al., Trends Immunol. (2005) 26(2):111 -7 and Muranski et al., Nat Clin Pract Oncol. (2006) (12):668-81 , both of which are hereby incorporated by reference in their entirety.
  • the aim of lymphodepleting chemotherapy is to deplete the recipient subject’s endogenous lymphocyte population.
  • lymphodepleting chemotherapy is typically administered prior to adoptive cell transfer, to condition the recipient subject to receive the adoptively transferred cells.
  • Lymphodepleting chemotherapy is thought to promote the persistence and activity of adoptively transferred cells by creating a permissive environment, e.g. through elimination of cells expressing immunosuppressive cytokines, and creating the ‘lymphoid space’ required for expansion and activity of adoptively transferred lymphoid cells.
  • Chemotherapeutic agents commonly used in lymphodepleting chemotherapy include e.g. fludarabine, cyclophosphamide, bedamustine and pentostatin.
  • doses of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition may be provided.
  • One or more, or each, of the doses may be accompanied by simultaneous or sequential administration of another therapeutic agent.
  • Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1 , 2, 3, 4, 5, or 6 months.
  • doses may be given once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).
  • a method of treating and/or preventing a disease/condition may comprise one or more of the following: reducing the number/proportion of cells expressing the target antigen for the CAR (e.g. CD30); inhibiting tumor growth (e.g. of a tumor expressing the target antigen for the CAR); reducing metastasis of a cancer (e.g. a cancer expressing the target antigen for the CAR); increasing survival of a subject having a cancer (e.g. a cancer expressing the target antigen for the CAR).
  • a subject in accordance with the various aspects of the present disclosure may be any animal or human.
  • Therapeutic and prophylactic applications may be in human or animals (veterinary use).
  • the subject to be administered with an article of the present disclosure may be a subject in need of such intervention.
  • the subject is preferably mammalian, more preferably human.
  • the subject may be a non-human mammal, but is more preferably human.
  • the subject may be male or female.
  • the subject may be a patient.
  • a subject may have (e.g. may have been diagnosed with) a disease or condition described herein, may be suspected of having such a disease/condition, or may be at risk of developing/contracting such a disease/condition.
  • a subject may be selected for treatment according to the methods based on characterisation for one or more markers of such a disease/condition.
  • a subject may be selected for therapeutic or prophylactic intervention as described herein based on the detection of cells/tissue expressing the target antigen for the CAR (e.g. CD30), or of cells/tissue overexpressing the target antigen for the CAR, e.g. in a sample obtained from the subject.
  • the target antigen for the CAR e.g. CD30
  • cells/tissue overexpressing the target antigen for the CAR e.g. in a sample obtained from the subject.
  • a subject may be an allogeneic subject with respect to an intervention in accordance with the present disclosure.
  • a subject to be treated/prevented in accordance with the present disclosure may be genetically non-identical to the subject from which the CAR-expressing immune cells are derived.
  • a subject to be treated/prevented in accordance with the present disclosure may be HLA mismatched with respect to the subject from which the CAR-expressing immune cells are derived.
  • a subject to be treated/prevented in accordance with the present disclosure may be HLA matched with respect to the subject from which the CAR-expressing immune cells are derived.
  • the subject to which cells are administered in accordance with the present disclosure may be allogeneic/non-autologous with respect to the source from which the cells are/were derived.
  • the subject to which cells are administered may be a different subject to the subject from which cells are/were obtained for the production of the cells to be administered.
  • the subject to which the cells are administered may be genetically non-identical to the subject from which cells are/were obtained for the production of the cells to be administered.
  • the subject to which cells are administered may comprise MHC/HLA genes encoding MHC/HLA molecules which are non-identical to the MHC/HLA molecules encoded by the MHC/HLA genes of the subject from which cells are/were obtained for the production of the cells to be administered.
  • the subject to which cells are administered may comprise MHC/HLA genes encoding MHC/HLA molecules which are identical to the MHC/HLA molecules encoded by the MHC/HLA genes of the subject from which cells are/were obtained for the production of the cells to be administered.
  • the subject to which cells are administered is HLA matched with respect to the subject from which cells are/were obtained for the production of the cells to be administered. In some embodiments, the subject to which cells are administered is a near or complete HLA match with respect to the subject from which cells are/were obtained for the production of the cells to be administered.
  • the subject is a >4/8 (i.e. 4/8, 5/8, 6/8, 7/8 or 8/8) match across HLA-A, -B, -C, and -DRB1 .
  • the subject is a >5/10 (i.e. 5/10, 6/10, 7/10, 8/10, 9/10 or 10/10) match across HLA-A, -B, -C, -DRB1 and -DQB1 .
  • the subject is a >6/12 (i.e.
  • the subject is an 8/8 match across HLA-A, -B, -C, and -DRB1 .
  • the subject is a 10/10 match across HLA-A, -B, -C, -DRB1 and -DQB1 .
  • the subject is a 12/12 match across HLA-A, -B, -C, -DRB1 , -DQB1 and -DPB1 .
  • kits of parts may have at least one container having a predetermined quantity of a CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.
  • the kit may comprise materials for producing a CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.
  • the kit may provide the CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition together with instructions for administration to a patient in order to treat a specified disease/condition.
  • kits may further comprise at least one container having a predetermined quantity of another therapeutic agent (e.g. as described herein).
  • the kit may also comprise a second medicament or pharmaceutical composition such that the two medicaments or pharmaceutical compositions may be administered simultaneously or separately such that they provide a combined treatment for the specific disease or condition.
  • Kits according to the present disclosure may include instructions for use, e.g. in the form of an instruction booklet or leaflet. The instructions may include a protocol for performing any one or more of the methods described herein.
  • sequence identity refers to the percent of nucleotides/amino acid residues in a subject sequence that are identical to nucleotides/amino acid residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for the purposes of determining
  • 10 percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951 -960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780) software.
  • ClustalOmega Soding, J. 2005, Bioinformatics 21 , 951 -960
  • T-coffee Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217
  • Kalign Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)
  • MAFFT K
  • the default parameters e.g. for gap penalty and extension penalty, are preferably used.
  • the present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.
  • an amino acid sequence, or a region of a polypeptide which ‘corresponds’ to a specified reference amino acid sequence or region of a polypeptide has at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the amino acid sequence/polypeptide/region.
  • An amino acid sequence/region/position of a polypeptide/amino acid sequence which ‘corresponds’ to a specified reference amino acid sequence/region/position of a polypeptide/amino acid sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g. using sequence alignment software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951 -960).
  • in vitro is intended to encompass procedures performed with cells in culture whereas the term ‘in vivo’ is intended to encompass procedures with/on intact multi-cellular organisms.
  • FIG. 1A to 1C Expression and functional assessment of novel alternative spacers in CD30 CAR.
  • FIG. 2A to 2C Functional assessment of novel alternative spacers in CD30 CAR with Raji expressing different CD30 densities.
  • 2A Flow cytometry analysis of transduced Raji clones 1 B1 , 1 C4 and 2C5 expressing varying levels of CD30, in comparison to CD30 expressing KM-H2 cells.
  • FIGS. 3A to 3C Expression and functional assessment of CARs expressing novel 4-1 BB spacer with humanized CD30 scFv.
  • FIGS 4A to 4G Co-culture assay of autologous primary monocytes and natural killer cells with CAR T cells expressing CARs with various spacers.
  • (4A) Experiment layout of assay assessing monocytes interaction with CAR T cells expressing CARs with various spacers.
  • (4C) Immune checkpoint molecules expressed by CD30 CAR T cells with different spacers following co-culture with autologous monocytes.
  • (4E) Experiment layout of assay assessing NK cells interaction with CAR T cells expressing CARs with various spacers.
  • (4F) Cytotoxicity against NK cells measured by flow cytometry.
  • (4G) CD16 expression on NK cells and number of CD16 high NK cells remaining after co-culture with CD30 CAR T cells bearing different spacers.
  • FIG. 5A to 5F In vivo persistence and efficacy of CD30 CAR ATCs with novel spacers against NK-T Lymphoma cell line.
  • 5A Experiment scheme for SNK6 model.
  • 5B IVIS monitoring twice a week to track T cells biodistribution.
  • 5C Average radiance of transferred T cells in tumor following treatment
  • 5D Endpoint average radiance measured by IVIS and flow cytometric quantification of T cells in tumors.
  • 5E T cell counts in blood, spleen, and liver quantified by flow cytometry.
  • 5F Endpoint tumor counts quantified by flow cytometry.
  • FIGS. 6A to 6E In vivo persistency, efficacy, and toxicity of CD30 CAR ATCs in humanized mice xenografted with peripheral T cell lymphoma.
  • Figures 7A to 7G In vivo persistency, efficacy, and toxicity of CD30 CAR ATCs in humanized mice supplemented with human GM-CSF and IL-3.
  • (7A) Experiment set up of CD30 high NALM-6 model.
  • (7B) Circulating IgG levels at baseline and 3 days post I VIG administration.
  • (7C) Body weight changes after T cell treatment.
  • (7D) IVIS image taken twice a week to track NALM-6 disease.
  • FIGS 8A to 8E Generation and characterisation of T cells bearing humanized CD30 CAR with the 4-1 BB spacer.
  • FIGS 9A to 9D In vitro anti-tumor activity of T cells bearing humanized CD30 CAR with the 4- 1 BB spacer.
  • FIGS 10A to 10F In vitro on-target, off-tumor activity of T cells bearing humanized CD30 CAR with the 4-1 BB spacer.
  • 10A CD30 expression on HSPCs from 2 donors over 12 days of stimulation with 10 ng/mL FLT3L, SCF and TPO.
  • 10B CD30 expression on KM-H2 cells.
  • 10C CD30 expression on HSPC subsets after 2 days of stimulation.
  • 10D Impact of CD30 CAR T cell exposure on the survival of HSPC subsets. Two HSPC donors and two T cell donors were combined to obtain four co-culture combinations for 10D to 10F.
  • 10E Effect of CD30 CAR T cell exposure on the erythroid and myeloid developmental potential of HSPCs.
  • 10F Effect of priming CD30 CAR T cells with CD30-high KM-H2 targets on cytolysis of CD30-low HSPCs.
  • FIGS 11 A to 11G In vivo persistency, efficacy, and toxicity of humanized CD30 CAR T cells in GM-
  • FIGS 13A to 13C Characterisation of EBVSTs transduced to express humanized CD30 CARs.
  • 13A Expression and transduction efficiency of CD30 CARs in EBVSTs.
  • 13B Masking efficiency of CD30 CARs in EBVSTs.
  • 13C Cytotoxic efficacy and specificity of CD30 CAR EBVSTs with CD30+ KM- 0 H2 and CD30- Daudi cells.
  • Murine HRS3 scFv and humanised versions thereof were cloned into a pSFG retrovirus vector upstream of either a wildtype IgG 1 Fc, 0X40 or 4-1 BB derived spacer, followed by CD28 0 transmembrane domain, a CD28 and CD3 signalling domains.
  • a truncated form of the CD30 molecule that consisted only of the CD30 extracellular domain was cloned into pSFG retrovirus vector.
  • Retroviruses carrying the CD30 CARs or truncated CD30 were produced in HEK293VG or RD114 packaging cell line (BioVec Pharma, Quebec, Canada) by transient transfection with the pSFG vector using PEIpro transfection reagent (Polyplus, lllkirch, FRANCE).
  • PEIpro transfection reagent Polyplus, lllkirch, FRANCE.
  • RetroX Concentrator Takara Bio, Kusatsu, Shiga, Japan. The retroviruses were either used immediately or snap frozen and stored at -80°C.
  • the stable RD114 retrovirus packaging cell line that produces high titers of GFP-Firefly Luciferase 0 (GFP-FFIuc) virus particles was a kind gift from Dr Masataka Suzuki (Baylor Center for Gene Therapy Baylor College of Medicine).
  • Enriched leukapheresis products collected from consented healthy donors by Spectra Optia® 5 Apheresis System CMNC collection protocol and frozen in ACD-A anticoagulant, was purchased from HemaCare (Northridge, California, U.S.A.). The frozen leukopaks were thawed and PBMCs were extracted by gradient centrifugation using Ficoll-Paque PLUS (Cytiva, MA, U.S.A.). The PBMCs were either used immediately for experiments or frozen in smaller aliquots of 30-50 x 10 6 cells per cryovial in CryoStor® CS10 Cell Freezing Medium (STEMCELL Technologies, Cambridge, 0 Massachusetts, U.S.A.).
  • Cord blood CD34+ cells isolated from cord blood mononuclear cells via positive immunomagnetic separation from consented donors, were purchased from Lonza (Walkersville, Maryland, U.S.A). Frozen vials of up to 1 x 10 6 cells were thawed and CD34+ cells were either used immediately for 5 experiments or frozen in aliquots of 5 x 10 4 cells per cryovial in CryoStor® CS10 Cell Freezing Medium (STEMCELL Technologies, Cambridge, Massachusetts, U.S.A.). 1 .3 Production of CAR T cells
  • PBMC For activated T cells (ATC) transduction, PBMC were thawed and plated onto cell culture plates precoated with anti-CD3/CD28, and cultured in 10% FBS, 45% Advanced RPMI and 45% Click’s media with 5% CO2 at 37°C to generate ATCs.
  • FBS 45% Advanced RPMI and 45% Click’s media with 5% CO2 at 37°C to generate ATCs.
  • IL-7 and IL-15 Two days after culture, IL-7 and IL-15 were added into cell culture.
  • retrovirus containing CD30.CAR with the indicated scFv and spacer was transduced into ATCs by spinfection. Retrovirus was washed off 24 hours later and ATCs were cultured with occasional media change to replenish the IL-7 and IL-15.
  • ATCs were additionally transduced on the fourth day of culture with a retroviral vector encoding GFP-Firefly Luciferase (GFP-FFIuc) to enable tracking of ATCs by IVIS in live mice.
  • GFP-FFIuc GFP-Firefly Luciferase
  • ATCs were either frozen down using CryoStor following manufacturer’s manual or injected into mice.
  • ATCs were generated from CD34- cord blood cells instead of PBMCs, and later infused into humanised mice reconstituted with CD34+ cord blood cells from the same donor.
  • CD45RA depletion of PBMCs was performed by negative selection using CD45RA MACS Beads (Miltenyi Biotec, Bergisch Gladbach, Germany).
  • Whole PBMCs or RAD-PBMCs were cultured 1 x10 6 cells/well with viral peptides consisting of overlapping peptide libraries (15-mers overlapping by 11 amino acids) from JPT Technologies (Berlin, Germany).
  • cells were transduced with humanised CD30 CAR constructs using RetroNectin (Takara Bio, Kusatsu, Shiga, Japan), according to the description above.
  • T cells were then stimulated with irradiated co-stimulatory cells expressing markers such as CD80, CD86, 4-1 BB four days post transduction. Seven-eight days later, VSTs were harvested, frozen or used for cell assays.
  • CD30 CAR expression was measured with recombinant human CD30 protein fused with a His tag (10777-H08H, Sino Biological) followed by PE-conjugated anti-His (clone J095G46, BioLegend), or with biotinylated recombinant human CD30 protein (ACROBiosystems, CD0-H82E6) followed by PE-conjugated streptavidin (BD Biosciences, 554061 ), or with FITC-labeled recombinant human CD30 protein (ACROBiosystems, CD0-HF2H3). Flow data were analyzed and gated in FlowJo v10.8.1 for Windows.
  • CAR potency via cytotoxicity assay Prior to assessment of CAR potency via cytotoxicity assay, the cell media is changed to 2% assay media containing RPMI and 2% FBS. Cytotoxicity assay was carried out using the xCelligence Real- Time Cell Analysis System with 5% CO2 at 37°C (Agilent). Target cells will be added onto PET plates tethered with anti-CD40 antibody following manufacturer’s manual (Aligent). 24 hours after target cell adherence, CAR T cells will be plated into the PET plates at CAR T: target cell ratio of 0.2:1 , 1 :1 or 5:1 . The PET plates will return into the xCelligence system and cytotoxicity will be monitored for 48 hours.
  • KM-H2 cells were used as the target cell line with an initial E:T ratio of 1 :2, with 50,000 effector and 100,000 target cells per well of a 96-well plate for Encounter 1 . Three sets of this set-up were prepared. After 48h, cells from 2 sets were harvested and added to 2 new sets of 100,000 KM-H2 target cells per well to set up Encounter 2. After a further 48h, cells from 1 set were harvested and added to a new set of 100,000 KM-H2 target cells per well for Encounter 3. To distinguish target cells from the different encounters, KM-H2 cells for Encounters 2 and 3 were labelled with the lipophilic membrane dyes PKH67 and PKH26 (Sigma-Aldrich) respectively.
  • PKH67 and PKH26 Sigma-Aldrich
  • the cell media Prior to assessment of cytokine release, the cell media is changed to 2% assay media containing RPMI and 2% FBS. Target cells will be added onto 96-well flat-bottom plates. CAR T cells will be plated on top of the target cells at CAR T: target cell ratio 1 :1 . The plates will be incubated at 5% CO2 at 37°C for 24 hours. After 24 hours, the cell-free media will be collect by centrifugation at 500 g for 5 mins. The cytokine released in the media, such as TNFa, GMCSF and IFNy will be assayed by ELISA kits from BioLegends.
  • Milliplex map human high sensitivity T cell panel premixed 13-plex (Millipore, HSTCMAG28SPMX13) was used to determine cytokine levels in co-cultures or plasma by FLEXMAP 3D® (Luminex). Analysis was performed using Bio-plex Manager software (Bio-Rad)
  • HSPCs hematopoietic stem and progenitor cells
  • CD34+ HSPCs were stimulated with 10 ng/mL each of Flt3-ligand (FLT3L), stem cell factor (SCF) and thrombopoietin (TPO) (all from Miltenyi Biotec) for the specified time period at 5,000 - 20,000 cells per 200pL per well of a 96-well U-bottom plate.
  • FLT3L Flt3-ligand
  • SCF stem cell factor
  • TPO thrombopoietin
  • CD34+ cells were isolated by depletion of T cells using magnetic beads conjugated to an anti-CD3 antibody (Miltenyi Biotec).
  • HSPC subsets were analyzed by flow cytometry after staining with Live/DeadTM NIR viability dye (Thermo Fisher) and fluorescently labelled monoclonal antibodies to CD34 (clone 561 , BD Biosciences), CD133 (clone 7, BioLegend), CD45RA (clone HI100, BD Biosciences), CD38 (clone HIT2, BD Biosciences), CD10 (clone HI10a, BD Biosciences) following the gating strategy described in Hornbach et al (18).
  • CFU-G colony forming units for granulocytes
  • CFU-M macrophages
  • BFU-E burst forming units for erythrocytes
  • mice were injected into right flank of NOD-sc/c/ IL2Rgamma nul1 (NSG) mice subcutaneously.
  • NSG NOD-sc/c/ IL2Rgamma nul1
  • mice were randomized into treatment groups, stratified by tumor volume.
  • 4x10 6 of GFP-luciferase tagged ATCs were injected into mice intravenously.
  • IL-2 1000 IU per mouse
  • Mouse Fc receptor blocking antibody (Bio X Cell, Cat#:BE0307) was administered to mice receiving lgG1 CH2CH3 spacer CD30 CAR at a dose of 8 pg drug per 1 g of body weight on alternate days.
  • Mouse Fc receptor blocking antibody was injected to prevent non-specific interaction between IgG 1 CH2CH3 domain with murine Fc receptor bearing immune cells. T cell biodistribution were evaluated using IVIS imaging system (Perkin Elmer). Region of interest (ROI) was drawn over the mice and the average radiance (p/s/cm 2 /sr) was quantified using Living lmage®4.7.4 software. On day 36, When mice were sacrificed on day 36, blood, spleen, liver, and tumor were collected for endpoint flow cytometric analysis.
  • mice 1 .1 1 IL-3/GM-CSF-supplemented humanised mice
  • mice NSG pups were irradiated and reconstituted with 1 x10 5 CD34+ cells from HLA-typed cord blood donors.
  • human IL-3/GM-CSF encoding plasmids were delivered via hydrodynamic tail vein injection to mice with more than 20% of hCD45 reconstitution, to support myeloid cells reconstitution. Cheek bleed was performed on humanised mice beforehand, for assessment of baseline human cytokine levels in serum.
  • mice 1 x10 6 of HuT-78 were injected subcutaneously into the right flank of humanised mice. On day 4 or 6, mice were injected intravenously with 1 x10 6 or 4x10 6 of CAR and luciferase double transduced ATCs T cells as specified in figures.
  • IL-2 and mouse FcR block injection were administered in a regimen similar to the NK/T lymphoma model. Cheek bleeds were done performed at 6 days post treatment to assess the cytokine levels in the plasma 6 days post ATCs treatment.
  • an additional cheek bleed was done at 6 days post treatment. T cell biodistribution was evaluated using the IVIS imaging system (Perkin Elmer).
  • Region of interest was drawn over the mice and measured the average radiance (p/s/cm2/sr) was quantified using Living lmage®4.7.4 software. Mice were sacrificed and blood, spleen, liver, and tumor were collected for endpoint flow cytometry analysis. For the CD30CAR scFv studies, bone marrow and lung were harvested as well
  • mice were cheek bled 9 days before T cell treatment to assess cytokine levels in plasma post tumor engraftment.
  • mice were injected intra-peritoneally with 35mg human MG (Sigma- Aldrich, I4506) and in some treatment groups with 8pg/g mouse FcR block (Bio X Cell, Cat#:BE0307) for 2 days before ATC treatment.
  • 8pg/g mouse FcR block Bio X Cell, Cat#:BE0307
  • Cheek bleeds were performed at 2 and 7 days post treatment for the CD30CAR spacer studies, and at 3 and 6 days post treatment for the CD30CAR scFv studies. At the experiment end point, mice were sacrificed and blood, spleen, liver and bone marrow were collected for flow cytometry analysis.
  • Novel 0X40 and 41 -BB derived spacers retain in vitro killing efficacy with lower cytokine production.
  • the spacer domain can significantly affect the safety and efficacy of the CAR T cell.
  • IgG Fc domain derived spacers have been commonly used in early designs of CARs.
  • the use of the wildtype Fc domains can result in undesired interactions with Fc receptor expressing cells.
  • Highly flexible linkers can facilitate the recognition of sterically challenging epitopes while the length of the linkers can optimize the synaptic distance between the CAR T cell and the target cell.
  • the alternative spacers were cloned into the HRS-3 CD30 CAR pSFG retroviral vector. Expression of the transduced CAR T cells with the different spacers was assessed by flow cytometry (Figure 1 A). All CARs regardless of spacer domains were transduced equally efficiently in activated T cells. Next, the efficiency of the various CARs to recognize and kill target cells was also compared using an assay with CD30+ KM-H2 cells at an effector to target ratio of 1 :1 . Aside from CD96 spacer, the alternative spacers exhibited equivalent killing efficiencies as the original lgG1 spacer (Figure 1 B). In addition, we first studies the basal level of cytokine secretion of the CAR T cells with different spacers.
  • CD30 negative Raji cells were retroviral ly transduced to express a truncated CD30 protein.
  • FACs sorting and single cell cloning was done to identify 3 distinct clones expressing various levels of CD30 (Figure 2A).
  • CAR T cells expressing either the IgG 1 , 0X40 and 4- 1 BB spacers were co-incubated with the different CD30 expressing Raji clones, untransduced Raji or the positive control cell line KM-H2 at an effector to target ratio of 1 :1 ( Figure 2B).
  • CARs bearing wildtype lgG1 derived spacers readily interact with Fc receptors expressing cells such and monocytes and NK cells.
  • the novel spacers are not known to interact with any receptors or ligands.
  • CD14+ monocytes were isolated from donor PBMCs and incubated with CAR T cells with the different spacers at a T cell: Monocyte ratio of 1 :5. Cytotoxicity of CARs with novel 0X40 and 41 -BB derived spacers against monocytes were assessed by flow cytometry 48hrs or 96 hrs post incubation.
  • CD30 CAR T cells with novel spacers have improved persistency and efficacy against human NK-T lymphoma.
  • mice The presence of the IgG 1 CH2CH3 linker in CAR-T cells has been described to result in CAR-T cell interactions with murine Fc receptor expressing immune cells in immunodeficient mice, resulting in lung sequestration and compromised anti-tumor efficacy (19).
  • This phenomenon is less pertinent in hosts with an intact capacity to produce physiological amounts of immunoglobulin as IgG can occupy Fc receptors on immune cells to reduce availability for interaction with the IgG 1 CH2CH3 domain.
  • mouse Fc receptor blocking antibody (clone 2.4.G2) was administered to mice receiving CD30 CAR T cells with lgG1 CH2CH3 domains.
  • mice were randomised to receive luciferase expressing CD30 CAR T with various spacers.
  • I VIS monitoring at day 1 post-treatment revealed that all CD30 CAR T cells initially traffic to the lung.
  • CD30 CAR T cells with the IgG 1 CH2CH3 spacer quickly disappeared in animals within 4 days post-treatment, consistent with published data (19) that lgG1 CH2CH3 domain interaction with Fc receptor expressing murine cells can greatly compromise CAR T cells in vivo lifespan (Figure 5B).
  • Administration of mouse FcR blocking antibody to mice receiving CD30 CAR T cells with IgG 1 CH2CH3 spacer prolonged persistence of the CAR T cells but did not completely prevent lung trapping and cell elimination (Figure 5B).
  • CD30 CAR T cells with IgG 1 CH2CH3 spacer also failed to migrate into tumors, even in the presence of mouse FcR blocking antibody ( Figures 5B and 5C).
  • CD30 CAR T cells with lgG2, 0X40, 41 BB and CD96 spacers persisted and expanded well in animals, with clearly increased numbers over IgG 1 CH2CH3 spacer CD30 CAR T cells up to 20 days post-treatment ( Figure 5B).
  • CD30 CAR T cells Compared to CD30 CAR T cells with IgG 1 CH2CH3 spacer, lgG2, 0X40, 41 BB and CD96 spacer CD30 CAR T cells had improved migration and expansion within tumors in-situ, with these effects observed up to 20 days post-treatment ( Figures 5B and 5C).
  • mice were sacrificed for endpoint flow analysis at day 20. Higher number of CD30 CAR T cells bearing the lgG2, 0X40, 41 BB and CD96 domains were observed in tumor samples at endpoint, which is congruent with the I VIS results ( Figure 5D). Sizeable populations of CD30 CAR T cells with lgG2, 0X40, 41 BB and CD96 spacers could also be detected in blood, spleen, liver and total body of mice ( Figure 5E). Lastly, SNK-6 cells defined as live CD3-, CD45+ and CD30+ cell populations in tumors were observed to trend towards lower numbers in mice which received with 0X40, 41 BB, CD96 and CD44 spacers CD30 CAR T cells ( Figure 5F).
  • mice which have been reconstituted with human CD34+ cells for a model of peripheral T cell lymphoma. Following tumor engraftment, mice were randomised to receive luciferase expressing CD30 CAR T cells that were generated from the CD34 negative fraction of the same donor used for humanisation of the mice. The CD30 CAR T cells were also transduced with luciferase to enable in vivo cell tracing ( Figure 6A; see Example 1 for details).
  • mouse Fc receptor blocking antibody was also administered to a group of mice receiving lgG1 CH2CH3 CD30 CAR T cells. Due to the encouraging readouts from earlier in vitro experiments, the inventors chose to focus on CD30 CAR T cells with IgG 1 CH2CH3, 0X40 and 41 BB spacers.
  • Plasma collected at day 6 post-treatment were assessed for cytokines levels using Luminex®.
  • Plasma levels of lnterleukin-6 (IL-6), Interleukin 8 (IL-8), Tumor Necrosis Factor Alpha (TNF- a), Interferon Gamma (IFN-y), Granulocyte macrophage-colony stimulating factor (GM-CSF) and Interleukin 2 (IL-2) remained low in all treatment arms (Figure 6E), demonstrating that 0X40 and 41 BB spacer CD30 CAR T cells in vivo activity and expansion did not induce systemic production of inflammatory cytokines (Figure 6E).
  • High tumor load is a known risk factor for the occurrence of cytokine release syndrome (CRS) associated with CAR T cell therapy in patients with haematological malignancies (20, 21 ).
  • CRS cytokine release syndrome
  • Myeloid cells including monocytes and macrophages have been delineated to be key players in the development of CRS and neurotoxicity after CAR-T cell therapy (22, 23).
  • mice utilised in this model were supplemented with human GM-CSF and interleukin-3 (IL-3) after humanisation in order to support the stable engraftment of myeloid lineages (refer to Methods and Materials).
  • Disease progression was monitored by bioluminescence imaging of CD30 + NALM-6 cells transduced with firefly luciferase. Treatment was administered when systemic tumor burden in mice was high (Figure 7A).
  • mice were randomised to receive no treatment, untransduced T cells + human IVIG, CD30 CAR T cells with IgG 1 CH2CH3 spacer + human IVIG + murine Fc receptor blocking antibody or CD30 CAR T cells with 4-1 BB spacer + human IVIG (Figure 7A).
  • Plasma IgG levels was assessed 3 days post-IVIG administration, at a time point after IVIG distribution into the extravascular compartment and IgG levels are at steady state.
  • Administration of human IVIG significantly increased plasma levels of IgG from baseline. ( Figure 7B).
  • VH3Vk3-Cys-41 BB and VH5Vk3-Cys-41 BB CD30 CAR ATCs expanded as well as, if not better than HRS3-41 BB CD30 CAR ATCs (Figure 8A).
  • the CD30 CAR variants had high transduction efficiencies of up to 98% ( Figure 8B). Where there was variation among the constructs, HRS3-41 BB CD30 CAR T cells had slightly lower frequencies of CAR+ cells. Most donors generated relatively more CD4 than CD8 T cells, with CD4/CD8 ratios largely equivalent among the CD30 CAR variants (Figure 8C).
  • T cells upregulate CD30, which may bind CD30 CAR-expressing cells and activate them, possibly resulting in fratricide.
  • CD30 CAR binding to CD30 in cis may help to protect cells from inadvertently activating their CD30 CAR-bearing brethren, and thus from fratricide as well.
  • the inventors employed two distinct commercially available anti-CD30 clones, BerH8 and BY88. BerH8 is thought to bind to a similar site of CD30 as the parental HRS3 scFv, as CD30 expression is virtually undetectable by BerH8 once T cells are transduced with HRS3-lgG1 .
  • CD4 T cells primarily comprised Tim3 single-positive cells, with a small proportion ( ⁇ 5-20%) of cells expressing both Tim3 and PD-1 , or none of the markers.
  • CD8 T cells were similarly largely Tim3 single-positive, particularly for donor 5056. The remaining cells consisted of cells bearing none of the markers, and Tim3-LAG3 double expressors. Within each donor, the frequencies of these populations were comparable among the different CD30CAR variants. The fraction of cells expressing all three proteins was very low, suggesting that cells were not terminally exhausted at the end of the expansion process.
  • Effective tumor clearance in vivo is predicated on the ability of CAR T cells to maintain killing activity throughout successive rounds of target encounters.
  • a flow cytometry-based serial killing assay was established in which CAR T cells underwent three consecutive target encounters, with each encounter lasting 48h ( Figure 9B; see Example 1 .6).
  • KM-H2 were labelled cells for Encounters 2 and 3 with the lipophilic membrane dyes PKH67 and PKH26 dyes respectively.
  • CD30 can also be upregulated on hematopoietic stem and progenitor cells (HSPC) upon cytokine stimulation, albeit at lower levels than lymphoma cells (21 ).
  • HSPC hematopoietic stem and progenitor cells
  • CD30 expression was reportedly heterogeneous on HSPC subsets (23), and this was also found to be true for the two donors assayed ( Figure 10C).
  • CD30 was upregulated on multipotent progenitors (MPPs) and lymphoid-primed multipotents (LMPPs), and to a lesser extent on multi-lymphoid primed progenitors (MLPs), but not on erythro-myeloid primed progenitors (EMPs).
  • MLPs multi-lymphoid primed progenitors
  • EMPs erythro-myeloid primed progenitors
  • CD34+ HSPCs were exposed to effector cells for 24h, then isolated the HSPCs for further culture over 9 days in the same cytokine cocktail of 10 ng/mL FLT3L, SCF and TPO before analyzing the cells by flow cytometry ( Figure 10D; see Example 1 ).
  • Encounter with any of the CD30CAR variants altered the progenitor population composition compared to untransduced T cells, suggesting there was some cytolysis of progenitors, especially the LMPP subset.
  • the EMP subset experienced a reciprocal increase as a proportion of all progenitor cells.
  • CD30CAR variants did not drastically affect HSPCs directly, the possibility that CD30CAR effectors might exhibit elevated cytolysis of HSPCs following encounter with CD30-high tumor cells was explored.
  • T cells bearing the CD30CAR variants were first primed by co-culturing them with KM- H2 target cells for 24h, then added in 2 day-stimulated HSPCs (Figure 10F). After a further 24h of culture, cells were harvested and analyzed by flow cytometry. HSPCs emerged unscathed with all CD30CAR variants for both T cell donors, while KM-H2-directed cytolysis remained high and comparable to that when only KM-H2 cells and T cells were co-incubated for 48h.
  • CD30 CAR T cells in an in vivo model that contains critical components of the human immune system such as myeloid, lymphoid and natural killer cells.
  • these candidates were evaluated in GM-CSF, IL-3 supplemented humanised mice which were subcutaneously xenografted with HuT-78 cells. Mice were randomised to receive either untransduced T cells, HRS3 or the humanised CD30CAR variants, stratified based on tumor volume, human CD45 and CD33 reconstitution levels and body weight. All infused T cells were also transduced with GFP-Luciferase (see Example 1 ) to enable in v/vo cell tracking and discrimination from endogenous human T cells ( Figure 11 A).
  • Luminex® analysis of mouse plasma drawn at Day 7 post treatment revealed that compared to untransduced T cells, inflammatory cytokines (IFN-y, GM-CSF, IL-8, IL-6, TNF-a and IL-2 ) were generally increased in mice from all 3 CD30 CAR T cells treatment groups.
  • inflammatory cytokines IFN-y, GM-CSF, IL-8, IL-6, TNF-a and IL-2
  • CAR T cell treatment group Figure 11 G, circles
  • mice utilised in this model were supplemented with human GM-CSF and interleukin-3 (IL-3) after human chimerism of >20% has been achieved in the peripheral blood (see Example 1 ).
  • IL-3 interleukin-3
  • Disease progression was monitored by bioluminescence imaging of CD30 + NALM-6 cells transduced with firefly luciferase. Treatment was administered when systemic tumor burden in mice was high ( Figure 12A).
  • mice were randomised to receive either untransduced T cells, HRS3 or the humanised CD30CAR variants, stratified based on tumor volume, human CD45 and CD33 reconstitution levels and body weight. Body weight changes were similar in mice across all treatment groups (Figure 12B). Treatment with VH3Vk3-Cys and VH5Vk3-Cys humanised CD30 CAR T cells significantly improved tumor control in mice compared to untransduced T cells. In addition, anti-tumor responses in VH3Vk3-Cys and VH5Vk3-Cys humanised CD30 CAR T cells treated mice appeared more durable compared to HRS- 3 CD30 CAR T cells (Figure 12C).
  • Luminex® analysis of mouse serum at Day 11 post treatment levels showed while that inflammatory cytokines (IFN-y, GM- CSF, IL-8, IL-6, TNF-a and IL-2) were higher in mice from CD30 CAR T cells treatment groups compared to untransduced T cells (Figure 12F), they were not at levels associated with the occurrence of CRS.
  • inflammatory cytokines IFN-y, GM- CSF, IL-8, IL-6, TNF-a and IL-2
  • CAR T cells have a good safety profile comparable to murine HRS3 CD30CAR. CAR T cells in mice carrying high tumor load.

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Abstract

The present application describes novel chimeric antigen receptor (CAR) constructs comprising novel spacer region sequences. Also provided are cells comprising the CAR, a nucleic acid or a plurality of nucleic acids encoding the CAR, an expression vector or a plurality of expression vectors comprising said nucleic acids, methods of making such molecules, and the use of such molecules in a method of medical treatment or prophylaxis.

Description

Chimeric Antigen Receptor Domains
Technical Field
The present disclosure relates to the fields of molecular biology, more specifically chimeric antigen receptor (CAR) technology. The present disclosure also relates to methods of medical treatment and prophylaxis.
Background
The development of chimeric antigen receptor (CAR) T cell therapy has significantly changed the treatment of patients suffering from certain B cell leukemia and lymphoma (7), providing effective and durable clinical responses by utilizing engineered T cells to eliminate target cells expressing a non-major histocompatibility complex (MHC) restricted antigen. CARs are synthetic receptors that comprise of 4 main domains: the antigen specific domain, commonly a single chain variable fragment of an antibody (scFv), the spacer domain that connects the scFv to the third or transmembrane domain of the receptor, and finally the signalling domains (8). Design and engineering of the CAR receptor is critical for the efficacy and safety of the CAR T cell strategy.
The spacer or hinge domain plays an important role in the function of the CAR, providing reach and flexibility for the scFv. Flexibility of the spacer allow the scFv to access otherwise sterically hindered epitopes, whilst the length of the spacer ensures that the optimal synapse distance between the CAR T cell and the target cell is maintained for efficient delivery of granzymes and perforins for potent cytotoxicity (8). The most commonly used spacer domains are derived from antibody Fc domains, i.e. IgG 1 , lgG2 and lgG4-Fc, CD8a and CD28. IgG-derived spacers can interact with Fc receptors on other immune cells and thus can result in the generation of immune responses mounted against T cells expressing the CARs. In addition, this interaction can also lead to nonantigen specific activation of CAR T cells, resulting in elevated cytokine release, exhaustion, activation induced cell death (11 ). Alternatively, CARs bearing CD8a- and CD28-derived spacers have been shown to be highly-effective and have been clinically approved in CAR T cell therapies. In addition, CARs with the CD28 spacer have been shown to lower the threshold of activation of CD19 CAR T cells compared to those with the CD8a spacer (12). It was recently shown that CARs with CD28 and CD8a spacers facilitate hetero-dimerization of endogenous CD28, and also with CARs CARs having CD28 transmembrane and endodomains (13), potentially lowering the threshold for CAR T activation, and producing a stronger activation signal that could increase the risk of CAR T therapy associated toxicities such as cytokine release syndrome and neurotoxicity (14).
Summary
The present disclosure provides a chimeric antigen receptor (CAR) comprising: (i) an antigen-binding domain which binds specifically to a target antigen, (ii) a spacer domain comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID N0s:100, 99, 101 and 102, (iii) a transmembrane domain, and (iv) a signalling domain comprising an amino acid sequence comprising an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the CAR comprises a spacer domain comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:100.
In some embodiments, the antigen-binding domain binds specifically to a target antigen selected from CD30, CD19, CD20, CD22, ROR1 R, CD4, CD7, CD38, BCMA, Mesothelin, EGFR, GPC3, MUC1 , HER2, GD2, CEA, EpCAM, LeY and PSCA.
In some embodiments, the antigen-binding domain binds specifically to CD30.
In some embodiments, the antigen-binding domain comprises:
(i) a heavy chain variable (VH) region incorporating the following CDRs:
HC-CDR1 having the amino acid sequence of SEQ ID NO:2 HC-CDR2 having the amino acid sequence of SEQ ID NO:3 HC-CDR3 having the amino acid sequence of SEQ ID NO:4; and
(ii) a light chain variable (VL) region incorporating the following CDRs:
LC-CDR1 having the amino acid sequence of SEQ ID NQ:10 LC-CDR2 having the amino acid sequence of SEQ ID NO:1 1 LC-CDR3 having the amino acid sequence of SEQ ID NO:12.
In some embodiments, the antigen-binding domain comprises: a VH region having an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs:1 , 45, 52, 17, 21 , 24, 26, 28; and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs:9, 49, 54, 30, 35, 38, 41 , 43, 147, 148, 149, 150, 151 or 152.
In some embodiments, the antigen-binding domain comprises:
(i) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:1 , and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:9; or
(ii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:52, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:54; or
(iii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:17, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NQ:30; or
(iv) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:21 , and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:35; or
(v) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:24, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:38; or (vi) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:26, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:41 ; or
(vii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:28, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:43; or
(viii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:1 , and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:147; or
(ix) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:17, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:148; or
(x) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:21 , and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:149; or
(xi) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:24, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NQ:150; or
(xii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:26, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:151 ; or
(xiii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:28, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:152.
In some embodiments, the antigen-binding domain is or comprises a single chain variable fragment (scFv) comprising the VH region and the VL region.
In some embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs:103, 104 or 105.
In some embodiments, the signalling domain comprises an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs:106, 107 or 108.
In some embodiments, the signalling domain comprises an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:111 .
In some embodiments, the CAR comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs:124, 144, 190, 210, 117, 118, 119, 120, 121 , 122, 123, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 145, 146, 183, 184, 185, 186, 187, 188, 189, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 211 or 212. The present disclosure also provides a nucleic acid, or a plurality of nucleic acids, optionally isolated, encoding a CAR according to the present disclosure.
The present disclosure also provides an expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to the present disclosure.
The present disclosure also provides a cell comprising a CAR, a nucleic acid or a plurality of nucleic acids, or an expression vector or a plurality of expression vectors according to the present disclosure.
The present disclosure also provides a method comprising culturing a cell according to the present disclosure under conditions suitable for expression of an CAR by the cell.
The present disclosure also provides a composition comprising a CAR, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, or a cell according to the present disclosure, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
The present disclosure also provides a CAR, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the present disclosure, for use in a method of medical treatment or prophylaxis.
The present disclosure also provides a CAR, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the present disclosure, for use in the treatment or prevention of a cancer.
In some embodiments, the cancer is selected from the group consisting of: a cancer expressing the target antigen for the CAR, a CD30-positive cancer, an EBV-associated cancer, a hematological cancer, a myeloid hematologic malignancy, a hematopoietic malignancy a lymphoblastic hematologic malignancy, myelodysplastic syndrome, leukemia, T cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, B cell nonHodgkin’s lymphoma, diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, EBV- associated lymphoma, EBV-positive B cell lymphoma, EBV-positive diffuse large B cell lymphoma, EBV- positive lymphoma associated with X-linked lymphoproliferative disorder, EBV-positive lymphoma associated with HIV infection/AIDS, oral hairy leukoplakia, Burkitt’s lymphoma, post-transplant lymphoproliferative disease, central nervous system lymphoma, anaplastic large cell lymphoma, T cell lymphoma, ALK-positive anaplastic T cell lymphoma, ALK-negative anaplastic T cell lymphoma, peripheral T cell lymphoma, cutaneous T cell lymphoma, NK-T cell lymphoma, extra-nodal NK-T cell lymphoma, thymoma, multiple myeloma, a solid cancer, epithelial cell cancer, gastric cancer, gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck cancer, head and neck squamous cell carcinoma, oral cavity cancer, oropharyngeal cancer, oropharyngeal carcinoma, oral cancer, laryngeal cancer, nasopharyngeal carcinoma, oesophageal cancer, colorectal cancer, colorectal carcinoma, colon cancer, colon carcinoma, cervical carcinoma, prostate cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, squamous lung cell carcinoma, bladder cancer, urothelial carcinoma, skin cancer, melanoma, advanced melanoma, renal cell cancer, renal cell carcinoma, ovarian cancer, ovarian carcinoma, mesothelioma, breast cancer, brain cancer, glioblastoma, prostate cancer, pancreatic cancer, mastocytosis, advanced systemic mastocytosis, germ cell tumor or testicular embryonal carcinoma.
The present disclosure also provides a CAR, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the present disclosure, for use in the treatment or prevention of a disease or condition characterised by an alloreactive immune response.
In some embodiments, the disease or condition characterised by an alloreactive immune response is selected from the group consisting of: a disease or condition associated with allotransplantation, graft versus host disease (GVHD) or graft rejection.
The present disclosure also provides the use of a CAR, a nucleic acid or a plurality of nucleic acids, an expression vector or a plurality of expression vectors, a cell, or a composition according to the present disclosure to deplete or increase killing of cells expressing the target antigen for the CAR.
The present disclosure also provides an in vitro complex, optionally isolated, comprising a CAR or a cell according to the present disclosure, bound to the target antigen for the CAR.
Description
The present disclosure provides novel chimeric antigen receptor (CAR) constructs comprising novel spacer region sequences.
Unexpectedly, T cells expressing the novel CAR constructs are shown to expand/proliferate similarly, to have a improved safety profile, to display similar cytotoxicity to cells expressing the target antigen for the CAR, to have improved persistence in vivo, and to display improved tumor growth inhibition of cancer comprising cells expressing the target antigen for the CAR, as compared to equivalent CAR constructs instead having the known IgG 1 CH2-CH3 spacer region.
Chimeric antigen receptors (CARs)
Aspects of the present disclosure relate to chimeric antigen receptors (CARs). CARs are recombinant receptors that provide both antigen-binding and T cell activating functions. CAR structure and engineering is reviewed, for example, in Dotti et al., Immunol Rev (2014) 257(1 ), hereby incorporated by reference in its entirety. CARs comprise an antigen-binding domain linked via a transmembrane domain to a signalling domain. An optional hinge or spacer domain may provide separation between the antigen-binding domain and transmembrane domain and may act as a flexible linker. When expressed by a cell, the antigenbinding domain is provided in the extracellular space, and the signalling domain is intracellular. The antigen-binding domain mediates binding to the target antigen for which the CAR is specific. The antigen-binding domain of a CAR may be based on the antigen-binding region of an antibody which is specific for the antigen to which the CAR is targeted. For example, the antigen-binding domain of a CAR may comprise amino acid sequences for the complementarity-determining regions (CDRs) of an antibody which binds specifically to the target antigen. The antigen-binding domain of a CAR may comprise or consist of the light chain and heavy chain variable region amino acid sequences of an antibody which binds specifically to the target antigen. The antigen-binding domain may be provided as a single chain variable fragment (scFv) comprising the sequences of the light chain and heavy chain variable region amino acid sequences of an antibody. Antigen-binding domains of CARs may target antigens based on other protein protein interactions, such as ligand:receptor binding; for example an IL-13Ra2-targeted CAR has been developed using an antigen-binding domain based on IL-13 (see e.g. Kahlon et al. 2004 Cancer Res 64(24): 9160-9166).
A spacer domain provides separation between the antigen-binding domain and the transmembrane domain, and may act as a flexible linker. Such domains may be or comprise flexible regions allowing the binding moiety to orient in different directions. Spacer domains are sometimes derived from constant regions of immunoglobulin molecules.
The transmembrane domain is provided between the antigen-binding domain and the signalling domain of the CAR. The transmembrane domain provides for anchoring the CAR to the cell membrane of a cell expressing a CAR, with the antigen-binding domain in the extracellular space and signalling domain inside the cell. Transmembrane domains of CARs may be derived from transmembrane region sequences for cell membrane-bound proteins (e.g. CD28, CD8, CD4, CD3-£, etc.).
The signalling domain comprises amino acid sequences required for activation of immune cell function. The CAR signalling domains may comprise the amino acid sequence of the intracellular domain of CD3- , which provides immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation and activation of the CAR-expressing cell. Signalling domains comprising sequences of other ITAM- containing proteins have also been employed in CARs, such as domains comprising the ITAM containing region of FcyRI (Haynes et al., 2001 J Immunol 166(1 ):182-187). CARs comprising a signalling domain derived from the intracellular domain of CD3-£ are often referred to as first generation CARs.
The signalling domains of CARs typically also comprise the signalling domain of a costimulatory protein (e.g. CD28, 4-1 BB etc.), for providing the costimulation signal necessary for enhancing immune cell activation and effector function. CARs having a signalling domain including additional costimulatory sequences are often referred to as second generation CARs. In some cases, CARs are engineered to provide for costimulation of different intracellular signalling pathways. For example, CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, whereas 4-1 BB costimulation triggers signalling is through TNF receptor associated factor (TRAF) adaptor proteins. Signalling domains of CARs therefore sometimes contain costimulatory sequences derived from signalling domains of more than one costimulatory molecule. CARs comprising a signalling domain with multiple costimulatory sequences are often referred to as third generation CARs. Throughout this specification, polypeptides, domains and amino acid sequences which are ‘derived from’ a reference polypeptide/domain/amino acid sequence have at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference polypeptide/domain/amino acid sequence. Polypeptides, domains and amino acid sequences which are ‘derived from’ a reference polypeptide/domain/amino acid sequence preferably retain the functional and/or structural properties of the reference polypeptide/domain/amino acid sequence.
By way of illustration, an amino acid sequence derived from the intracellular domain of CD28 may comprise an amino acid sequence having 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the intracellular domain of CD28, e.g. as shown in SEQ ID NO:106. Furthermore, an amino acid sequence derived from the intracellular domain of CD28 preferably retains the functional properties of the amino acid sequence of SEQ ID NO:106, i.e. the ability to activate CD28-mediated signalling.
The amino acid sequence of a given polypeptide or domain thereof can be retrieved from, or determined from a nucleic acid sequence retrieved from, databases known to the person skilled in the art. Such databases include GenBank, EMBL and UniProt.
Through engineering to express a CAR specific for a particular target antigen, immune cells (typically T cells, but also other immune cells such as NK cells) can be directed to kill cells expressing the target antigen. Binding of a CAR-expressing T cell (CAR-T cell) to the target antigen for which it is specific triggers intracellular signalling, and consequently activation of the T cell. The activated CAR-T cell is stimulated to divide and produce factors resulting in killing of the cell expressing the target antigen.
Antigen-binding domain
An ‘antigen-binding domain’ refers to a domain which is capable of binding to a target antigen. The target antigen may e.g. be a peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. Antigen-binding domains according to the present disclosure may be derived from antibodies (i.e. immunoglobulins (Igs)) and antigen-binding fragments thereof. As used herein, ‘antibodies’ include monoclonal antibodies, polyclonal antibodies, monospecific and multispecific (e.g., bispecific, trispecific, etc.) antibodies, and antibody-derived antigen-binding molecules such as scFv, scFab, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH), etc.). Antigen-binding fragments of antibodies include e.g. Fv, Fab, F(ab’)2 and F(ab’) fragments. In some embodiments, an antigen-binding domain according to the present disclosure may comprise or consist of an antibody or an antigen-binding fragment thereof.
In some embodiments, the antigen-binding domain comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specific binding to the target antigen. An antigen-binding domain formed by a VH and a VL may also be referred to herein as an Fv. In some embodiments, the domain capable of binding to a target antigen comprises or consists of an antigen-binding peptide/polypeptide, e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (/.e. a single-domain antibody (sdAb)), affilin, armadillo repeat protein (ArmRP), OBody or fibronectin - reviewed e.g. in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082-1101 , which is hereby incorporated by reference in its entirety (see also e.g. Boersma et al., J Biol Chem (2011 ) 286:41273-85 and Emanuel et al., Mabs (2011 ) 3:38-48).
The antigen-binding domains of the present disclosure generally comprise a VH and a VL of an antibody capable of specific binding to the target antigen. Antibodies generally comprise six complementaritydetermining regions CDRs; three in the heavy chain variable region (VH): HC-CDR1 , HC-CDR2 and HC- CDR3, and three in the light chain variable region (VL): LC-CDR1 , LC-CDR2, and LC-CDR3. The six CDRs together define the paratope of the antibody, which is the part of the antibody which binds to the target antigen. The VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, VHs comprise the following structure: N term-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C term; and VLs comprise the following structure: N term-[LC-FR1 ]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]- [LC-CDR3]-[LC-FR4]-C term.
VH and VL sequences may be provided in any suitable format provided that the antigen-binding domain can be linked to the other domains of the CAR. Formats contemplated in connection with the antigenbinding domain of the present disclosure include those described in Carter, Nat. Rev. Immunol (2006), 6: 343-357, such as scFv, dsFV, (scFv)2 diabody, triabody, tetrabody, Fab, minibody, and F(ab)2 formats.
In some embodiments, the antigen-binding domain comprises the CDRs of an antibody/antibody fragment which is capable of binding to the target antigen. In some embodiments, the antigen-binding domain comprises the VH region and the VL region of an antibody/antibody fragment which is capable of binding to the target antigen. A moiety comprised of the VH and a VL of an antibody may also be referred to herein as a variable fragment (Fv). The VH and VL may be provided on the same polypeptide chain, and joined via a linker sequence; such moieties are referred to as single-chain variable fragments (scFvs). Suitable linker sequences for the preparation of scFv are known to the skilled person, and may comprise serine and glycine residues.
In some embodiments, the antigen-binding domain comprises, or consists of, Fv capable of binding to the target antigen. The antigen-binding domain may be provided with any suitable format, e.g. scFv, scFab, etc. In some embodiments, the antigen-binding domain comprises, or consists of, a scFv capable of binding to the target antigen.
The target antigen for which the antigen-binding domain (and thus the CAR) is specific may be any target antigen. In some embodiments, the target antigen is an antigen whose expression/activity, or whose upregulated expression/activity, is positively associated with a disease or disorder (e.g. a cancer, an infectious disease or an autoimmune disease). The target antigen is preferably expressed at the cell surface of a cell expressing the target antigen. It will be appreciated that the CAR directs effector activity of the cell expressing the CAR against cells/tissues expressing the target antigen for which the CAR comprises a specific antigen-binding domain.
In some embodiments, a target antigen may be a cancer cell antigen. A cancer cell antigen is an antigen which is expressed or over-expressed by a cancer cell. A cancer cell antigen may be any peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. A cancer cell antigen’s expression may be associated with a cancer. A cancer cell antigen may be abnormally expressed by a cancer cell (e.g. the cancer cell antigen may be expressed with abnormal localisation), or may be expressed with an abnormal structure by a cancer cell. A cancer cell antigen may be capable of eliciting an immune response. In some embodiments, the antigen is expressed at the cell surface of the cancer cell (i.e. the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the part of the antigen which is bound by the antigen-binding molecule described herein is displayed on the external surface of the cancer cell (i.e. is extracellular). The cancer cell antigen may be a cancer- associated antigen. In some embodiments the cancer cell antigen is an antigen whose expression is associated with the development, progression or severity of symptoms of a cancer. The cancer- associated antigen may be associated with the cause or pathology of the cancer, or may be expressed abnormally as a consequence of the cancer. In some embodiments, the cancer cell antigen is an antigen whose expression is upregulated (e.g. at the RNA and/or protein level) by cells of a cancer, e.g. as compared to the level of expression of by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue/cell type). In some embodiments, the cancer-associated antigen may be preferentially expressed by cancerous cells, and not expressed by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue/cell type). In some embodiments, the cancer- associated antigen may be the product of a mutated oncogene or mutated tumor suppressor gene. In some embodiments, the cancer-associated antigen may be the product of an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein.
Cancer cell antigens are reviewed by Zarour HM, DeLeo A, Finn OJ, et al. Categories of Tumor Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003. Cancer cell antigens include oncofetal antigens: CEA, Immature laminin receptor, TAG-72; oncoviral antigens such as HPV E6 and E7; overexpressed proteins: BING-4, calcium-activated chloride channel 2, cyclin-B1 , 9D7, Ep-CAM, EphA3, HER2/neu, telomerase, mesothelin, SAP-1 , survivin; cancer-testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1 , PRAME, SSX-2; lineage restricted antigens: MARTI , Gp100, tyrosinase, TRP-1/2, MC1 R, prostate specific antigen; mutated antigens: 0-catenin, BRCA1/2, CDK4, CML66, Fibronectin, MART-2, p53, Ras, TGF-pRII; post-translationally altered antigens: MUC1 , idiotypic antigens: Ig, TCR. Other cancer cell antigens include heat-shock protein 70 (HSP70), heat-shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, feto-acinar pancreatic protein (FAPP), alkaline phosphatase placental-like 2 (ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1 ), protein tyrosine kinase 7 (PTK7), and cyclophilin B. In some embodiments the cancer cell antigen is a cancer cell antigen described in Zhao and Cao, Front Immunol. (2019) 10: 2250, which is hereby incorporated by reference in its entirety. In some embodiments, a cancer cell antigen is selected from CD30, CD19, CD20, CD22, ROR1 R, CD4, CD7, CD38, BCMA, Mesothelin, EGFR, GPC3, MUC1 , HER2, GD2, CEA, EpCAM, LeY and PSCA.
In some embodiments, a cancer cell antigen is an antigen expressed by cells of a hematological malignancy. In some embodiments, a cancer cell antigen is selected from CD30, CD19, CD20, CD22, ROR1 R, CD4, CD7, CD38 and BCMA. In some embodiments, a cancer cell antigen is an antigen expressed by cells of a solid tumor. In some embodiments, a cancer cell antigen is selected from Mesothelin, EGFR, GPC3, MUC1 , HER2, GD2, CEA, EpCAM, LeY and PSCA.
In some embodiments, the antigen-binding domain (and thus the CAR) is multispecific. By ‘multispecific’ it is meant that the antigen-binding domain displays specific binding to more than one target. In some embodiments the antigen-binding domain is a bispecific antigen-binding domain. In some embodiments the antigen-binding molecule comprises at least two different antigen-binding moieties (i.e. at least two antigen-binding moieties, e.g. comprising non-identical VHs and VLs). Individual antigen-binding moieties of multispecific antigen-binding domains may be connected, e.g. via linker sequences.
In some embodiments the antigen-binding domain binds to at least two, non-identical target antigens, and so is at least bispecific. The term ‘bispecific’ means that the antigen-binding domain is able to bind specifically to at least two distinct antigenic determinants. In some embodiments, at least one of the target antigens for the multispecific antigen-binding domain/CAR is CD30.
Each of the target antigens may independently be a target antigen as described herein. In some embodiments each target antigen is independently a cancer cell antigen as described herein.
It will be appreciated that an antigen-binding domain according to the present disclosure (e.g. a multispecific antigen-binding domain) comprises antigen-binding moieties capable of binding to the target(s) for which the antigen-binding domain is specific. For example, an antigen-binding domain which is capable of binding to CD30 and an antigen other than CD30 may comprise: (i) an antigen-binding moiety which is capable of binding to CD30, and (ii) an antigen-binding moiety which is capable of binding to a target antigen other than CD30.
In aspects and embodiments of the present disclosure, the target antigen is CD30. Accordingly, in some aspects and embodiments of the present disclosure the antigen-binding domain is a CD30-binding domain.
CD30 (also known as TNFRSF8) is the protein identified by UniProt: P28908. CD30 is a single pass, type I transmembrane glycoprotein of the tumor necrosis factor receptor superfamily. CD30 structure and function is described e.g. in van der Weyden et al., Blood Cancer Journal (2017) 7: e603 and Muta and Podack Immunol. Res. (2013) 57(1 -3) :151 -8, both of which are hereby incorporated by reference in their entirety. Alternative splicing of mRNA encoded by the human TNFRSF8 gene yields three isoforms: isoform 1 (‘long’ isoform; UniProt: P28908-1 , v1 ; SEQ ID NO:87), isoform 2 (‘cytoplasmic’, ‘short’ or ‘C30V’ isoform, UniProt: P28908-2; SEQ ID NO:88) in which the amino acid sequence corresponding to positions 1 to 463 of SEQ ID NO:87 are missing, and isoform 3 (UniProt: P28908-3; SEQ ID NO:89) in which the amino acid sequence corresponding to positions 1 to 111 and position 446 of SEQ ID NO:87 are missing. The N-terminal 18 amino acids of SEQ ID NO:87 form a signal peptide (SEQ ID NQ:90), which is followed by a 367 amino acid extracellular domain (positions 19 to 385 of SEQ ID NO:87, shown in SEQ ID NO:91 ), a 21 amino acid transmembrane domain (positions 386 to 406 of SEQ ID NO:87, shown in SEQ ID NO:92), and a 189 amino acid cytoplasmic domain (positions 407 to 595 of SEQ ID NO:87, shown in SEQ ID NO:93).
In this specification ‘CD30’ refers to CD30 from any species and includes CD30 isoforms, fragments, variants or homologues from any species. As used herein, a ‘fragment’, ‘variant’ or ‘homologue’ of a reference protein may optionally be characterised as having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference protein (e.g. a reference isoform). In some embodiments fragments, variants, isoforms and homologues of a reference protein may be characterised by ability to perform a function performed by the reference protein.
In some embodiments, the CD30 is from a mammal (e.g. a primate (rhesus, cynomolgous, or human) and/or a rodent (e.g. rat or murine) CD30). In preferred embodiments the CD30 is a human CD30. Isoforms, fragments, variants or homologues may optionally be characterised as having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of an immature or mature CD30 isoform from a given species, e.g. human. A fragment of CD30 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 590 amino acids, and may have a maximum length of one of 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 595 amino acids.
In some embodiments, the CD30 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:87, 88, 89 or 91 .
In some embodiments, the CD30 comprises an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:92 or 95. In some embodiments, a fragment of CD30 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:92 or 95.
The CD30-binding domain of the CAR of the present disclosure preferably displays specific binding to CD30 or a fragment thereof. The CD30-binding domain of the CAR of the present disclosure preferably displays specific binding to the extracellular domain of CD30. The CD30-binding domain may be derived from an anti-CD30 antibody or other CD30-binding agent, e.g. a CD30-binding peptide or CD30-binding small molecule.
The CD30-binding domain may be derived from the antigen-binding moiety of an anti-CD30 antibody.
Anti-CD30 antibodies include HRS3 and HRS4 (described e.g. in Hornbach et al., Scand J Immunol (1998) 48(5):497-501 ), HRS3 derivatives described in Schlapschy et al., Protein Engineering, Design and Selection (2004) 17(12): 847-860, BerH2 (MBL International Cat# K0145-3, RRID:AB_590975), SGN-30 (also known as cAC10, described e.g. in Forero-Torres et al., Br J Haematol (2009) 146:171-9), MDX- 060 (described e.g. in Ansell et al., J Clin Oncol (2007) 25:2764-9; also known as 5F11 , iratumumab), and MDX-1401 (described e.g. in Cardarelli et al., Clin Cancer Res. (2009) 15(10):3376-83), and anti- CD30 antibodies described in WO 2020/068764 A1 , WO 2003/059282 A2, WO 2006/089232 A2, WO 2007/084672 A2, WO 2007/044616 A2, WO 2005/001038 A2, US 2007/166309 A1 , US 2007/258987 A1 , WO 2004/010957 A2 and US 2005/009769 A1 .
In some embodiments a CD30-binding domain according to the present disclosure comprises the CDRs of an anti-CD30 antibody. In some embodiments a CD30-binding domain according to the present disclosure comprises the VH and VL regions of an anti-CD30 antibody. In some embodiments a CD30- binding domain according to the present disclosure comprises a scFv comprising the VH and VL regions of an anti-CD30 antibody.
There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991 ), Chothia et al., J. Mol. Biol. 196:901 -917 (1987), and VBASE2, as described in Retter et al., Nucl. Acids Res. (2005) 33 (suppl 1 ): D671 -D674. The CDRs and FRs of the VH regions and VL regions of the antibody clones described herein are defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77.
In some embodiments, the antigen-binding domain comprises a polypeptide or polypeptides comprising:
(i) a VH region comprising HC-CDR1 , HC-CDR2 and HC-CDR3 as indicated in column A of Table A, and
(ii) a VL region comprising LC-CDR1 , LC-CDR2 and LC-CDR3 as indicated in column B of Table A, wherein the sequences of columns A and B are selected from the same row of Table A. That is, in some embodiments, the antigen-binding molecule comprises a polypeptide or polypeptides comprising: (i) a VH region comprising HC-CDR1 = SEQ ID NO:2, HC-CDR2 = SEQ ID NO:3 and HC-CDR3 = SEQ ID NO:4, and (ii) a VL region comprising LC-CDR1 = SEQ ID NOU 0, LC-CDR2 = SEQ ID NOU 1 and LC-CDR3 SEQ ID NO:12.
In some embodiments, the antigen-binding domain comprises a polypeptide or polypeptides comprising a VH region comprising the heavy chain FRs, and a VL region comprising the light chain FRs, of an antibody selected from HRS3, VH1 VK1 , VH1 VK2, VH1VK3, VH1 VK4, VH1 VK5, VH2VK1 , VH2VK2, VH2VK3, VH2VK4, VH2VK5, VH3VK1 , VH3VK2, VH3VK3, VH3VK4, VH3VK5, VH4VK1 , VH4VK2, VH4VK3, VH4VK4, VH4VK5, VH5VK1 , VH5VK2, VH5VK3, VH5VK4, VH5VK5, HRS3Cys, VH1 VK1 Cys, VH1VK2Cys, VH1VK3Cys, VH1 VK4Cys, VH1VK5Cys, VH2VK1 Cys, VH2VK2Cys, VH2VK3Cys, VH2VK4Cys, VH2VK5Cys, VH3VK1 Cys, VH3VK2Cys, VH3VK3Cys, VH3VK4Cys, VH3VK5Cys, VH4VK1 Cys, VH4VK2Cys, VH4VK3Cys, VH4VK4Cys, VH4VK5Cys, VH5VK1 Cys, VH5VK2Cys, VH5VK3Cys, VH5VK4Cys, VH5VK5Cys, VH1 -5ConVK1 -3Con or VH3-5ConVK2-3Con, as shown in Table B herein. That is, in some embodiments, the antigen-binding molecule comprises a polypeptide or polypeptides comprising: (i) a VH region comprising HC-FR1 , HC-FR2, HC-FR3 and HC-FR4 as indicated in column A of Table B, and (ii) a VL region comprising LC-FR1 , LC-FR2, LC-FR3, and LC-FR4 as indicated in column B of Table B, wherein the sequences of columns A and B are selected from the same row of Table B.
In some embodiments the antigen-binding domain comprises: a VH comprising, or consisting of, an amino acid sequence having at least 70% sequence identity (e.g. at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to the amino acid sequence of SEQ ID NO:1 , 45, 52, 17, 21 , 24, 26, 28; and a VL comprising, or consisting of, an amino acid sequence having at least 70% sequence identity (e.g. at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to the amino acid sequence of SEQ ID NO:9, 49, 54, 30, 35, 38, 41 , 43, 147, 148, 149, 159, 151 or 152.
In some embodiments, the antigen-binding domain comprises a polypeptide or polypeptides comprising: (i) an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to an amino acid sequence indicated in column A of Table C, and (ii) an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to an amino acid sequence indicated in column B of Table C, wherein the sequences of columns A and B are selected from the same row of Table C.
In some embodiments the antigen-binding domain comprises: a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:1 , and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:9; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:45, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:49; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:52, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:54; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:17, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:30; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:21 , and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:35; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:24, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:38; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:26, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:41 ; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:28, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:43; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:1 , and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:147; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:17, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:148; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:21 , and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:149; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:24, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:150; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:26, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:151 ; or a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:28, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:152.
In some embodiments, the antigen-binding domain comprises a polypeptide or polypeptides comprising a VH region and a VL region of an antibody selected from HRS3, VH1 VK1 , VH1 VK2, VH1 VK3, VH1 VK4, VH1 VK5, VH2VK1 , VH2VK2, VH2VK3, VH2VK4, VH2VK5, VH3VK1 , VH3VK2, VH3VK3, VH3VK4, VH3VK5, VH4VK1 , VH4VK2, VH4VK3, VH4VK4, VH4VK5, VH5VK1 , VH5VK2, VH5VK3, VH5VK4, VH5VK5, HRS3Cys, VH1VK1 Cys, VH1VK2Cys, VH1VK3Cys, VH1VK4Cys, VH1 VK5Cys, VH2VK1 Cys, VH2VK2Cys, VH2VK3Cys, VH2VK4Cys, VH2VK5Cys, VH3VK1 Cys, VH3VK2Cys, VH3VK3Cys, VH3VK4Cys, VH3VK5Cys, VH4VK1 Cys, VH4VK2Cys, VH4VK3Cys, VH4VK4Cys, VH4VK5Cys, VH5VK1 Cys, VH5VK2Cys, VH5VK3Cys, VH5VK4Cys, VH5VK5Cys, VH1 -5ConVK1 -3Con or VH3- 5ConVK2-3Con, as shown in Table C herein. That is, in some embodiments, the antigen-binding molecule comprises a polypeptide or polypeptides comprising: (i) an amino acid sequence indicated in column A of Table C, and (ii) an amino acid sequence indicated in column B of Table C, wherein the sequences of columns A and B are selected from the same row of Table C.
In some embodiments, the antigen-binding domain comprises or consists of an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 or 182.
In some embodiments, the antigen-binding domain does not comprise, or does not consist of, a polypeptide having the amino acid sequence of SEQ ID NO:59.
In some embodiments, a VH region according to the present disclosure does not comprise, or does not consist of, the amino acid sequence of SEQ ID NO:1 . In some embodiments, a VL region according to the present disclosure does not comprise, or does not consist of, the amino acid sequence of SEQ ID NO:9.
In some embodiments, a VH region according to the present disclosure does not comprise HC-FR1 having the amino acid sequence of SEQ ID NO:5. In some embodiments, a VH region does not comprise HC-FR2 having the amino acid sequence of SEQ ID NO:6. In some embodiments, a VH region does not comprise HC-FR3 having the amino acid sequence of SEQ ID NO:7.
In some embodiments, a VL region according to the present disclosure does not comprise LC-FR1 having the amino acid sequence of SEQ ID NO:13. In some embodiments, a VL region does not comprise LC-FR2 having the amino acid sequence of SEQ ID NO:14. In some embodiments, a VL region does not comprise LC-FR3 having the amino acid sequence of SEQ ID NO:15.
In some embodiments, the antigen-binding domain comprises, or consists of, a CD30-binding Fv (/.e. a VH and VL pair) as described herein. In some embodiments, the antigen-binding domain comprises, or consists of, a CD30-binding Fv, in which the VH and VL are covalently linked. In some embodiments, the VH and VL of a CD30-binding Fv sequences are linked by a flexible linker sequence, e.g. a flexible linker sequence as described herein. The flexible linker sequence may be joined to ends of the VH sequence and VL sequence, thereby linking the VH and VL sequences.
In some embodiments, a CD30-binding domain may comprise or consist of a single chain variable fragment (scFv) comprising a VH sequence and a VL sequence as described herein. The VH sequence and VL sequence may be covalently linked. In some embodiments, the VH and the VL sequences are linked by a flexible linker sequence, e.g. a flexible linker sequence as described herein. The flexible linker sequence may be joined to ends of the VH sequence and VL sequence, thereby linking the VH and VL sequences.
In some embodiments, the VH and VL are joined via a linker sequence comprising one or more copies of an amino acid sequence according to SEQ ID NO:57. In some embodiments, the linker sequence comprises at least 1 , 2, 3 or 4 copies of an amino acid sequence according to SEQ ID NO:57. In some embodiments, the VH and VL are joined via a linker sequence comprising, or consisting of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:58.
In some embodiments, the antigen-binding molecule is capable of binding to the epitope of CD30 which is bound by antibody HRS3, e.g. within the region of amino acid positions 185-335 of human CD30 numbered according to SEQ ID NO:87, shown in SEQ ID NO:95 (Schlapschy et al., Protein Engineering, Design and Selection (2004) 17(12): 847-860, hereby incorporated by reference in its entirety). In some embodiments, the antigen-binding domain is capable of binding the same region of CD30, or an overlapping region of CD30, to the region of CD30 which is bound by an antibody comprising the VH and VL regions (see e.g. Table C) of one of HRS3, VH1 VK1 , VH1 VK2, VH1 VK3, VH1 VK4, VH1 VK5, VH2VK1 , VH2VK2, VH2VK3, VH2VK4, VH2VK5, VH3VK1 , VH3VK2, VH3VK3, VH3VK4, VH3VK5, VH4VK1 , VH4VK2, VH4VK3, VH4VK4, VH4VK5, VH5VK1 , VH5VK2, VH5VK3, VH5VK4, VH5VK5, HRS3Cys, VH1VK1 Cys, VH1VK2Cys, VH1 VK3Cys, VH1VK4Cys, VH1 VK5Cys, VH2VK1 Cys, VH2VK2Cys, VH2VK3Cys, VH2VK4Cys, VH2VK5Cys, VH3VK1 Cys, VH3VK2Cys, VH3VK3Cys, VH3VK4Cys, VH3VK5Cys, VH4VK1 Cys, VH4VK2Cys, VH4VK3Cys, VH4VK4Cys, VH4VK5Cys, VH5VK1 Cys, VH5VK2Cys, VH5VK3Cys, VH5VK4Cys, VH5VK5Cys, VH1 -5ConVK1 -3Con or VH3- 5ConVK2-3Con.
Spacer domain
In some embodiments, the CAR comprises a spacer domain. The spacer domain may be provided between the antigen-binding domain and the transmembrane domain. The spacer domain may also be referred to as a hinge domain. A spacer domain is an amino acid sequence which provides for flexible linkage of the antigen-binding and transmembrane domains of the CAR.
The presence, absence and length of spacer domains has been shown to influence CAR function (reviewed e.g. in Dotti et al., Immunol Rev (2014) 257(1 ) and Jayaraman et al., EBioMedicine (2020) 58:102931 , supra). Spacer domains include those derived from: the CH2-CH3 region of human lgG1 (e.g. as shown in SEQ ID NO:96), the CH2-CH3 region of human IgG 1 (e.g. as shown in SEQ ID NO:98), the CH1 -CH2 hinge region of human IgG 1 , CD8a, e.g. as described in WO 2012/031744 A1 , and CD28, e.g. as described in WO 2011/041093 A1 . Hornbach et al., Gene Therapy (2010) 17:1206-1213 describes a variant hlgG1 CH2-CH3 spacer region for reduced activation of FcyR-expressing cells such as monocytes and NK cells. The amino acid sequence of the variant CH2-CH3 region is shown in SEQ ID NO:97. Spacer domains can lend flexibility to the antigen-binding domain, and can thus enable recognition of target epitopes that would otherwise be sterically inaccessible. For example, in Wilkie et al., J Immunol. (2008) 180:4901 -4909 replacement of a CD28 spacer region with a highly-flexible IgD spacer improved binding of the CAR to a sterically hindered epitope of MUC1 .
Spacer domains can also be engineered to regulate synaptic cleft distances, and hence modulate signalling. To maintain optimal synapse distance, membrane-proximal epitopes typically require shorter spacers, while membrane-distal epitopes require longer spacers (Hudecek et al., Clin Cancer Res. (2013) 19:3153-3164). Increasing epitope-paratope distance permits the interference by inhibitory phosphatases, and can result in impaired delivery of perforins and granzymes to the target cell, thereby reducing the efficiency of cytolysis. A compact immunological synapse impairs diffusion of lytic granules, enhancing pore formation by perforins and delivery of granzymes (Woodsworth et al., Biophys J. (2015) 109:477-488).
Spacer lengths have been found to influence cytolytic activity and signalling by CAR-T cells. Introduction of an IgG 1 -Fc spacer into a first generation anti-CEA CAR was found to reduce secretion of IFNy without comprising lytic efficiency (Guest et al., J Immunother. (2005) 28:203-21 1 ). Spacer length has also been shown to affect mechano-transduction of ligand recognition. Chang et al., Nat Chem Biol. (2018)14:317— 324 found that cells expressing CARs specific for soluble homo-dimeric TGF-0 with longer lgG4-Fc spacers had a decreased activation profile compared to cells expressing CARs having a shorter lgG4 hinge only spacer.
Where T cell activation is too strong, it can impair CAR-T cell persistence and function in vivo due to activation-induced cell death (AICD) (Kuunkele et al., Cancer Immunol. Res. (2015) 3:368-379), and excessive activation of CAR-T cells can moreover contribute to undesirable side effects such as cytokine release syndrome. Spacer domain engineering can be useful to fine-tune activation of CAR-expressing immune cells to an optimal level.
Human IgG 1 Fc, lgG4 Fc and lgG2 Fc spacer domains have been employed in CARs, and variants of IgG 1 Fc and lgG4 Fc spacers designed to reduce/eliminate binding to FcyRs have been shown to reduce depletion of cells expressing the CARs and improve persistence in vivo.
Non-IgG Fc-based spacers such as CD8 and CD28 spacer domains have also been used in clinically- approved CAR-T cell therapies. A CD28-derived spacer has been shown to result in increased CAR activation compared to a CD8a-derived spacer, which might be a consequence of an increased propensity of the CD28-derived spacer to participate in homotypic associations (Alabanza et al., Mol. Ther. (2017) 25:2452-2465).
Spacer domains are also commonly used in the detection of CAR-expressing cells in vitro and in vivo. Fc- specific antibodies can be employed to detect CARs comprising a spacer domain comprising the appropriate Fc region, and specific epitope tags have also been incorporated into CAR spacer domains for such purposes (see e.g. Liu et al., Nat. Biotechnol. (2016) 34:430-434).
In some embodiments, a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence derived from the amino acid sequence of one of 41 BB, 0X40, CD96 CD44, lgG2 or IgG 1 . In some embodiments, a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence derived from the amino acid sequence of one of 41 BB, 0X40, CD96 or CD44. In some embodiments, a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence derived from the amino acid sequence of one of 41 BB.
In some embodiments, a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:96.
In some embodiments, a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:97.
In some embodiments, a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:98.
In some embodiments, a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:99.
In some embodiments, a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NQ:100.
In some embodiments, a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NQ:101 .
In some embodiments, a spacer domain according to the present disclosure comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NQ:102.
Transmembrane domain
The CAR of the present disclosure comprises a transmembrane domain. A transmembrane domain refers to any three-dimensional structure formed by a sequence of amino acids which is thermodynamically stable in a biological membrane, e.g. a cell membrane. In connection with the present disclosure, the transmembrane domain may be an amino acid sequence which spans the cell membrane of a cell expressing the CAR.
The transmembrane domain may comprise or consist of a sequence of amino acids which forms a hydrophobic alpha helix or beta-barrel. The amino acid sequence of the transmembrane domain of the CAR of the present disclosure may be, or may be derived from, the amino acid sequence of a transmembrane domain of a protein comprising a transmembrane domain. Transmembrane domains are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as TMHMM (Krogh et al., 2001 J Mol Biol 305: 567-580).
In some embodiments, the amino acid sequence of the transmembrane domain of the CAR of the present disclosure may be, or may be derived from, the amino acid sequence of the transmembrane domain of a protein expressed at the cell surface. In some embodiments the protein expressed at the cell surface is a receptor or ligand, e.g. an immune receptor or ligand. In some embodiments the amino acid sequence of the transmembrane domain may be, or may be derived from, the amino acid sequence of the transmembrane domain of one of ICOS, ICOSL, CD86, CTLA-4, CD28, CD80, MHC class I a, MHC class II a, MHC class II p, CD3e, CD35, CD3y, CD3 TCRa TCRp, CD4, CD8a, CD8p, CD40, CD40L, PD-1 , PD-L1 , PD-L2, 4-1 BB, 4-1 BBL, 0X40, OX40L, GITR, GITRL, TIM-3, Galectin 9, LAG3, CD27, CD70, LIGHT, HVEM, TIM-4, TIM-1 , ICAM1 , LFA-1 , LFA-3, CD2, BTLA, CD160, LILRB4, LILRB2, VTCN1 , CD2, CD48, 2B4, SLAM, CD30, CD30L, DR3, TL1 A, CD226, CD155, CD112 and CD276. In some embodiments, the transmembrane is, or is derived from, the amino acid sequence of the transmembrane domain of CD28, CD3- , CD8a, CD80 or CD4. In some embodiments, the transmembrane is, or is derived from, the amino acid sequence of the transmembrane domain of CD28.
In some embodiments, the transmembrane domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:103.
In some embodiments, the transmembrane domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:104.
In some embodiments, the transmembrane domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:105.
Signalling domain
The chimeric antigen receptor of the present disclosure comprises a signalling domain. The signalling domain provides sequences for initiating intracellular signalling in cells expressing the CAR. ITAM-containinq sequence:
The signalling domain comprises ITAM-containing sequence. An ITAM-containing sequence comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs comprise the amino acid sequence YXXL/I (SEQ ID NO:109), wherein ‘X’ denotes any amino acid. In ITAM-containing proteins, sequences according to SEQ ID NO:109 are often separated by 6 to 8 amino acids; YXXL/I (X)6-sYXXL/l (SEQ ID NO:110). When phosphate groups are added to the tyrosine residue of an ITAM by tyrosine kinases, a signalling cascade is initiated within the cell.
In some embodiments, the signalling domain comprises one or more copies of an amino acid sequence according to SEQ ID NO:109 or SEQ ID NO:110. In some embodiments, the signalling domain comprises at least 1 , 2, 3, 4, 5 or 6 copies of an amino acid sequence according to SEQ ID NO:109. In some embodiments, the signalling domain comprises at least 1 , 2, or 3 copies of an amino acid sequence according to SEQ ID NO:110.
In some embodiments, the signalling domain comprises an ITAM-containing sequence which is, or which is derived from, the amino acid sequence of an ITAM-containing sequence of a protein having an ITAM- containing amino acid sequence. In some embodiments the signalling domain comprises an ITAM- containing sequence which is, or which is derived from, the amino acid sequence of the intracellular domain of one of CD3 FcyRI, CD3e, CD35, CD3y, CD79a, CD79p, FcyRIIA, FcyRIIC, FcyRIIIA, FcyRIV or DAP12. In some embodiments the signalling domain comprises an ITAM-containing sequence which is, or which is derived from, the amino acid sequence of the intracellular domain of CD3-
In some embodiments, the signalling domain comprises an ITAM-containing sequence which comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:111.
Costimulatory sequence:
The signalling domain may additionally comprise one or more costimulatory sequences. A costimulatory sequence is an amino acid sequence which provides for costimulation of the cell expressing the CAR of the present disclosure. Costimulation promotes proliferation and survival of a CAR-expressing cell upon binding to the target antigen, and may also promote cytokine production, differentiation, cytotoxic function and memory formation by the CAR-expressing cell. Molecular mechanisms of T cell costimulation are reviewed in Chen and Flies, (2013) Nat Rev Immunol 13(4):227-242.
A costimulatory sequence may be, or may be derived from, the amino acid sequence of a costimulatory protein. In some embodiments the costimulatory sequence is an amino acid sequence which is, or which is derived from, the amino acid sequence of the intracellular domain of a costimulatory protein.
Upon binding of the CAR to the target antigen, the costimulatory sequence provides costimulation to the cell expressing the CAR of the kind which would be provided by the costimulatory protein from which the costimulatory sequence is derived upon ligation by its cognate ligand. By way of example in the case of a CAR comprising a signalling domain comprising a costimulatory sequence derived from CD28, binding to the target antigen triggers signalling in the cell expressing the CAR of the kind that would be triggered by binding of CD80 and/or CD86 to CD28. Thus, a costimulatory sequence is capable of delivering the costimulation signal of the costimulatory protein from which the costimulatory sequence is derived.
In some embodiments, the costimulatory protein may be a member of the B7-CD28 superfamily (e.g. CD28, ICOS), or a member of the TNF receptor superfamily (e.g. 4-1 BB, 0X40, CD27, DR3, GITR, CD30, HVEM). In some embodiments, the costimulatory sequence is, or is derived from, the intracellular domain of one of CD28, 4-1 BB, ICOS, CD27, 0X40, HVEM, CD2, SLAM, TIM-1 , CD30, GITR, DR3, CD226 and LIGHT. In some embodiments, the costimulatory sequence is, or is derived from, the intracellular domain of CD28.
In some embodiments the signalling domain comprises more than one costimulatory sequence. In some embodiments the signalling domain comprises 1 , 2, 3, 4, 5 or 6 costimulatory sequences. Plural costimulatory sequences may be provided in tandem.
Whether a given amino acid sequence is capable of initiating signalling mediated by a given costimulatory protein can be investigated e.g. by analysing a correlate of signalling mediated by the costimulatory protein (e.g. expression/activity of a factor whose expression/activity is upregulated or downregulated as a consequence of signalling mediated by the costimulatory protein).
Costimulatory proteins upregulate expression of genes promoting cell growth, effector function and survival through several transduction pathways. For example, CD28 and ICOS signal through phosphatidylinositol 3 kinase (PI3K) and AKT to upregulate expression of genes promoting cell growth, effector function and survival through NF-KB, mTOR, NFAT and AP1/2. CD28 also activates AP1/2 via CDC42/RAC1 and ERK1 Z2 via RAS, and ICOS activates C-MAF. 4-1 BB, 0X40, and CD27 recruit TNF receptor associated factor (TRAF) and signal through MAPK pathways, as well as through PI3K.
In some embodiments the signalling domain comprises a costimulatory sequence which is, or which is derived from CD28.
Kofler et al. Mol. Ther. (2011 ) 19: 760-767 describes a variant CD28 intracellular domain in which the lek kinase binding site is mutated in order to reduce induction of IL-2 production on CAR ligation, in order to minimise regulatory T cell-mediated suppression of CAR-T cell activity. The amino acid sequence of the variant CD28 intracellular domain is shown in SEQ ID NO:107.
In some embodiments, the signalling domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:106. In some embodiments, the signalling domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NQ:107. In some embodiments, the signalling domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:108.
In some embodiments, the signalling domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:115.
In some embodiments, the CAR comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:116.
Linkers and additional sequences
The CARs of the present disclosure may additionally comprise further amino acids or sequences of amino acids.
An amino acid sequence described herein (e.g. an amino acid sequence of a domain/region of a CAR described herein) may comprise one or more additional amino acids at one or both ends (i.e. the N- or C- terminus) of the reference amino acid sequence. In some embodiments, the peptide/polypeptide comprises e.g. 1 -5, 1 -10, 1 -20, 1 -30, 1 -40, 1 -50, 5-10, 5-20, 5-30, 5-40, 5-50, 10-20, 10-30, 10-40, 10-50, 20-30, 20-40 or 20-50 additional amino acids at one or both ends of the reference amino acid sequence. In some embodiments, the additional amino acids correspond to the amino acids provided at those positions relative to the reference amino acid sequence, in the context of the protein from which the reference amino acid sequence is derived. By way of illustration, where a reference amino acid sequence corresponds to amino acid positions 20 to 30 of the amino acid sequence of the protein from which the reference amino acid sequence is derived, and wherein the amino acid sequence an additional 5 amino acids at its N-terminus, those additional 5 amino acids may correspond to positions 15 to 19 of the amino acid sequence of the protein from which the reference amino acid sequence is derived.
The CARs of the present disclosure may comprise one or more linker sequences between sequences of amino acids. For example, a linker sequence may be provided between domains of a CAR (e.g. between the antigen-binding domain and spacer domain, and/or between the spacer domain and the transmembrane domain, and/or between the transmembrane domain and the signalling domain). By way of further example, a linker sequence may be provided between subsequences of the domains of a CAR (e.g. between VH and VL of an antigen-binding domain, and/or between the costimulatory and sequence and ITAM-containing sequence of a signalling domain).
Linker sequences are known to the skilled person, and are described, for example in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, a linker sequence may be a flexible linker sequence. Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence. Flexible linkers are known to the skilled person, and several are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and/or serine residues.
In some embodiments, the linker sequence comprises at least one glycine residue and/or at least one serine residue. In some embodiments, the linker sequence comprises or consists of glycine and serine residues. In some embodiments, the linker sequence has the structure: (GxS)n or (GxS)nGm; wherein G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5 or 6, and m = 0, 1 , 2 or 3. In some embodiments, the linker sequence comprises one or more (e.g. 1 , 2, 3, 4, 5 or 6) copies (e.g. in tandem) of the sequence motif G4S. In some embodiments, the linker sequence comprises or consists of (G4S)4 or (G4S)e. In some embodiments, the linker sequence has a length of 1 -2, 1 -3, 1 -4, 1 -5, 1 -10, 1 -15, 1 -20, 1 -25, or 1 -30 amino acids.
In some embodiments, the linker sequence comprises one or more copies of an amino acid sequence according to SEQ ID NO:57. In some embodiments, the linker sequence comprises at least 1 , 2, 3 or 4 copies of an amino acid sequence according to SEQ ID NO:57.
In some embodiments, the linker sequence comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:58.
The CARs of the present disclosure may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification or detection of the antigen-binding molecule/polypeptide. For example, antigen-binding molecules and polypeptides of the present disclosure may additionally comprise a sequence of amino acids forming a detectable moiety, e.g. as described hereinbelow.
The CARs of the present disclosure may additionally comprise a signal peptide (also known as a leader sequence or signal sequence). Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides. Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt and Ensembl, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).
The signal peptide may be present at the N-terminus of the CAR, and may be present in the newly synthesised CAR. The signal peptide provides for efficient trafficking of the CAR to the cell surface. Signal peptides are often removed by cleavage, and thus are not comprised in the mature CAR expressed at the cell surface.
Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and/or can be identified/predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 201 1 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176).
In some embodiments, the signal peptide comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:1 12. In some embodiments, the signal peptide comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:1 13. In some embodiments, the signal peptide comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:1 14.
Labels and conjugates
In some embodiments, the CAR of the present disclosure additionally comprise a detectable moiety.
In some embodiments, a detectable moiety is a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label (e.g. an epitope tag), radiolabel, chemical, nucleic acid or enzymatic label. The CAR may be covalently or non-covalently labelled with the detectable moiety.
Fluorescent labels include e.g. fluorescein, rhodamine, allophycocyanin, eosine and NDB, green fluorescent protein (GFP), chelates of rare earths such as europium (Eu), terbium (Tb) and samarium (Sm), tetramethyl rhodamine, Texas Red, 4-methyl umbelliferone, 7-amino-4-methyl coumarin, Cy3, and Cy5. Radiolabels include radioisotopes such as Hydrogen3, Sulfur35, Carbon14, Phosphorus32, Iodine123, Iodine125, Iodine126, Iodine131 , Iodine133, Bromine77, Technetiurn99m, Indium111 , Indium113"1, Gallium67, Gallium68, Ruthenium95, Ruthenium97, Ruthenium103, Ruthenium105, Mercury207, Mercury203, Rhenium99m, Rhenium101 , Rhenium105, Scandium47, Tellurium121"1, Tellurium122"1, Tellurium125"1, Thulium165, Thuliuml167, Thulium168, Copper67, Fluorine18, Yttrium90, Palladium100, Bismuth217 and Antimony211. Luminescent labels include as radioluminescent, chemiluminescent (e.g. acridinium ester, luminol, isoluminol) and bioluminescent labels. Immuno-detectable labels include haptens, peptides/polypeptides, antibodies, receptors and ligands such as biotin, avidin, streptavidin or digoxigenin. Nucleic acid labels include aptamers.
In some embodiments, the CAR comprises an epitope tag, e.g. a His, (e.g. 6XHis), FLAG, c-Myc, StrepTag, haemagglutinin, E, calmodulin-binding protein (CBP), glutathione-s-transferase (GST), maltose-binding protein (MBP), thioredoxin, S-peptide, T7 peptide, SH2 domain, avidin, streptavidin, and haptens (e.g. biotin, digoxigenin, dinitrophenol), optionally at the N- or C- terminus of the antigen-binding molecule/polypeptide/CAR.
In some embodiments, the CAR comprises a moiety having a detectable activity, e.g. an enzymatic moiety. Enzymatic moieties include e.g. luciferases, glucose oxidases, galactosidases (e.g. beta- galactosidase), glucorinidases, phosphatases (e.g. alkaline phosphatase), peroxidases (e.g. horseradish peroxidase) and cholinesterases.
In some embodiments, the CAR of the present disclosure is conjugated to a chemical moiety. The chemical moiety may be a moiety for providing a therapeutic effect, i.e. a drug moiety. A drug moiety may be a small molecule (e.g. a low molecular weight (< 1000 daltons, typically between -300-700 daltons) organic compound). Drug moieties are described e.g. in Parslow et al., Biomedicines. 2016 Sep; 4(3):14 (hereby incorporated by reference in its entirety). In some embodiments, a drug moiety may be or comprise a cytotoxic agent. In some embodiments, a drug moiety may be or comprise a chemotherapeutic agent. Drug moieties include e.g. calicheamicin, DM1 , DM4, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5 and PBD.
Particular exemplary CARs
In some embodiments, the CAR according to the present disclosure comprises, or consists of:
An antigen-binding domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 or 182;
A spacer domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:100, 98, 99, 101 , or 102;
A transmembrane domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:103, 104 or 105; and A signalling domain comprising:
A costimulatory sequence comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NQ:106, 107 or 108; and
An ITAM-containing sequence comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:1 1 1 .
In some embodiments, the CAR comprises, or consists of:
An antigen-binding domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 or 182;
A spacer domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:100, 98, 99, 101 , or 102;
A transmembrane domain comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:103; and
A signalling domain comprising:
A costimulatory sequence comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NQ:106; and
An ITAM-containing sequence comprising or consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:111.
In some embodiments, the CAR comprises, or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:117, 118, 119, 120, 121 ,
122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142,
143, 144, 145, 146, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199,
200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220,
221 , 222, 223 or 224.
In some embodiments, the CAR comprises, or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:117, 118, 119, 120, 121 ,
122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142,
143, 144, 145, 146, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199,
200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 or 212.
In some embodiments, the CAR comprises, or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:119, 124, 129, 134, 139,
144, 185, 190, 195, 200, 205 or 210.
In some embodiments, the CAR comprises, or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:124, 144, 190 or 210. Nucleic acids and vectors
The present disclosure provides a nucleic acid, or a plurality of nucleic acids, encoding a CAR according to the present disclosure. In some embodiments, the nucleic acid(s) comprise or consist of DNA and/or RNA.
In some embodiments, the nucleic acid(s) may be, or may be comprised in, a vector, or a plurality of vectors. That is, the nucleotide sequence(s) of the nucleic acid(s) may be contained in vector(s). The CAR according to the present disclosure may be produced within a cell by transcription from a vector encoding the antigen-binding molecule, polypeptide or CAR, and subsequent translation of the transcribed RNA.
Accordingly, the present disclosure also provides a vector, or plurality of vectors, comprising the nucleic acid or plurality of nucleic acids according to the present disclosure. The vector may facilitate delivery of the nucleic acid(s) encoding an CAR according to the present disclosure. The vector may be an expression vector comprising elements required for expressing nucleic acid(s) comprising/encoding an CAR according to the present disclosure.
Nucleic acids and vectors according to the present disclosure may be provided in purified or isolated form, i.e. from other nucleic acid, or naturally-occurring biological material.
The nucleotide sequence may be contained in a vector, e.g. an expression vector. A ‘vector’ as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the cell. Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.
The term ‘operably linked’ may include the situation where a selected nucleic acid sequence and regulatory nucleic acid sequence (e.g. promoter and/or enhancer) are covalently linked in such a way as to place the expression of nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcript(s) may then be translated into a desired peptide(s)/polypeptide(s).
Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g. retroviral vectors, e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors, e.g. SFG vector), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes), e.g. as described in Maus et al., Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines (2016) 4:9, which are both hereby incorporated by reference in their entirety. In some embodiments, the vector may be a eukaryotic vector, e.g. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.
Cells expressing the CARs of the disclosure
In some aspects and embodiments, the present disclosure provides a cell comprising a CAR according to the present disclosure. The CAR according to the present disclosure may be used to generate a CAR- expressing cell, e.g. a CAR-expressing immune cells(e.cj. CAR-T or CAR-NK cell).
It will be appreciated that where cells are referred to herein in the singular (i.e. ‘a/the cell’), pluralities/populations of such cells are also contemplated.
CAR-expressing cells may comprise or express nucleic acid encoding a CAR according to the present disclosure. It will be appreciated that a CAR-expressing cell comprises the CAR it expresses. It will also be appreciated that a cell expressing nucleic acid encoding a CAR also expresses and comprises the CAR encoded by the nucleic acid.
The cell may be a eukaryotic cell, e.g. a mammalian cell. The mammal may be a primate (rhesus, cynomolgous, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate).
CAR-expressing cell is preferably an immune cell. An immune cell may be a cell of hematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. A lymphocyte may be e.g. a T cell, B cell, NK cell, NKT cell or innate lymphoid cell (ILC), or a precursor thereof. The immune cell may express e.g. CD3 polypeptides (e.g. CD3y CD3e CD3 or CD35), TCR polypeptides (TCRa or TCR0), CD27, CD28, CD4 or CD8. In some embodiments, the immune cell is a T cell, e.g. a CD3+ T cell. In some embodiments, the T cell is a CD3+, CD4+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a T helper cell (TH cell). In some embodiments, the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)).
Aspects and embodiments of the present disclosure relate particularly to T cells comprising/expressing CD30-specific CARs according to the present disclosure.
In some aspects and embodiments, the immune cell may be a virus-specific immune cell. A ‘virus-specific immune cell’ as used herein refers to an immune cell which is specific for a virus. A virus-specific immune cell expresses/comprises a receptor (preferably a T cell receptor) capable of recognising a peptide of an antigen of a virus (e.g. when presented by an MHC molecule). The virus-specific immune cell may express/comprise such a receptor as a result of expression of endogenous nucleic acid encoding such antigen receptor, or as a result of having been engineered to express such a receptor. The virus-specific immune cell preferably expresses/comprises a TCR specific for a peptide of an antigen of a virus. A virusspecific T cell may display certain functional properties of a T cell in response to the viral antigen for which the T cell is specific, or in response a cell comprising/expressing the virus/antigen. In some embodiments, the properties are functional properties associated with effector T cells, e.g. cytotoxic T cells.
In some embodiments, a virus-specific T cell may display one or more of the following properties: cytotoxicity to a cell comprising/expressing the virus /the viral antigen for which the T cell is specific; proliferation, IFNy expression, CD107a expression, IL-2 expression, TNFa expression, perforin expression, granzyme expression, granulysin expression, and/or FAS ligand (FASL) expression in response to stimulation with the virus/the viral antigen for which the T cell is specific, or in response to exposure to a cell comprising/expressing the virus /the viral antigen for which the T cell is specific.
Virus-specific T cells express/comprise a TCR capable of recognising a peptide of the viral antigen for which the T cell is specific when presented by the appropriate MHC molecule. Virus-specific T cells may be CD4+ T cells and/or CD8+ T cells.
The virus for which the virus-specific immune cell is specific may be any virus. For example, the virus may be a dsDNA virus (e.g. adenovirus, herpesvirus, poxvirus), ssRNA virus (e.g. parvovirus), dsRNA virus (e.g. reovirus), (+)ssRNA virus (e.g. picornavirus, togavirus), (-)ssRNA virus (e.g. orthomyxovirus, rhabdovirus), ssRNA-RT virus (e.g. retrovirus) or dsDNA-RT virus (e.g. hepadnavirus). In particular, the present disclosure contemplates viruses of the families adenoviridae, herpesviridae, papillomaviridae, polyomaviridae, poxviridae, hepadnaviridae, parvoviridae, astroviridae, caliciviridae, picornaviridae, coronaviridae, flaviviridae, togaviridae, hepeviridae, retroviridae, orthomyxoviridae, arenaviridae, bunyaviridae, filoviridae, paramyxoviridae, rhabdoviridae and reoviridae. In some embodiments the virus is selected from Epstein-Barr virus, adenovirus, Herpes simplex type 1 virus, Herpes simplex type 2 virus, Varicella-zoster virus, Human cytomegalovirus, Human herpesvirus type 8, Human papillomavirus, BK virus, JC virus, Smallpox, Hepatitis B virus, Parvovirus B19, Human Astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, TBE virus, Rubella virus, Hepatitis E virus, Human immunodeficiency virus, influenza virus, lassa virus, Crimean-Congo hemorrhagic fever virus, Hantaan virus, ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, picornavirus, respiratory syncytial virus, rabies virus, hepatitis D virus, rotavirus, orbivirus, coltivirus, and banna virus.
In some embodiments, the virus is selected from Epstein-Barr virus (EBV), adenovirus, cytomegalovius (CMV), human papilloma virus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), or herpes simplex virus (HSV).
In some embodiments, the virus-specific immune cell may be specific for a peptide/polypeptide of a virus e.g. selected from Epstein-Barr virus (EBV), adenovirus, cytomegalovius (CMV), human papilloma virus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), or herpes simplex virus (HSV).
A T cell which is specific for an antigen of a virus may be referred to herein as a virus-specific T cell (VST). A T cell which is specific for an antigen of a particular virus may be described as being specific for the relevant virus; for example, a T cell which is specific for an antigen of EBV may be referred to as an EBV-specific T cell, or ‘EBVST’.
Accordingly, in some embodiments the virus-specific immune cell is an Epstein-Barr virus-specific T cell (EBVST), adenovirus-specific T cell (AdVST), cytomegalovius-specific T cell (CMVST), human papilloma virus (HPVST), influenza virus-specific T cell, measles virus-specific T cell, hepatitis B virus-specific T cell (HBVST), hepatitis C virus-specific T cell (HCVST), human immunodeficiency virus-specific T cell (HIVST), lymphocytic choriomeningitis virus-specific T cell (LCMVST), or herpes simplex virus-specific T cell (HSVST).
In some preferred embodiments, the virus-specific immune cell is specific for a peptide/polypeptide of an EBV antigen. In preferred embodiments the virus-specific immune cell is an Epstein-Barr virus-specific T cell (EBVST).
EBV virology is described e.g. in Stanfield and Luftiq, FI OOORes. (2017) 6:386 and Odumade et al., Clin Microbiol Rev (2011 ) 24(1 ):193-209, both of which are hereby incorporated by reference in their entirety.
EBV infects epithelial cells via binding of viral protein BMFR2 to pi integrins, and binding of viral protein gH/gL with integrins av06 and av08. EBV infects B cells through interaction of viral glycoprotein gp350 with CD21 and/or CD35, followed by interaction of viral gp42 with MHC class II. These interactions trigger fusion of the viral envelope with the cell membrane, allowing the virus to enter the cell. Once inside, the viral capsid dissolves and the viral genome is transported to the nucleus.
EBV has two modes of replication; latent and lytic. The latent cycle does not result in production of virions, and can take place in place B cells and epithelial cells. The EBV genomic circular DNA resides in the cell nucleus as an episome and is copied by the host cell’s DNA polymerase. In latency, only a fraction of EBV's genes are expressed, in one of three different patterns known as latency programs, which produce distinct sets of viral proteins and RNAs. The latent cycle is described e.g. in Amon and Farrell, Reviews in Medical Virology (2004) 15(3): 149-56, which is hereby incorporated by reference in its entirety.
EBNA1 protein and non-coding RNA EBER are expressed in each of latency programs l-lll. Latency programs II and III further involve expression of EBNALP, LMP1 , LMP2A and LMP2B proteins, and latency program III further involves expression of EBNA2, EBNA3A, EBNA3B and EBNA3C.
EBNA1 is multifunctional, and has roles in gene regulation, extrachromosomal replication, and maintenance of the EBV episomal genome through positive and negative regulation of viral promoters (Duellman et al., J Gen Virol. (2009); 90(Pt 9): 2251-2259). EBNA2 is involved in the regulation of latent viral transcription and contributes to the immortalisation of cells infected with EBV (Kempkes and Ling, Curr Top Microbiol Immunol. (2015) 391 :35-59). EBNA-LP is required for transformation of native B cells, and recruits transcription factors for viral replication (Szymula et al., PLoS Pathog.
(2018);14(2):e1006890). EBNA3A, 3B and 3C interact with RBPJ to influence gene expression, contributing to survival and growth of infected cells (Wang et al., J Virol. (2016) 90(6):2906-2919). LMP1 regulates expression of genes involved in B cell activation (Chang et al., J. Biomed. Sci. (2003) 10(5): 490-504). LMP2A and LMP2B inhibit normal B cell signal transduction by mimicking the activated B cell receptor (Portis and Longnecker, Oncogene (2004) 23(53): 8619-8628). EBERs form ribonucleoprotein complexes with host cell proteins, and are proposed to have roles in cell transformation.
The latent cycle can progress according to any of latency programs I to III in B cells, and usually progresses from III to II to I. Upon infection of a resting naive B cell, EBV enters latency program III. Expression of latency III genes activates the B cell, which becomes a proliferating blast. EBV then typically progresses to latency II by restricting expression to a subset of genes, which cause differentiation of the blast to a memory B cell. Further restriction of gene expression causes EBV to enter latency I. EBNA1 expression allows EBV to replicate when the memory B cell divides. In epithelial cells, only latency II occurs.
In primary infection, EBV replicates in oropharyngeal epithelial cells and establishes Latency III, II, and I infections in B-lymphocytes. EBV latent infection of B-lymphocytes is necessary for virus persistence, subsequent replication in epithelial cells, and release of infectious virus into saliva. EBV Latency III and II infections of B-lymphocytes, Latency II infection of oral epithelial cells, and Latency II infection of NK- or T cell can result in malignancies, marked by uniform EBV genome presence and gene expression.
Latent EBV in B cells can be reactivated to switch to lytic replication. The lytic cycle results in the production of infectious virions and can take place in place B cells and epithelial cells, and is reviewed e.g. by Kenney in Chapter 25 of Arvin et al., Human Herpesviruses: Biology, Therapy and Immunoprophylaxis; Cambridge University Press (2007), which is hereby incorporated by reference in its entirety.
Lytic replication requires the EBV genome to be linear. The latent EBV genome is episomal, and so it must be linearised for lytic reactivation. In B cells, lytic replication normally only takes place after reactivation from latency.
Immediate-early lytic gene products such as BZFL1 and BRLF1 act as transactivators, enhancing their own expression, and the expression of later lytic cycle genes.
Early lytic gene products have roles in viral replication (e.g. EBV DNA polymerase catalytic component BALF5; DNA polymerase processivity factor BMRF1 , DNA binding protein BALF2, helicase BBLF4, primase BSLF1 , and primase-associated protein BBLF2/3) and deoxynucleotide metabolism (e.g. thymidine kinase BXLF1 , dUTPase BORF2). Other early lytic gene products act transcription factors (e.g. BMRF1 , BRRF1 ), have roles in RNA stability and processing (e.g. BMLF1 ), or are involved in immune evasion (e.g. BHRF1 , which inhibits apoptosis).
Late lytic gene products are traditionally classed as those expressed after the onset of viral replication. They generally encode structural components of the virion such as nucleocapsid proteins, as well as glycoproteins which mediate EBV binding and fusion (e.g. gp350/220, gp85, gp42, gp25). Other late lytic gene products have roles in immune evasion; BCLF1 encodes a viral homologue of IL-10, and BALF1 encodes a protein with homology to the anti-apoptotic protein Bcl2.
An ‘EBV-specific immune cell’ as used herein refers to an immune cell which is specific for Epstein-Barr virus (EBV). An EBV-specific immune cell expresses/comprises a receptor (preferably a T cell receptor) capable of recognising a peptide of an antigen of EBV (e.g. when presented by an MHC molecule). The EBV-specific immune cell preferably expresses/comprises a TCR specific for a peptide of an EBV antigen presented by MHC class I.
In some embodiments, the EBV-specific immune cell is a T cell, e.g. a CD3+ T cell. In some embodiments, the T cell is a CD3+, CD4+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a T helper cell (TH cell)). In some embodiments, the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)).
EBV-specific T cells preferably express/comprise a TCR capable of recognising a peptide of the EBV antigen for which the T cell is specific when presented by the appropriate MHC molecule. EBV-specific T cells may be CD4+ T cells and/or CD8+ T cells.
An immune cell specific for EBV may be specific for any EBV antigen, e.g. an EBV antigen described herein. A population of immune cell specific for EBV, or a composition comprising a plurality of immune cells specific for EBV, may comprise immune cells specific for one or more EBV antigens.
In some embodiments, an EBV antigen is an EBV latent antigen, e.g. a type III latency antigen (e.g. EBNA1 , EBNA-LP, LMP1 , LMP2A, LMP2B, BARF1 , EBNA2, EBNA3A, EBNA3B or EBNA3C), a type II latency antigen (e.g. EBNA1 , EBNA-LP, LMP1 , LMP2A, LMP2B or BARF1 ), or a type I latency antigen, (e.g. EBNA1 or BARF1 ). In some embodiments, an EBV antigen is an EBV lytic antigen, e.g. an immediate-early lytic antigen (e.g. BZLF1 , BRLF1 or BMRF1 ), an early lytic antigen (e.g. BMLF1 , BMRF1 , BXLF1 , BALF1 , BALF2, BARF1 , BGLF5, BHRF1 , BNLF2A, BNLF2B, BHLF1 , BLLF2, BKRF4, BMRF2, FU or EBNA1 -FUK), or a late lytic antigen (e.g. BALF4, BILF1 , BILF2, BNFR1 , BVRF2, BALF3, BALF5, BDLF3 or gp350).
In some embodiments in accordance with the various aspects of the present disclosure, cells may comprise/express more than one (e.g. 2, 3, 4, etc.) CAR.
In some embodiments, the cells may comprise/express more than one, non-identical CAR. Cells comprising/expressing more than one non-identical CAR may comprise/express CARs specific for non- identical target antigens. In some embodiments, each non-identical target antigen is independently a cancer cell antigen as described herein.
Functional properties of cells expressing the CARs of the disclosure
Cells (e.g. immune cells, e.g. T cells) expressing a CAR according to the present disclosure may display certain functional properties in response to the target antigen for the CAR (e.g. CD30), or in response a cell comprising/expressing the target antigen for the CAR. In some embodiments, the properties are functional properties associated with effector T cells, e.g. cytotoxic T cells.
Cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure may display one or more of the following properties: expression of one or more cytotoxic/effector factors (e.g. IFNy, TNFa, GM-CSF), proliferation/population expansion, and/or growth factor (e.g. IL-2) expression in response to cells expressing the target antigen for the CAR (e.g. CD30); cytotoxicity to cells expressing the target antigen for the CAR (e.g. CD30); no cytotoxicity (i.e. above baseline) to cells which do not express the target antigen for the CAR (e.g. CD30); anti-cancer activity (e.g. cytotoxicity to cancer cells, tumor growth inhibition, reduction of metastasis, etc.) against cancer comprising cells expressing the target antigen for the CAR (e.g. CD30); cytotoxicity to alloreactive immune cells, e.g. alloreactive immune cells expressing the target antigen for the CAR (e.g. CD30).
In some embodiments, CAR-expressing T cell according to the present disclosure may display one or more of the following properties: cytotoxicity to a cell comprising/expressing the target antigen for the CAR; proliferation, IFNy expression, CD107a expression, IL-2 expression, TNFa expression, perforin expression, granzyme expression, granulysin expression, and/or FAS ligand (FASL) expression in response to stimulation with the target antigen for the CAR, or in response to exposure to a cell comprising/expressing the target antigen for the CAR; anti-cancer activity (e.g. cytotoxicity to cancer cells, tumor growth inhibition, reduction of metastasis, etc.) against cancer comprising cells expressing the target antigen for the CAR.
Cell proliferation/population expansion can be investigated by analysing cell division or the number of cells over a period of time. Cell division can be analysed, for example, by in vitro analysis of incorporation of 3H-thymidine or by CFSE dilution assay, e.g. as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564, hereby incorporated by reference in entirety. Proliferating cells can also be identified by analysis of incorporation of 5-ethynyl-2'-deoxyuridine (EdU) by an appropriate assay, as described e.g. in Buck et al., Biotechniques. 2008 Jun; 44(7):927-9, and Sali and Mitchison, PNAS USA 2008 Feb 19; 105(7): 2415-2420, both hereby incorporated by reference in their entirety.
As used herein, ‘expression’ may be gene or protein expression. Gene expression encompasses transcription of DNA to RNA, and can be measured by various means known to those skilled in the art, for example by measuring levels of mRNA by quantitative real-time PCR (qRT-PCR), or by reporterbased methods. Similarly, protein expression can be measured by various methods well known in the art, e.g. by antibody-based methods, for example by western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or reporter-based methods.
Cytotoxicity and cell killing can be investigated, for example, using any of the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (2011 ), 9(6):601 -616, hereby incorporated by reference in its entirety. Examples of in vitro assays of cytotoxicity/cell killing assays include release assays such as the 51Cr release assay, the lactate dehydrogenase (LDH) release assay, the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) release assay, and the calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cell killing based on the detection of factors released from lysed cells. Cell killing of a given test cell type by a given effector immune cell type can be analysed e.g. by co-culturing the test cells with the effector immune cells, and measuring the number/proportion of viable/dead (e.g. lysed) test cells after a suitable period of time. Other suitable assays include the xCELLigence real-time cytolytic in vitro potency assay described in Cerignoli et al., PLoS One. (2018) 13(3): e0193498 (hereby incorporated by reference in its entirety).
In some embodiments, cell killing of cells expressing the target antigen for the CAR by CAR-expressing cells may be evaluated by xCELLigence assay as described in Example 1 .5 herein. Cell killing by CAR- expressing cells can also be evaluated in vivo, e.g. by evaluating the number/proportion of cells expressing the target antigen for the CAR, and inferring their kil ling/depletion by CAR-expressing cells.
Cells may be evaluated for anti-cancer activity by analysis in an appropriate in vitro assays or in vivo models of the relevant cancer.
Cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure preferably possess novel and/or improved properties compared to cells comprising/comprising nucleic acid encoding a known CAR, e.g. a CAR comprising a known spacer domain.
In particular, the cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure preferably possess novel and/or improved properties compared to cells comprising a CAR/nucleic acid encoding an equivalent CAR construct identical in all respects, with the exception that the spacer domain consists of the sequence shown in SEQ ID NO:96, 97 or 98. By way of illustration, the CAR construct of SEQ ID NO:213 is identical to that of SEQ ID NO:119, with the exception that the CAR of SEQ ID NO:213 comprises the human IgG 1 CH2-CH3 spacer domain shown in SEQ ID NO:96, whereas the CAR of SEQ ID NO:119 comprises the 41 BB spacer domain shown in SEQ ID NO:100. In preferred embodiments, the cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure possess novel and/or improved properties compared to cells comprising a CAR/nucleic acid encoding an equivalent CAR construct identical in all respects, with the exception that the spacer domain consists of the sequence shown in SEQ ID NO:96. For conciseness, in the following paragraphs, such reference CARs comprising the human IgG 1 CH2- CH3 spacer domain may be referred to simply as ‘an equivalent CAR comprising the hlgG 1 spacer’.
In some embodiments, cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure: expand and/or proliferate to an extent which is similar to, or greater than, the extent to which cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer expand/proliferate; express cytotoxic/effector factors (e.g. IFNy, TNFa, GM-CSF) in response to stimulation with cells expressing the target antigen for the CAR to an extent which is similar to, or greater than, the extent to which cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer express such factors; kill cells expressing the target antigen for the CAR with potency/rate which is similar to, or greater than, the potency/rate of killing of such cells by cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG1 spacer; display off-target cytotoxicity (e.g. to NK cells) which is similar to, or less than, the off-target cytotoxicity displayed by cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG1 spacer; induce systemic inflammation to a recipient subject to an extent which is similar to, or less than, the systemic inflammation induced by administration of cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer; inhibit tumor growth, e.g. of a cancer expressing the target antigen for the CAR with a potency/to an extent which is similar to, or greater than, tumor growth inhibition by cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG1 spacer; increase survival of subjects having a cancer, e.g. a cancer expressing the target antigen for the CAR to an extent which is similar to, or greater than, the extent to which survival is increased by cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the h IgG 1 spacer; display persistence in vivo which is similar to, or greater than, the in vivo persistence of cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer.
Cell proliferation and cell population expansion can be measured as described hereinabove. The rate of cell proliferation/population expansion for a given cell type can be determined by analysing the number of such cells over time, e.g. at different time points. Cell proliferation/population expansion may be measured in vitro, or alternatively in vivo, e.g. following administration to a subject (e.g. a subject having a cancer expressing the target antigen for the cancer).
In some embodiments, the rate of cell proliferation or population expansion of cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure (e.g. in vitro or in vivo) is > 0.5 times and < 2 times, e.g. one of > 0.55 times and < 1 .9 times, > 0.6 times and < 1 .8 times, > 0.65 times and < 1 .7 times, > 0.7 times and < 1 .6 times, > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .4 times, > 0.85 times and < 1 .3 times, > 0.9 times and < 1 .2 times, > 0.95 times and < 1.1 times the rate of cell proliferation/population expansion of cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer, as determined in a given assay. In some embodiments, the rate of cell proliferation/population expansion of cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure (e.g. in vitro or in vivo) is greater than 1 times, e.g. one of >1 .01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1 .1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the rate of cell proliferation/population expansion of cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer, as determined in a given assay.
In some embodiments, the level of expression of one or more cytotoxic/effector factors (e.g. IFNy, TNFa, GM-CSF) by cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure in response to stimulation with cells expressing the target antigen for the CAR is > 0.5 times and < 2 times, e.g. one of > 0.55 times and < 1 .9 times, > 0.6 times and < 1 .8 times, > 0.65 times and < 1 .7 times, > 0.7 times and < 1 .6 times, > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .4 times, > 0.85 times and < 1 .3 times, > 0.9 times and < 1 .2 times, > 0.95 times and < 1 .1 times the level of expression of such factor(s) by cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer in response to stimulation with cells expressing the target antigen for the CAR, as determined in a given assay. In some embodiments, the level of expression of one or more cytotoxic/effector factors (e.g. IFNy, TNFa, GM-CSF) by cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure in response to stimulation with cells expressing the target antigen for the CAR is greater than 1 times, e.g. one of >1 .01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1 .1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the level of expression of such factor(s) by cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer in response to stimulation with cells expressing the target antigen for the CAR, as determined in a given assay.
In some embodiments, the potency or rate of cell killing of cells expressing the target antigen for the CAR by cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure (e.g. in vitro or in vivo) is > 0.5 times and < 2 times, e.g. one of > 0.55 times and < 1 .9 times, > 0.6 times and < 1 .8 times, > 0.65 times and < 1 .7 times, > 0.7 times and < 1 .6 times, > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .4 times, > 0.85 times and < 1 .3 times, > 0.9 times and < 1 .2 times, > 0.95 times and < 1.1 times the potency or rate of cell killing of such cells by cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer, as determined in a given assay. In some embodiments, the potency or rate of cell killing of cells expressing the target antigen for the CAR by cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure (e.g. in vitro or in vivo) is greater than 1 times, e.g. one of >1 .01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1 .1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the potency or rate of cell killing of such cells by cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG1 spacer, as determined in a given assay. In some embodiments, the cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure induce cell killing of cells not expressing the target antigen for the CAR to an extent which is similar to or less than the extent to which cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer induce cell killing of cells not expressing the target antigen for the CAR.
In some embodiments, the cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure induce cell killing of cells not expressing the target antigen for the CAR (e.g. in vitro or in vivo) to a level which is > 0.5 times and < 2 times, e.g. one of > 0.55 times and < 1 .9 times, > 0.6 times and < 1 .8 times, > 0.65 times and < 1 .7 times, > 0.7 times and < 1 .6 times, > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .4 times, > 0.85 times and < 1 .3 times, > 0.9 times and < 1 .2 times, > 0.95 times and < 1.1 times the level of cell killing of such cells by cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer, as determined in a given assay. In some embodiments, the cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure induce cell killing of cells not expressing the target antigen for the CAR (e.g. in vitro or in vivo) to a level which is less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level of cell killing of such cells by cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer, as determined in a given assay.
In some embodiments, cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure induce systemic inflammation (e.g. cytokine release syndrome) in a receipt subject of such cells to an extent which is similar to, or less than, that of cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer. Induction of systemic inflammation by CAR-expressing cells in a recipient subject can be evaluated by measuring one or more markers of systemic inflammation in a recipient subject following administration of such cells. Markers of systemic inflammation include e.g. levels of proinflammatory cytokines (e.g. IL-6, IL-8 and TNFa and GM-CSF) in the peripheral blood. In some embodiments, cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure induce the expression of one or more proinflammatory cytokines (e.g. selected from IL-6, IL-8 and TNFa and GM-CSF) to an extent which is similar to, or less than, that of cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer.
In some embodiments, the cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure induce the expression of one or more proinflammatory cytokines (e.g. selected from IL-6, IL-8 and TNFa and GM-CSF) to a level which is > 0.5 times and < 2 times, e.g. one of > 0.55 times and < 1.9 times, > 0.6 times and < 1 .8 times, > 0.65 times and < 1 .7 times, > 0.7 times and < 1 .6 times, > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .4 times, > 0.85 times and < 1 .3 times, > 0.9 times and < 1 .2 times, > 0.95 times and < 1.1 times the level to which their expression is induced by cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer, as determined in a given assay. In some embodiments, the cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure induce the expression of one or more proinflammatory cytokines (e.g. selected from IL-6, IL-8 and TNF- a and GM-CSF) to a level which is less than 1 times, e.g. <0.99 times, <0.95 times, <0.9 times, <0.85 times, <0.8 times, <0.75 times, <0.7 times, <0.65 times, <0.6 times, <0.55 times, <0.5 times, <0.45 times, <0.4 times, <0.35 times, <0.3 times, <0.25 times, <0.2 times, <0.15 times, <0.1 times, <0.05 times, or <0.01 times the level to which their expression is induced by cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer, as determined in a given assay.
In some embodiments, the level of inhibition of tumor growth (e.g. of a tumor expressing the target antigen for the CAR) achieved by cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure is > 0.5 times and < 2 times, e.g. one of > 0.55 times and < 1 .9 times, > 0.6 times and < 1 .8 times, > 0.65 times and < 1 .7 times, > 0.7 times and < 1 .6 times, > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .4 times, > 0.85 times and < 1 .3 times, > 0.9 times and < 1 .2 times, > 0.95 times and < 1.1 times the level achieved by administration of a comparable quantity of cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer, as determined in a given assay. In some embodiments, the level of inhibition of tumor growth (e.g. of a tumor expressing the target antigen for the CAR) achieved by cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure is greater than 1 times, e.g. one of >1 .01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1 .1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the level achieved by administration of a comparable quantity of cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG1 spacer, as determined in a given assay.
In some embodiments, administration of cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure increases survival of recipient subjects having a cancer expressing the target antigen for the CAR to > 0.5 times and < 2 times, e.g. one of > 0.55 times and < 1 .9 times, > 0.6 times and < 1 .8 times, > 0.65 times and < 1 .7 times, > 0.7 times and < 1 .6 times, > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .4 times, > 0.85 times and < 1 .3 times, > 0.9 times and < 1 .2 times, > 0.95 times and < 1 .1 times the level of survival achieved by administration of a comparable quantity of cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer, as determined in a given assay. In some embodiments, administration of cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure increases survival of recipient subjects having a cancer expressing the target antigen for the CAR to greater than 1 times, e.g. one of >1 .01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1 .1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the level of survival achieved by administration of a comparable quantity of cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer, as determined in a given assay.
In some embodiments, the in vivo persistence of cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure following administration to a subject (e.g. a subject having a cancer, e.g. a cancer expressing the target antigen for the CAR) is > 0.5 times and < 2 times, e.g. one of > 0.55 times and < 1 .9 times, > 0.6 times and < 1 .8 times, > 0.65 times and < 1 .7 times, > 0.7 times and < 1 .6 times, > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .4 times, > 0.85 times and < 1 .3 times, > 0.9 times and < 1 .2 times, > 0.95 times and < 1 .1 times the in vivo persistence observed for cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer, as determined in a given assay. In some embodiments, the in vivo persistence of cells comprising a CAR/nucleic acid encoding a CAR according to the present disclosure following administration to a subject (e.g. a subject having a cancer, e.g. a cancer expressing the target antigen for the CAR) is greater than 1 times, e.g. one of >1 .01 times, >1 .02 times, >1 .03 times, >1 .04 times, >1 .05 times, >1 .1 times, >1 .2 times, >1 .3 times, >1 .4 times, >1 .5 times, >1 .6 times, >1 .7 times, >1 .8 times, >1 .9 times, >2 times, >3 times, >4 times, >5 times, >6 times, >7 times, >8 times, >9 times or >10 times the in vivo persistence observed for cells comprising a CAR/nucleic acid encoding an equivalent CAR comprising the hlgG 1 spacer, as determined in a given assay.
Producing cells expressing the CARs of the disclosure
The present disclosure provides methods for producing a cell comprising a nucleic acid(s) or vector(s) according to the present disclosure, comprising introducing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present disclosure into a cell. In some embodiments, introducing an isolated nucleic acid(s) or vector(s) according to the present disclosure into a cell comprises transformation, transfection, electroporation or transduction (e.g. retroviral transduction).
The present disclosure also provides methods for producing a cell expressing/comprising a CAR according to the present disclosure, comprising introducing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present disclosure in a cell. In some embodiments, the methods additionally comprise culturing the cell under conditions suitable for expression of the nucleic acid(s) or vector(s) by the cell. In some embodiments, the methods are performed in vitro.
Methods for producing CAR-expressing cells are well known to the skilled person. They generally involve modifying cells (e.g. immune cells, e.g. T cells or NK cells) to express/comprise a CAR, e.g. introducing nucleic acid encoding a CAR into the immune cells.
Immune cells may be modified to comprise/express a CAR or nucleic acid encoding a CAR described herein according to methods that are well known to the skilled person. The methods generally comprise nucleic acid transfer for permanent (stable) or transient expression of the transferred nucleic acid.
Any suitable genetic engineering platform may be used to modify a cell according to the present disclosure. Suitable methods for modifying a cell include the use of genetic engineering platforms such as gammaretroviral vectors, lentiviral vectors, adenovirus vectors, DNA transfection, transposon-based gene delivery and RNA transfection, for example as described in Maus et al., Annu Rev Immunol (2014) 32:189-225, hereby incorporated by reference in its entirety.
Methods also include those described e.g. in Wang and Riviere Mol Ther Oncolytics. (2016) 3:16015, which is hereby incorporated by reference in its entirety. Suitable methods for introducing nucleic acid(s)/vector(s) into cells include transduction, transfection and electroporation. Methods for generating/expanding populations of CAR-expressing immune cells in vitro/ex vivo are well known to the skilled person. Suitable culture conditions (i.e. cell culture media, additives, stimulations, temperature, gaseous atmosphere), cell numbers, culture periods and methods for introducing nucleic acid encoding a CAR into cells, etc. can be determined by reference e.g. to Hornbach et al. J Immunol (2001 ) 167:6123-6131 , Ramos et al. J. Clin. Invest. (2017) 127(9):3462-3471 and WO 2015/028444 A1 , all of which are hereby incorporated by reference in their entirety.
Conveniently, cultures of cells according to the present disclosure may be maintained at 37°C in a humidified atmosphere containing 5% CO2. The cells of cell cultures can be established and/or maintained at any suitable density, as can readily be determined by the skilled person.
Cultures can be performed in any vessel suitable for the volume of the culture, e.g. in wells of a cell culture plate, cell culture flasks, a bioreactor, etc. In some embodiments cells are cultured in a bioreactor, e.g. a bioreactor described in Somerville and Dudley, Oncoimmunology (2012) 1 (8):1435-1437, which is hereby incorporated by reference in its entirety. In some embodiments cells are cultured in a GRex cell culture vessel, e.g. a GRex flask or a GRex 100 bioreactor.
Immune cells (e.g. T cells) may be activated prior to introduction of nucleic acid encoding the CAR. For example, T cells within a population of PBMCs may be non-specifically activated by stimulation in vitro with agonist anti-CD3 and agonist anti-CD28 antibodies, in the presence of IL-2.
Introducing nucleic acid(s)/vector(s) into a cell may comprise transduction, e.g. retroviral transduction. Accordingly, in some embodiments the nucleic acid(s) is/are comprised in a viral vector(s), or the vector(s) is/are a viral vector(s). Transduction of immune cells with viral vectors is described e.g. in Simmons and Alberola-lla, Methods Mol Biol. (2016) 1323:99-108, which is hereby incorporated by reference in its entirety.
Agents may be employed to enhance the efficiency of transduction. Hexadimethrine bromide (polybrene) is a cationic polymer which is commonly used to improve transduction, through neutralising charge repulsion between virions and sialic acid residues expressed on the cell surface. Other agents commonly used to enhance transduction include e.g. the poloxamer-based agents such as LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech) and also SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs).
In some embodiments the methods comprise centrifuging the cells into which it is desired to introduce nucleic acid encoding the CAR in the presence of cell culture medium comprising viral vector comprising the nucleic acid (referred to in the art as ‘spinfection’).
In some embodiments, the methods comprise introducing a nucleic acid or vector according to the present disclosure into an immune cell by electroporation, e.g. as described in Koh et al., Molecular Therapy - Nucleic Acids (2013) 2, e114, which is hereby incorporated by reference in its entirety. The methods generally comprise introducing a nucleic acid encoding a CAR into a cell, and culturing the cell under conditions suitable for expression of the nucleic acid/CAR by the cell. In some embodiments, the methods comprise culturing immune cells into which nucleic acid encoding a CAR has been introduced in order to expand their number. In some embodiments, the methods comprise culturing immune cells into which nucleic acid encoding a CAR has been introduced in the presence of IL-7 and/or IL-15 (e.g. recombinant IL-7 and/or IL-15).
In some embodiments the methods further comprise purifying/isolating CAR-expressing cells, e.g. from other cells (e.g. cells which do not express the CAR). Methods for purifying/isolating immune cells from heterogeneous populations of cells are well known in the art, and may employ e.g. FACS- or MACS- based methods for sorting populations of cells based on the expression of markers of the immune cells.
In some embodiments the methods purifying/isolating cells of a particular type, e.g. CAR-expressing CD8+ T cells, CAR-expressing CTLs.
In preferred embodiments, CAR-expressing T cells may be generated from T cells within populations of PBMCs by a process comprising: stimulating PBMCs with antagonist anti-CD3 and andti-CD28 antibodies, transducing the cells with a viral vector (e.g. a gamma-retroviral vector) encoding the CAR, and subsequently culturing the cells in the presence of IL-7 and IL-15.
The present disclosure also provides CAR-expressing cells obtained or obtainable by the methods according to the present disclosure.
Compositions
The present disclosure also provides compositions comprising the CARs, nucleic acids, expression vectors and cells described herein.
The cells, CARs, nucleic acids and expression vectors described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
The compositions of the present disclosure may comprise one or more pharmaceutically-acceptable carriers (e.g. liposomes, micelles, microspheres, nanoparticles), diluents/excipients (e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben), anti-oxidants (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g. magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g. sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents or colouring agents (e.g. titanium oxide). The term ‘pharmaceutically-acceptable’ as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed. A. Adejare), 23rd Edition (2020), Academic Press.
Compositions may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration. In some embodiments, a pharmaceutical composition/medicament may be formulated for administration by injection or infusion, or administration by ingestion.
Suitable formulations may comprise the relevant article in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the human or animal body.
In some embodiments, the composition is formulated for injection or infusion, e.g. into a blood vessel, tissue/organ of interest, or tumor.
The present disclosure also provides methods for the production of pharmaceutically useful compositions, such methods of production may comprise one or more steps selected from: producing a CAR or a cell comprising/expressing a CAR according to the present disclosure; isolating a CAR or a cell comprising/expressing a CAR according to the present disclosure; and/or mixing a CAR or a cell comprising/expressing a CAR according to the present disclosure with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.
For example, a further aspect the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in the treatment of a disease/condition (e.g. a cancer), the method comprising formulating a pharmaceutical composition or medicament by mixing a CAR or a cell comprising/expressing a CAR according to the present disclosure with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent. Therapeutic and prophylactic applications
The CARs, nucleic acids, expression vectors, cells and compositions described herein find use in therapeutic and prophylactic methods.
The present disclosure provides a CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein for use in a method of medical treatment or prophylaxis. Also provided is a CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein for use in a method of treating or preventing a disease or condition described herein. Also provided is the use of a CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition described herein. Also provided is a method of treating or preventing a disease or condition described herein, comprising administering to a subject a therapeutically or prophylactically effective amount of a CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.
The methods may be effective to reduce the development or progression of a disease/condition, alleviation of the symptoms of a disease/condition or reduction in the pathology of a disease/condition. The methods may be effective to prevent progression of the disease/condition, e.g. to prevent worsening of, or to slow the rate of development of, the disease/condition. In some embodiments, the methods may lead to an improvement in the disease/condition, e.g. a reduction in the symptoms of the disease/condition or reduction in some other correlate of the severity/activity of the disease/condition. In some embodiments, the methods may prevent development of the disease/condition a later stage (e.g. a chronic stage or metastasis).
It will be appreciated that the articles of the present disclosure may be used for the treatment/prevention of any disease/condition that would derive therapeutic or prophylactic benefit from a reduction in the level/activity of the target antigen for the CAR (e.g. CD30), or a reduction in the number or activity of cells comprising/expressing the target antigen for the CAR.
For example, the disease/condition may be a disease/condition in which the target antigen for the CAR, or cells comprising/expressing the target antigen for the CAR are pathologically-implicated, e.g. a disease/condition in which an increased level/activity of the target antigen for the CAR, or an increase in the number/proportion of cells comprising/expressing the target antigen for the CAR is positively associated with the onset, development or progression of the disease/condition, and/or severity of one or more symptoms of the disease/condition. In some embodiments, an increased level/activity of the target antigen for the CAR, or an increase in the number/proportion of cells comprising/expressing the target antigen for the CAR may be a risk factor for the onset, development or progression of the disease/condition.
In some embodiments, the disease/condition to be treated/prevented in accordance with the present disclosure is a disease/condition characterised by an increase in the level of expression or activity of the target antigen for the CAR (e.g. CD30), e.g. as compared to the level of expression/activity in the absence of the disease/condition. In some embodiments, the disease/condition to be treated/prevented is a disease/condition characterised by an increase in the number/proportion/activity of cells expressing the target antigen for the CAR, e.g. as compared to the level/number/proportion/activity in the absence of the disease/condition (e.g. in a healthy subject, or in equivalent non-diseased tissue). Where the disease/condition is a cancer, the level of expression or activity of the target antigen for the CAR may be greater than the level of expression or activity of the target antigen for the CAR in equivalent non- cancerous cells/non-tumor tissue. A cancer/cell thereof may comprise one or more mutations (e.g. relative to equivalent non-cancerous cells/non-tumor tissue) causing upregulation of expression or activity of the target antigen for the CAR.
Treatment in accordance with the methods of the present disclosure may achieve one or more of the following in a subject (compared to an equivalent untreated subject, or subject treated with an appropriate control): a reduction in the level of the target antigen for the CAR; a reduction in the activity of the target antigen for the CAR; and/or a reduction in the number/proportion of cells comprising/expressing the target antigen for the CAR.
In aspects and embodiments according to the present disclosure, cells (particularly immune cells, more particularly T cells) comprising/expressing a CAR according to the present disclosure are provided for therapeutic and prophylactic use. It will be appreciated that the methods generally comprise administering a population of immune cells expressing a CAR according to the present disclosure to a subject. In some embodiments, immune cells expressing a CAR according to the present disclosure may be administered in the form of a pharmaceutical composition comprising such cells.
In particular, use of immune cells expressing a CAR according to the present disclosure in methods to treat/prevent diseases/conditions by adoptive cell transfer (ACT) is contemplated.
Adoptive cell transfer generally refers to a process by which cells (e.g. immune cells) are obtained from a subject, typically by drawing a blood sample from which the cells are isolated. The cells are then typically modified and/or expanded, and then administered either to the same subject (in the case of adoptive transfer of autologous/autogeneic cells) or to a different subject (in the case of adoptive transfer of allogeneic cells). The treatment is typically aimed at providing a population of cells with certain desired characteristics to a subject, or increasing the frequency of such cells with such characteristics in that subject. Adoptive transfer may be performed with the aim of introducing a cell or population of cells into a subject, and/or increasing the frequency of a cell or population of cells in a subject.
Adoptive transfer of immune cells is described, for example, in Kalos and June (2013), Immunity 39(1 ): 49-60, and Davis et al. (2015), Cancer J. 21 (6): 486-491 , both of which are hereby incorporated by reference in their entirety. The skilled person is able to determine appropriate reagents and procedures for adoptive transfer of cells according to the present disclosure, for example by reference to Dai et al., 2016 J Nat Cancer Inst 108(7): djv439, which is incorporated by reference in its entirety. In some embodiments, the target antigen for the CAR is CD30. The biology of CD30 and intervention targeting CD30 for the treatment and prevention of disease is reviewed e.g. in van der Weyden et al., Blood Cancer Journal (2017) 7:e603 and Muta and Podack, Immunol Res (2013), 57(1 -3):151 -8, both of which are hereby incorporated by reference in their entirety.
The utility of immune cells expressing CD30-specific CARs in the treatment/prevention of cancer is described e.g. in Hornbach et al. Cancer Res. (1998) 58(6):1116-9, Hornbach et al. Gene Therapy (2000) 7:1067-1075, Hornbach et al. J Immunother. (1999) 22(6):473-80, Hornbach et al. Cancer Res. (2001 ) 61 :1976-1982, Hornbach et a/. J Immunol (2001 ) 167:6123-6131 , Savoldo et al. Blood (2007) 110(7):2620-30, Koehler et al. Cancer Res. (2007) 67(5):2265-2273, Di Stasi et al. Blood (2009) 113(25) :6392-402, Hornbach et al. Gene Therapy (2010) 17:1206-1213, Chmielewski et al. Gene Therapy (2011 ) 18:62-72, Kofler et al. Mol. Ther. (2011 ) 19(4):760-767, Gilham, Abken and Pule. Trends in Mol. Med. (2012) 18(7):377-384, Chmielewski et al. Gene Therapy (2013) 20:177-186, Hornbach et al. Mol. Ther. (2016) 24(8):1423-1434, Ramos et al. J. Clin. Invest. (2017) 127(9):3462-3471 , WO 2015/028444 A1 , WO 2016/008973 A1 , WO 2021/222927 A1 and WO 2021/222928 A1 , incorporated by reference hereinabove.
The immune cells expressing a CAR according to the present disclosure may be employed in the treatment/prevention of diseases/conditions by allotransplantation or autotransplantation.
As used herein, ‘allotransplantation’ refers to the transplantation to a recipient subject of cells, tissues or organs which are genetically non-identical to the recipient subject. The cells, tissues or organs may be from, or may be derived from, cells, tissues or organs of a donor subject that is genetically non-identical to the recipient subject. Allotransplantation is distinct from autotransplantation, which refers to the transplantation of cells, tissues or organs which are from/derived from a donor subject genetically identical to the recipient subject (i.e. autologous material). It will be appreciated that adoptive transfer of allogeneic immune cells is a form of allotransplantation, and that adoptive transfer of autologous immune cells is a form of autotransplantation.
The present disclosure provides methods comprising administering immune cells comprising/expressing a CAR according to the present disclosure, or immune cells comprising/expressing nucleic acid encoding a CAR according to the present disclosure, to a subject.
In some embodiments, the methods comprise modifying an immune cell to comprise/express a CAR according to the present disclosure. In some embodiments, the methods comprise modifying an immune cell specific for a virus to comprise/express nucleic acid encoding a CAR according to the present disclosure.
In some embodiments, the methods comprise:
(a) modifying an immune cell to express or comprise a CAR according to the present disclosure, or to express or comprise nucleic acid encoding a CAR according to the present disclosure, and (b) administering the immune cell specific for a virus modified to express or comprise a CAR according to the present disclosure, or modified to express or comprise a nucleic acid encoding a CAR according to the present disclosure, to a subject.
In some embodiments, the methods comprise:
(a) isolating or obtaining immune cells;
(b) modifying an immune cell to express or comprise a CAR according to the present disclosure, or to express or comprise nucleic acid encoding a CAR according to the present disclosure, and
(c) administering the immune cell modified to express or comprise a CAR according to the present disclosure, or modified to express or comprise a nucleic acid encoding a CAR according to the present disclosure, to a subject.
In some embodiments, the methods comprise:
(a) isolating immune cells (e.g. PBMCs) from a subject;
(b) generating/expanding a population of immune cells specific for a virus;
(c) modifying an immune cell specific for a virus to express or comprise a CAR according to the present disclosure, or to express or comprise nucleic acid encoding a CAR according to the present disclosure, and
(d) administering the immune cell specific for a virus modified to express or comprise a CAR according to the present disclosure, or modified to express or comprise a nucleic acid encoding a CAR according to the present disclosure, to a subject.
In some embodiments, the methods comprise administering to a subject an EBV-specific immune cell modified to express or comprise a CD30-specific CAR according to the present disclosure, or modified to express or comprise a nucleic acid encoding a CD30-specific CAR according to the present disclosure.
In some embodiments, the subject from which the immune cells (e.g. PBMCs) are isolated is the same subject to which cells are administered (i.e., adoptive transfer may be of autologous/autogeneic cells). In some embodiments, the subject from which the immune cells (e.g. PBMCs) are isolated is a different subject to the subject to which cells are administered (i.e., adoptive transfer may be of allogeneic cells).
In some embodiments the methods may comprise one or more of: obtaining a blood sample from a subject; isolating immune cells (e.g. PBMCs) from a blood sample which has been obtained from a subject; generating/expanding a population of immune cells; culturing the immune cells in in vitro or ex vivo cell culture; modifying an immune cell to express or comprise a CAR according to the present disclosure, or to express or comprise a nucleic acid encoding a CAR according to the present disclosure (e.g. by transduction with a viral vector encoding such CAR, or a viral vector comprising such nucleic acid); culturing immune cells expressing/comprising a CAR according to the present disclosure, or expressing/comprising a nucleic acid encoding a CAR according to the present disclosure in in vitro or ex vivo cell culture; collecting/isolating immune cells expressing/comprising a CAR according to the present disclosure, or expressing/comprising a nucleic acid encoding a CAR according to the present disclosure; formulating immune cells expressing/comprising a CAR according to the present disclosure, or a nucleic acid encoding a CAR according to the present disclosure to a pharmaceutical composition, e.g. by mixing the cells with a pharmaceutically acceptable adjuvant, diluent, or carrier; administering immune cells expressing/comprising a CAR according to the present disclosure, or expressing/comprising a nucleic acid encoding a CAR according to the present disclosure, or a pharmaceutical composition comprising such cells, to a subject.
In some embodiments, the methods may additionally comprise treating the cells or subject to induce/enhance expression of CAR and/or to induce/enhance proliferation or survival of virus-specific immune cells comprising/expressing the CAR.
Cancer
In some embodiments, the disease to be treated/prevented in accordance with the present disclosure is a cancer.
Cancer may refer to any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor. The cancer may be benign or malignant and may be primary or secondary (metastatic). A neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue. The cancer may be of tissues/cells derived from e.g. the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g. renal epithelia), gallbladder, oesophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, and/or white blood cells.
Tumors may be nervous or non-nervous system tumors. Nervous system tumors may originate either in the central or peripheral nervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma. Non-nervous system cancers/tumors may originate in any other non-nervous tissue, examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer , colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, NSCLC, hematologic cancer and sarcoma. In some embodiments the cancer is selected from the group consisting of: a solid cancer, a hematological cancer, gastric cancer (e.g. gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma), liver cancer (hepatocellular carcinoma, cholangiocarcinoma), head and neck cancer (e.g. head and neck squamous cell carcinoma), oral cavity cancer (e.g. oropharyngeal cancer (e.g. oropharyngeal carcinoma), oral cancer, laryngeal cancer, nasopharyngeal carcinoma, oesophageal cancer), colorectal cancer (e.g. colorectal carcinoma), colon cancer, colon carcinoma, cervical carcinoma, prostate cancer, lung cancer (e.g. NSCLC, small cell lung cancer, lung adenocarcinoma, squamous lung cell carcinoma), bladder cancer, urothelial carcinoma, skin cancer (e.g. melanoma, advanced melanoma), renal cell cancer (e.g. renal cell carcinoma), ovarian cancer (e.g. ovarian carcinoma), mesothelioma, breast cancer, brain cancer (e.g. glioblastoma), prostate cancer, pancreatic cancer, a myeloid hematologic malignancy, a lymphoblastic hematologic malignancy, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), lymphoma, non-Hodgkin’s lymphoma (NHL), thymoma or multiple myeloma (MM).
In some embodiments the cancer is a cancer in which the target antigen for the CAR (e.g. CD30) is pathologically implicated. That is, in some embodiments the cancer is a cancer which is caused or exacerbated by the expression of the target antigen for the CAR, a cancer for which expression of the target antigen for the CAR is a risk factor and/or a cancer for which expression of the target antigen for the CAR is positively associated with onset, development, progression, severity or metastasis of the cancer. The cancer may be characterised by expression of the target antigen for the CAR, e.g. the cancer may comprise cells expressing the target antigen for the CAR. Such cancers may be referred to as being positive for the target antigen for the CAR. A cancer which is ‘positive’ for the the target antigen for the CAR may be a cancer comprising cells expressing the target antigen for the CAR (e.g. at the cell surface). A cancer which is ‘positive’ for the target antigen for the CAR may overexpress the target antigen for the CAR.
CD30-positive cancers are described e.g. in van der Weyden eta!., Blood Cancer Journal (2017) 7:e603 and Muta and Podack, Immunol Res (2013), 57(1 -3) :151 -8. CD30 is expressed on small subsets of activated T and B lymphocytes, and by various lymphoid neoplasms including classical Hodgkin’s lymphoma and anaplastic large cell lymphoma. Variable expression of CD30 has also been shown for peripheral T cell lymphoma, not otherwise specified (PTCL-NOS), adult T cell leukemia/lymphoma, cutaneous T cell lymphoma (CTCL), extra-nodal NK-T cell lymphoma, various B cell non-Hodgkin’s lymphomas (including diffuse large B cell lymphoma, particularly EBV-positive diffuse large B cell lymphoma), and advanced systemic mastocytosis. CD30 expression has also been observed in some non-hematopoietic malignancies, including germ cell tumors and testicular embryonal carcinomas.
The transmembrane glycoprotein CD30, is a member of the tumor necrosis factor receptor superfamily (Falini et al., Blood (1995) 85(1 ):1 -14). Members of the TNF/TNF-receptor (TNF-R) superfamily coordinate the immune response at multiple levels and CD30 plays a role in regulating the function or proliferation of normal lymphoid cells. CD30 was originally described as an antigen recognised by a monoclonal antibody, Ki-1 , which was raised by immunizing mice with a HL-derived cell line, L428 (Muta and Podack, Immunol Res (2013) 57: 151 -158). CD30 antigen expression has been used to identify ALCL and Reed-Sternberg cells in Hodgkin's disease (Falini eta!., Blood (1995) 85(1 ):1 -14). With the wide expression in the lymphoma malignant cells, CD30 is therefore a potential target for developing both antibody-based immunotherapy and cellular therapies. Importantly, CD30 is not typically expressed on normal tissues under physiologic conditions, thus is notably absent on resting mature or precursor B or T cells (Younes and Ansell, Semin Hematol (2016) 53: 186-189). Brentuximab vedotin, an antibody-drug conjugate that targets CD30 was initially approved for the treatment of CD30-positive HL (Adcetris® US Package Insert 2018). Data from brentuximab vedotin trials support CD30 as a therapeutic target for the treatment of CD30-positive lymphoma.
Hodgkin lymphoma (HL) is an uncommon malignancy involving lymph nodes and the lymphatic system. The incidence of HL is bimodal with most patients diagnosed between 15 and 30 years of age, followed by another peak in adults aged 55 years or older. In 2019 it is estimated there will be 8,110 new cases (3,540 in females and 4570 in males) in the United States and 1 ,000 deaths (410 female and 590 males) from this disease (American Cancer Society 2019). Based on 2012-2016 cases in National Cancer Institute’s SEER database, the incidence rate for HL for the pediatric HL patients in US is as follows: Age 1 -4: 0.1 ; Age 5-9: 0.3; Age 10-14: 1 .3; Age 15-19: 3.3 per 100,000 (SEER Cancer Statistics Review, 1975-2016]). The World Health Organisation (WHO) classification divides HL into 2 main types: classical Hodgkin lymphoma (cHL) and nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). In Western countries, cHL accounts for 95% and NLPHL accounts for 5% of all HL (National Comprehensive Cancer Network Guidelines 2019).
First-line chemotherapy for cHL patients with advanced disease is associated with cure rates between 70% and 75% (Karantanos et al., Blood Lymphat Cancer (2017) 7:37-52). Salvage chemotherapy followed by Autologous Stem Cell Transplant (ASCT) is commonly used in patients who relapse after primary therapy. Unfortunately, up to 50% of the cHL patients experience disease recurrence after ASCT. The median overall survival of patients who relapse after ASCT is approximately two years (Alinari Blood (2016) 127:287-295). Despite aggressive combination chemotherapy, between 10% and 40% of patients do not achieve a response to salvage chemotherapy and there are no randomised clinical trial data supporting ASCT in non-responders. For patients who do not respond to salvage chemotherapy, relapse after ASCT or who are not candidates for this approach, the prognosis continues to be grave and new treatment approaches are urgently needed (Keudell British Journal of Haematology (2019) 184:105-112).
While a majority of the pediatric population (children, adolescents, and young adults) will be cured with currently available therapy, a small fraction of patients may have refractory or relapsed disease and require novel therapies that have an acceptable safety profile with improved efficacy benefit (Flerlage et al., Blood (2018) 132: 376-384; Kelly, Blood (2015) 126: 2452-2458; McClain and Kamdar, in UpToDate 2019; Moskowitz, ASCO Educational Book (2019) 477-486). HL patients treated with high dose chemotherapy during childhood commonly experience treatment-related long-term sequelae, such as cardiac, pulmonary, gonadal, and endocrine toxicity as well as second malignant neoplasms (Castellino etal., Blood (2011 ) 117(6): 1806-1816). In some embodiments, the cancer to be treated/prevented is an EBV-associated cancer. EBV infection is implicated in several cancers, as reviewed e.g. in Jha et al., Front Microbiol. (2016) 7:1602, which is hereby incorporated by reference in its entirety. In some embodiments, the cancer is a cancer which is caused or exacerbated by infection with EBV, a cancer for which infection with EBV is a risk factor and/or a cancer for which infection with EBV is positively associated with onset, development, progression, severity or metastasis of the cancer. The cancer may be characterised by EBV infection, e.g. the cancer may comprise cells infected with EBV. Such cancers may be referred to as EBV-positive cancers.
EBV-associated cancers which may be treated/prevented in accordance with the present disclosure include B cell-associated cancers such as Burkitt’s lymphoma, post-transplant lymphoproliferative disease (PTLD), central nervous system lymphoma (CNS lymphoma), Hodgkin’s lymphoma, nonHodgkin’s lymphoma, and EBV-associated lymphomas associated with immunodeficiency (including e.g. EBV-positive lymphoma associated with X-linked lymphoproliferative disorder, EBV-positive lymphoma associated with HIV infection/AIDS, and oral hairy leukoplakia), and epithelial cell-related cancers such as nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). In some embodiments, the cancer is selected from lymphoma (e.g. EBV-positive lymphoma), head and neck squamous cell carcinoma (HNSCC; e.g. EBV-positive HNSCC), nasopharyngeal carcinoma (NPC; e.g. EBV-positive NPC), and gastric carcinoma (GC; e.g. EBV-positive GC).
In some embodiments, a CD30-positive cancer may be selected from: a solid cancer, a hematological cancer, a hematopoietic malignancy, Hodgkin’s lymphoma (HL), anaplastic large cell lymphoma (ALCL), ALK-positive anaplastic T cell lymphoma, ALK-negative anaplastic T cell lymphoma, peripheral T cell lymphoma (e.g. PTCL-NOS), T cell leukemia, T cell lymphoma, cutaneous T cell lymphoma (CTCL), NK- T cell lymphoma (e.g. extra-nodal NK-T cell lymphoma), non-Hodgkin’s lymphoma (NHL), B cell nonHodgkin’s lymphoma, diffuse large B cell lymphoma (e.g. diffuse large B cell lymphoma-NOS), primary mediastinal B cell lymphoma, EBV-positive B cell lymphoma, EBV-positive diffuse large B cell lymphoma, advanced systemic mastocytosis, a germ cell tumor and testicular embryonal carcinoma.
In some embodiments, the cancer is selected from: a CD30-positive cancer, an EBV-associated cancer, a hematological cancer, a myeloid hematologic malignancy, a hematopoietic malignancy a lymphoblastic hematologic malignancy, myelodysplastic syndrome, leukemia, T cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, lymphoma, Hodgkin’s lymphoma, nonHodgkin’s lymphoma, B cell non-Hodgkin’s lymphoma, diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, EBV-associated lymphoma, EBV-positive B cell lymphoma, EBV-positive diffuse large B cell lymphoma, EBV-positive lymphoma associated with X-linked lymphoproliferative disorder, EBV- positive lymphoma associated with HIV infection/AIDS, oral hairy leukoplakia, Burkitt’s lymphoma, posttransplant lymphoproliferative disease, central nervous system lymphoma, anaplastic large cell lymphoma, T cell lymphoma, ALK-positive anaplastic T cell lymphoma, ALK-negative anaplastic T cell lymphoma, peripheral T cell lymphoma, cutaneous T cell lymphoma, NK-T cell lymphoma, extra-nodal NK-T cell lymphoma, thymoma, multiple myeloma, a solid cancer, epithelial cell cancer, gastric cancer, gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck cancer, head and neck squamous cell carcinoma, oral cavity cancer, oropharyngeal cancer, oropharyngeal carcinoma, oral cancer, laryngeal cancer, nasopharyngeal carcinoma, oesophageal cancer, colorectal cancer, colorectal carcinoma, colon cancer, colon carcinoma, cervical carcinoma, prostate cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, squamous lung cell carcinoma, bladder cancer, urothelial carcinoma, skin cancer, melanoma, advanced melanoma, renal cell cancer, renal cell carcinoma, ovarian cancer, ovarian carcinoma, mesothelioma, breast cancer, brain cancer, glioblastoma, prostate cancer, pancreatic cancer, mastocytosis, advanced systemic mastocytosis, germ cell tumor or testicular embryonal carcinoma.
In some embodiments, the cancer may be a relapsed cancer. As used herein, a ‘relapsed’ cancer refers to a cancer which responded to a treatment (e.g. a first line therapy for the cancer), but which has subsequently re-emerged/progressed, e.g. after a period of remission. For example, a relapsed cancer may be a cancer whose growth/progression was inhibited by a treatment (e.g. a first line therapy for the cancer), and which has subsequently grown/progressed.
In some embodiments, the cancer may be a refractory cancer. As used herein, a ‘refractory’ cancer refers to a cancer which has not responded to a treatment (e.g. a first line therapy for the cancer). For example, a refractory cancer may be a cancer whose growth/progression was not inhibited by a treatment (e.g. a first line therapy for the cancer). In some embodiments a refractory cancer may be a cancer for which a subject receiving treatment for the cancer did not display a partial or complete response to the treatment.
In embodiments where the cancer is anaplastic large cell lymphoma, the cancer may be relapsed or refractory with respect to treatment with chemotherapy, brentuximab vedotin, or crizotinib. In embodiments where the cancer is peripheral T cell lymphoma, the cancer may be relapsed or refractory with respect to treatment with chemotherapy or brentuximab vedotin. In embodiments where the cancer is extranodal NK-T cell lymphoma, the cancer may be relapsed or refractory with respect to treatment with chemotherapy (with or without asparaginase) or brentuximab vedotin. In embodiments where the cancer is diffuse large B cell lymphoma, the cancer may be relapsed or refractory with respect to treatment with chemotherapy (with or without rituximab) or CD19 CAR-T therapy. In embodiments where the cancer is primary mediastinal B cell lymphoma, the cancer may be relapsed or refractory with respect to treatment with chemotherapy, immune checkpoint inhibitor (e.g. PD-1 inhibitor) or CD19 CAR-T therapy.
Treatment of a cancer in accordance with the methods of the present disclosure achieves one or more of the following treatment effects: reduces the number of cancer cells in the subject, reduces the size of a cancerous tumor/lesion in the subject, inhibits (e.g. prevents or slows) growth of cancer cells in the subject, inhibits (e.g. prevents or slows) growth of a cancerous tumor/lesion in the subject, inhibits (e.g. prevents or slows) the development/progression of a cancer (e.g. to a later stage, or metastasis), reduces the severity of symptoms of a cancer in the subject, increases survival of the subject (e.g. progression free survival or overall survival), reduces a correlate of the number or activity of cancer cells in the subject, and/or reduces cancer burden in the subject. Subjects may be evaluated in accordance with the Revised Criteria for Response Assessment: The Lugano Classification (described e.g. in Cheson et al., J Clin Oncol (2014) 32: 3059-3068, incorporated by reference hereinabove) in order to determine their response to treatment. In some embodiments, treatment of a subject in accordance with the methods of the present disclosure achieves one of the following: complete response, partial response, or stable disease.
Applications relating to treatment/prevention of alloreactive immune responses
The CAR-expressing immune cells and compositions of the present disclosure can be used in methods involving allotransplantation, e.g. to treat/prevent a disease/condition in a subject. The CAR-expressing immune cells and compositions of the present disclosure are useful in methods to reduce/prevent alloreactive immune responses (particularly T cell-mediated alloreactive immune responses) and the deleterious consequences thereof.
Alloreactive T cells express CD30. Chan et al., J Immunol (2002) 169(4):1784-91 identify CD30- expressing T cells as a subset of activated T cells (also expressing CD25 and CD45RO) having an important role in CD30 alloimmune responses. CD30 expression and the proliferation of CD30- expressing T cells increases in response to alloantigen. Chen et al., Blood (2012) 120(3):691 -6 identifies CD30 expression on CD8+ T cell subsets as a potential biomarker for GVHD, and propose CD30 as a therapeutic target for GVHD.
The utility of immune cells expressing CD30-specific CARs in methods to reduce/prevent alloreactive immune responses is described e.g. in WO 2021/222929 A1 , which is hereby incorporated by reference in its entirety.
The CAR-expressing immune cells and compositions of the present disclosure are particularly useful in methods involving allotransplantation, and also in the processing/production of allotransplants.
In particular, the CAR-expressing immune cells and compositions are contemplated for use in the production and administration of ‘off-the-shelf’ materials for use in therapeutic and prophylactic methods comprising administration of allogeneic material.
As explained hereinabove, CAR-expressing immune cells of the present disclosure are useful for the treatment/prevention of diseases/conditions by adoptive cell transfer. CAR-expressing immune cells of the present disclosure are less susceptible to T cell-mediated alloreactive immune responses of the recipient following adoptive transfer, and thus exhibit enhanced proliferation/survival in the recipient after transfer, and superior therapeutic/prophylactic effects.
The CAR-expressing immune cells and compositions of the present disclosure are also useful in methods comprising allotransplantation of allogeneic cells other than the CAR-expressing immune cells of the present disclosure. In particular, the CAR-expressing immune cells and compositions of the present disclosure are useful for depleting allotransplants (populations of cells, tissues and organs) and subjects of alloreactive immune cells (e.g. alloreactive T cells). In such methods the CAR-expressing immune cells and compositions are useful for conditioning of donor and/or recipient subjects, and/or treatment of the allotransplant to reduce/prevent an alloreactive immune response following allotransplantation.
Cells, tissues and organs to be allotransplanted include e.g. immune cells (e.g. adoptive cell transfer), the heart, lung, kidney, liver, pancreas, intestine, face, cornea, skin, hematopoietic stem cells (bone marrow), blood, hands, leg, penis, bone, uterus, thymus, islets of Langerhans, heart valve and ovary. Populations of cells, tissues or organs to be allotransplanted may be referred to as ‘allotransplants’.
The disease/condition to be treated/prevented by the allotransplantation can be any disease/condition which would derive therapeutic or prophylactic benefit from the allotransplantation. In some embodiments, the disease/condition to be treated/prevented by allotransplantation may e.g. be a T cell dysfunctional disorder, a cancer, an infectious disease or an autoimmune disease.
A T cell dysfunctional disorder may be a disease/condition in which normal T cell function is impaired causing downregulation of the subject’s immune response to pathogenic antigens, e.g. generated by infection by exogenous agents such as microorganisms, bacteria and viruses, or generated by the host in some disease states such as in some forms of cancer (e.g. in the form of tumor-associated antigens). The T cell dysfunctional disorder may comprise T cell exhaustion or T cell anergy. T cell exhaustion comprises a state in which CD8+ T cells fail to proliferate or exert T cell effector functions such as cytotoxicity and cytokine (e.g. IFNy) secretion in response to antigen stimulation. Exhausted T cells may also be characterised by sustained expression of one or more markers of T cell exhaustion, e.g. PD-1 , CTLA-4, LAG-3, TIM-3. The T cell dysfunctional disorder may manifest as an infection, or inability to mount an effective immune response against an infection. The infection may be chronic, persistent, latent or slow, and may be the result of bacterial, viral, fungal or parasitic infection. As such, treatment may be provided to patients having a bacterial, viral or fungal infection. Examples of bacterial infections include infection with Helicobacter pylori. Examples of viral infections include infection with HIV, hepatitis B or hepatitis C. The T cell dysfunctional disorder may be associated with a cancer, such as tumor immune escape. Many human tumors express tumor-associated antigens recognised by T cells and capable of inducing an immune response.
An infectious disease may be e.g. bacterial, viral, fungal, or parasitic infection. In some embodiments, it may be particularly desirable to treat chronic/persistent infections, e.g. where such infections are associated with T cell dysfunction or T cell exhaustion. It is well established that T cell exhaustion is a state of T cell dysfunction that arises during many chronic infections (including viral, bacterial and parasitic), as well as in cancer (Wherry Nature Immunology Vol.12, No.6, p492-499, June 2011 ). Examples of bacterial infections that may be treated include infection by Bacillus spp., Bordetella pertussis, Clostridium spp., Corynebacterium spp., Vibrio chloerae, Staphylococcus spp., Streptococcus spp. Escherichia, Klebsiella, Proteus, Yersinia, Erwina, Salmonella, Listeria sp, Helicobacter pylori, mycobacteria (e.g. Mycobacterium tuberculosis) and Pseudomonas aeruginosa. For example, the bacterial infection may be sepsis or tuberculosis. Examples of viral infections that may be treated include infection by influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), Herpes simplex virus and human papilloma virus (HPV). Examples of fungal infections that may be treated include infection by Alternaria sp, Aspergillus sp, Candida sp and Histoplasma sp. The fungal infection may be fungal sepsis or histoplasmosis. Examples of parasitic infections that may be treated include infection by Plasmodium species (e.g. Plasmodium falciparum, Plasmodium yoeli, Plasmodium ovale, Plasmodium vivax, or Plasmodium chabaudi chabaudi). The parasitic infection may be a disease such as malaria, leishmaniasis and toxoplasmosis.
In some embodiments, the disease/condition is an autoimmune disease. In such embodiments the treatment may be aimed at reducing the number of autoimmune effector cells. In some embodiments the autoimmune disease is selected from: diabetes mellitus type 1 , celiac disease, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.
The CAR-expressing immune cells and compositions of the present disclosure are also useful for the treatment/prevention of an alloreactive immune response, and diseases/conditions characterised by an alloreactive immune response.
Diseases and conditions characterised by an alloreactive immune response include diseases/conditions caused or exacerbated by alloreactive immune responses associated with allotransplantation. Such diseases/conditions include graft versus host disease (GVHD) and graft rejection, and are described in detail in Perkey and Maillard Annu Rev Pathol. (2018) 13:219-245, which is hereby incorporated by reference in its entirety.
Graft-versus-host disease (GVHD) can occur following allotransplantation of large numbers of donor immune cells, and involves reactivity of donor-derived immune cells against allogeneic recipient cells/tissues/organs. Graft rejection refers to the destruction of transplanted cells/tissue/organs by a recipient’s immune system following transplantation. Where graft rejection is of an allotransplant, it may be referred to as allograft rejection.
The CAR-expressing immune cells and compositions of the present disclosure may be used to deplete alloreactive T cells in an allotransplant, which could otherwise lead to graft versus host disease (GVHD) in a recipient upon allotransplantation.
The CAR-expressing immune cells and compositions of the present disclosure may be used to deplete alloreactive T cells in a donor for an allotransplant (e.g. prior to harvesting/collecting the allotransplant), which could otherwise lead to GVHD in a recipient upon allotransplantation.
The CAR-expressing immune cells and compositions of the present disclosure may be used to deplete alloreactive T cells in the recipient for an allotransplant, which could otherwise cause/promote graft rejection. The present disclosure provides methods of treating/preventing graft-versus-host disease (GVHD) following allotransplantation, comprising administering a CAR-expressing immune cell or composition according to the present disclosure to a donor subject for an allotransplant. The present disclosure also provides methods of treating/preventing graft-versus-host disease (GVHD) following allotransplantation, comprising contacting an allotransplant with a CAR-expressing immune cell or composition according to the present disclosure. The aim for such methods is to reduce/remove the ability of alloreactive immune cells in the allograft to mount an alloreactive immune response to cells, tissue and/or organs of the recipient for the allotransplant.
The present disclosure provides methods of treating/preventing graft rejection following allotransplantation, comprising administering a CAR-expressing immune cell or composition according to the present disclosure to a recipient subject for an allotransplant. The aim for such methods is to reduce/remove the ability of the receipt subject to mount an alloreactive immune response to the allotransplant. The CAR-expressing immune cells are useful to eliminate immune cells in the recipient that would otherwise effect an alloreactive immune response against donor cells, tissue and/or organs.
The present disclosure provides methods comprising depleting an allotransplant of alloreactive immune cells (e.g. alloreactive T cells), comprising contacting an allotransplant (e.g. a population of cells, tissue or an organ to be transplanted) with a CAR-expressing immune cell or composition of the present disclosure. The methods may comprise administering a CAR-expressing immune cell or composition of the present disclosure to a donor subject for the allotransplant. The aim for such methods is to reduce/remove the ability of alloreactive immune cells in the allograft to mount an alloreactive immune response to cells, tissue and/or organs of the recipient for the allotransplant.
In some embodiments the methods comprise one or more of: obtaining/collecting a population of cells, tissue or organ from a subject; contacting a population of cells, tissue or organ with a CAR-expressing immune cell or composition according to the present disclosure; culturing a population of cells, tissue or organ in vitro or ex vivo in the presence of a CAR- expressing immune cell according to the present disclosure; harvesting/collecting a population of cells, tissue or organ depleted of alloreactive immune cells; and transplanting/administering a population of cells, tissue or organ depleted of alloreactive immune cells to a subject.
The present disclosure also provides methods comprising depleting a subject of alloreactive immune cells (e.g. alloreactive T cells), comprising administering a CAR-expressing immune cell or composition of the present disclosure to the subject. The subject may be a donor subject for an allotransplant, or may be an intended recipient subject for an allotransplant.
In some embodiments the methods comprise one or more of: administering a CAR-expressing immune cell or composition according to the present disclosure to a subject, in order to deplete alloreactive immune cells in the subject; obtaining/collecting a population of cells, tissue or organ from a subject to which a CAR- expressing immune cell or composition according to the present disclosure has been administered; and transplanting/administering a population of cells, tissue or organ depleted of alloreactive immune cells to a subject.
In some embodiments the methods comprise one or more of: administering a CAR-expressing immune cell or composition according to the present disclosure to a subject, in order to deplete alloreactive immune cells in the subject; and transplanting/administering a population of cells, tissue or organ to a subject to which a CAR- expressing immune cell or composition according to the present disclosure have previously been administered.
Depletion of alloreactive immune cells may result in e.g. a 2-fold, 10-fold, 100-fold, 1000-fold, 10000-fold or greater reduction in the quantity of alloreactive immune cells in the allotransplant or subject.
The methods may be performed in vitro or ex vivo, or in vivo in a subject. Method steps performed in vitro or ex vivo may comprise in vitro or ex vivo cell culture.
The methods may further comprise method steps for the production of CAR-expressing immune cells and compositions according to the present disclosure.
In some embodiments, administration of a CAR-expressing immune cell or composition according to the present disclosure to a recipient subject for an allotransplantation and allotransplantation are performed simultaneously (/.e. at the same time, or within e.g. 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 8 hrs, 12 hrs, 24 hrs, 36 hrs or 48 hrs).
In some embodiments, administration of a CAR-expressing immune cell or composition according to the present disclosure to a recipient subject for an allotransplantation and allotransplantation are performed sequentially. The time interval between administration of a CAR-expressing immune cell or composition and allotransplantation may be any time interval, including hours, days, weeks, months, or years. The CAR-expressing immune cell or composition may be administered to the recipient subject before or after allotransplantation. The CAR-expressing immune cell or composition are preferably administered to the recipient subject prior to allotransplantation.
In some embodiments, administration of a CAR-expressing immune cell or composition according to the present disclosure to a donor subject for an allotransplantation and collection of the allotransplant (i.e. collection of the cells, tissue and/or an organ) from the subject are performed simultaneously (i.e. at the same time, or within e.g. 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 8 hrs, 12 hrs, 24 hrs, 36 hrs or 48 hrs). In some embodiments administration of a CAR-expressing immune cell or composition according to the present disclosure to a donor subject for an allotransplantation and collection of the allotransplant (i.e. collection of the cells, tissue and/or an organ) from the subject are performed sequentially. The time interval between administration of a CAR-expressing immune cell or composition and collection of the allotransplant may be any time interval, including hours, days, weeks, months, or years. The CAR- expressing immune cell or composition may be administered to the donor subject before or after collection of the allotransplant. The CAR-expressing immune cell or composition are preferably administered to the donor subject prior to collection of the allotransplant.
In some embodiments, the methods comprise additional intervention to treat/prevent an alloreactive immune response, graft rejection and/or GVHD.
In some embodiments, the methods to treat/prevent alloreactivity, graft rejection and/or GVHD comprise administration of immunosuppressive and/or lymphodepletive therapy such as treatment with corticosteroids (e.g. prednisolone, hydrocortisone), calcineurin inhibitors (e.g. cyclosporin, tacrolimus) anti-proliferative agents (e.g. azathioprinem, mycophenolic acid) and/or mTOR inhibitors (e.g. sirolimus, everolimus).
In some embodiments, the methods to treat/prevent alloreactivity and/or graft rejection comprise antibody therapy, such as treatment with monoclonal anti-IL-2Ra receptor antibodies (e.g. basiliximab, daclizumab), anti-T cell antibodies (e.g. anti-thymocyte globulin, anti-lymphocyte globulin) and/or anti- CD20 antibodies (e.g. rituximab).
In some embodiments, the methods to treat/prevent alloreactivity and/or graft rejection comprise blood transfusion and/or bone marrow transplantation.
Where a method is disclosed herein, the present disclosure also provides the CAR-expressing immune cells and compositions of the present disclosure for use in such methods. Also provided is the use of the CAR-expressing immune cells or compositions of the present disclosure in the manufacture of products (e.g. medicaments) for use in such methods.
In some embodiments, the methods of various aspects of the present disclosure cause less depletion and/or increased survival of non-alloreactive immune cells as compared to methods employing immunosuppressive agent(s). For example, the present methods are useful for preserving/maintaining the non-alloreactive immune cell compartment in a recipient subject for an allotransplant, or in an allotransplant.
In some embodiments of the methods of the present disclosure comprising allotransplantation, the present methods are associated with an increased number/proportion of non-alloreactive immune cells in the recipient subject for the allotransplant as compared to methods involving treatment with an immunosuppressive agent. In some embodiments of the methods of the present disclosure comprising adoptive transfer of allogeneic immune cells, the present methods are associated with an increased number/proportion of non-alloreactive immune cells in the recipient subject for the allogeneic immune cells as compared to methods involving treatment with an immunosuppressive agent. In some embodiments of the methods of the present disclosure comprising allotransplantation, the present methods are associated with an increased number/proportion of non-alloreactive immune cells in the allotransplant as compared to methods involving treatment with an immunosuppressive agent.
The present disclosure also provides the CAR-expressing immune cell or composition of the present disclosure for use in a method of: killing a cell expressing the target antigen for which the CAR is specific (e.g. a cell expressing CD30); and/or killing an alloreactive immune cell (e.g. a T cell expressing CD30).
The present disclosure also provides the use of such CAR-expressing immune cells and compositions in such methods, and methods using the CAR-expressing immune cell and compositions to such ends.
Administration
Administration of the articles of the present disclosure is preferably in a ‘therapeutically-effective’ or ‘prophylactically-effective’ amount, this being sufficient to show therapeutic or prophylactic benefit to the subject. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease/condition and the particular article administered. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease/disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s ‘The Science and Practice of Pharmacy’ (ed. A. Adejare), 23rd Edition (2020), Academic Press.
Administration of the articles of the present disclosure may be topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal or transdermal. Administration may be by injection or infusion. Administration of the articles of the present disclosure may be intratumoral.
In some aspects and embodiments in accordance with the present disclosure there may be targeted delivery of articles of the present disclosure, i.e. wherein the concentration of the relevant agent in the subject is increased in some parts of the body relative to other parts of the body. In some embodiments, the methods comprise intravenous, intra-arterial, intramuscular or subcutaneous administration and wherein the relevant article is formulated in a targeted agent delivery system. Suitable targeted delivery systems include, for example, nanoparticles, liposomes, micelles, beads, polymers, metal particles, dendrimers, antibodies, aptamers, nanotubes or micro-sized silica rods. Such systems may comprise a magnetic element to direct the agent to the desired organ or tissue. Suitable nanocarriers and delivery systems will be apparent to one skilled in the art.
In some cases, the articles of the present disclosure are formulated for targeted delivery to specific cells, a tissue, an organ and/or a tumor. Further intervention
Administration may be alone or in combination with other treatments, either simultaneously or sequentially dependent upon the disease/condition to be treated. The antigen-binding molecule, CAR, cell or composition described herein and another prophylactic/therapeutic agent may be administered simultaneously or sequentially.
In some embodiments, the methods comprise additional therapeutic or prophylactic intervention, e.g. for the treatment/prevention of a cancer. In some embodiments, the therapeutic or prophylactic intervention is selected from chemotherapy, immunotherapy, radiotherapy, surgery, vaccination and/or hormone therapy. In some embodiments, the therapeutic or prophylactic intervention comprises leukapheresis. In some embodiments, the therapeutic or prophylactic intervention comprises a stem cell transplant.
Simultaneous administration refers to administration of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition and therapeutic agent together, for example as a pharmaceutical composition containing both agents (combined preparation), or immediately after each other and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel. Sequential administration refers to administration of one of the antigen-binding molecule/composition or therapeutic agent followed after a given time interval by separate administration of the other agent. It is not required that the two agents are administered by the same route, although this is the case in some embodiments. The time interval may be any time interval.
In some embodiments, treatment of cancer further comprises chemotherapy and/or radiotherapy. Chemotherapy and radiotherapy respectively refer to treatment of a cancer with a drug or with ionising radiation (e.g. radiotherapy using X-rays or y-rays). The drug may be a chemical entity, e.g. small molecule pharmaceutical, antibiotic, DNA intercalator, protein inhibitor (e.g. kinase inhibitor), or a biological agent, e.g. antibody, antibody fragment, aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide, or protein. The drug may be formulated as a pharmaceutical composition or medicament. The formulation may comprise one or more drugs (e.g. one or more active agents) together with one or more pharmaceutically acceptable diluents, excipients or carriers.
Chemotherapy may involve administration of more than one drug. A drug may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
The chemotherapy may be administered by one or more routes of administration, e.g. parenteral, intravenous injection, oral, subcutaneous, intradermal or intratumoral.
The chemotherapy may be administered according to a treatment regime. The treatment regime may be a pre-determined timetable, plan, scheme or schedule of chemotherapy administration which may be prepared by a physician or medical practitioner and may be tailored to suit the patient requiring treatment. The treatment regime may indicate one or more of: the type of chemotherapy to administer to the patient; the dose of each drug or radiation; the time interval between administrations; the length of each treatment; the number and nature of any treatment holidays, if any etc. For a co-therapy a single treatment regime may be provided which indicates how each drug is to be administered.
Chemotherapeutic drugs may be selected from: Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE- PC, AC, Acalabrutinib, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aliqopa (Copanlisib Hydrochloride), Alkeran for Injection (Melphalan Hydrochloride), Alkeran Tablets (Melphalan), Aloxi (Palonosetron Hydrochloride), Alunbrig (Brigatinib), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, Avastin (Bevacizumab), Avelumab, Axicabtagene Ciloleucel, Axitinib, Azacitidine, Bavencio (Avelumab), BEACOPP, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Besponsa (Inotuzumab Ozogamicin) , Bevacizumab, Bexarotene, Bexxar (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometyx (Cabozantinib-S- Malate), Cabozantinib-S-Malate, CAF, Calquence (Acalabrutinib), Campath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride), Capecitabine, CAPOX, Carac (Fluorouracil-Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine Implant, Casodex (Bicalutamide), CEM, Ceritinib, Cerubidine (Daunorubicin Hydrochloride), Cervarix (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), CMF, Cobimetinib, Cometriq (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine Liposome, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Daratumumab, Darzalex (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), DTIC-Dome (Dacarbazine), Durvalumab, Efudex (Fluorouracil-Topical), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Elotuzumab, Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Empliciti (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista (Raloxifene Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU (Fluorouracil Injection), 5-FU (Fluorouracil-Topical), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil-Topical), Fluorouracil Injection, Fluorouracil-Topical, Flutamide, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINEOXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (Carmustine Implant), Gliadel wafer (Carmustine Implant), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Hemangeol (Propranolol Hydrochloride), Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin (Topotecan Hydrochloride), Hydrea (Hydroxyurea), Hydroxyurea, Hyper-CVAD, Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Idamycin (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelal isib, Idhifa (Enasidenib Mesylate), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, lnterleukin-2 (Aldesleukin), Intron A (Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, Istodax (Romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), JEB, Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lartruvo (Olaratumab), Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Leustatin (Cladribine), Levulan (Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), Lomustine, Lonsurf (Trifluridine and Tipiracil Hydrochloride), Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide, Mexate (Methotrexate), Mexate-AQ (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate), Necitumumab, Nelarabine, Neosar (Cyclophosphamide), Neratinib Maleate, Nerlynx (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide, Ninlaro (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride, Onivyde (Irinotecan Hydrochloride Liposome), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-lntron (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Portrazza (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R- CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, Relistor (Methylnaltrexone Bromide), R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, R-ICE, Rituxan (Rituximab), Rituxan Hycela (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and Hyaluronidase Human, Rolapitant Hydrochloride, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, Rydapt (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa- 2b), Sylvant (Siltuximab), Synribo (Omacetaxine Mepesuccinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq (Atezolizumab), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, Tolak (Fluorouracil-Topical), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and Iodine I 131 Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Unituxin (Dinutuximab), Uridine Triacetate, VAC, Valrubicin, Valstar (Valrubicin), Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride), Vectibix (Panitumumab), VelP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, Venclexta (Venetoclax), Venetoclax, Verzenio (Abemaciclib), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, Vistogard (Uridine Triacetate), Voraxaze (Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride), Vyxeos (Daunorubicin Hydrochloride and Cytarabine Liposome), Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda (Capecitabine), XELIRI, XELOX, Xgeva (Denosumab), Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab), Yescarta (Axicabtagene Ciloleucel), Yondelis (Trabectedin), Zaltrap (Ziv-Aflibercept), Zarxio (Filgrastim), Zejula (Niraparib Tosylate Monohydrate), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zofran (Ondansetron Hydrochloride), Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), Zydelig (Idelalisib), Zykadia (Ceritinib) and Zytiga (Abiraterone Acetate).
In some embodiments, the treatment may comprise administration of a corticosteroid, e.g. dexamethasone and/or prednisone.
In some embodiments, a subject is administered lymphodepleting chemotherapy prior to administration of immune cells expressing/comprising a CAR described herein (or expressing/comprising nucleic acid encoding such a CAR).
That is, in some embodiments, methods of treating/preventing a disease/condition in accordance with the present disclosure comprise: (i) administering a lymphodepleting chemotherapy to a subject, and (ii) subsequently administering an immune cell expressing/comprising a CAR according to the present disclosure, or expressing/comprising a nucleic acid encoding a CAR according to the present disclosure.
As used herein, ‘lymphodepleting chemotherapy’ refers to treatment with a chemotherapeutic agent which results in depletion of lymphocytes (e.g. T cells, B cells, NK cells, NKT cells or innate lymphoid cell (ILCs), or precursors thereof) within the subject to which the treatment is administered. A ‘lymphodepleting chemotherapeutic agent’ refers to a chemotherapeutic agent which results in depletion of lymphocytes.
Lymphodepleting chemotherapy and its use in methods of treatment by adoptive cell transfer are described e.g. in Klebanoff et al., Trends Immunol. (2005) 26(2):111 -7 and Muranski et al., Nat Clin Pract Oncol. (2006) (12):668-81 , both of which are hereby incorporated by reference in their entirety. The aim of lymphodepleting chemotherapy is to deplete the recipient subject’s endogenous lymphocyte population.
In the context of treatment of disease by adoptive transfer of immune cells, lymphodepleting chemotherapy is typically administered prior to adoptive cell transfer, to condition the recipient subject to receive the adoptively transferred cells. Lymphodepleting chemotherapy is thought to promote the persistence and activity of adoptively transferred cells by creating a permissive environment, e.g. through elimination of cells expressing immunosuppressive cytokines, and creating the ‘lymphoid space’ required for expansion and activity of adoptively transferred lymphoid cells.
Chemotherapeutic agents commonly used in lymphodepleting chemotherapy include e.g. fludarabine, cyclophosphamide, bedamustine and pentostatin.
Multiple doses of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition may be provided. One or more, or each, of the doses may be accompanied by simultaneous or sequential administration of another therapeutic agent. Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1 , 2, 3, 4, 5, or 6 months. By way of example, doses may be given once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).
In accordance with various aspects of the present disclosure, a method of treating and/or preventing a disease/condition may comprise one or more of the following: reducing the number/proportion of cells expressing the target antigen for the CAR (e.g. CD30); inhibiting tumor growth (e.g. of a tumor expressing the target antigen for the CAR); reducing metastasis of a cancer (e.g. a cancer expressing the target antigen for the CAR); increasing survival of a subject having a cancer (e.g. a cancer expressing the target antigen for the CAR).
Subjects
A subject in accordance with the various aspects of the present disclosure may be any animal or human. Therapeutic and prophylactic applications may be in human or animals (veterinary use).
The subject to be administered with an article of the present disclosure (e.g. in accordance with therapeutic or prophylactic intervention) may be a subject in need of such intervention. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient.
A subject may have (e.g. may have been diagnosed with) a disease or condition described herein, may be suspected of having such a disease/condition, or may be at risk of developing/contracting such a disease/condition. In embodiments according to the present disclosure, a subject may be selected for treatment according to the methods based on characterisation for one or more markers of such a disease/condition.
In some embodiments, a subject may be selected for therapeutic or prophylactic intervention as described herein based on the detection of cells/tissue expressing the target antigen for the CAR (e.g. CD30), or of cells/tissue overexpressing the target antigen for the CAR, e.g. in a sample obtained from the subject.
A subject may be an allogeneic subject with respect to an intervention in accordance with the present disclosure. A subject to be treated/prevented in accordance with the present disclosure may be genetically non-identical to the subject from which the CAR-expressing immune cells are derived. A subject to be treated/prevented in accordance with the present disclosure may be HLA mismatched with respect to the subject from which the CAR-expressing immune cells are derived. A subject to be treated/prevented in accordance with the present disclosure may be HLA matched with respect to the subject from which the CAR-expressing immune cells are derived.
The subject to which cells are administered in accordance with the present disclosure may be allogeneic/non-autologous with respect to the source from which the cells are/were derived. The subject to which cells are administered may be a different subject to the subject from which cells are/were obtained for the production of the cells to be administered. The subject to which the cells are administered may be genetically non-identical to the subject from which cells are/were obtained for the production of the cells to be administered.
The subject to which cells are administered may comprise MHC/HLA genes encoding MHC/HLA molecules which are non-identical to the MHC/HLA molecules encoded by the MHC/HLA genes of the subject from which cells are/were obtained for the production of the cells to be administered. The subject to which cells are administered may comprise MHC/HLA genes encoding MHC/HLA molecules which are identical to the MHC/HLA molecules encoded by the MHC/HLA genes of the subject from which cells are/were obtained for the production of the cells to be administered.
In some embodiments, the subject to which cells are administered is HLA matched with respect to the subject from which cells are/were obtained for the production of the cells to be administered. In some embodiments, the subject to which cells are administered is a near or complete HLA match with respect to the subject from which cells are/were obtained for the production of the cells to be administered.
In some embodiments, the subject is a >4/8 (i.e. 4/8, 5/8, 6/8, 7/8 or 8/8) match across HLA-A, -B, -C, and -DRB1 . In some embodiments, the subject is a >5/10 (i.e. 5/10, 6/10, 7/10, 8/10, 9/10 or 10/10) match across HLA-A, -B, -C, -DRB1 and -DQB1 . In some embodiments, the subject is a >6/12 (i.e. 6/12, 7/12, 8/12, 9/12, 10/12, 1 1 /12 or 12/12) match across HLA-A, -B, -C, -DRB1 , -DQB1 and -DPB1 . In some embodiments, the subject is an 8/8 match across HLA-A, -B, -C, and -DRB1 . In some embodiments, the subject is a 10/10 match across HLA-A, -B, -C, -DRB1 and -DQB1 . In some embodiments, the subject is a 12/12 match across HLA-A, -B, -C, -DRB1 , -DQB1 and -DPB1 .
Kits
In some aspects of the present disclosure a kit of parts is provided. In some embodiments, the kit may have at least one container having a predetermined quantity of a CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.
In some embodiments, the kit may comprise materials for producing a CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.
The kit may provide the CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition together with instructions for administration to a patient in order to treat a specified disease/condition.
In some embodiments the kit may further comprise at least one container having a predetermined quantity of another therapeutic agent (e.g. as described herein). In such embodiments, the kit may also comprise a second medicament or pharmaceutical composition such that the two medicaments or pharmaceutical compositions may be administered simultaneously or separately such that they provide a combined treatment for the specific disease or condition. Kits according to the present disclosure may include instructions for use, e.g. in the form of an instruction booklet or leaflet. The instructions may include a protocol for performing any one or more of the methods described herein.
5 Sequence identity
As used herein, ‘sequence identity’ refers to the percent of nucleotides/amino acid residues in a subject sequence that are identical to nucleotides/amino acid residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignment for the purposes of determining
10 percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951 -960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780) software. When
15 using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.
Sequences Table A
92
SUBSTITUTE SHEET (RULE 26) Table B
5
94
SUBSTITUTE SHEET (RULE 26) Table C ***
The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.
The section headings used herein are for organisational purposes only and are not to be construed as limiting the subject matter described.
Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.
Throughout this specification, including the claims which follow, unless the context requires otherwise, the word ‘comprise,’ and variations such as ‘comprises’ and ‘comprising,’ will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
As used herein, an amino acid sequence, or a region of a polypeptide which ‘corresponds’ to a specified reference amino acid sequence or region of a polypeptide has at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the amino acid sequence/polypeptide/region. An amino acid sequence/region/position of a polypeptide/amino acid sequence which ‘corresponds’ to a specified reference amino acid sequence/region/position of a polypeptide/amino acid sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g. using sequence alignment software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951 -960).
It must be noted that, as used in the specification and the appended claims, the singular forms ‘a,’ ‘an,’ and ‘the’ include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from ‘about’ one particular value, and/or to ‘about’ another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about,’ it will be understood that the particular value forms another embodiment.
Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.
Methods described herein may preferably be performed in vitro. The term ‘in vitro’ is intended to encompass procedures performed with cells in culture whereas the term ‘in vivo’ is intended to encompass procedures with/on intact multi-cellular organisms.
Brief Description of the Figures
Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying figures. Figures 1A to 1C. Expression and functional assessment of novel alternative spacers in CD30 CAR. (1 A) Expression of CD30 CARs with the various spacers on surface of transduced primary T cells. (1B) Cytotoxic efficacy of CAR T cells expressing CARs with the different spacer with CD30+ KM-H2 cells. (1C) Basal GM-CSF, interferon-y and TNF-a cytokine secretion of CD30 CAR T cells with various spacers, with or without simulation with KM-H2 cells for 24hr.
Figure 2A to 2C. Functional assessment of novel alternative spacers in CD30 CAR with Raji expressing different CD30 densities. (2A) Flow cytometry analysis of transduced Raji clones 1 B1 , 1 C4 and 2C5 expressing varying levels of CD30, in comparison to CD30 expressing KM-H2 cells. (2B) Cytotoxicity assay of Raji clones expressing varying levels of CD30 with CD30 CAR T cells expressing IgG 1 , lgG2, 0X40, 4-1 BB, CD96 and CD33 spacers at effector target ratio of 1 :1 . (2C) Inteferon-y, GM- CSF and TNF-a secretion by CD30 CAR T cells expressing the different spacers upon activation with the different CD30+ cell lines Data is representative of 3 independent donors tested.
Figures 3A to 3C. Expression and functional assessment of CARs expressing novel 4-1 BB spacer with humanized CD30 scFv. (3A) Expression of humanized CD30 CARs with the 4-1 BB spacer on surface of transduced primary T cells. (3B) Cytotoxic efficacy of CAR T cells expressing CARs with 4-1 BB spacer. (3C) Levels of secretion of GM-CSF and TNF-a by resting CAR T cells expressing humanized CARs with 4-1 BB.
Figures 4A to 4G. Co-culture assay of autologous primary monocytes and natural killer cells with CAR T cells expressing CARs with various spacers. (4A) Experiment layout of assay assessing monocytes interaction with CAR T cells expressing CARs with various spacers. (4B) Cytotoxicity against monocytes measured by flow cytometry. (4C) Immune checkpoint molecules expressed by CD30 CAR T cells with different spacers following co-culture with autologous monocytes. (4D) Cytokines detected in co-cultures of monocytes and CD30 CAR T cells with different spacers. (4E) Experiment layout of assay assessing NK cells interaction with CAR T cells expressing CARs with various spacers. (4F) Cytotoxicity against NK cells measured by flow cytometry. (4G) CD16 expression on NK cells and number of CD16 high NK cells remaining after co-culture with CD30 CAR T cells bearing different spacers.
Figure 5A to 5F. In vivo persistence and efficacy of CD30 CAR ATCs with novel spacers against NK-T Lymphoma cell line. (5A) Experiment scheme for SNK6 model. (5B) IVIS monitoring twice a week to track T cells biodistribution. (5C) Average radiance of transferred T cells in tumor following treatment (5D) Endpoint average radiance measured by IVIS and flow cytometric quantification of T cells in tumors. (5E) T cell counts in blood, spleen, and liver quantified by flow cytometry. (5F) Endpoint tumor counts quantified by flow cytometry.
Figures 6A to 6E. In vivo persistency, efficacy, and toxicity of CD30 CAR ATCs in humanized mice xenografted with peripheral T cell lymphoma. (6A) Experiment set up of human peripheral T cell lymphoma model. (6B) IVIS monitoring twice a week to track T cells biodistribution and (6C) Endpoint average radiance measured by IVIS. (6D) Absolute tumor counts quantified by flow cytometry. (6E) Cytokine levels in mice plasma at day 6 post-treatment.
Figures 7A to 7G. In vivo persistency, efficacy, and toxicity of CD30 CAR ATCs in humanized mice supplemented with human GM-CSF and IL-3. (7A) Experiment set up of CD30 high NALM-6 model. (7B) Circulating IgG levels at baseline and 3 days post I VIG administration. (7C) Body weight changes after T cell treatment. (7D) IVIS image taken twice a week to track NALM-6 disease. (7E) CD30 CAR T cell present in organs at endpoint. (7F) Immune cell subsets present in organs at endpoint. (7G) Cytokine levels in mice plasma 8 days post treatment.
Figures 8A to 8E. Generation and characterisation of T cells bearing humanized CD30 CAR with the 4-1 BB spacer. (8A) Fold expansion in culture of untransduced T cells and T cells expressing HRS3- 41 BB, VH3Vk3-Cys-41 BB, or VH5Vk3-Cys-41 BB. (8B) Transduction efficiency of the CD30CAR variants at day 11 . (8C) Ratio of CD4 T cells to CD8 T cells in T cells transduced with the CD30CAR variants at day 11 . Each color represents a unique donor that is consistent across 8A to 8C. (8D) CD30 expression detected by the BerH8 and BY88 clones on total T cells over the culture period to demonstrate CD30 masking in cis by the CAR. (8E) Proportions of Tim3, PD-1 , LAG3 expressing cells and combinations thereof among CD4 and CD8 T cells in the day 11 final product.
Figures 9A to 9D. In vitro anti-tumor activity of T cells bearing humanized CD30 CAR with the 4- 1 BB spacer. (9A) Cytolysis of KM-H2 and HuT-78 cells were assessed using the xCELLigence Real-Time Cell Analysis system at 2:1 E:T ratio for 4 and 5 donors respectively. (9B) Serial killing assay set-up. See Example 1 . (9C) Serial killing cytolysis for 3 donors. (9D) Total T cell numbers at the end of each serial killing encounter.
Figures 10A to 10F. In vitro on-target, off-tumor activity of T cells bearing humanized CD30 CAR with the 4-1 BB spacer. (10A) CD30 expression on HSPCs from 2 donors over 12 days of stimulation with 10 ng/mL FLT3L, SCF and TPO. (10B) CD30 expression on KM-H2 cells. (10C) CD30 expression on HSPC subsets after 2 days of stimulation. (10D) Impact of CD30 CAR T cell exposure on the survival of HSPC subsets. Two HSPC donors and two T cell donors were combined to obtain four co-culture combinations for 10D to 10F. (10E) Effect of CD30 CAR T cell exposure on the erythroid and myeloid developmental potential of HSPCs. (10F) Effect of priming CD30 CAR T cells with CD30-high KM-H2 targets on cytolysis of CD30-low HSPCs.
Figures 11 A to 11G. In vivo persistency, efficacy, and toxicity of humanized CD30 CAR T cells in GM-
CSF and IL-3-supplemented humanized mice xenografted with peripheral T cell lymphoma. (11 A) Experiment set up of HuT-78 model. (11B) Tumor volume, tumor volume changes and endpoint tumor count. (11C) IVIS monitoring twice a week to track T cell biodistribution. (11D) Survival curves of mice following treatment. (11 E-11F) Immune cell subsets present in organs at endpoint. (11G) Cytokine levels in mice plasma at 8 days post treatment. Figures 12A to 12F. In vivo persistency, efficacy, and toxicity of humanized CD30 CAR T cells in GM- CSF and IL-3-supplemented humanized mice xenografted with CD30high NALM-6. (12A) Experiment set up of NALM-6 model. (12B) Body weight changes in mice. (12C) I VIS monitoring twice a week to track NALM-6 disease (12D-12E) Immune cell subsets present in organs at endpoint. (12F) Cytokine levels in 5 mice plasma at 11 days post treatment.
Figures 13A to 13C. Characterisation of EBVSTs transduced to express humanized CD30 CARs. (13A) Expression and transduction efficiency of CD30 CARs in EBVSTs. (13B) Masking efficiency of CD30 CARs in EBVSTs. (13C) Cytotoxic efficacy and specificity of CD30 CAR EBVSTs with CD30+ KM- 0 H2 and CD30- Daudi cells.
Examples
In the following Examples, the inventors describe the generation and characterisation of novel CAR constructs. 5
Example 1 : Materials and Methods
1 .1 Plasmid constructs and retrovirus production
Murine HRS3 scFv and humanised versions thereof were cloned into a pSFG retrovirus vector upstream of either a wildtype IgG 1 Fc, 0X40 or 4-1 BB derived spacer, followed by CD28 0 transmembrane domain, a CD28 and CD3 signalling domains. A truncated form of the CD30 molecule that consisted only of the CD30 extracellular domain was cloned into pSFG retrovirus vector.
Retroviruses carrying the CD30 CARs or truncated CD30 were produced in HEK293VG or RD114 packaging cell line (BioVec Pharma, Quebec, Canada) by transient transfection with the pSFG vector using PEIpro transfection reagent (Polyplus, lllkirch, FRANCE). Medium containing
5 retroviruses were harvested at 48h and 72h post transfection and concentrated 10-fold using RetroX Concentrator (Takara Bio, Kusatsu, Shiga, Japan). The retroviruses were either used immediately or snap frozen and stored at -80°C.
The stable RD114 retrovirus packaging cell line that produces high titers of GFP-Firefly Luciferase 0 (GFP-FFIuc) virus particles was a kind gift from Dr Masataka Suzuki (Baylor Center for Gene Therapy Baylor College of Medicine).
1 .2 Donors
Enriched leukapheresis products, collected from consented healthy donors by Spectra Optia® 5 Apheresis System CMNC collection protocol and frozen in ACD-A anticoagulant, was purchased from HemaCare (Northridge, California, U.S.A.). The frozen leukopaks were thawed and PBMCs were extracted by gradient centrifugation using Ficoll-Paque PLUS (Cytiva, MA, U.S.A.). The PBMCs were either used immediately for experiments or frozen in smaller aliquots of 30-50 x 106 cells per cryovial in CryoStor® CS10 Cell Freezing Medium (STEMCELL Technologies, Cambridge, 0 Massachusetts, U.S.A.).
Cord blood CD34+ cells, isolated from cord blood mononuclear cells via positive immunomagnetic separation from consented donors, were purchased from Lonza (Walkersville, Maryland, U.S.A). Frozen vials of up to 1 x 106 cells were thawed and CD34+ cells were either used immediately for 5 experiments or frozen in aliquots of 5 x 104 cells per cryovial in CryoStor® CS10 Cell Freezing Medium (STEMCELL Technologies, Cambridge, Massachusetts, U.S.A.). 1 .3 Production of CAR T cells
For activated T cells (ATC) transduction, PBMC were thawed and plated onto cell culture plates precoated with anti-CD3/CD28, and cultured in 10% FBS, 45% Advanced RPMI and 45% Click’s media with 5% CO2 at 37°C to generate ATCs. Two days after culture, IL-7 and IL-15 were added into cell culture. On the third day of culture, retrovirus containing CD30.CAR with the indicated scFv and spacer was transduced into ATCs by spinfection. Retrovirus was washed off 24 hours later and ATCs were cultured with occasional media change to replenish the IL-7 and IL-15. For cells to be used in in vivo solid tumor models, ATCs were additionally transduced on the fourth day of culture with a retroviral vector encoding GFP-Firefly Luciferase (GFP-FFIuc) to enable tracking of ATCs by IVIS in live mice. On day 11 post-transduction, ATCs were either frozen down using CryoStor following manufacturer’s manual or injected into mice. For humanised mouse studies, ATCs were generated from CD34- cord blood cells instead of PBMCs, and later infused into humanised mice reconstituted with CD34+ cord blood cells from the same donor.
For Epstein Barr virus specific T cells transduction, CD45RA depletion of PBMCs (RAD-PBMCs, optional) was performed by negative selection using CD45RA MACS Beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Whole PBMCs or RAD-PBMCs were cultured 1 x106 cells/well with viral peptides consisting of overlapping peptide libraries (15-mers overlapping by 11 amino acids) from JPT Technologies (Berlin, Germany). Five days later, cells were transduced with humanised CD30 CAR constructs using RetroNectin (Takara Bio, Kusatsu, Shiga, Japan), according to the description above. T cells were then stimulated with irradiated co-stimulatory cells expressing markers such as CD80, CD86, 4-1 BB four days post transduction. Seven-eight days later, VSTs were harvested, frozen or used for cell assays.
1 .4 Transduction efficiency and phenotypinq by flow cytometry and antibody staining
Flow cytometry in this study was performed with the Aurora cytometer (Cytek Biosciences) or with the FACSymphony A3 cell analyzer (BD Biosciences). Up to 200k T-cells were stained with Live/Dead™ NIR viability dye (Thermo Fisher) and assessed for surface presentation of epitopes using fluorescently labeled monoclonal antibodies to CD3 (clone SK7, BD Biosciences), CD4 (clone SK3, BD Biosciences), CD8 (clone SK1 , BioLegend), CD56 (clone B159, BD Biosciences), CD19 (clone SJ25C1 , BioLegend), CD30 (clone BerH8, BD Biosciences; clone BY88, BioLegend), PD-1 (clone EH12.1 , BD Biosciences), Tim3 (clone 7D3, BD Biosciences), and LAG3 (clone 11 C3C65, BioLegend). CD30 CAR expression was measured with recombinant human CD30 protein fused with a His tag (10777-H08H, Sino Biological) followed by PE-conjugated anti-His (clone J095G46, BioLegend), or with biotinylated recombinant human CD30 protein (ACROBiosystems, CD0-H82E6) followed by PE-conjugated streptavidin (BD Biosciences, 554061 ), or with FITC-labeled recombinant human CD30 protein (ACROBiosystems, CD0-HF2H3). Flow data were analyzed and gated in FlowJo v10.8.1 for Windows.
1 .5 Cytotoxicity Assay
Prior to assessment of CAR potency via cytotoxicity assay, the cell media is changed to 2% assay media containing RPMI and 2% FBS. Cytotoxicity assay was carried out using the xCelligence Real- Time Cell Analysis System with 5% CO2 at 37°C (Agilent). Target cells will be added onto PET plates tethered with anti-CD40 antibody following manufacturer’s manual (Aligent). 24 hours after target cell adherence, CAR T cells will be plated into the PET plates at CAR T: target cell ratio of 0.2:1 , 1 :1 or 5:1 . The PET plates will return into the xCelligence system and cytotoxicity will be monitored for 48 hours.
1 .6 Serial Killing Potency Assay
KM-H2 cells were used as the target cell line with an initial E:T ratio of 1 :2, with 50,000 effector and 100,000 target cells per well of a 96-well plate for Encounter 1 . Three sets of this set-up were prepared. After 48h, cells from 2 sets were harvested and added to 2 new sets of 100,000 KM-H2 target cells per well to set up Encounter 2. After a further 48h, cells from 1 set were harvested and added to a new set of 100,000 KM-H2 target cells per well for Encounter 3. To distinguish target cells from the different encounters, KM-H2 cells for Encounters 2 and 3 were labelled with the lipophilic membrane dyes PKH67 and PKH26 (Sigma-Aldrich) respectively. 48h after each encounter, 1 set of cells was stained with Live/Dead™ NIR viability dye (Thermo Fisher) and fluorescently labelled monoclonal antibodies to CD3 (clone UCHT1 , BD Biosciences), CD4 (clone SK3, BD Biosciences), CD8 (clone RPA-T8, BD Biosciences), CD40 (clone 5C3, BioLegend), and CD30 (clone BerH8, BD Biosciences) to enumerate the target cell populations remaining at each round of 48h culture. See Figure 9B for schematic diagram of assay set-up. Cytolysis was calculated as (KM-H2 count in target-only control well - KM-H2 count in assay well) / KM-H2 count in target- only control well.
1 .7 Cytokine Release Assay
Prior to assessment of cytokine release, the cell media is changed to 2% assay media containing RPMI and 2% FBS. Target cells will be added onto 96-well flat-bottom plates. CAR T cells will be plated on top of the target cells at CAR T: target cell ratio 1 :1 . The plates will be incubated at 5% CO2 at 37°C for 24 hours. After 24 hours, the cell-free media will be collect by centrifugation at 500 g for 5 mins. The cytokine released in the media, such as TNFa, GMCSF and IFNy will be assayed by ELISA kits from BioLegends.
1 .8 Luminex Multiplex Assay
Milliplex map human high sensitivity T cell panel premixed 13-plex (Millipore, HSTCMAG28SPMX13) was used to determine cytokine levels in co-cultures or plasma by FLEXMAP 3D® (Luminex). Analysis was performed using Bio-plex Manager software (Bio-Rad)
1 .9 In v/fro safety assays with hematopoietic stem and progenitor cells (HSPCs)
CD34+ HSPCs were stimulated with 10 ng/mL each of Flt3-ligand (FLT3L), stem cell factor (SCF) and thrombopoietin (TPO) (all from Miltenyi Biotec) for the specified time period at 5,000 - 20,000 cells per 200pL per well of a 96-well U-bottom plate.
In co-cultures of CD34+ HSPCs and T cells, CD34+ cells were isolated by depletion of T cells using magnetic beads conjugated to an anti-CD3 antibody (Miltenyi Biotec). HSPC subsets were analyzed by flow cytometry after staining with Live/Dead™ NIR viability dye (Thermo Fisher) and fluorescently labelled monoclonal antibodies to CD34 (clone 561 , BD Biosciences), CD133 (clone 7, BioLegend), CD45RA (clone HI100, BD Biosciences), CD38 (clone HIT2, BD Biosciences), CD10 (clone HI10a, BD Biosciences) following the gating strategy described in Hornbach et al (18).
Erythroid and myeloid developmental potential were assessed using the StemMACS™ HSC-CFU Assay kit (Miltenyi Biotec, 130-125-042). Cells were labelled with fluorescently conjugated antibodies to CD14, CD15 and CD235a as part of the StemMACS kit antibody cocktail, and colony types were identified as per the manufacturer’s protocol. Colony types include colony forming units for granulocytes (CFU-G) and macrophages (CFU-M), burst forming units for erythrocytes (BFU-E). For more primitive progenitors, CFU-GM give rise to both granulocytes and macrophages, while CFU- GEMM differentiate into all three cell populations.
1 .10 Extranodal NK-T lymphoma mouse model
In the extranodal NK-T cell lymphoma model, 2x106 of SNK-6 cells were injected into right flank of NOD-sc/c/ IL2Rgammanul1 (NSG) mice subcutaneously. On day 15, mice were randomized into treatment groups, stratified by tumor volume. On day 16, 4x106 of GFP-luciferase tagged ATCs were injected into mice intravenously. IL-2 (1000 IU per mouse) was delivered via intra-peritoneal injection for all mice. Mouse Fc receptor blocking antibody (Bio X Cell, Cat#:BE0307) was administered to mice receiving lgG1 CH2CH3 spacer CD30 CAR at a dose of 8 pg drug per 1 g of body weight on alternate days. Mouse Fc receptor blocking antibody was injected to prevent non-specific interaction between IgG 1 CH2CH3 domain with murine Fc receptor bearing immune cells. T cell biodistribution were evaluated using IVIS imaging system (Perkin Elmer). Region of interest (ROI) was drawn over the mice and the average radiance (p/s/cm2/sr) was quantified using Living lmage®4.7.4 software. On day 36, When mice were sacrificed on day 36, blood, spleen, liver, and tumor were collected for endpoint flow cytometric analysis.
1 .1 1 IL-3/GM-CSF-supplemented humanised mice
To generate humanised mice, NSG pups were irradiated and reconstituted with 1 x105 CD34+ cells from HLA-typed cord blood donors. On week 16, human IL-3/GM-CSF encoding plasmids were delivered via hydrodynamic tail vein injection to mice with more than 20% of hCD45 reconstitution, to support myeloid cells reconstitution. Cheek bleed was performed on humanised mice beforehand, for assessment of baseline human cytokine levels in serum.
1 .12 Peripheral T cell lymphoma mouse model
1 x106 of HuT-78 were injected subcutaneously into the right flank of humanised mice. On day 4 or 6, mice were injected intravenously with 1 x106 or 4x106 of CAR and luciferase double transduced ATCs T cells as specified in figures. For the CD30CAR spacer studies, IL-2 and mouse FcR block injection were administered in a regimen similar to the NK/T lymphoma model. Cheek bleeds were done performed at 6 days post treatment to assess the cytokine levels in the plasma 6 days post ATCs treatment. For the CD30CAR scFv studies, an additional cheek bleed was done at 6 days post treatment. T cell biodistribution was evaluated using the IVIS imaging system (Perkin Elmer). Region of interest (ROI) was drawn over the mice and measured the average radiance (p/s/cm2/sr) was quantified using Living lmage®4.7.4 software. Mice were sacrificed and blood, spleen, liver, and tumor were collected for endpoint flow cytometry analysis. For the CD30CAR scFv studies, bone marrow and lung were harvested as well
1 .13 CD30 positive NALM-6 systemic tumor model Nalm6-CD30high expressing GFP-FFIuc was generated by transducing Nalm6 with retroviral vectors encoding truncated CD30 and GFP-FFIuc. Nalm6 expressing both CD30 and GFP was sorted twice to generate >99% double positive clones. 2x106 of Nalm6-CD30high cells stably expressing GFP- Firefly Luciferase (GFP-FFIuc) were injected intravenously into humanised mice. Tumor engraftment was monitored using I VIS imaging system (Perkin Elmer) once a week. Mice were cheek bled 9 days before T cell treatment to assess cytokine levels in plasma post tumor engraftment. For the CD30CAR spacer studies, mice were injected intra-peritoneally with 35mg human MG (Sigma- Aldrich, I4506) and in some treatment groups with 8pg/g mouse FcR block (Bio X Cell, Cat#:BE0307) for 2 days before ATC treatment. At 20 or 21 days post NALM-6 injection, 5x106 T cells were administered to mice via retroorbital injection. Body weight and temperature were monitored daily and tumor burden was tracked by I VIS twice a week. Cheek bleeds were performed at 2 and 7 days post treatment for the CD30CAR spacer studies, and at 3 and 6 days post treatment for the CD30CAR scFv studies. At the experiment end point, mice were sacrificed and blood, spleen, liver and bone marrow were collected for flow cytometry analysis.
Example 2: Results
2.1 Novel 0X40 and 41 -BB derived spacers retain in vitro killing efficacy with lower cytokine production. The spacer domain can significantly affect the safety and efficacy of the CAR T cell. IgG Fc domain derived spacers have been commonly used in early designs of CARs. However, the use of the wildtype Fc domains can result in undesired interactions with Fc receptor expressing cells. Highly flexible linkers can facilitate the recognition of sterically challenging epitopes while the length of the linkers can optimize the synaptic distance between the CAR T cell and the target cell. Optimization of this synaptic distance can enhance CAR signalling through the exclusion of phosphatases such as CD45 and cytotoxic efficacy by facilitating the efficient delivery of mediators of cell killing such as perforins and granzymes. We thus seek to optimize the CD30 CAR by exploring novel spacers. We designed 4 spacers of different lengths and rigidity originating from various immune receptors, namely 0X40, 4-1 BB, CD44 and CD96 with 0X40 and 4-1 BB spacers are relatively shorter and less glycosylated, hence more flexible, whereas CD44 and CD96 spacers are known to be heavily glycosylated and thus are more rigid structure. None of these sequences are known to interact with their cognate ligands, or any other known receptors and thus are theoretically inert sequences.
Lastly, they do not contain unpaired cysteine residues and are not known to dimerize. For comparison, we included a lgG2 CH2CH3 spacer that is in length and rigidity to the IgG 1 spacer but has lower affinity for the Fc receptors.
The alternative spacers were cloned into the HRS-3 CD30 CAR pSFG retroviral vector. Expression of the transduced CAR T cells with the different spacers was assessed by flow cytometry (Figure 1 A). All CARs regardless of spacer domains were transduced equally efficiently in activated T cells. Next, the efficiency of the various CARs to recognize and kill target cells was also compared using an assay with CD30+ KM-H2 cells at an effector to target ratio of 1 :1 . Aside from CD96 spacer, the alternative spacers exhibited equivalent killing efficiencies as the original lgG1 spacer (Figure 1 B). In addition, we first studies the basal level of cytokine secretion of the CAR T cells with different spacers. In the absence of stimulation, elevated cytokine secretion would suggest instability of the CAR thus resulting tonic activation of the CAR T cells. All CARs with the alternative spacers did not exhibit significant increase in basal secretion of the GM-CSF, interferon-y or TNF-a, suggesting stability of the CARs was not compromised with the novel spacers (Figure 1 C). However, upon activation of CAR T cells with CD30+ KM-H2, secretion of the cytokines by CAR T cells expressing the 0X40, 41 -BB, CD96 spacers were notably lower in all 3 donors tested (Figure 1 C).
Next, the inventors investigated the influence of CD30 antigen density on the efficacy of CD30 CARs expressing the novel spacers. CD30 negative Raji cells were retroviral ly transduced to express a truncated CD30 protein. FACs sorting and single cell cloning was done to identify 3 distinct clones expressing various levels of CD30 (Figure 2A). CAR T cells expressing either the IgG 1 , 0X40 and 4- 1 BB spacers were co-incubated with the different CD30 expressing Raji clones, untransduced Raji or the positive control cell line KM-H2 at an effector to target ratio of 1 :1 (Figure 2B). All CARs expressing the alternative spacers exhibited similar cytotoxic efficiencies with the Raji clones expressing different levels of CD30 (Figure 2C), suggesting that the difference in ability to dimerize did not compromise the sensitivity of the CARs with the alternative spacers to CD30 density.
Secretion of the GM-CSF, interferon-y or TNF-a cytokines by the CARs with the various spacers stimulated by the different CD30+ target cells were compared. Similar to our previous observation with KM-H2 cells, cytokine secretion by CAR T cells expressing the 0X40, 41 -BB, CD96 spacers were all notably lower with the Raji cells expressing various levels of CD30 (Figure 2C).
We next cloned the 4-1 BB spacer into the humanized VH3VK3 and VH5VK3 CARs with and without the Cys23 mutation. We show that the humanized CD30 CARs with the 4-1 BB spacers are well expressed in the transduced primary T cells (Figure 3A) and similarly elicited potent cytotoxicity with CD30 expressing KM-H2 cells at a 1 :1 effector to target ratio (Figure 3B). Resting transduced cell expressing the humanized CARs with the 4-1 BB spacers also preserved their low tonic signalling in the absence of antigen stimulation (Figure 3C).
2.2 CARs with novel 0X40 and 41 -BB derived spacers do not interact non-specifically to monocytes or NK cells
CARs bearing wildtype lgG1 derived spacers readily interact with Fc receptors expressing cells such and monocytes and NK cells. The novel spacers are not known to interact with any receptors or ligands. Next, the potential of the novel spacers to interact with other immune cells was investigated. CD14+ monocytes were isolated from donor PBMCs and incubated with CAR T cells with the different spacers at a T cell: Monocyte ratio of 1 :5. Cytotoxicity of CARs with novel 0X40 and 41 -BB derived spacers against monocytes were assessed by flow cytometry 48hrs or 96 hrs post incubation. At 48hrs, a set of CAR T cells were re-stimulated with fresh monocytes at a T cell: Monocyte ratio of 1 :5. Cytotoxicity against monocytes were assessed by flow cytometry 48hrs post re-stimulation (Figure 4A). CD30 CAR T cells bearing the IgG 1 CH2CH3 domains were observed to induce high cytolysis of monocytes at both 48 and 96 hours post incubation and at 48 hours post- restimulation. In contrast, cytolysis of monocytes by CD30 CAR T cells bearing the lgG2, 0X40, 4- 1 BB, CD96 and CD44 spacer domains was low or absent at all above time points (Figure 4B). Furthermore, cytolysis of monocytes by CD30 CAR T cells bearing the IgG 1 CH2CH3 domain was partially attenuated in the presence of human Fc receptor blocking antibody (Figure 4B), suggesting that interaction between the IgG 1 CH2CH3 domain and the Fc receptor was key for monocyte cytolysis.
Interaction between CD30 CAR T cells with the IgG 1 CH2CH3 domains and monocytes led to increased exhaustion compared to their counterparts with 0X40 and 4-1 BB spacer domains, evident by the higher proportion of CD30 CAR T cells with IgG 1 CH2CH3 spacer co-expressing 3 immune checkpoint molecules (PD-1 , Lag-3, Tim-3) (Figure 4C). Interactions between CD30 CAR T cells with IgG 1 CH2CH3 domains and monocytes also resulted in production of vast amounts of inflammatory cytokines (G-CSF, GM-CSF, IL-6, IL-1 a, TNF-a) in the co-cultures and contrasts to the low amounts detected in monocyte co-cultures with CD30 CAR T cells of 0X40 and 4-1 BB spacers (Figure 4D).
Further experiments to evaluate interactions between CD30 CAR T cells and natural kill (NK) cells via the spacers (Figure 4E) revealed that CD30 CAR T cells with the IgG 1 CH2CH3 domain induced cytolysis of NK cells (Figure 4F), specifically the CD16 high fraction (Figure 4G). In contrast, CD30 CAR T cells with 0X40 and 4-1 BB spacer domains induced little NK cell cytolysis, sparing most CD16 high NK cells (Figures 4F and 4G).
2.3 CD30 CAR T cells with novel spacers have improved persistency and efficacy against human NK-T lymphoma.
We next performed studies in immunodeficient NSG mice to assess the effect of the various spacers on in vivo persistence and efficacy of CD30 CAR T cells against extranodal NK-T cell lymphoma (Figure 5A, Example 1 ).
The presence of the IgG 1 CH2CH3 linker in CAR-T cells has been described to result in CAR-T cell interactions with murine Fc receptor expressing immune cells in immunodeficient mice, resulting in lung sequestration and compromised anti-tumor efficacy (19). This phenomenon is less pertinent in hosts with an intact capacity to produce physiological amounts of immunoglobulin as IgG can occupy Fc receptors on immune cells to reduce availability for interaction with the IgG 1 CH2CH3 domain. To circumvent this phenomenon which may be unique to severely immunodeficient hosts, mouse Fc receptor blocking antibody (clone 2.4.G2) was administered to mice receiving CD30 CAR T cells with lgG1 CH2CH3 domains.
Following tumor engraftment, mice were randomised to receive luciferase expressing CD30 CAR T with various spacers. I VIS monitoring at day 1 post-treatment revealed that all CD30 CAR T cells initially traffic to the lung. However, CD30 CAR T cells with the IgG 1 CH2CH3 spacer quickly disappeared in animals within 4 days post-treatment, consistent with published data (19) that lgG1 CH2CH3 domain interaction with Fc receptor expressing murine cells can greatly compromise CAR T cells in vivo lifespan (Figure 5B). Administration of mouse FcR blocking antibody to mice receiving CD30 CAR T cells with IgG 1 CH2CH3 spacer prolonged persistence of the CAR T cells but did not completely prevent lung trapping and cell elimination (Figure 5B). CD30 CAR T cells with IgG 1 CH2CH3 spacer also failed to migrate into tumors, even in the presence of mouse FcR blocking antibody (Figures 5B and 5C). On the contrary, CD30 CAR T cells with lgG2, 0X40, 41 BB and CD96 spacers persisted and expanded well in animals, with clearly increased numbers over IgG 1 CH2CH3 spacer CD30 CAR T cells up to 20 days post-treatment (Figure 5B). Compared to CD30 CAR T cells with IgG 1 CH2CH3 spacer, lgG2, 0X40, 41 BB and CD96 spacer CD30 CAR T cells had improved migration and expansion within tumors in-situ, with these effects observed up to 20 days post-treatment (Figures 5B and 5C).
Mice were sacrificed for endpoint flow analysis at day 20. Higher number of CD30 CAR T cells bearing the lgG2, 0X40, 41 BB and CD96 domains were observed in tumor samples at endpoint, which is congruent with the I VIS results (Figure 5D). Sizeable populations of CD30 CAR T cells with lgG2, 0X40, 41 BB and CD96 spacers could also be detected in blood, spleen, liver and total body of mice (Figure 5E). Lastly, SNK-6 cells defined as live CD3-, CD45+ and CD30+ cell populations in tumors were observed to trend towards lower numbers in mice which received with 0X40, 41 BB, CD96 and CD44 spacers CD30 CAR T cells (Figure 5F).
2.4 In vivo persistency and efficacy of CD30 CAR T cells with QX-40 and 4-1 BB spacers in humanised mice xenoqrafted with human peripheral T cell lymphoma
In order to further examine the effect of spacers on in vivo persistence, expansion, anti-tumor activity and safety of CD30 CAR T cells, we performed studies in a system where human immune responses can be better recapitulated. We utilised humanised mice which have been reconstituted with human CD34+ cells for a model of peripheral T cell lymphoma. Following tumor engraftment, mice were randomised to receive luciferase expressing CD30 CAR T cells that were generated from the CD34 negative fraction of the same donor used for humanisation of the mice. The CD30 CAR T cells were also transduced with luciferase to enable in vivo cell tracing (Figure 6A; see Example 1 for details). For the same reasons stated above, mouse Fc receptor blocking antibody was also administered to a group of mice receiving lgG1 CH2CH3 CD30 CAR T cells. Due to the encouraging readouts from earlier in vitro experiments, the inventors chose to focus on CD30 CAR T cells with IgG 1 CH2CH3, 0X40 and 41 BB spacers.
Similar to observations in the SNK6 model, all CD30 CAR T cells initially traffic to the lungs on Day 1 . By day 10 post-treatment, CD30 CAR T cells with 0X40 and 41 BB spacers were observed to have undergone systemic expansion with good localisation in tumors (Figures 6B and 6C) In contrast, CD30 CAR T cells with lgG1 CH2CH3 domain showed limited systemic survival and localisation to tumors. Concurrent administration of Fc receptor blocking antibody did not rescue this poor persistence of IgG 1 CH2CH3 spacer CD30 CAR T cells (Figures 6B and 6C). Unsurprisingly, significantly better tumor control was observed in mice which received 0X40 and 41 BB spacer CD30 CAR T cells, evident by the lower number of live HuT-78 cells detected in primary tumors at endpoint (Figure 6D).
Plasma collected at day 6 post-treatment were assessed for cytokines levels using Luminex®. Plasma levels of lnterleukin-6 (IL-6), Interleukin 8 (IL-8), Tumor Necrosis Factor Alpha (TNF- a), Interferon Gamma (IFN-y), Granulocyte macrophage-colony stimulating factor (GM-CSF) and Interleukin 2 (IL-2) remained low in all treatment arms (Figure 6E), demonstrating that 0X40 and 41 BB spacer CD30 CAR T cells in vivo activity and expansion did not induce systemic production of inflammatory cytokines (Figure 6E).
2.5 CD30 CARs with novel 41 -BB spacer have an improved safety profile in high tumor burden disease compared to lqG1 CH2CH3 spacer
High tumor load is a known risk factor for the occurrence of cytokine release syndrome (CRS) associated with CAR T cell therapy in patients with haematological malignancies (20, 21 ). Myeloid cells including monocytes and macrophages have been delineated to be key players in the development of CRS and neurotoxicity after CAR-T cell therapy (22, 23).
With this in mind, we sought to examine safety of CD30 CARs with IgG 1 CH2CH3 and 41 -BB spacers in humanised NSG mice xenografted with CD30-transduced NALM-6 cells. Of note, the mice utilised in this model were supplemented with human GM-CSF and interleukin-3 (IL-3) after humanisation in order to support the stable engraftment of myeloid lineages (refer to Methods and Materials). Disease progression was monitored by bioluminescence imaging of CD30 + NALM-6 cells transduced with firefly luciferase. Treatment was administered when systemic tumor burden in mice was high (Figure 7A).
It has been hypothesised that interactions between the IgG 1 CH2CH3 spacer and Fc receptorexpressing immune cells in human subjects are minimised as physiological levels of circulating immunoglobulin can occupy Fc receptors and reduce their availability to interact with the lgG1 CH2CH3 linker in CAR-T cells. To set up a model recapitulative of patient conditions, we treated all humanised mice with human intravenous immunoglobulin (IVIG) prior to CAR T cell treatment. Humanised mice randomised to receive CD30 CAR T cells with IgG 1 CH2CH3 spacer also received mouse Fc receptor blocking antibody to abrogate interactions with murine Fc receptor-expressing immune cells. Mice were randomised to receive no treatment, untransduced T cells + human IVIG, CD30 CAR T cells with IgG 1 CH2CH3 spacer + human IVIG + murine Fc receptor blocking antibody or CD30 CAR T cells with 4-1 BB spacer + human IVIG (Figure 7A). Plasma IgG levels was assessed 3 days post-IVIG administration, at a time point after IVIG distribution into the extravascular compartment and IgG levels are at steady state. Administration of human IVIG significantly increased plasma levels of IgG from baseline. (Figure 7B).
There were minimal weight changes in untreated mice or mice treated with untransduced T cells, CD30 CAR T cells with 4-1 BB spacers. On the contrary, mice which received with IgG 1 CH2CH3 spacer CD30 CAR T cells experienced progressive weight loss of up to 30% of basal weight. (Figure 7C). Owing to the debilitating weight loss and rapid deterioration in mice that received CD30 CAR T cells with IgG 1 CH2CH3 spacers (Figure 7C), we terminated the experiment at Day 9. While tumor control at day 9 post treatment appeared to be improved in mice that received CD30 CAR T cells with either spacer, this difference compared to untreated or untransduced T cell treatment was not significant (Figure 7D). As tumor control with the NALM-6 leukemia model can take up to 2 weeks in our experience, the early termination of experiment precluded us from understanding if efficacy differences may exist between CD30 CAR T cells with IgG 1 CH2CH3 or 4-1 BB spacers.
We did not observe any differences in IgG 1 CH2CH3 or 4-1 BB spacers CD30 CAR T cell population at endpoint, suggesting that the combined use of human I VIG and murine Fc receptor blocking antibody abated interactions between human and murine Fc receptor expressing immune cells (Figure 7E). Endogenous human T, B, NK and myeloid cells in the femur, liver and spleen of mice were noted to be similar between all treatment groups (Figure 7F), indicating that the unfavourable safety profile observed with CD30 CAR T cells with IgG 1 CH2CH3 spacer in mice were unrelated to any alterations in immune cell populations. Along with the dramatic weight loss, we noted greatly elevated levels of human IL-6, IL-8, IL-10, IFN-y, TNF- a and GM-CSF in mice which received CD30 CAR T cells with IgG 1 CH2CH3 spacer. Of these cytokines, IL-6, IL-8 and TNF- a and GM-CSF have frequently been implicated as soluble mediators of CRS following CAR T cell therapy (22, 23). In comparison, levels of these cytokines remained low in mice from the other treatment groups. We note that these key observation of elevated systemic levels of multiple inflammatory cytokines and rapid deterioration in IgG 1 CH2CH3 spacer CD30 CAR T treated mice cells satisfy the unified criteria for CRS (24).
2.6 Generation and characterisation of T cells bearing humanised CD30 CAR with the 4-1 BB spacer Next the inventors evaluated the phenotype and function of T cells transduced with CAR constructs comprising the 4-1 BB spacer domain and the VH3Vk3-Cys or VH5Vk3-Cys antigen-binding domain. Untransduced or CD30 CAR ATCs were generated from PBMCs (4 donors) and from cord blood- derived CD34- cells (3 donors) for use in experiments involving mice humanised with the same cord blood donors. VH3Vk3-Cys-41 BB and VH5Vk3-Cys-41 BB CD30 CAR ATCs expanded as well as, if not better than HRS3-41 BB CD30 CAR ATCs (Figure 8A). The CD30 CAR variants had high transduction efficiencies of up to 98% (Figure 8B). Where there was variation among the constructs, HRS3-41 BB CD30 CAR T cells had slightly lower frequencies of CAR+ cells. Most donors generated relatively more CD4 than CD8 T cells, with CD4/CD8 ratios largely equivalent among the CD30 CAR variants (Figure 8C).
Upon activation, T cells upregulate CD30, which may bind CD30 CAR-expressing cells and activate them, possibly resulting in fratricide. CD30 CAR binding to CD30 in cis may help to protect cells from inadvertently activating their CD30 CAR-bearing brethren, and thus from fratricide as well. To evaluate the extent of CD30 masking by the CAR molecule, the inventors employed two distinct commercially available anti-CD30 clones, BerH8 and BY88. BerH8 is thought to bind to a similar site of CD30 as the parental HRS3 scFv, as CD30 expression is virtually undetectable by BerH8 once T cells are transduced with HRS3-lgG1 . In contrast, staining CD30 with BY88 yields detectable populations in both untransduced and CD30 CAR T cells. Before transduction (day 0), CD30 expression detected by both BerH8 and BY88 were equivalent on ATCs, and remained similar on untransduced T cells over the course of expansion (Figure 8D). In CD30CAR transduced cells, CD30 detection by BerH8 was undetectable as early as day 3 post-transduction. On the other hand, staining using BY-88 revealed that cell surface CD30 expression often spiked early on and gradually declined to under 10% in most conditions at the harvest time point (day 11 ), demonstrating that all of the scFv constructs effectively masked CD30 in cis.
To evaluate the extent of activation and/or exhaustion of the CD30CAR-expressing cells, the inventors stained cells with antibodies to PD-1 , Tim3 and LAG3 and analyzed the proportions of cells expressing one or a combination of these proteins in 3 donors (Figure 8E). CD4 T cells primarily comprised Tim3 single-positive cells, with a small proportion (~5-20%) of cells expressing both Tim3 and PD-1 , or none of the markers. CD8 T cells were similarly largely Tim3 single-positive, particularly for donor 5056. The remaining cells consisted of cells bearing none of the markers, and Tim3-LAG3 double expressors. Within each donor, the frequencies of these populations were comparable among the different CD30CAR variants. The fraction of cells expressing all three proteins was very low, suggesting that cells were not terminally exhausted at the end of the expansion process.
2.7 In vitro anti-tumor activity of T cells bearing humanised CD30 CARs
The inventors assessed the cytolytic function of T cells transduced with the humanised CD30CAR variants by co-culturing T cells at a 2:1 E:T ratio with two target tumor cell lines, KM-H2 and HuT-78, both of which express high levels of CD30. All CD30CAR variants achieved close to 100% cytolysis of KM-H2 targets by 60h in three of the four tested donors (Figure 9A). With HuT-78 as target cells, there was more heterogeneity in the killing capacity of the CD30CAR variants, both in terms of maximum cytolysis and in the kinetics of cytolysis. Most constructs attained at least 60% cytolysis of HuT-78 cells by 60h.
Effective tumor clearance in vivo is predicated on the ability of CAR T cells to maintain killing activity throughout successive rounds of target encounters. To model such a scenario in vitro, a flow cytometry-based serial killing assay was established in which CAR T cells underwent three consecutive target encounters, with each encounter lasting 48h (Figure 9B; see Example 1 .6). To distinguish target cells from the different encounters, KM-H2 were labelled cells for Encounters 2 and 3 with the lipophilic membrane dyes PKH67 and PKH26 dyes respectively. The HRS3 and VH3Vk3-Cys CD30CAR variants exhibited good serial killing potency in the three donors tested, while VH5Vk3-Cys maintained cytolytic function through three successive rounds of target encounter only in one of the donors, 8584 (Figure 9C).
T cell expansion accompanied target cytolysis, with HRS3 and VH3Vk3-Cys cells proliferating well in all three donors (Figure 9D), whereas VH5Vk3-Cys cells multiplied only in donor 8584. HRS3- bearing cells increased steadily in numbers with each successive target encounter. VH3Vk3-Cys cells exhibited a peak in cell count at the end of Encounter 2, but declined following Encounter 3 with no apparent impact on killing potency. 2.8 In vitro on-target, off-tumor activity of T cells bearing humanised CD30 CAR with the 4-1 BB spacer Aside from being expressed on malignant lymphoid cells and on activated lymphocytes, CD30 can also be upregulated on hematopoietic stem and progenitor cells (HSPC) upon cytokine stimulation, albeit at lower levels than lymphoma cells (21 ). This presents a potential safety concern should CD30CAR T cells inadvertently attack CD30-expressing HSPCs in recipients. While one group observed minimal killing of HSPCs by HRS3scFv CAR T cells (21 ), the inventors wanted to ensure the same was true of our humanised CD30CAR variants.
The inventors first assessed the CD30 expression on cord blood CD34+ cells from two donors following the same cytokine stimulation protocol employed by Hornbach et al. CD30 expression peaked at 2-3 days of stimulation for both donors following culture in 10 ng/mL of FLT3L, SCF and TPO (Figure 10A), but did not reach the high levels on KM-H2 cells (Figure 10B). CD30 surface expression is reportedly heterogeneous on HSPC subsets (23), and this was also found to be true for the two donors assayed (Figure 10C). With 2 days of stimulation, CD30 was upregulated on multipotent progenitors (MPPs) and lymphoid-primed multipotents (LMPPs), and to a lesser extent on multi-lymphoid primed progenitors (MLPs), but not on erythro-myeloid primed progenitors (EMPs). These data are consistent with the trends reported by Hornbach et al. (23). HSPCs from 2 days post cytokine stimulation were thus used to capture the population at its peak of CD30 expression.
To determine if the CD30CAR variants had any impact on the survival of progenitor populations, CD34+ HSPCs were exposed to effector cells for 24h, then isolated the HSPCs for further culture over 9 days in the same cytokine cocktail of 10 ng/mL FLT3L, SCF and TPO before analyzing the cells by flow cytometry (Figure 10D; see Example 1 ). Encounter with any of the CD30CAR variants altered the progenitor population composition compared to untransduced T cells, suggesting there was some cytolysis of progenitors, especially the LMPP subset. In turn, the EMP subset experienced a reciprocal increase as a proportion of all progenitor cells. Humanisation of the CD30CAR appeared to confer a benefit, in that the reduction in LMPP frequencies tended to be more pronounced with the murine CD30CAR HRS3. Nevertheless, none of the populations was completely ablated. Furthermore, the most primitive MPP subset was not adversely affected, suggesting that even if some of the more differentiated progeny are killed, MPPs are still present to replenish these compartments.
Since alterations in the progenitor composition following co-culture with the CD30CAR variants were observed, the inventors next examined if these changes were functionally meaningful in terms of erythroid and myeloid developmental potential. Following co-culture with effector cells for 24h, a multi-well plate-based colony forming assay was set up using the isolated CD34+ HSPCs (24; Figure 10E; see Example 1 ). After 12 days of culture, cells were harvested and analyzed by flow cytometry for the presence of erythrocytes, granulocytes and monocytes to determine the original progenitor from which they arose on a per well basis. No differences in colony frequencies were observed among the CD30CAR variants for both HSPC donors when exposed to the T cell donor 0165. However, with T cells derived from the second donor 8584, the VH3Vk3-Cys and VH5Vk3-Cys humanised CD30CAR variants resulted in lower proportions of CFU-G in comparison to HRS3 upon co-culture with HSPC donor 41878. When cultured with the second HSPC donor 41817, VH5Vk3- Cys cells gave rise to more CFU-M than HRS3. These effects were not consistent across donors, but since there was no complete elimination of any colony type, the HRS3 and humanised CD30CAR variants do not appear to have a major functional impact on CD30-expressing HSPCs.
While the CD30CAR variants did not drastically affect HSPCs directly, the possibility that CD30CAR effectors might exhibit elevated cytolysis of HSPCs following encounter with CD30-high tumor cells was explored. T cells bearing the CD30CAR variants were first primed by co-culturing them with KM- H2 target cells for 24h, then added in 2 day-stimulated HSPCs (Figure 10F). After a further 24h of culture, cells were harvested and analyzed by flow cytometry. HSPCs emerged unscathed with all CD30CAR variants for both T cell donors, while KM-H2-directed cytolysis remained high and comparable to that when only KM-H2 cells and T cells were co-incubated for 48h. For reasons unknown, untransduced T cells from donor 0165 had high background killing of KM-H2 targets, while still sparing HSPCs. Prior exposure to CD30-high target cells thus did not appear to result in collateral damage of CD30-low HSPCs, even when the effectors outnumbered the HSPCs at an E:T ratio of 1 :0.4.
2.9 In vivo efficacy and safety of T cells bearing humanised CD30 CAR with the 4-1 BB spacer in a peripheral T cell lymphoma model
It was imperative in our next experiments to evaluate efficacy and safety of our CD30 CAR T cells in an in vivo model that contains critical components of the human immune system such as myeloid, lymphoid and natural killer cells. To this end, these candidates were evaluated in GM-CSF, IL-3 supplemented humanised mice which were subcutaneously xenografted with HuT-78 cells. Mice were randomised to receive either untransduced T cells, HRS3 or the humanised CD30CAR variants, stratified based on tumor volume, human CD45 and CD33 reconstitution levels and body weight. All infused T cells were also transduced with GFP-Luciferase (see Example 1 ) to enable in v/vo cell tracking and discrimination from endogenous human T cells (Figure 11 A).
Measurement of HuT-78 tumors indicated tumor growth and fold change was similar in mice from all 4 treatment groups. Our earlier experience suggests that HuT78 tumor size can increase following treatment as a result of CAR T cell infiltration and human immune cell responses, similar to the experience with immune checkpoint inhibitors (27, 28). Tumor size monitoring may therefore be a less accurate indicator of response. Indeed, endpoint flow cytometric analysis of flank tumors revealed significantly lower numbers of live HuT-78 cells in mice treated with any CD30 CAR T cells, with no appreciable difference observed between the different variants (Figure 11 B). Tumor control was accompanied by IVIS detection of high numbers of HRS3, VH3Vk3-Cys and VH5Vk3-Cys humanised CD30CAR. CAR T cells in the dorsal and ventral plane as well as tumors of mice (Figure 11 C). In contrast, bioluminescence signals from untransduced T cells were barely detectable in mice. Mice treated with VH5Vk3-Cys humanised CD30CAR. CAR T cells experienced a sudden deterioration between 8 to 11 days after treatment with 4 of 8 mice succumbing quickly. Mice that were treated with untransduced T cells, HRS3 or VH3Vk3-Cys humanised CD30CAR. CAR T cells remained well till experiment endpoint (Figure 11 D). The inventors probed for possible differences in human immune cell composition. Human CD3+, CD4+ and CD8+ T cell populations were noted to be similar in mice from all treatment arms (Figure 11 E). Other immune cell subsets including CD19+ B cells, CD56+ NK cells, CD33+ monocyte and subsets based on CD14 and CD16 expression, CD15+ neutrophils in various organs were noted to be also grossly normal across the different treatment groups. There were a few exceptions where the frequencies of CD19+ B cell in the liver, CD33+ CD14- monocytes in the lung, CD15+ neutrophils in the liver and lung in CD30 CAR T cells treated mice was different compared to untransduced T cells (Figure 11 F). These differences are thought to represent compensatory immune cell responses to tumor cell elimination. It should be highlighted that there was no observed ablation of any immune cell subsets in mice which received either of the three CD30 CAR T cell variants. Most importantly, human immune cell frequencies in mice which received the new humanised CD30CAR variants were similar to mice treated with HRS3 CD30 CAR T cells, which has been demonstrated in human clinical trials to have an excellent safety profile (29).
Luminex® analysis of mouse plasma drawn at Day 7 post treatment revealed that compared to untransduced T cells, inflammatory cytokines (IFN-y, GM-CSF, IL-8, IL-6, TNF-a and IL-2 ) were generally increased in mice from all 3 CD30 CAR T cells treatment groups. Several inflammatory cytokines were found to be highly elevated in 2 mice from the VH5Vk3-Cys humanised CD30CAR. CAR T cell treatment group (Figure 11 G, circles), both which eventually died at day 8 and 11 post treatment. It is noteworthy that similar levels of these cytokines were detected in mice which received HRS-3 and VH3Vk3-Cys humanised CD30CAR T cells and these mice remained well till termination of experiment. Altogether, these data demonstrates that VH3Vk3-Cys humanised CD30CAR. CAR T cells have a good safety profile comparable to murine HRS3 CD30CAR. CAR T cells.
2.10 In vivo efficacy and safety of humanised CD30 CAR T cells in a model with high tumor burden High tumor load is a known risk factor for the occurrence of cytokine release syndrome (CRS) associated with CAR T cell therapy in patients with haematological malignancies (20, 21 ). Myeloid cells including monocytes and macrophages have been delineated to be key players in the development of CRS and neurotoxicity after CAR-T cell therapy (22, 23).
With this in mind, the inventors sought to examine safety of T cells bearing humanised CD30 CAR in humanised NSG mice xenografted with truncated CD30-transduced NALM-6 cells. In order to support the stable engraftment of myeloid lineages, the mice utilised in this model were supplemented with human GM-CSF and interleukin-3 (IL-3) after human chimerism of >20% has been achieved in the peripheral blood (see Example 1 ). Disease progression was monitored by bioluminescence imaging of CD30 + NALM-6 cells transduced with firefly luciferase. Treatment was administered when systemic tumor burden in mice was high (Figure 12A). Mice were randomised to receive either untransduced T cells, HRS3 or the humanised CD30CAR variants, stratified based on tumor volume, human CD45 and CD33 reconstitution levels and body weight. Body weight changes were similar in mice across all treatment groups (Figure 12B). Treatment with VH3Vk3-Cys and VH5Vk3-Cys humanised CD30 CAR T cells significantly improved tumor control in mice compared to untransduced T cells. In addition, anti-tumor responses in VH3Vk3-Cys and VH5Vk3-Cys humanised CD30 CAR T cells treated mice appeared more durable compared to HRS- 3 CD30 CAR T cells (Figure 12C). An early death in a mouse treated with VH3Vk3-Cys humanised CD30 CAR T cells occurred 2 days post treatment and is believed to be precipitated by the incidental presence of NALM-6 in a major vessel rather than progressive disease (Figure 12C). While it was not possible to discern infused T cells from endogenous human T cells, CD3+, CD4+ and CD8+ T cell populations were similar between all treatment arms (Figure 12D). Immune cell subsets including CD19+ B cells, CD56+ NK cells (CD16 bright and dim), CD33+ monocyte and subsets based on CD14 and CD16 expression, CD66+ neutrophils were noted to be grossly normal in various organs across the different treatment. There were a few exceptions where the frequencies of CD19+ B cell in the spleen, CD33+ monocytes in the liver and CD33+ subsets in the lung in CD30 CAR T cells treated mice was different compared to untransduced T cells (Figure 12E). These differences likely represent compensatory immune cell responses in the various organs to NALM-6 cell killing. There was no observed ablation of any subsets in mice which received CD30 CAR T cells. Most importantly, human immune cell frequencies in mice which received the new humanised CD30CAR variants were similar to mice treated with HRS3 CD30 CAR T cells, which has been demonstrated in human clinical trials to have an excellent safety profile (27). Luminex® analysis of mouse serum at Day 11 post treatment levels showed while that inflammatory cytokines (IFN-y, GM- CSF, IL-8, IL-6, TNF-a and IL-2) were higher in mice from CD30 CAR T cells treatment groups compared to untransduced T cells (Figure 12F), they were not at levels associated with the occurrence of CRS.
Altogether, these data demonstrates that VH3Vk3-Cys and VH5Vk3-Cys humanised CD30CAR. CAR T cells have a good safety profile comparable to murine HRS3 CD30CAR. CAR T cells in mice carrying high tumor load.
2.11 Expression and functional characterisation of humanised variant VH3VK3 and VH5VK3 on EBVSTs Lastly, the functionality of humanised CARs with the 4-1 BB expressed by EBV specific T cells (EBVSTs) was investigated. Whole PBMCs were depleted of CD45RA positive cells by a negative selection step using CD45RA MACs beads. The depleted cells (RAD-PBMCs) were then cultured with EBV peptide libraries and expanded cells were then transduced to express the original parental HRS-3, or the humanised variants VH3VK3, VH5VK3 CARs. Expression and transduction efficiency of the CARs were comparable in EBVSTs (Figure 13A). Masking of CD30 molecules expressed by the CAR T cells in cis by the CD30 CAR will affect the degree of fratricide and hence expansion of the CD30 CAR T cells. Efficiency of CD30 masking by the CD30 CAR was assessed by flow using the pair of CD30 specific antibodies BY88 and BerH8. BY88 does not share the same epitope as HRS-3 and thus the level of staining with BY88 will be indicative of total CD30 expression on the CAR T cells. BerH8 binds to the same epitope as HRS-3 and the humanised variants, thus the lack of BerH8 binding to the CAR T cells will indicate the masking of CD30 by the expressed CARs. All three CARs showed similar efficiency of CD30 masking (Figure 13B). Lastly, cytotoxicity of target cells was assessed with CD30+ KM-H2 cells and CD30- Daudi cells (Figure 13C). The humanised CARs demonstrated similar killing efficacy as the original HRS-3 CAR with similarly low nonspecificity.
References
1 ) Durkop H, Latza U, Hummel M, et al. Molecular cloning and expression of a new member of the nerve growth factor receptor family that is characteristic for Hodgkin’s disease. Cell. 1992; 68:421-427
2) Stein H, Mason DY, Gerdes J, et al. The expression of the Hodgkin’s disease associated antigen Ki-1 in reactive and neoplastic lymphoid tissue: Evidence that Reed-Sternberg cells and histiocytic malignancies are derived from activated lymphoid cells. Blood. 1985; 66:848- 858.
3) Foyil KV, Bartlett NL. Anti-CD30 Antibodies for Hodgkin lymphoma. Curr Hematol Malig Rep. 2010 Jul;5(3):140-7.
4) Younes A, Gopal AK, Smith SE, Ansell SM, Rosenblatt JD, Savage KJ, Ramchandren R, Bartlett NL, Cheson BD, de Vos S, Forero-Torres A, Moskowitz CH, Connors JM, Engert A, Larsen EK, Kennedy DA, Sievers EL, Chen R. Results of a pivotal phase II study of brentuximab vedotin for patients with relapsed or refractory Hodgkin's lymphoma. J Clin Oncol. 2012 Jun 20;30(18):2183-9.
5) Velasco R, Domingo-Domenech E, Sureda A. Brentuximab-lnduced Peripheral Neurotoxicity: A Multidisciplinary Approach to Manage an Emerging Challenge in Hodgkin Lymphoma Therapy. Cancers (Basel). 2021 Dec 5;13(23):6125.
6) Voorhees TJ, Beaven AW. Therapeutic Updates for Relapsed and Refractory Classical Hodgkin Lymphoma. Cancers (Basel). 2020;12(10):2887.
7) Sterner RC, Sterner RM. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 2021 Apr 6;11 (4):69
8) Jayaraman J, Mellody MP, Hou AJ, Desai RP, Fung AW, Pham AHT, Chen YY, Zhao W. CAR-T design: Elements and their synergistic function. EBioMedicine. 2020 Aug;58:102931 . doi: 10.1016/j.ebiom.2020.102931 .
9) Guedan S, Calderon H, Posey AD Jr, Maus MV. Engineering and Design of Chimeric Antigen Receptors. Mol Ther Methods Clin Dev. 2018 Dec 31 ;12:145-156.
10) Wagner DL, Fritsche E, Pulsipher MA, Ahmed N, Hamieh M, Hegde M, Ruella M, Savoldo B, Shah NN, Turtle CJ, Wayne AS, Abou-EI-Enein M. Immunogenicity of CAR T cells in cancer therapy. Nat Rev Clin Oncol. 2021 Jun;18(6):379-393.
11 ) Hornbach A, Hornbach AA, Abken H. Adoptive immunotherapy with genetically engineered T cells: modification of the IgG 1 Fc 'spacer' domain in the extracellular moiety of chimeric antigen receptors avoids 'off-target' activation and unintended initiation of an innate immune response. Gene Ther. 2010 Oct;17(10):1206-13.
12) Majzner RG, Rietberg SP, Sotillo E, Dong R, Vachharajani VT, Labanieh L, Myklebust JH, Kadapakkam M, Weber EW, Tousley AM, Richards RM, Heitzeneder S, Nguyen SM, Wiebking V, Theruvath J, Lynn RC, Xu P, Dunn AR, Vale RD, Mackall CL. Tuning the Antigen Density Requirement for CAR T-cell Activity. Cancer Discov. 2020 May;10(5):702- 723. 13) Muller YD, Nguyen DP, Ferreira LMR, Ho P, Raffin C, Valencia RVB, Congrave-Wilson Z, Roth TL, Eyquem J, Van Gool F, Marson A, Perez L, Wells JA, Bluestone JA, Tang Q. The CD28-Transmembrane Domain Mediates Chimeric Antigen Receptor Heterodimerisation With CD28. Front Immunol. 2021 Mar 23;12:639818.
14) Ferreira LMR, Muller YD. CAR T-Cell Therapy: Is CD28-CAR Heterodimerisation Its Achilles' Heel? Front Immunol. 2021 Nov 17;12:766220.
15) G. Winter, F.J. Carr and W.J. Harris, Altered Antibodies, Products and Processes Relating Thereto, EP0629240
16) F.J. Carr, G. Carter, A.A. Hamilton, F. Z. Adair. Method for the production of non- immunogenic proteins, EP0983303
17) Perry LC, Jones TD, Baker MP. New approaches to prediction of immune responses to therapeutic proteins during preclinical development. Drugs R D. 2008;9(6):385-96.
18) Hornbach AA, Gorgens A, Chmielewski M, Murke F, Janine Kimpel , Giebel B2 and Abken H. Superior Therapeutic Index in Lymphoma Therapy: CD30+ CD34+ Hematopoietic Stem Cells Resist a Chimeric Antigen Receptor T-cell Attack. Mol Ther. 2016 Aug; 24(8): 1423- 1434.
19) Watanabe N, Bajgain P, Sukumaran Oncoimmunology. 2016; 5(12): e1253656. S, Ansari S, Heslop HE, Rooney CM, Fine-tuning the CAR spacer improves T-cell potency.
20) Vipul S Sheth, Jordan Gauthier. Taming the beast: CRS and ICANS after CAR T-cell therapy for ALL. Bone Marrow Transplantation (2021 ) 56:552-566
21 ) Zhiling Yan, Huanxin Zhang, Jiang Cao, Cheng Zheng, Hui Liu, Hongming Huang et al. Characteristics and Risk Factors of Cytokine Release Syndrome in Chimeric Antigen Receptor T Cell Treatment. Front Immunol. 2021 Feb 23;12 :61 1366.
22) Norelli M, Camisa B, Barbiera G, Falcone L, Purevdorj A, Genua M et al. Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells. Nat Med. 2018 Jun;24(6):739-748
23) Giavridis T, C van der Stegen S, Eyquem J, Hamieh M, Piersig illi A, Sadelain M. CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade. Nat Med. 2018 Jun;24(6):731 -738.
24) David C. Fajgenbaum and Carl H. June. Cytokine Storm. N Engl J Med. 2020 Dec 3;383(23):2255-2273
25) Hornbach AA, Gorgens A, Chmielewski M, Murke F, Kimpel J, Giebel B, Abken H. Superior Therapeutic Index in Lymphoma Therapy: CD30(+) CD34(+) Hematopoietic Stem Cells Resist a Chimeric Antigen Receptor T-cell Attack. Mol Ther. 2016 Aug ;24(8):1423-34.
26) Bissels U, Aivazidou F, Knobel S, Bosio A, Papanikolaou. A novel user-independent CFU assay for hematopoietic stem and progenitor cells. Poster by Miltenyi Biotec GmbH. 2019. https ://www. miltenyibiotec.com/ Resources/Persistent/43ed61 c2b210b8873c1 d5b1648e53a f0d9fe675a/A7o20novel%20user- independent7o20CFU7o20assav7o20for7o20hematopoietic7o20stem7o20and7o20proqenitor7o 20cells.pdf. Date accessed: 20th January 2021 .
27) Chiou VL, Burotto M. Pseudoprogression and Immune-Related Response in Solid Tumors. J Clin Oncol. 2015 Nov 1 ;33(31 ):3541 -3 ) Borcoman E, Nandikolla A, Long G, Goel S and Le Tourneau C. Patterns of Response and Progression to Immunotherapy. American Society of Clinical Oncology Educational Book 38 (May 23, 2018) 169-178. ) Ramos CA, Grover NS, Beaven AW, Lulla PD, Wu MF, Ivanova A et al. Anti-CD30 CAR-T Cell Therapy in Relapsed and Refractory Hodgkin Lymphoma. J Clin Oncol. 2020 Nov
10;38(32):3794-3804. ) nt. Drugs R D. 2008;9(6):385-96.

Claims

Claims:
1 . A chimeric antigen receptor (CAR) comprising: (i) an antigen-binding domain which binds specifically to a target antigen, (ii) a spacer domain comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs:100, 99, 101 and 102, (Hi) a transmembrane domain, and (iv) a signalling domain comprising an amino acid sequence comprising an immunoreceptor tyrosine-based activation motif (ITAM).
2. The CAR according to claim 1 , wherein the CAR comprises a spacer domain comprising or consisting of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NQ:100.
3. The CAR according to claim 1 or claim 2, wherein the antigen-binding domain binds specifically to a target antigen selected from CD30, CD19, CD20, CD22, ROR1 R, CD4, CD7, CD38, BCMA, Mesothelin, EGFR, GPC3, MUC1 , HER2, GD2, CEA, EpCAM, LeY and PSCA.
4. The CAR according to any one of claims 1 to 3, wherein the antigen-binding domain binds specifically to CD30.
5. The CAR according to any one of claims 1 to 4, wherein the antigen-binding domain comprises:
(i) a heavy chain variable (VH) region incorporating the following CDRs:
HC-CDR1 having the amino acid sequence of SEQ ID NO:2 HC-CDR2 having the amino acid sequence of SEQ ID NO:3 HC-CDR3 having the amino acid sequence of SEQ ID NO:4; and
(ii) a light chain variable (VL) region incorporating the following CDRs:
LC-CDR1 having the amino acid sequence of SEQ ID NQ:10 LC-CDR2 having the amino acid sequence of SEQ ID NO:11 LC-CDR3 having the amino acid sequence of SEQ ID NO:12.
6. The CAR according to any one of claims 1 to 5, wherein the antigen-binding domain comprises: a VH region having an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs:1 , 45, 52, 17, 21 , 24, 26, 28; and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs:9, 49, 54, 30, 35, 38, 41 , 43, 147, 148, 149, 150, 151 or 152.
7. The CAR according to any one of claims 1 to 7, wherein the antigen-binding domain comprises:
(i) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:1 , and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:9; or
(ii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:45, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:42; or (iii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:52, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:54; or
(iv) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:17, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NQ:30; or
(v) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:21 , and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:35; or
(vi) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:24, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:38; or
(vii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:26, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:41 ; or
(viii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:28, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:43; or
(ix) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:1 , and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:147; or
(x) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:17, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:148; or
(xi) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:21 , and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:149; or
(xii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:24, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:150; or
(xiii) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:26, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:151 ; or
(xiv) a VH region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:28, and a VL region having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:152.
8. The CAR according to any one of claims 5 to 7, wherein the antigen-binding domain is or comprises a single chain variable fragment (scFv) comprising the VH region and the VL region.
9. The CAR according to any one of claims 1 to 8, wherein the transmembrane domain comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NQs:103, 104 or 105.
10. The CAR according to any one of claims 1 to 9, wherein the signalling domain comprises an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs:106, 107 or 108.
11 . The CAR according to any one of claims 1 to 10, wherein the signalling domain comprises an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO:111 .
12. The CAR according to any one of claims 1 to 11 , wherein the CAR comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to any one of SEQ ID NOs:124, 144,
190, 210, 117, 118, 119, 120, 121 , 122, 123, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136,
137, 138, 139, 140, 141 , 142, 143, 145, 146, 183, 184, 185, 186, 187, 188, 189, 191 , 192, 193, 194, 195,
196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 211 or 212.
13. A nucleic acid, or a plurality of nucleic acids, optionally isolated, encoding a CAR according to any one of claims 1 to 12.
14. An expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to claim 13.
15. A cell comprising a CAR according to any one of claims 1 to 12, a nucleic acid or a plurality of nucleic acids according to claim 13, or an expression vector or a plurality of expression vectors according to claim 14.
16. A method comprising culturing a cell according to claim 15 under conditions suitable for expression of an CAR by the cell.
17. A composition comprising a CAR according to any one of claims 1 to 12, a nucleic acid or a plurality of nucleic acids according to claim 13, an expression vector or a plurality of expression vectors according to claim 14, or a cell according to claim 15, and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
18. A CAR according to any one of claims 1 to 12, a nucleic acid or a plurality of nucleic acids according to claim 13, an expression vector or a plurality of expression vectors according to claim 14, a cell according to claim 15, or a composition according to claim 17, for use in a method of medical treatment or prophylaxis.
19. A CAR according to any one of claims 1 to 12, a nucleic acid or a plurality of nucleic acids according to claim 13, an expression vector or a plurality of expression vectors according to claim 14, a cell according to claim 15, or a composition according to claim 17, for use in the treatment or prevention of a cancer.
20. The CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use according to claim 19, wherein the cancer is selected from the group consisting of: a cancer expressing the target antigen for the CAR, a CD30-positive cancer, an EBV- associated cancer, a hematological cancer, a myeloid hematologic malignancy, a hematopoietic malignancy a lymphoblastic hematologic malignancy, myelodysplastic syndrome, leukemia, T cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, B cell non-Hodgkin’s lymphoma, diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, EBV-associated lymphoma, EBV-positive B cell lymphoma, EBV-positive diffuse large B cell lymphoma, EBV-positive lymphoma associated with X-linked lymphoproliferative disorder, EBV-positive lymphoma associated with HIV infection/AIDS, oral hairy leukoplakia, Burkitt’s lymphoma, post-transplant lymphoproliferative disease, central nervous system lymphoma, anaplastic large cell lymphoma, T cell lymphoma, ALK-positive anaplastic T cell lymphoma, ALK-negative anaplastic T cell lymphoma, peripheral T cell lymphoma, cutaneous T cell lymphoma, NK-T cell lymphoma, extra-nodal NK-T cell lymphoma, thymoma, multiple myeloma, a solid cancer, epithelial cell cancer, gastric cancer, gastric carcinoma, gastric adenocarcinoma, gastrointestinal adenocarcinoma, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck cancer, head and neck squamous cell carcinoma, oral cavity cancer, oropharyngeal cancer, oropharyngeal carcinoma, oral cancer, laryngeal cancer, nasopharyngeal carcinoma, oesophageal cancer, colorectal cancer, colorectal carcinoma, colon cancer, colon carcinoma, cervical carcinoma, prostate cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, squamous lung cell carcinoma, bladder cancer, urothelial carcinoma, skin cancer, melanoma, advanced melanoma, renal cell cancer, renal cell carcinoma, ovarian cancer, ovarian carcinoma, mesothelioma, breast cancer, brain cancer, glioblastoma, prostate cancer, pancreatic cancer, mastocytosis, advanced systemic mastocytosis, germ cell tumor or testicular embryonal carcinoma.
21 . A CAR according to any one of claims 1 to 12, a nucleic acid or a plurality of nucleic acids according to claim 13, an expression vector or a plurality of expression vectors according to claim 14, a cell according to claim 15, or a composition according to claim 17, for use in the treatment or prevention of a disease or condition characterised by an alloreactive immune response.
22. The CAR, nucleic acid or plurality of nucleic acids, expression vector or plurality of expression vectors, cell or composition for use according to claim 21 , wherein the disease or condition characterised by an alloreactive immune response is selected from the group consisting of: a disease or condition associated with allotransplantation, graft versus host disease (GVHD) or graft rejection.
23. Use of a CAR according to any one of claims 1 to 12, a nucleic acid or a plurality of nucleic acids according to claim 13, an expression vector or a plurality of expression vectors according to claim 14, a cell according to claim 15, or a composition according to claim 17 to deplete or increase killing of cells expressing the target antigen for the CAR.
24. An in vitro complex, optionally isolated, comprising a CAR according to any one of claims 1 to 12, or a cell according to claim 15, bound to the target antigen for the CAR.
EP23853152.9A 2022-08-08 2023-08-07 Chimeric antigen receptor domains Pending EP4568999A1 (en)

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