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WO2025191154A1 - Therapeutic agents - Google Patents

Therapeutic agents

Info

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
French (fr)
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/en
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

Provided herein are chimeric co-stimulatory receptor (CCRs) comprising tumour necrosis receptor (TNFR) co-stimulatory domains and trimerisation motifs. These CCRs may be used in parallel CARs (pCARs) and parallel adaptor CARs (pACARs) to provide optimal signalling. Also provided herein are compositions and methods for improving anti-tumour efficacy and restimulation capacity of CAR T-cells, pCAR T-cells, and pACAR T-cells.

Description

THERAPEUTIC AGENTS TECHNICAL FIELD Provided herein are chimeric co-stimulatory receptor (CCRs) comprising tumour necrosis receptor (TNFR) co-stimulatory domains and trimerisation motifs. These CCRs may be used in parallel CARs (pCARs) and parallel adaptor CARs (pACARs) to provide optimal signalling. Also provided herein are compositions and methods for improving anti-tumour efficacy and restimulation capacity of CAR T-cells, pCAR T-cells, and pACAR T-cells. BACKGROUND CAR T cell therapy has shown impressive efficacy for treatment of haematological malignancies. However solid tumours present several obstacles which enable them to resist CAR T cell therapy. Within the field, considerable research focuses on engineering CAR T cells to overcome resistance of solid tumours. Approaches include armouring CAR T cells with cytokines and chemokines, blocking immune checkpoints, downregulating inhibitory receptors and proteins, and improving CAR T cell metabolism (Glover, Maya et al. “How Can We Engineer CAR T Cells to Overcome Resistance?.” Biologics : targets & therapy vol. 15 175-198. 19 May. 2021). Nevertheless, there is still a need to optimise co-stimulation to improve CAR T cell persistence within the hostile tumour microenvironment. 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). This pCAR structure recapitulates the positioning of the costimulatory molecules found naturally in immune cells in relation to their distance from the cell membrane. It was demonstrated that this structure allows superior signalling from both costimulatory domains in both the CAR and CCR compared to when they are positioned in a linear structure (Muliaditan, Tamara et al. “Synergistic T cell signaling by 4-1BB and CD28 is optimally achieved by membrane proximal positioning within parallel chimeric antigen receptors.” Cell reports. Medicine vol. 2,12 100457. 21 Dec. 2021; and Halim, Leena et al. “Engineering of an Avidity-Optimized CD19-Specific Parallel Chimeric Antigen Receptor That Delivers Dual CD28 and 4-1BB Co-Stimulation.” Frontiers in immunology vol. 13 836549. 9 Feb. 2022). This current pCAR structure includes a 4-1BB containing CCR which is constitutively dimeric due to the presence of the CD8a hinge. However, natural 4-1BB ligand (4-1BBL) is a trimer and induces 4-1BB trimerisation following binding. SUMMARY OF THE INVENTION The inventors have surprisingly shown that a more natural and superior 4-1BB signal can be induced by encouraging the 4-1BB containing CCR to trimerise. To induce trimerisation in the CCRs, the inventors incorporated a natural coiled coil trimersation motif found in the coronin 1A protein. The trimeric CCRs surprisingly produced fitter CAR T cells which can persistently control solid tumours in comparison to CAR T cells containing monomeric or dimeric versions of the CCRs. The work also confirms the trimerisation technology can be applied to different CCRs with different binding domains. Furthermore, the inventors surprisingly confirmed the addition of trimeric CCRs not only have superior efficacy in the context of pCARs but can also be used to further improve adaptor CAR technology. The present disclosure provides a chimeric co-stimulatory receptor (CCR) polypeptide comprising at least one tumour necrosis receptor (TNFR) co-stimulatory domain, wherein the CCR polypeptide further comprises at least one trimerisation motif. The present disclosure also provides: - a trimeric CCR comprising three or more CCR polypeptides provided in this disclosure; - a lateral CAR comprising (a) a CCR comprising a CCR polypeptide provided in this disclosure or a trimeric CCR provided in this disclosure and (b) a second-generation chimeric antigen receptor (CAR) or an adaptor CAR; - one or more polynucleotides which encode a CCR provided in this disclosure, a trimeric CCR provided in this disclosure or a lateral CAR provided in this disclosure; - one or more vectors comprising one or more polynucleotides provided in this disclosure; - a host cell expressing a CCR polypeptide provided in this disclosure, a trimeric CCR provided in this disclosure or a lateral CAR provided in this disclosure; - an immunoresponsive cell which expresses a CCR polypeptide provided in this disclosure, a trimeric CCR provided in this disclosure or a lateral CAR provided in this disclosure; - a pharmaceutical composition 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 and a pharmaceutically or physiologically acceptable diluent, carrier and/or excipient; - a method of preparing the immunoresponsive cell provided in this disclosure, the method comprising transfecting or transducing an immunoresponsive cell with one or more polynucleotides provided in this disclosure or one or more vectors provided in this disclosure; - a method for directing a T cell-mediated immune response to a target cell or treating cancer in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of immunoresponsive cells provided in this disclosure; and - 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, an immunoresponsive cell provided in this disclosure or a pharmaceutical composition provided in this disclosure for use (i) in therapy or as a medicament or (ii) in the treatment of cancer. BRIEF DESCRIPTION OF THE FIGURES The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention. Figure 1. Design of CCRs and CARs used in Example 1. Figure 2. In vitro benefit of trimeric CCRs. Restimulation assays on BXPC3 monolayers comparing N1012 and N1012 expressed alongside either the PD-1 (top panel of graphs) or NKp44 (bottom panel of graphs) binding trimeric CCR. Graphs presenting tumour cell viability following each stimulation (upper left), CAR T cell counts (upper right) and total number of successful restimulation numbers (bottom). n=8-12. Figure 3. 4-1BB signalling is essential for CCR function. 4-1BB truncated trimeric CCR and 4-1BB mutant CCRs were compared to fully intact trimeric CCR. Graphs presenting restimulation assays on BXPC3 monolayers including tumour cell viability (upper left), CAR T cell counts (upper right) and total number of successful restimulation numbers (bottom). n=5. Figure 4. Confirmation of CCR structure. Native PAGE and western blotting for PD-1 for clarification of CCR structure. Bands show size of monomeric, dimeric and trimeric CCRs. Figure 5. Trimeric structure of 4-1BB containing CCRs is essential for its optimal function. Dimeric and monomeric CCRs were generated, and their functional benefit was compared to the trimeric CCR in restimulation assays on BXPC3 monolayers with graphs presenting total number of successful stimulations (left graph) and peak fold cell expansion during restimulation assay (right graph). N=5. Figure 6. Trimeric NKp44 CCR superior to monomeric CCR. N1012 with monomeric and trimeric NKp44 CCRs were tested in restimulation assays on BXPC3 monolayers. Graphs presenting tumour cell viability following each stimulation (upper left), CAR T cell counts (upper right) and total number of successful restimulation numbers (bottom). n=8. Figure 7. CCR binding domain essential for function. The construct myc-bb N1012 was generated to confirm whether CCR function was dependent on ligand binding. Additionally, CARs were armoured with CXCR2 for future in vivo testing. Graphs presenting tumour cell viability following each stimulation (left), CAR T cell counts (right), n=1. Figure 8. In vivo superiority of trimeric CCR containing CARs. NSG mice were S.C. injected with 1x105 BXPC3 tumour cells, after 14 days mice were treated with 4x106 CAR+ T cells, I.V. Tumours were measured weekly using callipers. Graphs presenting tumour volume (upper) and overall survival (bottom). Figure 9. In vivo superiority of trimeric CCR containing MUC1 CARs. NSG mice were I.P. injected with 1x105 BXPC3 tumour cells, after 12 days mice were treated with 1x107 CAR+ T cells I.P. Tumours were measured by BLI. Graphs presenting whole body BLI (upper) and overall survival (bottom). Figure 10. Schematic of constructs used in Example 2. The trimeric CCRs used in Example 2 included NKG2Dbb(trimer) and Pbb(trimer). These CCRs were also used in Example 1 and are shown in Figure 1. N(di)bb_X28z includes the NKG2D_bb CCR. The CAR (X28z) was a standard CD28-based second generation format (CD28 hinge, transmembrane domain and CD3z intracellular domain fused in frame). The extracellular antigen-binding domain used in the CARs was an scFv. Figure 11. Results of the degranulation assay in Example 2. Figure 12. Results of the T-cell exhaustion experiments in Example 2. DETAILED DESCRIPTION Definitions As used herein, the term “variant” refers to a polypeptide sequence which is a naturally occurring polymorphic form of the basic sequence as well as synthetic variants, in which one or more amino acids within the basic sequence are inserted, removed or replaced. However, the variant produces a biological effect which is similar to that of the basic sequence. For example, a variant of the intracellular domain of human CD3 zeta (CD3ζ) chain will act in a manner similar to that of the intracellular domain of human CD3ζ chain. Amino acid substitutions may be regarded as “conservative” where an amino acid is replaced with a different amino acid in the same class with broadly similar properties. Non-conservative substitutions are where amino acids are replaced with amino acids of a different type or class. As is well known to those skilled in the art, altering the primary structure of a peptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptide's conformation. 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. In general, 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. As used herein, the term “antigen” refers to any member of a specific binding pair that will bind to the binding elements. The term includes receptors on target cells. As used herein and with regard to the binding element to a target molecule, the terms “bind,” “specific binding,” “specifically binds to,” “specifically interacts with,” “specific for,” “selectively binds,” “selectively interacts with,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. The term “chimeric co-stimulatory receptor” or “CCR” as used herein refers to a molecule comprising an extracellular antigen-binding domain that is fused to a co- stimulatory signalling region and a transmembrane domain. When the CCR is used in a pCAR or parallel adaptor CAR, it comprises a co-stimulatory signalling region which is different from the co-stimulatory signalling region of the CAR or adaptor CAR, a transmembrane domain, and at least one binding element that specifically binds an epitope on a target antigen. 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. Typically, the CCR lacks a TCR-like signalling region such as CD3ζ. The CCR provided in this disclosure is typically a trimer formed from three or more CCR polypeptides. The term “linear CAR” as used herein 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. The term “chimeric antigen receptor” or “CAR” as used herein refers to a molecule comprising an extracellular antigen-binding domain that is fused to an intracellular signalling domain that is capable of activating or stimulating an immunoresponsive cell and a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of a CAR comprises a scFv. The term “parallel chimeric antigen receptor” or “pCAR” as used herein refers to a parallel chimeric antigen receptor which comprises the combination of a second-generation chimeric antigen receptor (CAR) and, in parallel, a chimeric co-stimulatory receptor (CCR). pCAR has been described in WO 2017/021701, which is incorporated by reference in its entirety herein. The term “adaptor CAR” as used herein refers to an adaptor chimeric antigen receptor which comprises the combination of (i) at least one adaptor protein comprising an activation signalling domain and (ii) at least one antigen binding domain (targeting moiety) that specifically binds a target antigen. In some embodiments, the adaptor comprises in (i) at least one adaptor protein comprising an activation signalling domain and a co-stimulatory signalling region. The at least one adaptor protein (i) and the at least one targeting moiety (ii) are expressed as two or more separate polypeptides that associate via non-covalent interaction(s). The term “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. It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of” is thus also encompassed and disclosed. Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. "About" as used herein when referring to a measurable value such as an amount, a percentage, a temporal duration, and the like, is meant to encompass variations of ± 20 % or ± 10 %, ± 5 %, ± 1 %, or ± 0.1 % from the specified value, as such variations are appropriate to perform the disclosure. Any disclosure using the term “about” can also be read as if the term is not present. For instance, a sequence having at least about 85% sequence identity to the sequence shown in SEQ ID NO: 1 covers a sequence having at least 85% sequence identity to the sequence shown in SEQ ID NO: 1. All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall be in control. Section and table headings are not intended to be limiting. Chimeric co-stimulatory receptor (CCR) polypeptide 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. In some embodiments, the CCR polypeptide comprises a co- stimulatory signalling region which comprises or consists of at least one TNFR co- stimulatory domain. In some embodiments, 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. In some embodiments, the at least one TNFR co-stimulatory domain is selected from 4-1BB, OX40, CD27, and variants thereof. In some embodiments, 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. In particular, 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. In some embodiments, 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. In certain embodiments, the at least one 4-1BB co-stimulatory domain comprises or consists of the the sequence shown in SEQ ID NO: 37 as shown below: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 37). In some embodiments, 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. In the CCR polypeptide, 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 α subunits, the trimeric catalytic domain of Bruton's tyrosine kinase, some domain-swapping and Interlocking β-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). In some embodiments, the at least one trimerisation motif is encoded and/or located upstream of the at least one TNFR co-stimulatory domain. In some embodiments, 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 In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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, CD8α, CD28, CD4, CD3ζ, FcεR1γ or a variant thereof. In certain embodiments, the transmembrane domain of the CCR polypeptide is the transmembrane domain of CD8α. In some embodiments, the CCR polypeptide comprises a portion of the extracellular domain and transmembrane domain of CD8α. In certain embodiments, 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) In particular embodiments, the CCR polypeptide comprises the transmembrane domain of 4-1BB. In some embodiments, the 4-1BB transmembrane domain comprises or consists of the 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. In various embodiments, the binding element of the CCR polypeptide specifically binds to an epitope. In various embodiments, the first binding element of the CCR polypeptide binds to a first target antigen. In certain embodiments, the first target antigen is associated with a disease, such as a cancer. Thus, 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. In various embodiments, the binding element used in the CCR polypeptides described herein are antigen binding sites (ABS) of antibodies. In typical embodiments, 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. In some embodiments, 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. Alternatively, a binding element of a CCR polypeptide may comprise ligands that bind to a surface protein of interest. In some embodiments, the binding element is associated with a leader sequence which facilitates expression on the cell surface. Many leader sequences are known in the art, and these include the CD8α leader sequence, macrophage colony-stimulating factor receptor (FMS) leader sequence or CD124 leader sequence. 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. 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. In some embodiments, the first binding element specifically binds or interacts with a first epitope on a first target antigen selected from an NKG2D ligand, MUC1, αvβ6 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-β (TGF-β), PD-L1, CD155, CD112, death receptor 4 (DR4), death receptor 5 (DR5), thrombopoietin (TPO), Eltrombopag, and an ErbB family member. In some embodiments, the NKG2D ligand is selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, the first binding element specifically binds or interacts with a first epitope on a first target antigen selected from an NKG2D ligand, MUC1, αvβ6 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-β (TGF-β), PD-L1, CD155, CD112, death receptor 4 (DR4), death receptor 5 (DR5), thrombopoietin (TPO), Eltrombopag, DLL3, and an ErbB family member. In some embodiments, the NKG2D ligand is selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, the NKp30 ligand is selected from BAG6 and B7-H6. In some embodiments, the NKp44 ligand is selected from NKp44L (MLL5), PCNA, viral haemagglutinins, nidogen-1, galectin-3, and other proteoglycans. In some embodiments, the ErbB family member is selected from ErbB1, ErbB2, ErbB3 and ErbB4. In some embodiments, the first binding element specifically binds an NKG2D ligand or specifically binds a first epitope on a NKG2D ligand. In some embodiments, the NKG2D ligand is selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, the first binding element is an NKG2D polypeptide or a fragment or functional variant thereof. In some embodiments, the first binding element comprises or consists of the sequence shown in SEQ ID NO: 75. In some embodiments, the first binding element specifically binds a PD-1 ligand or specifically binds a first epitope on a PD-1 ligand. In some embodiments, the binding element or first binding element is a PD-1 polypeptide or a fragment or functional variant thereof. In some embodiments, 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. MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNW YRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIK ESLRAELRVTERRAEVPTAH (SEQ ID NO: 86) In some embodiments, the trimerized CCR comprises or consists of the sequence shown in SEQ ID NO: 81. In some embodiments, the first binding element specifically binds a NKp44 ligand or a first epitope on a NKp44 ligand. In some embodiments, the NKp44 ligand is selected from NKp44L (MLL5), PCNA, viral haemagglutinins, nidogen-1, galectin-3, and other proteoglycans. In some embodiments, the first binding element is a NKp44 polypeptide or a fragment or functional variant thereof. In some embodiments, 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). MAWRALHPLLLLLLLFPGSQAQSKAQVLQSVAGQTLTVRCQYPPTGSLYEKKGWCKEASALVCIRLVTS SKPRTMAWTSRFTIWDDPDAGFFTVTMTDLREEDSGHYWCRIYRPSDNSVSKSVRFYLVVSPASASTQ TSWTPRDLVSSQTQTQSCVPPTAGARQAPESPSTIPVPSQPQNSTLRPGPAAPIA (SEQ ID NO: 87) In some embodiments, the trimerized CCR comprises or consists of the sequence shown in SEQ ID NO: 83. In some embodiments, the first binding element specifically binds an ErbB antigen or specifically binds a first epitope on an ErbB antigen. In some embodiments, 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. In some embodiments, the first binding element is a T1E peptide or a fragment or functional variant thereof. In some embodiments, 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-α (TGF-α) 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-α 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). In some embodiments, the first binding element comprises or consists of the sequence shown in SEQ ID NO: 18. In some embodiments, the first binding element is ICR62, which binds to EGFR. In particular embodiments, the first binding element comprises the antigen binding site of the ICR62 antibody formatted as scFv. In certain embodiments, 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). In some embodiments, the first binding element specifically binds an NKp30 ligand or specifically binds a first epitope on an NKp30 ligand. In some embodiments, the NKp30 ligand is selected from BAG6 and B7-H6. In some embodiments, the first binding element is selected from: (a) an element that specifically binds a PD-1 ligand or specifically binds a first epitope on a PD-1 ligand or a PD-1 polypeptide or a fragment or functional variant thereof; (b) an element that specifically binds a NKp44 ligand or a first epitope on a NKp44 ligand or a NKp44 polypeptide or a fragment or functional variant thereof; (c) an element that specifically binds an NKG2D ligand or specifically binds a first epitope on a NKG2D ligand or an NKG2D polypeptide or a fragment or functional variant thereof. Suitable elements, polypeptides, fragments, and variants for each of (a)-(c) are discussed above. Exemplary CCR polypeptides In some embodiments, the CCR polypeptide comprises 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)”). In some embodiments the CCR polypeptide comprises or consists of the sequence shown in SEQ ID NO: 81. In some embodiments, the CCR polypeptide comprises 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)”). In some embodiments the CCR polypeptide comprises or consists of the sequence shown in SEQ ID NO: 83. In some embodiments, the CCR polypeptide comprises 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)”). In some embodiments, the CCR polypeptide comprises or consists of the sequence shown in SEQ ID NO: 79. Provided herein is a CCR polypeptide comprising or consisting of the sequence shown in SEQ ID NO: 81, 83 or 79 or or a variant sequence having at least about 85% identity to the sequence shown in SEQ ID NO: 81, 83 or 79. Trimeric chimeric co-stimulatory receptor (CCR) The present disclosure also provides a trimeric chimeric co-stimulatory receptor (CCR) comprising three or more CCR polypeptides provided in this disclosure. The CCR polypeptides may be any of those described below. The trimeric CCR may comprise any number of three or more CCR polypeptides, such as 3, 4, 5 or more CCR polypeptides. The CCR polypeptides in the trimeric CCR may be different. The CCR polypeptides in the trimeric CCR are typically the same. In some embodiments, the three or more CCR polypeptide trimerise on ligand binding. Lateral chimeric antigen receptors (pCARs and pACARs) The present disclosure also provides a lateral chimeric antigen receptor comprising a CCR comprising a CCR polypeptide provided in this disclosure. In some embodiments, the CCR is three polypeptides. In some embodiments, the CCR is more than three polypeptides. In some embodiments, the CCR is trimeric. The present disclosure also provides a lateral chimeric antigen receptor comprising a trimeric CCR provided in this disclosure. The lateral CAR also comprises a second-generation chimeric antigen receptor (CAR) or an adaptor CAR. The present disclosure also provides a parallel chimeric antigen receptor (pCAR) comprising (a) a CCR comprising a CCR polypeptide provided in this disclosure or a trimeric CCR provided in this disclosure and (b) a second-generation chimeric antigen receptor (CAR). The present disclosure also provides a parallel adaptor CAR (pACAR) comprising (a) a CCR comprising a CCR polypeptide provided in this disclosure or a trimeric CCR provided in this disclosure and (b) an adaptor CAR. In some embodiments, the second-generation CAR is a single polypeptide. Second-generation CARs Second-generation CARs can transduce a functional antigen-dependent co-stimulatory signal in human primary T-cells in addition to antigen-dependent TCR-like signal, permitting T-cell proliferation in addition to tumouricidal activity. Second-generation CARs most commonly provide co­stimulation using co-stimulatory domains (synonymously, co- stimulatory signalling regions) derived from CD28 or 4-1BB. The combined delivery of co- stimulation plus a CD3ζ signal can render second-generation CARs functionally superior to their first-generation counterparts. Exemplary second-generation CARs that can be used as described herein are disclosed in US Patent No 7,446,190; Finney et al., “Chimeric receptors providing both primary and costimulatory signaling in T cells from a single gene product,” J. Immunol 161(6):2791-7 (1998); Maher et al., “Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor,” Nat. Biotechnol. 20(1):70-5 (2002); Finney et al., “Activation of resting human primary T cells with chimeric receptors: costimulation from CD28, inducible costimulator, CD134, and CD137 in series with signals from the TCR zeta chain,” J. Immunol. 172(1):104-13 (2004); and Imai et al., “Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia,” Leukemia 18(4):676-84 (2004), incorporated herein by reference in their entireties. In particular embodiments, a second-generation CAR, denoted “H,” “H2”, or “H28z”, is used. The H2 CAR comprises, from extracellular to intracellular domain, a MUC-1 targeting HMFG2 scFv, CD28 hinge, transmembrane and co-stimulatory domains, and a CD3ζ signalling region. The H2 CAR is described in Wilkie et al., “Retargeting of human T cells to tumor-associated MUC1: the evolution of a chimeric antigen receptor,” J. Immunol. 180:4901-9 (2008), incorporated herein by reference in its entirety. In particular embodiments, a second-generation CAR, called T1E28z, is used. The T1E28z CAR comprises, from extracellular to intracellular domain, the ErbB targeting T1E peptide, CD28 hinge, transmembrane and co-stimulatory domains, and a CD3ζ 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. In some embodiments, the second-generation CAR comprises, from intracellular to extracellular domain, (a) an intracellular signalling region; (b) a second co-stimulatory signalling region; (c) a transmembrane domain; (d) an optional hinge region; and (e) a second binding element that specifically interacts with a second epitope on a second target antigen. In some embodiments, the second-generation CAR comprises, from intracellular to extracellular domain, (a), (b), (c) and (e). In some embodiments, the second-generation CAR comprises (a) an intracellular signalling domain comprising a modified CD3ζ polypeptide; (b) a second co-stimulatory co-signalling region; (c) a transmembrane domain; (d) an optional hinge region; and (e) a second binding element that specifically interacts with a second epitope on a second target antigen, wherein the modified CD3ζ polypeptide comprises an unmodified ITAM1, and a mutation in ITAM2 and/or ITAM3. In some embodiments, the second-generation CAR comprises, from intracellular to extracellular domain, (a), (b), (c) and (e). The second target antigen may be present on a target cell, such as a target cancer cell. 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). In some embodiments, 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. In some embodiments, the intracellular signalling region comprises or consists of an intracellular signalling domain. In some embodiments, the signalling region comprises the intracellular domain of human CD3 zeta chain (CD3ζ), as described for example in US Patent No. 7,446,190, incorporated by reference herein, or a variant thereof. In particular embodiments, the signalling region comprises the domain which spans amino acid residues 52-163 of the full-length human CD3ζ chain (e.g., SEQ ID NO: 1 or 2). The CD3ζ chain has a number of known polymorphic forms, (e.g. Sequence ID: gb|AAF34793.1 and gb|AAA60394.1), all of which are useful herein: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 1); RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 2). Alternative signalling regions to the CD3ζ domain include, e.g., FcεR1γ, CD3ε, DAP12, and multi-ITAM. See Eshhar Z et al., Proc Natl Acad Sci U S A 90:720-724 (1993); Nolan et al., Clin Cancer Res 5: 3928-3941 (1999); Zhao et al., J Immunol 183: 5563-5574 (2009); Topfer et al., J Immunol 194: 3201-3212 (2015); and James JR, Sci Signal 11(531) eaan1088 (2018), the disclosures of which are incorporated herein by reference in their entireties. In certain embodiments, the signalling region comprises FcεR1γ endodomain or a variant thereof. In particular embodiments, the FcεR1γ signalling region comprises the sequence shown in SEQ ID NO: 20 as shown below: RLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO: 20). In certain embodiments, the signalling region comprises DAP12 endodomain or a variant thereof. In particular embodiments, the DAP12 signalling region comprises the sequence shown in SEQ ID NO: 58. YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO: 58). Immunoreceptor Tyrosine-based Activation Motif (ITAM) In some embodiments, the intracellular signalling region comprises at least one Immunoreceptor Tyrosine-based Activation Motif (ITAM). In some embodiments, the intracellular signalling domain is TCRζ, FcRγ, FcRβ, CD3γ, CD3θ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In particular embodiments, the intracellular signalling domain is FcεR1γ, DAP12, or CD3ζ. In typical embodiments, the intracellular signalling domain is CD3ζ. The CD3ζ domain has three ITAMs named ITAM1, ITAM2 and ITAM3 from membrane proximal to distal direction. Primary signalling domains that regulate activation of the TCR complex may contain ITAMs. Non-limiting examples of ITAM-containing intracellular signalling domains include those derived from DAP12, TCRζ, FcRγ, FcRβ, CD3γ, CD3θ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. Modified CD3ζ intracellular signalling domains In some embodiments, the CD3ζ intracellular signalling domain is modified. In some embodiments, the CD3ζ intracellular signalling domain includes modifications in at least one of the three ITAMs. In some embodiments, the CD3ζ intracellular signalling domain is truncated. In some embodiments, the CD3ζ intracellular signalling domain is truncated to remove one or more of the three ITAMs. In some embodiments, the CD3ζ intracellular signalling domain is modified by alternative splicing. In some embodiments, the CD3ζ intracellular signalling domain is engineered to express a fusion polypeptide between CD3ε, CD3δ or CD3θ and CD3ζ. In various embodiments, at least one, at least two, or all three ITAMs are modified. In some embodiments, at least two of the ITAMs of CD3ζ are modified. In some embodiments, three ITAMs of CD3ζ are modified. In certain embodiments, one ITAM of CD3ζ is unmodified and at least one of the two other two ITAMs is modified. In certain embodiments, the modified CD3ζ intracellular signalling domain comprises an ITAM1 variant comprising one or more loss-of-function mutations. In certain embodiments, the modified CD3ζ intracellular signalling domain comprises an ITAM1 variant comprising two loss-of-function mutations. In certain embodiments, the loss-of-function mutation comprises a mutation of a tyrosine residue in ITAM1. In certain embodiments, the loss-of- function mutation comprises a mutation of a tyrosine residue to a phenylalanine residue in ITAM1. In certain embodiments, the modified CD3ζ intracellular signalling domain comprises an ITAM2 variant comprising one or more loss-of-function mutations. In certain embodiments, the modified CD3ζ 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 CD3ζ intracellular signalling domain comprises an ITAM3 variant comprising one or more loss-of-function mutations. In certain embodiments, the modified CD3ζ intracellular signalling domain comprises an ITAM3 variant comprising two loss-of-function mutations. In certain embodiments, the loss-of-function mutation comprises a mutation of a tyrosine residue in ITAM3. In certain embodiments, the loss-of- function mutation comprises a mutation of a tyrosine residue to a phenylalanine residue in ITAM3. In certain embodiments, the modified CD3ζ intracellular signalling domain comprises an ITAM2 variant and an ITAM3 variant comprising one or more loss-of-function mutations. In certain embodiments, the modified CD3ζ intracellular signalling domain comprises an ITAM2 variant and an ITAM3 variant comprising two loss-of-function mutations. In certain embodiments, 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. In certain embodiments, the modified CD3ζ intracellular signalling domain comprises ITAM1 and one or more modifications on at least one of ITAM2 and/or ITAM3. In some embodiments, the modified CD3ζ intracellular signalling domain comprises ITAM1 and one or more loss-of-function mutations in ITAM2 and/or ITAM3. In some embodiments, the modified CD3ζ intracellular signalling domain comprises ITAM1 and two loss-of-function mutations in each of ITAM2 and ITAM3. In some embodiments, the modified CD3ζ comprises one or more ITAMs comprising two tyrosine (Y) to phenylalanine (F) mutations. In some embodiments, the modified CD3ζ intracellular signalling domain comprises unmodified ITAM1 and two tyrosine (Y) to phenylalanine (F) mutations in ITAM2 and/or ITAM3. In some embodiments, the modified CD3ζ intracellular signalling domain comprises unmodified ITAM1 and two tyrosine (Y) to phenylalanine (F) mutations in each of ITAM2 and ITAM3. In some embodiments, the modified CD3ζ 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). In various embodiments, the modified CD3ζ polypeptide comprises at least one functional ITAM. In some embodiments, the modified CD3ζ polypeptide comprises mutations in both ITAM2 and ITAM3. In certain embodiments, ITAM2 and ITAM3 in the modified CD3ζ are non-functional. In some embodiments, each of ITAM2 and ITAM3 comprises at least one Tyr to Phe mutation. In typical embodiments, each of ITAM2 and ITAM3 comprises two Tyr to Phe mutations. In certain embodiments, the CD3ζ 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 CD3ζ) are double underlined. RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLFNE LQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID NO: 42) RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQ KDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID NO: 43) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNEL QKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID NO: 48). In some embodiments, the CAR comprises a truncated CD3ζ intracellular signalling domain. In some embodiments, the CD3ζ is truncated to remove at least one of the three ITAMs (e.g., ITAM1, ITAM2, ITAM3), or combinations thereof. In some embodiments, the CD3ζ is truncated to remove ITAM1. In some embodiments, the CD3ζ is truncated to remove ITAM2. In some embodiments, the CD3ζ is truncated to remove ITAM3. In some embodiments, the CD3ζ is truncated to remove ITAM2 and ITAM3. In certain embodiments, the truncated CD3ζ intracellular signalling domain has the sequence shown in SEQ ID NO: 49 as shown below: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG (SEQ ID NO: 49). In various embodiments, the modified CD3ζ domain comprises deletion of ITAM2 or a portion thereof and deletion of ITAM3 or a portion thereof. In some embodiments, the modified CD3ζ polypeptide comprises deletion of ITAM3 or a portion thereof and deletion of ITAM2 or a portion thereof. In some embodiments, the modified CD3ζ polypeptide comprises deletion of both ITAM2 and ITAM3. In some embodiments, the modified CD3ζ 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 CD3ζ 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 CD3ζ polypeptide further comprises deletion of one or more amino acid residues N-terminal to ITAM1. In some embodiments, the modified CD3ζ polypeptide comprises a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the sequence shown in SEQ ID NOs: 50-53. In some embodiments, the modified CD3ζ 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, CD2F-10, CD48, CD8, CD84, CD229, CRACC, NTB-A or SLAM; or members of the TIM family such as TIM-1, TIM-3 or TIM-4; or other co-stimulatory molecules such as CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP10, DAP12, Dectin-1, DPPIV, EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), CD7, NKG2X, CD83 or a variant thereof. See Mondino A et al., J Leukoc Biol. 55:805-815 (1994); Thompson CB, Cell. 81:979-982 (1995); Somoza C et al., Res Immunol. 146:171-176 (1995); Rhodes DA et al., Annu Rev Immunol. 34:151-172 (2016); Foell J et al., Curr Cancer Drug Targets. 7:55-70 (2007); Greenwald RJ et al., Annu Rev Immunol. 23:515-548 (2005); Flem-Karlsen K et al., Trends Cancer. 4:401-404 (2018); Flies DB et al., J Immunother. 30:251-260 (2007); Gavrieli M et al., Adv Immunol. 92:157- 185 (2006); Zhu Y et al., Nat Commun. 4:2043 (2013); Omar HA et al., Crit Rev Oncol Hematol. 135:21-29 (2019); Hashemi M et al., Oncotarget. 9:24857-24868 (2018); Kang X et al., Cell Cycle. 15:25-40 (2016); Watts TH, Annu Rev Immunol.23:23-68 (2005); Bryceson YT et al., Immunol Rev. 214:73-91 (2006); Sharpe AH, Curr Opin Immunol. 7:389-395 (1995); Wingren AG et al., Crit Rev Immunol. 15:235-253 (1995), the disclosures of which are incorporated herein by reference in their entireties. The co-stimulatory signalling regions may be selected depending upon the particular use intended for the immunoresponsive cell. In some embodiments, 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. In certain embodiments, 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. In a particular embodiment, the co-stimulatory signalling region of the CAR is the co- stimulatory signalling region of CD28. In some embodiments, the CD28 co-stimulatory signalling region comprises modification of one or more tyrosine residues in the CD28 cytoplasmic domain. In some embodiments, the CD28 co-stimulatory signalling region comprises a mutation at the C-terminal most tyrosine residue. In some embodiments, the CD28 co-stimulatory signalling region comprises a modified YRS motif. In some embodiments, 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. In certain embodiments, 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). In certain embodiments, 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). In a particular embodiment, 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. In certain embodiments, the co-stimulatory signalling region of the CAR is the DAP10 co- stimulatory signalling region. In certain embodiments, 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. In currently preferred embodiments, when the CAR and CCR constructs are expressed from a single vector, 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. Suitable transmembrane domains are known in the art and include for example, the transmembrane domains of CD8α, CD28, CD4, CD3ζ, FcεR1γ, 4-1BB or a variant thereof. Selection of CD3ζ 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. In certain embodiments, the transmembrane domain of the CAR is selected from the transmembrane domain of CD28 and the transmembrane domain of CD8α. In a particular CAR embodiment, the transmembrane domain of the CAR is the transmembrane domain of CD28. In some embodiments, the CAR comprises a portion of the extracellular domain and transmembrane domain of CD28. In certain embodiments, 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). Co-stimulatory signalling domain and transmembrane domain In some embodiments in which 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). In some embodiments, one of the co-stimulatory signalling regions is based upon the hinge region and suitably also the transmembrane domain and endodomain of CD28. In some embodiments, 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). In some embodiments in which the co-stimulatory signalling region of the CAR is, or comprises, the co-stimulatory signalling region of DAP10, 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) CAR second binding element The second binding element of the second-generation CAR binds at least one epitope. In various embodiments, 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. Thus, 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. In various embodiments, the binding element used in the second-generation CARs described herein are antigen binding sites (ABS) of antibodies. In typical embodiments, the ABS used as the binding element is formatted into an scFv or is single domain antibody from a camelid, human, or other species. In some embodiments, 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. Alternatively, a binding element of a second-generation CAR may comprise ligands that bind to a surface protein of interest. In some embodiments, the binding element is associated with a leader sequence which facilitates expression on the cell surface. Many 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. 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 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. In typical embodiments, the binding elements of the second-generation CAR and CCR constructs are different from one another. In various embodiments, the binding elements of the second-generation CAR and CCR specifically bind to a second epitope and first epitope of the same antigen. In some embodiments, the first and second antigens are the same. In certain of these embodiments, the binding elements of the 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 CAR and CCR can compete in their binding. In various embodiments, the binding elements of the second-generation 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 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. In various embodiments, the binding elements used in CCRs of the pCARs described herein are antigen binding sites (ABS) of antibodies. In typical embodiments, the 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. In some embodiments, 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. Alternatively, a binding element of a second-generation CAR of a pCAR disclosed herein may comprise ligands that bind to a surface protein of interest. In some embodiments, the binding element specifically binds a second epitope on a second target antigen selected from any of the antigens listed above for the CCRs. In some embodiments, the binding element specifically binds a second epitope on a second target antigen selected from an NKG2D ligand, MUC1, αvβ6 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). In some embodiments, the binding element specifically binds a second epitope on a second target antigen selected from an NKG2D ligand, MUC1, αvβ6 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). In some embodiments, the NKG2D ligand is selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, the binding element specifically binds an epitope on the antigen MUC1. MUC1 CAR In particular embodiments, the binding element specifically interacts with an epitope on a MUC1 target antigen. In currently preferred embodiments, the binding element of the second-generation CAR specifically interacts with a second epitope on a MUC1 target antigen. In some embodiments, the second-generation CAR binding element comprises the antigen binding site of an antibody specific to MUC1. In some embodiments, the second-generation CAR binding element comprises CDRs of an antibody specific to MUC1. In some embodiments, the second-generation CAR binding element comprises VH and VL sequences of an antibody specific to MUC1. In some embodiments, the second-generation CAR binding element comprises the antigen binding site of the HMFG2 antibody. In certain embodiments, 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); VH 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). In certain embodiments, 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). In particularly preferred embodiments, the second-generation CAR binding element comprises the antigen binding site of the HMFG2 antibody formatted as a scFv. In certain embodiments, 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 (SEQ ID NO: 16). In certain embodiments, 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 GGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGG CCGTGGTCACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTC AAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCA CTGGTCTAATAGGTGGTACCAACAACCGAGCACCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGAT TGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGC TCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGATCAGAG (SEQ ID NO: 17). NKG2DL CAR In particular embodiments, the binding element specifically interacts with an epitope on an NKG2D receptor ligand. In some embodiments, the second-generation CAR binding element comprises human NKG2D receptor polypeptide. In some embodiments, the second-generation CAR binding element comprises a fragment, portion, or variant of human NKG2D receptor polypeptide. The NKG2D-CD3ζ linear CAR (SEQ ID NO: 69) comprises an NKG2D ligand binding domain (SEQ ID NO: 75) fused to a CD3ζ polypeptide (SEQ ID NO: 1). The protein sequence of the NKG2D-CD3ζ linear CAR is shown below as SEQ ID NO: 69. CD3ζ sequence is italicized and ITAMs of CD3ζ are in bold; NKG2D sequence is double underlined. MRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGWIRGRRSRHS WEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNS LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLV KSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV (SEQ ID NO: 69). Exemplary second-generation CARs In some embodiments, 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 CD3ζ 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 CD3ζ polypeptide are italicized and in bold: MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGL EWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYW GQGTTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANW VQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWV FGGGTKLTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 21). In particular embodiments, 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 CD3ζ 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. In some embodiments, the second-generation CAR is H2-1XX. H2-1XX CAR is a second- generation (2G) CAR comprising, from intracellular to extracellular, a modified CD3ζ 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 CD3ζ are italicized and in bold. Residues mutated to phenylalanine (F) from tyrosine (Y) (with reference to unmodified CD3ζ) are double underlined. MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGL EWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYW GQGTTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANW VQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWV FGGGTKLTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIG MKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID NO: 59) In some embodiments, the second-generation CAR is H2BB CAR. H2BB is a 2G CAR comprising from intracellular to extracellular, a CD3ζ signalling region, a 4-1BB co- stimulatory domain, a CD8α transmembrane and extracellular spacer domain, and a human MUC1-targeting HMFG2 scFv domain. In some embodiments, 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 CD3ζ signalling region (“H-1”). The modified CD3ζ signalling region is a truncated CD3ζ 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 CD3ζ polypeptide in bold and underlined: MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGL EWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYW GQGTTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANW VQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWV FGGGTKLTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRG (SEQ ID NO: 22). In some embodiments, 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 CD3ζ 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. The protein sequence of the H-1∆3 CAR is shown below as SEQ ID NO: 62 with the VH and the VL sequences of HMFG2 underlined and in bold and ITAM1 of CD3ζ italicized and in bold. MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGL EWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYW GQGTTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANW VQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWV FGGGTKLTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRAPAYQQGQ NQLYNELNLGRREEYDVLDKRRG (SEQ ID NO:62). The protein sequence of H-1∆3 comprises a truncated CD3ζ polypeptide that has the 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 CD3ζ italicized and in bold. MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGL EWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYW GQGTTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANW VQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWV FGGGTKLTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKAPAYQQGQNQL YNELNLGRREEYDVLDKRRG (SEQ ID NO: 63). The protein sequence of H-1∆6 comprises a truncated CD3ζ 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 CD3ζ italicized and in bold. MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGL EWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYW GQGTTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANW VQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWV FGGGTKLTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSAPAYQQGQNQLYN ELNLGRREEYDVLDKRRG (SEQ ID NO: 64). The protein sequence of H-1∆9 comprises a truncated CD3ζ 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 VH 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. MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGL EWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYW GQGTTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANW VQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWV FGGGTKLTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAAPAYQQGQNQLYNELN LGRREEYDVLDKRRG (SEQ ID NO: 65). The protein sequence of H-1∆9 YRS comprises a truncated CD3ζ 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 (pCARs) 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 pCAR preferably comprises a H2-1XX CAR. The co-stimulatory domains in the pCAR provided in this disclosure may be selected depending upon the particular use intended for the immunoresponsive cell. In particular, 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. In certain pCAR embodiments, 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 4-1BB co-stimulatory domain. In certain pCAR embodiments, 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. In a particular pCAR embodiment, 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. In certain pCAR embodiments, 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. In certain pCAR embodiments, 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. Exemplary pCARs 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 CD3ζ signalling region (“H2-1XX”) (SEQ ID NO: 59). In some embodiments, 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). The modified CD3ζ 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. The CCR and the CAR are linked by a furin cleavage site (RRKR)-SGSG linker- T2A ribosomal skip peptide. 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 CD3ζ polypeptide are italicized and in bold with the mutated residues double underlined. MGWLCSGLLFPVSCLVLLQVASSGNLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQ ASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALY ASSFKGYIENCSTPNTYICMQRTVGGGGSGGGGDGGGGSGTPSSDAVSRLEEEMRKLQATVQELQKR LDRLEETVQAKGGGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCELRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAEVQLQQSGGGLVQ PGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSS VYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPG ETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTED EAIYFCALWYSNHWVFGGGTKLTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQ KDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID NO: 78). 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. MGWLCSGLLFPVSCLVLLQVASSGNLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNW YESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTI IEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVGGGGSGGGGDGGGGSGTPSSDAVSRLEEE MRKLQATVQELQKRLDRLEETVQAKGGGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 79). 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 CD3ζ signalling region (“H2-1XX”) (SEQ ID NO: 59). The modified CD3ζ 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. The CCR and the CAR are linked by a furin cleavage site (RRKR)-SGSG linker- T2A ribosomal skip peptide. 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 CD3ζ polypeptide are italicized and in bold with the mutated residues double underlined. MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNW YRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIK ESLRAELRVTERRAEVPTAHGGGGSGTPSSDAVSRLEEEMRKLQATVQELQKRLDRLEETVQAKGGGGI ISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRKR SGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGFTF SNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYY CTFGNSFAYWGQGTTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTS NYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFG GGTKLTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLL VTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGE RRRGKGHDGLFQGLSTATKDTFDALHMQALPPR (SEQ ID NO: 80). 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. MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTS ESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGT YLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHGGGGSGTPSSDAVSRLEEEMRKLQATVQEL QKRLDRLEETVQAKGGGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDG CSCRFPEEEEGGCEL (SEQ ID NO: 81). 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 CD3ζ signalling domain (“H2-1XX”) (SEQ ID NO: 59). The modified CD3ζ 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. The CCR and the CAR are linked by a furin cleavage site (RRKR)-SGSG linker- T2A ribosomal skip peptide. 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 CD3ζ polypeptide are italicized and in bold with the mutated residues double underlined. MAWRALHPLLLLLLLFPGSQAQSKAQVLQSVAGQTLTVRCQYPPTGSLYEKKGWCKEASALVCIRLVTS SKPRTMAWTSRFTIWDDPDAGFFTVTMTDLREEDSGHYWCRIYRPSDNSVSKSVRFYLVVSPASASTQ TSWTPRDLVSSQTQTQSCVPPTAGARQAPESPSTIPVPSQPQNSTLRPGPAAPIAGGGGSGTPSSDAVS RLEEEMRKLQATVQELQKRLDRLEETVQAKGGGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPL ALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATH YAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGGSG GGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARF SGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEAAAIEVMYPPPYLDNEKSNGTI IHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPT RKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQ ALPPR (SEQ ID NO: 82). 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. MAWRALHPLLLLLLLFPGSQAQSKAQVLQSVAGQTLTVRCQYPPTGSLYEKKGWCKEASALV CIRLVTSSKPRTMAWTSRFTIWDDPDAGFFTVTMTDLREEDSGHYWCRIYRPSDNSVSKSV RFYLVVSPASASTQTSWTPRDLVSSQTQTQSCVPPTAGARQAPESPSTIPVPSQPQNSTLRP GPAAPIAGGGGSGTPSSDAVSRLEEEMRKLQATVQELQKRLDRLEETVQAKGGGGIISFFLALTSTALL FLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 83). The protein sequence of the CAR (“H2-1XX”) of 44bb(trimer)/H-1XX is shown above as SEQ ID NO: 59. Provided herein is 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. Adaptor CARs In some embodiments, 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. In some embodiments, the adaptor CAR comprises (i) an adaptor protein comprising a CD3ζ activation signalling domain and a DAP10 polypeptide; and (ii) an NKG2D ligand binding domain polypeptide. In some embodiments, the CD3ζ activation signalling domain is modified. In some embodiments, the modified CD3ζ activation signalling domain comprises at least one ITAM variant comprising one or more loss-of-function mutations. In certain embodiments, the modified CD3ζ 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. In certain embodiments, the ITAM2 variant comprises two loss-of-function mutations and the ITAM3 variant comprises two loss-of-function mutations. In certain embodiments, each of the loss-of-function mutations comprises a tyrosine to phenylalanine substitution. In some embodiments, 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. As described elsewhere herein, 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 The adaptor CAR comprises one or more adaptor proteins. In some embodiments, 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, CD3ζ, or a variant thereof (e.g., CD3ζ-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, BLAME, CD2, CD2F-10, CD48, CD8, CD84, CD229, CRACC, NTB-A or SLAM; or members of the TIM family such as TIM-1, TIM-3 or TIM-4; or other co-stimulatory molecules such as CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP10, DAP12, Dectin-1, DPPIV, EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), CD7, NKG2X, CD83 or a variant thereof. In some embodiments, the co-stimulatory signalling region is DAP10 or a variant thereof. In some embodiments, 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. In some embodiments, 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. 65(10): 135701369 and Wriggers W et al., 2005, Biopolymers 80:736-746.) Linkers may also be used to join the fusion polypeptide of the disclosure to another polypeptide. In some embodiments, the adaptor protein is a fusion of a DAP10 polypeptide or functional variant thereof and a CD3ζ polypeptide or a functional variant thereof. In some embodiments, the adaptor protein is a fusion of DAP10 and CD3ζ (e.g., DAP10-CD3ζ in the NKG2D / DAP10-CD3ζ adaptor CAR). In particular embodiments, the adaptor protein is a fusion of DAP10 and CD3ζ-1XX (e.g., DAP10-CD3ζ-1XX in the NKG2D / DAP10-CD3ζ-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. 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. Thus, a suitable binding element may be any element which provides the adaptor CAR with the ability to recognize a target of interest. The target to which the adaptor CAR of the disclosure directed can be any target of clinical interest to which it would be desirable to direct a T cell response. In various embodiments, the binding element used in the adaptor CARs described herein are antigen binding sites (ABS) of antibodies. In typical embodiments, 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. In some embodiments, 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. Alternatively, a binding element of an adaptor CAR may comprise ligands that bind to a surface protein of interest. In some embodiments, the binding element is associated with a leader sequence which facilitates expression on the cell surface. Many 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. 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 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. In typical embodiments, the binding elements of the adaptor CAR and CCR constructs are different from one another. In various embodiments, the binding elements of the adaptor CAR and CCR specifically bind to a second epitope and first epitope of the same antigen. In some embodiments, the first and second antigens are the same. In certain of these embodiments, 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. In some embodiments, the adaptor CAR binds specifically to an NKG2D ligand. In some embodiments, 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. Exemplary adaptor 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. MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLI VGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQG QRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNY NLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPL TESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLV HIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV (SEQ ID NO: 67). 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-CD3ζ 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 CD3ζ 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 CD3ζ 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). The protein sequence of the NKG2D / DAP10-CD3ζ adaptor CAR is shown below as SEQ ID NO: 72. DAP10 sequence is bold; CD3ζ sequence is single underlined with the ITAMs of CD3ζ 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 STRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCP KNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSW QWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV (SEQ ID NO: 72). The NKG2D / DAP10-CD3ζ-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 CD3ζ 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 CD3ζ 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). The protein sequence of the NKG2D / DAP10-CD3ζ-1XX adaptor CAR is shown below as SEQ ID NO: 74. DAP10 sequence is bold; CD3ζ sequence is single underlined and ITAMs of CD3ζ are in italics, mutated residues of CD3ζ are double underlined; NKG2D sequence is double underlined and follows an intervening furin cleavage site (RRKR), SGSG linker and P2A ribosomal skip peptide. MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLI VGAVFLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFD ALHMQALPPRRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDF STRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCP KNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSW QWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV (SEQ ID NO: 74). Parallel adaptor CARs (pACARs) 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. MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNW YRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIK ESLRAELRVTERRAEVPTAHGGGGSGTPSSDAVSRLEEEMRKLQATVQELQKRLDRLEETVQAKGGGGI ISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRKR SGSGEGRGSLLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLP LLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITE TESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSW EMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLF NQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKS YHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV (SEQ ID NO: 95). 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. MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTS ESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGT YLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHGGGGSGTPSSDAVSRLEEEMRKLQATVQEL QKRLDRLEETVQAKGGGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDG CSCRFPEEEEGGCEL (SEQ ID NO: 81). The protein sequence of the N1012 adaptor CAR is shown above as SEQ ID NO: 67. 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. MAWRALHPLLLLLLLFPGSQAQSKAQVLQSVAGQTLTVRCQYPPTGSLYEKKGWCKEASALVCIRLVTS SKPRTMAWTSRFTIWDDPDAGFFTVTMTDLREEDSGHYWCRIYRPSDNSVSKSVRFYLVVSPASASTQ TSWTPRDLVSSQTQTQSCVPPTAGARQAPESPSTIPVPSQPQNSTLRPGPAAPIAGGGGSGTPSSDAVS RLEEEMRKLQATVQELQKRLDRLEETVQAKGGGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRKRSGSGEGRGSLLTCGDVEENPGPMIHLGHILFLLL LPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQED GKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSG SGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSK CRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDE SKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEM QKGDCALYASSFKGYIENCSTPNTYICMQRTV (SEQ ID NO: 97). 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. MAWRALHPLLLLLLLFPGSQAQSKAQVLQSVAGQTLTVRCQYPPTGSLYEKKGWCKEASALV CIRLVTSSKPRTMAWTSRFTIWDDPDAGFFTVTMTDLREEDSGHYWCRIYRPSDNSVSKSV RFYLVVSPASASTQTSWTPRDLVSSQTQTQSCVPPTAGARQAPESPSTIPVPSQPQNSTLRP GPAAPIAGGGGSGTPSSDAVSRLEEEMRKLQATVQELQKRLDRLEETVQAKGGGGIISFFLALTSTALL FLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 83). The protein sequence of the N1012 adaptor CAR is shown above as SEQ ID NO: 67. The CXCR2 Pbb(trimer)_N1012 pACAR (SEQ ID NO: 109) is a CXCR2 armoured pACAR. It comprises (i) a 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). The protein sequence of CXCR2 Pbb(trimer)_N1012 pACAR is shown below as SEQ ID NO: 109. MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVIIYALVFLLSLL GNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCKVVSLLKE VNFYSGILLLACISVDRYLAIVHATRTLTQKRYLVKFICLSIWGLSLLLALPVLLFRRTVYSSNV SPACYEDMGNNTANWRMLLRILPQSFGFIVPLLIMLFCYGFTLRTLFKAHMGQKHRAMRVIF AVVLIFLLCWLPYNLVLLADTLMRTQVIQETCERRNHIDRALDATEILGILHSCLNPLIYAFIG QKFRHGLLKILAIHGLISKDSLPKDSRPSFVGSSSGHTSTTLRRKRSGSGATNFSLLKQAGDVEE NPGPMQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFV LNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPK GGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RRKRSGSGEGRGSLLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGS LSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQ RITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSR HSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFL NSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLK LVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV (SEQ ID NO: 109). 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. MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTS ESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGT YLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHGGGGSGTPSSDAVSRLEEEMRKLQATVQEL QKRLDRLEETVQAKGGGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDG CSCRFPEEEEGGCEL (SEQ ID NO: 81). The protein sequence of the N1012 adaptor CAR is shown above as SEQ ID NO: 67. Provided herein is 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. In typical embodiments, the immunoresponsive cell is a T cell. In certain embodiments, the immunoresponsive cell is an αβ T cell. In particular embodiments, the immunoresponsive cell is a cytotoxic αβ T cell. In particular embodiments, the immunoresponsive cell is an αβ helper T cell. In particular embodiments, the immunoresponsive cells is a regulatory αβ T cell (Treg). In some embodiments, the T cell is a TCR-re-directed T-cell. In certain embodiments, the immunoresponsive cell is a γδ T cell. In particular embodiments, the immunoresponsive cell is a Vδ2+ γδ T cell. In particular embodiments, the immunoresponsive cell is a Vδ2– T cell. In specific embodiments, the Vδ2– T cell is a Vδ1+ cell. In certain embodiments, the immunoresponsive cell is a Natural Killer (NK) cell. In some embodiments, the immunoresponsive cell is an αβ T cell, γδ 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. In some embodiments, 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 αβ T cells. In other specific embodiments, the TIL cells are γδ 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). For instance, 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. In some embodiments, 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. As indicated above, for convenience herein, 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. Suitable sequences for the polynucleotides will be apparent to a skilled person based on the description of the CAR and CCR above. In some embodiments, 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. The single polynucleotide may comprise or consist of the sequence shown in any one of SEQ ID NOs: 90, 91, 92, 94, 96 and 108. The single polynucleotide may be in a single vector. The sequences may be optimized for use in the required immunoresponsive cell. However, in some cases, as discussed above, codons may be varied from the optimum or “wobbled” in order to avoid repeat sequences. Particular examples of such polynucleotides encode the preferred embodiments described above. In order to achieve transduction, the polynucleotide(s) are suitably introduced into one or more vectors, such as a plasmid or a retroviral or lentiviral vector. 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. In typical embodiments, 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. In some embodiments, the immunoresponsive cells are γδ T cells and the γδ T cells are activated by an anti-^^ TCR antibody prior to the genetic modification. In some embodiments, 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. In some embodiments, 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. In some embodiments, the kit further comprises a reagent for use in genetic modification of immunoresponsive cells. In some embodiments, 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. Pharmaceutical composition The present disclosure further provides pharmaceutical 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. Methods of treatment In certain embodiments, 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. Thus, in another aspect, 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. In typical embodiments, the target cell expresses MUC1 and/or one or more NKG2D ligands. In some embodiments, the one or more NKG2D ligands are selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In another aspect, methods for treating cancer in a patient in need thereof are provided. The method 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. In typical embodiments, the target cell expresses MUC1 and/or one or more NKG2D ligands. In some embodiments, the one or more NKG2D ligands are selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, the patient is human. In various embodiments, 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. In some embodiments, the patient has colon, breast, ovarian, lung, or pancreatic cancer. In some embodiments, the patient has breast cancer. In some embodiments, the patient has tumour cells expressing MUC1 and/or one or more NKG2D ligands. In some embodiments, the patient has been determined to have tumour cells expressing MUC1 and/or one or more NKG2D ligands. In some embodiments, the one or more NKG2D ligands are selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, 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. In various embodiments, a therapeutically effective number of the immunoresponsive cells is administered to the patient. In certain embodiments, the immunoresponsive cells are administered by intravenous infusion. In certain embodiments, the immunoresponsive cells are administered by intratumoural injection. In certain embodiments, the immunoresponsive cells are administered by peri-tumoural injection. In certain embodiments, the immunoresponsive cells are administered by intraperitoneal injection. In certain embodiments, the immunoresponsive cells are administered by a plurality of routes selected from intravenous infusion, intra-tumoural injection, and peri-tumoural injection. In another aspect, there is provided 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. There is also provided 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. Also provided is the use 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 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. In some embodiments, 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 106 to about 1 x 1011 cells are administered to the patient. In one embodiment, about 1 x 107 to about 1 x 1010 cells, or about 1 x 108 to about 1 x 109 cells are administered to the patient. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims. SEQUENCES SEQ ID Description Sequence NO: 1 CD3z (“DA”) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG (ITAMs in HDGLYQGLSTATKDTYDALHMQALPPR bold) 2 CD3z variant 1 RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP (“EP”) EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR 3 full length MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFS CD28 protein REFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNES (transmembra VTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPS ne domain in PLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLH bold) SDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 4 CD28 co- IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGG stimulatory VLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY region, APPRDFAAYRS transmembran e domain, and a portion of the extracellular domain (spacer) 5 c-myc tag EQKLISEEDL 6 CD28 co- IEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVV costimulatory VGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKH region with c- YQPYAPPRDFAAYRS myc tag 7 TBB/H pCAR MGPGVLLLLLVATAWHGQGGVVSHFNDCPLSHDGYCLHDGVCM (VH and VL YIEALDKYACNCVVGYIGERCQYRDLKWWELRAAAPTTTPAPR sequences of PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT HMFG2 are CGVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF underlined PEEEEGGCELRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPL and in bold, ALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMN T1E peptide WVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDS sequence is KSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGG underlined, GGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVT italicized and TSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDK in bold, and AALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEAAA ITAMs of CD3z IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGG are italicized VLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY and bold) APPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR HMFG2 VH GFTFSNY CDR1 HMFG2 VH RLKSNNYA CDR2 HMFG2 VH GNSFAY CDR3 HMFG2 VL RSSTGAVTTSNYAN CDR1 HMFG2 VL GTNNRAP CDR2 HMFG2 VL ALWYSNHWV CDR3 HMFG2 VH EVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGL EWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAED TGIYYCTFGNSFAYWGQGTTVTVSS HMFG2 VL QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFT GLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYS NHWVFGGGTKLTVLGSE HMFG2 scFv EVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGL EWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAED TGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGGSGGGGSQAVVT QESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGG TNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWV FGGGTKLTVLGSE HMFG2 scFv GAGGTGCAGCTGCAGCAGTCTGGAGGAGGCTTGGTGCAACCTGG nucleic acid AGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGT AACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTT GAGTGGGTTGCTGAAATTAGATTGAAATCTAATAATTATGCAACAC ATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATG ATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGCTGA AGACACTGGCATTTATTACTGTACCTTTGGTAACTCCTTTGCTTACT GGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGT TCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGGCCGTGGT CACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACA CTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATG CCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAAT AGGTGGTACCAACAACCGAGCACCAGGTGTTCCTGCCAGATTCTC AGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGC ACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGC AACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGA TCAGAG T1E peptide VVSHFNDCPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYR DLKWWELR T1E nucleic GTGGTGAGCCACTTCAACGACTGCCCTCTGAGCCACGACGGCTAC acid TGCCTGCACGACGGCGTGTGCATGTACATCGAGGCCCTGGACAAG TACGCCTGCAACTGCGTGGTGGGCTACATCGGCGAGAGATGCCAG TACAGAGACCTGAAGTGGTGGGAGCTGAGA FcεR1γ RLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ signalling region H2 (2G CAR) MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGF (VH and VL TFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKG sequences of RFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQG HMFG2 italics TTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLT and bold; CRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPAR ITAMs of CD3ζ FSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK are underlined LTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSK and bold in PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR sequence) PGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNE LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR H-1 CAR MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGF TFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKG (VH and VL RFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQG sequences of TTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLT HMFG2 in CRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPAR italics and FSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK bold; ITAM1 of LTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSK CD3ζ are PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR underlined PGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNE and in bold) LNLGRREEYDVLDKRRG TBB/H-1XX MGPGVLLLLLVATAWHGQGGVVSHFNDCPLSHDGYCLHDGVCMYIE (VH and VL ALDKYACNCVVGYIGERCQYRDLKWWELRAAAPTTTPAPRPPTPAPTI sequences of ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL HMFG2 italics VITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC and bold; ELRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAEV ITAMs QLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPE underlined KGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQ and bold in MNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGG sequence) SGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYAN WVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTIT GAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEAAAIEVMYP PPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR RGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGER RRGKGHDGLFQGLSTATKDTFDALHMQALPPR TBB CCR (T1E MGPGVLLLLLVATAWHGQGGVVSHFNDCPLSHDGYCLHDGVCM underlined YIEALDKYACNCVVGYIGERCQYRDLKWWELRAAAPTTTPAPR and in bold) PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT CGVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCEL CD28 co- IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGG stimulatory VLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY region with C- APPRDFAA term. YRS deleted TBB/H-1 (VH MGPGVLLLLLVATAWHGQGGVVSHFNDCPLSHDGYCLHDGVCMYIE and VL ALDKYACNCVVGYIGERCQYRDLKWWELRAAAPTTTPAPRPPTPAPTI sequences of ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL HMFG2 italics VITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC and bold; ELRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAEV ITAM1 of CD3ζ QLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPE underlined KGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQ and bold) MNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGG SGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYAN WVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTIT GAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEAAAIEVMYP PPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR RG TBB/H-1 Δ3 MGPGVLLLLLVATAWHGQGGVVSHFNDCPLSHDGYCLHDGVCMYIE (VH and VL ALDKYACNCVVGYIGERCQYRDLKWWELRAAAPTTTPAPRPPTPAPTI sequences of ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL HMFG2 italics VITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC and bold; ELRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAEV ITAM1 of CD3ζ QLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPE is underlined KGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQ and bold) MNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGG SGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYAN WVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTIT GAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEAAAIEVMYP PPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSRVKFSRAPAYQQGQNQLYNELNLGRREEYDVLDKRRG TBB/H-1 Δ6 MGPGVLLLLLVATAWHGQGGVVSHFNDCPLSHDGYCLHDGVCMYIE (VH and VL ALDKYACNCVVGYIGERCQYRDLKWWELRAAAPTTTPAPRPPTPAPTI sequences of ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL HMFG2 italics VITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC and bold; ELRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAEV ITAM1 of CD3ζ QLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPE KGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQ is underlined MNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGG and bold) SGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYAN WVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTIT GAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEAAAIEVMYP PPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSRVKAPAYQQGQNQLYNELNLGRREEYDVLDKRRG TBB/H-1 Δ9 MGPGVLLLLLVATAWHGQGGVVSHFNDCPLSHDGYCLHDGVCMYIE ALDKYACNCVVGYIGERCQYRDLKWWELRAAAPTTTPAPRPPTPAPTI (VH and VL ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL sequences of VITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC HMFG2 italics ELRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAEV and bold; QLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPE ITAM1 of CD3ζ KGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQ is underlined MNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGG and bold) SGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYAN WVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTIT GAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEAAAIEVMYP PPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSAPAYQQGQNQLYNELNLGRREEYDVLDKRRG TBB/H-1 Δ9 MGPGVLLLLLVATAWHGQGGVVSHFNDCPLSHDGYCLHDGVCMYIE ALDKYACNCVVGYIGERCQYRDLKWWELRAAAPTTTPAPRPPTPAPTI YRS ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL (VH and VL VITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC sequences of ELRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAEV HMFG2 italics QLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPE and bold; KGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQ ITAM1 of CD3z MNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGG underlined SGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYAN and bold) WVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTIT GAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEAAAIEVMYP PPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAAPAYQQGQNQLYNELNLGRREEYDVLDKRRG furin cleavage RRKR site Ser-Gly linker SGSG T2A peptide EGRGSLLTCGDVEENPGP P2A peptide ATNFSLLKQAGDVEENPGP CD28 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVG extracellular GVLACYSLLVTVAFIIFWV domain and transmembran e domain (transmembra ne domain in bold) CD8α PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI extracellular WAPLAGTCGVLLLSLVITLYCNH domain and transmembran e domain (including 5 intracellular domain residues) 4-1BB co- KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL stimulatory region CD27 co- QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP stimulatory region OX40 co- ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI stimulatory region ICOS co- CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL stimulatory region CD3z variant 2 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP (UniProt EMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK P20963) GHDGLYQGLSTATKDTYDALHMQALPPR (ITAMs in bold) CD3ζ variant RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP 2-1XX with Y EMGGKPQRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGK to F mutations GHDGLFQGLSTATKDTFDALHMQALPPR in ITAM2 and ITAM3 (ITAMs in bold; mutated residues are double underlined) CD3ζ variant RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP 1-1XX with Y EMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKG to F mutations HDGLFQGLSTATKDTFDALHMQALPPR in ITAM2 and ITAM3 (ITAMs in bold; mutated residues are double underlined) ICR62 scFv QVNLLQSGAALVKPGASVKLSCKGSGFTFTDYKIHWVKQSHGKSLE WIGYFNPNSGYSTYNEKFKSKATLTADKSTDTAYMELTSLTSEDSATY YCTRLSPGGYYVMDAWGQGASVTVSSAQTTAPSVYPLAPGSGGGGS GGGGSGGGGSDIQMTQSPSFLSASVGDRVTINCKASQNINNYLNW YQQKLGEAPKRLIYNTNNLQTGIPSRFSGSGSGTDYTLTISSLQPEDF ATYFCLQHNSFPTFGAGTKLELKRADAAPTVSIFPPSKS I62BB/H-1XX MGPGVLLLLLVATAWHGQGGQVNLLQSGAALVKPGASVKLSCK pCAR (VH and GSGFTFTDYKIHWVKQSHGKSLEWIGYFNPNSGYSTYNEKFK VL sequences SKATLTADKSTDTAYMELTSLTSEDSATYYCTRLSPGGYYVMD of HMFG2 and AWGQGASVTVSSAQTTAPSVYPLAPGSGGGGSGGGGSGGGGS ICR62 DIQMTQSPSFLSASVGDRVTINCKASQNINNYLNWYQQKLGE underlined APKRLIYNTNNLQTGIPSRFSGSGSGTDYTLTISSLQPEDFATY and in bold; FCLQHNSFPTFGAGTKLELKRADAAPTVSIFPPSKSAAAPTTTPA CD3z PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLA sequence is GTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF italicized and PEEEEGGCELRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPL ITAMs of CD3z ALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMN are in bold WVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDS and italics; KSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGG mutated GGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVT residues are TSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDK double AALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEAAA underlined) IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGG VLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY APPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIG MKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR I62BB CCR (VH MGPGVLLLLLVATAWHGQGGQVNLLQSGAALVKPGASVKLSCK and VL GSGFTFTDYKIHWVKQSHGKSLEWIGYFNPNSGYSTYNEKFK sequences of SKATLTADKSTDTAYMELTSLTSEDSATYYCTRLSPGGYYVMD ICR62 AWGQGASVTVSSAQTTAPSVYPLAPGSGGGGSGGGGSGGGGS underlined DIQMTQSPSFLSASVGDRVTINCKASQNINNYLNWYQQKLGE and in bold) APKRLIYNTNNLQTGIPSRFSGSGSGTDYTLTISSLQPEDFATY FCLQHNSFPTFGAGTKLELKRADAAPTVSIFPPSKSAAAPTTTPA This sequence PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLA may lack the GTCGVLLLSLVITLYCHNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC double RFPEEEEGGCEL underlined HN Human DAP10 MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPL sequence LAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRG CD3ζ domain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP in H2-1XX, EMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKG TBB/H-1XX, HDGLFQGLSTATKDTFDALHMQALPPR and NKG2D / DAP10- CD3ζ(1XX) (ITAMs in bold; mutated residues are underlined) CD3ζ domain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG in TBBH-1 (ITAM1 underlined and in bold) CD3ζ domain RVKFSRAPAYQQGQNQLYNELNLGRREEYDVLDKRRG in TBBH-1∆3 (ITAM1 underlined and in bold) CD3ζ domain RVKAPAYQQGQNQLYNELNLGRREEYDVLDKRRG in TBBH-1∆6 (ITAM1 underlined and in bold) CD3ζ domain APAYQQGQNQLYNELNLGRREEYDVLDKRRG in TBBH-1∆9 (ITAM1 underlined and in bold) Intentionally Intentionally blank blank TBB/H-FcεR1γ MGPGVLLLLLVATAWHGQGGVVSHFNDCPLSHDGYCLHDGVCMYIE (VH and VL ALDKYACNCVVGYIGERCQYRDLKWWELRAAAPTTTPAPRPPTPAPTI sequences of ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL HMFG2 are VITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC underlined ELRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAEV and in bold, QLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPE the ITAM of KGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQ FcεR1γ is MNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGG underlined SGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYAN and in italics) WVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTIT GAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEAAAIEVMYP PPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ H-FcεR1γ MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGF (MUC1 TFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKG targeting CAR) RFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQG (VH and VL TTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLT sequences of CRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPAR HMFG2 are FSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK underlined LTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSK and in bold, PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR the ITAM of PGPTRKHYQPYAPPRDFAAYRSRLKIQVRKAAITSYEKSDGVYTGL FcεR1γ is in STRNQETYETLKHEKPPQ bold and italics) TBB/H-DAP12 MGPGVLLLLLVATAWHGQGGVVSHFNDCPLSHDGYCLHDGVCMYIE (VH and VL ALDKYACNCVVGYIGERCQYRDLKWWELRAAAPTTTPAPRPPTPAPTI sequences of ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL HMFG2 are VITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC underlined ELRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAEV and in bold, QLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPE the ITAM of KGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQ DAP12 is in MNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGG bold and SGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYAN italics) WVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTIT GAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEAAAIEVMYP PPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDV YSDLNTQRPYYK H-DAP12 MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGF (MUC1 TFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKG targeting CAR) RFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQG (VH and VL TTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLT sequences of CRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPAR HMFG2 are FSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK underlined LTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSK and in bold, PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR the ITAM of PGPTRKHYQPYAPPRDFAAYRSYFLGRLVPRGRGAAEAATRKQRITE DAP12 is in TESPYQELQGQRSDVYSDLNTQRPYYK bold and italics) DAP12 YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQ signalling RPYYK region H2-1XX MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGF TFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKG (VH and VL RFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQG sequences of TTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLT HMFG2 italics CRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPAR and bold; FSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK ITAMs LTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSK underlined PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR and bold in PGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNE sequence, LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKD mutated KMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQ residues are ALPPR double underlined) T27 (T1E MGPGVLLLLLVATAWHGQGGVVSHFNDCPLSHDGYCLHDGVCM underlined YIEALDKYACNCVVGYIGERCQYRDLKWWELRAAAPTTTPAPR and in bold) PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT CGVLLLSLVITLYCNHQRRKYRSNKGESPVEPAEPCHYSCPREEEGST IPIQEDYRKPEPACSP TOX40 (T1E MGPGVLLLLLVATAWHGQGGVVSHFNDCPLSHDGYCLHDGVCM underlined YIEALDKYACNCVVGYIGERCQYRDLKWWELRAAAPTTTPAPR and in bold) PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT CGVLLLSLVITLYCNHALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQ ADAHSTLAKI H-1∆3 (MUC1 MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGF targeting CAR) TFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKG RFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQG (VH and the VL TTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLT sequences of CRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPAR HMFG2 FSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK underlined LTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSK and in bold) PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR PGPTRKHYQPYAPPRDFAAYRSRVKFSRAPAYQQGQNQLYNELNL GRREEYDVLDKRRG H-1∆6 (MUC1 MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGF targeting CAR) TFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKG RFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQG (VH and the VL TTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLT sequences of CRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPAR HMFG2 FSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK underlined LTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSK and in bold; PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR ITAM1 of CD3z PGPTRKHYQPYAPPRDFAAYRSRVKAPAYQQGQNQLYNELNLGR in italics) REEYDVLDKRRG H-1∆9 (MUC1 MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGF targeting CAR) TFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKG RFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQG (VH and the VL TTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLT sequences of CRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPAR HMFG2 FSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK underlined LTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSK and in bold; PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR ITAM1 of CD3z PGPTRKHYQPYAPPRDFAAYRSAPAYQQGQNQLYNELNLGRREE in italics) YDVLDKRRG H-1∆9 YRS MALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGF (MUC1 TFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKG targeting CAR) RFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQG TTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLT (VH and the VL CRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPAR sequences of FSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTK HMFG2 LTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSK underlined PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR and in bold; PGPTRKHYQPYAPPRDFAAAPAYQQGQNQLYNELNLGRREEYDV ITAM1 of CD3z LDKRRG in italics) NKG2D / atgatccacctgggccacatcctgttcctgctgctgctgcccgtggccgctgcccagacc DAP10-12 acccctggcgagcggagcagcctgcctgccttctaccctggcaccagcggcagctgca nucleic acid gcggctgcggcagcctgagcctgcccctgctggccggcctggtggccgccgacgccgt ggccagcctgctgatcgtgggcgccgtgttcctgtgcgccaggcccaggcggagccct gcccaggaggacggcaaggtgtacatcaacatgcccggccggggctacttcctgggc aggctggtgcccaggggcaggggcgctgccgaggctgccacccggaagcagcggat caccgagaccgagagcccctaccaggagctgcagggccagcggagcgacgtgtaca gcgacctgaacacccagaggccctactacaagaggcggaaaaggtctgggagtggg gctaccaatttctctctcctcaagcaagccggagacgttgaggaaaaccctggacccat gggctggatccggggacggaggagccggcacagctgggagatgagcgagttccaca actacaacctggacctgaagaagagcgacttcagcacccggtggcagaagcagcggt gccccgtggtgaagagcaagtgccgggagaacgccagccccttcttcttctgctgcttc atcgccgtggctatgggcatccggttcatcatcatggtggccatctggagcgccgtgttc ctgaacagcctgttcaaccaggaggtgcagatccccctgaccgagagctactgcggcc cctgccccaagaactggatctgctacaagaacaactgctaccagttcttcgacgagagc aagaactggtacgagagccaggccagctgcatgagccagaacgccagcctgctgaa ggtgtacagcaaggaggaccaggacctgctgaagctggtgaagagctaccactggat gggcctggtgcacatccccaccaacggcagctggcagtgggaggacggcagcatcct gagccccaacctgctgaccatcatcgagatgcagaagggcgactgcgccctgtacgcc agcagcttcaagggctacatcgagaactgcagcacccccaacacctacatctgcatgc agcggaccgtgtaa NKG2D / MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGS DAP10-12 LSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYI (DAP10 NMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRS sequence is DVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWI bold and not RGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCREN italicized; ASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGP DAP12 CPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQ sequence is DLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDC italicized and ALYASSFKGYIENCSTPNTYICMQRTV underlined, the ITAM of DAP12 is bold; NKG2D sequence is double underlined) NKG2D-CD3ζ atgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaa nucleic acid ccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaag sequence agacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcagg aaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattg ggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctc agtacagccaccaaggacacctacgatgcattgcacatgcaggccctgccccctcgcg gctggatccgcggccgcaggagccggcacagctgggagatgagcgagttccacaact acaacctggacctgaagaagagcgacttcagcacccggtggcagaagcagcggtgc cccgtggtgaagagcaagtgccgggagaacgccagccccttcttcttctgctgcttcatc gccgtggctatgggcatccggtttataatcatggtggccatctggagcgccgtgttcctg aacagcctgttcaaccaggaggtgcagatccccctgaccgagagctactgcggcccct gccccaagaactggatctgctacaagaacaactgctaccagttcttcgacgagagcaa gaactggtacgagagccaggccagctgcatgagccagaacgccagcctgctgaaggt gtacagcaaggaggaccaggacctgctgaagctggtgaagagctaccactggatgg gcctggtgcacatccccaccaacggcagctggcagtgggaggacggcagcatcctga gccccaacctgctgaccatcatcgagatgcagaagggcgactgcgccctgtacgccag cagcttcaagggctacatcgagaactgcagcacccccaacacctacatctgcatgcag cggaccgtgtaa NKG2D-CD3ζ MRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR CAR (CD3z DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG sequence is KGHDGLYQGLSTATKDTYDALHMQALPPRGWIRGRRSRHSWEM italicized and SEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVA ITAMs of CD3z MGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNC are bold; YQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWM NKG2D GLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIEN sequence is CSTPNTYICMQRTV double underlined) Intentionally Intentionally blank blank NKG2D / atgatccacctgggccacatcctgttcctgctgctgctgcccgtggccgctgcccagacc DAP10-CD3ζ acccctggcgagcggagcagcctgcctgccttctaccctggcaccagcggcagctgca nucleic acid gcggctgcggcagcctgagcctgcccctgctggccggcctggtggccgccgacgccgt sequence ggccagcctgctgatcgtgggcgccgtgttcctgtgcgccaggcccaggcggagccct gcccaggaggacggcaaggtgtacatcaacatgcccggccggggcagagtgaagtt cagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacg agctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccggg accctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaat gaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcga gcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaa ggacacctacgatgcattgcacatgcaggccctgccccctcgcaggcggaaaaggtct gggagtggggctaccaatttctctctcctcaagcaagccggagacgttgaggaaaacc ctggacccatgggctggatccgcggccgcaggagccggcacagctgggagatgagc gagttccacaactacaacctggacctgaagaagagcgacttcagcacccggtggcag aagcagcggtgccccgtggtgaagagcaagtgccgggagaacgccagccccttcttct tctgctgcttcatcgccgtggctatgggcatccggtttataatcatggtggccatctggag cgccgtgttcctgaacagcctgttcaaccaggaggtgcagatccccctgaccgagagct actgcggcccctgccccaagaactggatctgctacaagaacaactgctaccagttcttc gacgagagcaagaactggtacgagagccaggccagctgcatgagccagaacgcca gcctgctgaaggtgtacagcaaggaggaccaggacctgctgaagctggtgaagagct accactggatgggcctggtgcacatccccaccaacggcagctggcagtgggaggacg gcagcatcctgagccccaacctgctgaccatcatcgagatgcagaagggcgactgcgc cctgtacgccagcagcttcaagggctacatcgagaactgcagcacccccaacacctac atctgcatgcagcggaccgtgtaa NKG2D / MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGS DAP10-CD3ζ LSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYI (DAP10 NMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR sequence is GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK bold; CD3z GHDGLYQGLSTATKDTYDALHMQALPPRRRKRSGSGATNFSLLKQA sequence is GDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQK single QRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFN underlined QEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQ with ITAMs of NASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSP CD3z NLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV italicized; NKG2D sequence is double underlined) NKG2D / atgatccacctgggccacatcctgttcctgctgctgctgcccgtggccgctgcccagacc DAP10-CD3ζ- acccctggcgagcggagcagcctgcctgccttctaccctggcaccagcggcagctgca 1XX nucleic gcggctgcggcagcctgagcctgcccctgctggccggcctggtggccgccgacgccgt acid sequence ggccagcctgctgatcgtgggcgccgtgttcctgtgcgccaggcccaggcggagccct gcccaggaggacggcaaggtgtacatcaacatgcccggccggggcagagtgaagtt cagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacg agctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccggg accctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgttcaat gaactgcagaaagataagatggcggaggccttcagtgagattgggatgaaaggcga gcgccggaggggcaaggggcacgatggccttttccagggtctcagtacagccaccaa ggacaccttcgatgcattgcacatgcaggccctgccccctcgcaggcggaaaaggtct gggagtggggctaccaatttctctctcctcaagcaagccggagacgttgaggaaaacc ctggacccatgggctggatccgcggccgcaggagccggcacagctgggagatgagc gagttccacaactacaacctggacctgaagaagagcgacttcagcacccggtggcag aagcagcggtgccccgtggtgaagagcaagtgccgggagaacgccagccccttcttct tctgctgcttcatcgccgtggctatgggcatccggtttataatcatggtggccatctggag cgccgtgttcctgaacagcctgttcaaccaggaggtgcagatccccctgaccgagagct actgcggcccctgccccaagaactggatctgctacaagaacaactgctaccagttcttc gacgagagcaagaactggtacgagagccaggccagctgcatgagccagaacgcca gcctgctgaaggtgtacagcaaggaggaccaggacctgctgaagctggtgaagagct accactggatgggcctggtgcacatccccaccaacggcagctggcagtgggaggacg gcagcatcctgagccccaacctgctgaccatcatcgagatgcagaagggcgactgcgc cctgtacgccagcagcttcaagggctacatcgagaactgcagcacccccaacacctac atctgcatgcagcggaccgtgtaa NKG2D / MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGS DAP10-CD3ζ- LSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYI 1XX (DAP10 NMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR sequence is GRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKG bold; CD3z HDGLFQGLSTATKDTFDALHMQALPPRRRKRSGSGATNFSLLKQAG sequence is DVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQ single RCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQE underlined VQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNA with ITAMs of SLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNL CD3z italicized LTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV and mutated residues of CD3z double underlined; NKG2D sequence is double underlined) Human MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSK NKG2D CRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTES sequence YCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSK EDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQK GDCALYASSFKGYIENCSTPNTYICMQRTV NKG2D_bb/H- MGWLCSGLLFPVSCLVLLQVASSGNLFNQEVQIPLTESYCGPCPKNW 1XX pCAR ICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLV sequence. The KSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASS NKG2D FKGYIENCSTPNTYICMQRTVGGGGSGGGGDEPKSCDKTHTCPPCPI sequence is YIWAPLAGTCGVLLLSLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQE single EDGCSCRFPEEEEGGCELRRKRSGSGEGRGSLLTCGDVEENPGPMA underlined LPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNY and the ITAMs WMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSK of the CD3ζ SSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGG polypeptide GGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWV are italicized QEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEA and in bold IYFCALWYSNHWVFGGGTKLTVLGSEAAAIEVMYPPPYLDNEKSNGT with the IIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVR mutated SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRS residues ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP double RRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQ underlined GLSTATKDTFDALHMQALPPR NKG2D_bb MGWLCSGLLFPVSCLVLLQVASSGNLFNQEVQIPLTESYCGPCPK CCR NWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKED sequence. The QDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEM NKG2D QKGDCALYASSFKGYIENCSTPNTYICMQRTVGGGGSGGGGD polypeptide EPKSCDKTHTCPPCPIYIWAPLAGTCGVLLLSLVITLYCNHKRGRKKLL sequence in YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL bold and the 4-1BB co- stimulatory signalling region sequence underlined. NKG2Dbb(trim MGWLCSGLLFPVSCLVLLQVASSGNLFNQEVQIPLTESYCGPCPKNW er)/H-1XX ICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLV sequence. The KSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASS NKG2D FKGYIENCSTPNTYICMQRTVGGGGSGGGGDGGGGSGTPSSDAVS sequence is RLEEEMRKLQATVQELQKRLDRLEETVQAKGGGGIISFFLALTSTALLF single LLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE underlined GGCELRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLH and the ITAMs AEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKG of the CD3ζ LEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAE polypeptide DTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGGSGGGGSQAVV are italicized TQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIG and in bold GTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHW with the VFGGGTKLTVLGSEAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSP mutated LFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYM residues NMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQ double NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLF underlined. NELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTF DALHMQALPPR NKG2Dbb(trim MGWLCSGLLFPVSCLVLLQVASSGNLFNQEVQIPLTESYCGPCPK er) CCR NWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKED sequence. The QDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEM NKG2D QKGDCALYASSFKGYIENCSTPNTYICMQRTVGGGGSGGGGD polypeptide GGGGSGTPSSDAVSRLEEEMRKLQATVQELQKRLDRLEETVQAKGG sequence in GGIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQT bold and the TQEEDGCSCRFPEEEEGGCEL 4-1BB co- stimulatory signalling region sequence underlined. Pbb(trimer)/H MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTE -1XX pCAR GDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQD sequence. The CRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR PD-1 AELRVTERRAEVPTAHGGGGSGTPSSDAVSRLEEEMRKLQATVQELQ sequence is KRLDRLEETVQAKGGGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRK single KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRKRSGSGEG underlined RGSLLTCGDVEENPGPMALPVTALLLPLALLLHAEVQLQQSGGGLVQP and the ITAMs GGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYA of the CD3ζ THYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAY polypeptide WGQGTTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVT are italicized LTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSG and in bold SLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEA with the AAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVV mutated GGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHY residues QPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRRE double EYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFS underlined. EIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR Pbb(trimer) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPA CCR LLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAF sequence. The PEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLC PD-1 GAISLAPKAQIKESLRAELRVTERRAEVPTAHGGGGSGTPSSDA polypeptide VSRLEEEMRKLQATVQELQKRLDRLEETVQAKGGGGIISFFLALTSTA sequence is in LLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE bold and the EEEGGCEL 4-1BB transmembran e and co- stimulatory signalling region sequence is underlined. 44bb(trimer)/ MAWRALHPLLLLLLLFPGSQAQSKAQVLQSVAGQTLTVRCQYPPTGS H-1XX pCAR. LYEKKGWCKEASALVCIRLVTSSKPRTMAWTSRFTIWDDPDAGFFTV The NKp44 TMTDLREEDSGHYWCRIYRPSDNSVSKSVRFYLVVSPASASTQTSW sequence is TPRDLVSSQTQTQSCVPPTAGARQAPESPSTIPVPSQPQNSTLRPGP single AAPIAGGGGSGTPSSDAVSRLEEEMRKLQATVQELQKRLDRLEETVQ underlined AKGGGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMR and the ITAMs PVQTTQEEDGCSCRFPEEEEGGCELRRKRSGSGEGRGSLLTCGDVEE of the CD3ζ NPGPMALPVTALLLPLALLLHAEVQLQQSGGGLVQPGGSMKLSCVAS polypeptide GFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFT are italicized ISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSS and in bold GGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTT with the SNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTIT mutated GAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEAAAIEVMYPPPYL residues DNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVT double VAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY underlined. RSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR DPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRG KGHDGLFQGLSTATKDTFDALHMQALPPR 44bb(trimer) MAWRALHPLLLLLLLFPGSQAQSKAQVLQSVAGQTLTVRCQY CCR PPTGSLYEKKGWCKEASALVCIRLVTSSKPRTMAWTSRFTIW sequence. The DDPDAGFFTVTMTDLREEDSGHYWCRIYRPSDNSVSKSVRFY NKp44 LVVSPASASTQTSWTPRDLVSSQTQTQSCVPPTAGARQAPES polypeptide PSTIPVPSQPQNSTLRPGPAAPIAGGGGSGTPSSDAVSRLEEEM sequence is in RKLQATVQELQKRLDRLEETVQAKGGGGIISFFLALTSTALLFLLFFLTL bold and the RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BB co- stimulatory signalling region sequenced is underlined. Human DAP12 MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIV full length MGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQ sequence GQRSDVYSDLNTQRPYYK Human DAP12 YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQ intracellular RPYYK domain (residues 62- 113) PD-1 protein MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTE sequence GDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQD CRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR AELRVTERRAEVPTAH Amino acids 1-23 are the signal peptide NKp44 protein MAWRALHPLLLLLLLFPGSQAQSKAQVLQSVAGQTLTVRCQYPPTGS sequence LYEKKGWCKEASALVCIRLVTSSKPRTMAWTSRFTIWDDPDAGFFTV TMTDLREEDSGHYWCRIYRPSDNSVSKSVRFYLVVSPASASTQTSW Amino acids TPRDLVSSQTQTQSCVPPTAGARQAPESPSTIPVPSQPQNSTLRPGP 1-21 are the AAPIA signal peptide DAP10- CD3 ζ MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGS (1XX) fusion LSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYI protein NMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR sequence GRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKG DAP10 HDGLFQGLSTATKDTFDALHMQALPPR sequence is bold; CD3ζ (1XX) sequence is single underlined and ITAMs of CD3ζ are in italics, mutated residues of CD3ζ are double underlined. NKG2D_bb atgggatggctgtgcagcggactgctgttccccgtgtcctgtctggtgctgctgcaggtg /H-1XX pCAR gccagctccggaaacctgtttaatcaggaggtgcagatccctctgaccgagtcctactg cggcccatgtcccaagaactggatctgctacaagaacaactgttaccagttctttgatga nucleic acid gtctaagaactggtatgagagccaggcctcctgcatgtctcagaatgcctccctgctga sequence aggtgtactctaaggaggaccaggatctgctgaagctggtgaagagctatcactggat gggcctggtgcacatccctacaaacggctcctggcagtgggaggacggcagcatcctg tccccaaatctgctgaccatcatcgagatgcagaagggcgattgcgccctgtacgcctc tagctttaagggctatatcgagaactgctccacccctaatacatacatctgtatgcagag gaccgtgggaggaggaggaagcggaggaggaggcgacgagccaaagtcctgtgat aagacccacacatgcccaccctgtcctatctacatctgggcaccactggcaggaacatg cggcgtgctgctgctgagcctggtcatcaccctgtattgtaaccacaagagaggcaga aagaagctgctgtacatcttcaagcagcccttcatgagacccgtgcagaccacccagg aggaggacggctgcagctgcagattccccgaggaggaggagggcggctgcgagctg agaagaaagagaagcggcagcggcgagggcagaggcagcctgctgacctgcggcg acgtggaggagaaccccggacccatggctctcccagtgactgccctactgcttccccta gcgcttctcctgcatgcagaggtgcagctgcagcagtctggaggaggcttggtgcaac ctggaggatccatgaaactctcctgtgttgcctctggattcactttcagtaactactggat gaactgggtccgccagtctccagagaaggggcttgagtgggttgctgaaattagattg aaatctaataattatgcaacacattatgcggagtctgtgaaagggaggttcaccatctc aagagatgattccaaaagtagtgtctacctgcaaatgaacaacttaagagctgaagac actggcatttattactgtacctttggtaactcctttgcttactggggccaagggaccacgg tcaccgtctcctcaggtggaggcggttcaggcggaggtggctctggcggtggcggatc gcaggccgtggtcactcaggaatctgcactcaccacatcacctggtgaaacagtcaca ctcacttgtcgctcaagtactggggctgttacaactagtaactatgccaactgggtccaa gaaaaaccagatcatttattcactggtctaataggtggtaccaacaaccgagcaccag gtgttcctgccagattctcaggctccctgattggagacaaggctgccctcaccatcacag gggcacagactgaggatgaggcaatatatttctgtgctctatggtacagcaaccattgg gtgttcggtggaggaaccaaactgactgtcctaggatcagaggcggccgcaattgaa gttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtg aaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgc tggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattatt ttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactc cccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttc gcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccag cagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgat gttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaa gaaccctcaggaaggcctgttcaatgaactgcagaaagataagatggcggaggcctt cagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggccttt tccagggtctcagtacagccaccaaggacaccttcgacgcccttcacatgcaggccctg ccccctcgctaa NKG2Dbb(trim atgggatggctgtgcagcggactgctgttccccgtgtcctgtctggtgctgctgcaggtg er)/H-1XX gccagctccggaaacctgtttaatcaggaggtgcagatccctctgaccgagtcctactg pCAR cggcccatgtcccaagaactggatctgctacaagaacaactgttaccagttctttgatga gtctaagaactggtatgagagccaggcctcctgcatgtctcagaatgcctccctgctga nucleic acid aggtgtactctaaggaggaccaggatctgctgaagctggtgaagagctatcactggat sequence gggcctggtgcacatccctacaaacggctcctggcagtgggaggacggcagcatcctg tccccaaatctgctgaccatcatcgagatgcagaagggcgattgcgccctgtacgcctc tagctttaagggctatatcgagaactgctccacccctaatacatacatctgtatgcagag gaccgtgggaggaggaggaagcggaggaggaggcgacggcggagggggcagcg gcacccctagcagcgacgccgtgagccggctggaggaggagatgcggaagctgcag gccaccgtgcaggagctgcagaagcggctggaccggctggaggagaccgtgcaggc caagggcggaggaggcatcatcagcttcttcctggccctgaccagcaccgccctgctgt tcctgctgttcttcctgaccctgcggttcagcgtggtgaagagaggcagaaagaagctg ctgtacatcttcaagcagcccttcatgagacccgtgcagaccacccaggaggaggacg gctgcagctgcagattccccgaggaggaggagggcggctgcgagctgagaagaaag agaagcggcagcggcgagggcagaggcagcctgctgacctgcggcgacgtggagg agaaccccggacccatggctctcccagtgactgccctactgcttcccctagcgcttctcct gcatgcagaggtgcagctgcagcagtctggaggaggcttggtgcaacctggaggatc catgaaactctcctgtgttgcctctggattcactttcagtaactactggatgaactgggtc cgccagtctccagagaaggggcttgagtgggttgctgaaattagattgaaatctaataa ttatgcaacacattatgcggagtctgtgaaagggaggttcaccatctcaagagatgatt ccaaaagtagtgtctacctgcaaatgaacaacttaagagctgaagacactggcatttat tactgtacctttggtaactcctttgcttactggggccaagggaccacggtcaccgtctcct caggtggaggcggttcaggcggaggtggctctggcggtggcggatcgcaggccgtg gtcactcaggaatctgcactcaccacatcacctggtgaaacagtcacactcacttgtcgc tcaagtactggggctgttacaactagtaactatgccaactgggtccaagaaaaaccag atcatttattcactggtctaataggtggtaccaacaaccgagcaccaggtgttcctgcca gattctcaggctccctgattggagacaaggctgccctcaccatcacaggggcacagact gaggatgaggcaatatatttctgtgctctatggtacagcaaccattgggtgttcggtgga ggaaccaaactgactgtcctaggatcagaggcggccgcaattgaagttatgtatcctcc tccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacac ctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgg tggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgagg agtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccg ggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgc tccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaa ccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaag agacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcagg aaggcctgttcaatgaactgcagaaagataagatggcggaggccttcagtgagattg ggatgaaaggcgagcgccggaggggcaaggggcacgatggccttttccagggtctc agtacagccaccaaggacaccttcgacgcccttcacatgcaggccctgccccctcgcta a Pbb(trimer)/H atgcagatcccccaggctccctggcccgtggtgtgggccgtgctgcagctgggctggc -1XX pCAR ggcctggctggttcctggacagccccgaccggccctggaaccctcccaccttcagcccc gccctgctggtggtgaccgagggcgacaacgccaccttcacctgcagcttcagcaaca nucleic acid ccagcgagagcttcgtgctgaactggtaccggatgagccccagcaaccagaccgaca sequence agctggctgccttccccgaggaccggagccagcctggccaggactgccggttccgggt gacccagctgcccaacggacgggacttccacatgagcgtggtgcgggctcggaggaa cgacagcggcacctacctgtgcggagccatcagcctggctcccaaggcccagatcaag gagagcctgcgggctgagctgcgggtgaccgagcggagggccgaggtgcccaccgc ccacggcggagggggcagcggcacccctagcagcgacgccgtgagccggctggag gaggagatgcggaagctgcaggccaccgtgcaggagctgcagaagcggctggaccg gctggaggagaccgtgcaggccaagggcggaggaggcatcatcagcttcttcctggc cctgaccagcaccgccctgctgttcctgctgttcttcctgaccctgcggttcagcgtggtg aagaggggcaggaagaagctgctgtacatcttcaagcagcccttcatgcggcctgtgc agaccacccaggaggaggacggctgcagctgccggttccccgaggaggaggaggg cggatgcgagctgagaagaaagagaagcggcagcggcgagggcagaggcagcct gctgacctgcggcgacgtggaggagaaccccggacccatggctctcccagtgactgcc ctactgcttcccctagcgcttctcctgcatgcagaggtgcagctgcagcagtctggagga ggcttggtgcaacctggaggatccatgaaactctcctgtgttgcctctggattcactttca gtaactactggatgaactgggtccgccagtctccagagaaggggcttgagtgggttgc tgaaattagattgaaatctaataattatgcaacacattatgcggagtctgtgaaaggga ggttcaccatctcaagagatgattccaaaagtagtgtctacctgcaaatgaacaactta agagctgaagacactggcatttattactgtacctttggtaactcctttgcttactggggcc aagggaccacggtcaccgtctcctcaggtggaggcggttcaggcggaggtggctctg gcggtggcggatcgcaggccgtggtcactcaggaatctgcactcaccacatcacctgg tgaaacagtcacactcacttgtcgctcaagtactggggctgttacaactagtaactatgc caactgggtccaagaaaaaccagatcatttattcactggtctaataggtggtaccaaca accgagcaccaggtgttcctgccagattctcaggctccctgattggagacaaggctgcc ctcaccatcacaggggcacagactgaggatgaggcaatatatttctgtgctctatggta cagcaaccattgggtgttcggtggaggaaccaaactgactgtcctaggatcagaggcg gccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaac cattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaag cccttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacag tggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactac atgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgcccc accacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcc cccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaaga gaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaa gccgagaaggaagaaccctcaggaaggcctgttcaatgaactgcagaaagataaga tggcggaggccttcagtgagattgggatgaaaggcgagcgccggaggggcaaggg gcacgatggccttttccagggtctcagtacagccaccaaggacaccttcgacgcccttc acatgcaggccctgccccctcgctaa 44bb(trimer)/ atggcctggagggccctgcaccctctgctgctgctgctgctgctgttccccggcagccag H-1XX pCAR gcccagagcaaggcccaggtgctgcagagcgtggctggccagaccctgaccgtgcgg tgccagtaccctcccaccggcagcctgtacgagaagaagggctggtgcaaggaggcc nucleic acid agcgccctggtgtgcatccggctggtgaccagcagcaagcctcggacaatggcctgga sequence ccagccggttcaccatctgggacgaccccgacgctggcttcttcaccgtgaccatgacc gacctgcgggaggaggacagcggccactactggtgccggatctacaggcccagcga caacagcgtgagcaagagcgtgcggttctacctggtggtgagccctgccagcgccagc acccagaccagctggacccctcgggacctggtgagcagccagacccagacccagagc tgcgtgcctcccaccgccggagcccggcaggctcccgagagccccagcaccatccccg tgcccagccagccccagaacagcaccctgcggcctggacccgctgcccctatcgccgg cggagggggcagcggcacccctagcagcgacgccgtgagccggctggaggaggag atgcggaagctgcaggccaccgtgcaggagctgcagaagcggctggaccggctgga ggagaccgtgcaggccaagggcggaggaggcatcatcagcttcttcctggccctgacc agcaccgccctgctgttcctgctgttcttcctgaccctgcggttcagcgtggtgaagagg ggcaggaagaagctgctgtacatcttcaagcagcccttcatgcggcctgtgcagacca cccaggaggaggacggctgcagctgccggttccccgaggaggaggagggcggatgc gagctgagaagaaagagaagcggcagcggcgagggcagaggcagcctgctgacct gcggcgacgtggaggagaaccccggacccatggctctcccagtgactgccctactgct tcccctagcgcttctcctgcatgcagaggtgcagctgcagcagtctggaggaggcttgg tgcaacctggaggatccatgaaactctcctgtgttgcctctggattcactttcagtaacta ctggatgaactgggtccgccagtctccagagaaggggcttgagtgggttgctgaaatt agattgaaatctaataattatgcaacacattatgcggagtctgtgaaagggaggttcac catctcaagagatgattccaaaagtagtgtctacctgcaaatgaacaacttaagagctg aagacactggcatttattactgtacctttggtaactcctttgcttactggggccaagggac cacggtcaccgtctcctcaggtggaggcggttcaggcggaggtggctctggcggtggc ggatcgcaggccgtggtcactcaggaatctgcactcaccacatcacctggtgaaacag tcacactcacttgtcgctcaagtactggggctgttacaactagtaactatgccaactggg tccaagaaaaaccagatcatttattcactggtctaataggtggtaccaacaaccgagca ccaggtgttcctgccagattctcaggctccctgattggagacaaggctgccctcaccatc acaggggcacagactgaggatgaggcaatatatttctgtgctctatggtacagcaacc attgggtgttcggtggaggaaccaaactgactgtcctaggatcagaggcggccgcaat tgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatcca tgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgg gtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggccttta ttattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatg actccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcg acttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgta ccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagt acgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgaga aggaagaaccctcaggaaggcctgttcaatgaactgcagaaagataagatggcgga ggccttcagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatg gccttttccagggtctcagtacagccaccaaggacaccttcgacgcccttcacatgcag gccctgccccctcgctaa Coronin 1A SGTPSSDAVSRLEEEMRKLQATVQELQKRLDRLEETVQAK trimerisation motif Pbb(trimer)_N atgcagatcccccaggctccctggcccgtggtgtgggccgtgctgcagctgggctggc 1012 ggcctggctggttcctggacagccccgaccggccctggaaccctcccaccttcagcccc gccctgctggtggtgaccgagggcgacaacgccaccttcacctgcagcttcagcaaca ccagcgagagcttcgtgctgaactggtaccggatgagccccagcaaccagaccgaca agctggctgccttccccgaggaccggagccagcctggccaggactgccggttccgggt gacccagctgcccaacggacgggacttccacatgagcgtggtgcgggctcggaggaa cgacagcggcacctacctgtgcggagccatcagcctggctcccaaggcccagatcaag gagagcctgcgggctgagctgcgggtgaccgagcggagggccgaggtgcccaccgc ccacggcggagggggcagcggcacccctagcagcgacgccgtgagccggctggag gaggagatgcggaagctgcaggccaccgtgcaggagctgcagaagcggctggaccg gctggaggagaccgtgcaggccaagggcggaggaggcatcatcagcttcttcctggc cctgaccagcaccgccctgctgttcctgctgttcttcctgaccctgcggttcagcgtggtg aagaggggcaggaagaagctgctgtacatcttcaagcagcccttcatgcggcctgtgc agaccacccaggaggaggacggctgcagctgccggttccccgaggaggaggaggg cggatgcgagctgcggaggaagcggagcggcagcggcgagggccggggcagcctg ctgacctgcggcgatgtggaagagaatcccggccctatgatccacctgggccacatcct gttcctgctgctgctgcccgtggccgctgcccagaccacccctggcgagcggagcagcc tgcctgccttctaccctggcaccagcggcagctgcagcggctgcggcagcctgagcctg cccctgctggccggcctggtggccgccgacgccgtggccagcctgctgatcgtgggcg ccgtgttcctgtgcgccaggcccaggcggagccctgcccaggaggacggcaaggtgt acatcaacatgcccggccggggctacttcctgggcaggctggtgcccaggggcaggg gcgctgccgaggctgccacccggaagcagcggatcaccgagaccgagagcccctacc aggagctgcagggccagcggagcgacgtgtacagcgacctgaacacccagaggccc tactacaagaggcggaaaaggtctgggagtggggctaccaatttctctctcctcaagca agccggagacgttgaggaaaaccctggacccatgggctggatccggggacggagga gccggcacagctgggagatgagcgagttccacaactacaacctggacctgaagaaga gcgacttcagcacccggtggcagaagcagcggtgccccgtggtgaagagcaagtgcc gggagaacgccagccccttcttcttctgctgcttcatcgccgtggctatgggcatccggtt catcatcatggtggccatctggagcgccgtgttcctgaacagcctgttcaaccaggagg tgcagatccccctgaccgagagctactgcggcccctgccccaagaactggatctgctac aagaacaactgctaccagttcttcgacgagagcaagaactggtacgagagccaggcc agctgcatgagccagaacgccagcctgctgaaggtgtacagcaaggaggaccagga cctgctgaagctggtgaagagctaccactggatgggcctggtgcacatccccaccaac ggcagctggcagtgggaggacggcagcatcctgagccccaacctgctgaccatcatc gagatgcagaagggcgactgcgccctgtacgccagcagcttcaagggctacatcgag aactgcagcacccccaacacctacatctgcatgcagcggaccgtg Pbb(trimer)_N MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTE 1012 GDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQD CRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR AELRVTERRAEVPTAHGGGGSGTPSSDAVSRLEEEMRKLQATVQELQ KRLDRLEETVQAKGGGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRKRSGSGEG RGSLLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYP GTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQE DGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQG QRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMG WIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCR ENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYC GPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKED QDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGD CALYASSFKGYIENCSTPNTYICMQRTV 44bb(trimer)_ atggCCTGGAGGGCCCTGCACCCTCTGCTGCTGCTGCTGCTGCTGT N1012 TCCCCGGCAGCCAGGCCCAGAGCAAGGCCCAGGTGCTGCAGAGC GTGGCTGGCCAGACCCTGACCGTGCGGTGCCAGTACCCTCCCACC GGCAGCCTGTACGAGAAGAAGGGCTGGTGCAAGGAGGCCAGCGC CCTGGTGTGCATCCGGCTGGTGACCAGCAGCAAGCCTCGGACaAT GGCCTGGACCAGCCGGTTCACCATCTGGGACGACCCCGACGCTGG CTTCTTCACCGTGACCATGACCGACCTGCGGGAGGAGGACAGCGG CCACTACTGGTGCCGGATCTACAGGCCCAGCGACAACAGCGTGAG CAAGAGCGTGCGGTTCTACCTGGTGGTGAGCCCTGCCAGCGCCAG CACCCAGACCAGCTGGACCCCTCGGGACCTGGTGAGCAGCCAGAC CCAGACCCAGAGCTGCGTGCCTCCCACCGCCGGAGCCCGGCAGG CTCCCGAGAGCCCCAGCACCATCCCCGTGCCCAGCCAGCCCCAGA ACAGCACCCTGCGGCCTGGACCCGCTGCCCCTATCGCCGGCGGAG GGGGCagcggcacccctAGCAGCGACGCCGTGAGCCGGCTGGAGGA GGAGATGCGGAAGCTGCAGGCCACCGTGCAGGAGCTGCAGAAGC GGCTGGACCGGCTGGAGGAGACCGTGCAGGCCAAGGGCGGAGG AGGCATCATCAGCTTCTTCCTGGCCCTGACCAGCACCGCCCTGCTG TTCCTGCTGTTCTTCCTGACCCTGCGGTTCAGCGTGGTGAAGAGG GGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGG CCTGTGCAGACCACCCAGGAGGAGGACGGCTGCAGCTGCCGGTT CCCCGAGGAGGAGGAGGGCGGATGCGAGCTGCGGAGGAAGCGG AGCGGCAGCGGCGAGGGCCGGGGCAGCCTGCTGACCTGCGGCGA TGTGGAAGAGAATCCCGGCCCTATGATCCACCTGGGCCACATCCT GTTCCTGCTGCTGCTGCCCGTGGCCGCTGCCCAGACCACCCCTGG CGAGCGGAGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAG CTGCAGCGGCTGCGGCAGCCTGAGCCTGCCCCTGCTGGCCGGCC TGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCC GTGTTCCTGTGCGCCAGGCCCAGGCGGAGCCCtGCCCAGGAGGAC GGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGC AGGCTGGTGCCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCG GAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAGCTGC AGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGG CCCTACTACAAGAGGCGGAAAAGGTCTGGGAGTGGGGCTACCAAT TTCTCTCTCCTCAAGCAAGCCGGAGACGTTGAGGAAAACCCTGGaC CcATGGGCTGGATCCGGGGACGGAGGAGCCGGCACAGCTGGGAG ATGAGCGAGTTCCACAACTACAACCTGGACCTGAAGAAGAGCGAC TTCAGCACCCGGTGGCAGAAGCAGCGGTGCCCCGTGGTGAAGAG CAAGTGCCGGGAGAACGCCAGCCCCTTCTTCTTCTGCTGCTTCATC GCCGTGGCtATGGGCATCCGGTTCATCATCATGGTGGCCATCTGGA GCGCCGTGTTCCTGAACAGCCTGTTCAACCAGGAGGTGCAGATCC CCCTGACCGAGAGCTACTGCGGCCCCTGCCCCAAGAACTGGATCT GCTACAAGAACAACTGCTACCAGTTCTTCGACGAGAGCAAGAACTG GTACGAGAGCCAGGCCAGCTGCATGAGCCAGAACGCCAGCCTGCT GAAGGTGTACAGCAAGGAGGACCAGGACCTGCTGAAGCTGGTGA AGAGCTACCACTGGATGGGCCTGGTGCACATCCCCACCAACGGCA GCTGGCAGTGGGAGGACGGCAGCATCCTGAGCCCCAACCTGCTG ACCATCATCGAGATGCAGAAGGGCGACTGCGCCCTGTACGCCAGC AGCTTCAAGGGCTACATCGAGAACTGCAGCACCCCCAACACCTACA TCTGCATGCAGCGGACCGTG 44bb(trimer)_ MAWRALHPLLLLLLLFPGSQAQSKAQVLQSVAGQTLTVRCQYPPTGS N1012 LYEKKGWCKEASALVCIRLVTSSKPRTMAWTSRFTIWDDPDAGFFTV TMTDLREEDSGHYWCRIYRPSDNSVSKSVRFYLVVSPASASTQTSW TPRDLVSSQTQTQSCVPPTAGARQAPESPSTIPVPSQPQNSTLRPGP AAPIAGGGGSGTPSSDAVSRLEEEMRKLQATVQELQKRLDRLEETVQ AKGGGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCELRRKRSGSGEGRGSLLTCGDVEE NPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGS LSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGR GYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNT QRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHS WEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCF IAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYK NNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYH WMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYI ENCSTPNTYICMQRTV 44bb(monome ATGATCCACCTGGGCCACATCCTGTTCCTGCTGCTGCTGCCCGTGG r)N1012 CCGCTGCCCAGACCACCCCTGGCGAGCGGAGCAGCCTGCCTGCCT TCTACCCTGGCACCAGCGGCAGCTGCAGCGGCTGCGGCAGCCTGA GCCTGCCCCTGCTGGCCGGCCTGGTGGCCGCCGACGCCGTGGCC AGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGG CGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCC GGCCGGGGCTACTTCCTGGGCAGGCTGGTGCCCAGGGGCAGGGG CGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCG AGAGCCCCTACCAGGAGCTGCAGGGCCAGCGGAGCGACGTGTAC AGCGACCTGAACACCCAGAGGCCCTACTACAAGAGGCGGAAAAGG TCTGGGAGTGGGGCTACCAATTTCTCTCTCCTCAAGCAAGCCGGA GACGTTGAGGAAAACCCTGGaCCCatggCCTGGAGGGCCCTGCACC CTCTGCTGCTGCTGCTGCTGCTGTTCCCCGGCAGCCAGGCCCAGA GCAAGGCCCAGGTGCTGCAGAGCGTGGCTGGCCAGACCCTGACC GTGCGGTGCCAGTACCCTCCCACCGGCAGCCTGTACGAGAAGAAG GGCTGGTGCAAGGAGGCCAGCGCCCTGGTGTGCATCCGGCTGGT GACCAGCAGCAAGCCTCGGACaATGGCCTGGACCAGCCGGTTCAC CATCTGGGACGACCCCGACGCTGGCTTCTTCACCGTGACCATGAC CGACCTGCGGGAGGAGGACAGCGGCCACTACTGGTGCCGGATCT ACAGGCCCAGCGACAACAGCGTGAGCAAGAGCGTGCGGTTCTACC TGGTGGTGAGCCCTGCCAGCGCCAGCACCCAGACCAGCTGGACCC CTCGGGACCTGGTGAGCAGCCAGACCCAGACCCAGAGCTGCGTG CCTCCCACCGCCGGAGCCCGGCAGGCTCCCGAGAGCCCCAGCAC CATCCCCGTGCCCAGCCAGCCCCAGAACAGCACCCTGCGGCCTGG ACCCGCTGCCCCTATCGCCATCTACATCTGGGCTCCCCTGGCCGG CACCTGCGGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTG CAACCACAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCA GCCCTTCATGAGACCCGTGCAGACCACCCAGGAGGAGGACGGCTG CAGCTGCAGATTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTGA GAAGAAAGAGAAGCGGCAGCGGCGAGGGCAGAGGCAGCCTGCTG ACCTGCGGCGACGTGGAGGAGAACCCCGGACccATGGGCTGGATC CGGGGACGGAGGAGCCGGCACAGCTGGGAGATGAGCGAGTTCCA CAACTACAACCTGGACCTGAAGAAGAGCGACTTCAGCACCCGGTG GCAGAAGCAGCGGTGCCCCGTGGTGAAGAGCAAGTGCCGGGAGA ACGCCAGCCCCTTCTTCTTCTGCTGCTTCATCGCCGTGGCtATGGG CATCCGGTTCATCATCATGGTGGCCATCTGGAGCGCCGTGTTCCTG AACAGCCTGTTCAACCAGGAGGTGCAGATCCCCCTGACCGAGAGC TACTGCGGCCCCTGCCCCAAGAACTGGATCTGCTACAAGAACAACT GCTACCAGTTCTTCGACGAGAGCAAGAACTGGTACGAGAGCCAGG CCAGCTGCATGAGCCAGAACGCCAGCCTGCTGAAGGTGTACAGCA AGGAGGACCAGGACCTGCTGAAGCTGGTGAAGAGCTACCACTGGA TGGGCCTGGTGCACATCCCCACCAACGGCAGCTGGCAGTGGGAG GACGGCAGCATCCTGAGCCCCAACCTGCTGACCATCATCGAGATG CAGAAGGGCGACTGCGCCCTGTACGCCAGCAGCTTCAAGGGCTAC ATCGAGAACTGCAGCACCCCCAACACCTACATCTGCATGCAGCGG ACCGTG 44bb(monome MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPL r)N1012 LAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLG RLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYY KRRKRSGSGATNFSLLKQAGDVEENPGPMAWRALHPLLLLLLLFPGS QAQSKAQVLQSVAGQTLTVRCQYPPTGSLYEKKGWCKEASALVCIRL VTSSKPRTMAWTSRFTIWDDPDAGFFTVTMTDLREEDSGHYWCRIY RPSDNSVSKSVRFYLVVSPASASTQTSWTPRDLVSSQTQTQSCVPPT AGARQAPESPSTIPVPSQPQNSTLRPGPAAPIAIYIWAPLAGTCGVLLL SLVITLYCNHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRRKRSGSGEGRGSLLTCGDVEENPGPMGWIRGRRSRHSWEM SEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVA MGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNC YQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWM GLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIEN CSTPNTYICMQRTV Pbb(mut) ATGCAGATCCCCCAGGCTCCCTGGCCCGTGGTGTGGGCCGTGCTG N1012 CAGCTGGGCTGGCGGCCTGGCTGGTTCCTGGACAGCCCCGACCG GCCCTGGAACCCTCCCACCTTCAGCCCCGCCCTGCTGGTGGTGAC CGAGGGCGACAACGCCACCTTCACCTGCAGCTTCAGCAACACCAG CGAGAGCTTCGTGCTGAACTGGTACCGGATGAGCCCCAGCAACCA GACCGACAAGCTGGCTGCCTTCCCCGAGGACCGGAGCCAGCCTG GCCAGGACTGCCGGTTCCGGGTGACCCAGCTGCCCAACGGACGG GACTTCCACATGAGCGTGGTGCGGGCTCGGAGGAACGACAGCGG CACCTACCTGTGCGGAGCCATCAGCCTGGCTCCCAAGGCCCAGAT CAAGGAGAGCCTGCGGGCTGAGCTGCGGGTGACCGAGCGGAGGG CCGAGGTGCCCACCGCCCACGGCGGAGGGGGCagcggcacccctAG CAGCGACGCCGTGAGCCGGCTGGAGGAGGAGATGCGGAAGCTGC AGGCCACCGTGCAGGAGCTGCAGAAGCGGCTGGACCGGCTGGAG GAGACCGTGCAGGCCAAGGGCGGAGGAGGCATCATCAGCTTCTTC CTGGCCCTGACCAGCACCGCCCTGCTGTTCCTGCTGTTCTTCCTGA CCCTGCGGTTCAGCGTGGTGAAGAGGGGCAGGAAGAAGCTGCTG TACATCTTCAAGCAGCCCTTCATGCGGCCTGTGCAGACCACCgcGg cGgcagcCGGCTGCAGCTGCCGGTTCCCCgcGgctgccgcgGGCGGAT GCGAGCTGCGGAGGAAGCGGAGCGGCAGCGGCGAGGGCCGGGG CAGCCTGCTGACCTGCGGCGATGTGGAAGAGAATCCCGGCCCTAT GATCCACCTGGGCCACATCCTGTTCCTGCTGCTGCTGCCCGTGGC CGCTGCCCAGACCACCCCTGGCGAGCGGAGCAGCCTGCCTGCCTT CTACCCTGGCACCAGCGGCAGCTGCAGCGGCTGCGGCAGCCTGA GCCTGCCCCTGCTGGCCGGCCTGGTGGCCGCCGACGCCGTGGCC AGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGG CGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCC GGCCGGGGCTACTTCCTGGGCAGGCTGGTGCCCAGGGGCAGGGG CGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCG AGAGCCCCTACCAGGAGCTGCAGGGCCAGCGGAGCGACGTGTAC AGCGACCTGAACACCCAGAGGCCCTACTACAAGAGGCGGAAAAGG TCTGGGAGTGGGGCTACCAATTTCTCTCTCCTCAAGCAAGCCGGA GACGTTGAGGAAAACCCTGGaCCcATGGGCTGGATCCGGGGACGG AGGAGCCGGCACAGCTGGGAGATGAGCGAGTTCCACAACTACAAC CTGGACCTGAAGAAGAGCGACTTCAGCACCCGGTGGCAGAAGCAG CGGTGCCCCGTGGTGAAGAGCAAGTGCCGGGAGAACGCCAGCCC CTTCTTCTTCTGCTGCTTCATCGCCGTGGCtATGGGCATCCGGTTCA TCATCATGGTGGCCATCTGGAGCGCCGTGTTCCTGAACAGCCTGTT CAACCAGGAGGTGCAGATCCCCCTGACCGAGAGCTACTGCGGCCC CTGCCCCAAGAACTGGATCTGCTACAAGAACAACTGCTACCAGTTC TTCGACGAGAGCAAGAACTGGTACGAGAGCCAGGCCAGCTGCATG AGCCAGAACGCCAGCCTGCTGAAGGTGTACAGCAAGGAGGACCA GGACCTGCTGAAGCTGGTGAAGAGCTACCACTGGATGGGCCTGGT GCACATCCCCACCAACGGCAGCTGGCAGTGGGAGGACGGCAGCA TCCTGAGCCCCAACCTGCTGACCATCATCGAGATGCAGAAGGGCG ACTGCGCCCTGTACGCCAGCAGCTTCAAGGGCTACATCGAGAACT GCAGCACCCCCAACACCTACATCTGCATGCAGCGGACCGTG Pbb(mut) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTE N1012 GDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQD CRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR AELRVTERRAEVPTAHGGGGSGTPSSDAVSRLEEEMRKLQATVQELQ KRLDRLEETVQAKGGGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRK KLLYIFKQPFMRPVQTTAAAAGCSCRFPAAAAGGCELRRKRSGSGEG RGSLLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYP GTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQE DGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQG QRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMG WIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCR ENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYC GPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKED QDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGD CALYASSFKGYIENCSTPNTYICMQRTV Pbb(Tr) N1012 ATGCAGATCCCCCAGGCTCCCTGGCCCGTGGTGTGGGCCGTGCTG CAGCTGGGCTGGCGGCCTGGCTGGTTCCTGGACAGCCCCGACCG GCCCTGGAACCCTCCCACCTTCAGCCCCGCCCTGCTGGTGGTGAC CGAGGGCGACAACGCCACCTTCACCTGCAGCTTCAGCAACACCAG CGAGAGCTTCGTGCTGAACTGGTACCGGATGAGCCCCAGCAACCA GACCGACAAGCTGGCTGCCTTCCCCGAGGACCGGAGCCAGCCTG GCCAGGACTGCCGGTTCCGGGTGACCCAGCTGCCCAACGGACGG GACTTCCACATGAGCGTGGTGCGGGCTCGGAGGAACGACAGCGG CACCTACCTGTGCGGAGCCATCAGCCTGGCTCCCAAGGCCCAGAT CAAGGAGAGCCTGCGGGCTGAGCTGCGGGTGACCGAGCGGAGGG CCGAGGTGCCCACCGCCCACGGCGGAGGGGGCagcggcacccctAG CAGCGACGCCGTGAGCCGGCTGGAGGAGGAGATGCGGAAGCTGC AGGCCACCGTGCAGGAGCTGCAGAAGCGGCTGGACCGGCTGGAG GAGACCGTGCAGGCCAAGGGCGGAGGAGGCATCATCAGCTTCTTC CTGGCCCTGACCAGCACCGCCCTGCTGTTCCTGCTGTTCTTCCTGA CCCTGCGGTTCAGCGTGGTGAAGAGGGGCCGGAGGAAGCGGAGC GGCAGCGGCGAGGGCCGGGGCAGCCTGCTGACCTGCGGCGATGT GGAAGAGAATCCCGGCCCTATGATCCACCTGGGCCACATCCTGTT CCTGCTGCTGCTGCCCGTGGCCGCTGCCCAGACCACCCCTGGCGA GCGGAGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAGCTG CAGCGGCTGCGGCAGCCTGAGCCTGCCCCTGCTGGCCGGCCTGG TGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTG TTCCTGTGCGCCAGGCCCAGGCGGAGCCCtGCCCAGGAGGACGGC AAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGCAGG CTGGTGCCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAA GCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAGCTGCAGG GCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCT ACTACAAGAGGCGGAAAAGGTCTGGGAGTGGGGCTACCAATTTCT CTCTCCTCAAGCAAGCCGGAGACGTTGAGGAAAACCCTGGaCCcAT GGGCTGGATCCGGGGACGGAGGAGCCGGCACAGCTGGGAGATGA GCGAGTTCCACAACTACAACCTGGACCTGAAGAAGAGCGACTTCA GCACCCGGTGGCAGAAGCAGCGGTGCCCCGTGGTGAAGAGCAAG TGCCGGGAGAACGCCAGCCCCTTCTTCTTCTGCTGCTTCATCGCCG TGGCtATGGGCATCCGGTTCATCATCATGGTGGCCATCTGGAGCGC CGTGTTCCTGAACAGCCTGTTCAACCAGGAGGTGCAGATCCCCCT GACCGAGAGCTACTGCGGCCCCTGCCCCAAGAACTGGATCTGCTA CAAGAACAACTGCTACCAGTTCTTCGACGAGAGCAAGAACTGGTAC GAGAGCCAGGCCAGCTGCATGAGCCAGAACGCCAGCCTGCTGAA GGTGTACAGCAAGGAGGACCAGGACCTGCTGAAGCTGGTGAAGA GCTACCACTGGATGGGCCTGGTGCACATCCCCACCAACGGCAGCT GGCAGTGGGAGGACGGCAGCATCCTGAGCCCCAACCTGCTGACCA TCATCGAGATGCAGAAGGGCGACTGCGCCCTGTACGCCAGCAGCT TCAAGGGCTACATCGAGAACTGCAGCACCCCCAACACCTACATCTG CATGCAGCGGACCGTG Pbb(Tr) N1012 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTE GDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQD CRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR AELRVTERRAEVPTAHGGGGSGTPSSDAVSRLEEEMRKLQATVQELQ KRLDRLEETVQAKGGGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRR KRSGSGEGRGSLLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGE RSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCA RPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITET ESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGD VEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQR CPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEV QIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNAS LLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLL TIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV Pbb(dimer)N1 ATGCAGATCCCCCAGGCTCCCTGGCCCGTGGTGTGGGCCGTGCTG 012 CAGCTGGGCTGGCGGCCTGGCTGGTTCCTGGACAGCCCCGACAG GCCCTGGAACCCCCCTACCTTCAGCCCCGCCCTGCTGGTGGTGAC CGAGGGCGACAACGCCACCTTCACCTGCAGCTTCAGCAACACCAG CGAGAGCTTCGTGCTGAACTGGTACCGGATGAGCCCCAGCAACCA GACCGACAAGCTGGCTGCCTTCCCCGAGGACCGGAGCCAGCCTG GCCAGGACTGCCGGTTCCGGGTGACCCAGCTGCCCAACGGCAGG GACTTCCACATGAGCGTGGTGCGGGCTCGGAGGAACGACAGCGG CACCTACCTGTGCGGAGCCATCAGCCTGGCTCCCAAGGCCCAGAT CAAGGAGAGCCTGAGGGCCGAGCTGCGGGTGACCGAGCGGAGG GCCGAGGTGCCCACCGCCCACCCCAGCCCCGAGCCAAAGTCCTGT GATAAGACCCACACATGCCCACCCTGTCCTctagacaatgagaagagca atggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggac cttctaagcccATCATCAGCTTCTTCCTGGCCCTGACCAGCACCGCCCT GCTGTTCCTGCTGTTCTTCCTGACCCTGCGGTTCAGCGTGGTGAAG AGGGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATG CGGCCTGTGCAGACCACCCAGGAGGAGGACGGCTGCAGCTGCCG GTTCCCCGAGGAGGAGGAGGGCGGATGCGAGCTGCGGAGGAAGC GGAGCGGCAGCGGCGAGGGCCGGGGCAGCCTGCTGACCTGCGG CGATGTGGAAGAGAATCCCGGCCCTatgatccacctgggccacatcctgttc ctgctgctgctgcccgtggccgctgcccagaccacccctggcgagcggagcagcctgc ctgccttctaccctggcaccagcggcagctgcagcggctgcggcagcctgagcctgccc ctgctggccggcctggtggccgccgacgccgtggccagcctgctgatcgtgggcgccg tgttcctgtgcgccaggcccaggcggagccctgcccaggaggacggcaaggtgtacat caacatgcccggccggggctacttcctgggcaggctggtgcccaggggcaggggcgc tgccgaggctgccacccggaagcagcggatcaccgagaccgagagcccctaccagg agctgcagggccagcggagcgacgtgtacagcgacctgaacacccagaggccctact acaagaggcggaaaaggtctgggagtggggctaccaatttctctctcctcaagcaagc cggagacgttgaggaaaaccctggacccatgggctggatccggggacggaggagcc ggcacagctgggagatgagcgagttccacaactacaacctggacctgaagaagagc gacttcagcacccggtggcagaagcagcggtgccccgtggtgaagagcaagtgccgg gagaacgccagccccttcttcttctgctgcttcatcgccgtggctatgggcatccggttca tcatcatggtggccatctggagcgccgtgttcctgaacagcctgttcaaccaggaggtg cagatccccctgaccgagagctactgcggcccctgccccaagaactggatctgctaca agaacaactgctaccagttcttcgacgagagcaagaactggtacgagagccaggcca gctgcatgagccagaacgccagcctgctgaaggtgtacagcaaggaggaccaggac ctgctgaagctggtgaagagctaccactggatgggcctggtgcacatccccaccaacg gcagctggcagtgggaggacggcagcatcctgagccccaacctgctgaccatcatcg agatgcagaagggcgactgcgccctgtacgccagcagcttcaagggctacatcgaga actgcagcacccccaacacctacatctgcatgcagcggaccgtg Pbb(dimer)N1 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTE 012 GDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQD CRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR AELRVTERRAEVPTAHPSPEPKSCDKTHTCPPCPLDNEKSNGTIIHVK GKHLCPSPLFPGPSKPIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRKRSGSGEGRGS LLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTS GSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDG KVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQR SDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWI RGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCREN ASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGP CPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQ DLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDC ALYASSFKGYIENCSTPNTYICMQRTV Pbb(monomer ATGCAGATCCCCCAGGCTCCCTGGCCCGTGGTGTGGGCCGTGCTG )N1012 CAGCTGGGCTGGCGGCCTGGCTGGTTCCTGGACAGCCCCGACAG GCCCTGGAACCCCCCTACCTTCAGCCCCGCCCTGCTGGTGGTGAC CGAGGGCGACAACGCCACCTTCACCTGCAGCTTCAGCAACACCAG CGAGAGCTTCGTGCTGAACTGGTACCGGATGAGCCCCAGCAACCA GACCGACAAGCTGGCTGCCTTCCCCGAGGACCGGAGCCAGCCTG GCCAGGACTGCCGGTTCCGGGTGACCCAGCTGCCCAACGGCAGG GACTTCCACATGAGCGTGGTGCGGGCTCGGAGGAACGACAGCGG CACCTACCTGTGCGGAGCCATCAGCCTGGCTCCCAAGGCCCAGAT CAAGGAGAGCCTGAGGGCCGAGCTGCGGGTGACCGAGCGGAGG GCCGAGGTGCCCACCGCCCACCCCAGCCCCAGCCCTCGGCCTGCC GGCattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaacca ttatccatgtgaaagggaaacaccttAgtccaagtcccctatttcccggaccttctaagc ccATCATCAGCTTCTTCCTGGCCCTGACCAGCACCGCCCTGCTGTTC CTGCTGTTCTTCCTGACCCTGCGGTTCAGCGTGGTGAAGAGGGGC AGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCT GTGCAGACCACCCAGGAGGAGGACGGCTGCAGCTGCCGGTTCCC CGAGGAGGAGGAGGGCGGATGCGAGCTGCGGAGGAAGCGGAGC GGCAGCGGCGAGGGCCGGGGCAGCCTGCTGACCTGCGGCGATGT GGAAGAGAATCCCGGCCCTatgatccacctgggccacatcctgttcctgctgct gctgcccgtggccgctgcccagaccacccctggcgagcggagcagcctgcctgccttct accctggcaccagcggcagctgcagcggctgcggcagcctgagcctgcccctgctggc cggcctggtggccgccgacgccgtggccagcctgctgatcgtgggcgccgtgttcctgt gcgccaggcccaggcggagccctgcccaggaggacggcaaggtgtacatcaacatg cccggccggggctacttcctgggcaggctggtgcccaggggcaggggcgctgccgag gctgccacccggaagcagcggatcaccgagaccgagagcccctaccaggagctgca gggccagcggagcgacgtgtacagcgacctgaacacccagaggccctactacaaga ggcggaaaaggtctgggagtggggctaccaatttctctctcctcaagcaagccggaga cgttgaggaaaaccctggacccatgggctggatccggggacggaggagccggcaca gctgggagatgagcgagttccacaactacaacctggacctgaagaagagcgacttca gcacccggtggcagaagcagcggtgccccgtggtgaagagcaagtgccgggagaac gccagccccttcttcttctgctgcttcatcgccgtggctatgggcatccggttcatcatcat ggtggccatctggagcgccgtgttcctgaacagcctgttcaaccaggaggtgcagatcc ccctgaccgagagctactgcggcccctgccccaagaactggatctgctacaagaacaa ctgctaccagttcttcgacgagagcaagaactggtacgagagccaggccagctgcatg agccagaacgccagcctgctgaaggtgtacagcaaggaggaccaggacctgctgaa gctggtgaagagctaccactggatgggcctggtgcacatccccaccaacggcagctgg cagtgggaggacggcagcatcctgagccccaacctgctgaccatcatcgagatgcag aagggcgactgcgccctgtacgccagcagcttcaagggctacatcgagaactgcagc acccccaacacctacatctgcatgcagcggaccgtg Pbb(monomer MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTE )N1012 GDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQD CRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR AELRVTERRAEVPTAHPSPSPRPAGIEVMYPPPYLDNEKSNGTIIHVK GKHLSPSPLFPGPSKPIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLL YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRKRSGSGEGRGS LLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTS GSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDG KVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQR SDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWI RGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCREN ASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGP CPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQ DLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDC ALYASSFKGYIENCSTPNTYICMQRTV CXCR2 atggaggatttcaatatggagagcgactccttcgaggatttttggaagggcgaggacc Pbb(trimer) tgtctaactacagctatagctccacactgcccccttttctgctggatgccgccccttgtga N1012 gccagagtccctggagatcaacaagtacttcgtggtcatcatctatgccctggtgtttctg ctgtctctgctgggcaatagcctggtcatgctggtcatcctgtactccagggtgggccgc tctgtgaccgacgtgtatctgctgaatctggccctggccgatctgctgttcgcactgacac tgccaatctgggcagcaagcaaggtgaacggctggatcttcggcacctttctgtgcaag gtggtgtctctgctgaaggaggtgaacttctacagcggcatcctgctgctggcctgtatc tccgtggaccggtatctggccatcgtgcacgccaccaggacactgacccagaagcggt acctggtgaagttcatctgcctgagcatctggggactgtccctgctgctggccctgcctgt gctgctgtttaggcgcacagtgtactctagcaacgtgtctccagcctgttatgaggatat gggcaacaataccgccaattggaggatgctgctgcgcatcctgccacagagcttcggc tttatcgtgcccctgctgatcatgctgttctgctacggctttacactgcggaccctgttcaa ggcccacatgggccagaagcaccgggccatgagagtgatcttcgccgtggtgctgatc tttctgctgtgctggctgccctataacctggtgctgctggccgacacactgatgcggacc caggtcatccaggagacatgcgagcggagaaaccacatcgacagagccctggatgcc accgagatcctgggcatcctgcactcctgtctgaatcctctgatctatgccttcatcggcc agaagtttaggcacggcctgctgaagatcctggccatccacggcctgatctccaagga ctctctgcccaaggatagccgcccttccttcgtgggctcctctagcggccacacctctacc acactgcGgcGgAAacGgtcCGGatctGGgGctacaAacttCtctCTctTGAag caggccGGaGAtGTcGAaGAaAAtCCaGGcCctATGCAGATCCCCCAG GCTCCCTGGCCCGTGGTGTGGGCCGTGCTGCAGCTGGGCTGGCG GCCTGGCTGGTTCCTGGACAGCCCCGACCGGCCCTGGAACCCTCC CACCTTCAGCCCCGCCCTGCTGGTGGTGACCGAGGGCGACAACGC CACCTTCACCTGCAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTG AACTGGTACCGGATGAGCCCCAGCAACCAGACCGACAAGCTGGCT GCCTTCCCCGAGGACCGGAGCCAGCCTGGCCAGGACTGCCGGTTC CGGGTGACCCAGCTGCCCAACGGACGGGACTTCCACATGAGCGTG GTGCGGGCTCGGAGGAACGACAGCGGCACCTACCTGTGCGGAGC CATCAGCCTGGCTCCCAAGGCCCAGATCAAGGAGAGCCTGCGGGC TGAGCTGCGGGTGACCGAGCGGAGGGCCGAGGTGCCCACCGCCC ACGGCGGAGGGGGCagcggcacccctAGCAGCGACGCCGTGAGCCG GCTGGAGGAGGAGATGCGGAAGCTGCAGGCCACCGTGCAGGAGC TGCAGAAGCGGCTGGACCGGCTGGAGGAGACCGTGCAGGCCAAG GGCGGAGGAGGCATCATCAGCTTCTTCCTGGCCCTGACCAGCACC GCCCTGCTGTTCCTGCTGTTCTTCCTGACCCTGCGGTTCAGCGTGG TGAAGAGGGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCT TCATGCGGCCTGTGCAGACCACCCAGGAGGAGGACGGCTGCAGC TGCCGGTTCCCCGAGGAGGAGGAGGGCGGATGCGAGCTGCGGAG GAAGCGGAGCGGCAGCGGCGAGGGCCGGGGCAGCCTGCTGACCT GCGGCGATGTGGAAGAGAATCCCGGCCCTATGATCCACCTGGGCC ACATCCTGTTCCTGCTGCTGCTGCCCGTGGCCGCTGCCCAGACCA CCCCTGGCGAGCGGAGCAGCCTGCCTGCCTTCTACCCTGGCACCA GCGGCAGCTGCAGCGGCTGCGGCAGCCTGAGCCTGCCCCTGCTG GCCGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGT GGGCGCCGTGTTCCTGTGCGCCAGGCCCAGGCGGAGCCCtGCCCA GGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTT CCTGGGCAGGCTGGTGCCCAGGGGCAGGGGCGCTGCCGAGGCTG CCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAG GAGCTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACAC CCAGAGGCCCTACTACAAGAGGCGGAAAAGGTCTGGGAGTGGGG CTACCAATTTCTCTCTCCTCAAGCAAGCCGGAGACGTTGAGGAAAA CCCTGGaCCcATGGGCTGGATCCGGGGACGGAGGAGCCGGCACA GCTGGGAGATGAGCGAGTTCCACAACTACAACCTGGACCTGAAGA AGAGCGACTTCAGCACCCGGTGGCAGAAGCAGCGGTGCCCCGTG GTGAAGAGCAAGTGCCGGGAGAACGCCAGCCCCTTCTTCTTCTGC TGCTTCATCGCCGTGGCtATGGGCATCCGGTTCATCATCATGGTGG CCATCTGGAGCGCCGTGTTCCTGAACAGCCTGTTCAACCAGGAGG TGCAGATCCCCCTGACCGAGAGCTACTGCGGCCCCTGCCCCAAGA ACTGGATCTGCTACAAGAACAACTGCTACCAGTTCTTCGACGAGAG CAAGAACTGGTACGAGAGCCAGGCCAGCTGCATGAGCCAGAACGC CAGCCTGCTGAAGGTGTACAGCAAGGAGGACCAGGACCTGCTGAA GCTGGTGAAGAGCTACCACTGGATGGGCCTGGTGCACATCCCCAC CAACGGCAGCTGGCAGTGGGAGGACGGCAGCATCCTGAGCCCCA ACCTGCTGACCATCATCGAGATGCAGAAGGGCGACTGCGCCCTGT ACGCCAGCAGCTTCAAGGGCTACATCGAGAACTGCAGCACCCCCA ACACCTACATCTGCATGCAGCGGACCGTG CXCR2 MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINK Pbb(trimer) YFVVIIYALVFLLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFA N1012 LTLPIWAASKVNGWIFGTFLCKVVSLLKEVNFYSGILLLACISVDRYLA IVHATRTLTQKRYLVKFICLSIWGLSLLLALPVLLFRRTVYSSNVSPACY EDMGNNTANWRMLLRILPQSFGFIVPLLIMLFCYGFTLRTLFKAHMGQ KHRAMRVIFAVVLIFLLCWLPYNLVLLADTLMRTQVIQETCERRNHID RALDATEILGILHSCLNPLIYAFIGQKFRHGLLKILAIHGLISKDSLPKD SRPSFVGSSSGHTSTTLRRKRSGSGATNFSLLKQAGDVEENPGPMQI PQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGD NATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCR FRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAE LRVTERRAEVPTAHGGGGSGTPSSDAVSRLEEEMRKLQATVQELQK RLDRLEETVQAKGGGGIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRKRSGSGEGRG SLLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGT SGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQED GKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQ RSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGW IRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRE NASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCG PCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQ DLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDC ALYASSFKGYIENCSTPNTYICMQRTV CXCR2 atggaggatttcaatatggagagcgactccttcgaggatttttggaagggcgaggacc Pbb(dimer) tgtctaactacagctatagctccacactgcccccttttctgctggatgccgccccttgtga N1012 gccagagtccctggagatcaacaagtacttcgtggtcatcatctatgccctggtgtttctg ctgtctctgctgggcaatagcctggtcatgctggtcatcctgtactccagggtgggccgc tctgtgaccgacgtgtatctgctgaatctggccctggccgatctgctgttcgcactgacac tgccaatctgggcagcaagcaaggtgaacggctggatcttcggcacctttctgtgcaag gtggtgtctctgctgaaggaggtgaacttctacagcggcatcctgctgctggcctgtatc tccgtggaccggtatctggccatcgtgcacgccaccaggacactgacccagaagcggt acctggtgaagttcatctgcctgagcatctggggactgtccctgctgctggccctgcctgt gctgctgtttaggcgcacagtgtactctagcaacgtgtctccagcctgttatgaggatat gggcaacaataccgccaattggaggatgctgctgcgcatcctgccacagagcttcggc tttatcgtgcccctgctgatcatgctgttctgctacggctttacactgcggaccctgttcaa ggcccacatgggccagaagcaccgggccatgagagtgatcttcgccgtggtgctgatc tttctgctgtgctggctgccctataacctggtgctgctggccgacacactgatgcggacc caggtcatccaggagacatgcgagcggagaaaccacatcgacagagccctggatgcc accgagatcctgggcatcctgcactcctgtctgaatcctctgatctatgccttcatcggcc agaagtttaggcacggcctgctgaagatcctggccatccacggcctgatctccaagga ctctctgcccaaggatagccgcccttccttcgtgggctcctctagcggccacacctctacc acactgcGgcGgAAacGgtcCGGatctGGgGctacaAacttCtctCTctTGAag caggccGGaGAtGTcGAaGAaAAtCCaGGcCctATGCAGATCCCCCAG GCTCCCTGGCCCGTGGTGTGGGCCGTGCTGCAGCTGGGCTGGCG GCCTGGCTGGTTCCTGGACAGCCCCGACCGGCCCTGGAACCCTCC CACCTTCAGCCCCGCCCTGCTGGTGGTGACCGAGGGCGACAACGC CACCTTCACCTGCAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTG AACTGGTACCGGATGAGCCCCAGCAACCAGACCGACAAGCTGGCT GCCTTCCCCGAGGACCGGAGCCAGCCTGGCCAGGACTGCCGGTTC CGGGTGACCCAGCTGCCCAACGGCAGGGACTTCCACATGAGCGTG GTGCGGGCTCGGAGGAACGACAGCGGCACCTACCTGTGCGGAGC CATCAGCCTGGCTCCCAAGGCCCAGATCAAGGAGAGCCTGAGGGC CGAGCTGCGGGTGACCGAGCGGAGGGCCGAGGTGCCCACCGCCC ACCCCAGCCCCGAGCCAAAGTCCTGTGATAAGACCCACACATGCC CACCCTGTCCTctagacaatgagaagagcaatggaaccattatccatgtgaaagg gaaacacctttgtccaagtcccctatttcccggaccttctaagcccATCATCAGCTTC TTCCTGGCCCTGACCAGCACCGCCCTGCTGTTCCTGCTGTTCTTCC TGACCCTGCGGTTCAGCGTGGTGAAGAGGGGCAGGAAGAAGCTG CTGTACATCTTCAAGCAGCCCTTCATGCGGCCTGTGCAGACCACCC AGGAGGAGGACGGCTGCAGCTGCCGGTTCCCCGAGGAGGAGGAG GGCGGATGCGAGCTGCGGAGGAAGCGGAGCGGCAGCGGCGAGG GCCGGGGCAGCCTGCTGACCTGCGGCGATGTGGAAGAGAATCCC GGCCCTatgatccacctgggccacatcctgttcctgctgctgctgcccgtggccgctg cccagaccacccctggcgagcggagcagcctgcctgccttctaccctggcaccagcgg cagctgcagcggctgcggcagcctgagcctgcccctgctggccggcctggtggccgcc gacgccgtggccagcctgctgatcgtgggcgccgtgttcctgtgcgccaggcccaggc ggagccctgcccaggaggacggcaaggtgtacatcaacatgcccggccggggctact tcctgggcaggctggtgcccaggggcaggggcgctgccgaggctgccacccggaag cagcggatcaccgagaccgagagcccctaccaggagctgcagggccagcggagcga cgtgtacagcgacctgaacacccagaggccctactacaagaggcggaaaaggtctgg gagtggggctaccaatttctctctcctcaagcaagccggagacgttgaggaaaaccct ggacccatgggctggatccggggacggaggagccggcacagctgggagatgagcg agttccacaactacaacctggacctgaagaagagcgacttcagcacccggtggcaga agcagcggtgccccgtggtgaagagcaagtgccgggagaacgccagccccttcttctt ctgctgcttcatcgccgtggctatgggcatccggttcatcatcatggtggccatctggag cgccgtgttcctgaacagcctgttcaaccaggaggtgcagatccccctgaccgagagct actgcggcccctgccccaagaactggatctgctacaagaacaactgctaccagttcttc gacgagagcaagaactggtacgagagccaggccagctgcatgagccagaacgcca gcctgctgaaggtgtacagcaaggaggaccaggacctgctgaagctggtgaagagct accactggatgggcctggtgcacatccccaccaacggcagctggcagtgggaggacg gcagcatcctgagccccaacctgctgaccatcatcgagatgcagaagggcgactgcgc cctgtacgccagcagcttcaagggctacatcgagaactgcagcacccccaacacctac atctgcatgcagcggaccgtg CXCR2 MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINK Pbb(dimer) YFVVIIYALVFLLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFA N1012 LTLPIWAASKVNGWIFGTFLCKVVSLLKEVNFYSGILLLACISVDRYLA IVHATRTLTQKRYLVKFICLSIWGLSLLLALPVLLFRRTVYSSNVSPACY EDMGNNTANWRMLLRILPQSFGFIVPLLIMLFCYGFTLRTLFKAHMGQ KHRAMRVIFAVVLIFLLCWLPYNLVLLADTLMRTQVIQETCERRNHID RALDATEILGILHSCLNPLIYAFIGQKFRHGLLKILAIHGLISKDSLPKD SRPSFVGSSSGHTSTTLRRKRSGSGATNFSLLKQAGDVEENPGPMQI PQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGD NATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCR FRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAE LRVTERRAEVPTAHPSPEPKSCDKTHTCPPCPLDNEKSNGTIIHVKGK HLCPSPLFPGPSKPIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRKRSGSGEGRGSLLT CGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGS CSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVY INMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDV YSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGR RSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPF FFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKN WICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKL VKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYAS SFKGYIENCSTPNTYICMQRTV CXCR2 atggaggatttcaatatggagagcgactccttcgaggatttttggaagggcgaggacc Pbb(monomer tgtctaactacagctatagctccacactgcccccttttctgctggatgccgccccttgtga ) N1012 gccagagtccctggagatcaacaagtacttcgtggtcatcatctatgccctggtgtttctg ctgtctctgctgggcaatagcctggtcatgctggtcatcctgtactccagggtgggccgc tctgtgaccgacgtgtatctgctgaatctggccctggccgatctgctgttcgcactgacac tgccaatctgggcagcaagcaaggtgaacggctggatcttcggcacctttctgtgcaag gtggtgtctctgctgaaggaggtgaacttctacagcggcatcctgctgctggcctgtatc tccgtggaccggtatctggccatcgtgcacgccaccaggacactgacccagaagcggt acctggtgaagttcatctgcctgagcatctggggactgtccctgctgctggccctgcctgt gctgctgtttaggcgcacagtgtactctagcaacgtgtctccagcctgttatgaggatat gggcaacaataccgccaattggaggatgctgctgcgcatcctgccacagagcttcggc tttatcgtgcccctgctgatcatgctgttctgctacggctttacactgcggaccctgttcaa ggcccacatgggccagaagcaccgggccatgagagtgatcttcgccgtggtgctgatc tttctgctgtgctggctgccctataacctggtgctgctggccgacacactgatgcggacc caggtcatccaggagacatgcgagcggagaaaccacatcgacagagccctggatgcc accgagatcctgggcatcctgcactcctgtctgaatcctctgatctatgccttcatcggcc agaagtttaggcacggcctgctgaagatcctggccatccacggcctgatctccaagga ctctctgcccaaggatagccgcccttccttcgtgggctcctctagcggccacacctctacc acactgcGgcGgAAacGgtcCGGatctGGgGctacaAacttCtctCTctTGAag caggccGGaGAtGTcGAaGAaAAtCCaGGcCctATGCAGATCCCCCAG GCTCCCTGGCCCGTGGTGTGGGCCGTGCTGCAGCTGGGCTGGCG GCCTGGCTGGTTCCTGGACAGCCCCGACCGGCCCTGGAACCCTCC CACCTTCAGCCCCGCCCTGCTGGTGGTGACCGAGGGCGACAACGC CACCTTCACCTGCAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTG AACTGGTACCGGATGAGCCCCAGCAACCAGACCGACAAGCTGGCT GCCTTCCCCGAGGACCGGAGCCAGCCTGGCCAGGACTGCCGGTTC CGGGTGACCCAGCTGCCCAACGGCAGGGACTTCCACATGAGCGTG GTGCGGGCTCGGAGGAACGACAGCGGCACCTACCTGTGCGGAGC CATCAGCCTGGCTCCCAAGGCCCAGATCAAGGAGAGCCTGAGGGC CGAGCTGCGGGTGACCGAGCGGAGGGCCGAGGTGCCCACCGCCC ACCCCAGCCCCAGCCCTCGGCCTGCCGGCattgaagttatgtatcctcctc cttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacct tAgtccaagtcccctatttcccggaccttctaagcccATCATCAGCTTCTTCCTG GCCCTGACCAGCACCGCCCTGCTGTTCCTGCTGTTCTTCCTGACCC TGCGGTTCAGCGTGGTGAAGAGGGGCAGGAAGAAGCTGCTGTAC ATCTTCAAGCAGCCCTTCATGCGGCCTGTGCAGACCACCCAGGAG GAGGACGGCTGCAGCTGCCGGTTCCCCGAGGAGGAGGAGGGCGG ATGCGAGCTGCGGAGGAAGCGGAGCGGCAGCGGCGAGGGCCGG GGCAGCCTGCTGACCTGCGGCGATGTGGAAGAGAATCCCGGCCCT atgatccacctgggccacatcctgttcctgctgctgctgcccgtggccgctgcccagacc acccctggcgagcggagcagcctgcctgccttctaccctggcaccagcggcagctgca gcggctgcggcagcctgagcctgcccctgctggccggcctggtggccgccgacgccgt ggccagcctgctgatcgtgggcgccgtgttcctgtgcgccaggcccaggcggagccct gcccaggaggacggcaaggtgtacatcaacatgcccggccggggctacttcctgggc aggctggtgcccaggggcaggggcgctgccgaggctgccacccggaagcagcggat caccgagaccgagagcccctaccaggagctgcagggccagcggagcgacgtgtaca gcgacctgaacacccagaggccctactacaagaggcggaaaaggtctgggagtggg gctaccaatttctctctcctcaagcaagccggagacgttgaggaaaaccctggacccat gggctggatccggggacggaggagccggcacagctgggagatgagcgagttccaca actacaacctggacctgaagaagagcgacttcagcacccggtggcagaagcagcggt gccccgtggtgaagagcaagtgccgggagaacgccagccccttcttcttctgctgcttc atcgccgtggctatgggcatccggttcatcatcatggtggccatctggagcgccgtgttc ctgaacagcctgttcaaccaggaggtgcagatccccctgaccgagagctactgcggcc cctgccccaagaactggatctgctacaagaacaactgctaccagttcttcgacgagagc aagaactggtacgagagccaggccagctgcatgagccagaacgccagcctgctgaa ggtgtacagcaaggaggaccaggacctgctgaagctggtgaagagctaccactggat gggcctggtgcacatccccaccaacggcagctggcagtgggaggacggcagcatcct gagccccaacctgctgaccatcatcgagatgcagaagggcgactgcgccctgtacgcc agcagcttcaagggctacatcgagaactgcagcacccccaacacctacatctgcatgc agcggaccgtg CXCR2 MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINK Pbb(monomer YFVVIIYALVFLLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFA ) N1012 LTLPIWAASKVNGWIFGTFLCKVVSLLKEVNFYSGILLLACISVDRYLA IVHATRTLTQKRYLVKFICLSIWGLSLLLALPVLLFRRTVYSSNVSPACY EDMGNNTANWRMLLRILPQSFGFIVPLLIMLFCYGFTLRTLFKAHMGQ KHRAMRVIFAVVLIFLLCWLPYNLVLLADTLMRTQVIQETCERRNHID RALDATEILGILHSCLNPLIYAFIGQKFRHGLLKILAIHGLISKDSLPKD SRPSFVGSSSGHTSTTLRRKRSGSGATNFSLLKQAGDVEENPGPMQI PQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGD NATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCR FRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAE LRVTERRAEVPTAHPSPSPRPAGIEVMYPPPYLDNEKSNGTIIHVKGK HLSPSPLFPGPSKPIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRRKRSGSGEGRGSLLT CGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGS CSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVY INMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDV YSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGR RSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPF FFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKN WICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKL VKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYAS SFKGYIENCSTPNTYICMQRTV CXCR2 myc- ATGCAGATCCCCCAGGCTCCCTGGCCCGTGGTGTGGGCCGTGCTG bb N1012 CAGCTGGGCTGGCGGCCTGGCTGGgagcagaagctgatcagcgaggagg acctgGGCGGAGGGGGCagcggcacccctAGCAGCGACGCCGTGAGCC GGCTGGAGGAGGAGATGCGGAAGCTGCAGGCCACCGTGCAGGAG CTGCAGAAGCGGCTGGACCGGCTGGAGGAGACCGTGCAGGCCAA GGGCGGAGGAGGCATCATCAGCTTCTTCCTGGCCCTGACCAGCAC CGCCCTGCTGTTCCTGCTGTTCTTCCTGACCCTGCGGTTCAGCGTG GTGAAGAGGGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCC TTCATGCGGCCTGTGCAGACCACCCAGGAGGAGGACGGCTGCAG CTGCCGGTTCCCCGAGGAGGAGGAGGGCGGATGCGAGCTGCGGA GGAAGCGGAGCGGCAGCGGCGAGGGCCGGGGCAGCCTGCTGAC CTGCGGCGATGTGGAAGAGAATCCCGGCCCTATGATCCACCTGGG CCACATCCTGTTCCTGCTGCTGCTGCCCGTGGCCGCTGCCCAGAC CACCCCTGGCGAGCGGAGCAGCCTGCCTGCCTTCTACCCTGGCAC CAGCGGCAGCTGCAGCGGCTGCGGCAGCCTGAGCCTGCCCCTGC TGGCCGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATC GTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGGCGGAGCCCtGCC CAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCTA CTTCCTGGGCAGGCTGGTGCCCAGGGGCAGGGGCGCTGCCGAGG CTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACC AGGAGCTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAAC ACCCAGAGGCCCTACTACAAGAGGCGGAAAAGGTCTGGGAGTGG GGCTACCAATTTCTCTCTCCTCAAGCAAGCCGGAGACGTTGAGGAA AACCCTGGaCCcatggaggatttcaatatggagagcgactccttcgaggatttttg gaagggcgaggacctgtctaactacagctatagctccacactgcccccttttctgctgga tgccgccccttgtgagccagagtccctggagatcaacaagtacttcgtggtcatcatcta tgccctggtgtttctgctgtctctgctgggcaatagcctggtcatgctggtcatcctgtact ccagggtgggccgctctgtgaccgacgtgtatctgctgaatctggccctggccgatctg ctgttcgcactgacactgccaatctgggcagcaagcaaggtgaacggctggatcttcg gcacctttctgtgcaaggtggtgtctctgctgaaggaggtgaacttctacagcggcatcc tgctgctggcctgtatctccgtggaccggtatctggccatcgtgcacgccaccaggaca ctgacccagaagcggtacctggtgaagttcatctgcctgagcatctggggactgtccct gctgctggccctgcctgtgctgctgtttaggcgcacagtgtactctagcaacgtgtctcca gcctgttatgaggatatgggcaacaataccgccaattggaggatgctgctgcgcatcct gccacagagcttcggctttatcgtgcccctgctgatcatgctgttctgctacggctttaca ctgcggaccctgttcaaggcccacatgggccagaagcaccgggccatgagagtgatct tcgccgtggtgctgatctttctgctgtgctggctgccctataacctggtgctgctggccga cacactgatgcggacccaggtcatccaggagacatgcgagcggagaaaccacatcga cagagccctggatgccaccgagatcctgggcatcctgcactcctgtctgaatcctctgat ctatgccttcatcggccagaagtttaggcacggcctgctgaagatcctggccatccacg gcctgatctccaaggactctctgcccaaggatagccgcccttccttcgtgggctcctctag cggccacacctctaccacactgcGgcGgAAacGgtcCGGatctGGgGctacaAac ttCtctCTctTGAagcaggccGGaGAtGTcGAaGAaAAtCCaGGcCccATG GGCTGGATCCGGGGACGGAGGAGCCGGCACAGCTGGGAGATGAG CGAGTTCCACAACTACAACCTGGACCTGAAGAAGAGCGACTTCAG CACCCGGTGGCAGAAGCAGCGGTGCCCCGTGGTGAAGAGCAAGT GCCGGGAGAACGCCAGCCCCTTCTTCTTCTGCTGCTTCATCGCCGT GGCtATGGGCATCCGGTTCATCATCATGGTGGCCATCTGGAGCGC CGTGTTCCTGAACAGCCTGTTCAACCAGGAGGTGCAGATCCCCCT GACCGAGAGCTACTGCGGCCCCTGCCCCAAGAACTGGATCTGCTA CAAGAACAACTGCTACCAGTTCTTCGACGAGAGCAAGAACTGGTAC GAGAGCCAGGCCAGCTGCATGAGCCAGAACGCCAGCCTGCTGAA GGTGTACAGCAAGGAGGACCAGGACCTGCTGAAGCTGGTGAAGA GCTACCACTGGATGGGCCTGGTGCACATCCCCACCAACGGCAGCT GGCAGTGGGAGGACGGCAGCATCCTGAGCCCCAACCTGCTGACCA TCATCGAGATGCAGAAGGGCGACTGCGCCCTGTACGCCAGCAGCT TCAAGGGCTACATCGAGAACTGCAGCACCCCCAACACCTACATCTG CATGCAGCGGACCGTG CXCR2 myc- MQIPQAPWPVVWAVLQLGWRPGWEQKLISEEDLGGGGSGTPSSDA bb N1012 VSRLEEEMRKLQATVQELQKRLDRLEETVQAKGGGGIISFFLALTSTA LLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELRRKRSGSGEGRGSLLTCGDVEENPGPMIHLGHILFLLLLP VAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASL LIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEA ATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGATN FSLLKQAGDVEENPGPMEDFNMESDSFEDFWKGEDLSNYSYSSTLPP FLLDAAPCEPESLEINKYFVVIIYALVFLLSLLGNSLVMLVILYSRVGRS VTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCKVVSLLKEVNF YSGILLLACISVDRYLAIVHATRTLTQKRYLVKFICLSIWGLSLLLALPV LLFRRTVYSSNVSPACYEDMGNNTANWRMLLRILPQSFGFIVPLLIML FCYGFTLRTLFKAHMGQKHRAMRVIFAVVLIFLLCWLPYNLVLLADTL MRTQVIQETCERRNHIDRALDATEILGILHSCLNPLIYAFIGQKFRHGL LKILAIHGLISKDSLPKDSRPSFVGSSSGHTSTTLRRKRSGSGATNFS LLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFST RWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLN SLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASC MSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGS ILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV H2-1XX ATGGCTCTCCCagTgactgccCTaCTgCttCcccTAGCGCTTCTCCTGCA (sequence TGCAGAGGTGCAGCTGCAGcAGTCTGGAGGAGGCTTGGTGCAACC encoding SEQ TGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTC ID NO: 59) AGTAACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGG CTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAATTATGCAA CACATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAG ATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGC TGAAGACACTGGCATTTATTACTGTACctttGgtaactccTTTGCTTACT GGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGT TCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGcAggcCGtGgTC ACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACAC TCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGC CAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATA GGTGGTACCAACAACCGAGCACCAGGTGTTCCTGCCAGATTCTCA GGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCA CAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCA ACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGAT CAGAGGCGGCCGCaattgaagttatgtatcctcctccttacctagacaatgagaa gagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcc cggaccttctaagcccttttgggtgctggtggtggttggtggagtcctggcttgctatagc ttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctg cacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattacc agccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcagg agcgcagaCGcccccgcgtaccagcagggccagaaccagctctataacgagctcaat ctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctga gatggggggaaagccgagaaggaagaaccctcaggaaggcctgttcaatgaactgc agaaagataagatggcggaggccttcagtgagattgggatgaaaggcgagcgccgg aggggcaaggggcacgatggccttttccagggtctcagtacagccaccaaggacacct tcgacgcccttcacatgcaggccctgCCCCCTCGC 4-1BB IISFFLALTSTALLFLLFFLTLRFSVV transmembran e domain NKG2Dbb(trim ATGGGATGGCTGTGCAGCGGACTGCTGTTCCCCGTGTCCTGTCTG er) CCR GTGCTGCTGCAGGTGGCCAGCTCCGGAAACCTGTTTAATCAGGAG polynucleotide GTGCAGATCCCTCTGACCGAGTCCTACTGCGGCCCATGTCCCAAG sequence AACTGGATCTGCTACAAGAACAACTGTTACCAGTTCTTTGATGAGT CTAAGAACTGGTATGAGAGCCAGGCCTCCTGCATGTCTCAGAATG CCTCCCTGCTGAAGGTGTACTCTAAGGAGGACCAGGATCTGCTGA AGCTGGTGAAGAGCTATCACTGGATGGGCCTGGTGCACATCCCTA CAAACGGCTCCTGGCAGTGGGAGGACGGCAGCATCCTGTCCCCAA ATCTGCTGACCATCATCGAGATGCAGAAGGGCGATTGCGCCCTGT ACGCCTCTAGCTTTAAGGGCTATATCGAGAACTGCTCCACCCCTAA TACATACATCTGTATGCAGAGGACCGTGGGAGGAGGAGGAAGCG GAGGAGGAGGCGACGGCGGAGGGGGCagcggcacccctAGCAGCGA CGCCGTGAGCCGGCTGGAGGAGGAGATGCGGAAGCTGCAGGCCA CCGTGCAGGAGCTGCAGAAGCGGCTGGACCGGCTGGAGGAGACC GTGCAGGCCAAGGGCGGAGGAGGCATCATCAGCTTCTTCCTGGCC CTGACCAGCACCGCCCTGCTGTTCCTGCTGTTCTTCCTGACCCTGC GGTTCAGCGTGGTGAAGAGAGGCAGAAAGAAGCTGCTGTACATCT TCAAGCAGCCCTTCATGAGACCCGTGCAGACCACCCAGGAGGAGG ACGGCTGCAGCTGCAGATTCCCCGAGGAGGAGGAGGGCGGCTGC GAGCTG Pbb(trimer) ATGCAGATCCCCCAGGCTCCCTGGCCCGTGGTGTGGGCCGTGCTG CCR CAGCTGGGCTGGCGGCCTGGCTGGTTCCTGGACAGCCCCGACCG polynucleotide GCCCTGGAACCCTCCCACCTTCAGCCCCGCCCTGCTGGTGGTGAC sequence CGAGGGCGACAACGCCACCTTCACCTGCAGCTTCAGCAACACCAG CGAGAGCTTCGTGCTGAACTGGTACCGGATGAGCCCCAGCAACCA GACCGACAAGCTGGCTGCCTTCCCCGAGGACCGGAGCCAGCCTG GCCAGGACTGCCGGTTCCGGGTGACCCAGCTGCCCAACGGACGG GACTTCCACATGAGCGTGGTGCGGGCTCGGAGGAACGACAGCGG CACCTACCTGTGCGGAGCCATCAGCCTGGCTCCCAAGGCCCAGAT CAAGGAGAGCCTGCGGGCTGAGCTGCGGGTGACCGAGCGGAGGG CCGAGGTGCCCACCGCCCACGGCGGAGGGGGCagcggcacccctAG CAGCGACGCCGTGAGCCGGCTGGAGGAGGAGATGCGGAAGCTGC AGGCCACCGTGCAGGAGCTGCAGAAGCGGCTGGACCGGCTGGAG GAGACCGTGCAGGCCAAGGGCGGAGGAGGCATCATCAGCTTCTTC CTGGCCCTGACCAGCACCGCCCTGCTGTTCCTGCTGTTCTTCCTGA CCCTGCGGTTCAGCGTGGTGAAGAGGGGCAGGAAGAAGCTGCTG TACATCTTCAAGCAGCCCTTCATGCGGCCTGTGCAGACCACCCAGG AGGAGGACGGCTGCAGCTGCCGGTTCCCCGAGGAGGAGGAGGGC GGATGCGAGCTG 120 44bb(trimer) atggCCTGGAGGGCCCTGCACCCTCTGCTGCTGCTGCTGCTGCTGT CCR TCCCCGGCAGCCAGGCCCAGAGCAAGGCCCAGGTGCTGCAGAGC polynucleotide GTGGCTGGCCAGACCCTGACCGTGCGGTGCCAGTACCCTCCCACC sequence. GGCAGCCTGTACGAGAAGAAGGGCTGGTGCAAGGAGGCCAGCGC CCTGGTGTGCATCCGGCTGGTGACCAGCAGCAAGCCTCGGACaAT GGCCTGGACCAGCCGGTTCACCATCTGGGACGACCCCGACGCTGG CTTCTTCACCGTGACCATGACCGACCTGCGGGAGGAGGACAGCGG CCACTACTGGTGCCGGATCTACAGGCCCAGCGACAACAGCGTGAG CAAGAGCGTGCGGTTCTACCTGGTGGTGAGCCCTGCCAGCGCCAG CACCCAGACCAGCTGGACCCCTCGGGACCTGGTGAGCAGCCAGAC CCAGACCCAGAGCTGCGTGCCTCCCACCGCCGGAGCCCGGCAGG CTCCCGAGAGCCCCAGCACCATCCCCGTGCCCAGCCAGCCCCAGA ACAGCACCCTGCGGCCTGGACCCGCTGCCCCTATCGCCGGCGGAG GGGGCagcggcacccctAGCAGCGACGCCGTGAGCCGGCTGGAGGA GGAGATGCGGAAGCTGCAGGCCACCGTGCAGGAGCTGCAGAAGC GGCTGGACCGGCTGGAGGAGACCGTGCAGGCCAAGGGCGGAGG AGGCATCATCAGCTTCTTCCTGGCCCTGACCAGCACCGCCCTGCTG TTCCTGCTGTTCTTCCTGACCCTGCGGTTCAGCGTGGTGAAGAGG GGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGG CCTGTGCAGACCACCCAGGAGGAGGACGGCTGCAGCTGCCGGTT CCCCGAGGAGGAGGAGGGCGGATGCGAGCTG EXAMPLES Example 1 This study demonstrates that addition of a trimeric CCR produces fitter CAR T cells which can persistently control solid tumours in comparison to CAR T cells containing monomeric or dimeric CCRs. It also confirms the 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. To generate additional trimeric CCR with alternative binding domains, 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. To generate the truncated or mutated Pbb(trimer) CCRs either the full 4-1BB intracellular domain was removed or the two TRAF binding domains were mutated from QEED or EEEE to AAAA. T cell culture and transduction Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor blood samples by density gradient centrifugation using Ficoll-Paque. PBMCs were activated with 10µl/ml TransAct reagent and cultured in RPMI with GlutaMax supplemented with 5% human AB serum for 24-48hrs. Cells were supplemented with IL-2 (100IU/ml) 24 hours prior to T cell retroviral transduction of CAR constructs. CAR T cells were expanded in RPMI with GlutaMax supplemented with 5% human AB serum plus IL-2 (100U/ml) for 10-14 days before use in functional assays. Transduced cells are identified according to the CAR and CCR construct with which they were transduced. Repeated antigen stimulation assays Efficacy of CAR T cells was tested in repeated antigen stimulation assays. BXPC3 tumour cells and CAR T cells were cocultured at an effector target ratio of 1:1 for 72-96 hours. T cells were removed, resuspended in fresh medium, counted, and added to a new BXPC3 tumour monolayer. Residual tumour cell viability was measured by MTT assay after each restimulation. 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. CAR T cell in vivo efficacy 1x105 Firefly luciferase-tagged BXPC3 tumour cells were inoculated in to NSG mice by intraperitoneal (I.P.) injection. Engraftment was monitored by bioluminescence imaging (BLI). At 12 days post tumour injection, mice were treated with 1x107 CAR+ T cells or received PBS control, following mouse randomisation and blinding. Tumour progression was monitored by BLI. For a more stringent tumour model, 1x105 BXPC3 were subcutaneously (s.c.) injected in to NSG mice. Fourteen days after tumour engraftment, mice were treated with 4x106 CAR+ T cells via intravenous (I.V.) injection following mice randomisation and blinding. Tumour volume was measured weekly by calliper measurements. Results Enhanced efficacy of NKG2D adaptor CAR following addition of trimeric CCR Trimeric 4-1BB CCRs containing either the PD-1 binding domain or NKp44 binding domain were expressed alongside N1012 to create the parallel adaptor CAR (pACAR) constructs “Pbb(trimer) N1012” and “44bb(trimer) N1012” respectively. Restimulation assays revealed enhanced efficacy of both Pbb(trimer) N1012 and 44bb(trimer) N1012 compared to N1012 alone on BXPC3 monolayers. Enhanced efficacy included increased functional persistence i.e. number of stimulations CAR T cells can continue to kill fresh monolayers and increased proliferation (Figure 2). Benefit of trimeric CCR is dependent on 4-1BB intracellular domain To confirm the functional benefit from the trimeric CCRs was due to enhanced 4-1BB signalling and not due to a docking or decoy effect from the PD-1 binding domain, two new constructs were made. Either the 4-1BB intracellular domain was removed (Pbb(Tr) N1012) or the TRAF signalling motifs were mutated (Pbb(mut)N1012). Removal or mutation of the 4-1BB domain resulted in a loss of the enhanced function gained by the intact CCR. Both Pbb(Tr) N1012 and Pbb(mut) N1012 had similar function on BXPC3 restimulation assays compared to N1012 whereas Pbb(trimer) N1012 had superior function (Figure 3). Trimeric CCRs superior to dimeric or monomeric CCRs To confirm the trimerisation of 4-1BB within the CCR results in optimal 4-1BB signalling, dimeric and monomeric versions were also generated. To confirm the CCRs were forming their expected structure, a native PAGE gel and western blotting was completed. Western blotting revealed the trimeric CCR had the largest band followed by the dimeric and monomeric CCRs (Figure 4). A restimulation assay was completed to determine the efficacy of various CCR structures. On BXPC3 monolayers, Pbb(trimer) N1012 was able to destroy monolayers for the most rounds of stimulation, followed by the Pbb(dimer) N1012, Pbb(monomer) N1012 and finally N1012 (Figure 5). Additionally, a monomeric form of the CCR containing the NKp44 binding domain was compared with the trimeric form when expressed alongside N1012. In restimulation assays, 44bb(trimer) N1012 was superior to both 44bb(monomer) N1012 and N1012 (Figure 6). CCR binding domain essential for function For future in vivo models, CARs were armoured with CXCR2. An additional CCR was generated to determine whether ligand binding to the CCR was essential for its function or whether the trimerisation of the CCR induced constitutive signalling of the 4-1BB. To do this, the PD-1 binding domain was removed and replaced by a myc tag in the trimeric CCR. Myc-bb N1012 had similar function to N1012 on BXPC3 monolayers where no CCR was present, confirming ligand binding was essential for CCR signalling (Figure 7). Furthermore, the CXCR2 armoured CARs again demonstrated that the trimeric CCR improved function of N1012 in BXPC3 restimulation assays to a greater extent compared to the dimeric and monomeric CCR. Enhanced in vivo efficacy of trimeric CCR containing CARs To confirm the superior function of trimeric CCRs compared to dimeric and monomeric CCRs, an in vivo experiment was completed. pACARs were armoured with CXCR2 to allow a more stringent in vivo model to be tested. BXPC3 tumour cells were subcutaneously injected in to NSG mice, following engraftment, mice were treated with 4x106 CAR T cell by i.v. inoculation. Tumour progression was monitored by calliper measurements. Interestingly, only the Pbb(trimer) N1012 was able to control tumour growth whereas Pbb(dimer) N1012, Pbb(monomer) N1012 and Myc-bb N1012 all had similar tumour growth to the PBS control group. Additionally, the Pbb(trimer) N1012 treated group had the best overall survival (Figure 8). Beneficial function of trimeric CCRs in Muc1 targeting pCARs To confirm the beneficial function of trimeric CCRs in the context of alternative CAR structures and targets, the Pbb(trimer) CCR was expressed alongside the second-generation (2G) Muc1 targeting CAR (H-1XX), to produce the construct Pbb(trimer)/H-1XX. An additional trimeric CCR was generated containing the NKG2D binding domain to produce the pCAR NKG2Dbb(trimer)/H-1XX. Both CCRs significantly improved the function of the 2G Muc1 CAR as demonstrated in an in vivo experiment where mice bearing intraperitoneal BXPC3 tumours were treated with 1x107 CAR T cells I.P. Both Pbb(trimer)/H-1XX and NKG2D(trimer)/H-1XX treated groups were able to control tumour growth and had superior survival compared to H-1XX and PBS control groups (Figure 9). Discussion Our current pCAR technology features a dimeric CCR co-expressed alongside a 2G CAR. 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. To generate trimeric CCRs we made use of the coiled coil domain from coronin 1A and inserted it into the extracellular domain of our CCRs. We demonstrated improved function of both N1012 and H1-XX CARs when a trimeric CCR was included in both in vitro and in vivo experiments. Furthermore, trimeric CCRs were superior to their dimeric or monomeric counterparts confirming the importance of maintaining the natural structure of co-stimulatory domains. We have also demonstrated that both the binding domain and the signalling domain of the trimeric CCR are essential for proper function as removal of either results in a non-functional CCR. Functionality of trimeric CCRs has been shown using several different binding domains and can be applied to CCRs containing other TNF family co-stimulatory molecules. 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. Primary human T-cells expressing the detailed second generation (X28z) or parallel CARs (pCARs, Pbb(trimer)_X28z, NKG2Dbb(trimer)_X28z and N(di)bb_X28z) were added to each well. After 24 hours the T-cells were harvested and degranulation assessed by virtue of the surface expression of surface expression of the lysosomal protein, CD107a (LAMP-1), using flow cytometry. The results are shown in Figure 11. These data demonstrated that T-cells expressing the pCARs were able to respond to lower concentrations of target antigen than a second generation CAR targeting the same target antigen (X28z). Furthermore, T-cells co-expressing a trimeric CCR (NKG2Dbb(trimer)_X28z) 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. The results are shown in Figure 12. These data demonstrated that T-cells expressing the pCAR co-expressing the NKG2D-based CCR were less exhausted than those expressing the second- or third-generation CARs (X28z and X28bbz, respectively) as demonstrated by a higher percentage of cells expressing fewer exhaustion markers. Furthermore, T-cells co-expressing the trimeric CCR demonstrated less exhaustion than those expressing the dimeric CCR. All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference in its entirety, for all purposes. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. §1.57(b)(1), to relate to each and every individual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. §1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or document. While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMS A chimeric co-stimulatory receptor (CCR) polypeptide comprising at least one tumour necrosis receptor (TNFR) co-stimulatory domain, wherein the CCR polypeptide further comprises at least one trimerisation motif. A CCR polypeptide according to claim 1, wherein the TNFR 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. A CCR polypeptide according to claim 1 or 2, wherein the at least one TNFR co- stimulatory domain is at least one 4-1BB co-stimulatory domain or a variant thereof. A CCR polypeptide according to claim 3, wherein the at least one 4-1BB co-stimulatory domain comprises or consists of the sequence shown in SEQ ID NO: 37. A CCR polypeptide according to any one of the preceding claims, wherein the at least one trimerisation motif is derived from the coronin 1A protein. A CCR polypeptide according to any one of the preceding claims, wherein the at least one trimerisation motif comprises or consists of the sequence shown in SEQ ID NO: 93. A CCR polypeptide according to any one of the preceding claims, wherein the at least one trimerisation motif is located upstream of the at least one TNFR co-stimulatory domain. A CCR polypeptide according to any one of the preceding claims, wherein the CCR polypeptide further comprises a transmembrane domain and a first binding element that specifically binds a first epitope on a first target antigen. A CCR polypeptide according to claim 8, wherein the first target antigen is selected from an NKG2D ligand, MUC1, αvβ6 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-β (TGF-β), PD-L1, PD-L2, CD155, CD112, death receptor 4 (DR4), death receptor 5 (DR5), thrombopoietin (TPO), Eltrombopag, and an ErbB family member. A CCR polypeptide according to claim 8 or 9, wherein the first target antigen is MUC1. A CCR polypeptide according to any one of the preceding claims, wherein CCR polypeptide comprises or consists of the sequence shown in SEQ ID NO: 81, 83 or 79. A trimeric chimeric co-stimulatory receptor (CCR) comprising three or more CCR polypeptides according to any one of the preceding claims. A lateral chimeric antigen receptor (CAR) comprising (a) a CCR comprising a CCR polypeptide according to any one of claims 1-11 or a trimeric CCR according to claim 12 and (b) a second-generation chimeric antigen receptor (CAR) or an adaptor CAR. A lateral CAR according to claim 13, wherein the second-generation CAR comprises an intracellular signalling region, a second co-stimulatory signalling region, a transmembrane domain and a second binding element that specifically interacts with a second epitope on a second target antigen or the adaptor CAR comprises at least one adaptor protein comprising an activation signalling domain and a co-stimulatory signalling region and at least one second binding element that specifically binds a second epitope on a second antigen. A lateral CAR according to claim 14, wherein the second target antigen is selected from the antigens listed in claim 9. A lateral CAR according to any one of claims 13-15, wherein the first and second target antigens are different and/or the co-stimulatory signalling regions in the CCR polypeptide and CAR are different. One or more polynucleotides which encode a CCR polypeptide according to any one of claims 1-11, a trimeric CCR according to claim 12 or a lateral CAR according to any one of claims 13-16. One or more vectors comprising one or more polynucleotides according to claim 17. A host cell expressing a CCR polypeptide according to any one of claims 1-11, a trimeric CCR according to claim 12 or a lateral CAR according to any one of claims 13-16. An immunoresponsive cell which expresses a CCR polypeptide according to any one of claims 1-11, a trimeric CCR according to claim 12 or a lateral CAR according to any one of claims 13-16. An immunoresponsive cell according to claim 20, wherein the immunoresponsive cell is an αβ T cell, γδ T cell, or a Natural Killer (NK) cell. A pharmaceutical composition comprising a CCR polypeptide according to any one of claims 1-11, a trimeric CCR according to claim 12, a lateral CAR according to any one of claims 13-16, one or more polynucleotides according to claim 17, one or more vectors according to claim 18, a host cell according to claim 19 or an immunoresponsive cell according to claim 20 or 21 and a pharmaceutically or physiologically acceptable diluent, carrier and/or excipient. A method of preparing the immunoresponsive cell according to claim 20 or 21, the method comprising transfecting or transducing an immunoresponsive cell with one or more polynucleotides according to claim 17 or one or more vectors according to claim 18. A method for directing a T cell-mediated immune response to a target cell or treating cancer in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of immunoresponsive cells according to claim 20 or 21. A CCR polypeptide according to any one of claims 1-11, a trimeric CCR according to claim 12, a lateral CAR according to any one of claims 13-16, one or more polynucleotides according to claim 17, one or more vectors according to claim 18, a host cell according to claim 19, an immunoresponsive cell according to claim 20 or 21 or a pharmaceutical composition according to claim 22 for use (i) in therapy or as a medicament or (ii) in the treatment of cancer.
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