WO2015063069A1 - Chimeric antigen receptors with antigen binding domains derived from gamma delta t cell receptors - Google Patents

Chimeric antigen receptors with antigen binding domains derived from gamma delta t cell receptors Download PDF

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WO2015063069A1
WO2015063069A1 PCT/EP2014/073087 EP2014073087W WO2015063069A1 WO 2015063069 A1 WO2015063069 A1 WO 2015063069A1 EP 2014073087 W EP2014073087 W EP 2014073087W WO 2015063069 A1 WO2015063069 A1 WO 2015063069A1
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amino acid
acid sequence
seq
protein
cells
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PCT/EP2014/073087
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French (fr)
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Benjamin Felder
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Benjamin Felder
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    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the present invention relates to engineered membrane proteins comprising: (i) a signal peptide, (ii) an extracellular antigen recognition domain, derived from the variable domains of ⁇ T cell receptor chains (iii) an (optional) spacer region (iv) a transmembrane region and (v) an intracellular effector domain.
  • the present invention furthermore relates to nucleic acids encoding the proteins, expression constructs for expressing the protein in a host cell and host cells.
  • the proteins of the invention are chimeric antigen receptors, that due to their antigen binding region allow to redirect the effector functions of immune cells against a broad spectrum of cancer or virally infected cells.
  • the chimeric antigen receptors of the invention can be used to generate therapeutic cells, suitable for the immunological treatment of malignant or viral disease.
  • the present invention also relates to methods for generating antigen-specific effector cells as well as to methods for the treatment of diseases, in particular cancer, and methods of immunotherapy, preferably including adoptive, target-cell specific immunotherapy.
  • T cell based therapies currently limited due to a variety of factors, including the lack of suitable tumor antigens, the tolerance against tumor antigens, the small repertoire of endogenous tumor antigen specific T cells, and a variety of evasion mechanisms cancer cells employ to prevent their immunological removal (for reviews see Topfer et al., 2011; Capietto et al., 2011). The importance of these limiting factors is exemplified by the unconvincing clinical efficacy of therapeutic cancer vaccines up to date (Huber et al., 2012).
  • Synthetic biology offers new ways to improve the immunologic recognition and elimination of cancer cells.
  • One promising strategy involves the transduction of polyclonal T cells with DNA constructs, encoding engineered membrane receptors that couple the binding of an antigen specific targeting molecule, usually a single-chain variable fragment (scFv), to the delivery of a tailored T cell activating signal.
  • scFv single-chain variable fragment
  • Such artificial T cell receptors due to their composite make-up, commonly referred to as chimeric antigen receptors (CARs), endow their host cells with a predefined antigen specificity (for a review see Sadelain at al., 2013). Thereby the barriers and incremental kinetics of immunization can be bypassed and an immune reaction raised against any surface antigen on target cells in an MHC-independent manner.
  • CAR T cells gain the ability to overcome tumor immune suppression (Loskog et al., 2006). Because T cells are capable of proliferation, the adoptive transfer of tumor-reactive T cells has the additional advantage of an amplification of the anti-tumor response in vivo.
  • the CAR approach is shown to be functionally superior to antibody-dependent cell-mediated cytotoxicity (ADCC), especially within the context of the tumor microenvironment where exogenously introduced anti-tumor antibodies show only weak penetration and short persistence (Boissel et al., 2013). Those key properties single the CAR approach out to provide effective cancer therapeutics.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic T cells can be grouped into two distinct subpopulations. While 95% of human CD8 cells express a receptor constituted of a and ⁇ subunits, a small proportion express an alternate receptor consisting of ⁇ and ⁇ chains. Short protein fragments presented by MHC complex on the cell surface are the antigens recognized by ⁇ T cells. In contrast, ⁇ T cells possess a restricted T cell receptor diversity, recognizing a variety of non-protein antigens (for a review see Carding and Egan., 2002). One prominent example is the recognition of microbial phosphoantigens like isopentenyl pyrophosphate (IPP) by Vy9/V52 T cells.
  • IPP isopentenyl pyrophosphate
  • IPP is also displayed on the surface of many cancer cells but not normal cells, a circumstance thought to be caused by metabolic aberrations which are common in transformation (Kabelitz et al., 2004). Indeed Marcu-Malina et al. (2011) demonstrated the interesting ability of Vy9/V82 TCR transduced lymphocytes to specifically lyse 17 of 20 cancer cell lines, while untransformed cells remained untouched. This capability of Vy9/V62 T cells to discern a broad spectrum of cancer cells over their healthy counterparts is remarkable in so far that universal cancer-specific antigens otherwise remain at large. Therefore any immunological cancer therapy directed against IPP as an antigen may be suitable for a very large number of patients, including those for whom no effective treatment options exist today.
  • WO 00/31239 describes immune cells having a predefined specificity, wherein the cell is complexed either with an antigen-specific MHC-restricted chimeric T cell receptor or is transfected with an antigen-specific MHC-restricted chimeric TCR gene.
  • WO 2013/147606 describes Vy9/V52 T cell receptor chains with enhanced affinity and target recognition, due to point mutations in their CDR sequences.
  • Zheng et al. (2013) describe soluble TCRs, derived from a fusion of Yy9 and V52 variable domains to an Fc antibody fragment.
  • the fusion protein is shown to couple the ability of the ⁇ -TCR to recognize many cancer cell lines with the ability of the Fc fragment to induce antibody dependent cytotoxicity.
  • the present invention aims to use CAR technology for targeting the IPP antigen, in order to raise potent immune responses against a broad-spectrum of cancer cells.
  • proteins comprising:
  • an antigen recognition domain which is derived from the variable domains of Vy9 V52 T cell receptors (TCRs), (iii) optionally, a spacer region, connecting domain (ii) and domain (iv),
  • nucleic acids encoding the proteins of the present invention.
  • this objective is solved by an expression construct for expressing the protein of the present invention in a cell.
  • this object is solved by a host cell expressing a protein of the present invention or comprising a nucleic acid of the present invention or an expression construct of the present invention.
  • this object is solved by using a protein of the present invention, a nucleic acid of the present invention or an expression construct of the present invention for generating target-specific effector cells.
  • this object is solved by providing the protein of the present invention, the nucleic acid of the present invention, the expression construct of the present invention or the host cell of the present invention for use as a medicament.
  • this object is solved by providing the protein of the present invention, the nucleic acid of the present invention, the expression construct of the present invention or the host cell of the present invention for use in the treatment of cancer or for use in adoptive, target-cell specific immunotherapy.
  • lymphocytes including but not limited to cytotoxic lymphocytes, T cells, cytotoxic T cells, T helper cells, Thl7 T cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages, mast cells, dendritic cells, killer dendritic cells, B cells;
  • lymphocytes including but not limited to cytotoxic lymphocytes, T cells, cytotoxic T cells, T helper cells, Thl7 T cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages, mast cells, dendritic cells, killer dendritic cells, B cells;
  • step (c) transferring the protein, nucleic acid, or expression construct provided in step (a) into the host cell or cell line provided in step (b);
  • this object is solved by a method for the treatment of diseases, in particular cancer, comprising the step of
  • a protein of the present invention a nucleic acid of the present invention, an expression construct of the present invention, a host cell of the present invention or an antigen-specific effector cell as obtained according to the method for generating antigen-specific effector cells, and (b) optionally, respective excipient(s).
  • a protein of the present invention a nucleic acid of the present invention, an expression construct of the present invention, a host cell of the present invention or an antigen-specific effector cell as obtained according to the method for generating antigen-specific effector cells, and (b) optionally, respective excipient(s).
  • the present invention provides proteins, in particular chimeric antigen receptors, whose antigen binding domains are derived from endogenous Vy9/V52 TCRs.
  • the proteins of the present invention comprise several domains:
  • TCRs Vy9/V52 T cell receptors
  • the proteins of the invention are preferably cell surface receptor proteins and, thus, comprise:
  • the functionality of the proteins of the invention within a host cell is detectable in an assay suitable for demonstrating the signaling potential of said protein upon binding of a particular ligand. Such assays are available to the skilled artisan.
  • chimeric antigen receptors Upon binding to the target, such chimeric antigen receptors link to endogenous signaling pathways in a cell (an effector cell) and generate certain activating signals (depending on the effector domain).
  • CAR chimeric antigen receptors
  • target specificity such as target-cell specificity
  • NK natural killer
  • CARs generates antigen-specific effector cells for the use in adoptive, target-cell specific immunotherapy.
  • CARs are composed of a target or antigen specific recognition domain or cell recognition domain (domain (ii), such as an scFv antibody fragment) for recognition of an antigen or a target (such as a tumor-cell surface antigen) fused via a flexible spacer region to a transmembrane domain and an effector domain comprising one or more intracellular signaling domains (like the zeta-chain of the CD3 complex of the T- cell receptor).
  • CAR expression retargets the cytotoxic activity of the effector cells (lymphocytes) to antigens/targets (tumor cells) and triggers their cytolysis by the CAR expressing immune effector cells.
  • binding of the antigen / target specific recognition domain of the CAR to its cognate antigen/target on the surface of target cells/viruses transmits a signal into the CAR expressing immune effector cells via the intracellular signaling domain(s) of the CAR which activates the endogenous cytotoxic activity of such immune effector cells.
  • a “signal peptide” refers to a short peptide sequence that destines a protein to a specific location within the cell.
  • the signal peptide of the invention can be any signal peptide of a human secreted or type I transmembrane protein, enabling the correct localization of the CARs of the invention to the cell membrane, in particular the extracellular portion (domains)
  • transmembrane portion (iv) inserted into the plasma membrane and the cytoplasmic portion (v) within the host cell.
  • the signal peptide is cleavable and thereby eliminated before the CAR reaches the self-surface.
  • the signal peptide (i) comprises or is derived from human Alpha- 1 Antitrypsin signal peptide (liAAT) (uniprot accession P01009, amino acid residues 1-24) [SEQ ID NO. 1]:
  • the antigen recognition or binding domain (ii) binds the phosphoantigen IPP on the surface of a target cell.
  • the antigen binding region is understood to be essential for the functionality of the proteins of the invention.
  • antigen recognition domain refers to domain (ii) of the proteins of the invention.
  • the antigen recognition domain is derived from the variable domains of Vy9 V52 T cell receptors (TCRs).
  • TCRs T cell receptors
  • the target is a cell or a virus, more preferably a malignant or a virally infected cell.
  • the antigen recognition domain (ii) comprises or consists of
  • variable domain of Vy9 comprises or consists of an amino acid sequence of SEQ ID NO. 3 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 3.
  • variable domain of V62 comprises or consists of an amino acid sequence of SEQ ID NO. 4 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 4.
  • the antigen recognition domain (ii) comprises the variable domain of Vy9 and the variable domain of V52, which are covalently coupled or fused to each other.
  • variable domain of Vy9 and V62 is via a (flexible) linker (L) yielding a single chain, wherein the variable d domains are either positioned in the sequence of from N- to C-terminus:
  • the orientation is Vy9-L-V52.
  • a preferred linker comprises or consists of the amino acid sequence of SEQ ID NO. 2:
  • the protein of the present invention (in particular the domain (ii) thereof) comprises an amino acid sequence of SEQ ID NO. 5 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 5.
  • the antigen recognition domain (ii) comprises the variable domain of Vy9 and the variable domain of V52, which are not covalently coupled or fused to each other.
  • variable domain of Vy9 and the variable domain of V52 are preferably co-expressed in the host cell.
  • the protein of the present invention comprises two protein chains preferably not covalently coupled to each other and/or each comprising
  • TCRs Vy9 V52 T cell receptors
  • the antigen recognition domain (ii) of one protein chain comprises or consists of the variable domain of Vy9 and the antigen recognition domain (ii) of the other protein chain comprises or consists of the variable domain of V62.
  • the target specific recognition domain serves for the targeting of the protein of the present invention or a respective cell expressing/carrying the protein of the present invention on its surface to a specific target. Binding of the target specific recognition domain of the protein of the present invention (CAR) to its cognate target on the surface of target cells/viruses furthermore transmits a signal into the protein (CAR)-expressing immune effector cells via the intracellular signaling domain(s) of the protein of the present invention which activates the endogenous cytotoxic activity of such immune effector cells.
  • CAR target specific recognition domain of the protein of the present invention
  • a CAR comprising Vy9 (a) and a CAR comprising V62 can be co-expressed within the same host cell.
  • variable domains are mutated at key residues to enhance their stability, this might allow to omit T cell receptor constant regions as a stabilizing element.
  • the antigen recognition domain (ii) comprises one or more CDR sequences derived from the variable domains of Vy9 and/or V62, or an amino acid sequence that has at least 70%, preferably 80% or more preferably 90% or 95% sequence identity to said sequences.
  • the CDR sequences comprise or consist of
  • the antigen recognition domain (ii) may comprise or consist of CDR sequences derived from the variable domains of Vy9 and/or V52 grafted onto protein scaffolds. These scaffolds can be distinct from the native variable domains the CDR sequences are derived from. Any protein which allows to exchange certain unstructured portions, while maintaining an overall stable tertiary structure may be suitable as a scaffold. Examples of such scaffolds which might be suitable to accept CDR sequences as grafts are variable domains of immunoglobulin or the extracellular type III domains of fibronectin.
  • the spacer region (iii) connects the antigen recognition domain (ii) and the transmembrane domain (iv).
  • the spacer region serves as a flexible link between the antigen recognition domain (ii) and the transmembrane domain (iv) and the effector domain (v). It ensures the necessary accessibility and flexibility of the antigen recognition domain (ii).
  • the spacer region (iii) comprises or consists of the constant domains of any TCR chain, in order to enhance the stability and surface expression of the variable domains comprises in the antigen recognition domain (ii).
  • the spacer region (iii) comprises constant domain(s) of a T cell receptor.
  • said constant domain(s) of a T cell receptor are selected from Cy, C5, Ca and/or Cp.
  • the constant domain Gy comprises an amino acid sequence of SEQ ID NO. 6 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 6;
  • the constant domain C6 comprises an amino acid sequence of SEQ ID NO. 7 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 7;
  • the constant domain Ca comprises an amino acid sequence of SEQ ID NO. 8 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 8; and/or
  • the constant domain ⁇ comprises an amino acid sequence of SEQ ID NO. 9 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 9.
  • a transmembrane domain comprises a stretch of hydrophobic amino acids which by virtue of its hydrophobicity can thermodynamically stable integrate into the cell membrane.
  • the transmembrane domain (iv) within the protein of the invention anchors it to the cell membrane, whereby antigen binding (ii) and the spacer domains (iii) are positioned extracellularly and the effector domain (v) on the cytoplasmatic site.
  • the transmembrane domain (iv) comprises or consists of the transmembrane domain of human CD28, preferably comprising an amino acid sequence of SEQ ID NO. 10 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 10. human CD28.
  • the effector domain (v) connects to the C-terminus of the transmembrane domain (iv).
  • the effector domain (v) comprises one or more intracellular signaling domains and activates the intracellular signalling machinery upon antigen binding, activating the cytotoxic/effector function of the host cell and/or promoting its proliferation and/or cytokine secretion.
  • the at least one effector domain (v) of the protein of the invention preferably comprises endodomains/domain(s) of at least one of the following:
  • CD3 polypeptides ⁇ , ⁇ , ⁇
  • FceRIy B29
  • CD28 such as CD28, OX40 (CD134), 4-1BB (CD137).
  • At least one effector domain (v) that comprises or consists of (is):
  • the LL motif of the CD28 fragment is inactivated by mutation (such as to a diglycine motif, other functional similar mutations are possible as well) to enhance protein stability and surface of expression of the protein of the invention.
  • the respective CD28 fragment preferably comprises or consists of the amino acid sequence of SEQ ID NO. 13, or a functional equivalent thereof;
  • the term "functional equivalent” defines a protein or nucleotide sequence, having a different amino acid or base sequence, compared to the sequences disclosed herein, but exhibiting the same function in vitro and in vivo.
  • An example of a functional equivalent is a modified or synthetic gene, encoding the expression of a protein identical or highly homologous to that encoded by the wildtype gene or a sequence disclosed herein.
  • the effector domain (v) (preferably) comprises or consists of (is) an amino acid sequence with the amino acid sequence of SEQ ID NO. 15 or a functional equivalent thereof, wherein a "functional equivalent” has less sequence identity (such as at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity or 99% sequence identity) but is a functional fusion of the costimulatory CD28 receptor (diglycine motif mutated) fused to a fragment of the zeta-chain of the CD3 complex of the T- cell receptor fused to a fragment of the costimulatory OX40 receptor.
  • a "functional equivalent” has less sequence identity (such as at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity or 99% sequence identity) but is a functional fusion of the costimulatory CD28 receptor (diglycine motif mutated) fused to a fragment of the zeta-chain of the CD3 complex of the T- cell receptor fused to a fragment
  • the according to the invention comprises or consists of the amino acid sequence of:
  • an antigen binding domain that comprises or consists of (is) a variable domain of a Vy9-L-V82 (SEQ ID NO. 5)
  • a spacer region preferably comprising a constant region of a T cell receptor (SEQ ID NOs. 6 to 9)
  • transmembrane domain as defined herein, preferably of SEQ ID NO. 10
  • the protein comprises or consists of the amino acid sequence of SEQ ID NO. 16; or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to SEQ ID NO. 16.
  • amino acid sequence of SEQ ID NO. 16 refers to the amino acid sequence of the multifunctional protein with the domains:
  • the protein comprises or consists of the amino acid sequence of SEQ ID NO. 17; or an amino acid sequence that has at least 70%) sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to SEQ ID NO. 17.
  • amino acid sequence of SEQ ID NO. 17 refers to the amino acid sequence of the multifunctional protein with the domains:
  • the protein comprises or consists of the amino acid sequence of SEQ ID NO. 18; or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to SEQ ID NO. 18.
  • amino acid sequence of SEQ ID NO. 18 refers to the amino acid sequence of the multifunctional protein with the domains:
  • the protein of the present invention comprises or consists of the amino acid sequence of any of SEQ ID NOs. 16 to 18 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% or 99% sequence identity to the amino acid sequence of any of SEQ ID NOs. 16 to 18.
  • the present invention provides nucleic acids/nucleic acid molecules/isolated nucleic acid molecules encoding the proteins of the invention.
  • the nucleic acids according to this invention comprise DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA), combinations thereof or derivatives (such as PNA) thereof.
  • a nucleic acid of the invention comprises or consists of the nucleic acid encoding for the amino acid sequence of SEQ ID NO. 16,
  • nucleic acid sequence of SEQ ID NO. 19 or comprises or consists of the nucleic acid sequence of SEQ ID NO. 19 or their complementary sequences or sequences that have at least 95 % sequence identity.
  • a nucleic acid of the invention comprises or consists of the nucleic acid encoding for the amino acid sequence of SEQ ID NO. 17,
  • nucleic acid sequence of SEQ ID NO. 20 or comprises or consists of the nucleic acid sequence of SEQ ID NO. 20 or their complementary sequences or sequences that have at least 95 % sequence identity.
  • a nucleic acid of the invention comprises or consists of the nucleic acid encoding for the amino acid sequence of SEQ ID NO. 18,
  • nucleic acid sequence of SEQ ID NO. 21 or their complementary sequences or sequences that have at least 95 % sequence identity.
  • nucleic acids comprising or consisting of nucleic acid sequences of SEQ ID NOs. 16 and 17 (or respective sequences with at least 90% identity) are for co-expression in a host cell (to yield a functional receptor/CAR).
  • the nucleic acid sequences of the present invention are codon-optimized for expression in mammalian cells, preferably for expression in human cells.
  • Codon-optimization refers to the exchange in a sequence of interest of codons that are generally rare in highly expressed genes of a given species by codons that are generally frequent in highly expressed genes of such species,- such codons encoding the same amino acids as the codons that are being exchanged.
  • nucleotide sequences obtained due to the degeneration of the genetic code of the above nucleotide sequences are also the nucleotide sequences obtained due to the degeneration of the genetic code of the above nucleotide sequences.
  • the present invention provides expression constructs for expressing the protein of the invention in a cell.
  • the expression constructs further comprise promoter and terminator sequences.
  • An "expression construct” or “gene construct” refers to a nucleic acid construct, usually an expression vector or plasmid, that is used to introduce a specific gene sequence into a target cell. Once the expression or gene construct is inside the cell, the protein that is encoded by the gene is produced by the cellular transcription and translation macliinery.
  • the expression or gene construct is designed to contain respective regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene earned on the construct, including promoter and terminator sequences. The goal of a well-designed expression or gene construct is the production of suitable amounts of stable mRNA, and therefore proteins.
  • nucleic acids and/or in particulai- expression constructs of the invention are capable of directing the synthesis/expression of the engineered membrane proteins of the invention.
  • nucleic acids and/or expression constructs of the invention are dsDNA, ssD A, RNA or mRNA or combinations thereof.
  • the present invention provides host cells which express a protein of the invention or which comprise a nucleic acid or an expression construct of the invention.
  • the host cell is selected from effector cells of the immune system, such as lymphocytes including but not limited to cytotoxic lymphocytes, T cells, cytotoxic T cells, T helper cells, Thl7 T cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, neutrophils, macrophages, dendritic cells, killer dendritic cells, B cells.
  • lymphocytes including but not limited to cytotoxic lymphocytes, T cells, cytotoxic T cells, T helper cells, Thl7 T cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, neutrophils, macrophages, dendritic cells, killer dendritic cells, B cells.
  • Effective cells of the immune system or “immune effector cells” refers to cells of hematopoietic origin including but not limited to the cell types mentioned above that are functionally involved in the initiation and/or execution of innate and/or adaptive immune responses.
  • the protein of the invention comprises only the variable domain Vy9 or V52 as antigen binding domain (ii),
  • the host cell co-expresses two proteins of the invention or comprises two nucleic acids or expression constructs of the invention
  • first protein comprising the variable domain Vy9 as antigen binding domain (ii) and a second protein comprising the variable domain V82 as antigen binding domain (ii), respective nucleic acids/expression constructs of said two proteins,
  • proteins comprising or consisting of the amino acid sequences of SEQ ID NOs. 16 and 17 (or respective sequences with at least 90% identity), or nucleic acids comprising or consisting of the nucleic acid sequences of SEQ ID NOs. 19 and 20 (or respective sequences with at least 90% identity).
  • the invention provides the use of the protein, nucleic acid, or expression construct of the present invention for generating antigen-specific effector cells.
  • Antigen-specific effector cells or “target-specific effector cells” refer to effector cells of the immune system or immune effector cells genetically modified to express the multi-functional protein of the invention by transfer of an expression construct or nucleic acid encoding said multi-functional protein.
  • Such antigen-specific or target-specific effector cells are versatile means, in particular in the treatment of diseases (as described below for ACT and cancer treatment).
  • the invention provides the protein, nucleic acid, expression construct or host cell for use as a medicament.
  • the invention provides the protein, nucleic acid, expression construct or host cell for use in the treatment of cancer.
  • the invention provides the protein, nucleic acid, expression construct or host cell for use in adoptive, target-cell specific immunotherapy.
  • “Adoptive, target-cell specific immunotherapy” refers to a form of therapy in which immune cells are transferred to tumor-bearing hosts.
  • the immune cells have antitumor reactivity and can mediate direct or indirect antitumor effects.
  • ACT adoptive cell therapy
  • immune effector cells such as lymphocytes with anti-tumour activity
  • lymphocytes with anti-tumour activity expanded in vitro and infused into the patient with cancer.
  • ACT using autologous tumour- infiltrating lymphocytes has emerged as the most effective treatment for patients with metastatic melanoma and can mediate objective cancer regression in approximately 50% of patients.
  • the use of donor lymphocytes for ACT is an effective treatment for immunosuppressed patients who develop post-transplant lymphomas (reviewed in Rosenberg et al., 2008).
  • the invention provides methods for generating antigen-specific effector cells.
  • lymphocytes including but not limited to cytotoxic lymphocytes, T cells, cytotoxic T cells, T helper cells, Thl7 T cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages, mast cells, dendritic cells, killer dendritic cells, B cells;
  • lymphocytes including but not limited to cytotoxic lymphocytes, T cells, cytotoxic T cells, T helper cells, Thl7 T cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages, mast cells, dendritic cells, killer dendritic cells, B cells;
  • step (c) transferring the protein, nucleic acid, or expression construct provided in step (a) into the host cell or cell line provided in step (b);
  • the present invention also provides methods for the treatment of diseases, in particular cancer, and methods of immunotherapy, preferably including adoptive, target-cell specific immunotherapy.
  • the method for the treatment of diseases, in particular cancer comprises the step of administering to a subject in need thereof a therapeutically effective amount of (a) a protein, a nucleic acid, an expression construct or a host cell (in particular an antigen-specific effector cell) as obtained and defined herein, and (b) optionally, respective excipient(s).
  • the method of immunotherapy preferably including or utilizing adoptive, target-cell specific immunotherapy, according to the present invention comprises the step of administering to a subject in need thereof a therapeutically effective amount of (a) a protein, a nucleic acid, an expression construct or a host cell (in particular an antigen-specific effector cell) as obtained and defined herein, and (b) optionally, respective excipient(s).
  • a therapeutically effective amount of a protein, a nucleic acid, an expression construct or a host cell (in particular an antigen-specific effector cell) of this invention refers to the amount that is sufficient to treat the respective disease or achieve the respective outcome of the adoptive, target-cell specific immunotherapy.
  • FIG. 1 Schematic structure of the expression vectors for single-chain constructs.
  • V - promoter SP - signal peptide
  • V - variable domain Vy9 or V52
  • L - linker C - constant domain
  • TM - transmembrane domain TM - transmembrane domain
  • ED - effector domain pA - polyadenylation signal (terminator)
  • ABR - antigen binding region V - promoter, SP - signal peptide, V - variable domain (Vy9 or V52), L - linker, C - constant domain, TM - transmembrane domain, ED - effector domain, pA - polyadenylation signal (terminator), ABR - antigen binding region
  • Figure 2 Dotplot representation of flow -cytometric CAR expression analysis.
  • NC-E - only T cells NC-T only target cell
  • mock Vy9/V52 coexpression of both Yj9 and
  • V52 comprising CARs, sc_Vy9V62-expression of CAR with fusion ABR
  • Vy9, V52 and single-chain Vy9V62 mock CARs, codon optimized for human cells, were synthesized by Life Teclmologies (GeneArt® StringsTM DNA Fragments).
  • V62 eIF4G promoter- 5'-UTR-V52-C6-CD28 TM-CD28-CD3zeta-OX40-3'UTR-pA
  • SC_VY9V52 eIF4G promoter- 5*-UTR-SP-VY9-L-V52-C6-CD28 TM-CD28-CD3zeta-OX40- 3'UTR-pA (Fig. 1 B).
  • the mock construct omitted the antigen binding region, yielding the construct:
  • Linear DNA templates were circularized via digestion EcoRI (Biozym, Germany) and subsequent incubation with T4 DNA ligase (Biozym, Germany).
  • 10 ng of circular template was denatured at 95° C for 3 min and cooled to room temperature.
  • the template solution was then added to 1 ml of amplification solution containing phi29 DNA polymerase (10 U, Biozym, Germany), 0,2 mM dNTPs, 100 ig BSA in 50 mM Tri-HCl pH 7,5, 10 mM MgCl, 10 mM (NH) SO, DTT.
  • T cells were transduced with the amplified nucleic acid mock, Vy9, V62, dual Vy9/V2 and the single-chain construct (sc_Vy9V82).
  • sc_Vy9V82 the amplified nucleic acid mock, Vy9, V62, dual Vy9/V2 and the single-chain construct (sc_Vy9V82).
  • phosphate buffered 1 ml Leibovitz LI 5 medium Sigma- Aldrich, Germany
  • 100 g/ml ampicillin Sigma-Aldrich, Germany.
  • GenePORTER 2 transfection reagent BioCat, Heidelberg, Germany was used for cell transfection as directed by the manufacturer.
  • Target cells were provided in 96 well plates with 100,000 cells per well in Leibovitz L15 medium (150 ⁇ ), containing pamidronate (10 ⁇ , Sigma-Aldrich, Germany) and ampicillin (100 ⁇ g/ml).
  • the cytotoxicity of Vy9/V62 is generally higher than that of sc_Vy9 V62, potentially owing to a more native protein configuration. No cytotoxicity of Vy9/V62 and the sc_Vy9V52 can be detected against untransformed PBMC and CD 19+ cells.
  • SEQ ID NO. 1 shows the amino acid sequence of the signal peptide of human Alpha- 1 Antitrypsin
  • SEQ ID NO. 2 shows the amino acid sequence of the preferred linker sequence.
  • SEQ ID NO. 3 shows the amino acid sequence of the variable domain human Vy9 T cell receptor chain.
  • SEQ ID NO. 4 shows the amino acid sequence of the variable domain human V52 T cell receptor chain.
  • SEQ ID NO. 5 shows the amino acid sequence of the fusion of Vy9 to V62, via the linker described under SEQ ID NO. 2.
  • SEQ ID NO. 6 shows the amino acid sequence of the constant domain of the human ⁇ T cell receptor chain.
  • SEQ ID NO. 7 shows the amino acid sequence of the constant domain of the human ⁇ T cell receptor chain.
  • SEQ ID NO. 8 shows the amino acid sequence of the constant domain of the human a T cell receptor chain (uniprot accession number: P01848).
  • SEQ ID NO. 9 shows the amino acid sequence of the constant domain of the human ⁇ T cell receptor chain (uniprot accession number: P01850).
  • SEQ ID NO. 10 shows the amino acid sequence of the transmembrane domain of human CD28 (uniprot accession number: P01850, amino acid residues: 153-179).
  • SEQ ID NO. 11 shows the amino acid sequence of the intracellular domain of human T-cell surface glycoprotein CD3 zeta chain (UniProt accession number P20963-1 (CD3Z_HUMAN); Isoform 1, (amino acid residues 52-164)).
  • SEQ ID NO. 12 shows the amino acid sequence of the intracellular domain of human CD28 costimulatory receptor (UniProt accession number PI 0747-1 (CD28_HUMAN), isoform 1, (amino acid residues 180-220)).
  • SEQ ID NO. 13 shows the amino acid sequence described under SEQ ID NO. 12 wherein the diglycine motif has been inactivated by mutation.
  • SEQ ID NO. 14 shows the amino acid sequence of the intracellular domain of the human costimulatory OX40 receptor (amino acid residues 236-277]) (UniProt accession number P43489 (TN-FRSF4JHUMAN)).
  • SEQ ID NO. 15 shows the amino acid sequence of the effector domain comprising a fusion of CD28 (SEQ ID NO. 13), CD3 zeta (SEQ ID NO. 11) and OX40 (SEQ ID NO. 14), in the mentioned order.
  • SEQ ID NO. 16 shows the amino acid sequence of the multifunctional protein with the domains:
  • SEQ ID NO. 17 shows the amino acid sequence of the multifunctional protein with the domains:
  • SEQ ED NO. 18 shows the amino acid sequence of the multifunctional protein with the domains:
  • SEQ ID NO. 19 shows the nucleotide sequence encoding for the multi-functional protein with the domains:
  • SEQ ID NO. 20 shows the nucleotide sequence encoding for the multi-functional protein with the domains:
  • SEQ ID NO. 21 shows the nucleotide sequence encoding for the multi-functional protein with the domains:
  • SEQ ID NO. 22 shows the amino acid sequence of the CDR3 region of Vy9.
  • SEQ ID NO. 23 shows the amino acid sequence of the CDR3 region of V52.

Abstract

The present invention relates to engineered membrane proteins comprising: (i) a signal peptide, (ii) an extracellular antigen recognition domain, derived from the variable domains of γδ T cell receptor chains (iii) an (optional) spacer region (iv) a transmembrane region and (v) an intracellular effector domain. The present invention furthermore relates to nucleic acids encoding the proteins, expression constructs for expressing the protein in a host cell and host cells. The proteins of the invention are chimeric antigen receptors, that due to their antigen binding region allow to redirect the effector functions of immune cells against a broad spectrum of cancer or virally infected cells. Hence the chimeric antigen receptors of the invention can be used to generate therapeutic cells, suitable for the immunological treatment of malignant or viral disease. The present invention also relates to methods for generating antigen-specific effector cells as well as to methods for the treatment of diseases, in particular, cancer, and methods of immunotherapy, preferably including adoptive, target-cell specific immunotherapy.

Description

Chimeric antigen receptors with antigen binding domains derived from gamma delta T cell receptors
The present invention relates to engineered membrane proteins comprising: (i) a signal peptide, (ii) an extracellular antigen recognition domain, derived from the variable domains of γδ T cell receptor chains (iii) an (optional) spacer region (iv) a transmembrane region and (v) an intracellular effector domain. The present invention furthermore relates to nucleic acids encoding the proteins, expression constructs for expressing the protein in a host cell and host cells. The proteins of the invention are chimeric antigen receptors, that due to their antigen binding region allow to redirect the effector functions of immune cells against a broad spectrum of cancer or virally infected cells. Hence the chimeric antigen receptors of the invention can be used to generate therapeutic cells, suitable for the immunological treatment of malignant or viral disease. The present invention also relates to methods for generating antigen-specific effector cells as well as to methods for the treatment of diseases, in particular cancer, and methods of immunotherapy, preferably including adoptive, target-cell specific immunotherapy.
BACKGROUND OF THE INVENTION
The prowess of T cells for antigen-specific cell-removal makes them a promising venture for new treatments against cancer and viral disease. Nonetheless is the success of T cell based therapies, currently limited due to a variety of factors, including the lack of suitable tumor antigens, the tolerance against tumor antigens, the small repertoire of endogenous tumor antigen specific T cells, and a variety of evasion mechanisms cancer cells employ to prevent their immunological removal (for reviews see Topfer et al., 2011; Capietto et al., 2011). The importance of these limiting factors is exemplified by the unconvincing clinical efficacy of therapeutic cancer vaccines up to date (Huber et al., 2012).
Synthetic biology offers new ways to improve the immunologic recognition and elimination of cancer cells. One promising strategy involves the transduction of polyclonal T cells with DNA constructs, encoding engineered membrane receptors that couple the binding of an antigen specific targeting molecule, usually a single-chain variable fragment (scFv), to the delivery of a tailored T cell activating signal. Such artificial T cell receptors, due to their composite make-up, commonly referred to as chimeric antigen receptors (CARs), endow their host cells with a predefined antigen specificity (for a review see Sadelain at al., 2013). Thereby the barriers and incremental kinetics of immunization can be bypassed and an immune reaction raised against any surface antigen on target cells in an MHC-independent manner. Especially when the signalling domain is extended to provide costimulatory signals, CAR T cells gain the ability to overcome tumor immune suppression (Loskog et al., 2006). Because T cells are capable of proliferation, the adoptive transfer of tumor-reactive T cells has the additional advantage of an amplification of the anti-tumor response in vivo. The CAR approach is shown to be functionally superior to antibody-dependent cell-mediated cytotoxicity (ADCC), especially within the context of the tumor microenvironment where exogenously introduced anti-tumor antibodies show only weak penetration and short persistence (Boissel et al., 2013). Those key properties single the CAR approach out to provide effective cancer therapeutics. Indeed administration of CD19-specific CAR-modified T cells that target B cell non-Hodgkin lymphomas and leukemia has been remarkably effective in recent clinical trials (Grupp et al., 2013; Brentjens et al., 2013). These early successes of CAR therapy raises the hope to enable novel therapies for other cancer indications, too.
Depending on the type of T cell receptor, cytotoxic T cells can be grouped into two distinct subpopulations. While 95% of human CD8 cells express a receptor constituted of a and β subunits, a small proportion express an alternate receptor consisting of γ and δ chains. Short protein fragments presented by MHC complex on the cell surface are the antigens recognized by αβ T cells. In contrast, γδ T cells possess a restricted T cell receptor diversity, recognizing a variety of non-protein antigens (for a review see Carding and Egan., 2002). One prominent example is the recognition of microbial phosphoantigens like isopentenyl pyrophosphate (IPP) by Vy9/V52 T cells. Importantly IPP is also displayed on the surface of many cancer cells but not normal cells, a circumstance thought to be caused by metabolic aberrations which are common in transformation (Kabelitz et al., 2004). Indeed Marcu-Malina et al. (2011) demonstrated the interesting ability of Vy9/V82 TCR transduced lymphocytes to specifically lyse 17 of 20 cancer cell lines, while untransformed cells remained untouched. This capability of Vy9/V62 T cells to discern a broad spectrum of cancer cells over their healthy counterparts is remarkable in so far that universal cancer-specific antigens otherwise remain at large. Therefore any immunological cancer therapy directed against IPP as an antigen may be suitable for a very large number of patients, including those for whom no effective treatment options exist today.
WO 00/31239 describes immune cells having a predefined specificity, wherein the cell is complexed either with an antigen-specific MHC-restricted chimeric T cell receptor or is transfected with an antigen-specific MHC-restricted chimeric TCR gene.
Marcu-Malina et al. (2011) describe the transduction of αβ T cells with a Vy9/V52 T cell receptor clone, enabling them to lyse a broad range of cancer cells specifically over normal cells.
WO 2013/147606 describes Vy9/V52 T cell receptor chains with enhanced affinity and target recognition, due to point mutations in their CDR sequences.
Zheng et al. (2013) describe soluble TCRs, derived from a fusion of Yy9 and V52 variable domains to an Fc antibody fragment. The fusion protein is shown to couple the ability of the γδ-TCR to recognize many cancer cell lines with the ability of the Fc fragment to induce antibody dependent cytotoxicity.
The present invention aims to use CAR technology for targeting the IPP antigen, in order to raise potent immune responses against a broad-spectrum of cancer cells.
It is a further objective of the present invention to provide improved means and methods for generating antigen-specific effector cells as well as means and methods for the use in adoptive, target-cell specific immunotherapy and for treatment of cancer.
SUMMARY OF THE INVENTION
According to the present invention this objective of providing a broad-spectrum anti-cancer immunotherapy is solved by proteins comprising:
(i) a signal peptide;
(ii) an antigen recognition domain, which is derived from the variable domains of Vy9 V52 T cell receptors (TCRs), (iii) optionally, a spacer region, connecting domain (ii) and domain (iv),
(iv) a transmembrane domain,
(v) at least one effector domain comprising one or more intracellular signaling domains.
According to the present invention this objective is solved by nucleic acids encoding the proteins of the present invention.
According to the present invention this objective is solved by an expression construct for expressing the protein of the present invention in a cell.
According to the present invention this object is solved by a host cell expressing a protein of the present invention or comprising a nucleic acid of the present invention or an expression construct of the present invention.
According to the present invention this object is solved by using a protein of the present invention, a nucleic acid of the present invention or an expression construct of the present invention for generating target-specific effector cells.
According to the present invention this object is solved by providing the protein of the present invention, the nucleic acid of the present invention, the expression construct of the present invention or the host cell of the present invention for use as a medicament.
According to the present invention this object is solved by providing the protein of the present invention, the nucleic acid of the present invention, the expression construct of the present invention or the host cell of the present invention for use in the treatment of cancer or for use in adoptive, target-cell specific immunotherapy.
According to the present invention this object is solved by a method for generating antigen- specific effector cells, which comprises the steps of:
(a) providing a protein of the present invention, a nucleic acid of the present invention or an expression construct of the present invention;
(b) providing a host cell or cell line, which is selected from effector cells of the immune system, such as lymphocytes including but not limited to cytotoxic lymphocytes, T cells, cytotoxic T cells, T helper cells, Thl7 T cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages, mast cells, dendritic cells, killer dendritic cells, B cells;
(c) transferring the protein, nucleic acid, or expression construct provided in step (a) into the host cell or cell line provided in step (b);
(d) optional, selection of the transgenic cells.
According to the present invention this object is solved by a method for the treatment of diseases, in particular cancer, comprising the step of
administering to a subject in need thereof a therapeutically effective amount of (a) a protein of the present invention, a nucleic acid of the present invention, an expression construct of the present invention, a host cell of the present invention or an antigen-specific effector cell as obtained according to the method for generating antigen-specific effector cells, and (b) optionally, respective excipient(s).
According to the present invention this object is solved by a method of immunotherapy, comprising the step of
administering to a subject in need thereof a therapeutically effective amount of (a) a protein of the present invention, a nucleic acid of the present invention, an expression construct of the present invention, a host cell of the present invention or an antigen-specific effector cell as obtained according to the method for generating antigen-specific effector cells, and (b) optionally, respective excipient(s).
DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
Before the present invention is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. For the purpose of the present invention, all references cited herein are incorporated by reference in their entireties.
CARs derived fi'om yd ICRs
As described above, the present invention provides proteins, in particular chimeric antigen receptors, whose antigen binding domains are derived from endogenous Vy9/V52 TCRs.
The proteins of the present invention comprise several domains:
(i) a signal peptide;
(ii) an antigen recognition domain;
which is derived from the variable domains of Vy9/V52 T cell receptors (TCRs),
(iii) optionally, a spacer region, connecting domain (ii) and domain (iv),
(iv) a transmembrane domain,
(v) at least one effector domain comprising one or more intracellular signaling domains.
The proteins of the invention are preferably cell surface receptor proteins and, thus, comprise:
- an extracellular portion (domains (i) and (ii) and, optionally, (iii)),
- a transmembrane portion (transmembrane domain (iv)) and
- a cytoplasmic portion (domain (v)),
and can thus be inserted into the plasma membrane of the host cell. The functionality of the proteins of the invention within a host cell is detectable in an assay suitable for demonstrating the signaling potential of said protein upon binding of a particular ligand. Such assays are available to the skilled artisan.
Upon binding to the target, such chimeric antigen receptors link to endogenous signaling pathways in a cell (an effector cell) and generate certain activating signals (depending on the effector domain). The expression of chimeric antigen receptors (CAR) with defined target specificity (such as target-cell specificity) in lymphocytes and other effector cells of the immune system (such as T cells or natural killer (NK) cells) results in genetically modified variants of said cells that selectively target and eliminate defined targets, including but not limited to malignant or virus infected cells displaying the phospho-antigen IPP on their surface.
The expression of CARs generates antigen-specific effector cells for the use in adoptive, target-cell specific immunotherapy. CARs are composed of a target or antigen specific recognition domain or cell recognition domain (domain (ii), such as an scFv antibody fragment) for recognition of an antigen or a target (such as a tumor-cell surface antigen) fused via a flexible spacer region to a transmembrane domain and an effector domain comprising one or more intracellular signaling domains (like the zeta-chain of the CD3 complex of the T- cell receptor). CAR expression retargets the cytotoxic activity of the effector cells (lymphocytes) to antigens/targets (tumor cells) and triggers their cytolysis by the CAR expressing immune effector cells. Thereby binding of the antigen / target specific recognition domain of the CAR to its cognate antigen/target on the surface of target cells/viruses transmits a signal into the CAR expressing immune effector cells via the intracellular signaling domain(s) of the CAR which activates the endogenous cytotoxic activity of such immune effector cells.
(i) the signal peptide
A "signal peptide" refers to a short peptide sequence that destines a protein to a specific location within the cell. The signal peptide of the invention can be any signal peptide of a human secreted or type I transmembrane protein, enabling the correct localization of the CARs of the invention to the cell membrane, in particular the extracellular portion (domains
(i) and (ii) and (iii)) on the cell surface; the transmembrane portion (iv) inserted into the plasma membrane and the cytoplasmic portion (v) within the host cell.
Preferably, the signal peptide is cleavable and thereby eliminated before the CAR reaches the self-surface.
In preferred embodiments, the signal peptide (i) comprises or is derived from human Alpha- 1 Antitrypsin signal peptide (liAAT) (uniprot accession P01009, amino acid residues 1-24) [SEQ ID NO. 1]:
1 PSSVSWGIL LLAGLCCLVP VSLA
(ii) the antigen recognition domain
The antigen recognition or binding domain (ii) binds the phosphoantigen IPP on the surface of a target cell. The antigen binding region is understood to be essential for the functionality of the proteins of the invention.
The terms "antigen recognition domain", "antigen binding domain" and "target specific recognition domain" or "target binding domain" are used interchangeably herein and refer to domain (ii) of the proteins of the invention.
The antigen recognition domain is derived from the variable domains of Vy9 V52 T cell receptors (TCRs). Preferably, the target is a cell or a virus, more preferably a malignant or a virally infected cell.
Preferably, the antigen recognition domain (ii) comprises or consists of
(a) the variable domain of Vy9 of the γδ T cell receptor,
(b) the variable domain of V52 of the γδ T cell receptor,
(c) both the variable domain of Vy9 and the variable domain of V62,
(d) parts of the variable domain of Vy9 and/or the variable domain of V52, such as CDRs.
Preferably, the variable domain of Vy9 comprises or consists of an amino acid sequence of SEQ ID NO. 3 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 3.
Preferably, the variable domain of V62 comprises or consists of an amino acid sequence of SEQ ID NO. 4 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 4.
In one embodiment, the antigen recognition domain (ii) comprises the variable domain of Vy9 and the variable domain of V52, which are covalently coupled or fused to each other.
Preferably, the fusion of the variable domain of Vy9 and V62 is via a (flexible) linker (L) yielding a single chain, wherein the variable d domains are either positioned in the sequence of from N- to C-terminus:
Vy9-L-V52 or V62-L-Vy9.
Preferably the orientation is Vy9-L-V52.
A preferred linker comprises or consists of the amino acid sequence of SEQ ID NO. 2:
1 GSADDAKKDA AKKDGK
Preferably, the protein of the present invention (in particular the domain (ii) thereof) comprises an amino acid sequence of SEQ ID NO. 5 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 5.
(a) The sequence of the variable domain human Vy9 T cell receptor chain [SEQ ID NO. 3]
1 AGHLEQPQIS STKTLSKTAR LECVVSGITI SATSVY YRE RPGEVIQFLV SISYDGTVRK ESGIPSGKFE 71 VDRIPETSTS TLTIHNVEKQ DIATYYCALW EAQQELGKKI KVFGPGTKLI IT
(b) The sequence of the variable domain human V52 T cell receptor chain [SEQ ID NO. 4]
1 MQRISSLIHL SLFWAGVMSA IELVPEHQTV PVSIGVPATL RCS KGEAIG NYYINWYRKT QGNTMTFIYR 71 EKDIYGPGFK DNFQGDIDIA KNLAVLKILA PSERDEGSYY CACDALKRTD TDKLIFGKGT RVTVE
(c) The sequence of a fusion of Vy9 to V62 via a flexible linker (linker underlined)
[SEQ ID NO. 5]
1 AGHLEQPQIS STKTLSKTAR LECVVSGITI SATSVYWYRE RPGEVIQFLV SISYDGTVRK ESGIPSGKFE 71 VDRIPETSTS TLTIHNVEKQ DIATYYCALW EAQQELGKKI KVFGPGTKLI ITGSADDAKK DAAKKDGKMQ 141 RISSLIHLSL FWAGV SAIE LVPEHQTVPV SIGVPATLRC SMKGEAIGNY YINWYRKTQG NT TFIYREK 211 DIYGPGFKDN FQGDIDIAK LAVLKILAPS ERDEGSYYCA CDALKRGGEY TDKLIFGKGT RVTVE
In one embodiment, the antigen recognition domain (ii) comprises the variable domain of Vy9 and the variable domain of V52, which are not covalently coupled or fused to each other.
The variable domain of Vy9 and the variable domain of V52 are preferably co-expressed in the host cell.
In said embodiment, the protein of the present invention comprises two protein chains preferably not covalently coupled to each other and/or each comprising
(ii) an antigen recognition domain;
comprising one domain which is derived from the variable domains of Vy9 V52 T cell receptors (TCRs),
(iii) optionally, a spacer region, connecting domain (ii) and domain (iv),
(iv) a transmembrane domain,
(v) at least one effector domain comprising one or more intracellular signaling domains. In this embodiment, the antigen recognition domain (ii) of one protein chain comprises or consists of the variable domain of Vy9 and the antigen recognition domain (ii) of the other protein chain comprises or consists of the variable domain of V62.
The target specific recognition domain serves for the targeting of the protein of the present invention or a respective cell expressing/carrying the protein of the present invention on its surface to a specific target. Binding of the target specific recognition domain of the protein of the present invention (CAR) to its cognate target on the surface of target cells/viruses furthermore transmits a signal into the protein (CAR)-expressing immune effector cells via the intracellular signaling domain(s) of the protein of the present invention which activates the endogenous cytotoxic activity of such immune effector cells.
As both variable domains are thought to be required for binding ΓΡΡ, in one embodiment a CAR comprising Vy9 (a) and a CAR comprising V62 can be co-expressed within the same host cell.
In one embodiment, the variable domains are mutated at key residues to enhance their stability, this might allow to omit T cell receptor constant regions as a stabilizing element.
In one embodiment, the antigen recognition domain (ii) comprises one or more CDR sequences derived from the variable domains of Vy9 and/or V62, or an amino acid sequence that has at least 70%, preferably 80% or more preferably 90% or 95% sequence identity to said sequences.
Preferably, the CDR sequences comprise or consist of
- the third CDR (CDR3) of Vy9
[SEQ ID NO. 22]
1 ALWEAQQELG KKIKVF and/or
- the third CDR (CDR3) of V62
[SEQ ID NO. 23]
1 ACDTLGMGGE YTDKLI or an amino acid sequence that has at least 70%, preferably 80% or more preferably 90% or 95% sequence identity to said sequences
In one embodiment, the antigen recognition domain (ii) may comprise or consist of CDR sequences derived from the variable domains of Vy9 and/or V52 grafted onto protein scaffolds. These scaffolds can be distinct from the native variable domains the CDR sequences are derived from. Any protein which allows to exchange certain unstructured portions, while maintaining an overall stable tertiary structure may be suitable as a scaffold. Examples of such scaffolds which might be suitable to accept CDR sequences as grafts are variable domains of immunoglobulin or the extracellular type III domains of fibronectin.
(iii) the spacer region
The spacer region (iii) connects the antigen recognition domain (ii) and the transmembrane domain (iv).
The spacer region serves as a flexible link between the antigen recognition domain (ii) and the transmembrane domain (iv) and the effector domain (v). It ensures the necessary accessibility and flexibility of the antigen recognition domain (ii).
In an embodiment, the spacer region (iii) comprises or consists of the constant domains of any TCR chain, in order to enhance the stability and surface expression of the variable domains comprises in the antigen recognition domain (ii).
Preferably, the spacer region (iii) comprises constant domain(s) of a T cell receptor.
In a preferred embodiment, said constant domain(s) of a T cell receptor are selected from Cy, C5, Ca and/or Cp.
Cy (SEQ ID NO. 6)
1 DKQLDADVSP KPTIFLPSIA ETKLQKAGTY LCLLEKFFPD VIKIH EEKK SNTILGSQEG NTMKTNDTYM 71 KFSWLTVPEK SLDKEHRCIV RHENNK GVD QEIIFPPIKT DVITMDPKDN
C6 (SEQ ID NO. 7)
1 SQPHT PSVF VMKNGTNVAC LVKEFYPKDI RINLVSSKKI TEFDPAIVIS PSGKYNAVKL GKYEDSNSVT 71 CSVQHDNKTV HSTDFEVKTD STDHVKPKET ENTKQPSKSC HKPKAIVHTE KVNMMSLTV Ca (uniprot: P01848) (SEQ ID NO. 8)
1 PNIQNPDPAV YQLRDSKSSD KSVCLFTDFD SQTNVSQSKD SDVYITDKTV LDMRSMDFKS NSAVAWSNKS 71 DFACANAFNN SIIPEDTFFP SPESSCDVKL VEKSFETDTN LNFQNLSVIG FRILLLKVAG FNLLMTLRLW 141 SS
Cp (uniprot: P01850) (SEQ ID NO. 9)
1 EDLNKVFPPE VAVFEPSEAE ISHTQKATLV CLATGFFPDH VELSWWVNGK EVHSGVSTDP QPLKEQPALN 71 DSRYCLSSRL RVSATFWQNP RNHFRCQVQF YGLSENDEWT QDRAKPVTQI VSAEA GRAD CGFTSVSYQQ 141 GVLSATILYE ILLGKATLYA VLVSALVLMA MVKR DF
For example,
- the constant domain Gy comprises an amino acid sequence of SEQ ID NO. 6 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 6;
and/or
- the constant domain C6 comprises an amino acid sequence of SEQ ID NO. 7 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 7;
and/or
- the constant domain Ca comprises an amino acid sequence of SEQ ID NO. 8 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 8; and/or
- the constant domain Οβ comprises an amino acid sequence of SEQ ID NO. 9 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 9.
(iv) the transmembrane domain
A transmembrane domain comprises a stretch of hydrophobic amino acids which by virtue of its hydrophobicity can thermodynamically stable integrate into the cell membrane. The transmembrane domain (iv) within the protein of the invention anchors it to the cell membrane, whereby antigen binding (ii) and the spacer domains (iii) are positioned extracellularly and the effector domain (v) on the cytoplasmatic site.
Preferably, the transmembrane domain (iv) comprises or consists of the transmembrane domain of human CD28, preferably comprising an amino acid sequence of SEQ ID NO. 10 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to the amino acid sequence of SEQ ID NO. 10. human CD28.
(uniprot: P01850, residues: 153-179) (SEQ ID NO. 10)
1 F VLVVVGGV LACYSLLVTV AFIIF V
(v) the effector domain
The effector domain (v) connects to the C-terminus of the transmembrane domain (iv).
The effector domain (v) comprises one or more intracellular signaling domains and activates the intracellular signalling machinery upon antigen binding, activating the cytotoxic/effector function of the host cell and/or promoting its proliferation and/or cytokine secretion.
The at least one effector domain (v) of the protein of the invention preferably comprises endodomains/domain(s) of at least one of the following:
- proteins with an ITAM motif (YXXL/IX(6-8)YXXL/I),
such as CD3 polypeptides (δ, ε, ζ), FceRIy, B29;
- proteins with ITSM Motif (TXYXXV/I),
such as CD 150
- proteins with YINM Motif (ΥΓΝΜ),
such as DAP 10;
- immune kinases,
such as syk, ZAP70, lck, csk;
- costimulatory receptors,
such as CD28, OX40 (CD134), 4-1BB (CD137).
Preferably, at least one effector domain (v) that comprises or consists of (is):
(1) the zeta-chain of the human CD3 complex of the T-cell receptor or fragment(s) thereof,
preferably comprising or consisting of the amino acid sequence of SEQ ID NO. 11, or a functional equivalent thereof;
(2) the human costimulatory CD28 receptor or fragment(s) thereof,
preferably comprising or consisting of the amino acid sequence of SEQ ID NO. 12, or a functional equivalent thereof;
In a particularly preferred embodiment, the LL motif of the CD28 fragment is inactivated by mutation (such as to a diglycine motif, other functional similar mutations are possible as well) to enhance protein stability and surface of expression of the protein of the invention. In this embodiment, the respective CD28 fragment preferably comprises or consists of the amino acid sequence of SEQ ID NO. 13, or a functional equivalent thereof;
(3) the human costimulatory OX40 receptor or fragment(s) thereof,
preferably comprising or consisting of the amino acid sequence of SEQ ID NO. 14, or a functional equivalent thereof;
or
a fusion of (2) a fragment of the human costimulatory CD28 receptor, (1) a fragment of the zeta-chain of the human CD3 complex of the T-cell receptor, and (3) a fragment of the human costimulatory OX40 receptor,
preferably a fusion of (1) to (3) in the order (2), (1) to (3) from N- to C-terminus, more preferably comprising or consisting of the amino acid sequence of SEQ ID NO. 15; or a functional equivalent thereof.
The term "functional equivalent" defines a protein or nucleotide sequence, having a different amino acid or base sequence, compared to the sequences disclosed herein, but exhibiting the same function in vitro and in vivo. An example of a functional equivalent is a modified or synthetic gene, encoding the expression of a protein identical or highly homologous to that encoded by the wildtype gene or a sequence disclosed herein.
(a) The sequence of the intracellular domain of human T-cell surface glycoprotein CD3 zeta chain (UniProt accession number P20963-1 (CD3Z_HUMAN); Isoform 1, ' (amino acid residues 52-164))
[SEQ ID NO. 11]
1 RVKFSRSADA PAYQQGQNQL Y ELNLGRRE EYDVLDKRRG RDPEMGGKPQ RRK PQEGLY NELQKDKMAE 71 AYSEIGMKGE RRRGKGHDGL YQGLSTATKD TYDALHMQAL PPR
(b) The sequence of the intracellular domain of human CD28 costimulatory receptor (UniProt accession number PI 0747-1 (CD28_HUMAN), isoform 1, (amino acid residues 180-220)) [SEQ ID NO.12]
1 RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR S (bl) the protein sequence described under [SEQ ID NO. 12]
wherein the LL motif was inactivated by mutation to a diglycine motif (GG).
[SEQ ID NO. 13]
1 RSKRSRGGHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR S
(c) The sequence of the intracellular domain of the human costimulatory OX40 receptor (amino acid residues 236-277]) (UniProt accession number P43489 (TNFRSF4_HUMAN)) [SEQ ID NO. 14]
1 ALYLLRRDQR LPPDAHKPPG GGSFRTPIQE EQADAHSTLA KI
(d) The sequence of the effector domain comprising a fusion of CD28 (bl), CD3 zeta (a) and OX40 (c), in the mentioned order. [SEQ ID NO. 15]
1 RSKRSRGGHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR SRVKFSRSAD APAYQQGQNQ LYNELNLGRR 71 EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA EAYSEIGMKG ERRRGKGHDG LYQGLSTATK 141 DTYDALHMQA LPPRALYLLR RDQRLPPDAH KPPGGGSFRT PIQEEQADAH STLAKI
The effector domain (v) (preferably) comprises or consists of (is) an amino acid sequence with the amino acid sequence of SEQ ID NO. 15 or a functional equivalent thereof, wherein a "functional equivalent" has less sequence identity (such as at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity or 99% sequence identity) but is a functional fusion of the costimulatory CD28 receptor (diglycine motif mutated) fused to a fragment of the zeta-chain of the CD3 complex of the T- cell receptor fused to a fragment of the costimulatory OX40 receptor.
- preferred CARs of the invention
Preferably, the according to the invention comprises or consists of the amino acid sequence of:
(i) a (cleavable) signal peptide (such as SEQ ID NO. 1),
(ii) an antigen binding domain that comprises or consists of (is) a variable domain of a Vy9-L-V82 (SEQ ID NO. 5)
(iiii) a spacer region, preferably comprising a constant region of a T cell receptor (SEQ ID NOs. 6 to 9)
(iv) a transmembrane domain (as defined herein, preferably of SEQ ID NO. 10), and
(v) the CD3 zeta chain or fragment(s) thereof or a fusion of fragment(s) of CD28 with fragment(s) of CD3 zeta-chain and fragments of OX40
(wherein the signal peptide is at the N-terminus and the zeta chain/fusion is at the C- terminus).
In a preferred embodiment, the protein comprises or consists of the amino acid sequence of SEQ ID NO. 16; or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to SEQ ID NO. 16.
The amino acid sequence of SEQ ID NO. 16 refers to the amino acid sequence of the multifunctional protein with the domains:
(i)[hAAT signal peptide] -(ii)[V62]-(iii)[C5]-(iv)[CD28 transmembrane] -(v)[CD28 ICD (LL- >GG)-CD3 zeta-OX40 ICD] (antigen-binding region in bold)
1 MPSSVSWGIL LLAGLCCLVP VSLAMQRISS LIHLSLFWAG VMSAIELVPE HQTVPVSIGV PATLRCSMKG
71 EAIGNYYI W YRKTQGNTMT FIYREKDIYG PGFKDNFQGD IDIAKNLAVL KILAPSERDE GSYYCACDAL
141 KRTDTDKLIF GKGTRVTVES QPHTKPSVFV MKNGTNVACL VKEFYPKDIR INLVSSKKIT EFDPAIVISP 211 SGKY AVKLG KYEDSNSVTC SVQHDNKTVH STDFEVKTDS TDHVKPKETE NTKQPSKSCH KPKAIVHTEK 281 VNMMSLTVFW VLWVGGVLA CYSLLVTVAF IIFWVRSKRS RGGHSDYM M TPRRPGPTRK HYQPYAPPRD 351 FAAYRSRV F SRSADAPAYQ QGQNQLY EL NLGRREEYDV LDKRRGRDPE MGGKPQRRK PQEGLYNELQ 421 KDKMAEAYSE IG GERRRG KGHDGLYQGL STATKDTYDA LHMQALPPRA LYLLRRDQRL PPDAHKPPGG 491 GSFRTPIQEE QADAHSTLAK I
In a preferred embodiment, the protein comprises or consists of the amino acid sequence of SEQ ID NO. 17; or an amino acid sequence that has at least 70%) sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to SEQ ID NO. 17.
The amino acid sequence of SEQ ID NO. 17 refers to the amino acid sequence of the multifunctional protein with the domains:
(i)[hAAT signal
Figure imgf000017_0001
transmembrane]-(v)[CD28 ICD (LL- >GG)-CD3 zeta-OX40 ICD] (antigen-binding region in bold)
1 MPSSVSWGIL LLAGLCCLVP VSLAAGHLEQ PQISSTKTLS KTARLECWS GITISATSVY YRERPGEVI
71 QFLVSISYDG TVRKESGIPS GKFEVDRIPE TSTSTLTIH VEKQDIATYY CALWEAQQEL GKKIKVFGPG
141 TKLIITDKQL DADVSPKPTI FLPSIAETKL QKAGTYLCLL EKFFPDVIKI HWEEKKSNTI LGSQEGNTMK 211 TNDTYMKFSW LTVPEKSLDK EHRCIVRHEN NKNGVDQEII FPPIKTDVIT MDPKDNFWVL VWGGVLACY 281 SLLVTVAFII FWVRSKRSRG GHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRSRVKFSR SADAPAYQQG 351 QNQLYNELNL GRREEYDVLD RRGRDPEMG GKPQRRKNPQ EGLYNELQKD KMAEAYSEIG MKGERRRGKG 421 HDGLYQGLST ATKDTYDALH MQALPPRALY LLRRDQRLPP DAHKPPGGGS FRTPIQEEQA DAHSTLAKI
In a preferred embodiment, the protein comprises or consists of the amino acid sequence of SEQ ID NO. 18; or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% sequence identity to SEQ ID NO. 18.
The amino acid sequence of SEQ ID NO. 18 refers to the amino acid sequence of the multifunctional protein with the domains:
(i)[hAAT signal peptide] -(ii)[V 9-L-V62]-(iii)[C6]-(iv)[CD28 transmembrane]-(v)[CD28 ICD (LL->GG)-CD3 zeta-OX40 ICD] (antigen-binding region in bold)
1 MPSSVSWGIL LLAGLCCLVP VSLAAGHLEQ PQISSTKTLS KTARLECWS GITISATSVY YRERPGEVI
71 QFLVSISYDG TVRKESGIPS GKFEVDRIPE TSTSTLTIHN VEKQDIATYY CALWEAQQEL GKKIKVFGPG
1 1 TKLIITGSAD DAKKDAAKKD GKMQRISSLI HLSLFWAGVM SAIELVPEHQ TVPVSIGVPA TLRCSMKGEA 211 IGNYYINWYR KTQGNTMTFI YREKDIYGPG FKDNFQGDID IAKNLAVLKI LAPSERDEGS YYCACDALKR 281 GGEYTDKLIF GKGTRV VES QPHT PSVFV MK GTNVACL VKEFYPKDIR INLVSSKKIT EFDPAIVISP 351 SGKYNAV LG KYEDSNSVTC SVQHDNKTVH STDFEVKTDS TDHVKPKETE NTKQPSKSCH KPKAIVHTEK 421 VN SLTVFW VLVWGGVLA CYSLLVTVAF IIFWVRSKRS RGGHSDYMNM TPRRPGPTRK HYQPYAPPRD 491 FAAYRSRVKF SRSADAPAYQ QGQNQLYNEL NLGRREEYDV LDKRRGRDPE MGGKPQRR N PQEGLYNELQ 561 KDKMAEAYSE IGMKGERRRG KGHDGLYQGL STATKDTYDA LHMQALPPRA LYLLRRDQRL PPDAHKPPGG 631 GSFRTPIQEE QADAHSTLAK I
Generally, a person skilled in the art is aware of the fact that some amino acid exchanges in the amino acid sequence of a protein or peptide do not have any influence on the (secondary or tertiary) structure, function and activity of the protein or peptide (at all). Amino acid sequences with such "neutral" amino acid exchanges as compared to the amino acid sequences disclosed herein fall within the scope of the present invention.
In one embodiment, the protein of the present invention comprises or consists of the amino acid sequence of any of SEQ ID NOs. 16 to 18 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, more preferably 90% or more preferably 95% or 99% sequence identity to the amino acid sequence of any of SEQ ID NOs. 16 to 18.
Nucleic acids, expression constructs and host cells
As described above, the present invention provides nucleic acids/nucleic acid molecules/isolated nucleic acid molecules encoding the proteins of the invention.
The nucleic acids according to this invention comprise DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA), combinations thereof or derivatives (such as PNA) thereof.
Preferably, a nucleic acid of the invention comprises or consists of the nucleic acid encoding for the amino acid sequence of SEQ ID NO. 16,
or comprises or consists of the nucleic acid sequence of SEQ ID NO. 19 or their complementary sequences or sequences that have at least 95 % sequence identity.
Preferably, a nucleic acid of the invention comprises or consists of the nucleic acid encoding for the amino acid sequence of SEQ ID NO. 17,
or comprises or consists of the nucleic acid sequence of SEQ ID NO. 20 or their complementary sequences or sequences that have at least 95 % sequence identity.
Preferably, a nucleic acid of the invention comprises or consists of the nucleic acid encoding for the amino acid sequence of SEQ ID NO. 18,
or comprises or consists of the nucleic acid sequence of SEQ ID NO. 21 or their complementary sequences or sequences that have at least 95 % sequence identity.
SEQ ID NO. 19
1 ATGCCATCCA GCGTCTCATG GGGTATCCTT CTGCTCGCCG GATTATGCTG TCTCGTGCCA GTATCCCTGG 71 CCATGCAACG CATTTCGTCT CTCATCCACT TGTCCCTCTT CTGGGCCGGG GTTATGAGTG CTATTGAGTT
141 AGTACCAGAA CACCAAACTG TCCCAGTGTC CATAGGCGTT CCCGCCACCC TCCGGTGTTC AATGAAGGGA
211 GAAGCCATAG GAAATTACTA CATTAACTGG TACAGAAAGA CTCAGGGGAA CACTATGACC TTCATCTATA
281 GGGAGAAAGA CATCTACGGG CCAGGTTTCA AAGACAATTT TCAGGGCGAC ATTGATATCG CAAAGAACCT
351 GGCTGTGCTA AAAATACTCG CACCATCGGA GCGCGACGAG GGGTCCTACT ACTGTGCCTG TGATGCACTC
421 AAGCGGACGG ATACCGACAA GCTTATCTTT GGGAAGGGGA CCAGGGTCAC AGTGGAGTCT CAGCCGCATA
491 CTAAGCCTTC AGTGTTCGTG ATGAAGAATG GCACAAATGT CGCTTGTCTA GTTAAAGAAT TTTACCCTAA
561 AGACATAAGG ATTAATCTGG TGTCAAGCAA AAAAATCACA GAATTCGACC CAGCTATAGT TATCTCCCCA
631 TCCGGAAAAT ACAATGCCGT AAAACTGGGT AAGTACGAGG ATTCAAATTC AGTCACGTGC TCGGTGCAAC
701 ATGATAACAA AACTGTGCAC TCAACGGATT TCGAGGTCAA GACAGATTCA ACCGACCACG TGAAACCGAA
771 GGAAACCGAG AACACAAAGC AGCCTTCAAA GAGTTGTCAC AAGCCAAAGG CTATTGTTCA CACCGAGAAA
841 GTGAACATGA TGTCTCTGAC CGTATTTTGG GTGCTCGTGG TCGTAGGGGG AGTGCTCGCG TGCTATTCTC
911 TGCTCGTGAC AGTCGCCTTC ATCATTTTTT GGGTGAGGAG TAAACGATCG CGCGGCGGCC ACTCAGACTA
981 CATGAACATG ACTCCCCGAA GACCTGGCCC GACAAGGAAA CACTACCAGC CCTACGCACC CCCTAGAGAT
1051 TTCGCTGCTT ACCGGTCACG AGTGAAGTTT TCAAGAAGTG CCGACGCGCC CGCTTACCAG CAGGGCCAGA 1121 ACCAGCTGTA CAATGAGCTG AACCTCGGAC GGCGAGAGGA GTATGATGTG CTGGACAAGC GCCGTGGCCG 1191 AGATCCTGAA ATGGGCGGGA AGCCCCAGAG GAGAAAGAAT CCCCAGGAAG GCCTGTACAA CGAGCTCCAG
1261 AAGGACAAAA TGGCGGAAGC CTATTCAGAA ATCGGAATGA AGGGCGAACG CCGCAGAGGA AAGGGGCATG
1331 ACGGGCTCTA TCAGGGATTG TCTACCGCAA CCAAAGATAC TTACGATGCT CTCCACATGC AGGCCCTTCC
1401 ACCAAGAGCC CTATATCTGT TGCGTCGCGA CCAGCGCCTT CCGCCCGATG CTCACAAGCC GCCAGGCGGT
1471 GGGAGCTTCA GAACTCCCAT TCAGGAGGAG CAGGCAGACG CCCACAGCAC ACTGGCAAAA ATCTAGTAAA
SEQ ID NO. 20
1 ATGCCATCAT CTGTCAGTTG GGGGATCCTA CTGCTTGCTG GCCTCTGCTG TCTCGTGCCA GTGTCGCTAG 71 CCGCTGGCCA CCTGGAGCAG CCTCAGATCT CATCCACGAA GACTCTGTCT AAGACCGCCC GGTTAGAGTG
141 TGTGGTTTCA GGCATCACGA TAAGCGCGAC TTCAGTTTAT TGGTACCGCG AGAGGCCGGG CGAGGTAATC
211 CAGTTTCTGG TCAGTATAAG CTATGACGGT ACCGTGCGGA AGGAAAGTGG AATCCCTAGC GGGAAGTTTG
281 AGGTGGACCG CATCCCTGAA ACCAGCACTT CTACCCTGAC TATTCACAAC GTGGAAAAGC AGGATATCGC
351 TACTTATTAC TGCGCATTGT GGGAGGCACA GCAGGAATTA GGGAAAAAGA TCAAAGTCTT CGGGCCTGGT
421 ACCAAACTCA TCATCACAGA CAAACAACTG GATGCCGACG TGAGCCCCAA GCCCACGATC TTTCTGCCAT
491 CTATTGCGGA GACAAAGCTT CAGAAAGCTG GGACATATCT CTGCCTGCTT GAAAAGTTTT TCCCTGACGT
561 GATTAAGATT CATTGGGAGG AAAAGAAGAG TAACACCATC TTGGGAAGTC AGGAGGGTAA CACCATGAAA
631 ACCAATGACA CCTATATGAA GTTCTCATGG CTTACAGTTC CCGAGAAGAG CCTTGACAAG GAGCACAGGT
701 GTATAGTGCG GCACGAGAAT AACAAAAACG GAGTGGACCA AGAAATTATC TTCCCTCCTA TCAAGACAGA
771 CGTTATTACC ATGGATCCCA AGGATAACTT CTGGGTACTG GTTGTAGTGG GCGGAGTGCT GGCCTGCTAT
841 TCATTGCTGG TGACCGTGGC CTTTATCATA TTCTGGGTTA GGTCCAAAAG AAGCAGGGGC GGCCATAGCG
911 ACTACATGAA CATGACACCA CGCAGACCTG GCCCTACACG CAAACACTAC CAACCATACG CACCACCGCG
981 TGACTTCGCG GCCTACCGTA GTAGAGTGAA GTTTTCCCGT AGCGCCGATG CCCCCGCATA TCAGCAGGGG
1051 CAGAACCAGC TGTACAACGA GCTGAATCTG GGACGCCGAG AGGAGTACGA TGTCTTGGAC AAACGGCGAG
1121 GCCGTGATCC TGAGATGGGC GGAAAGCCGC AACGGAGAAA GAATCCCCAG GAAGGCCTCT ACAACGAATT
1191 GCAGAAGGAT AAAATGGCAG AGGCGTATTC TGAAATTGGT ATGAAGGGCG AGCGACGCAG GGGTAAAGGA
1261 CACGATGGCT TATACCAGGG CCTTTCCACG GCAACTAAAG ATACTTATGA CGCGCTGCAC ATGCAGGCTC
1331 TGCCTCCCCG CGCCCTGTAT CTGCTCCGAC GTGACCAGAG ACTGCCACCC GACGCCCACA AACCGCCCGG
1401 TGGCGGCTCA TTCCGTACAC CGATCCAGGA GGAACAGGCC GACGCCCACT CCACTTTAGC AAAAATCTAG
1471 TAAA
SEQ ID NO. 21
1 ATGCCATCTT CCGTAAGTTG GGGCATTTTG CTGCTGGCGG GCCTGTGTTG CCTCGTGCCA GTGAGCCTCG 71 CAGCCGGGCA CCTAGAGCAA CCTCAGATCA GCAGCACGAA GACCCTTTCA AAAACCGCTC GTCTCGAGTG
141 TGTGGTGAGC GGTATAACAA TTTCTGCCAC TAGTGTCTAC TGGTATCGGG AGAGGCCTGG CGAAGTCATC
211 CAGTTTTTAG TCTCCATCAG TTATGACGGT ACAGTTCGCA AGGAAAGCGG GATACCCAGC GGAAAGTTTG
281 AGGTGGATAG AATACCCGAG ACTTCAACCA GCACCTTAAC AATTCATAAT GTCGAAAAAC AGGATATCGC
351 TACTTACTAT TGCGCTTTAT GGGAAGCTCA GCAGGAGCTG GGGAAGAAGA TTAAGGTTTT TGGTCCTGGA
421 ACAAAACTGA TTATTACAGG GTCTGCGGAT GACGCTAAAA AGGACGCCGC GAAAAAGGAC GGAAAGATGC
491 AGCGAATTAG CTCCCTGATC CACCTCTCCT TGTTCTGGGC AGGCGTAATG TCCGCAATCG AACTCGTGCC
561 TGAGCATCAG ACCGTGCCGG TAAGTATTGG GGTGCCAGCA ACTCTAAGAT GTAGTATGAA GGGTGAAGCA
631 ATCGGGAACT ACTACATCAA TTGGTATAGA AAAACCCAAG GTAACACAAT GACCTTCATC TACCGGGAAA
701 AAGATATATA CGGGCCGGGA TTCAAGGACA ACTTCCAAGG GGACATAGAT ATCGCAAAAA ACCTCGCTGT
771 GCTGAAAATC CTGGCTCCCT CGGAAAGGGA CGAGGGCTCA TACTACTGCG CTTGCGATGC TCTGAAGCGC
841 GGAGGCGAAT ACACCGACAA GCTGATCTTC GGAAAAGGGA CACGAGTCAC CGTGGAAAGT CAGCCACACA
911 CTAAGCCTTC CGTTTTCGTG ATGAAGAACG GTACAAACGT GGCTTGCCTC GTAAAAGAAT TTTACCCAAA 981 GGATATCAGG ATAAACCTGG TGTCGTCAAA GAAAATCACA GAGTTCGACC CAGCCATTGT CATCTCCCCA
1051 TCAGGCAAGT ACAATGCCGT CAAACTTGGG AAGTATGAGG ACTCAAACTC AGTTACATGC TCTGTTCAGC
1121 ATGACAATAA AACAGTGCAT TCTACCGACT TTGAGGTGAA GACGGACTCG ACTGATCACG TTAAGCCCAA
1191 GGAGACTGAA AACACTAAGC AGCCAAGCAA GTCATGCCAT AAACCGAAGG CGATAGTGCA CACCGAGAAG
1261 GTCAACATGA TGTCCCTGAC TGTGTTTTGG GTGCTCGTCG TGGTTGGCGG AGTGCTGGCG TGTTATAGCC
1331 TGCTGGTGAC CGTTGCCTTC ATCATCTTCT GGGTGCGCTC CAAAAGAAGC CGGGGTGGTC ACTCTGACTA
1401 CATGAATATG ACCCCCAGGC GTCCCGGTCC CACAAGAAAG CACTACCAGC CATATGCGCC CCCTCGAGAC
1471 TTCGCCGCCT ATCGAAGCAG AGTGAAGTTC TCCAGGTCAG CCGACGCACC CGCATATCAA CAGGGACAGA
1541 ACCAGTTATA CAATGAGCTT AACCTCGGCC GGCGCGAAGA ATACGATGTC TTGGACAAGC GGCGTGGGAG
1611 GGACCCTGAG ATGGGCGGGA AGCCTCAGAG GCGAAAGAAC CCACAGGAGG GACTTTATAA TGAGCTCCAA
1681 AAGGATAAAA TGGCTGAAGC CTACAGCGAG ATCGGGATGA AGGGGGAACG TAGGAGAGGA AAAGGGCATG
1751 ACGGGCTCTA TCAGGGCCTG AGTACCGCCA CCAAAGATAC TTATGATGCC TTGCACATGC AGGCGCTGCC
1821 TCCTCGAGCA TTGTATCTGC TGAGAAGAGA TCAAAGGCTC CCTCCAGATG CACACAAACC GCCAGGCGGA
1891 GGTAGCTTCA GAACACCAAT TCAAGAAGAA CAGGCCGATG CACACAGTAC CCTAGCAAAG ATCTAGTAAA
In one embodiment, nucleic acids comprising or consisting of nucleic acid sequences of SEQ ID NOs. 16 and 17 (or respective sequences with at least 90% identity) are for co-expression in a host cell (to yield a functional receptor/CAR).
Preferably, the nucleic acid sequences of the present invention are codon-optimized for expression in mammalian cells, preferably for expression in human cells.
Codon-optimization refers to the exchange in a sequence of interest of codons that are generally rare in highly expressed genes of a given species by codons that are generally frequent in highly expressed genes of such species,- such codons encoding the same amino acids as the codons that are being exchanged.
Within the scope of this invention are also the nucleotide sequences obtained due to the degeneration of the genetic code of the above nucleotide sequences.
As described above, the present invention provides expression constructs for expressing the protein of the invention in a cell.
Preferably, the expression constructs further comprise promoter and terminator sequences. An "expression construct" or "gene construct" (wherein both terms are used interchangeably throughout this specification) refers to a nucleic acid construct, usually an expression vector or plasmid, that is used to introduce a specific gene sequence into a target cell. Once the expression or gene construct is inside the cell, the protein that is encoded by the gene is produced by the cellular transcription and translation macliinery. The expression or gene construct is designed to contain respective regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene earned on the construct, including promoter and terminator sequences. The goal of a well-designed expression or gene construct is the production of suitable amounts of stable mRNA, and therefore proteins.
The skilled artisan can select further suitable components of expression or gene constructs. The nucleic acids and/or in particulai- expression constructs of the invention are capable of directing the synthesis/expression of the engineered membrane proteins of the invention.
The nucleic acids and/or expression constructs of the invention are dsDNA, ssD A, RNA or mRNA or combinations thereof.
As described above, the present invention provides host cells which express a protein of the invention or which comprise a nucleic acid or an expression construct of the invention.
Preferably, the host cell is selected from effector cells of the immune system, such as lymphocytes including but not limited to cytotoxic lymphocytes, T cells, cytotoxic T cells, T helper cells, Thl7 T cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, neutrophils, macrophages, dendritic cells, killer dendritic cells, B cells.
"Effector cells" of the immune system or "immune effector cells" refers to cells of hematopoietic origin including but not limited to the cell types mentioned above that are functionally involved in the initiation and/or execution of innate and/or adaptive immune responses.
In the embodiment, where the protein of the invention comprises only the variable domain Vy9 or V52 as antigen binding domain (ii),
the host cell co-expresses two proteins of the invention or comprises two nucleic acids or expression constructs of the invention,
in particular a first protein comprising the variable domain Vy9 as antigen binding domain (ii) and a second protein comprising the variable domain V82 as antigen binding domain (ii), respective nucleic acids/expression constructs of said two proteins,
such as proteins comprising or consisting of the amino acid sequences of SEQ ID NOs. 16 and 17 (or respective sequences with at least 90% identity), or nucleic acids comprising or consisting of the nucleic acid sequences of SEQ ID NOs. 19 and 20 (or respective sequences with at least 90% identity).
Uses of the proteins, nucleic acids, expression constructs and host cells
As described above, the invention provides the use of the protein, nucleic acid, or expression construct of the present invention for generating antigen-specific effector cells.
"Antigen-specific effector cells" or "target-specific effector cells" refer to effector cells of the immune system or immune effector cells genetically modified to express the multi-functional protein of the invention by transfer of an expression construct or nucleic acid encoding said multi-functional protein. Such antigen-specific or target-specific effector cells are versatile means, in particular in the treatment of diseases (as described below for ACT and cancer treatment).
As described above, the invention provides the protein, nucleic acid, expression construct or host cell for use as a medicament.
As described above, the invention provides the protein, nucleic acid, expression construct or host cell for use in the treatment of cancer.
As described above, the invention provides the protein, nucleic acid, expression construct or host cell for use in adoptive, target-cell specific immunotherapy.
"Adoptive, target-cell specific immunotherapy" refers to a form of therapy in which immune cells are transferred to tumor-bearing hosts. The immune cells have antitumor reactivity and can mediate direct or indirect antitumor effects.
"Adoptive, target-cell specific immunotherapy" or "adoptive cell therapy (ACT)" is a treatment that uses immune effector cells, such as lymphocytes with anti-tumour activity, expanded in vitro and infused into the patient with cancer. ACT using autologous tumour- infiltrating lymphocytes has emerged as the most effective treatment for patients with metastatic melanoma and can mediate objective cancer regression in approximately 50% of patients. The use of donor lymphocytes for ACT is an effective treatment for immunosuppressed patients who develop post-transplant lymphomas (reviewed in Rosenberg et al., 2008). However, the ability to genetically engineer human lymphocytes and use them to mediate cancer regression in patients, which has recently been demonstrated (see Morgan et al, 2006), has opened possibilities for the extension of ACT immunotherapy to patients with a wide variety of cancer types and is a promising new approach to cancer treatment. Thus, genetically engineering of lymphocytes with chimeric antigen receptors (CAR), such as provided by this invention, is very suitable for ACT and opens more possibilities in the treatment of cancer.
Especially, since studies have clearly demonstrated that the administration of highly avid anti- tumour T cells directed against a suitable target can mediate the regression of large, vascularized, metastatic cancers in humans and provide guiding principles as well as encouragement for the further development of immunotherapy for the treatment of patients with cancer.
Methods of treatment
As discussed above, the invention provides methods for generating antigen-specific effector cells.
The method for generating antigen-specific effector cells according to the present invention comprises the steps of:
(a) providing a protein, nucleic acid, or expression construct according to the invention;
(b) providing a host cell or cell line, which is selected from effector cells of the immune system, such as lymphocytes including but not limited to cytotoxic lymphocytes, T cells, cytotoxic T cells, T helper cells, Thl7 T cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages, mast cells, dendritic cells, killer dendritic cells, B cells;
(c) transferring the protein, nucleic acid, or expression construct provided in step (a) into the host cell or cell line provided in step (b);
(d) optional, selection of the transgenic (gene-modified) cells.
The present invention also provides methods for the treatment of diseases, in particular cancer, and methods of immunotherapy, preferably including adoptive, target-cell specific immunotherapy.
The method for the treatment of diseases, in particular cancer, according to the present invention comprises the step of administering to a subject in need thereof a therapeutically effective amount of (a) a protein, a nucleic acid, an expression construct or a host cell (in particular an antigen-specific effector cell) as obtained and defined herein, and (b) optionally, respective excipient(s).
The method of immunotherapy, preferably including or utilizing adoptive, target-cell specific immunotherapy, according to the present invention comprises the step of administering to a subject in need thereof a therapeutically effective amount of (a) a protein, a nucleic acid, an expression construct or a host cell (in particular an antigen-specific effector cell) as obtained and defined herein, and (b) optionally, respective excipient(s).
A therapeutically effective amount of a protein, a nucleic acid, an expression construct or a host cell (in particular an antigen-specific effector cell) of this invention refers to the amount that is sufficient to treat the respective disease or achieve the respective outcome of the adoptive, target-cell specific immunotherapy.
The following examples and drawings illustrate the present invention without, however, limiting the same thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 Schematic structure of the expression vectors for single-chain constructs.
P - promoter, SP - signal peptide, V - variable domain (Vy9 or V52), L - linker, C - constant domain, TM - transmembrane domain, ED - effector domain, pA - polyadenylation signal (terminator), ABR - antigen binding region
(A) structure of expression vectors comprising only one variable domain in their antigen binding region;
(B) structure of expression vectors comprising a fusion of Vy9 and V52 variable domains in their antigen binding region;
(C) structure of mock expression vector, omitting ABR.
Figure 2 Dotplot representation of flow -cytometric CAR expression analysis.
x-axis V52-FITC, y-axis Υγ9-ΡΕ (A) mock
(B) Vy9
(C) V52
(D) Vy9/V52 coexpression
(E) sc_Vy9V52
Figure 3 Cytotoxicity of CAR transduced T cells against cancer cell lines.
NC-E - only T cells, NC-T only target cell, mock, Vy9/V52 coexpression of both Yj9 and
V52 comprising CARs, sc_Vy9V62-expression of CAR with fusion ABR
(A) Daudi cells
(B) K562 cells
(C) Jurkat cells
(D) PBMC
(E) B cells
EXAMPLES
Example 1 Materials & Methods
1. Construction of CAR receptors
Three expression constructs for Vy9, V52 and single-chain Vy9V62 (sc_Vy9V52) mock CARs, codon optimized for human cells, were synthesized by Life Teclmologies (GeneArt® Strings™ DNA Fragments).
Vy9:eIF4G promoter- 5*-UTR-SP-VY9-CY-CD28 TM-CD28-CD3zeta-OX40-3'UTR-pA (Fig. 1 A),
V62: eIF4G promoter- 5'-UTR-V52-C6-CD28 TM-CD28-CD3zeta-OX40-3'UTR-pA
(Fig. 1 A),
SC_VY9V52: eIF4G promoter- 5*-UTR-SP-VY9-L-V52-C6-CD28 TM-CD28-CD3zeta-OX40- 3'UTR-pA (Fig. 1 B).
The mock construct omitted the antigen binding region, yielding the construct:
eIF4G promoter- 5'-UTR-SP-C6-CD28 TM-CD28-CD3zeta-OX40-3'UTR-pA (Fig. 1 C).
EcoRI restriction sites were included flanking the linear expression cassettes on both sites. 2. In vitro DNA amplification
Linear DNA templates were circularized via digestion EcoRI (Biozym, Germany) and subsequent incubation with T4 DNA ligase (Biozym, Germany). In 200 μΐ PCR tubes containing 10 mM Tris pH 8, and 200 pmol random hexamers (phosphothioate protected), 10 ng of circular template was denatured at 95° C for 3 min and cooled to room temperature. The template solution was then added to 1 ml of amplification solution containing phi29 DNA polymerase (10 U, Biozym, Germany), 0,2 mM dNTPs, 100 ig BSA in 50 mM Tri-HCl pH 7,5, 10 mM MgCl, 10 mM (NH) SO, DTT. Amplification was carried out at 32°C in for 16 hours. Following amplification the phi29 DNA polymerase was heat inactivated (5 min, 65°C). The amplified DNA concatamers were ethanol/salt precipitated and monomerized with EcoRI according to the manual. DNA was isolated using DCC-100 DNA Clean & Concentrator (TM) (Zymo Research Corporation, Irvine, California), according to manufacturer instructions. Spectrometric analysis (Pharmacia GeneQuant Pro) at 260 nm and agarose gel electrophoresis with a nucleic acid ladder (500 bp Molecular Ruler, BIORAD, Miinchen, Germany) were conducted to analyze quantitity and purity of the obtained DNA product.
3. Non-viral transduction of T cells
T cells were transduced with the amplified nucleic acid mock, Vy9, V62, dual Vy9/V2 and the single-chain construct (sc_Vy9V82). For each construct 2*106 freshly isolated T cells from human peripheral blood samples were suspended in 2 ml Eppendorf tubes containing phosphate buffered 1 ml Leibovitz LI 5 medium (Sigma- Aldrich, Germany) with 100 g/ml ampicillin (Sigma-Aldrich, Germany). GenePORTER 2 transfection reagent (BioCat, Heidelberg, Germany) was used for cell transfection as directed by the manufacturer.
Briefly, 8 μg of the amplified and digested DNA construct 200 ml diluent B DNA solution was then mixed with 40 μΐ of GENEPORTER 2 reagent prediluted in 160 μΐ of serum-free LI 5 medium and incubated at room temperature for an additional 5 min. The resulting transfection volume of 400 μΐ was then added to the T cell suspension. Following 4 hours of incubation transduced T cells were washed with PBS and topped with 0.9 ml of L15 medium. For the Vy9/V62 co-expression, the constructs were transduced after another with a washing step in between as described for the single transductants.
4. Surface expression of CARs Transduced T cells were analyzed by two-colour flourescence flow-cytometry on a FACSCalibur instrument (Becton Dickinson (BD), Mountain View, CA), using the fluorophor marked antibodies Anti-Vy9-PE, anti-V52-FITC (Beckman Coultier, Marseille Cedex, France). Cells were collected and washed once with phosphate buffered saline containing 1% fetal bovine serum (Hy clone, FACS buffer) prior to the addition of antibodies. Cell suspension (100 μΐ) was incubated for 45 minutes on ice in the dark with 1 μg of each antibody, washed once, and fixed in 0.5% paraformaldehyde/FACS buffer prior to analysis. Data was plotted and analysed with Flowing Software (www.flowingsoftware.com). Forward and side scatter gating were used to discriminate live from dead cells.
5. Cytotoxic activity of CAR T cells
All experiments were conducted in triplicate. The cancer cell lines Daudi, K562, Jurkat and the normal PBMC, and B cells (Marcu-Malina, et al, 2011; B cells freshly isolated) were used as targets. Target cells were provided in 96 well plates with 100,000 cells per well in Leibovitz L15 medium (150 μΐ), containing pamidronate (10 μΜ, Sigma-Aldrich, Germany) and ampicillin (100 μg/ml). After 48 hours incubation the apoptosis indicator NucView-488 (in DMSO, Cambridge Bioscience, Cambridge, United Kingdom) and 20,000 or 100,000 of Vy9/V52 CAR, sc_Vy9V62 CAR or mock transduced T cells were added, yielding effector to target ratios of 0,2:1 and 1:1 respectively.
Cultures using only effector or only target cells were used as negative controls.
Co-culture was continued for 8 hours. Micrographic images under UV illumination were taken of each well (modified Bresser Biolux NV). Apoptosis quantification was conducted via CellProfiler image analysis software, using the inbuilt machine learning function to discern live from dead cells (www.cellprofiler.org, BROAD institute).
Example 2 Results
FACS analysis shows no expression of CARs for mock, Vy9 or V52 transduced T cells (Fig 2 A, B, C), while expression can be detected for the co-expression of Vy9/V52 and the sc_Vy9V62 transduced effector cells (Fig. 2 D and E). Apparently Vy9, and V52 require each other for efficient expression, either in the form of coexpression on two separate protein chains or fused to a single chain. While mock transfected T cells do not show any cytotoxic activity against any cancer cell line tested, Vy9 V62 and the sc_Vy9V52 efficiently lyse every cancer cell line tested (Fig 3). The cytotoxicity of Vy9/V62 is generally higher than that of sc_Vy9 V62, potentially owing to a more native protein configuration. No cytotoxicity of Vy9/V62 and the sc_Vy9V52 can be detected against untransformed PBMC and CD 19+ cells. These results demonstrate the ability of CARs with antigen binding domains comprising Vy9V62 domains to lyse several cancer cell lines of distinct origin. The results are therefore in accordance with the objective of the invention to provide CAR receptors with a broad-spectrum anti-cancer activity.
Sequences:
SEQ ID NO. 1 shows the amino acid sequence of the signal peptide of human Alpha- 1 Antitrypsin
(uniprot accession number: P01009, amino acid residues: 1-24).
SEQ ID NO. 2 shows the amino acid sequence of the preferred linker sequence.
SEQ ID NO. 3 shows the amino acid sequence of the variable domain human Vy9 T cell receptor chain.
SEQ ID NO. 4 shows the amino acid sequence of the variable domain human V52 T cell receptor chain.
SEQ ID NO. 5 shows the amino acid sequence of the fusion of Vy9 to V62, via the linker described under SEQ ID NO. 2.
SEQ ID NO. 6 shows the amino acid sequence of the constant domain of the human γ T cell receptor chain.
SEQ ID NO. 7 shows the amino acid sequence of the constant domain of the human δ T cell receptor chain.
SEQ ID NO. 8 shows the amino acid sequence of the constant domain of the human a T cell receptor chain (uniprot accession number: P01848).
SEQ ID NO. 9 shows the amino acid sequence of the constant domain of the human β T cell receptor chain (uniprot accession number: P01850). SEQ ID NO. 10 shows the amino acid sequence of the transmembrane domain of human CD28 (uniprot accession number: P01850, amino acid residues: 153-179).
SEQ ID NO. 11 shows the amino acid sequence of the intracellular domain of human T-cell surface glycoprotein CD3 zeta chain (UniProt accession number P20963-1 (CD3Z_HUMAN); Isoform 1, (amino acid residues 52-164)).
SEQ ID NO. 12 shows the amino acid sequence of the intracellular domain of human CD28 costimulatory receptor (UniProt accession number PI 0747-1 (CD28_HUMAN), isoform 1, (amino acid residues 180-220)).
SEQ ID NO. 13 shows the amino acid sequence described under SEQ ID NO. 12 wherein the diglycine motif has been inactivated by mutation.
SEQ ID NO. 14 shows the amino acid sequence of the intracellular domain of the human costimulatory OX40 receptor (amino acid residues 236-277]) (UniProt accession number P43489 (TN-FRSF4JHUMAN)).
SEQ ID NO. 15 shows the amino acid sequence of the effector domain comprising a fusion of CD28 (SEQ ID NO. 13), CD3 zeta (SEQ ID NO. 11) and OX40 (SEQ ID NO. 14), in the mentioned order.
SEQ ID NO. 16 shows the amino acid sequence of the multifunctional protein with the domains:
(i)[hAAT signal peptide]-(ii)[V52]-(iii)[C5]-(iv)[CD28 transmembrane] -(v)[CD28 ICD (LL- >GG)-CD3 zeta-OX40 ICD].
SEQ ID NO. 17 shows the amino acid sequence of the multifunctional protein with the domains:
(i)[hAAT signal peptide]-(ii)[Vy9]-(iii)[CY]-(iv)[CD28 transmembrane] -(v)[CD28 ICD (LL- >GG)-CD3 zeta-OX40 ICD].
SEQ ED NO. 18 shows the amino acid sequence of the multifunctional protein with the domains:
(i)[hAAT signal peptide] -(ii)[Vy9-L-V52]-(iii)[C5]-(iv)[CD28 transmembrane] -(v)[CD28 ICD (LL->GG)-CD3 zeta-OX40 ICD].
SEQ ID NO. 19 shows the nucleotide sequence encoding for the multi-functional protein with the domains:
(i)[hAAT signal peptide] -(ii)[V82]-(iii)[C5]-(iv)[CD28 transmembrane]-(v)[CD28 ICD (LL- >GG)-CD3 zeta-OX40 ICD] (SEQ ID NO. 16) in a codon-optimized form.
SEQ ID NO. 20 shows the nucleotide sequence encoding for the multi-functional protein with the domains:
(i)[hAAT signal peptide] -(ii)[VY9]-(iii)[Cy]-(iv)[CD28 transmembrane] -(v)[CD28 ICD (LL- >GG)-CD3 zeta-OX40 ICD] (SEQ ID NO. 17) in a codon-optimized form.
SEQ ID NO. 21 shows the nucleotide sequence encoding for the multi-functional protein with the domains:
(i)[hAAT signal peptide]-(ii)rVy9-L-V52]-(iii)[C5]-(iv)[CD28 transmembrane]-(v)[CD28 ICD (LL->GG)-CD3 zeta-OX40 ICD] (SEQ ID NO. 18) in a codon-optimized form.
SEQ ID NO. 22 shows the amino acid sequence of the CDR3 region of Vy9.
SEQ ID NO. 23 shows the amino acid sequence of the CDR3 region of V52.
REFERENCES
Boissel, L. et al. (2013) Retargeting NK-92 cells by means of CD 19- and CD20-specific chimeric antigen receptors compares favorably with antibody-dependent cellular cytotoxicity. Oncoimmunology, 2, e26527.
Brentjens, R. et al. (2013) CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med. 2013 Mar 20;5(177):177ra38.
Carding, S.R. and Egan, P.J. (2002) Gamma delta T cells: functional plasticity and heterogeneity. Nature reviews. Immunology, 2, 336-45.
Capietto, A.-H.H. et al. (2011) How tumors might withstand γδ T-cell attack. Cellular and molecular life sciencesU: CMLS, 68, 2433-42.
Grupp, S.A. et al. (2013) Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. The New England journal of medicine, 368, 1509-18.
Huber, M.L. et al. (2012) Interdisciplinary critique of sipuleucel-T as immunotherapy in castration-resistant prostate cancer. Journal of the National Cancer Institute, 104, 273-9. abelitz, D. et al. (2004) Potential of human gammadelta T lymphocytes for immunotherapy of cancer. International journal of cancer. Journal international du cancer, 112, 727-32.
Loskog, A. et al. (2006) Addition of the CD28 signaling domain to chimeric T-cell receptors enhances chimeric T-cell resistance to T regulatory cells. Leukemia, 20, 1819-28.
Marcu-Malina,V. et al. (2011) Redirecting αβ T cells against cancer cells by transfer of a broadly tumor-reactive γδΤ-cell receptor. Blood, 118, 50-9.
Morgan, R.A. et al. (2006) Cancer regression in patients after transfer of genetically engineered lymphocytes. Science (New York, NY.), 314, 126-9.
Rosenberg, S.A. et al. (2008) Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nature reviews. Cancer, 8, 299-308.
Sadelain, M. et al. (2013) The basic principles of chimeric antigen receptor design. Cancer discovery,?!, 388-98.
Topfer, . et al. (2011) Tumor evasion from T cell surveillance. Journal of biomedicine & biotechnology, 2011, 918471.
Zheng, J et al. (2013) A novel antibody-like TCRy5-Ig fusion protein exhibits antitumor activity against human ovarian carcinoma. Cancer Letters, 341,2, 150-158.

Claims

Claims
1. A protein comprising:
(i) a signal peptide;
(ii) an antigen recognition domain;
which is derived from the variable domains of Vy9/V52 T cell receptors (TCRs),
(iii) optionally, a spacer region, connecting domain (ii) and domain (iv),
(iv) a transmembrane domain,
(v) at least one effector domain comprising one or more intracellular signaling domains.
2. The protein of claim 1 , wherein the target is a cell or a virus, preferably a malignant or virus-infected cell.
3. The protein of claim 1 or 2, wherein the antigen recognition domain (ii) comprises the variable domain of Vy9 and/or the variable domain of V52 and/or parts thereof.
4. The protein of any one of claims 1 to 3, wherein the variable domain of Vy9 comprises an amino acid sequence of SEQ ID NO. 3 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO. 3.
5. The protein of any one of claims 1 to 4, wherein the variable domain of V62 comprises an amino acid sequence of SEQ ID NO. 4 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO. 4.
6. The protein of any one of claims 3 to 5, wherein the antigen recognition domain (ii) comprises the variable domain of Vy9 and the variable domain of V52,
which are not covalently coupled or fused to each other.
7. The protein of any one of claims 3 to 5, wherein the antigen recognition domain (ii) comprises the variable domain of Vy9 and the variable domain of V52,
which are covalently coupled or fused to each other,
preferably via a linker, more preferably a linker comprising or consisting of the amino acid sequence of SEQ ID NO. 2.
8. The protein of claim 7, comprising an amino acid sequence of SEQ ID NO. 5 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO. 5.
9. The protein of claim 6, comprising two protein chains,
preferably not covalently coupled to each other and/or each comprising
(ii) an antigen recognition domain;
comprising one domain which is derived from the variable domains of Vy9/V52 T cell receptors (TCRs),
(iii) optionally, a spacer region, connecting domain (ii) and domain (iv),
(iv) a transmembrane domain,
(v) at least one effector domain comprising one or more intracellular signaling domains,
wherein preferably the antigen recognition domain (ii) of one protein chain comprises or consists of the variable domain of Vy9 and the antigen recognition domain (ii) of the other protein chain comprises or consists of the variable domain of V52.
10. The protein of any one of claims 1 to 3, wherein the antigen recognition domain (ii) comprises one or more CDR sequences derived from the variable domains of Vy9 and/or V52, such as the antigen recognition domain (ii) comprises or consists of an amino acid sequence of SEQ ID NO. 22 (CDR3 of Vy9) and/or SEQ ID NO. 22 (CDR3 of V62),
or an amino acid sequence that has at least 70%, preferably 80% or more preferably 90% or 95% sequence identity to said sequences.
11. The protein of any one of claims 1 to 3 and 10, wherein the antigen recognition domain (ii) comprises CDR sequences derived from the variable domains of Vy9 and/or V62 grafted onto protein scaffolds.
12. The protein of any one of the preceding claims, wherein the spacer region (iii) comprises constant domain(s) of a T cell receptor.
13. The protein of claim 12, wherein the constant domain(s) of a T cell receptor are selected from Cy, C5, Ca and/or Cp.
14. The protein of claim 12 or 13, wherein
- the constant domain Cy comprises an amino acid sequence of SEQ ID NO. 6 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO. 6;
and/or
- the constant domain C5 comprises an amino acid sequence of SEQ ID NO. 7 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, 90% or 95%» sequence identity to the amino acid sequence of SEQ ID NO. 7;
and/or
- the constant domain Ca comprises an amino acid sequence of SEQ ID NO. 8 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, 90% or 95%> sequence identity to the amino acid sequence of SEQ ID NO. 8;
and/or
- the constant domain Οβ comprises an amino acid sequence of SEQ ID NO. 9 or an amino acid sequence that has at least 70% sequence identity, preferably 80%), 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO. 9.
15. The protein of any one of the preceding claims, wherein the transmembrane domain (iv) comprises or consists of the transmembrane domain of human CD28,
preferably comprising an amino acid sequence of SEQ ID NO. 10 or an amino acid sequence that has at least 70% sequence identity, preferably 80%), 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO. 10.
16. The protein of any one of the preceding claims, wherein the at least one effector domain (v) comprises domain(s) of at least one of the following:
- proteins with an ITAM motif (YXXL/IX(6-8)YXXL/I),
such as CD3 polypeptides (δ, ε, ζ), FceRIy, B29; - proteins with ITSM Motif (TXYXXV/I),
such as CD 150;
- proteins with ΥΓΝΜ Motif (YINM),
such as DAP 10;
- immune kinases,
such as syk, ZAP70, lck, csk;
- costimulatory receptors,
such as CD28, OX40 (CD134), 4-1BB (CD137).
17. The protein of any one of the preceding claims, wherein the at least one effector domain (v) comprises or is:
(1) the zeta-chain of the human CD3 complex of the T-cell receptor or fragment(s) thereof,
preferably comprising or consisting of the amino acid sequence of SEQ ID NO. 11, or a functional equivalent thereof;
(2) the human costimulatory CD28 receptor or fragment(s) thereof,
preferably comprising or consisting of the amino acid sequence of SEQ ID NO. 12, or a functional equivalent thereof;
more preferably comprising or consisting of the amino acid sequence of SEQ ID NO. 13, or a functional equivalent thereof;
(3) the human costimulatory OX40 receptor or fragment(s) thereof,
preferably comprising or consisting of the amino acid sequence of SEQ ID NO. 14, or a functional equivalent thereof;
or
a fusion of (2) a fragment of the human costimulatory CD28 receptor, (1) a fragment of the zeta-chain of the human CD3 complex of the T-cell receptor, and (3) a fragment of the human costimulatory OX40 receptor,
preferably a fusion of (1) to (3) in the order (2), (1) to (3) from N- to C- terminus,
more preferably comprising or consisting of the amino acid sequence of SEQ ID NO. 15; or a functional equivalent thereof.
18. The protein of any one of the preceding claims, comprising the amino acid sequence of any of SEQ ID NOs. 16 to 18 or an amino acid sequence that has at least 70% sequence identity, preferably 80%, 90% or 95% sequence identity to the amino acid sequence of any of SEQ ID NOs. 16 to 18.
19. A nucleic acid encoding the protein of any one of claims 1 to 18.
20. The nucleic acid of claim 19, comprising the nucleic acid encoding for the amino acid sequence of SEQ ID NO. 16,
or comprising of the nucleic acid sequence of SEQ ID NO. 19 or their complementary sequences or sequences that have at least 95 % sequence identity.
21. The nucleic acid of claim 19, comprising the nucleic acid encoding for the amino acid sequence of SEQ ID NO. 17,
or comprising of the nucleic acid sequence of SEQ ID NO. 20 or their complementary sequences or sequences that have at least 95 % sequence identity.
22. The nucleic acid of claim 19, comprising the nucleic acid encoding for the amino acid sequence of SEQ ID NO. 18,
or comprising of the nucleic acid sequence of SEQ ID NO. 21 or their complementary sequences or sequences that have at least 95 % sequence identity.
23. An expression construct for expressing the protein of any one of claims 1 to 16 in a cell, preferably further comprising promoter and terminator sequences.
24. A host cell expressing a protein of any one of claims 1 to 16 or comprising a nucleic acid of any one of claim 19 or 22 or an expression construct of claim 21.
25. The host cell of claim 24, which is selected from effector cells of the immune system, such as lymphocytes including but not limited to cytotoxic lymphocytes, T cells, cytotoxic T cells, T helper cells, Thl7 T cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, neutrophils, macrophages, dendritic cells, killer dendritic cells, B cells.
26. Use of a protein of any one of claims 1 to 18, a nucleic acid of any one of claim 19 or 22 or an expression construct of claim 23 for generating target-specific effector cells.
27. The protein of any one of claims 1 to 18, the nucleic acid of any one of claim 19 or 22, the expression construct of claim 23 or the host cell of claim 24 or 25 for use as a medicament.
28. The protein of any one of claims 1 to 18, the nucleic acid of any one of claim 19 or 22, the expression construct of claim 23 or the host cell of claim 24 or 25 for use in the treatment of cancer or for use in adoptive, target-cell specific immunotherapy.
29. A method for generating antigen-specific effector cells, which comprises the steps f
(a) providing a protein of any one of claims 1 to 18, a nucleic acid of any one of claim 19 or 22 or an expression construct of claim 23;
(b) providing a host cell or cell line,
which is selected from effector cells of the immune system, such as lymphocytes including but not limited to cytotoxic lymphocytes, T cells, cytotoxic T cells, T helper cells, Thl7 T cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages, mast cells, dendritic cells, killer dendritic cells, B cells;
(c) transferring the protein, nucleic acid, or expression construct provided in step (a) into the host cell or cell line provided in step (b);
(d) optional, selection of the transgenic cells.
The present invention also provides methods for the treatment of diseases, in particular cancer, and methods of immunotherapy, preferably including adoptive, target-cell specific immunotherapy.
30. A method for the treatment of diseases, in particular cancer, comprising the step of administering to a subject in need thereof a therapeutically effective amount of (a) a protein of any one of claims 1 to 18, a nucleic acid of any one of claim 19 or 22, an expression constmct of claim 23, a host cell of claim 24 or 25 or an antigen-specific effector cell as obtained in claim 29, and (b) optionally, respective excipient(s).
31. A method of immunotherapy, comprising the step of
administering to a subject in need thereof a therapeutically effective amount of (a) a protein of any one of claims 1 to 18, a nucleic acid of any one of claim 19 or 22, an expression construct of claim 23, a host cell of claim 24 or 25 or an antigen-specific effector cell as obtained in claim 29, and (b) optionally, respective excipient(s).
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