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WO2025191154A1 - Agents thérapeutiques - Google Patents

Agents thérapeutiques

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Publication number
WO2025191154A1
WO2025191154A1 PCT/EP2025/057064 EP2025057064W WO2025191154A1 WO 2025191154 A1 WO2025191154 A1 WO 2025191154A1 EP 2025057064 W EP2025057064 W EP 2025057064W WO 2025191154 A1 WO2025191154 A1 WO 2025191154A1
Authority
WO
WIPO (PCT)
Prior art keywords
ccr
car
domain
seq
stimulatory
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
PCT/EP2025/057064
Other languages
English (en)
Inventor
Maya GLOVER
John Maher
David Marc DAVIES
Chelsea Alice TAYLOR
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.)
Kings College London
Leucid Bio Ltd
Original Assignee
Kings College London
Leucid Bio Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kings College London, Leucid Bio Ltd filed Critical Kings College London
Publication of WO2025191154A1 publication Critical patent/WO2025191154A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K40/4256—Tumor associated carbohydrates
    • A61K40/4257—Mucins, e.g. MUC-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/28—Expressing multiple CARs, TCRs or antigens
    • 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
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/54—Pancreas
    • 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
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • CCRs chimeric co-stimulatory receptor
  • TNFR tumour necrosis receptor
  • pCARs parallel CARs
  • pACARs parallel adaptor CARs
  • compositions and methods for improving anti-tumour efficacy and restimulation capacity of CAR T-cells, pCAR T-cells, and pACAR T-cells are provided herein.
  • BACKGROUND CAR T cell therapy has shown impressive efficacy for treatment of haematological malignancies.
  • solid tumours present several obstacles which enable them to resist CAR T cell therapy.
  • a parallel CAR (pCAR) technology has been developed which consists of a CD28 second- generation CAR combined with a TNFR co-stimulatory domain-containing chimeric costimulatory receptor (CCR).
  • CCR costimulatory receptor
  • Non-conservative substitutions may also be possible provided that these do not interrupt the function of the polypeptide as described above. Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptides.
  • variants will have amino acid sequences that will be at least about 70%, for instance at least about 71%, about 75%, about 79%, about 81%, about 84%, about 87%, about 90%, about 93%, about 95%, about 96% or about 98% identical to the basic sequence.
  • Identity in this context may be determined using the BLASTP computer program. Identity is typically measured over the entire length of the reference sequence.
  • the term “antigen” refers to any member of a specific binding pair that will bind to the binding elements.
  • the CCR binding element may bind the same epitope in the same antigen, a different epitope in the same antigen, or an entirely different antigen as/from the CAR or adaptor CAR.
  • the CCR lacks a TCR-like signalling region such as CD3 ⁇ .
  • the CCR provided in this disclosure is typically a trimer formed from three or more CCR polypeptides.
  • linear CAR refers to a chimeric antigen receptor which comprises at least one extracellular binding domain (targeting moiety) and one or more intracellular signalling domains in a single polypeptide configured to span a cell’s plasma membrane. Examples of linear CARs include, but are not limited to, second-generation CARs.
  • parallel adaptor CAR or “pACAR” as used herein refers to a parallel chimeric antigen receptor which comprises the combination of an adaptor CAR and, in parallel, a chimeric co-stimulatory receptor (CCR).
  • Parallel CARs (pCARs), adaptor CARs and parallel adaptor CARs (pACARs) may collectively be called lateral CARs.
  • Other interpretational conventions In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context.
  • Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
  • the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps.
  • CCR co-stimulatory receptor
  • the present disclosure provides a chimeric co-stimulatory receptor (CCR) polypeptide capable of forming a trimer, i.e., capable of trimerisation.
  • the CCR polypeptide is capable of forming a trimer/trimerisation in the context of a cell membrane, especially when present or expressed in an immunoresponsive cell.
  • the CCR polypeptide is capable of forming a trimeric CCR.
  • TNFR co-stimulatory domain The CCR polypeptide comprises at least one tumour necrosis receptor (TNFR) co-stimulatory domain.
  • the CCR polypeptide may comprise any number of TNFR co-stimulatory domains, such as 2, 3 or 4 or more TNFR co-stimulatory domains.
  • the at least one TNFR co-stimulatory domain is or forms a co-stimulatory signalling region in the CCR polypeptide.
  • the CCR polypeptide comprises a co- stimulatory signalling region which comprises or consists of at least one TNFR co- stimulatory domain.
  • the at least one TNFR co-stimulatory domain is selected from 4-1BB, OX40, CD27, BAFFR, TACI and CD40, glucocorticoid-induced tumour necrosis factor (GITR), herpesvirus entry mediator (HVEM), death receptor 3 (DR3), CD30, TNFR2, and variants thereof.
  • the at least one TNFR co-stimulatory domain is selected from 4-1BB, OX40, CD27, and variants thereof.
  • the at least one TNFR co-stimulatory domain is selected from 4-1BB, CD27, and variants thereof.
  • the at least one TNFR co-stimulatory domain may be selected depending upon the particular use intended for the immunoresponsive cell.
  • the at least one TNFR co-stimulatory domain of the CCR polypeptide can be selected to work additively or synergistically together with the co-stimulatory signalling region of the CAR.
  • the at least one TNFR co-stimulatory domain of the CCR polypeptide comprises or consists of at least one 4-1BB co-stimulatory domain or a variant thereof.
  • the at least one 4-1BB co-stimulatory domain comprises or consists of the sequence shown in SEQ ID NO: 37 as shown below: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 37).
  • the at least one 4-1BB co-stimulatory domain comprises or consists of a variant sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% identity to the sequence shown in SEQ ID NO: 37. Identity is typically measured over the entire length of SEQ ID NO: 37.
  • the at least one TNFR co-stimulatory domain is suitably located adjacent to the transmembrane domain and remote from the binding element. Trimerisation motif
  • the CCR polypeptide further comprises at least one trimerisation motif.
  • the CCR polypeptide may comprise any number of trimerisation motifs, such as 2, 3 or 4 or more trimerisation motifs.
  • a trimerisation motif is a sequence within the CCR polypeptide which promotes trimerisation.
  • Such motifs are known in the art (Kammerer RA, Kostrewa D, Progias P, Honnappa S, Avila D, Lustig A, Winkler FK, Pieters J, Steinmetz MO. Proc Natl Acad Sci U S A. 2005 Sep 27;102(39):13891-6). Examples include, but are not limited to, the GCN4 leucine zipper, coronin proteins, trimeric G protein ⁇ subunits, the trimeric catalytic domain of Bruton's tyrosine kinase, some domain-swapping and Interlocking ⁇ -sheet motifs.
  • the at least one trimerisation motif is preferably derived from the coronin 1A protein.
  • the at least one trimerisation motif preferably comprises or consists of the sequence shown in SEQ ID NO: 93 shown below. SGTPSSDAVSRLEEEMRKLQATVQELQKRLDRLEETVQAK (SEQ ID NO: 93).
  • the at least one trimerisation motif is encoded and/or located upstream of the at least one TNFR co-stimulatory domain.
  • the trimerisation domain is encoded and/or located downstream of the at least one TNFR co- stimulatory domain.
  • the at least one trimerisation motif may be located at any position in the CCR polypeptide.
  • the at least one trimerisation motif may be intracellular when the CCR polypeptide is expressed in a cell or may be in the transmembrane domain. In some embodiments, the at least one trimerisation motif is extracellular when the CCR polypeptide is expressed in a cell, such as an immunoresponsive cell.
  • the location of the various domains, regions, and motifs in the CCR polypeptide are discussed in more detail below. Additional domains/regions/elements
  • the CCR polypeptide further comprises a transmembrane domain and a first binding element that specifically binds or interacts with a first epitope on a first target antigen.
  • the first target antigen may be present on a target cell, such as a target cancer cell.
  • the first target antigen may be soluble or secreted.
  • the chimeric co-stimulatory receptor (CCR) polypeptide comprises, from C-terminus to N-terminus (from intracellular to extracellular as expressed within the immunoresponsive cell), (a) at least one tumour necrosis receptor (TNFR) co-stimulatory domain; (b) at least one first transmembrane domain, (c) at least one trimerisation motif and (d) at least one first binding element that specifically binds or interacts with a first epitope on a first target antigen.
  • TNFR tumour necrosis receptor
  • the chimeric co-stimulatory receptor (CCR) polypeptide comprises, from C-terminus to N-terminus (from intracellular to extracellular as expressed within the immunoresponsive cell), (a) at least one tumour necrosis receptor (TNFR) co-stimulatory domain; (b) at least one first transmembrane domain, (c) at least one trimerisation motif (d) an optional hinge/spacer region and (e) at least one first binding element that specifically binds or interacts with a first epitope on a first target antigen.
  • the at least one tumour necrosis receptor (TNFR) co-stimulatory domain is different from the co-stimulatory signalling region of the CAR.
  • Transmembrane domains Suitable transmembrane domains are known in the art and include for example, the transmembrane domains of 4-1BB, CD8 ⁇ , CD28, CD4, CD3 ⁇ , Fc ⁇ R1 ⁇ or a variant thereof.
  • the transmembrane domain of the CCR polypeptide is the transmembrane domain of CD8 ⁇ .
  • the CCR polypeptide comprises a portion of the extracellular domain and transmembrane domain of CD8 ⁇ .
  • a portion of the CD8a extracellular domain and transmembrane domain comprises the sequence shown in SEQ ID NO: 36 as shown below.
  • the final 5 amino acids in SEQ ID NO: 36 are the first 5 amino acids of the CD8a intracellular domain and are typically included to ensure stability.
  • PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNH SEQ ID NO: 36
  • the CCR polypeptide comprises the transmembrane domain of 4-1BB.
  • the 4-1BB transmembrane domain comprises or consists of the sequence shown in SEQ ID NO: 117 as shown below: IISFFLALTSTALLFLLFFLTLRFSVV (SEQ ID NO: 117)
  • First binding element The first binding element of the CCR binds at least one epitope.
  • the binding element of the CCR polypeptide specifically binds to an epitope.
  • the first binding element of the CCR polypeptide binds to a first target antigen.
  • the first target antigen is associated with a disease, such as a cancer.
  • a suitable binding element may be any element which provides the CCR polypeptide with the ability to recognize a target of interest.
  • the target to which the CCR polypeptide of the disclosure is directed can be any target of clinical interest to which it would be desirable to direct a T cell response.
  • the binding element used in the CCR polypeptides described herein are antigen binding sites (ABS) of antibodies.
  • the ABS used as the binding element is formatted into an scFv or is a single domain antibody from a camelid, human, or other species.
  • the binding element is an alternative binding moiety, such as an aptamer (an RNA molecule that forms a secondary structure capable of mediating binding) or a ‘man-made’ binder, such as a DARPin.
  • a binding element of a CCR polypeptide may comprise ligands that bind to a surface protein of interest.
  • the binding element is associated with a leader sequence which facilitates expression on the cell surface.
  • the binding element specifically binds a tumour antigen or tumour- associated antigen, providing a means to target tumour cells while limiting damage to non- tumour cells or tissues. In some embodiments, the binding element specifically binds a tumour antigen or tumour- associated antigen, providing a means to target tumour cells while limiting damage to non- tumour cells or tissues.
  • the first binding element specifically binds or interacts with a first epitope on a first target antigen selected from an NKG2D ligand, MUC1, ⁇ v ⁇ 6 integrin, HER2, CD19, B7-H3, GD2, Claudin 18.2, Claudin 6, Glypican 3, anaplastic lymphoma kinase (ALK), CD70, prostate-specific membrane antigen (PSMA), mesothelin, ROR1, MUC16, folate receptor alpha, IL-13 receptor alpha 2, prostate stem cell antigen (PSCA), carcinoembryonic antigen (CEA), CD133, CD33, CD123, CLL, fibroblast activation protein (FAP), BCMA, ROBO1, NKp30 ligand, NKp44 ligand, NKp46 ligand, erythropoietin (EPO), interleukin-6 (IL-6), transforming growth factor- ⁇ (TGF- ⁇ ), PD-L1, CD155, CD112,
  • the NKG2D ligand is selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the first binding element specifically binds or interacts with a first epitope on a first target antigen selected from an NKG2D ligand, MUC1, ⁇ v ⁇ 6 integrin, HER2, CD19, B7-H3, GD2, Claudin 18.2, Claudin 6, Glypican 3, anaplastic lymphoma kinase (ALK), CD70, prostate-specific membrane antigen (PSMA), mesothelin, ROR1, MUC16, folate receptor alpha, IL-13 receptor alpha 2, prostate stem cell antigen (PSCA), carcinoembryonic antigen (CEA), CD133, CD33, CD123, CLL, fibroblast activation protein (FAP), BCMA, ROBO1, NKp30 ligand, NKp44 ligand, NKp46
  • the NKG2D ligand is selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the NKp30 ligand is selected from BAG6 and B7-H6.
  • the NKp44 ligand is selected from NKp44L (MLL5), PCNA, viral haemagglutinins, nidogen-1, galectin-3, and other proteoglycans.
  • the ErbB family member is selected from ErbB1, ErbB2, ErbB3 and ErbB4.
  • the first binding element specifically binds an NKG2D ligand or specifically binds a first epitope on a NKG2D ligand.
  • the NKG2D ligand is selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the first binding element is an NKG2D polypeptide or a fragment or functional variant thereof.
  • the first binding element comprises or consists of the sequence shown in SEQ ID NO: 75.
  • the first binding element specifically binds a PD-1 ligand or specifically binds a first epitope on a PD-1 ligand.
  • the binding element or first binding element is a PD-1 polypeptide or a fragment or functional variant thereof.
  • the first binding element comprises or consists of the sequence shown in SEQ ID NO: 86 or SEQ ID NO: 86 lacking the signal peptide (shown below with the signal peptide underlined).
  • SEQ ID NO: 86 is amino acids 1-155 of PD-1.
  • the trimerized CCR comprises or consists of the sequence shown in SEQ ID NO: 81.
  • the first binding element specifically binds a NKp44 ligand or a first epitope on a NKp44 ligand.
  • the NKp44 ligand is selected from NKp44L (MLL5), PCNA, viral haemagglutinins, nidogen-1, galectin-3, and other proteoglycans.
  • the first binding element is a NKp44 polypeptide or a fragment or functional variant thereof.
  • the first binding element comprises or consists of the sequence shown in SEQ ID NO: 87 or SEQ ID NO: 87 lacking the signal peptide (shown below with the signal peptide underlined).
  • the trimerized CCR comprises or consists of the sequence shown in SEQ ID NO: 83.
  • the first binding element specifically binds an ErbB antigen or specifically binds a first epitope on an ErbB antigen.
  • the binding element of the CCR polypeptide is a T1E peptide, an antigen binding site of an antibody that specifically binds HER2, an antigen binding site of an antibody that specifically binds another ErbB family member or a fragment or functional variant thereof.
  • the first binding element is a T1E peptide or a fragment or functional variant thereof.
  • the first binding element is the T1E peptide, which binds ErbB homo- and hetero-dimers.
  • T1E is a chimeric peptide derived from transforming growth factor- ⁇ (TGF- ⁇ ) and epidermal growth factor (EGF) and is a promiscuous ErbB ligand.
  • the T1E peptide is a chimeric fusion protein composed of the entire mature human EGF protein, excluding the five most N­terminal amino acids (amino acids 971-975 of pro-epidermal growth factor precursor (NP 001954.2)), which have been replaced by the seven most N- terminal amino acids of the mature human TGF- ⁇ protein (amino acids 40-46 of pro- transforming growth factor alpha isoform 1 (NP 003227.1)). See Wingens et al., J. Biol. Chem. 278:39114-23 (2003) and Davies et al., Mol. Med. 18:565-576 (2012), the disclosures of which are incorporated herein by reference in their entireties.
  • the sequence of T1E is shown below as SEQ ID NO: 18: VVSHFNDCPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR (SEQ ID NO: 18).
  • the first binding element comprises or consists of the sequence shown in SEQ ID NO: 18.
  • the first binding element is ICR62, which binds to EGFR.
  • the first binding element comprises the antigen binding site of the ICR62 antibody formatted as scFv.
  • the amino acid sequence of the scFv of the ICR62 antibody is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, or about 100% identical to the sequence shown in SEQ ID NO: 44 shown below: QVNLLQSGAALVKPGASVKLSCKGSGFTFTDYKIHWVKQSHGKSLEWIGYFNPNSGYSTYNEKFKSKA TLTADKSTDTAYMELTSLTSEDSATYYCTRLSPGGYYVMDAWGQGASVTVSSAQTTAPSVYPLAPGSG GGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRVTINCKASQNINNYLNWYQQKLGEAPKRLIYNTNN LQTGIPSRFSGSGSGTDYTLTISSLQPEDFATYFCLQHNSFPTFGAGTKLELKRADAAPTVSIFPPSKS (SEQ ID NO: 44).
  • the first binding element specifically binds an NKp30 ligand or specifically binds a first epitope on an NKp30 ligand.
  • the NKp30 ligand is selected from BAG6 and B7-H6.
  • the second target antigen may be soluble or secreted.
  • Intracellular signalling region The CAR construct comprises a signalling region (i.e. a TCR-like signalling region).
  • the signalling region comprises an Immune-receptor­Tyrosine-based- Activation-Motif (ITAM), as reviewed for example by Love et al., Cold Spring Harbor Perspect. Biol 2010 2(6)1 a002485.
  • the intracellular signalling region comprises or consists of an intracellular signalling domain.
  • the signalling region comprises the intracellular domain of human CD3 zeta chain (CD3 ⁇ ), as described for example in US Patent No. 7,446,190, incorporated by reference herein, or a variant thereof.
  • the modified CD3 ⁇ intracellular signalling domain comprises an ITAM2 variant comprising one or more loss-of-function mutations. In certain embodiments, the modified CD3 ⁇ intracellular signalling domain comprises an ITAM2 variant comprising two loss-of-function mutations. In certain embodiments, the loss-of-function mutation comprises a mutation of a tyrosine residue in ITAM2. In certain embodiments, the loss-of- function mutation comprises a mutation of a tyrosine residue to a phenylalanine residue in ITAM2. In certain embodiments, the modified CD3 ⁇ intracellular signalling domain comprises an ITAM3 variant comprising one or more loss-of-function mutations.
  • the modified CD3 ⁇ intracellular signalling domain comprises an ITAM3 variant comprising two loss-of-function mutations.
  • the loss-of-function mutation comprises a mutation of a tyrosine residue in ITAM3.
  • the loss-of- function mutation comprises a mutation of a tyrosine residue to a phenylalanine residue in ITAM3.
  • the modified CD3 ⁇ intracellular signalling domain comprises an ITAM2 variant and an ITAM3 variant comprising one or more loss-of-function mutations.
  • the modified CD3 ⁇ intracellular signalling domain comprises an ITAM2 variant and an ITAM3 variant comprising two loss-of-function mutations.
  • the loss-of-function mutation comprises a mutation of a tyrosine residue in each of ITAM2 and ITAM3. In certain embodiments, the loss-of-function mutation comprises a mutation of a tyrosine residue to a phenylalanine residue in each of ITAM2 and ITAM3.
  • the modified CD3 ⁇ intracellular signalling domain comprises ITAM1 and one or more modifications on at least one of ITAM2 and/or ITAM3. In some embodiments, the modified CD3 ⁇ intracellular signalling domain comprises ITAM1 and one or more loss-of-function mutations in ITAM2 and/or ITAM3.
  • the modified CD3 ⁇ intracellular signalling domain comprises ITAM1 and two loss-of-function mutations in each of ITAM2 and ITAM3.
  • the modified CD3 ⁇ comprises one or more ITAMs comprising two tyrosine (Y) to phenylalanine (F) mutations.
  • the modified CD3 ⁇ intracellular signalling domain comprises unmodified ITAM1 and two tyrosine (Y) to phenylalanine (F) mutations in ITAM2 and/or ITAM3.
  • the modified CD3 ⁇ intracellular signalling domain comprises unmodified ITAM1 and two tyrosine (Y) to phenylalanine (F) mutations in each of ITAM2 and ITAM3.
  • the modified CD3 ⁇ intracellular signalling domain comprises tyrosine (Y) to phenylalanine (F) mutations in at least one, at least two, or all three ITAMs (e.g., ITAM1, ITAM2, and ITAM3).
  • the modified CD3 ⁇ polypeptide comprises at least one functional ITAM.
  • the modified CD3 ⁇ polypeptide comprises mutations in both ITAM2 and ITAM3.
  • ITAM2 and ITAM3 in the modified CD3 ⁇ are non-functional.
  • each of ITAM2 and ITAM3 comprises at least one Tyr to Phe mutation.
  • each of ITAM2 and ITAM3 comprises two Tyr to Phe mutations.
  • the CD3 ⁇ intracellular signalling domain has the sequence shown in SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 48 as shown below (ITAM1 bold and single underlined). Residues mutated to phenylalanine (F) from tyrosine (Y) (with reference to unmodified CD3 ⁇ ) are double underlined.
  • RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLFNE LQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQ KDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID NO: 43) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNEL QKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID NO: 48).
  • the CAR comprises a truncated CD3 ⁇ intracellular signalling domain.
  • the CD3 ⁇ is truncated to remove at least one of the three ITAMs (e.g., ITAM1, ITAM2, ITAM3), or combinations thereof.
  • the CD3 ⁇ is truncated to remove ITAM1.
  • the CD3 ⁇ is truncated to remove ITAM2.
  • the CD3 ⁇ is truncated to remove ITAM3.
  • the CD3 ⁇ is truncated to remove ITAM2 and ITAM3.
  • the truncated CD3 ⁇ intracellular signalling domain has the sequence shown in SEQ ID NO: 49 as shown below: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG (SEQ ID NO: 49).
  • the modified CD3 ⁇ domain comprises deletion of ITAM2 or a portion thereof and deletion of ITAM3 or a portion thereof.
  • the modified CD3 ⁇ polypeptide comprises deletion of ITAM3 or a portion thereof and deletion of ITAM2 or a portion thereof.
  • the modified CD3 ⁇ polypeptide comprises deletion of both ITAM2 and ITAM3.
  • the modified CD3 ⁇ polypeptide comprises the amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the N-terminal 40 amino acids of the sequence shown in SEQ ID NO: 41 or SEQ ID NO: 48. In some embodiments, the modified CD3 ⁇ polypeptide comprises the N- terminal 40 amino acids of the sequence shown in SEQ ID NO: 41 or SEQ ID NO: 48. In some embodiments, the modified CD3 ⁇ polypeptide further comprises deletion of one or more amino acid residues N-terminal to ITAM1. In some embodiments, the modified CD3 ⁇ polypeptide comprises a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the sequence shown in SEQ ID NOs: 50-53.
  • the modified CD3 ⁇ polypeptide comprises a sequence selected from SEQ ID NOs: 50-53.
  • One skilled in the art will be capable of introducing mutations into the polynucleotide sequence of a gene or gene product, for example an ITAM, using standard techniques. For example, point mutations can be introduced via site-directed point mutagenesis using PCR.
  • Co-stimulatory signalling region In the CAR, the co-stimulatory signalling region is suitably located between the signalling region and transmembrane domain, and remote from the binding element.
  • Suitable co-stimulatory signalling regions are well known in the art, and include the co- stimulatory signalling regions of members of the B7/CD28 family such as B7-1, B7-2, B7- H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD-1, PD-L2 or PDCD6; or ILT/CD85 family proteins such as LILRA3, LILRA4, LILRB1, LILRB2, LILRB3 or LILRB4; or tumour necrosis factor (TNF) superfamily members such as 4-1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, Lymphotoxin-alpha, OX40, RELT, TACI, TL1A, TNF-alpha, or TNF RII; or members of the SLAM family such as 2B4, BLAME, CD2, CD
  • co-stimulatory signalling regions may be selected depending upon the particular use intended for the immunoresponsive cell.
  • the co-stimulatory signalling regions are selected from the co-stimulatory signalling regions of CD28, CD27, ICOS, 4-1BB, OX40, DAP10, CD30, GITR, HVEM, DR3 and CD40 or a variant thereof.
  • the co-stimulatory signalling regions are selected from the co- stimulatory signalling regions of CD28, 4-1BB, CD27, OX40, DAP10 and ICOS or a variant thereof.
  • the co-stimulatory signalling region of the CAR is the co- stimulatory signalling region of CD28.
  • the CD28 co-stimulatory signalling region comprises modification of one or more tyrosine residues in the CD28 cytoplasmic domain.
  • the CD28 co-stimulatory signalling region comprises a mutation at the C-terminal most tyrosine residue.
  • the CD28 co-stimulatory signalling region comprises a modified YRS motif.
  • the CD28 co-stimulatory signalling region comprises a mutation in the YRS motif. In some embodiments, the CD28 co-stimulatory signalling region lacks a YRS motif. In certain embodiments, the co-stimulatory signalling region of the CAR is the CD28 co- stimulatory signalling region.
  • the co-stimulatory signalling region of CD28 comprises the sequence shown in SEQ ID NO: 4 as shown below where the endodomain is shown in bold: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKR SRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 4).
  • the co-stimulatory signalling region of CD28 comprises the sequence shown in SEQ ID NO: 25 as shown below: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRS RLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA (SEQ ID NO: 25).
  • the co-stimulatory signalling region of the CAR is the co- stimulatory signalling region of CD28 and the at least one TNFR co-stimulatory domain in the CCR polypeptide comprises or consists of at least one 4-1BB co-stimulatory domain.
  • the co-stimulatory signalling region of the CAR is the DAP10 co- stimulatory signalling region.
  • the co-stimulatory signalling region of DAP10 comprises the sequence underlined in SEQ ID NO: 47 below: MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCA (SEQ ID NO: 47).
  • Transmembrane domain The transmembrane domains for the CAR and CCR constructs may be the same or different.
  • the transmembrane domains of the CAR and CCR are different, to ensure separation of the constructs on the surface of the cell. Selection of different transmembrane domains may also enhance stability of the expression vector since inclusion of a direct repeat polynucleotide sequence in the viral vector renders it prone to rearrangement, with deletion of sequences between the direct repeats. In embodiments in which the transmembrane domains of the CAR and CCR of the pCAR are chosen to be the same, this risk can be reduced by modifying or “wobbling” the codons selected to encode the same protein sequence.
  • transmembrane domains are known in the art and include for example, the transmembrane domains of CD8 ⁇ , CD28, CD4, CD3 ⁇ , Fc ⁇ R1 ⁇ , 4-1BB or a variant thereof. Selection of CD3 ⁇ as transmembrane domain may lead to the association of the CAR with other elements of TCR/CD3 complex. This association may recruit more ITAMs but may also lead to the competition between the CAR and the endogenous TCR/CD3.
  • the transmembrane domain of the CAR is selected from the transmembrane domain of CD28 and the transmembrane domain of CD8 ⁇ .
  • the transmembrane domain of the CAR is the transmembrane domain of CD28.
  • the CAR comprises a portion of the extracellular domain and transmembrane domain of CD28.
  • a portion of the CD28 extracellular domain and transmembrane domain comprises the sequence shown in SEQ ID NO: 35 as shown below, where the transmembrane domain is shown in bold type: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 35).
  • the co-stimulatory signalling region of the CAR is, or comprises, the co-stimulatory signalling region of CD28
  • the CD28 transmembrane domain represents a suitable, often preferred, option for the transmembrane domain.
  • the full length CD28 protein is a 220 amino acid protein of SEQ ID NO: 3, where the transmembrane domain is shown in bold type: MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYG NYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHL CPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHY QPYAPPRDFAAYRS (SEQ ID NO: 3).
  • one of the co-stimulatory signalling regions is based upon the hinge region and suitably also the transmembrane domain and endodomain of CD28.
  • the co-stimulatory signalling region comprises amino acids 114-220 of the sequence shown in SEQ ID NO: 3, shown below as SEQ ID NO: 4, where the transmembrane domain is shown in bold type: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRS KRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 4).
  • the DAP10 transmembrane domain represents a suitable, often preferred, option for the transmembrane domain.
  • the full length DAP10 protein is a 93 amino acid protein of SEQ ID NO: 47, where the transmembrane domain is shown in bold type: MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVF LCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO: 47)
  • the second binding element of the second-generation CAR binds at least one epitope.
  • the second binding element of the second-generation CAR specifically binds to an epitope. In various embodiments, the second binding element of the second-generation CAR binds to a second target antigen. In certain embodiments, the second antigen is associated with a disease, such as a cancer. In some embodiments, the first and second antigens are associated with the same disease, such as the same cancer.
  • a suitable binding element may be any element which provides the second-generation CAR with the ability to recognize a target of interest.
  • the target to which the second- generation CAR of the disclosure directed can be any target of clinical interest to which it would be desirable to direct a T cell response.
  • the binding element used in the second-generation CARs described herein are antigen binding sites (ABS) of antibodies.
  • ABS used as the binding element is formatted into an scFv or is single domain antibody from a camelid, human, or other species.
  • the binding element is an alternative binding moiety, such as an aptamer (an RNA molecule that forms a secondary structure capable of mediating binding) or a ‘man-made’ binder, such as a DARPin.
  • a binding element of a second-generation CAR may comprise ligands that bind to a surface protein of interest.
  • the binding element is associated with a leader sequence which facilitates expression on the cell surface.
  • the binding element specifically binds a tumour antigen or tumour- associated antigen, providing a means to target tumour cells while limiting damage to non- tumour cells or tissues.
  • the binding elements of the second-generation CAR and CCR constructs of the pCAR respectively bind or interact with a second epitope and a first epitope.
  • the first epitope can be identical to or distinct from the second epitope.
  • the binding elements of the second-generation CAR and CCR constructs are different from one another.
  • the binding elements of the second-generation CAR and CCR specifically bind to a second epitope and first epitope of the same antigen.
  • the first and second antigens are the same.
  • the binding elements of the CAR and CCR specifically bind to the same, overlapping, or different epitopes of the same antigen.
  • the binding elements on the CAR and CCR can compete in their binding.
  • the binding elements of the second-generation CAR and CCR constructs of the pCAR bind to different antigens.
  • the antigens are different but may be associated with the same disease, such as the same specific cancer.
  • a suitable binding element may be any element which provides the CCR with the ability to recognize a target of interest.
  • the target to which the CCRs of the disclosure are directed can be any target of clinical interest to which it would be desirable to direct a T cell response.
  • the binding elements used in CCRs of the pCARs described herein are antigen binding sites (ABS) of antibodies.
  • ABS used as the binding element is formatted into a single chain antibody (scFv) or is single domain antibody from a camelid, human or other species.
  • the binding element is an alternative binding moiety, such as an aptamer (an RNA molecule that forms a secondary structure capable of mediating binding) or a ‘man-made’ binder, such as a DARPin.
  • a binding element of a second-generation CAR of a pCAR disclosed herein may comprise ligands that bind to a surface protein of interest.
  • the binding element specifically binds a second epitope on a second target antigen selected from any of the antigens listed above for the CCRs.
  • the binding element specifically binds a second epitope on a second target antigen selected from an NKG2D ligand, MUC1, ⁇ v ⁇ 6 integrin, ErbB1-4, HER2, B7-H3, Claudin 18.2, Claudin 6, Glypican 3, ALK, CD70, GD2, CD19, BCMA, CSF1R and prostate- specific membrane antigen (PSMA).
  • a second target antigen selected from an NKG2D ligand, MUC1, ⁇ v ⁇ 6 integrin, ErbB1-4, HER2, B7-H3, Claudin 18.2, Claudin 6, Glypican 3, ALK, CD70, GD2, CD19, BCMA, CSF1R and prostate- specific membrane antigen (PSMA).
  • the binding element specifically binds a second epitope on a second target antigen selected from an NKG2D ligand, MUC1, ⁇ v ⁇ 6 integrin, ErbB1-4, HER2, B7-H3, Claudin 18.2, Claudin 6, Glypican 3, ALK, CD70, GD2, CD19, BCMA, CSF1R, DLL3 and prostate-specific membrane antigen (PSMA).
  • the NKG2D ligand is selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the binding element specifically binds an epitope on the antigen MUC1.
  • the binding element specifically interacts with an epitope on a MUC1 target antigen.
  • the binding element of the second-generation CAR specifically interacts with a second epitope on a MUC1 target antigen.
  • the second-generation CAR binding element comprises the antigen binding site of an antibody specific to MUC1.
  • the second-generation CAR binding element comprises CDRs of an antibody specific to MUC1.
  • the second-generation CAR binding element comprises VH and VL sequences of an antibody specific to MUC1.
  • the second-generation CAR binding element comprises the antigen binding site of the HMFG2 antibody.
  • the second-generation CAR binding element comprises the CDRs of the HMFG2 antibody.
  • the CDR sequences of the HMFG2 antibody were determined using the tools provided on www.abysis.org and are shown below as SEQ ID NOs: 8-13: VH CDR1 GFTFSNY (SEQ ID NO: 8); V H CDR2 RLKSNNYA (SEQ ID NO: 9); VH CDR3 GNSFAY (SEQ ID NO: 10); VL CDR1 RSSTGAVTTSNYAN (SEQ ID NO: 11); VL CDR2 GTNNRAP (SEQ ID NO: 12); VL CDR3 ALWYSNHWV (SEQ ID NO: 13).
  • the second-generation CAR binding element comprises the VH and VL domains of the HMFG2 antibody.
  • the VH and VL domain sequences of the HMFG2 antibody are shown below as SEQ ID NOs: 14-15: EVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVK GRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSS (SEQ ID NO: 14); QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLI GDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSE (SEQ ID NO: 15).
  • the second-generation CAR binding element comprises the antigen binding site of the HMFG2 antibody formatted as a scFv.
  • the amino acid sequence of the scFv of the HMGF2 antibody is about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, or about 100% identical to the sequence shown in SEQ ID NO: 16 shown below: EVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVK GRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGGSGGGGSQ AVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIG DKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSE
  • the polynucleotide encoding the scFv of the HMGF2 antibody has the sequence shown in SEQ ID NO: 17 shown below: GAGGTGCAGCTGCAGCAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGT GTTGCCTCTGGATTCACTTTCAGTAACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGC TTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAATTATGCAACACATTATGCGGAGTCTGTGAAA GGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAG CTGAAGACACTGGCATTTATTACTGTACCTTTGGTAACTCCTTTGCTTACTGGGGCCAAGGGACCAC GGTCACCGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGG CCGTGGATCGCAGG CCGTGGATCGCAGGCCGTGGATCGCAGGCCGTGG
  • the binding element specifically interacts with an epitope on an NKG2D receptor ligand.
  • the second-generation CAR binding element comprises human NKG2D receptor polypeptide.
  • the second-generation CAR binding element comprises a fragment, portion, or variant of human NKG2D receptor polypeptide.
  • the NKG2D-CD3 ⁇ linear CAR (SEQ ID NO: 69) comprises an NKG2D ligand binding domain (SEQ ID NO: 75) fused to a CD3 ⁇ polypeptide (SEQ ID NO: 1).
  • SEQ ID NO: 69 The protein sequence of the NKG2D-CD3 ⁇ linear CAR is shown below as SEQ ID NO: 69.
  • CD3 ⁇ sequence is italicized and ITAMs of CD3 ⁇ are in bold; NKG2D sequence is double underlined.
  • the second-generation CAR is H or H2.
  • H2 CAR is a second- generation (2G) CAR originally described in Wilkie et al., J. Immunol. 180:4901-9 (2008), incorporated herein by reference in its entirety. It comprises, from intracellular to extracellular, a CD3 ⁇ signalling region, CD28 co-stimulatory, transmembrane and extracellular spacer domains, and a human MUC1-targeting HMFG2 scFv domain.
  • the protein sequence H2 is shown below as SEQ ID NO: 21 with the VH and the VL sequences of HMFG2 underlined and in bold and the ITAMs of the CD3 ⁇ polypeptide are italicized and in bold: MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGL EWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYW GQGTTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANW VQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWV FGGGTKLTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRS
  • the CAR is the T1E28z second-generation CAR, which comprises, from extracellular to intracellular domain, the ErbB targeting T1E peptide, CD28 hinge, transmembrane and co-stimulatory domains, and a CD3 ⁇ signalling region.
  • the T1E28z second-generation CAR is described in Davies, “Flexible targeting of ErbB dimers that drive tumourigenesis by using genetically engineered T cells,” Mol. Med. 18:565-576 (2012), incorporated herein by reference in its entirety.
  • the second-generation CAR is H2-1XX.
  • H2-1XX CAR is a second- generation (2G) CAR comprising, from intracellular to extracellular, a modified CD3 ⁇ signalling region, CD28 co-stimulatory, transmembrane and extracellular spacer domains, and a human MUC1-targeting HMFG2 scFv domain.
  • the protein sequence of H2-1XX is shown below as SEQ ID NO: 59 with the VH and VL sequences of HMFG2 are underlined and in bold, the ITAMs of CD3 ⁇ are italicized and in bold. Residues mutated to phenylalanine (F) from tyrosine (Y) (with reference to unmodified CD3 ⁇ ) are double underlined.
  • H2BB is a 2G CAR comprising from intracellular to extracellular, a CD3 ⁇ signalling region, a 4-1BB co- stimulatory domain, a CD8 ⁇ transmembrane and extracellular spacer domain, and a human MUC1-targeting HMFG2 scFv domain.
  • the second-generation CAR is a human MUC1-targeting HMFG2 scFv fused to a portion of the CD28 ectodomain, followed by a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a modified CD3 ⁇ signalling region (“H-1”).
  • the modified CD3 ⁇ signalling region is a truncated CD3 ⁇ polypeptide.
  • the protein sequence of the H-1 is shown below as SEQ ID NO: 22 with VH and VL sequences of HMFG2 in italics and bold and ITAM1 of the CD3 ⁇ polypeptide in bold and underlined: MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGL EWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYW GQGTTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANW VQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWV FGGGTKLTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLL
  • the second-generation CAR comprises a human MUC1-targeting HMFG2 scFv fused to a portion of the CD28 ectodomain, followed by a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a modified CD3 ⁇ signalling region (“H-1 ⁇ ”) that lacks each of ITAM2 and ITAM3 and has three (“H-1 ⁇ 3”), six (“H-1 ⁇ 6”) or nine (“H-1 ⁇ 9”) amino acid residues deleted N-terminal to ITAM1.
  • H-1 ⁇ modified CD3 ⁇ signalling region
  • H-1 ⁇ 3 CAR The protein sequence of the H-1 ⁇ 3 CAR is shown below as SEQ ID NO: 62 with the V H and the VL sequences of HMFG2 underlined and in bold and ITAM1 of CD3 ⁇ italicized and in bold.
  • the protein sequence of H-1 ⁇ 3 comprises a truncated CD3 ⁇ polypeptide that has the sequence shown in SEQ ID NO: 50 as shown below.
  • the ITAM1 sequence is italicized and in bold.
  • RVKFSRAPAYQQGQNQLYNELNLGRREEYDVLDKRRG (SEQ ID NO: 50)
  • the protein sequence of H-1 ⁇ 6 CAR is shown below as SEQ ID NO: 63 with the VH and the VL sequences of HMFG2 underlined and in bold and ITAM1 of CD3 ⁇ italicized and in bold.
  • the protein sequence of H-1 ⁇ 6 comprises a truncated CD3 ⁇ polypeptide that has the amino acid sequence of SEQ ID NO: 51 as shown below.
  • the ITAM1 sequence is italicized and in bold.
  • RVKAPAYQQGQNQLYNELNLGRREEYDVLDKRRG (SEQ ID NO: 51).
  • the protein sequence of the H-1 ⁇ 9 CAR is shown below as SEQ ID NO: 64 with the VH and the VL sequences of HMFG2 underlined and in bold and ITAM1 of CD3 ⁇ italicized and in bold.
  • the protein sequence of H-1 ⁇ 9 comprises a truncated CD3 ⁇ polypeptide that has the sequence shown in SEQ ID NO: 52 as shown below.
  • the ITAM1 sequence is italicized and in bold.
  • APAYQQGQNQLYNELNLGRREEYDVLDKRRG (SEQ ID NO: 52).
  • the protein sequence of the H-1 ⁇ 9 YRS CAR is shown below as SEQ ID NO: 65 with the V H and the VL sequences of HMFG2 underlined and in bold, the sequence of the CD28 co- stimulatory region double underlined, and the ITAM1 sequence of CD3 italicized and in bold.
  • the protein sequence of H-1 ⁇ 9 YRS comprises a truncated CD3 ⁇ polypeptide that has the sequence shown in SEQ ID NO: 52 as shown below.
  • the ITAM1 sequence is italicized and in bold.
  • APAYQQGQNQLYNELNLGRREEYDVLDKRRG SEQ ID NO: 52.
  • Parallel chimeric antigen receptors The present disclosure provides parallel chimeric antigen receptors (pCARs).
  • the pCAR comprises a second-generation CAR and a chimeric co-stimulatory receptor (CCR) provided in this disclosure.
  • the pCAR may comprise any of the CCRs provided in this disclosure.
  • the pCAR may comprise any of the second-generation CARs described above.
  • the co-stimulatory signalling region of the CAR is the co- stimulatory signalling region of ICOS and the at least one TNFR co-stimulatory domain in the CCR polypeptide comprises or consists of at least one 4-1BB co-stimulatory domain.
  • the co-stimulatory signalling region of the CAR is the co- stimulatory signalling region of CD28 and the at least one TNFR co-stimulatory domain in the CCR polypeptide comprises or consists of at least one 4-1BB co-stimulatory domain.
  • the co-stimulatory signalling region in the CAR is the co- stimulatory signalling region of CD28 and the at least one TNFR co-stimulatory domain in the CCR polypeptide comprises or consists of at least one CD27 co-stimulatory domain.
  • the co-stimulatory signalling region of the CAR is the co- stimulatory signalling region of ICOS and the at least one TNFR co-stimulatory domain in the CCR polypeptide comprises or consists of at least one CD27 co-stimulatory domain.
  • the NKG2Dbb(trimer)/H-1XX pCAR (SEQ ID NO: 78) comprises (i) a trimeric CCR comprising three CCR polypeptides, each comprising a CD124 leader peptide, NKG2D extracellular domain (amino acids 82-216) fused to a G4S-G4D-G4 linker, coiled coil domain of Coronin 1A, G4 linker, followed by a 4-1BB transmembrane domain and a 4-1BB co stimulatory domain (“NKG2Dbb(trimer)”) (SEQ ID NO: 79) and (ii) a second-generation CAR comprising a human MUC1-targeting HMFG2 scFv fused to a portion of the CD28 ectodomain, followed by a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a modified CD3 ⁇ signalling region (“H2-1XX”) (SEQ ID NO: 78) comprises
  • the CCR may comprise three or more CCR polypeptides, each comprising a CD124 leader peptide, NKG2D extracellular domain (amino acids 82-216) fused to a G4S-G4D-G4 linker, coiled coil domain of Coronin 1A, G4 linker, followed by a 4-1BB transmembrane domain and a 4-1BB co stimulatory domain (“NKG2Dbb(trimer)”) (SEQ ID NO: 79).
  • CCR polypeptides each comprising a CD124 leader peptide, NKG2D extracellular domain (amino acids 82-216) fused to a G4S-G4D-G4 linker, coiled coil domain of Coronin 1A, G4 linker, followed by a 4-1BB transmembrane domain and a 4-1BB co stimulatory domain (“NKG2Dbb(trimer)”) (SEQ ID NO: 79).
  • the modified CD3 ⁇ polypeptide (SEQ ID NO: 48) comprises a wild-type immunoreceptor tyrosine-based activation motif 1 (ITAM1) and two tyrosine to phenylalanine mutations in each of ITAM2 and ITAM3.
  • ITAM1 immunoreceptor tyrosine-based activation motif 1
  • RRKR furin cleavage site
  • RRKR furin cleavage site
  • the protein sequence of NKG2Dbb(trimer)/H-1XX pCAR is shown below as SEQ ID NO: 78.
  • the NKG2D sequence is single underlined and the ITAMs of the CD3 ⁇ polypeptide are italicized and in bold with the mutated residues double underlined.
  • NKG2Dbb(trimer) The protein sequence of the CCR (“NKG2Dbb(trimer)”) of NKG2Dbb(trimer)/H-1XX is shown below as SEQ ID NO: 79 with the NKG2D polypeptide sequence in bold and the 4-1BB co- stimulatory signalling region sequence underlined.
  • the protein sequence of the CAR (“H2-1XX”)-1XX is shown above as SEQ ID NO: 59.
  • the Pbb(trimer)/H-1XX pCAR (SEQ ID NO: 80) comprises (i) a trimeric CCR comprising three CCR polypeptides, each comprising a PD-1 polypeptide fused to a G4 linker, coiled coil domain of Coronin 1A, G4 linker, followed by a 4-1BB transmembrane domain and a 4-1BB co-stimulatory domain (“Pbb(trimer)”) (SEQ ID NO: 81) and (ii) a second-generation CAR comprising a human MUC1-targeting HMFG2 scFv fused to a portion of the CD28 ectodomain, followed by a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a modified CD3 ⁇ signalling region (“H2-1XX”) (SEQ ID NO: 59).
  • the modified CD3 ⁇ polypeptide (SEQ ID NO: 48) comprises a wild-type immunoreceptor tyrosine-based activation motif 1 (ITAM1) and two tyrosine to phenylalanine mutations in each of ITAM2 and ITAM3.
  • ITAM1 immunoreceptor tyrosine-based activation motif 1
  • RRKR furin cleavage site
  • RRKR furin cleavage site
  • the protein sequence of Pbb(trimer)/H-1XX pCAR is shown below as SEQ ID NO: 80.
  • the PD-1 sequence is single underlined and the ITAMs of the CD3 ⁇ polypeptide are italicized and in bold with the mutated residues double underlined.
  • Pbb(trimer) The protein sequence of the CCR (“Pbb(trimer)”) of Pbb(trimer)/H-1XX is shown below as SEQ ID NO: 81 with the PD-1 polypeptide sequence in bold and the 4-1BB co-stimulatory signalling region sequence underlined.
  • the protein sequence of the CAR (“H2-1XX”) of Pbb(trimer)/H-1XX is shown above as SEQ ID NO: 59.
  • the 44bb(trimer)/H-1XX pCAR (SEQ ID NO: 82) comprises (i) a trimeric CCR comprising three CCR polypeptides, each comprising an NKp44 polypeptide fused to a G4 linker, coiled coil domain of Coronin 1A, G4 linker, followed by a 4-1BB transmembrane domain and a 4- 1BB co-stimulatory domain (“44bb(trimer)”) (SEQ ID NO: 83) and (ii) a second-generation CAR comprising a human MUC1-targeting HMFG2 scFv fused to a portion of the CD28 ectodomain, followed by a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a modified CD3 ⁇ signalling domain (“H2-1XX
  • the modified CD3 ⁇ polypeptide (SEQ ID NO: 48) comprises a wild-type immunoreceptor tyrosine-based activation motif 1 (ITAM1) and two tyrosine to phenylalanine mutations in each of ITAM2 and ITAM3.
  • ITAM1 immunoreceptor tyrosine-based activation motif 1
  • RRKR furin cleavage site
  • RRKR furin cleavage site
  • the protein sequence of the 44bb(trimer) pCAR is shown below as SEQ ID NO: 82.
  • the NKp44 sequence is single underlined and the ITAMs of the CD3 ⁇ polypeptide are italicized and in bold with the mutated residues double underlined.
  • the protein sequence of the CCR (“44bb(trimer)”) of 44bb(trimer)/H-1XX is shown below as SEQ ID NO: 83 with the NKp44 polypeptide sequence in bold and the 4-1BB co-stimulatory signalling region sequenced underlined.
  • the protein sequence of the CAR (“H2-1XX”) of 44bb(trimer)/H-1XX is shown above as SEQ ID NO: 59.
  • a pCAR comprising or consisting of the sequence shown in SEQ ID NO: 80, 82 or 78 or or a variant sequence having at least about 85% identity to the sequence shown in SEQ ID NO: 80, 82 or 78.
  • the adaptor CAR comprises, from C-terminus to N-terminus (from intracellular to extracellular as expressed within an immunoresponsive cell) (i) at least one adaptor protein comprising an activation signalling domain and a co-stimulatory signalling region; and (ii) at least one second binding element that specifically binds a second epitope on a second antigen.
  • the at least one adaptor protein (i) and the at least one second binding element (ii) are expressed as two or more separate polypeptides that associate via non-covalent interaction(s) in the plasma membrane driven by complementary charged amino acids in each polypeptide.
  • the second target antigen may be present on a target cell.
  • the second target antigen may be soluble or secreted.
  • the adaptor CAR comprises (i) an adaptor protein comprising a CD3 ⁇ activation signalling domain and a DAP10 polypeptide; and (ii) an NKG2D ligand binding domain polypeptide.
  • the CD3 ⁇ activation signalling domain is modified.
  • the modified CD3 ⁇ activation signalling domain comprises at least one ITAM variant comprising one or more loss-of-function mutations.
  • the modified CD3 ⁇ activation signalling domain comprises an ITAM2 variant comprising one or more loss-of-function mutations and an ITAM3 variant comprising one or more loss-of-function mutations.
  • the ITAM2 variant comprises two loss-of-function mutations and the ITAM3 variant comprises two loss-of-function mutations.
  • each of the loss-of-function mutations comprises a tyrosine to phenylalanine substitution.
  • the adaptor CAR comprises (i) an adaptor protein comprising a DAP10 co-stimulatory polypeptide and a DAP12 activation signalling domain; and (ii) an NKG2D ligand binding domain polypeptide.
  • adaptor CARs are named according to the following convention: (ii) targeting moiety (comprising a binding element that specifically interacts with a target antigen) / (i) co-stimulatory signalling region-activation signalling domain wherein use of a forward slash (/) denotes an adaptor (non-covalent) association between the targeting moiety (ii) and the adaptor protein (signalling unit) (i).
  • the dash (-) in the adaptor CAR name indicates a direct fusion between the two components (e.g. between the co-stimulatory signalling region and the activation signalling domain).
  • Adaptor proteins comprises one or more adaptor proteins.
  • the adaptor protein comprises an activation signalling domain. In some embodiments, the adaptor protein comprises a co-stimulatory signalling region. In some embodiments, the adaptor protein is a fusion of an activation signalling domain and a co-stimulatory region. In some embodiments, the adaptor protein is a fusion of a modified activation signalling domain and a co-stimulatory region. In some embodiments, the activation signalling domain of the adaptor protein comprises at least one ITAM. In particular embodiments, the activation signalling domain is DAP12, CD3 ⁇ , or a variant thereof (e.g., CD3 ⁇ -1XX).
  • the co-stimulatory signalling region in the adaptor CAR may be any of those listed above, including the co-stimulatory signalling regions of members of the B7/CD28 family such as B7-1, B7-2, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD-1, PD-L2 or PDCD6; or ILT/CD85 family proteins such as LILRA3, LILRA4, LILRB1, LILRB2, LILRB3 or LILRB4; or tumour necrosis factor (TNF) superfamily members such as 4- 1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, Lymphotoxin-alpha, OX40, RELT, TACI, TL1A, TNF-alpha, or TNF RII; or members of the SLAM family such as 2B4,
  • the co-stimulatory signalling region is DAP10 or a variant thereof.
  • the adaptor protein is a fusion of an activation signalling domain and a co-stimulatory region that are directly bonded to each other in a contiguous polypeptide chain.
  • the adaptor protein is a fusion of an activation signalling domain and a co-stimulatory region that are indirectly bonded to each other through a suitable linker (e.g., peptide linker).
  • Peptide linkers are commonly used in fusion polypeptides and methods for selecting or designing such linkers are well-known. (See, e.g., Chen X et al., 2013, Adv. Drug Deliv. Rev.
  • the adaptor protein is a fusion of a DAP10 polypeptide or functional variant thereof and a CD3 ⁇ polypeptide or a functional variant thereof.
  • the adaptor protein is a fusion of DAP10 and CD3 ⁇ (e.g., DAP10-CD3 ⁇ in the NKG2D / DAP10-CD3 ⁇ adaptor CAR).
  • the adaptor protein is a fusion of DAP10 and CD3 ⁇ -1XX (e.g., DAP10-CD3 ⁇ -1XX in the NKG2D / DAP10-CD3 ⁇ -1XX adaptor CAR).
  • Adaptor proteins are further described in PCT/EP2020/076566, published as WO 2021/058563, which is incorporated by reference in its entirety.
  • Adaptor CAR binding element The second binding element of the adaptor CAR binds at least one epitope. In various embodiments, the second binding element of the adaptor CAR specifically binds to an epitope. In various embodiments, the second binding element of the adaptor CAR binds to a second target antigen.
  • the binding element is an alternative binding moiety, such as an aptamer (an RNA molecule that forms a secondary structure capable of mediating binding) or a ‘man-made’ binder, such as a DARPin.
  • a binding element of an adaptor CAR may comprise ligands that bind to a surface protein of interest.
  • the binding element is associated with a leader sequence which facilitates expression on the cell surface.
  • leader sequences are known in the art, and these include the CD8a leader sequence, macrophage colony-stimulating factor receptor (FMS) leader sequence or CD124 leader sequence.
  • the binding element specifically binds a tumour antigen or tumour- associated antigen, providing a means to target tumour cells while limiting damage to non- tumour cells or tissues.
  • the binding elements of the adaptor CAR and CCR constructs of the pACAR respectively bind or interact with a second epitope and a first epitope.
  • the first epitope can be identical to or distinct from the second epitope.
  • the binding elements of the adaptor CAR and CCR constructs are different from one another.
  • the binding elements of the adaptor CAR and CCR specifically bind to a second epitope and first epitope of the same antigen.
  • the first and second antigens are the same.
  • the binding elements of the adaptor CAR and CCR specifically bind to the same, overlapping, or different epitopes of the same antigen. In embodiments in which the second and first epitopes are the same or overlapping, the binding elements on the adaptor CAR and CCR can compete in their binding. In various embodiments, the binding elements of the adaptor CAR and CCR constructs of the pCAR bind to different antigens. In certain embodiments, the antigens are different but may be associated with the same disease, such as the same specific cancer. Thus, a suitable binding element may be any element which provides the adaptor CCR with the ability to recognize a target of interest.
  • the target to which the CCRs of the disclosure are directed can be any target of clinical interest to which it would be desirable to direct a T cell response.
  • the adaptor CAR binds specifically to an NKG2D ligand.
  • the NKG2D ligand is selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the second binding element of the adaptor CAR may be any of the NKG2D polypeptides discussed above for the second-generation CARs.
  • the NKG2D / DAP10-12 adaptor CAR (SEQ ID NO: 67) comprises a first polypeptide that is an NKG2D ligand binding domain (SEQ ID NO: 75) and a second polypeptide that is a fusion of the full length DAP10 polypeptide (SEQ ID NO: 47) and a DAP12 activation signalling domain (SEQ ID NO: 85).
  • the first and second polypeptides are initially expressed from a single polynucleotide/mRNA transcript in which the N-terminus of NKG2D is linked to the C- terminus of the DAP12 activation signalling domain by a furin cleavage site (SEQ ID NO: 31), a Ser-Gly linker (SEQ ID NO: 32), and a P2A ribosomal skip peptide (SEQ ID NO: 34).
  • the protein sequence of the NKG2D / DAP10-12 adaptor CAR is shown below as SEQ ID NO: 67. This construct is also known as N1012.
  • DAP10 sequence is bold; DAP12 sequence is underlined with the DAP12 ITAM in bold; NKG2D sequence is double underlined and follows an intervening furin cleavage site (RRKR), SGSG linker and P2A ribosomal skip peptide.
  • RRKR furin cleavage site
  • the NKG2D / DAP10-12 adaptor CAR is described in more detail in PCT/EP2020/076566, published as WO 2021/058563, which is incorporated by reference herein in its entirety.
  • the NKG2D / DAP10-CD3 ⁇ adaptor CAR (SEQ ID NO: 72) comprises a first polypeptide that is an NKG2D ligand binding domain (SEQ ID NO: 75) and a second polypeptide that is a fusion of the full-length DAP10 polypeptide (SEQ ID NO: 47) and a CD3 ⁇ activation signalling domain (SEQ ID NO: 1).
  • the first and second polypeptides are initially expressed from a single polynucleotide/mRNA transcript in which the N-terminus of NKG2D is linked to the C-terminus of CD3 ⁇ by a furin cleavage site (SEQ ID NO: 31), a Ser-Gly linker (SEQ ID NO: 32), and a P2A skip peptide (SEQ ID NO: 34).
  • SEQ ID NO: 31 furin cleavage site
  • SEQ ID NO: 32 Ser-Gly linker
  • P2A skip peptide SEQ ID NO: 34
  • DAP10 sequence is bold; CD3 ⁇ sequence is single underlined with the ITAMs of CD3 ⁇ in italics; NKG2D sequence is double underlined and follows an intervening furin cleavage site (RRKR), SGSG linker and P2A ribosomal skip peptide: MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLI VGAVFLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPRRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDF STRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVA
  • the NKG2D / DAP10-CD3 ⁇ -1XX adaptor CAR (SEQ ID NO: 74) comprises a first polypeptide that is an NKG2D ligand binding domain (SEQ ID NO: 75) and a second polypeptide that is a fusion of the full-length DAP10 polypeptide (SEQ ID NO: 47) and a modified CD3 ⁇ activation signalling domain (SEQ ID NO: 88).
  • the first and second polypeptides are initially expressed from a single polynucleotide/mRNA transcript in which the N-terminus of NKG2D is linked to the C-terminus of CD3 ⁇ by a furin cleavage site (SEQ ID NO: 31), a Ser-Gly linker (SEQ ID NO: 32), and P2A skip peptide (SEQ ID NO: 34).
  • SEQ ID NO: 31 furin cleavage site
  • SEQ ID NO: 32 Ser-Gly linker
  • P2A skip peptide SEQ ID NO: 34.
  • the protein sequence of the NKG2D / DAP10-CD3 ⁇ -1XX adaptor CAR is shown below as SEQ ID NO: 74.
  • DAP10 sequence is bold; CD3 ⁇ sequence is single underlined and ITAMs of CD3 ⁇ are in italics, mutated residues of CD3 ⁇ are double underlined; NKG2D sequence is double underlined and follows an intervening furin cleavage site (RRKR), SGSG linker and P2A ribosomal skip peptide.
  • RRKR furin cleavage site
  • SGSG linker P2A ribosomal skip peptide
  • the present disclosure provides parallel adaptor CARs (pACARs).
  • the pACAR comprises an adaptor CAR and a chimeric co-stimulatory receptor (CCR) provided in this disclosure.
  • the pACAR may comprise any of the CCRs provided in this disclosure.
  • the pACAR may comprise any of the adaptor CARs described above.
  • the pACAR preferably comprises a N1012 CAR. An example of this is shown in SEQ ID NO: 67.
  • the Pbb(trimer)_N1012 pACAR (SEQ ID NO: 95) comprises (i) a trimeric CCR comprising three CCR polypeptides, each comprising a PD-1 polypeptide fused to a G4 linker, coiled coil domain of Coronin 1A, G4 linker, followed by a 4-1BB transmembrane domain and a 4-1BB co-stimulatory domain (“Pbb(trimer)”) (SEQ ID NO: 81) and (ii) a N1012 adaptor CAR (SEQ ID NO: 67).
  • the protein sequence of Pbb(trimer)_N1012 pACAR is shown below as SEQ ID NO: 95.
  • the PD-1 sequence is single underlined.
  • Pbb(trimer) The protein sequence of the CCR (“Pbb(trimer)”) of Pbb(trimer)_N1012 pACAR is shown below as SEQ ID NO: 81 with the PD-1 polypeptide sequence in bold and the 4-1BB transmembrane and co-stimulatory signalling region sequence underlined.
  • the 44bb(trimer)_N1012 pACAR (SEQ ID NO: 97) comprises (i) a trimeric CCR comprising three CCR polypeptides, each comprising an NKp44 polypeptide fused to a G4 linker, coiled coil domain of Coronin 1A, G4 linker, followed by a 4-1BB transmembrane domain and a 4- 1BB co-stimulatory domain (“44bb(trimer)”) (SEQ ID NO: 83) and (ii) the N1012 adaptor CAR (SEQ ID NO: 67).
  • the protein sequence of the 44bb(trimer)_N1012 pACAR is shown below as SEQ ID NO: 97.
  • the NKp44 sequence is single underlined.
  • the protein sequence of the CCR (“44bb(trimer)”) of 44bb(trimer)_N1012 pACAR is shown below as SEQ ID NO: 83 with the NKp44 polypeptide sequence in bold and the 4-1BB co- stimulatory signalling region sequenced underlined.
  • CXCR2 polypeptide (bold), (ii) a furin cleavage site plus P2A ribosomal skip peptide (italicized); (iii) a CCR polypeptide, comprising a PD-1 polypeptide (single underlined) fused to a G4 linker, coiled coil domain of Coronin 1A, G4 linker, followed by a 4-1BB transmembrane domain and a 4-1BB co-stimulatory domain (“Pbb(trimer)”) (SEQ ID NO: 81) (iv) a furin cleavage site plus T2A ribosomal skip peptide (italicized) and (v) a N1012 adaptor CAR (SEQ ID NO: 67).
  • CXCR2 Pbb(trimer)_N1012 pACAR The protein sequence of CXCR2 Pbb(trimer)_N1012 pACAR is shown below as SEQ ID NO: 109.
  • SEQ ID NO: 109 The protein sequence of CXCR2 Pbb(trimer)_N1012 pACAR is shown below as SEQ ID NO: 109.
  • Pbb(trimer) The protein sequence of the CCR (“Pbb(trimer)”) of Pbb(trimer)_N1012 pACAR is shown below as SEQ ID NO: 81 with the PD-1 polypeptide sequence in bold and the 4-1BB transmembrane and co-stimulatory signalling region sequence underlined.
  • a pACAR comprising or consisting of the sequence shown in SEQ ID NO: 95, 97 or 109 or or a variant sequence having at least about 85% identity to the sequence shown in SEQ ID NO: 95, 97 or 109.
  • Cells The present disclosure provides host cells. The present disclosure provides immunoresponsive cells. In some embodiments, the host cell or immunoresponsive cell expresses a CCR provided in this disclosure, a trimeric CCR provided in this disclosure, or a lateral CAR provided in this disclosure. The CRR may be any of those described above. The trimeric CCR may be any of those described above.
  • the lateral CAR may be a pCAR or pACAR. The pCAR or pACAR may be any of those described above.
  • the immunoresponsive cell is a T cell. In certain embodiments, the immunoresponsive cell is an ⁇ T cell. In particular embodiments, the immunoresponsive cell is a cytotoxic ⁇ T cell. In particular embodiments, the immunoresponsive cell is an ⁇ helper T cell. In particular embodiments, the immunoresponsive cells is a regulatory ⁇ T cell (Treg). In some embodiments, the T cell is a TCR-re-directed T-cell. In certain embodiments, the immunoresponsive cell is a ⁇ T cell. In particular embodiments, the immunoresponsive cell is a V ⁇ 2 + ⁇ T cell. In particular embodiments, the immunoresponsive cell is a V ⁇ 2 – T cell. In specific embodiments, the V ⁇ 2 – T cell is a V ⁇ 1 + cell.
  • the immunoresponsive cell is a Natural Killer (NK) cell. In some embodiments, the immunoresponsive cell is an ⁇ T cell, ⁇ T cell, or a Natural Killer (NK) cell. In some embodiments, the host cell or immunoresponsive cell expresses no additional exogenous proteins. In other embodiments, the host cell or immunoresponsive cell is engineered to express additional exogenous proteins, such as a protease, an engineered T cell receptor (TCR) or chimeric antigen receptor (CAR). In some embodiments, the immunoresponsive cell is a cell from a cell line. In some embodiments, the immunoresponsive cell is a primary T-cell. In some embodiments, the immunoresponsive cell is a human cell, optionally a human primary T-cell.
  • the immunoresponsive cell is obtained from peripheral blood mononuclear cells (PBMCs). In some embodiments, the immunoresponsive cell is obtained from a tumour. In particular embodiments, the immunoresponsive cell obtained from a tumour is a tumour infiltrating lymphocyte (TIL). In specific embodiments, the TIL cells are ⁇ T cells. In other specific embodiments, the TIL cells are ⁇ T cells. In some embodiments, the immunoresponsive cell is a macrophage or a neutrophil. In any of the embodiments discussed above, the immunoresponsive cell may be derived from a progenitor cell or a stem cell, such as an induced pluripotent stem cell (iPSC).
  • iPSC induced pluripotent stem cell
  • the progenitor cell or stem cell may be transduced according to the disclosure and then differentiated into an immunoresponsive cell.
  • Polynucleotides and methods of making cells Also provided herein is one or more polynucleotides which encode a CCR polypeptide provided in this disclosure, a trimeric CCR provided in this disclosure, or a lateral CAR provided in this disclosure.
  • the one or more polynucleotides comprise a first polynucleotide which encodes a CCR polypeptide provided in this disclosure.
  • the one or more polynucleotides or the first polynucleotide may encode a polypeptide which comprises or consists of the sequence shown in any one of SEQ ID NOs: 79, 81 and 83.
  • the one or more polynucleotides or the first polynucleotide may comprise or consist of the sequence shown in any one of SEQ ID NOs: 118, 119 and 120.
  • Also provided herein is a combination of a first polynucleotide encoding a CCR polypeptide or trimeric CCR provided in this disclosure and a second polynucleotide encoding a CAR as described above.
  • the first polynucleotide may be any of those described above.
  • the second polynucleotide may encode a polypeptide which comprises or consists of the sequence shown in any one of SEQ ID NOs: 21, 59, 22, 62, 63, 64, 65, 67, 69, 72, and 74.
  • the second polynucleotide may comprise or consist of the sequence shown in any one of SEQ ID NOs: 66, 71, 73 and 116.
  • the CAR and CCR combination is referred to in the singular as a lateral CAR, such as a pCAR or pACAR, although the CAR and CCR are separate, co-expressed, proteins.
  • the first and second polynucleotides can be expressed from a single vector or two or more vectors.
  • the first polynucleotide and the second polynucleotide are in a single vector. In some embodiments, the first polynucleotide and the second polynucleotide are expressed from a single vector. Also provided herein is a single polynucleotide which encodes a lateral CAR provided in this disclosure.
  • the single polynucleotide may encode a polypeptide which comprises or consists of the sequence shown in any one of SEQ ID NOs: 78, 80, 82, 95, 97 and 109.
  • Such vectors including plasmid vectors, or cell lines containing them, are also provided by this disclosure.
  • the polynucleotide(s) may also be introduced using any other method, such as electroporation, nanoparticle delivery or transfection.
  • the host cells or immunoresponsive cells are subjected to genetic modification, for example by retroviral or lentiviral mediated transduction, to introduce CCR and CAR coding polynucleotides into the host T-cell genome, thereby permitting stable CCR and CAR expression.
  • the host cells or immunoresponsive cells may be subjected to genetic modification by any other method known in the art. They may then be reintroduced into the patient, optionally after expansion, to provide a beneficial therapeutic effect, as described below.
  • the immunoresponsive cells are ⁇ T cells and the ⁇ T cells are activated by an anti- ⁇ TCR antibody prior to the genetic modification.
  • an immobilised anti- ⁇ TCR antibody is used for activation.
  • the first and second polynucleotides encoding the CCR and CAR can be expressed from the same vector or different vectors.
  • the present disclosure further provides a kit for the generation of immunoresponsive cells, such as pCAR T-cells described herein.
  • the kit can comprise a combination of a first polynucleotide encoding a CCR provided in this disclosure and a second polynucleotide encoding a CAR as described above.
  • the kit comprises a combination of one or more vectors comprising the first polynucleotide encoding a CCR provided in this disclosure and a second polynucleotide encoding a CAR as described above.
  • the kit further comprises a reagent for use in genetic modification of immunoresponsive cells.
  • the method comprises, (i) obtaining T-cells and/or NK cells from a subject, (ii) transducing a polynucleotide(s) or one or more vector(s) encoding the CCR and CAR peptides provided in this disclosure into the T-cells and/or NK cells, and (iii) culturing the T-cells and/or NK cells such that the CCR and CAR are expressed.
  • compositions comprising a CCR polypeptide provided in this disclosure, a trimeric CCR provided in this disclosure, a lateral CAR provided in this disclosure, one or more polynucleotides provided in this disclosure, one or more vectors provided in this disclosure, a host cell provided in this disclosure or an immunoresponsive cell provided in this disclosure.
  • the pharmaceutical compositions can further comprise a pharmaceutically or physiologically acceptable diluent, carrier and/or excipient.
  • the physiologically acceptable diluent, carrier and/or excipient is generally selected to be suitable for the intended mode of administration and can include agents for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition.
  • These carriers can include aqueous or alcoholic/aqueous solutions, emulsions, or suspensions, including saline and/or buffered media.
  • a CCR polypeptide provided in this disclosure a trimeric CCR provided in this disclosure, a lateral CAR provided in this disclosure, a one or more polynucleotides provided in this disclosure, a one or more vectors provided in this disclosure, a host cell provided in this disclosure or an immunoresponsive cell provided in this disclosure is useful in therapy to direct a T cell-mediated immune response to a target cell.
  • methods for directing a T cell-mediated immune response to a target cell in a patient in need thereof are provided.
  • the method comprises administering to the patient a therapeutically effective amount of a CCR polypeptide provided in this disclosure, a trimeric CCR provided in this disclosure, a lateral CAR provided in this disclosure, a one or more polynucleotides provided in this disclosure, a one or more vectors provided in this disclosure, a host cell provided in this disclosure or an immunoresponsive cell provided in this disclosure, wherein the binding elements are specific for the target cell.
  • the target cell expresses MUC1 and/or one or more NKG2D ligands.
  • the one or more NKG2D ligands are selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • methods for treating cancer in a patient in need thereof comprises administering to the patient a therapeutically effective amount of a CCR polypeptide, a trimeric CCR, a lateral CAR, a one or more polynucleotides, a one or more vectors, a host cell or an immunoresponsive cell, wherein the binding elements are specific for the target cancer.
  • the target cell expresses MUC1 and/or one or more NKG2D ligands.
  • the one or more NKG2D ligands are selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the patient is human.
  • the patient has breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, gastric cancer, bladder cancer, myeloma, Hodgkin or non- Hodgkin lymphoma, prostate cancer, oesophageal cancer, endometrial cancer, hepatobiliary cancer, duodenal carcinoma, thyroid carcinoma, melanoma, or renal cell carcinoma.
  • the patient has colon, breast, ovarian, lung, or pancreatic cancer.
  • the patient has breast cancer.
  • the patient has tumour cells expressing MUC1 and/or one or more NKG2D ligands.
  • the patient has been determined to have tumour cells expressing MUC1 and/or one or more NKG2D ligands.
  • the one or more NKG2D ligands are selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the treatment method further comprises the preceding steps of (i) obtaining immunoresponsive cells from a subject, (ii) transducing the immunoresponsive cells with one or more polynucleotides or one or more vectors encoding the CCR and CAR polypeptides provided in this disclosure, and (iii) culturing the immunoresponsive cells such that the CCR and CAR are expressed.
  • a therapeutically effective number of the immunoresponsive cells is administered to the patient.
  • the immunoresponsive cells are administered by intravenous infusion.
  • the immunoresponsive cells are administered by intratumoural injection.
  • the immunoresponsive cells are administered by peri-tumoural injection.
  • the immunoresponsive cells are administered by intraperitoneal injection.
  • the immunoresponsive cells are administered by a plurality of routes selected from intravenous infusion, intra-tumoural injection, and peri-tumoural injection.
  • a CCR polypeptide provided in this disclosure a trimeric CCR provided in this disclosure, a lateral CAR provided in this disclosure, a one or more polynucleotides provided in this disclosure, a one or more vectors provided in this disclosure, a host cell provided in this disclosure or an immunoresponsive cell provided in this disclosure for use in therapy.
  • a CCR polypeptide provided in this disclosure a trimeric CCR provided in this disclosure, a lateral CAR provided in this disclosure, a one or more polynucleotides provided in this disclosure, a one or more vectors provided in this disclosure, a host cell provided in this disclosure or an immunoresponsive cell provided in this disclosure for use in a method for treating cancer.
  • the cancer may be any of those described above.
  • the method may be for treating cancer in a human patient.
  • a CCR polypeptide provided in this disclosure a trimeric CCR provided in this disclosure, a lateral CAR provided in this disclosure, a one or more polynucleotides provided in this disclosure, a one or more vectors provided in this disclosure, a host cell provided in this disclosure or an immunoresponsive cell provided in this disclosure for the manufacture of a medicament for the treatment of cancer.
  • the cancer may be any of those described above.
  • the medicament may be for the treatment of cancer in a human patient. In some embodiments, the patient has been pre-treated with a chemotherapeutic agent.
  • the administration of immunoresponsive cells to the patient results in a decrease in tumour size of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or even about 100%, when compared to an untreated tumour.
  • the amount of immunoresponsive cells administered to the patient should take into account the route of administration, the cancer being treated, the weight of the patient and/or the age of the patient. In general, about 1 x 10 6 to about 1 x 10 11 cells are administered to the patient. In one embodiment, about 1 x 10 7 to about 1 x 10 10 cells, or about 1 x 10 8 to about 1 x 10 9 cells are administered to the patient.
  • the KSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASS NKG2D FKGYIENCSTPNTYICMQRTVGGGGSGGGGDEPKSCDKTHTCPPCPI sequence is YIWAPLAGTCGVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQE single EDGCSCRFPEEEEGGCELRRKRSGSGEGRGSLLTCGDVEENPGPMA underlined LPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNY and the ITAMs WMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSK of the CD3 ⁇ SSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGG polypeptide GGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWV are italicized QEKPDHLFT
  • the KSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASS NKG2D FKGYIENCSTPNTYICMQRTVGGGGSGGGGDGGGGSGTPSSDAVS sequence is RLEEEMRKLQATVQELQKRLDRLEETVQAKGGGGIISFFLALTSTALLF single LLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEEE underlined GGCELRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLH and the ITAMs
  • AEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKG of the CD3 ⁇ LEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAE polypeptide DTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGGSGGGGSQAVV are italicized TQES
  • DALHMQALPPR NKG2Dbb(trim MGWLCSGLLFPVSCLVLLQVASSGNLFNQEVQIPLTESYCGPCPK er)
  • CCR NWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKED sequence.
  • the CRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR PD-1 AELRVTERRAEVPTAHGGGGSGTPSSDAVSRLEEEMRKLQATVQELQ sequence is KRLDRLEETVQAKGGGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRK single KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRKRSGSGEG underlined RGSLLTCGDVEENPGPMALPVTALLLPLALLLHAEVQLQQSGGGLVQP and the ITAMs GGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYA of the CD3 ⁇ THYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAY polypeptide WGQGTTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVT are italicized LTCRSSTGAVTTS
  • NKp44 TMTDLREEDSGHYWCRIYRPSDNSVSKSVRFYLVVSPASASTQTSW sequence is TPRDLVSSQTQTQSCVPPTAGARQAPESPSTIPVPSQPQNSTLRPGP single AAPIAGGGGSGTPSSDAVSRLEEEMRKLQATVQELQKRLDRLEETVQ underlined AKGGGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMR and the ITAMs PVQTTQEEDGCSCRFPEEEEGGCELRRKRSGSGEGRGSLLTCGDVEE of the CD3 ⁇ NPGPMALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVAS polypeptide GFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFT are italicized ISRDDSKSSVYLQ
  • the DDPDAGFFTVTMTDLREEDSGHYWCRIYRPSDNSVSKSVRFY NKp44 LVVSPASASTQTSWTPRDLVSSQTQTQSCVPPTAGARQAPES polypeptide PSTIPVPSQPQNSTLRPGPAAPIAGGGGSGTPSSDAVSRLEEEM sequence is in RKLQATVQELQKRLDRLEETVQAKGGGGIISFFLALTSTALLFLLFFLTL bold and the RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BB co- stimulatory signalling region sequenced is underlined.
  • trimerisation technology can be applied to CCRs containing a variety of binding domains. Furthermore, the addition of trimeric CCRs are efficacious in both a pCAR and pAdaptor structure.
  • Methods Generation of trimeric CCR CARs The Pbb(trimer) CCR contains the PD-1 extracellular domain followed by the coiled coil domain from coronin 1A (flanked by G4 linkers), the 4-1BB transmembrane domain and the 4-1BB intracellular domain. The CCR was synthetically synthesised and expressed alongside N1012 or a second-generation MUC1 targeting CAR.
  • the PD-1 extracellular domain was removed and replaced with either the extracellular domain of NKp44, NKG2D or a myc tag.
  • Generation of alternative CCRs To generate a dimeric form of the Pbb CCR, the coronin 1A domain was removed from the trimeric CCR and replaced with the IgG1 hinge and a portion of the CD28 extracellular domain. To generate the monomeric CCR, a portion of the CD28 extracellular domain containing a C>S (C123S) mutation replaced the coiled coil domain in the trimeric CCR.
  • CAR T cells were added to fresh monolayer until the tumour cell monolayer reached 70% viability compared to untreated monolayer. Confirmation of CCR structure by Western Blot Cell lysates from UT and T cells transduced with N1012, Pbb(trimer) N1012, Pbb(dimer) N1012 and Pbb(monomer) N1012 were generated. Proteins were separated by native polyacrylamide electrophoresis (PAGE) and transferred to a nitrocellulose membrane for immunoblotting with an anti-PD-1 antibody.
  • BLI bioluminescence imaging
  • the co-stimulatory domain 4-1BB belongs to the TNF family of proteins which naturally are induced to trimerise following binding to their trimeric receptors. Therefore, we hypothesised a superior, more natural 4-1BB signal could be induced by utilising trimeric 4- 1BB containing CCRs.
  • trimeric CCRs we made use of the coiled coil domain from coronin 1A and inserted it into the extracellular domain of our CCRs.
  • trimeric CCRs were superior to their dimeric or monomeric counterparts confirming the importance of maintaining the natural structure of co-stimulatory domains.
  • Example 2 This Example tested additional pCARs using similar methods to those described above for Example 1. The pCARs and control constructs are shown in Figure 10. Recombinant human PD-L1 or recombinant human MIC-A were immobilised at a concentration of 1ug/mL on non-tissue culture-treated plates. Recombinant target antigen was then immobilised in the same well at the concentrations indicated on the x-axis of Figure 11.
  • T-cells co-expressing a trimeric CCR displayed greater antigen sensitivity than those expressing a dimeric CCR (N(di)bb_28z), as demonstrated by greater CD107a expression at lower concentrations of recombinant target antigen.
  • T-cells expressing the CARs and pCARs shown in Figure 10 were phenotyped by flow cytometry at the end of the 12 day ex vivo expansion period for the expression of markers associated with exhaustion. The markers assessed were PD-1, LAG-3 and TIM-3. The percentage of cells expressing each marker and the number of markers expressed simultaneously on individual cells were calculated using simplified presentation of incredibly complex evaluations (SPICE) analysis.

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Abstract

L'invention concerne des récepteurs co-stimulateurs schimériques (CCR) comprenant des domaines co-stimulateurs du récepteur de nécrose tumorale (TNFR) et des motifs de trimérisation. Ces CCR peuvent être utilisés dans des CAR parallèles (PCAR) et des CAR adaptateurs parallèles (pACAR) pour assurer une signalisation optimale. L'invention concerne également des compositions et des procédés pour améliorer l'efficacité antitumorale et la capacité de restimulation de lymphocytes T CAR, de lymphocytes T pCAR et de lymphocytes T pACAR.
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