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WO2025191067A1 - Récepteur transmembranaire chimérique comprenant au moins immunorécepteur de lymphocytes t avec une region polypeptidique des domaines ig et itim (tigit), des lymphocytes t exprimant le récepteur de commutateur tigit humain chimérique, des vecteurs avec des acides nucléiques codant pour le récepteur tigit, des kits pour préparer les lymphocytes t, ainsi que des compositions pharmaceutiques correspondantes et des méthodes pour traiter un patient ayant une maladie et pour augmenter la cytotoxicité d'un lymphocyte t dans une thérapie cellulaire adoptive - Google Patents

Récepteur transmembranaire chimérique comprenant au moins immunorécepteur de lymphocytes t avec une region polypeptidique des domaines ig et itim (tigit), des lymphocytes t exprimant le récepteur de commutateur tigit humain chimérique, des vecteurs avec des acides nucléiques codant pour le récepteur tigit, des kits pour préparer les lymphocytes t, ainsi que des compositions pharmaceutiques correspondantes et des méthodes pour traiter un patient ayant une maladie et pour augmenter la cytotoxicité d'un lymphocyte t dans une thérapie cellulaire adoptive

Info

Publication number
WO2025191067A1
WO2025191067A1 PCT/EP2025/056870 EP2025056870W WO2025191067A1 WO 2025191067 A1 WO2025191067 A1 WO 2025191067A1 EP 2025056870 W EP2025056870 W EP 2025056870W WO 2025191067 A1 WO2025191067 A1 WO 2025191067A1
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WO
WIPO (PCT)
Prior art keywords
cell
tigit
polypeptide
receptor
region
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/056870
Other languages
English (en)
Inventor
Mikhail STEKLOV
Josephine KEMNA
Panagiota A. SOTIROPOULOU
Marleen Van Loenen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
T Knife GmbH
Original Assignee
T Knife GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by T Knife GmbH filed Critical T Knife GmbH
Publication of WO2025191067A1 publication Critical patent/WO2025191067A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/36—Immune checkpoint inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4267—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K40/4268—MAGE
    • 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
    • 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 a chimeric transmembrane receptor comprising a polypeptide, wherein the polypeptide comprises at least one T-cell immunoreceptor with Ig and ITIM domains (TIGIT) - polypeptide region comprising a TIGIT extracellular ligand binding domain, and further wherein the polypeptide comprises at least one non-TIGIT polypeptide region.
  • TIGIT T-cell immunoreceptor with Ig and ITIM domains
  • the present invention further relates to an isolated nucleic acid comprising a nuclear acid sequence encoding for the chimeric transmembrane receptor, a vector comprising the nucleic acid, and to (isolated) T-cells expressing the chimeric transmembrane receptor and/or comprising the vector or nucleic acid encoding for the chimeric transmembrane receptor.
  • the invention further relates to a kit for preparing the (isolated) T-cell of the present invention, as well as to a pharmaceutical composition comprising the T-cells.
  • the invention also relates to a method for preparing a T-cell for immunotherapy, and to methods for treating a patient having a disease comprising administering the pharmaceutical composition, and/or for increasing cytotoxicity of a T-cell in adoptive cell therapy, comprising introducing the vector into the T-cell.
  • T-cells are known to be important mediators of adaptive cell-mediated immune responses.
  • Adoptive T-cell therapy (ACT) with T-cells expressing native or transgenic ap-T-cell receptors (TCRs) is a promising treatment for cancer, as TCRs cover a wide range of potential target antigens [Chandran and Klebanoff, 2019].
  • Native TCR specificities have successfully been exploited for ACT with tumor infiltrating lymphocytes (TILs) for melanoma [Dafni et al., 2019] and other tumors [Chandran and Klebanoff, 2019], or with virus-specific T-cells (VSTs) for viral-associated malignancies [Leung and Heslop, 2019]
  • Transgenic TCR-based ACT allows the genetic redirection of T-cell specificity in a highly specific and reproducible manner, and has produced promising results in melanoma and several solid tumors [Robbins et al., 2015], multiple myeloma (
  • T-cell antigen recognition and subsequent T-cell activation is known to depend on the interaction between the T-cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) molecules [Davis and Bjdrkman, 1988],
  • TCR T-cell receptor
  • pMHC peptide-major histocompatibility complex
  • the CD8 co-receptor plays a major role in CD8 T-cell activation
  • the CD4 co-receptor stabilizes the interaction between the TCR on CD4 T-cells and the MHC class II molecule on antigen-presenting cells (APCs).
  • APCs antigen-presenting cells
  • T-cells need to receive positive signals.
  • co-signaling molecules have a crucial role in regulating T-cell activation, subset differentiation, effector function and survival.
  • CD28 is constitutively expressed on naive CD4 and CD8 T- cells and has been shown to act as positive co-stimulatory molecule.
  • CD28 engagement in the immunological synapse decreases the amount of antigen necessary to elicit T-cell activation [Kamphorst et al., 2015], [006]
  • many other costimulatory molecules have been identified.
  • TLRs Toll like receptors
  • innate immune cells particularly antigen-presenting cells
  • TLR agonists can enhance cytokine production by activated T-cells, increase T- cell sensitivity to T-cell receptor stimulation, promote long-lived T-cell memory, and reduce the suppressive activity of regulatory T-cells.
  • ACT’S targeting tumor cell specific antigen genes
  • solid tumors can effectively evade the immune response, including the promising T cell therapies, through the expression of various inhibitory molecules that can hinder the function of T cells.
  • IgSF immunoglobulin superfamily
  • TNFRSF tumor necrosis factor receptor superfamily
  • chimeric switch receptors i.e.
  • chimeric receptors comprising the extracellular domain of an inhibitory receptor and the cytoplasmic domain of an activating receptor
  • This object is inter alia accomplished by the chimeric transmembrane receptor, the (isolated) nucleic acid, the vectors, the (isolated) T-cells, the pharmaceutical compositions, the kits and the methods, having the features of the respective independent claims.
  • the invention provides a chimeric transmembrane receptor comprising a polypeptide, wherein the polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a TIGIT wildtype polypeptide as set forth in SEQ ID No.
  • TIGIT T cell immunoreceptor with Ig and ITIM domains
  • the TIGIT polypeptide region comprises a TIGIT extracellular ligand binding domain; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30 , and wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30.
  • the invention provides a chimeric transmembrane receptor comprising a polypeptide, wherein the polypeptide comprises at least one TIGIT polypeptide region, wherein the TIGIT polypeptide region comprises a TIGIT extracellular ligand binding domain; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, and wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30.
  • a chimeric TIGIT-receptor including the costimulatory domain of CD2, CD40, HVEM, or CD30, and further comprising a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, together with a TIGIT extracellular ligand binding domain, thus being engineered for use as a switch receptor, is able to turn negative signals e.g. present in a tumor microenvironment into positive signals for T-cell activation.
  • the chimeric transmembrane receptors comprising the co-stimulatory domains as herein described which are linked to the at least one TIGIT polypeptide region that is still capable to bind to its natural ligand, are conveying resistance to the T-cell to the immunosuppressive tumor microenvironment.
  • the T-cells expressing the chimeric transmembrane receptor as herein provided exhibit less TCR-T exhaustion and depletion through apoptosis, and show stimulated TCR-T proliferation and functional activity.
  • the chimeric TIGIT switch receptors as herein provided lack the cytoplasmic inhibitory motif/domain/region of the wildtype TIGIT receptor, the binding of a natural ligand to the TIGIT switch receptor, e.g. in tumor microenvironment, does no longer lead to e.g. inhibition of activation, promotion of exhaustion and/or induction of apoptosis of the T-cell expressing the chimeric TIGIT switch receptor, but - instead - even costimulates the T-cell, thereby enhancing its cytotoxic effect.
  • the invention provides a (isolated) nucleic acid encoding for the chimeric transmembrane receptor as herein provided.
  • the invention provides a vector comprising a nucleic acid comprising a nuclear acid sequence encoding for a chimeric TIGIT switch receptor as herein provided.
  • the vector in addition to comprising the nucleic acid sequence encoding for the chimeric TIGIT receptor, may further comprise the nucleic acid sequence encoding for an engineered T-cell receptor. According to a further embodiment, the vector may additionally comprise a nucleic acid encoding for a CD8 Co-receptor, such as a wildtype CD8 Co-receptor or a chimeric CD8 Co-receptor.
  • a CD8 Co-receptor such as a wildtype CD8 Co-receptor or a chimeric CD8 Co-receptor.
  • overexpression of a chimeric human TIGIT switch receptor as herein described in the T-cells as herein provided, e.g. alongside an engineered transgenic ap-T-cell receptor TCR and, in some embodiments, for example, alongside a CD8 Co-receptor, may offer several advantages and expands the therapeutic potential of this approach.
  • the optional, additional provision of the CD8 Co-receptor together with the chimeric TIGIT switch receptor and e.g. together with the engineered T-cell receptor in the T-cell of the present invention may e.g. allow for the efficient incorporation of CD4 T cells into TCR-T-cell therapy, such that it becomes possible to harness their unique properties to augment the antitumor immune response.
  • CD4 T cells possess the ability to regulate the function of other immune cells, such as CD8 cytotoxic T-cells, dendritic cells, macrophages and B cells, by providing vital signals through the secretion of cytokines and direct cell-cell interactions.
  • This known helper function may be crucial for enhancing the persistence and potency of TCR-T-cells within the tumour microenvironment.
  • the invention provides an isolated T-cell, the T-cell comprising the vector or the nucleic acid according to the present invention.
  • the invention provides an isolated T-cell, the T-cell being introduced with, such as transfected (e.g. electroporated), transduced or transformed with the vector or the nucleic acid according to the present invention.
  • the invention provides an isolated T-cell, the T-cell being treated, such as transfected (e.g. electroporated), transduced or transformed to express the chimeric TIGIT receptor as herein described.
  • the T- cell may be treated, such as transfected, transduced or transformed to express the chimeric TIGIT receptor as herein described together with an engineered T-cell receptor.
  • the invention provides a kit comprising means to prepare the isolated and/or engineered T-cells according to the present invention.
  • the invention provides a pharmaceutical composition comprising the isolated T-cell according to the present invention.
  • the invention provides a method for preparing a T-cell for immunotherapy, comprising isolating T-cells from a human subject, introducing the vector or the nucleic acid, as herein provided, into the T-cell, and expanding the T-cells in which the vector or nucleic acid has been introduced.
  • the invention provides a pharmaceutical composition comprising T-cells expressing the chimeric TIGIT receptor as herein described.
  • the composition may comprise T-cells expressing the chimeric TIGIT receptor as herein described, and further expressing an engineered T-cell receptor.
  • the invention provides a method for treating a patient having a disease, comprising administering to the patient the pharmaceutical composition according to the present invention.
  • the invention provides a method for treating a patient having a disease, comprising introducing in vivo the nucleic acid as herein described, or the vector as herein provided into a T-cell of the patient.
  • the invention provides a method for increasing cytotoxicity of a T-cell in adoptive cell therapy, comprising - introducing a vector into the T- cell, wherein the vector comprises a nucleic acid encoding for a chimeric TIGIT-receptor as herein described, and, for example, further encoding an engineered T-cell receptor.
  • the invention provides an isolated T-cell wherein the T- cell expresses a chimeric transmembrane receptor comprising a polypeptide, wherein said polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a TIGIT wildtype polypeptide as set forth in SEQ ID No.
  • TIGIT T cell immunoreceptor with Ig and ITIM domains
  • the TIGIT - polypeptide region comprises a TIGIT extracellular ligand binding domain, and further wherein the TIGIT polypeptide region comprises a TIGIT transmembrane polypeptide domain or region; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, or CD28; wherein the T-cell further expresses a recombinant T-cell receptor.
  • the invention provides an isolated T-cell wherein the T- cell expresses a chimeric transmembrane receptor comprising a polypeptide, wherein said polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region comprising a TIGIT extracellular ligand binding domain, and further wherein the TIGIT polypeptide region comprises a TIGIT transmembrane polypeptide domain or region; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, or CD28; wherein the T-cell further expresses a recombinant T-cell receptor.
  • TIGIT T cell immunoreceptor with Ig and ITIM domains
  • the T-cells comprising both an engineered T-cell receptor, and a chimeric TIGIT receptor including the costimulatory domain of CD2 or CD28, comprising a TIGIT extracellular ligand binding domain, and further comprising a TIGIT transmembrane polypeptide domain or region, thus being engineered for use as a switch receptor, are able to turn negative signals e.g. present in a tumor microenvironment into positive signals for T-cell activation.
  • the T-cells as provided according to the fifteenth or sixteenth aspect of the invention by expressing both an engineered T-cell receptor and specifically engineered recombinant chimeric TIGIT receptors, linking the co-stimulatory domains of CD2 or CD28 to at least one TIGIT polypeptide region that is still capable to bind to its natural ligand and to a TIGIT transmembrane polypeptide domain or region, is having resistance to the immunosuppressive tumor microenvironment.
  • the T-cells exhibit less TCR-T exhaustion and depletion through apoptosis, and show stimulated TCR-T proliferation and functional activity.
  • the binding of a natural ligand to the TIGIT switch receptor e.g.
  • this invention therefore provides T-cells comprising the chimeric TIGIT receptors according to the fifteenth or sixteenth aspect, and it is herewith contemplated that the chimeric TIGIT receptors according to the fifteenth or sixteenth aspect may also be used in the compositions, kits and methods of the present invention. [0037] All aspects of the invention provide the above described advantages and improvements related to the provision of
  • a chimeric transmembrane receptor comprising a polypeptide, wherein the polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region (having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a TIGIT wildtype polypeptide as set forth in SEQ ID No.
  • TIGIT T cell immunoreceptor with Ig and ITIM domains
  • the TIGIT polypeptide region comprises a TIGIT extracellular ligand binding domain; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30 , and wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30, or the provision of
  • T- T-cells comprising both an engineered/recombinant T-cell receptor and a chimeric transmembrane receptor comprising a polypeptide
  • said polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region (having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a TIGIT wildtype polypeptide as set forth in SEQ ID No.
  • TIGIT T cell immunoreceptor with Ig and ITIM domains
  • the switch receptors and T-cells herein provided are capable of turning negative signals (with respect to e.g. the T-cell’s activation status and/or cytotoxic capacity) into positive signals.
  • Fig. 1 shows a graphical representation of results from a flow cytometric analysis of T-cells transduced with chimeric TIGIT receptor polypeptides. For better clarity, Fig. 1 is depicted on four pages (drawing sheets 1/8 - 4/8).
  • Fig. 2 shows a graphical representation of results from an in-vitro T-cell killing assay of T-cells according to the invention transduced with an engineered T-cell receptor and a chimeric TIGIT receptor polypeptide in CorL23-A2-NLR cells overexpressing TIGIT ligands CD155- and Nectin4.
  • Fig. 2 is depicted on four pages (drawing sheets 5/8 - 8/8).
  • the invention is directed to a chimeric transmembrane receptor comprising a polypeptide, wherein the polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region having at least 60% sequence identity with a polypeptide domain, a polypeptide region or a polypeptide motif of a TIGIT wildtype polypeptide as set forth in SEQ ID No.
  • TIGIT T cell immunoreceptor with Ig and ITIM domains
  • the TIGIT polypeptide region comprises a TIGIT extracellular ligand binding domain; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, and wherein the at least one non- TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30.
  • the invention provides a chimeric transmembrane receptor comprising a polypeptide, wherein the polypeptide comprises at least one TIGIT polypeptide region, wherein the TIGIT polypeptide region comprises a TIGIT extracellular ligand binding domain; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, and wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30.
  • the TIGIT polypeptide region may be a human TIGIT polypeptide region from a human wildtype TIGIT receptor.
  • human wildtype TIGIT receptor relates to a protein having an amino acid sequence according to UniProtKB database entry No. Q495A1 ⁇ TIGIT_HUMAN, as set forth e.g. in SEQ ID No. 1.
  • TIGIT polypeptide region refers to a polypeptide containing at least a functional portion (e.g., an extracellular ligand binding domain) of a wild-type TIGIT protein or a variant thereof, such as a variant that has at least 60% sequence identity (e.g., at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence of SEQ ID NO: 1 and that retains the ability to bind an endogenous TIGIT ligand.
  • a functional portion e.g., an extracellular ligand binding domain
  • TIGIT immunoglobulin variable domain refers to a portion of wild-type TIGIT comprising the corresponding protein domain (i.e., the immunoglobulin variable domain, co-stimulatory cytoplasmic polypeptide domain, or homodimerization motif, respectively, of wild-type TIGIT) or a sequence variant thereof, such as a sequence variant recited herein.
  • non-TIGIT polypeptide domains, regions, or motifs refers to a polypeptide domain, region, and motif, respectively, that is neither obtained from wild-type TIGIT nor is a functional variant thereof.
  • non-TIGIT polypeptide domains, regions, or motifs are those that are obtained from, e.g., CD2, CD40, HVEM, CD28 or CD30, as well as sequence variants thereof, such as sequence variants recited herein.
  • Other Examples of non-TIGIT polypeptide domains, regions, or motifs are e.g. those that may be obtained from other costimulatory molecules, as well as sequence variants thereof.
  • the extracellular ligand binding domain may comprise a TIGIT immunoglobulin variable (IgV) domain, wherein the TIGIT IgV domain may have at least 85% sequence identity to the amino acid sequence of SEQ ID No. 2.
  • IgV immunoglobulin variable
  • the at least one TIGIT IgV domain may have at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID No. 2.
  • sequence identity means the percentage of pair-wise identical residues, following homology alignment of a sequence of a polypeptide and or nucleic acid of the present invention with a sequence in question, with respect to the number of residues in the longer of these two sequences.
  • the percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP, version blastp 2.2.5 (November 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res. 25, 3389-3402).
  • the percentage of homology is based on the alignment of the entire polypeptide sequences (matrix: BLOSIIM 62; gap costs: 11.1 ; cutoff value set to 10-3) including the respective sequences. It is calculated as the percentage of numbers of "positives" (homologous amino acids) indicated as result in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.
  • the chimeric TIGIT receptor comprising both a functional extracellular TIGIT receptor ligand binding domain and at least one non-TIGIT co-stimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30, as well as a non-TIGIT transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, is able to turn negative signals (e.g. present in a tumor microenvironment) into positive signals for T-cell activation.
  • replacing e.g. at least the cytoplasmic “ITIM” motif of wildtype TIGIT amino acids 229-243 of SEQ ID No.
  • the at least one TIGIT polypeptide region may have at least 70%, or at least 71 %, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the functional polypeptide domain or a functional polypeptide motif of a wildtype human TIGIT receptor (e.g. Seq ID No. 1).
  • a wildtype human TIGIT receptor e.g. Seq ID
  • the (human) TIGIT polypeptide region comprising a TIGIT extracellular ligand binding domain may have -in general- a sufficient portion of the human wildtype TIGIT extracellular ligand binding domain to be functional in binding at least one TIGIT ligand.
  • said at least one TIGIT polypeptide region having at least 60% sequence identity with TIGIT ligand binding domain of a human wildtype TIGIT receptor may comprise the complete or a considerable part of the human wildtype TIGIT ligand binding domain and/or all amino acids at respective amino acid positions of the wildtype TIGIT receptor that are necessary and sufficient for binding of at least one ligand to the TIGIT receptor.
  • the TIGIT polypeptide region may comprise a complete wildtype TIGIT receptor extracellular domain.
  • the expression “wildtype TIGIT receptor extracellular domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 22-141 of UniProtKB database entry No. Q495A1 ⁇ TIGIT_HUMAN, as set forth e.g. in SEQ ID No. 1.
  • the wildtype TIGIT receptor extracellular domain as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 4.
  • the TIGIT receptor extracellular domain as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the extracellular domain of the wildtype TIGIT receptor.
  • all amino acid substitutions that maintain the functional activity of the wildtype TIGIT extracellular domain are envisaged.
  • domain region As used herein are understood to relate to e.g. a region of the chimeric transmembrane receptor polypeptide which is necessary and/or sufficient for a biological function of the chimeric receptor, or to a region of the chimeric TIGIT receptor which is defined e.g. by a localization with respect to a cell, or to a structurally defined unit of the chimeric TIGIT receptor polypeptide.
  • chimeric transmembrane receptor as herein provided may comprise
  • CD2 a transmembrane region from CD2 and a costimulatory cytoplasmic polypeptide domain, region or motif from CD2, or
  • HVEM transmembrane region from HVEM and a costimulatory cytoplasmic polypeptide domain, region or motif from HVEM, or
  • CD30 a transmembrane region from CD30 and a costimulatory cytoplasmic polypeptide domain, region or motif from CD30.
  • the at least one non-TIGIT polypeptide region may comprise a transmembrane polypeptide region of CD2.
  • the transmembrane domain of wildtype CD2 as referred to herein may relate to a polypeptide comprising amino acid sequence 210-235 of UniProtKB database entry P06729 ⁇ CD2_HUMAN, as set forth e.g. in SEQ ID No. 5.
  • the transmembrane domain of wildtype CD2 as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 6.
  • transmembrane domain as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the transmembrane domain of the wildtype CD2 receptor.
  • all amino acid substitutions that maintain the functional activity of the wildtype transmembrane domain are envisaged.
  • the non TIGIT transmembrane polypeptide region may have at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 6.
  • the at least one non-TIGIT polypeptide region may comprise a transmembrane polypeptide region of CD40.
  • the transmembrane domain of wildtype CD40 as referred to herein may relate to a polypeptide comprising amino acid sequence 194-215 of UniProtKB database entry No. P25942- TNR5_HUMAN, as set forth e.g. in SEQ ID No. 7.
  • the transmembrane domain of wildtype CD40 as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 8.
  • transmembrane domain as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the transmembrane domain of the wildtype CD40 receptor.
  • all amino acid substitutions that maintain the functional activity of the wildtype transmembrane domain are envisaged.
  • the non TIGIT transmembrane polypeptide region may have at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 8.
  • the at least one non-TIGIT polypeptide region may comprise a transmembrane polypeptide region of HVEM.
  • the transmembrane domain of wildtype HVEM as referred to herein may relate to a polypeptide comprising amino acid sequence 203-223 of UniProtKB database entry No. Q92956- TNR14_HUMAN, as set forth e.g. in SEQ ID No. 9.
  • the transmembrane domain of wildtype HVEM as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 10.
  • transmembrane domain as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the transmembrane domain of the wildtype HVEM receptor.
  • all amino acid substitutions that maintain the functional activity of the wildtype transmembrane domain are envisaged.
  • the non TIGIT transmembrane polypeptide region may have at least 70%, or at least 71 %, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 10.
  • the at least one non-TIGIT polypeptide region may comprise a transmembrane polypeptide region of CD30.
  • the transmembrane domain of wildtype CD30 as referred to herein may relate to a polypeptide comprising amino acid sequence 386-406 of UniProtKB database entry P28908 ⁇ TNR8_HUMAN, as set forth e.g. in SEQ ID No. 11.
  • the transmembrane domain of wildtype CD30 as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 12.
  • transmembrane domain as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the transmembrane domain of the wildtype CD30 receptor.
  • all amino acid substitutions that maintain the functional activity of the wildtype transmembrane domain are envisaged.
  • the non TIGIT transmembrane polypeptide region may have at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the amino acid sequence as set forth in SEQ ID NO: 12.
  • the transmembrane domain of the chimeric transmembrane receptor of the T-cell as herein provided may be from other proteins which comprise a transmembrane domain, except TIGIT.
  • the transmembrane domain may be from a further co-stimulatory molecule known in the art.
  • any transmembrane domain which is functional and allows surface detectable expression of the chimeric transmembrane receptor is herewith envisaged.
  • the chimeric transmembrane receptor as herein provided may further comprise at least one linker region.
  • This may be e.g. a polypeptide linker region.
  • Such linker(s) may be included e.g. between functional domains/regions/motifs of the chimeric transmembrane receptor. It may be a linker region naturally occurring e.g. in wildtype TIGIT receptor, or e.g. in co-stimulatory proteins, e.g. in costimulatory proteins from which the costimulatory domain of the receptor is derived.
  • polypeptide linker regions may be included between the transmembrane domain and the ligand binding domain, and/or between the transmembrane domain and the IgV domain of the chimeric TIGIT receptor, and/or between the transmembrane domain and the at least one intracellular co-stimulatory domain, and/or between individual co-stimulatory domains (in embodiments comprising more than one co-stimulatory domains).
  • Such linker region may comprise 1-100 amino acids, or e.g. 1-80 amino acids, or e.g. 1-50 amino acids, or e.g. 5-100 amino acids.
  • a linker region of the chimeric transmembrane receptor as herein provided may comprise the amino acid sequence as set forth in SEQ ID No. 13 (GGGS)n or as set forth in Seq ID No. 14 (GGGGS) n , wherein n is between 0 and 20, or wherein n is between 0 and 10, or where n is between 0 and 5, or where n is between 3 and 5.
  • each (polypeptide) linker known in the art is herewith envisaged as being potentially included in the chimeric transmembrane switch receptor of the present invention.
  • the chimeric transmembrane receptor polypeptide as herein provided may comprise at least one costimulatory cytoplasmic polypeptide domain or cytoplasmic polypeptide motif of CD2, CD40, HVEM, or CD30.
  • cytoplasmic co-stimulatory domains of CD2, CD40, HVEM, or CD30 may be fused to the at least one TIGIT polypeptide region comprising a functional TIGIT receptor extracellular ligand binding domain in order to generate a functional chimeric TIGIT switch receptor capable of redirecting the signaling pathways triggered by TIGIT receptor engagement with at least one TIGIT ligand such that - instead of inducing inhibitory pathways in the cell - binding of the at least one TIGIT ligand promotes T-cell activation, persistence and enhanced anti-tumor responses of a T-cell expressing the chimeric transmembrane receptors as herein provided, for example when the T-cell at the same time comprises/expresses e.g. an engineered T-cell receptor.
  • the chimeric TIGIT receptor as herein provided may comprise e.g. at least one complete cytoplasmic domain of CD2, CD40, HVEM, or CD30.
  • the at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif selected from the group consisting of CD2, CD40, HVEM, and CD30 may have an amino acid sequence having at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the respective functional polypeptide domain or a functional polypeptide
  • the chimeric transmembrane receptor as herein provided may be able to sustain or enhance cytotoxicity and/or cytokine secretion of a T-cell upon binding a TIGIT ligand.
  • the chimeric transmembrane receptor as herein provided may be capable of increasing resistance of T-cells to TIGIT ligand expressing cancer cells.
  • the chimeric TIGIT receptor as herein provided may comprise any functional combination of co-stimulatory cytoplasmic polypeptide domain(s) motif(s) and/or region(s) of the cytoplasmic polypeptide domain, region or motif selected from the group consisting of CD2, CD40, HVEM, and CD30.
  • the chimeric transmembrane receptor as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of wildtype CD40.
  • the expression “wildtype human CD40 cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 216- 277 of UniProtKB database entry No. P25942- TNR5_HUMAN, as set forth e.g. in SEQ ID No. 7.
  • the cytoplasmic polypeptide domain, region or motif of wildtype CD40 as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID No. 15.
  • all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD40 are envisaged.
  • the polypeptide of the chimeric transmembrane receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO: 16.
  • the chimeric transmembrane receptor as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of HVEM.
  • a cytoplasmic polypeptide region of HVEM may comprise the complete cytoplasmic domain of wildtype human HVEM.
  • the cytoplasmic polypeptide region of HVEM included in the chimeric transmembrane receptor may comprise at least one functional, co-stimulatory motif/domain/region of the complete wildtype human HVEM cytoplasmic domain.
  • the expression “wildtype human HVEM cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 224-283 of UniProtKB database entry No.
  • the cytoplasmic polypeptide domain, region or motif of wildtype HVEM as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID No. 17.
  • all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype HVEM are envisaged.
  • the polypeptide of the chimeric transmembrane receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO: 18.
  • the chimeric transmembrane receptor as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of CD30.
  • a cytoplasmic polypeptide region of CD30 may comprise the complete cytoplasmic domain of wildtype human CD30.
  • the cytoplasmic polypeptide region of CD30 included in the chimeric transmembrane receptor may comprise at least one functional, co-stimulatory motif/domain/region of the complete wildtype human CD30 cytoplasmic domain.
  • the polypeptide comprises a truncated cytoplasmic domain of CD30, optionally wherein the truncated cytoplasmic domain of CD30 comprises or consists of at least one CD30 TRAF binding motif.
  • the expression “wildtype human CD30 cytoplasmic domain” as referred to herein may relate to a polypeptide comprising amino acid sequence 407- 595 of UniProtKB database entry P28908 ⁇ TNR8_HUMAN, as set forth e.g. in SEQ ID No. 11.
  • the cytoplasmic polypeptide domain, region or motif of wildtype CD30 as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID No. 19.
  • all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD30 are envisaged.
  • the polypeptide of the chimeric transmembrane receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO.: 20.
  • the chimeric transmembrane receptor as herein provided may comprise at least one cytoplasmic polypeptide domain, region or motif of CD2.
  • a cytoplasmic polypeptide region of CD2 may comprise the complete cytoplasmic domain of wildtype human CD2.
  • the cytoplasmic polypeptide region of CD2 included in the chimeric transmembrane receptor may comprise at least one functional, co-stimulatory motif/domain/region of the complete wildtype human CD2 cytoplasmic domain.
  • wildtype human CD2 cytoplasmic domain as referred to herein may relate to a polypeptide comprising amino acid sequence 236-351 of UniProtKB database entry P06729 ⁇ CD2_HUMAN, as set forth e.g. in SEQ ID No. 5. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype CD2 as included in the chimeric transmembrane receptor of the T-cell as herein provided may have one or more conservative amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID No. 21 . In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD2 are envisaged.
  • the polypeptide of the chimeric transmembrane receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO.: 22.
  • a chimeric transmembrane receptor as herein provided may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in any one of the SEQ ID No’s selected from the group consisting of SEQ ID No. 16, 18, 20, and 22.
  • a chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to an amino acid sequence as set forth in any one of the SEQ ID No’s selected from the group consisting of SEQ ID No. 16, 18, 20, and 22.
  • the invention provides a (isolated) nucleic acid encoding for the chimeric transmembrane receptor as herein provided.
  • the invention provides a vector comprising a nucleic acid comprising a nuclear acid sequence encoding for a chimeric transmembrane switch receptor (chimeric TIGIT switch receptor) as herein provided.
  • a chimeric transmembrane switch receptor chimeric TIGIT switch receptor
  • polynucleotide or “nucleic acid” as used herein comprises a sequence of polyribonucleotides and polydeoxribonucleotides, e.g. modified or unmodified RNA or DNA, each in single-stranded and/or double-stranded form linear or circular, or mixtures thereof, including hybrid molecules.
  • the nucleic acids according to this invention thus comprise DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA ivtRNA), combinations thereof or derivatives (such as RNA) thereof.
  • a polynucleotide may comprise a conventional phosphodiester bond or a non- conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (RNA)).
  • the polynucleotides of the invention may also contain one or more modified bases, such as, for example, tritylated bases and unusual bases such as inosine. Other modifications, including chemical, enzymatic, or metabolic modifications, are also conceivable, as long as a binding molecule of the invention can be expressed from the polynucleotide.
  • the polynucleotide may be provided in isolated form as defined elsewhere herein.
  • a polynucleotide may include regulatory sequences such as transcription control elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding site, introns, or the like.
  • the present invention provides a polynucleotide comprising or consisting of a nucleic acid that is at least about 80 %, about 85 %, about 90 %, about 91 %, about 92 %, about 93 %, about 94 %, about 95 %, about 96 %, about 97 %, about 98 %, about 99 %, or 100 % identical to a reference polynucleotide sequence selected from the group consisting of sequences as depicted in SEQ ID NOs: 23 - 26.
  • polynucleotides described above may or may not comprise additional or altered nucleotide sequences encoding e.g., altered amino acid residues.
  • the polynucleotides may further encode fusion polypeptides, fragments, variants and other derivatives of the chimeric transmembrane receptors described herein.
  • the nucleic acid sequences of the vectors of the present invention may be codon-optimized for optimal expression in the desired host T-cell, e.g. a human lymphocyte; or for expression in bacterial, yeast or insect T-cells that are particularly envisaged for the expression of a soluble TCR of the invention.
  • 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. Selection of optimum codons thus depends on codon usage of the host genome and the presence of several desirable and undesirable sequence motifs.
  • a “vector” as understood herein relates to a nucleic acid molecule used as a vehicle to transfer (foreign) genetic material into a host T-cell where it can for instance be replicated and/or expressed.
  • the vector may be a viral vector or a non-viral vector.
  • Viral vectors may be selected from adenoviruses, poxviruses, alphaviruses, arenaviruses, flaviruses, rhabdoviruses, retroviruses, lentiviruses, herpesviruses, paramyxoviruses, picornaviruses, and combinations thereof.
  • Viruses used for transfection of T-cells may include naturally occurring viruses as well as artificial viruses. Viruses may be either an enveloped or non-enveloped virus. Parvoviruses (such as AAVs) are examples of non-enveloped viruses.
  • the viruses may be enveloped viruses.
  • the viruses used for transfection of T-cells may be retroviruses and in particular lentiviruses.
  • Viral envelope proteins that can promote viral infection of eukaryotic cells may comprise HIV- 1 derived lentiviral vectors (LVs) pseudotyped with envelope glycoproteins (GPs) from the vesicular stomatitis virus (VSV-G), the modified feline endogenous retrovirus (RD114TR), and the modified gibbon ape leukemia virus (GALVTR).
  • LVs HIV- 1 derived lentiviral vectors
  • GPs envelope glycoproteins
  • VSV-G vesicular stomatitis virus
  • RD114TR modified feline endogenous retrovirus
  • GALVTR gibbon ape leukemia virus
  • viruses such as parvoviruses, including adeno- associated viruses (AAV), thereby demonstrating their broad efficiency.
  • viruses such as parvoviruses, including adeno- associated viruses (AAV), thereby demonstrating their broad efficiency.
  • other viral envelop proteins may be used including Moloney murine leukemia virus (MLV)
  • RD114 env chimeric envelope protein RD114pro or RDpro (which is an RD114-HIV chimera that was constructed by replacing the R peptide cleavage sequence of RD114 with the HIV-1 matrix/capsid (MA/CA) cleavage sequence, such as described in Bell et al. Experimental Biology and Medicine 2010; 235: 1269-1276; the content of which is incorporated herein by reference), baculovirus GP64 env (such as described in Wang et al. J. Virol.
  • Non-viral vectors may comprise a naked nucleic acid such as naked plasmid DNA, cationic lipids, synthetic polycationic polymers, dendrimers, synthetic peptides such as cell-penetrating peptides (CPP’s), p-1 ,3- glucans, or combinations thereof
  • a naked nucleic acid such as naked plasmid DNA, cationic lipids, synthetic polycationic polymers, dendrimers, synthetic peptides such as cell-penetrating peptides (CPP’s), p-1 ,3- glucans, or combinations thereof
  • vector encompasses, without limitation, plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors, and adenovirus-associated vectors (AAV)), phages, phagemids, cosmids and artificial chromosomes (including BACs and YACs).
  • viral vectors including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors, and adenovirus-associated vectors (AAV)
  • phages phagemids
  • cosmids and artificial chromosomes including BACs and YACs.
  • the vector itself is generally a nucleotide sequence, commonly a DNA sequence that comprises an insert (transgene) and a larger sequence that serves as the “backbone” of the vector.
  • Engineered vectors typically comprise an origin for autonomous replication in the host-cells (if stable expression of the polynucleotide is desired), selection markers, and restriction enzyme cleavage sites (e.g. a multiple cloning site, MCS).
  • the vector may additionally comprise promoters, genetic markers, reporter genes, targeting sequences, other regulatory elements, and/or protein purification tags. As known to those skilled in the art, large numbers of suitable vectors are known to those of skill in the art and many are commercially available.
  • the vector may further comprise a nucleic acid encoding a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the vector may further comprise a nucleic acid encoding a T-cell receptor comprising a TCRa chain and a TCRp chain.
  • the engineered T-cell receptor may be a recombinant/engineered T-cell receptor.
  • the vector may further comprise a nucleic acid encoding for a CD8 Co-receptor.
  • the CD 8 Co-receptor may be a wildtype CD 8 Co-receptor. It is contemplated that the nucleic acid may encode e.g. a CD8a and a CD8p Co-receptor.
  • An advantage of incorporation of CD8 co-receptor into the vector is the resulting option of achieving a coordinated CD4+ and CD8+ TCR-T cell response in adoptive cell therapy which broadens and deepens clinical responses.
  • the CD8 Co-receptor may be a chimeric CD8 Co-receptor with CD8 Co-receptor functionality.
  • the expression human wildtype CD8a Co-receptor relates to a protein having an amino acid sequence according to UniProtKB database entry No. P01732 ⁇ CD8A_HUMAN, as set forth e.g. in SEQ ID No. 27. It is further understood that the expression human wildtype CD8p Co-receptor relates to a protein having an amino acid sequence according to UniProtKB database entry No. P10966 ⁇ CD8B_HUMAN, as set forth e.g. in SEQ ID No. 28.
  • a chimeric human CD8 Co-receptor polypeptide as referred to herein may comprise a single chain polypeptide comprising both an CD8a IG-like domain region together with an CD8p IG-like domain region, and may be able to maintain the function of an CD8a IG-like domain region and an CD8p IG-like domain region being present on individual, separate polypeptides in a wildtype CD8a co-receptor.
  • T-cells as herein provided comprising the chimeric transmembrane receptor, the engineered T-cell receptor and, in addition, a chimeric CD8 Co-receptor therefore exhibit enhanced T-cell activation, proliferation, cytokine production, and cytotoxicity, ultimately improving the therapeutic efficacy of TCR-T-cell therapy.
  • the vector may further include one or more multicistronic element(s) and the multicistronic element(s) may be positioned, for example, between any two nucleic acid sequences encoding for the chimeric transmembrane receptor, and the optional TCRa, TCRp, and CD8 Coreceptor.
  • the multicistronic element(s) may include a sequence encoding a ribosome skip element selected from among a T2A, a P2A, a E2A or a F2A or an internal ribosome entry site (IRES).
  • self-cleaving 2A peptide refers to relatively short peptides (of the order of 20 amino acids long, depending on the virus of origin) acting co- translationally, by preventing the formation of a normal peptide bond between the glycine and last proline, resulting in the ribosome skipping to the next codon, and the nascent peptide cleaving between the Gly and Pro. After cleavage, the short 2A peptide remains fused to the C-terminus of the 'upstream’ protein, while the proline is added to the N- terminus of the 'downstream’ protein.
  • Self-cleaving 2A peptide may be selected from porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), foot- and-mouth disease virus (F2A), or any combination thereof.
  • P2A porcine teschovirus-1
  • E2A equine rhinitis A virus
  • T2A Thosea asigna virus
  • F2A foot- and-mouth disease virus
  • linker sequences GGSG or SGSG [SEQ ID No.: 48]
  • this may enable efficient synthesis of biologically active proteins, e.g., TCRs and chimeric transmembrane receptors as described herein.
  • an isolated T-cell comprising a nucleic acid encoding for the chimeric human transmembrane receptor of the present invention.
  • the T-cell may further comprise a nucleic acid encoding for an engineered T-cell receptor or a CAR.
  • the T-cell may further comprise a nucleic acid encoding for a recombinant CD8 Co-receptor.
  • the one or more nucleic acid may have been stably integrated into the genome of the T-cell, e.g. by targeted knock-in utilizing e.g. CRISPR/Cas9.
  • a T-cell may express the chimeric transmembrane receptor as herein described.
  • the T-cell may be a CD4 T-cell, and further the CD4 T-cell may additionally expresses a recombinant human CD8 co-receptor, such as e.g. a CD8a and CD8p receptor, or a chimeric CD8 co-receptor.
  • a recombinant human CD8 co-receptor such as e.g. a CD8a and CD8p receptor, or a chimeric CD8 co-receptor.
  • the vector and/or the nucleic acid as herein described may have been introduced into the T-cell.
  • the (isolated) T-cells may be generated using various methods, including those recognized in the literature.
  • a polynucleotide encoding an expression cassette that comprises a tumor recognition, or another type of recognition moiety, and that also encodes for a chimeric transmembrane receptor as herein described and, optionally, the engineered T-cell receptor or a CAR and, optionally, a CD8 Co-receptor may be stably introduced into the T-cell by a transposon/transposase system or a viralbased gene transfer system, such as a lentiviral or a retroviral system, or another suitable method, such as transfection, electroporation, transduction, lipofection, calcium phosphate (CaPCll), nanoengineered substances, such as Ormosil, mRNA-based therapy, viral delivery methods, including adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, or another suitable method. It is envisaged that
  • the T-cells may be transfected by means known in the art including lipofection (liposome-based transfection), electroporation, calcium phosphate transfection, biolistic particle delivery (e.g., gene guns), microinjection, or combinations thereof.
  • lipofection liposome-based transfection
  • electroporation calcium phosphate transfection
  • biolistic particle delivery e.g., gene guns
  • microinjection microinjection
  • Various methods of transfecting cells are known in the art. See, e.g., Sambrook & Russell (Eds.) Molecular Cloning: A Laboratory Manual (3rd Ed.) Volumes 1-3 (2001) Cold Spring Harbor Laboratory Press; Ramamoorth & Narvekar “Non Viral Vectors in Gene Therapy- An Overview.” JCIinDiagn Res. (2015) 9(1): GE01-GE06.
  • the cell may be an p T-cell, y8 T-cell, and/or a natural killer T-cell.
  • the ap T-cell may be a CD4 T-cell, or the ap T-cell may be a CD8 T-cell, or the y8 T-cell may comprise e.g. a Vy1 chain or a Vy2 chain, or may be e.g. a Vy9V82+ T-cell.
  • the T-cell may express a chimeric transmembrane receptor as herein described.
  • the T-cell may further express an engineered T-cell receptor or a CAR.
  • the T-cell may be a CD 4 T-cell that further expresses a CD8 Co-receptor, e.g. both CD8a and CD8p Co-receptor, or any engineered protein exhibiting CD8 Co-receptor functionality.
  • the T-cells may further express an engineered T-cell receptor.
  • Engineered T-cells of the present disclosure can be used to treat a subject in need of treatment for a condition, for example, a cancer described herein.
  • the T-cells may be ap T-cells or y8 T-cells that express the chimeric transmembrane receptor polypeptide as described herein, and furthermore an engineered TCR.
  • the T- cells may further express a CD8 Co-receptor such as a wildtype or chimeric CD8 co- receptor.
  • T-cells described herein may be used to treat a cancer, including solid tumors and hematologic malignancies. For example, “hot” tumors or “cold” tumors may be treated by the T-cells herewith provided.
  • the engineered T-cell receptor as herein described may specifically bind a MAGE antigen family member, such as MAGE-A1 or MAGE-A4, or wherein the engineered T-cell receptor may specifically bind an antigen selected from the group consisting of a PRAME antigen, a NY-ESO-1 antigen, a GP100 antigen, an AFP antigen, a Col6A3 antigen, an HPV-16 antigen, a WT1 antigen, an HA1 antigen, an HA2 antigen, a mutated KRAS antigen, a mutated NRAS antigen, a mutated HRAS antigen, a mutated TP53 antigen, and an EGFR antigen.
  • a PRAME antigen such as MAGE-A1 or MAGE-A4
  • a GP100 antigen such as MAGE-A1 or MAGE-A4
  • an antigen selected from the group consisting of a PRAME antigen, a NY-ESO-1 antigen,
  • the expression “mutated” with respect to specific tumor antigens as herein used relates to well-known mutations within the epitope region of the respective protein, polypeptide or peptide that has been correlated with expression in a human cancer.
  • the T-cells described herein may also be used to treat an infectious disease.
  • the T-cells described herein may be used to treat an infectious disease; an infectious disease may be caused a virus.
  • the T-cells described herein may be used to treat an immune disease, such as an autoimmune disease.
  • the T- cells may be ap T-cells or y8 T-cells that express a chimeric transmembrane receptor as described herein, and optionally an engineered TCR, and optionally a CD8 Co-receptor such as a wildtype or chimeric CD8 co-receptor.
  • the T-cell may be derived from a healthy subject, or the T-cell may be derived from a patient suffering from a disease.
  • the T-cell may be derived from an induced pluripotent stem cell (iPSCs).
  • iPSCs induced pluripotent stem cell
  • the TIGIT polypeptide region comprises a TIGIT extracellular ligand binding domain, and further wherein the TIGIT polypeptide region comprises a TIGIT transmembrane polypeptide domain or region; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2 or CD28, wherein the T-cell further expresses a recombinant T-cell receptor.
  • an isolated T-cell wherein the T-cell expresses a chimeric transmembrane receptor comprising a polypeptide, wherein said polypeptide comprises at least one T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide region comprising a TIGIT extracellular ligand binding domain, and further wherein the TIGIT polypeptide region comprises a TIGIT transmembrane polypeptide domain or region; further wherein the polypeptide comprises at least one non-TIGIT polypeptide region, wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2 or CD28, wherein the T-cell further expresses a recombinant T-cell receptor.
  • TIGIT T cell immunoreceptor with Ig and ITIM domains
  • T-cells comprising a chimeric TIGIT-receptor including the costimulatory domain of CD2 or CD28, and further comprising a transmembrane polypeptide region from TIGIT, as well as a TIGIT extracellular ligand binding domain, thus being engineered for use as a switch receptor, is able to turn negative signals e.g. present in a tumor microenvironment into positive signals for T-cell activation.
  • the inventors could show for the first time that, if expressed in T-cells which further express an engineered T-cell receptor, the chimeric transmembrane receptors comprising the at least one co-stimulatory domain of CD2 or CD28 which are linked to the TIGIT transmembrane domain and to the at least one TIGIT polypeptide region that is still capable to bind to its natural ligand, the chimeric transmembrane receptors are conveying resistance to the T-cell to the immunosuppressive tumor microenvironment.
  • the T-cells according to the fifteenth or sixteenth aspect of the invention exhibit less TCR-T exhaustion and depletion through apoptosis, and show stimulated TCR-T proliferation and functional activity.
  • the TIGIT polypeptide region of the chimeric receptor of the T- cell comprises a TIGIT transmembrane polypeptide domain or region.
  • the transmembrane domain of wildtype TIGIT as referred to herein may relate to a polypeptide comprising amino acid sequence 142-162 of UniProtKB database entry No. Q495A1 ⁇ TIGIT_HUMAN, as set forth e.g. in SEQ ID No. 1.
  • the transmembrane domain of wildtype TIGIT as referred to herein may relate to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 29.
  • transmembrane domain as included in the chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to the transmembrane domain of the wildtype TIGIT receptor.
  • all amino acid substitutions that maintain the functional activity of the wildtype transmembrane domain are envisaged.
  • the chimeric transmembrane receptor of the T-cell in accordance with the fifteenth or sixteenth aspect may further comprise at least one linker region.
  • This may be e.g. a polypeptide linker region.
  • Such linker(s) may be included e.g. between functional domains/regions/motifs of the chimeric transmembrane receptor. It may be a linker region naturally occurring e.g. in wildtype TIGIT receptor, or e.g. in co-stimulatory proteins, e.g. in costimulatory proteins from which the costimulatory domain of the receptor is derived.
  • polypeptide linker regions may be included between the transmembrane domain and the ligand binding domain, and/or between the transmembrane domain and the IgV domain of the chimeric TIGIT receptor, and/or between the transmembrane domain and the at least one intracellular co-stimulatory domain, and/or between individual co-stimulatory domains (in embodiments comprising more than one co-stimulatory domains).
  • Such linker region may comprise 1-100 amino acids, or e.g. 1-80 amino acids, or e.g. 1-50 amino acids, or e.g. 5-100 amino acids.
  • a linker region of the chimeric transmembrane receptor as herein provided may comprise the amino acid sequence as set forth in SEQ ID No.13 (GGGS)n or as set forth in Seq ID No. 14 (GGGGS) n , wherein n is between O and 20, or wherein n is between 0 and 10, or where n is between 0 and 5, or where n is between 3 and 5.
  • each (polypeptide) linker known in the art is herewith envisaged as being potentially included in the chimeric transmembrane switch receptor of the T-cells according to the fifteenth or sixteenth aspect.
  • the chimeric transmembrane receptor polypeptide of the T-cells may comprise at least one costimulatory cytoplasmic polypeptide domain or cytoplasmic polypeptide motif of CD2, or CD28.
  • cytoplasmic co-stimulatory domains of CD2, or CD28 may be fused to the at least one TIGIT polypeptide region comprising a functional TIGIT receptor extracellular ligand binding domain, and further comprising a functional TIGIT transmembrane polypeptide region, in order to generate a functional chimeric TIGIT switch receptor capable of redirecting the signaling pathways triggered by TIGIT receptor engagement with at least one TIGIT ligand such that - instead of inducing inhibitory pathways in the cell - binding of the at least one TIGIT ligand promotes T-cell activation, persistence and enhanced anti-tumor responses of a T-cell expressing the chimeric transmembrane receptors as herein provided, if the T-cell at the same time comprises/expresses an engineered T-cell receptor.
  • the T-cells expressing the chimeric TIGIT receptor according to the fifteenth or sixteenth aspect may comprise e.g. at least one complete cytoplasmic domain of CD2 or CD28.
  • the at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif selected from the group consisting of transmembrane polypeptide region of CD2 or CD28 may have an amino acid sequence having at least 70%, or at least 71 %, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81 %, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the respective functional polypeptide domain or a
  • T-cell comprising the chimeric transmembrane receptor together with the engineered TOR according to the fifteenth or sixteenth aspect may exhibit sustained or enhanced cytotoxicity and/or cytokine secretion upon binding a TIGIT ligand.
  • T-cells comprising the chimeric transmembrane receptor in accordance with the fifteenth or sixteenth aspect may be capable of increasing resistance to TIGIT ligand expressing cancer cells.
  • the chimeric TIGIT receptors of the T-cells according to the fifteenth or sixteenth aspect may comprise any functional combination of costimulatory cytoplasmic polypeptide domain(s) motif(s) and/or region(s) of the cytoplasmic polypeptide domain, region or motif selected from the group consisting of CD2 and CD 28.
  • the chimeric transmembrane receptor of the T- cells according to the fifteenth or sixteenth aspect may comprise at least one cytoplasmic polypeptide domain, region or motif of CD2.
  • a cytoplasmic polypeptide region of CD2 may comprise the complete cytoplasmic domain of wildtype human CD2.
  • the cytoplasmic polypeptide region of CD2 included in the chimeric transmembrane receptor may comprise at least one functional, co-stimulatory motif/domain/region of the complete wildtype human CD2 cytoplasmic domain.
  • wildtype human CD2 cytoplasmic domain as referred to herein may relate to a polypeptide comprising amino acid sequence 236-351 of UniProtKB database entry P06729 ⁇ CD2_HUMAN, as set forth e.g. in SEQ ID No. 5.
  • the cytoplasmic polypeptide domain, region or motif of wildtype CD2 as included in the chimeric transmembrane receptor of the T-cell as herein provided may have one or more conservative amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID No. 21.
  • all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD2 are envisaged.
  • the polypeptide of the chimeric transmembrane receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO.: 30.
  • the chimeric transmembrane receptor of the T- cell may comprise at least one cytoplasmic polypeptide domain, region or motif of CD28.
  • a cytoplasmic polypeptide region of CD28 may comprise the complete cytoplasmic domain of wildtype human CD28.
  • the cytoplasmic polypeptide region of CD28 included in the chimeric transmembrane receptor may comprise at least one functional, co-stimulatory motif/domain/region of the complete wildtype human CD28 cytoplasmic domain.
  • wildtype human CD28 cytoplasmic domain may relate to a polypeptide comprising amino acid sequence 180-220 of UniProtKB database entry No. P10747- CD28_HUMAN, as set forth e.g. in SEQ ID No. 31. It is herewith envisaged that the cytoplasmic polypeptide domain, region or motif of wildtype CD28 as included in the chimeric transmembrane receptor of the T-cell as herein provided may have one or more conservative amino acid substitutions relative to the amino acid sequence as set forth in SEQ ID No. 32. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region or motif of wildtype CD28 are envisaged.
  • the polypeptide of the chimeric transmembrane receptor may have an amino acid sequence with at least 85% identity to the amino acids as set forth in SEQ ID NO.: 33.
  • a chimeric transmembrane receptor as herein provided may e.g. comprise a polypeptide having an amino acid sequence with at least 85% or at least 86%, or at least 87%, or at least 88%, or at least 89% or at least 90%, or at least 91 %, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96% or at least 97%, or at least 98%, or at least 99%, or 100% identity to the amino acids as set forth in any one of the SEQ ID No’s selected from the group consisting of SEQ ID No. 30 and 33.
  • a chimeric transmembrane receptor as herein provided may have one or more conservative amino acid substitutions relative to an amino acid sequence as set forth in any one of the SEQ ID No’s selected from the group consisting of SEQ ID No. 30 and 33.
  • the cell may be an p T-cell, y8 T-cell, and/or a natural killer T-cell.
  • the ap T-cell may be a CD4 T-cell, or the ap T-cell may be a CD8 T-cell, or the y8 T-cell may comprise e.g. a Vy1 chain or a Vy2 chain, or may be e.g. a Vy9V82+ T-cell.
  • the T-cell may express a chimeric transmembrane receptor as herein described.
  • the T-cell may further express an engineered T-cell receptor or a CAR.
  • the T-cell may be a CD 4 T-cell that further expresses a CD8 Co-receptor, e.g. both CD8a and CD8p Co-receptor, or any engineered protein exhibiting CD8 Co-receptor functionality.
  • the T-cells further express an engineered T-cell receptor.
  • Engineered T-cells of the present disclosure can be used to treat a subject in need of treatment for a condition, for example, a cancer described herein.
  • the T-cells may be ap T-cells or y8 T-cells that express the chimeric transmembrane receptor polypeptide as described herein, and furthermore an engineered TCR.
  • the T-cells may further express a CD8 Co-receptor such as a wildtype or chimeric CD8 co- receptor.
  • T-cells described herein may be used to treat a cancer, including solid tumors and hematologic malignancies. For example, “hot” tumors or “cold” tumors may be treated by the T-cells herewith provided.
  • the engineered T-cell receptor as herein described may specifically bind a MAGE antigen family member, such as MAGE-A1 or MAGE-A4, or wherein the engineered T-cell receptor may specifically bind an antigen selected from the group consisting of a PRAME antigen, a NY-ESO-1 antigen, a GP100 antigen, an AFP antigen, a Col6A3 antigen, an HPV-16 antigen, a WT1 antigen, an HA1 antigen, an HA2 antigen, a mutated KRAS antigen, a mutated NRAS antigen, a mutated HRAS antigen, a mutated TP53 antigen, and an EGFR antigen.
  • a PRAME antigen such as MAGE-A1 or MAGE-A4
  • a GP100 antigen such as MAGE-A1 or MAGE-A4
  • an antigen selected from the group consisting of a PRAME antigen, a NY-ESO-1 antigen,
  • the expression “mutated” with respect to specific tumor antigens as herein used relates to well-known mutations within the epitope region of the respective protein, polypeptide or peptide that has been correlated with expression in a human cancer.
  • the T-cells described herein may also be used to treat an infectious disease.
  • the T-cells described herein may be used to treat an infectious disease; an infectious disease may be caused a virus.
  • the T-cells described herein may be used to treat an immune disease, such as an autoimmune disease.
  • the T-cells may be ap T-cells or y8 T-cells that express a chimeric transmembrane receptor as described herein, and an engineered TCR, and optionally a CD8 Co-receptor such as a wildtype or chimeric CD8 co-receptor.
  • the T-cell of the fifteenth or sixteenth aspect may be derived from a healthy subject, or the T-cell may be derived from a patient suffering from a disease.
  • the T-cell may be derived from an induced pluripotent stem cell (iPSCs).
  • iPSCs induced pluripotent stem cell
  • kits comprising means to prepare the T-cells as herein provided.
  • this invention relates to a pharmaceutical composition comprising the T-cell provided by the present invention.
  • the pharmaceutical composition may further comprise an adjuvant, excipient, buffer, diluent, carrier, stabilizer or combination thereof.
  • a pharmaceutical composition comprising T-cells which express the chimeric transmembrane receptor as herein described.
  • the T-cells may further express an engineered T-cell receptor or a CAR.
  • the pharmaceutical composition may further comprise CD4 T-cells expressing said chimeric transmembrane receptor, expressing an engineered T-cell receptor, and further expressing a recombinant CD8 Co-receptor, such as e.g. a CD8a receptor and a CD8p receptor, or a chimeric CD8 receptor.
  • the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers.
  • Any pharmaceutically acceptable carrier can be used, as long as the carrier does not impact the viability of the T-cells to be administered is suitable for the chosen route of administration of the pharmaceutical composition.
  • the pharmaceutical acceptable carrier may be a physiological saline solution, optionally with components such as human serum albumin that can improve the viability of the T-cells that express the chimeric transmembrane receptor. It is also possible that the chimeric transmembrane receptor expressing T-cells are stored, after their manufacture, in frozen form, for example at a temperature of between -20°C and -80 °C. In this case, the pharmaceutical composition may contain cryo-protectants that have been added to protect the cells from being damaged by the freezing process.
  • cryoprotectants examples include glycerol, DMSO. These cryoprotectants can be used together with crystalloid solutions such as commercially available HypoThermosol® or PlasmaLyte- A solution which are both approved for infusion and are available in pharmaceutical grade.
  • media of the “CryoStor family” include media of the “CryoStor family”, commercially available animal protein-free defined cryopreservation media from Biolife Solutions such as CyroStor2 (CS2, an optimized freeze media pre-formulated with 2% DMSO), CyroStor5 (CS5, an optimized freeze media pre-formulated with 5% DMSO), or CyroStorlO (CS10, an optimized freeze media preformulated with 10% DMSO).
  • a method for preparing a T-cell for immunotherapy comprising isolating T-cells from a human subject, introducing the vector as herein provided, or introducing the nucleic acid as herein provided into the T-cell, and expanding the transduced T-cells.
  • the method may comprise transforming, transfecting or transducing the isolated T-cells with the vector.
  • a method for treating a patient having a disease comprising administering to the patient the pharmaceutical composition according to the present invention.
  • a method for treating a patient having a disease comprising introducing in vivo the vector as herein disclosed into a T-cell of the patient.
  • the nucleic acid may be a DNA or an mRNA.
  • the vector may be - for example - a nonreplicating viral vector.
  • the nucleic acid may be mRNA, and the mRNA may be in vivo introduced into the T-cell of the patient using nanoparticles, such as lipid nanoparticles.
  • the disease may be e.g. an autoimmune disease or a cancer.
  • the cancer treated may be a solid tumor.
  • the lung cancer may be, but is not limited to, non-small cell lung cancer (NSCLC), including squamous cell carcinoma of the lung, adenocarcinoma of the lung, large cell carcinoma of the lung and other histologic types of NSCLC) or small cell lung cancer.
  • NSCLC non-small cell lung cancer
  • the breast cancer may be, but is not limited to, ductal breast cancer, ductal-invasive breast cancer, invasive breast cancer, tubular breast cancer, medullary breast cancer or combinations thereof.
  • the gastric cancer may be gastric adenocarcinoma or squamous cell cancer.
  • the sarcoma cancer may be, but is not limited to, chondrosarcoma cancer, osteosarcoma cancer or combinations thereof.
  • the adenoma cancer may include, but is also not limited to, gastric adenocarcinoma, pancreatic adenocarcinoma or combinations thereof.
  • the T-cell receptor may be a recombinant T-cell receptor that specifically binds a tumor specific antigen.
  • this may be a MAGE antigen such as e.g. a MAGE-A1 antigen.
  • Example 1 In-vitro T-cell killing analysis of T-cells according to the present invention transduced with chimeric TIGIT receptor polypeptides according to the present invention and an engineered T-cell receptor
  • chimeric TIGIT receptor constructs have been used to transduce CD8 T-cells together with a HLA-I restricted TCR raised against MAGE-A1.
  • Purified transduced T-cells were used in an in- vitro T-cell killing assay with CorL23-A2-NLR cells expressing TIGIT ligands CD155- and Nectin4 for evaluating cytotoxicity of the transduced T-cells.
  • CYP relates to the origin of the cytoplasmic domain of the chimeric receptor encoded by the plasmid created by the Inventors.
  • TM relates to the origin of the transmembrane domain of the chimeric receptor encoded by the plasmid, and
  • EC relates to the origin of the extracellular domain of the chimeric receptor encoded by the plasmid created by the Inventors.
  • PBMCs from a healthy donor buffy coat were isolated by density gradient centrifugation with Lymphoprep.
  • Purified polyclonal CD8 T-cells were obtained by positive selection with anti-CD8+ microbeads.
  • CD3 T-cells were activated using TransAct in presence of IL-7/IL-15.
  • CD8 T-cells were separately transduced with either HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) alone, or together with different versions of the TIGIT Switch receptors.
  • HLA-I restricted TCR raised against MAGE-A1 MAGE-A1_TCR
  • Transduced CD8 T-cells were further expanded, and at Day 9 the transduced fraction was positively selected using CD34 microbeads.
  • Purified transduced T-cells were cultured for further expansion and were harvested and cryopreserved at Day 10.
  • T-cell characterization was based on transgene expression levels using FACS and killing assay.
  • the in-vitro T-cell killing assay was performed according to the method described e.g. by Kalbasi, A., Siurala, M., Su, L.L. et al. “Potentiating adoptive cell therapy using synthetic IL-9 receptors”. Nature 607, 360-365 (2022).
  • the human TCR T-cell repetitive killing assay was conducted using IncuCyte Live Cell Analysis. CD155- and Nectin4- overexpressing CorL23-A2-NLR cells 1x10 4 tumor cells were plated per well in 96-well plates.
  • Transduced human T-cells (transduced with either MAGE_TCR alone, or transduced with MAGE_TCR together with either dominant negative TIGIT receptor or a chimeric TIGIT receptor) were added in triplicates at 1 to 6 E:T ratio.
  • TIGIT receptor (TB-M070-1) was added together with cell surface staining antibodies. Zombie YellowTM Fixable Viability Kit was used to discriminate between live and dead cells. The expression of the chimeric TIGIT receptor has been determined for CD8 cells transduced with different chimeric TIGIT receptors as herein provided.
  • Fig. 2 shows the results of the killing assay with the chimeric TIGIT receptors in CD155- and Nectin4-overexpressing CorL23-A2-NLR cells.
  • TCR only relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR);
  • TCR+TIGIT DN relates to a CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a dominant negative version of a TIGIT receptor (a truncated version of the TIGIT receptor which consists of the TIGIT extracellular and transmembrane domains, but which lacks the cytoplasmic domain),
  • TCR+SwR_TIGIT(EC+TM)-CD2(CYP) relates to CD8 T-cell fraction transduced with HLA-I restricted TCR raised against MAGE-A1 (MAGE-A1_TCR) together with a chimeric TIGIT receptor comprising a TIGIT extracellular and transmembrane
  • CD8 T cells transduced with vectors comprising nucleic acids encoding for different chimeric TIGIT receptors as herein provided have been checked for expression of the chimeric TIGIT receptor.
  • the respective chimeric TIGIT receptors used in Flow Cytometry Analysis correspond to the respective constructs according to Table 2 and Table 3 as shown above.
  • TIGIT DN and Mock transduces cells have been used as controls.
  • the chimeric TIGIT receptors comprising cytoplasmic domains of TLR2 and TLR4, respectively, were not expressed.
  • Fig. 1 the chimeric TIGIT receptors comprising cytoplasmic domains of TLR2 and TLR4, respectively, were not expressed.
  • Co-transduction of CD8 cells with an engineered HLA-I restricted TCR raised against MAGE-A1 together with a chimeric TIGIT receptor comprising a non - TIGIT co-stimulatory cytoplasmic polypeptide domain, motif or region of CD2 or CD28, and comprising a TIGIT transmembrane region also results in an increased killing activity of the engineered T-cells compared with mock transduced T-cells and/or T-cells only transduced with the HLA-I restricted TCR raised against MAGE-A1 , as visible in CD155- and Nectin4-overexpressing CorL23-A2-NLR cells.
  • the results described above demonstrate - in principle - suitability of the chimeric TIGIT switch receptor polypeptides as herein provided for improving adoptive cell therapy (ACT).
  • the chimeric TIGIT receptor polypeptides of the present invention e.g. in combination with an engineered T-cell receptor, may be functional in providing improved resistance to the T-cell in immunosuppressive tumor microenvironment, in preventing T-cell exhaustion and/or depletion through apoptosis; and in stimulating T-cell proliferation and functional activity, such as increased cytotoxicity.
  • fusion of at least one non-TIGIT polypeptide region wherein the at least one non-TIGIT polypeptide region comprises a transmembrane polypeptide region of CD2, CD40, HVEM, or CD30, and wherein the at least one non-TIGIT polypeptide region comprises at least one costimulatory cytoplasmic polypeptide domain, region or motif of CD2, CD40, HVEM, or CD30 to a TIGIT polypeptide region that comprises the TIGIT ligand binding domain, is able to act like a “switch” receptor, by turning negative signals into positive signals, thereby enhancing cytotoxicity of a T-cell in presence of tumor cells that express at least one TIGIT ligand.
  • Engineered T-cells expressing the chimeric TIGIT receptor polypeptides together with an engineered T-cell receptor as provided herein exhibit an improved killing activity compared to control samples in presence of a TIGIT ligand.
  • Engineered T-cells expressing the chimeric TIGIT receptor polypeptides together with an engineered T-cell receptor as provided herein exhibit an improved killing activity compared to control samples in presence of a TIGIT ligand.

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Abstract

La présente invention concerne, entre autres, un récepteur transmembranaire chimérique comprenant un polypeptide, le polypeptide comprenant au moins un immunorécepteur de lymphocytes T avec une région polypeptidique des domaines Ig et ITIM (TIGIT) comprenant un domaine de liaison de ligand extracellulaire TIGIT ; en outre le polypeptide comprenant au moins une région polypeptidique non-TIGIT, l'au moins une région polypeptidique non-TIGIT comprenant une région polypeptidique transmembranaire de CD2, CD40, HVEM ou CD30, et l'au moins une région polypeptidique non-TIGIT comprenant au moins un domaine, une région ou un motif polypeptidique cytoplasmique costimulateur de CD2, CD40, HVEM ou CD30, ou le domaine transmembranaire provenant de TIGIT, et l'au moins une région polypeptidique non-TIGIT comprenant au moins un domaine, une région ou un motif polypeptidique cytoplasmique costimulateur de CD2 ou CD28. L'invention concerne également des acides nucléiques, des vecteurs et des lymphocytes T correspondants comprenant ou exprimant les récepteurs chimériques, une composition pharmaceutique comprenant les lymphocytes T, et des méthodes de préparation d'un lymphocyte T pour une immunothérapie et pour le traitement d'une maladie, respectivement, dans lequel le récepteur transmembranaire chimérique est utilisé.
PCT/EP2025/056870 2024-03-13 2025-03-13 Récepteur transmembranaire chimérique comprenant au moins immunorécepteur de lymphocytes t avec une region polypeptidique des domaines ig et itim (tigit), des lymphocytes t exprimant le récepteur de commutateur tigit humain chimérique, des vecteurs avec des acides nucléiques codant pour le récepteur tigit, des kits pour préparer les lymphocytes t, ainsi que des compositions pharmaceutiques correspondantes et des méthodes pour traiter un patient ayant une maladie et pour augmenter la cytotoxicité d'un lymphocyte t dans une thérapie cellulaire adoptive Pending WO2025191067A1 (fr)

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