WO2024251981A1 - Détection et/ou modulation de la sensibilité à des agents thérapeutiques ciblant btn2a1 et/ou btn3a1 - Google Patents

Détection et/ou modulation de la sensibilité à des agents thérapeutiques ciblant btn2a1 et/ou btn3a1 Download PDF

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WO2024251981A1
WO2024251981A1 PCT/EP2024/065794 EP2024065794W WO2024251981A1 WO 2024251981 A1 WO2024251981 A1 WO 2024251981A1 EP 2024065794 W EP2024065794 W EP 2024065794W WO 2024251981 A1 WO2024251981 A1 WO 2024251981A1
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btn3a1
cells
cell
binding peptide
cancer
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Zsolt SEBESTYEN
Jürgen Herbert Ernst KUBALL
Astrid CLEVEN
Thijs KOORMAN
Patrick William Bernd DERKSEN
Angelo Dominique MERINGA
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UMC Utrecht Holding BV
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UMC Utrecht Holding BV
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Vg9Vd2T cells which are considered the most innate-like subset of gamma delta T cells in general (1), are activated by intermediate metabolites of the isoprenoid/mevalonate pathway, such as isopentenyl-5-pyrophosphate (IPP) (8), also referred to as phosphoantigens (pAgs) which can build up in cancerous- or virally-infected cells due to disruption of the mevalonate pathway.
  • Aminobiphosphonate (ABP) drugs such as pamidronate (PAM) can also further increase cellular pAg levels, by inhibiting farnesyl diphosphate synthase (FPPS), an essential enzyme in this pathway (9).
  • FPPS farnesyl diphosphate synthase
  • BTN3A1 phosphorylation of BTN3A1 (in particular S296 and T297 within the juxtamembrane region of BTN3A1), increases BTN2A1 and/or BTN3A1 expression and thus enhances recognition by BTN2A1 and/or BTN3A1 targeting therapy such as by Vg9Vd2TCR T cells.
  • the present disclosure provides for a method for determining susceptibility, of a cancer patient (or infectious disease/auto-immune disease patient), to BTN2A1 and/or BTN3A1 targeting therapy, the method comprising determining in a sample that has been obtained from a patient: i) presence or absence, in ErbB2, of an amino acid other than valine at a position corresponding to position 777 in SEQ ID NO:1; ii) level of PI3K-AKT1-mTOR pathway activity; and/or iii) phosphorylation status of butyrophilin subfamily 3 member A1 (BTN3A1).
  • the present disclosure also provides for a BTN2A1 and/or BTN3A1 targeting therapeutic for use in the treatment of cancer (or auto-immune disease or infectious disease), wherein the BTN2A1 and/or BTN3A1 targeting therapeutic is administered separately, sequentially or simultaneously to
  • the present invention relates to a method for determining susceptibility to a BTN2A1 and/or BTN3A1 targeting agent (e.g. therapeutic), the method comprising determining in a sample that has been obtained from a patient: i) presence or absence, in Erb-B2 receptor tyrosine kinase 2 (ErbB2), of an amino acid other than valine at a position corresponding to position 777 in SEQ ID NO:1; ii) level of PI3K-AKT1-mTOR pathway activity; and/or iii) phosphorylation status of BTN3A1.
  • ErbB2 receptor tyrosine kinase 2 ErbB2 receptor tyrosine kinase 2
  • BTN2A1 and BTN3A1 are a butyrophilins and members of the butyrophilin family of transmembrane I proteins of which 8 members (BTN1A1, BTN2A1/2A2, BTN3A1/3A2/3A3, MOG, and BTNL2) are located in the major histocompatibility complex (MHC) class I region of human chromosome 6.
  • MHC major histocompatibility complex
  • BTN2A1 and/or BTN3A1 binding is important for recognizing intracellular phosphoantigens that originate either from microbial pathogens or from a dysregulated mevalonate pathway in the case of stressed or malignant cells, e.g. as occurring in cancer, infectious disease or auto-immune disease. These phosphoantigens bind to the intracellular domain B30.2 of BTN3A1 resulting in the formation of a complex between the intracellular domains of BTN3A1 and BTN2A1.
  • the cancer cells or cancer tissue may be for example from leukemia, multiple myeloma, lymphoma, breast cancer, head and neck cancer, lung cancer, colorectal cancer, prostate cancer, skin cancer, bladder cancer, non-Hodgkin lymphoma, kidney cancer, pancreatic cancer, liver cancer, ovarian cancer, brain and central nervous system (CNS) tumors, stomach cancer, esophageal cancer.
  • the sample preferably is from a mammalian subject, preferably a human patient.
  • the method according to the present disclosure is preferably ex vivo and/or does not involve a diagnostic method practised on the human or animal body.
  • the method may comprise determining i) presence or absence, in Erb-B2 receptor tyrosine kinase 2 (ErbB2), of an amino acid other than valine at a position corresponding to position 777 in SEQ ID NO:1.
  • ErbB2 Erb-B2 receptor tyrosine kinase 2
  • ERBB2 Erb-B2 receptor tyrosine kinase 2
  • ERBB2 Erb-B2 receptor tyrosine kinase 2
  • absence in Erb-B2 receptor tyrosine kinase 2 (ERBB2), of an amino acid other than valine at a position corresponding to position 777 in SEQ ID NO:1
  • the said amino acid other than valine may be glutamic acid.
  • Sequencing may be performed by extracting the DNA from the source material (sample) that contains the nucleic acid sequence. PCR amplification may be used to amplify the specific region of interest that includes the target protein coding sequence.
  • Sanger sequencing also known as chaintermination sequencing
  • NGS Next-Generation Sequencing
  • determining presence or absence, in Erb-B2 receptor tyrosine kinase 2 (ErbB2), of an amino acid other than valine at a position corresponding to position 777 in SEQ ID NO:1 as under i) may be performed by detecting binding (or no binding) of an antibody that specifically binds ErbB2 with an amino acid other than valine at a position corresponding to position 777 in SEQ ID NO:1 (and that does not bind in case of valine at said position).
  • KRAS is a gene that plays a critical role in cell signalling and regulation of cell growth and division. It is a member of the RAS gene family, which also includes NRAS and HRAS.
  • the KRAS gene provides instructions for producing the KRAS protein, which is a small GTPase protein involved in transmitting signals from cell surface receptors to the cell nucleus.
  • Protein 53 is well-known to a skilled person and is also known as tumor protein 53 (TP53), a crucial tumor suppressor protein that plays a pivotal role in regulating cell cycle progression, DNA repair, apoptosis (programmed cell death), and genomic stability.
  • TP53 tumor protein 53
  • determining presence or absence of one or more of the above indicated amino acids can also be used under ii) of the present method, in order to determine level of PI3K-AKT1-mTOR pathway activity, preferably wherein presence is indicative of susceptibility to a BNT2A1 and/or BTN3A1 targeting agent/therapeutic (and/or increased level of PI3K-AKT1-mTOR pathway activity relative to absence) wherein absence is indicative of reduced susceptibility to a BNT2A1 and/or BTN3A1 targeting agent/therapeutic (and/or no increased level of PI3K-AKT1-mTOR pathway activity) (relative to said presence).
  • Determining level of PI3K-AKT1-mTOR pathway activity, as under ii) in the present method, may alternatively be performed e.g. by transcriptomic analysis, preferably by determining, relative to healthy cells or tissue, downregulation or preferably upregulation (e.g.
  • EGF EGF, EGFR, PIK3CA (PI3KCA), PIK3CB, PIK3CD, AKT1 , AKT2, AKT3, BAD, IGF1, IGFR, TSC1, TSC2, RHEB, mTOR, EIF4EBP1, CHUK, IKBKB, IKBKG, NFKBIA, NFKB1, RELA, GRB2, SOS1 , SOS2, HRAS, KRAS, NRAS; and MAP2K1, MAP2K2, BRAF.
  • the above-mentioned genes are found to be upregulated upon upregulation of PI3K-AKT1- mTOR pathway activity.
  • the level of PI3K-AKT1-mTOR pathway activity under ii) may preferably be, by determining, relative to healthy tissue, downregulation or upregulation of expression of one or more of the following genes: ACACA, ACTR2, ACTR3, ADCY2, ADRBK1 , AKT1 , AKT1S1 , AP2M1, ARF1, ARHGDIA, ARPC3, ATF1 , CAB39, CAB39L, e.g.
  • upregulation of PI3K-AKT1- mTOR pathway activity, relative to healthy tissue is indicative of susceptibility to a BNT2A1 binding peptide and/or BTN3A1 binding peptide and wherein no upregulation of PI3K-AKT1- mTOR activity, relative to healthy tissue, is indicative of reduced susceptibility to a BNT2A1 binding peptide and/or BTN3A1 binding peptide.
  • ii) may be performed by determining, relative to healthy tissue, downregulation or upregulation of expression of one or more of the following genes: RPS6KB1, PDPK1 , PIK3CA, TSC1, PTEN, EIF4B, PRKCA, PAK1, AKT2, GRB2 (upregulated) and PIK3R1 , MTOR, TSC2, PRKCZ, AKT1 , GRB2, EIF4A, HSPB1, RHEB (downregulated), wherein upregulation of PI3K-AKT1-mTOR pathway activity, relative to healthy tissue, is indicative of susceptibility to a BNT2A1 binding peptide and/or BTN3A1 binding peptide and wherein no upregulation of PI3K-AKT1-mTOR activity, relative to healthy tissue, is indicative of reduced susceptibility to a BNT2A1 binding peptide and/or BTN3A1 binding peptide, preferably upregulation by more than 10% of expression of one or more of the following genes: RPS6KB
  • the method according to the present disclosure may additionally comprise determining in a sample that has been obtained from a patient of iv) regulation of BTN3A1 expression and/or formation of BTN3A1 and BTN2A1 heterodimers, preferably by determining (such as by transcriptomic analysis), relative to healthy tissue, downregulation or upregulation of expression of one or more of the following genes (e.g. by more than 1 , 2, 3, 4, 5, 10, 15, 20, 25% in number of mRNA molecules): RHOB, PHLDB2, SYNJ2 and CARMIL-1 , preferably upregulation of one or more of the following genes: RHOB, SYNJ2 and CARMIL-1 and/or downregulation of PHLDB2.
  • the genes RHOB, PHLDB2, SYNJ2 and CARMIL1 were found to be involved in orchestrating BTN3A1 expression, ultimately leading to increased BTN2A1 surface expression (leading to Vg9Vd2TCR T cell activation).
  • ADCY2 108 ADCY2 adenylate cyclase 2
  • AKT1 207 AKT1 AKT serine/threonine kinase 1
  • ARF1 375 ARF1 ADP ribosylation factor 1
  • ATF1 466 ATF1 activating transcription factor 1
  • CAB39 51719 CAB39 calcium binding protein 39 CAB39L 81617 CAB39L calcium binding protein 39 like
  • FGF6 2251 FGF6 fibroblast growth factor 6
  • GSK3B 2932 GSK3B glycogen synthase kinase 3 beta
  • ITPR2 3709 ITPR2 inositol 1 ,4, 5-trisphosphate receptor type 2
  • MAPK9 5601 MAPK9 mitogen-activated protein kinase 9
  • PAK4 10298 PAK4 p21 (RAC1) activated kinase 4
  • PPP2R1 B 5519 PPP2R1 B protein phosphatase 2 scaffold subunit Abeta
  • PRKAG1 5571 PRKAG1 protein kinase AMP-activated non-catalytic subunit
  • PRKAR2A 5576 PRKAR2A protein kinase cAMP-dependent type II regulatory
  • TRIB3 57761 TRIB3 tribbles pseudokinase 3
  • TSC2 7249 TSC2 TSC complex subunit 2
  • EGF 1950 EGF phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha
  • PIK3CB 5291 PIK3CB phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta
  • PIK3CD 5293 PIK3CD phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta
  • AKT1 207 AKT1 AKT serine/threonine kinase 1
  • AKT3 10000 AKT3 AKT serine/threonine kinase 3 BAD 572 BAD BCL2 associated agonist of cell death
  • TSC2 7249 TSC2 TSC complex subunit 2
  • EIF4EBP1 1978 EIF4EBP1 eukaryotic translation initiation factor 4E binding protein
  • RELA 5970 RELA RELA proto-oncogene, NF-kB subunit
  • MAP2K1 5640 MAP2K1 mitogen-activated protein kinase kinase 1
  • SYNJ2 8871 SYNJ2 Synaptic Inositol 1 ,4, 5-Trisphosphate 5-Phosphatase 2
  • determining level of PI3K-AKT1-mT0R pathway activity may be performed e.g. by transcriptom ic analysis, by determining, relative to healthy cells or tissue, upregulation (e.g. by more than 5, 10, 15% in number of mRNA molecules) of expression of one or more of the following genes: PI3K(CA), AKT 1 , mTOR, KRAS, BRAF, MAP2K1, and MAP2K2 and/or downregulation (e.g. by more than 1 , 2, 3, 4, 5, 10, 15, 20, 25% in number of mRNA molecules) of PTEN and/or MAP2K1.
  • upregulation or upregulation may be determined by determining the number of mRNA molecules transcribed from said genes.
  • Transcriptomic analysis typically involves measuring and comparing the levels of RNA transcripts (i.e. mRNA molecules) in cells or tissues in the respective samples, e.g. comparing a sample comprising cancer cells (or infected cells) with a sample comprising healthy cells/tissue.
  • Microarrays are one of the platforms used for transcriptomic analysis. Typically it involves extraction of RNA from the sample, converting the RNA into complementary DNA (cDNA) using reverse transcription, labelling the cDNA with fluorescent dyes, commonly Cy3 and Cy5, to distinguish between different samples or conditions, hybridizing the labelled cDNA and applying to the microarray chip, and binding to complementary probes on the chip. The binding is specific to the sequences represented by the probes.
  • the microarray chip can then be scanned using a microarray scanner to measure the fluorescent signal emitted by the labelled cDNA. The intensity of the fluorescence indicates the abundance of the corresponding RNA transcripts in the samples.
  • determining phosphorylation status of BTN3A1 may be performed by detecting binding (or no binding) of an antibody that specifically binds BTN3A1 having a phosphorylated serine at a position corresponding to position 296 in SEQ ID NO:4 and/or a phosphorylated threonine at a position corresponding to position 297 in SEQ ID NO:4.
  • iii) may be performed by detecting upregulation of kinase (PRKCQ, Protein kinase C theta (PKC-0)) expression, relative to healthy tissue (see Figure 3G).
  • PRKCQ Protein kinase C theta
  • the phosphorylation status of BTN3A1 can for example be determined using immunoblotting, also known as Western blotting, which may involve the use of primary and secondary antibodies.
  • immunoblotting also known as Western blotting
  • the respective protein may be extracted from the sample, and transferred to a membrane, which facilitates antibody binding and detection.
  • the membrane can be combined with a (primary) antibody that specifically recognizes the phosphorylated form of BTN3A1.
  • the (primary) antibody preferably is validated for its specificity and selectivity in detecting the phosphorylated epitopes.
  • a secondary antibody conjugated to a label may then be used.
  • the secondary antibody should be raised against the species in which the primary antibody was produced (e.g., if the primary antibody is raised in rabbit, use an anti-rabbit secondary antibody).
  • the appropriate detection method may be applied.
  • the method according to the present disclosure can be useful for selecting patients for who i), ii), iii) and/or vi) indicate susceptibility to a BTN2A1 and/or BTN3A1 targeting therapeutic and/or for excluding patients for who i), ii), iii) and/or vi) do not indicate susceptibility to a BTN2A1 and/or BTN3A1 targeting therapeutic.
  • the method may comprise a further step of administering a BTN2A1 and/or BTN3A1 targeting therapeutic to selected patients.
  • the present disclosure provides for a BTN2A1 and/or BTN3A1 targeting therapeutic for use in the treatment of cancer (or treatment of an infectious disease or auto-immune disease), wherein the BTN2A1 and/or BTN3A1 targeting therapeutic is administered
  • RhoB phosphorylation-inducing agent
  • CN01 Rho activator
  • the cancer may for example be leukaemia, multiple myeloma, lymphoma, breast cancer, head and neck cancer, lung cancer, colorectal cancer, prostate cancer, skin cancer, bladder cancer, non-Hodgkin lymphoma, kidney cancer, pancreatic cancer, liver cancer, ovarian cancer, brain and central nervous system (CNS) tumour, stomach cancer, esophageal cancer.
  • leukaemia multiple myeloma, lymphoma, breast cancer, head and neck cancer
  • lung cancer colorectal cancer
  • prostate cancer skin cancer, bladder cancer, non-Hodgkin lymphoma, kidney cancer, pancreatic cancer, liver cancer, ovarian cancer, brain and central nervous system (CNS) tumour, stomach cancer, esophageal cancer.
  • CNS central nervous system
  • the infectious disease may be or may be caused by e.g. bacterial infection, fungal infection, viral infection (e.g. COVID-19, or Hanta virus) (which can cause) sepsis, pneumonia, meningitis, acute respiratory distress syndrome, necrotizing fasciitis.
  • the auto-immune disease may be chosen e.g. from rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type 1 diabetes, inflammatory bowel disease, psoriasis, Hashimoto’s thyroiditis, Sjogren’s syndrome, autoimmune hepatitis, pemphigus vulgaris and graft versus host disease after allogeneic stem cell transplantation or rejection of a transplant.
  • BTN2A1 and/or BTN3A1 targeting therapeutic can be dramatically improved by co-administering at least one PI3K-AKT1-mTOR pathway activator and/or at least one phosphorylation-inducing agent (and/or to a modulator of one or more of RHOB, PHLDB2, SYNJ2 and CARMIL-1).
  • the present disclosure for example allows to improve recognition by therapies aiming to enhancing vg9vd2T cell therapies with autologous or allogeneic vg9vd2 T cells or Vg9Vd2TCR engineered T cells or soluble fragments like bispecific antibodies that e.g. bind BTN2A1.
  • (prior) administering of at least one PI3K-AKT1-mTOR pathway activator and/or at least one phosphorylation-inducing agent (and/or to a modulator of one or more of RHOB, PHLDB2, SYNJ2 and CARMIL-1) may sensitize patients to a BTN2A1 and/or BTN3A1 targeting therapeutic according to the present disclosure.
  • the at least one PI3K-AKT1-mTOR pathway activator preferably is at least one growth factor ligand, preferably Epidermal Growth Factor (EGF).
  • a growth factor ligand is a type of signaling molecule that triggers cellular responses involved in cell growth, proliferation, differentiation, and survival. Growth factors play crucial roles in various physiological processes during development, tissue repair, and maintenance of normal cellular function. Growth factor ligands are typically small proteins or peptides that are secreted by cells and act on nearby or distant target cells. In the present disclosure, the growth factor ligand may be epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), or transforming growth factor (TGF).
  • EGF Epidermal Growth Factor
  • FGF fibroblast growth factor
  • PDGF platelet-derived growth factor
  • IGF insulin-like growth factor
  • TGF transforming growth factor
  • the at least one PI3K-AKT1-mTOR pathway activator may be at least one agent that upregulates expression of one or more of the following genes ACACA, ACTR2, ACTR3, ADCY2, ADRBK1 , AKT1 , AKT1S1, AP2M1, ARF1 , ARHGDIA, ARPC3, ATF1, CAB39, CAB39L, CALR, CAMK4, CDK1, CDK2, CDK4, CDKN1A, CDKN1B, CFL1, CLTC, CSNK2B, CXCR4, DAPP1, DDIT3, DUSP3, E2F1 , ECSIT, EGFR, EIF4E, FASLG, FGF17, FGF22, FGF6, GNA14, GNGT1 , GRB2, GSK3B, HRAS, HSP90B1, IL2RG, IL4, IRAK4, ITPR2, LCK, MAP2K3, MAP2K6, MAP3K7, MAPK1
  • EGF EGF, EGFR, PIK3CA (PI3KCA), PIK3CB, PIK3CD, AKT1, AKT2, AKT3, BAD, IGF1 , IGFR, TSC1, TSC2, RHEB, mTOR, EIF4EBP1, CHUK, IKBKB, IKBKG, NFKBIA, NFKB1, RELA, GRB2, SOS1 , SOS2, HRAS, KRAS, NRAS; and MAP2K1, MAP2K2, BRAF.
  • the modulator of one or more of RHOB, PHLDB2, SYNJ2 and CARMIL-1 may be at least one agent that upregulates expression of one or more of RHOB, PHLDB2, SYNJ2 and CARMIL-1.
  • the at least one phosphorylation-inducing agent preferably is at least one aminobisphosphonate, preferably pamidronate and/or zoledronate.
  • Bisphosphonates are a group of compounds characterized by a two-phosphonate (bisphosphonate) structure. Aminobisphosphonates have an additional nitrogen-containing side chain, which distinguishes them from non-nitrogen-containing bisphosphonates.
  • Co-administration of aminobisphosphonates may also increase the intracellular levels of phosphoantigens, e.g. in tumor cells, which can further contribute to recognition of cancer cells by the BTN2A1 and/or BTN3A1 targeting therapeutic, e.g. thereby further contribute to activation of Vy9V ⁇ 52 T cells or Vy9V52-TCR based therapies.
  • the BTN2A1 and/or BTN3A1 targeting therapeutic according to the present disclosure may be or comprise a BTN2A1 binding peptide and/or BTN3A1 binding peptide, which preferably is a (human) T-cell receptor y-chain (variable) domain, preferably a (human) T-cell receptor y9-chain (variable) domain.
  • the BTN2A1 binding peptide according to the present disclosure may combined with a BTN3A1 binding peptide, e.g. a delta (2) chain, for example together in a y5 TCR or extracellular domain thereof (e.g. Vy9V52 TCR or extracellular domain thereof).
  • the BTN2A1 and/or BTN3A1 binding therapeutic according to the present disclosure may be a (human) y952 T-cell receptor or extracellular domain thereof.
  • the BTN2A1 and/or BTN3A1 binding peptide according to the present disclosure may be a (TCR-like) antibody and/or may be able to bind a heterodimer of BTN2A1 and BTN3A1, e.g. in an activating conformation.
  • the BTN2A1 and/or BTN3A1 binding peptide according to the present disclosure functions in vivo as a (y5) TCR.
  • the BTN2A1 and/or BTN3A1 binding peptide may be comprised in an (exogeneous) immune receptor or extracellular domain thereof, for example a human (exogeneous) immune receptor or extracellular domain thereof.
  • the immune receptor may be a T cell receptor or chimeric antigen receptor, preferably a y5 T-cell receptor or extracellular domain thereof, more preferably a y952 T-cell receptor or extracellular domain thereof.
  • the y(9)-chain (variable) domain comprising a BTN2A1 binding peptide may pair with any 51-8 chain.
  • the (exogenous) immune receptor, the T cell receptor or chimeric antigen receptor, preferably y5 T-cell receptor or extracellular domain thereof, is capable of binding, or binds, a tumor cell (or infected cell), e.g. an antigen present on the surface of a tumor cell (or infected cell).
  • a tumor cell or infected cell
  • the BTN2A1 and/or BTN3A1 binding peptide may be expressed by a cell, preferably an immune cell, more preferably a human T cell or human NK cell, more preferably an op T-cell or a y5 T-cell.
  • Such cell may be autologous, or allogenic to the patient receiving the treatment.
  • TCR T cell receptors
  • a alpha
  • beta P
  • Y gamma
  • delta (5) chains TCR chains are typically composed of two extracellular domains: a Variable (V) domain and a Constant (C) domain, both of Immunoglobulin superfamily (IgSF) forming antiparallel p-sheets.
  • the Constant domain is proximal to the cell membrane, followed by a transmembrane domain and a short cytoplasmic tail, while the Variable domain can bind an antigen or target moiety.
  • An exogenous immune receptor according to the disclosure is preferably defined as not being an endogenous T cell receptor.
  • an exogenous immune receptor may be a particular selected y ⁇ 5 T cell receptor that is useful in the treatment of a cancer. Said sequence may be similar to an endogenous y ⁇ 5 T cell receptor. The difference being that the exogenous immune receptor has been purposively selected for a specific target e.g. an antigen present on the surface of a tumor cell.
  • the exogenous immune receptor is e.g.
  • An exogenous immune receptor may be of a different origin, i.e. from another species, as compared to the origin of the T cells that were engineered to provide for the engineered T cells with exogenous immune receptors.
  • An exogenous immune receptor may be of the same origin, i.e. from the same species, as compared to the origin of the T cells that were engineered to provide for the engineered T cells with exogenous immune receptors.
  • An exogenous immune receptor may also be an engineered y ⁇ 5 T cell receptor or an engineered op T cell receptor.
  • any of the immune receptors according to the present disclosure may be a chimeric antigen receptor (CAR).
  • Chimeric antigen receptors are recombinant receptors that combine the specificity of an antigen-specific antibody with the T-cell’s activating functions.
  • a CAR may be a fusion molecule between an antibody and a trans-membrane domain allowing expression of an antibody at the cell surface of an immune cell as well as signalling into the cell.
  • any of the immune receptor according to the present disclosure may be selected from the group consisting of an (engineered) y ⁇ 5 T cell receptor, an (engineered) op T cell receptor, or a chimeric antigen receptor (CAR).
  • the present disclosure also provides that a i) y ⁇ 5 T-cell receptor or extracellular domain thereof as disclosed herein (comprising the BTN2A1 and/or BTN3A1 binding peptide according to the present disclosure) is combined with ii) a toxin and/or a label.
  • the toxin preferably is any compound or combination of compounds effective to kill a cancer cell or an infected cell.
  • the toxin may for example be a diptheria toxin, pseudomonas toxin, and/or saporin.
  • the toxin and/or the label may be fused to the y ⁇ 5 T-cell receptor or extracellular domain thereof, for example via a linker.
  • the label may be any label useful in diagnostic setting.
  • the label may allow for visualizing binding of the BTN2A1 binding peptide, to BTN2A1.
  • the label may for example be a fluorophore. Fluorophores are very sensitive and generally do not affect the properties of the target molecule.
  • the process may involves the binding of the fluorophore via the BTN2A1 peptide of the invention to a BTN2A1 protein as expressed e.g. by a cancer cell or infected cell. When the binding is complete, the fluorescence can be viewed by excitation, through a fluorescent microscope, for example. Fluorescent labelling can be used in assays such as: ELISA, FISH, and fluorescent microscopy.
  • the label may be linked to the BTN2A1 peptide of the invention by means of any linker.
  • the present disclosure further provides that a i) yb T-cell receptor or extracellular domain thereof as disclosed herein (comprising the BTN2A1 and/or BTN3A1 binding peptide according to the present disclosure) is combined with ii) an (effector) cell binding domain, preferably an immune cell binding domain, preferably B-cell binding domain, macrophage binding domain or fibroblast binding domain, more preferably a T-cell binding domain and/or Natural Killer (NK) cell-binding domain.
  • an immune cell binding domain preferably B-cell binding domain, macrophage binding domain or fibroblast binding domain, more preferably a T-cell binding domain and/or Natural Killer (NK) cell-binding domain.
  • NK Natural Killer
  • the T-cell receptor or extracellular domain thereof preferably is a Y9 ⁇ 52 T-cell receptor or extracellular domain thereof.
  • extracellular domain of a gamma or delta TCR chain comprises the V gamma and extracellular part of the C gamma domains, or the V delta and extracellular part of the C delta domains.
  • the above construct combines (low) affinity TCR interaction with its ligand on tumor cells with (high) affinity interaction with e.g. T lymphocytes and/or with NK cells, preferably by binding to CD3 on T lymphocytes and/or CD 16 on NK cells.
  • This concept may be elaborated by generating trispecific constructs for which tumor binding depends on ybTCR and a second molecule like a checkpoint ligand.
  • the construct according to the disclosure can attach to infected cells or cancer cells, as is indicated herein, and couple to immune cells, e.g. T-cells and/or Natural Killer (NK) cells to thereby elucidate an immune response against the infected cells or cancer cells that will reduce or even eliminate said cells.
  • immune cells e.g. T-cells and/or Natural Killer (NK) cells
  • NK Natural Killer
  • GABs ybTCR anti-CD3 bispecific molecules
  • the T-cell binding domain may bind cluster of differentiation 3 (CD3), CD4, CD8, CD 16, CD56, CD103, CD134, CD154 and/or CD314; and/or is a single chain Fv anti- CD3, CD4, CD8, CD 16, CD56, CD103, CD134, CD154; and/or
  • the Natural Killer (NK) cell-binding domain may bind CD16, NKG2D, NKp30, NKp44, NKp46, and/or DNAM, and/or is a single chain Fv anti- CD16, NKG2D, NKp30, NKp44, NKp46, and/or DNAM.
  • the binding domain might be modified so as to inhibit T cell activation.
  • the construct may bind inhibitory domain(s) of the T cell.
  • the T cell binding domain may bind PD1 (expressed on the surface of T cells), e.g. to reduce T cell activation.
  • This embodiment may be useful in prevention or treatment of autoimmune disease, e.g. wherein it is desired to block T cell activity and not enhance it (e.g. gdT cell or abT cell).
  • the T-cell binding domain may bind PD1 , LAG3, CTLA4, TIGIT, CD96, BTLA, VISTA, TIM3, LAIR1 , (inhibitory) KIR, CD160 and/or immune receptor with an intracellular ITIM or ITSM motif; and/or may be a single chain Fv anti- PD1, LAG3, CTLA4, TIGIT, CD96, BTLA, VISTA, TIM3, LAIR1, (inhibitory) KIR, CD160 and/or immune receptor with an intracellular ITIM or ITSM motif binding domain; and/or
  • the Natural Killer (NK) cell binding domain may bind NKG2A, CD96, TIGIT, (inhibitory) KIR, PD1, TIM3, LAG3, CD112R, CD160, LAIR1 and/or immune receptor with an intracellular ITIM or ITSM motif and/or may be a single chain Fv anti-NKG2A, CD96, TIGIT, (inhibitory) KIR, PD1, TIM3, LAG3, CD112R, CD160, LAIR1 and/or immune receptor with an intracellular ITIM or ITSM motif binding domain.
  • the y ⁇ 5 T-cell receptor (or extracellular domain thereof) and the immune cell-, T-cell- and/or Natural Killer (NK) cell-binding domain are preferably fused through a linker or linking group which preferably provides conformational flexibility so that the extracellular domain of a gamma-delta TCR can interact with its epitope, while the T-cell- and/or NK cell binding domain can interact with its cognate epitope.
  • a linker or linking group which preferably provides conformational flexibility so that the extracellular domain of a gamma-delta TCR can interact with its epitope, while the T-cell- and/or NK cell binding domain can interact with its cognate epitope.
  • a preferred linker group is a linker polypeptide comprising from 1 to 60 amino acid residues, preferably from 5 to 40 amino acid residues, most preferred about 15 amino acid residues such as 10 amino acid residues, 11 amino acid residues, 12 amino acid residues, 13 amino acid residues, 14 amino acid residues, 15 amino acid residues, 16 amino acid residues, 17 amino acid residues, 18 amino acid residues, 19 amino acid residues or 20 amino acid residues.
  • Gly-Ser linkers for example of the type (Glyx Sery )z such as, for example, (Gly4 Ser)3 (SEQ ID NO:5), (Gly4 Ser)7 (SEQ ID NO:6) or (Gly3 Ser2)3 (SEQ ID NO:7), as described in WO 99/42077, and the GS30 (SEQ ID NO:8), GS15 (SEQ ID NO:9), GS9 (SEQ ID NQ:10) and GS7 (SEQ ID NO:11) linkers described in, for example, WQ06/040153 and WO 06/122825, as well as hinge-like regions, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (such as described in WO 94/04678).
  • a most preferred linker is a (Gly4 Ser)3 (SEQ ID NO:5) linker.
  • the immune cell -, T-cell- and/or Natural Killer (NK) cell-binding domain preferably is an antibody, preferably a single heavy chain variable domain antibody such as a camelid VHH, a shark immunoglobulin-derived variable new antigen receptor, a scFv, a tandem scFv, a scFab, an improved scFab, or an antibody mimetic such as a designed ankyrin repeat protein, a binding protein that is based on a Z domain of protein A, a binding protein that is based on a fibronectin type III domain, engineered lipocalin, and a binding protein that is based on a human Fyn SH3 domain.
  • single-chain antibodies (scFv) against CD3 that are expressed on the plasma membrane of tumor cells may be used.
  • Single chain antibodies against CD3 are commercially available, for example from Creative Biolabs.
  • a preferred single chain antibody against CD3 that is present in the (bispecific) construct according to the disclosure comprises a single chain Fv anti-CD3 binding domain.
  • Said single chain Fv anti-CD3 binding domain preferably is derived from a chimeric mouse-human OKT3 antibody.
  • the construct according to the present disclosure preferably is a bispecific fusion protein, e.g. the construct comprising i) y ⁇ 5 T-cell receptor or extracellular domain thereof as disclosed herein (comprising the BTN2A1 binding peptide according to the present disclosure); and ii) a toxin and/or a label, or more preferably an (effector) cell binding domain, preferably an immune cell binding domain, more preferably a T-cell binding domain and/or Natural Killer (NK) cell-binding domain.
  • the y ⁇ 5 T-cell receptor or extracellular domain thereof is fused to the T-cell- and/or Natural Killer (NK) cell-binding domain.
  • a preferred recombinant bispecific protein according to the disclosure comprises the extracellular domains of a gamma delta TCR, preferably gamma 9 delta 2 TCR.
  • a preferred recombinant bispecific protein according to the disclosure comprises the extracellular domains of a TCR gamma chain, preferably gamma 9 (comprising the BTN2A1 binding peptide as disclosed herein), that is coupled at its C-terminus to a CD3-binding domain, preferably a scFv derived from the OKT3 antibody, and preferably an extracellular domain of a delta TCR, preferably a delta 2 TCR (comprising the BTN3A1 binding peptide as disclosed herein).
  • a TCR gamma chain preferably gamma 9 (comprising the BTN2A1 binding peptide as disclosed herein)
  • CD3-binding domain preferably a scFv derived from the OKT3 antibody
  • an extracellular domain of a delta TCR preferably a delta 2 TCR (comprising the BTN3A1 binding peptide as disclosed herein).
  • the extracellular domain of gamma and/or delta TCR preferably of the delta TCR, preferably of the delta 2 TCR, may be fused at the N- terminus or C-terminus to the extracellular domain of checkpoint-related molecule such as the extracellular domain of a PD-1 receptor.
  • the present disclosure also provides for an nucleic acid or nucleic acid combination encoding the BTN2A1 and/or BN3A1 targeting therapeutic according to the disclosure or (any of the) construct(s) according to the disclosure.
  • the nucleic acid may be comprised in a vector and/or comprised in a cell which may or may not be an immune cell.
  • the nucleic acid is expressed in said cell.
  • An immune receptor (or extracellular domain thereof) according to the present disclosure may be e.g. a gamma delta T cell receptor (or extracellular domain thereof) that comprises a first chain which is gamma and a second chain which is the delta chain. These may be provided on a single nucleic acid or on two separate nucleic acids. A first nucleic acid encoding the first chain, and a second nucleic acid encoding the second chain, or a single nucleic acid encoding both the first and second chains. Said nucleic acid or nucleic acids may be DNA or RNA. As long as when it is introduced in a cell and expressed such that the amino acid sequence of the exogenous immune receptor it encodes is expressed on the surface of the cell.
  • the nucleic acid encoding the immune receptor encodes an immune receptor (or extracellular domain thereof) wherein the different chains, e.g. gamma and delta chains, are expressed as a single translated protein product that comprising the F2A or T2A peptide linker sequence in between the encoding sequences of the both chains resulting in self-cleavage of the translated protein such that separate chains are formed.
  • the different chains e.g. gamma and delta chains
  • the nucleic acid or nucleic acids that encode the immune receptor (or extracellular domain thereof) may be mRNA that can be translated directly in the immune receptor (or extracellular domain thereof) when introduced in the cytoplasm of a T cell, e.g. via transfection.
  • the nucleic acid (or nucleic acids) encoding e.g. a T-cell receptor chain is comprised in a genetic construct.
  • the genetic construct (or constructs) may allow the expression of mRNA that encodes the immune receptor (or extracellular domain thereof) such that it is expressed on the surface of the engineered T cell.
  • a genetic construct may be comprised in a DNA vector or in a viral vector.
  • the genetic construct may consist of DNA or RNA.
  • the genetic construct when a genetic construct is incorporated in a retroviral or lentiviral vector the genetic construct is comprised in an RNA vector genome (i.e. the sequence that encodes the genetic construct).
  • Retroviral and lentiviral vectors are well known in the art having an RNA genome which, when entered in a cell, is reverse transcribed into DNA that is subsequently integrated into the host genome. Reverse transcription thus results in the genetic information, i.e.
  • a genetic construct may also be comprised in a DNA vector, e.g. plasmid DNA.
  • a suitable DNA vector may be a transposon. Suitable transposon systems (e.g. class I or class II based) are well known in the art.
  • an immune receptor comprises two chains, e.g. a gamma and delta T cell receptor chain
  • two separate genetic constructs can be provided e.g. on a single or two separate retroviral or DNA vectors.
  • a single genetic construct may also express a single mRNA encoding the two chains.
  • Such an mRNA may encode the two chains separately, e.g. via an IRES, or via using self-cleavable peptide sequences as described herein.
  • the nucleic acid or nucleic acids that are used may provide for expression of the encoded immune receptor (or extracellular domain thereof). This is achieved e.g. via high levels of expression of the immune receptor (or extracellular domain thereof) by using e.g. a strong promoter.
  • the present disclosure also provides for a cell expressing the BTN2A1 and/or BTN3A1 targeting therapeutic according to the disclosure, the construct according to the disclosure, and/or the nucleic acid or nucleic acid combination according to the disclosure as described above.
  • Said cell may be a bacterial cell, for example an Escherichia coli cell, or a eukaryotic cell such as a fungal cell including a yeast cell, for example Saccharomyces cerevisiae or a methylotrophic yeast such as Pichia pastoris, or a mammalian cell.
  • Said eukaryotic cell preferably is a cell that can easily be infected and/or transfected using standard methods known to the skilled person, such as, for example, yeast cells and chicken fibroblast cells.
  • Said eukaryotic cell preferably is an insect cell or a mammalian cell.
  • Suitable insect cells comprise, for example, ovarian Spodoptera frugiperda cells such as Sf9 and Sf21, Drosophila Schneider 2 cells and Aedes albopictus C6/36 cells.
  • Suitable mammalian cells comprise, for example, Baby Hamster Kidney cells, Human Embryonic Kidney cells such as HEK293 and freestyle HEK293FTM cells (ThermoFisher Scientific), VERO cells, MDCK cells, CHO cells, HeLa and PER.C6 cells.
  • Preferred cells are Human Embryonic Kidney cells such as HEK293 and freestyle HEK293FTM cells.
  • the cell expressing the BTN2A1 and/or BTN3A1 targeting therapeutic according to the disclosure may be an immune cell, more preferably a human T cell or human NK cell, more preferably an op T-cell or a y ⁇ 5 T-cell.
  • the immune cells according to the present disclosure may be immune cells that are engineered to comprise and preferably express an exogenous immune receptor.
  • the immune cell according to the present disclosure may be a human immune cell, preferably a human T cell or human NK cell.
  • the exogenous immune receptor may have the same function as a corresponding endogenous T cell receptor with regard to antigen recognition and T cell action.
  • Non-engineered immune cells are cells that express an endogenous immune receptor, i.e. T cell receptor.
  • Cells such as immune cells may be isolated or established immune cell lines may be used.
  • the subject may suffer from cancer (a patient) or may be a healthy subject.
  • These immune cells can be genetically modified in vitro to express the immune receptor (or extracellular domain thereof) as disclosed herein.
  • These engineered cells may be activated and expanded in vitro to a therapeutically effective population of expressing cells.
  • these engineered cells may be infused to a recipient in need thereof as a pharmaceutical composition.
  • the infused cells in the recipient may be able to kill (or at least stop growth of) cancerous cells expressing the antigen which is recognized by the immune receptor as disclosed herein.
  • the recipient may be the same subject from which the cells were obtained (autologous cell therapy) or may be from another subject of the same species (allogeneic cell therapy).
  • op T-cells with y ⁇ 5 TCRs which combine the strong proliferation capacity of op T cells (which are active even in late stage cancer patients, with the broad tumor- reactivity of y ⁇ 5 TCRs.
  • the present disclosure further relates to a method of producing the BTN2A1 and/or BTN3A1 targeting therapeutic according to the disclosure, or the construct according to the disclosure, wherein the method comprises expressing the BTN2A1 and/or BTN3A1 targeting therapeutic according to the disclosure, or the construct according to the disclosure in a host cell thereby producing the BTN2A1 and/or BTN3A1 targeting therapeutic according to the disclosure, or the construct according to the disclosure.
  • the BTN2A1 and/or BTN3A1 targeting therapeutic according to the disclosure, or the construct according to the disclosure may be secreted into the growth medium of the host cell.
  • a pharmaceutical composition comprising the BTN2A1 and/or BTN3A1 targeting therapeutic according to the present disclosure (or a y ⁇ 5 TCR or extracellular domain thereof comprising said peptide), the construct according to the present disclosure, or the cell according to the present disclosure.
  • a pharmaceutical composition preferably comprises a pharmaceutically acceptable carrier.
  • a carrier as used herein, means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient.
  • physiologically acceptable refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration.
  • Formulations comprising therapeutically effective population(s) of cells or constructs according to the present disclosure may include pharmaceutically acceptable excipient(s) (carrier or diluents).
  • Excipients included in the formulations will have different purposes depending, for example, on the nature of the construct, the (sub)population of immune cells used, and the mode of administration.
  • Examples of generally used excipients include, without limitation: saline, buffered saline, dextrose, water-for-injection, glycerol, ethanol, and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents.
  • the formulations comprising therapeutically effective population(s) of cells or constructs according to the present disclosure may be administered to a subject using modes and techniques known to the skilled artisan.
  • Exemplary modes include, but are not limited to, intravenous injection.
  • Other modes include, without limitation, intratumoral, intradermal, subcutaneous (s.c, s.q., sub-Q, Hypo), intramuscular (i.m.), intraperitoneal (i.p.), intra-arterial, intramedulary, intracardiac, intra- articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids).
  • formulations may be administered that comprise between about 1 x 10 4 and about
  • formulations may be administered that comprise between 0.1-10, or 1 -100, 10-1000 mg construct.
  • formulations may be administered that comprise between 0.1-10, or 1 -100, 10- 1000 mg EGF.
  • formulations may be administered that comprise between 0.1-10, or 1 -100, 10-1000 mg phosphorylation inducing agent.
  • the formulation may comprise between about 1 x 10 5 and about 1 x 10 9 immune cells, from about 5 x 10 5 to about 5 x 10 8 immune cells, or from about 1 x 10 6 to about 1 x 10 7 immune cells.
  • a physician may ultimately determine appropriate dosages to be used.
  • the BTN2A1 and/or BTN3A1 targeting therapeutic according to the present disclosure can be administered by injection or by (gradual) infusion over time.
  • the administration of said construct preferably is parenteral such as, for example, intravenous, intraperitoneal, intranasal, or intramuscular. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions suspensions, and emulsions.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, aleoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishes, electrolyte replenishes (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • a method for treatment of a cancer and/or an infection comprising administering an effective amount of the BTN2A1 and/or BTN3A1 targeting therapeutic according to the disclosure (e.g. a (yb)TCT or extracellular domain thereof comprising a BTN2A1 binding peptide), any one of the constructs according to the present disclosure, or the cell according to the present disclosure, e.g. to a subject in need thereof.
  • the subject is human.
  • the ybTCR or extracellular domain thereof preferably is capable of binding, or binds, a tumor cell, e.g. an antigen present on the surface of a tumor cell. Occasional off- target toxicity (binding on healthy tissue) can be overcome by lowering dose.
  • BTN2A1 and/or BTN3A1 targeting therapeutic may be administered to an individual that is suspected of suffering from a cancer or an infection, or may be administered to an individual already evidencing active infection or cancer in order to lessen signs and symptoms of said cancer or infection.
  • a patient undergoing an allogeneic stem cell transplantation may also benefit from an infusion of the BTN2A1 and/or BTN3A1 targeting therapeutic according to the disclosure. This way, elimination of leukaemia may be promoted.
  • the present disclosure also provides for use, e.g. in a patient in need thereof, of at least one PI3K-AKT1-mTOR pathway inhibitor in the treatment of side effect(s) (e.g. toxicity for example due to off-target binding) of (prior) treatment with a BTN2A1 and/or BTN3A1 targeting therapeutic according to the present disclosure.
  • the use may comprise administering said at least one PI3K-AKT1-mTOR pathway inhibitor.
  • the use may also comprise administering a modulator of one or more of RHOB, PHLDB2, and CARMIL-1, which may be at least one agent that upregulates expression of one or more of RHOB, and PHLDB2, and/or at least one agent that downregulates expression of CARMIL-1.
  • a modulator of one or more of RHOB, PHLDB2, and CARMIL-1 which may be at least one agent that upregulates expression of one or more of RHOB, and PHLDB2, and/or at least one agent that downregulates expression of CARMIL-1. See Figure 5D, RHOB, CARMIL-1 is unregulated in cancer, and also upon PAM, PHLDB2 is down regulated.
  • Said at least one PI3K-AKT1-mTOR pathway inhibitor may be at least one agent that downregulates expression of one or more of the following genes ACACA, ACTR2, ACTR3, ADCY2, ADRBK1, AKT1 , AKT1S1, AP2M1, ARF1 , ARHGDIA, ARPC3, ATF1 , CAB39, CAB39L, CALR, CAMK4, CDK1, CDK2, CDK4, CDKN1A, CDKN1 B, CFL1 , CLTC, CSNK2B, CXCR4, DAPP1, DDIT3, DUSP3, E2F1 , ECSIT, EGFR, EIF4E, FASLG, FGF17, FGF22, FGF6, GNA14, GNGT1 , GRB2, GSK3B, HRAS, HSP90B1, IL2RG, IL4, IRAK4, ITPR2, LCK, MKNK1, MKNK2, MYD88, NCK1 , NFKBI
  • EGF EGF, EGFR, PIK3CA (PI3KCA), PIK3CB, PIK3CD, AKT1 , AKT2, AKT3, BAD, IGF1, IGFR, TSC1, TSC2, RHEB, mTOR, EIF4EBP1, CHUK, IKBKB, IKBKG, NFKBIA, NFKB1, RELA, GRB2, SOS1 , SOS2, HRAS, KRAS, NRAS; and BRAF.
  • the term “(poly)peptide” is equivalent to the term “protein” and/or a (poly)peptide may be part of a protein, i.e. comprised in a protein or protein domain.
  • a (poly)peptide has a particular amino acid sequence.
  • a peptide according to the present disclosure may have a length of between 1-500 bp, 10-500 bp, 50-250 bp, or at least 5, 10, 20, 30, 40, 50 bp and/or at most 100, 150, 200, 300, 400, 500, 1000 bp.
  • T cells or T lymphocytes, belong to a group of white blood cells named lymphocytes, which play a role in cell-mediated immunity.
  • T cells originate from hematopoietic stem cells in the bone marrow, mature in the thymus (that is where the T is derived from), and gain their full function in peripheral lymphoid tissues.
  • CD4"CD8" T-cells (negative for both the CD4 and CD8 co-receptor) are committed either to an op (alpha beta) or y ⁇ 5 (gamma delta) fate as a result of an initial p or 5 TCR gene rearrangement.
  • Cells that undergo early p chain rearrangement express a pre-TCR structure composed of a complete p chain and a pre-TCRa chain on the cell surface. Such cells switch to a CD4 + CD8 + state, rearrange the TCRa chain locus, and express an apTCR on the surface.
  • CD4 + CD8 + state rearrange the TCRa chain locus
  • CD4CD8 + state rearrange the TCRa chain locus
  • CD4 + CD8 + state rearrange the TCRa chain locus
  • an apTCR on the surface CD4 + CD8 + state
  • CD4 + CD8 + state rearrange the TCRa chain locus
  • CD4 + CD8 + state rearrange the TCRa chain locus
  • the yCT-cells constitute about 1-5% of the total population of T cells.
  • the extracellular region of a T cell receptor chain comprises a variable region.
  • the variable region of a T cell receptor chain three complementarity determining regions (CDR1 , CDR2, CDR3) are located. These regions are in general the most variable and contribute to diversity among TCRs. CDR regions are composed during the development of a T-cell where so-called Variable-(V), Diverse-(D), and Joining-(J)-gene segments are randomly combined to generate diverse TCRs.
  • the constant region of a T cell receptor chain i.e. being either an alpha, beta, gamma or delta chain, does not substantially vary.
  • the framework regions of a T cell receptor chain i.e. being either an alpha, beta, gamma or delta chain, do not substantially vary either.
  • ybT cells or “gamma delta T cells” represent a small subset of T cells for which the antigenic molecules that trigger their activation is largely unknown.
  • Gamma delta T cells may be considered a component of adaptive immunity in that they rearrange TCR genes to produce junctional diversity and will develop a memory phenotype.
  • various subsets may also be considered part of the innate immunity where a restricted TCR is used as a pattern recognition receptor.
  • Vy9/V ⁇ 52 T cells are specifically and rapidly activated by a set of non-peptidic phosphorylated isoprenoid precursors, collectively named phosphoantigens.
  • yCT-cells may be identified using an antibody specific for the y ⁇ 5 T-cell receptor.
  • Antibodies suitable for FACS are widely available. Conditions are selected, such as provided by the antibody manufacturer that allows the selection of negative and/or positive cells. Examples of antibodies that may be suitable are available from BD Pharmingen (BD, 1 Becton Drive, Franklin Lakes, NJ USA), ybTCR-APC (clone B1 , #555718) or as available from Beckman Coulter, pan-ybTCR-PE (clone IMMU510, # IM1418U). Also, from such selected cells, the nucleic acid (or amino acid sequence) sequence corresponding to the yT cell receptor chain and/or the ST cell receptor chain may be determined. Hence, y6T cells may also be defined as being cells comprising a nucleic acid (or amino acid) sequence corresponding to a yT-cell receptor chain and/or a 52T-cell receptor chain.
  • Vy9V52 TCR T cells recognize early transformed CRC organoids.
  • PDO Patient derived CRC organoids
  • IFNy release of T cells was determined by ELISA.
  • B IFNy production by VY9V52TCR T cells after co-culture with either healthy colon organoids (normal) or CRC organoids mutated for APC, p53, KRAS and SMAD (AKPS) in the presence of 100uM PAM.
  • C In vivo efficacy of VY9V52TCR T cells against AKPS CRC organoids in the presence of PAM.
  • mice were treated with either PBS, T cells expressing a non-functional VY9V52TCR (LM1) or a high affinity VY9V52TCR (TEG001).
  • Tumor burden of AKPS CRC organoids assessed by in vivo bioluminescence imaging (BLI) measuring integrated density per entire tumor area of mice. Statistical significances were calculated by mixed-effects model with repeated measures; *, P ⁇ 0.05; ***, P ⁇ 0.001.
  • GS A Cluster heatmap displaying Gene Set Variation Analysis
  • ES enrichment scores
  • HALLMARK gene sets in normal and AKPS samples with or without 100pM PAM.
  • the heatmap is presented in two grayscale plots: one for positive scores, scaled from -1 to 1 with white representing zero and black representing the highest positive scores, and one for negative scores, scaled from -1 to 1 with white representing zero and black representing the lowest negative scores.
  • E Venn diagram illustrating differentially expressed genes (DEGs) with log fold change >1 and adjusted p-value ⁇ 0.001 , derived from pairwise comparisons of normal and AKPS samples, with or without 100pM PAM .
  • DEGs differentially expressed genes
  • PAM +/- AKPS models Heatmap representation of the expression of genes from the Murad 'killer signature' across wild-type and PAM +/- AKPS models. Scaled from -1 to 1 with white representing negative, (grey representing zero) and black representing positive Z- scores.
  • G Expression of BTNx genes in the WT-AKPS model, with and without PAM (normalized mean expression counts)
  • H IFNy production by Vy9V52TCR T cells after coculture with either healthy colon organoids (normal) or CRC organoids single mutated for APC, p53 and KRAS in the presence of 100uM PAM.
  • WT Healthy colon organoids
  • CRC organoids mutated for APC KO or AKPS mutant CRC were stained with microbeads coated with either non-functional LM1 soluble Vy9V52TCR (LM1) or with high affinity soluble Vy9V52TCR (CI5) in the absence of PAM. Data show MFI of bead binding.
  • FIG. 1 Transformed cells upregulate BTN2A1 surface expression via PI3K kinase activity.
  • MCF10a cells were transduced with different ErbB2 variants and co-cultured with either Vy9V52TCR or HER2-CAR transduced T cells. Tumor cells were pre-treated with the PI3K kinase inhibitor Pictilisib at 2uM overnight. After an overnight co-culture, supernatant was used to determine IFNy production by the Y9 52TCR T cells.
  • B Furthermore, tumor cells were isolated and stained for BTN2A1 via TCR tetramer staining and
  • C BTN3A cell surface expression.
  • D Protein expression of HER2, phosphorylated AKT (pAKT) and total AKT in MCF10a mutant lines cultured for 24h in full culture medium (F) or medium without additional growth factors (S).
  • E Multiple tumor cell lines were co-cultured with Y9 52TCR T-cells after pre-treatment with either the PI3K kinase inhibitor, AKT inhibitor or MEK inhibitor. After an overnight co-culture, supernatant was used to determine IFNy production by the Vy9V52TCR T cells.
  • A Previously published proteomic data including phospho-S/T/Y as variable modification on lysine identifies potential BTN3A1 phospho-sites enriched after PAM treatment.
  • D IFNy production of Vy9V52TCR T cells was measured upon co-culture with either the Phospho-deficient or the phospho-mimic mutated HEK293FT cells in the presence of PAM.
  • FIG. 4 BiolD identifies BTN3A-interacting proteins involved in T-cell tumor targeting.
  • A Schematic representation of the BTN3A1 proteome characterization pipeline.
  • B Genes as indicated have been transiently knocked-down using siRNA. After 48h Vy9V52TCR T cells and 100 pM PAM were added. After overnight co-culture INFy production of Vy9V52TCR T cells was measured.
  • B Box-and-whisker plots depicting the levels of activation after siRNA knock-downs. Percentage of activation was normalized at each experiment to wild type target cells HEK 293T or MZ1851rc.
  • Each gene is ranked according to the fold change in mean activation level relative to the control condition denoted as 'Scrambled'.
  • the genes are displayed in descending order of this fold change. Color coding indicates the direction of change compared to the 'Scrambled' baseline, (median is represented in box-and-whisker plots).
  • a linear model (lm()) was employed to assess the differences in activation levels across the (knocked-down) genes.
  • EMMs estimated marginal means
  • pairwise comparisons were conducted employing Dunnett's test. The results from these pairwise comparisons were summarized to include confidence intervals and adjusted p-values:
  • FIG. 5 Candidate proteins differentially colocalize with BTN3A in membrane clusters.
  • A Protein expression on 5 different targeted tumor cell lines was determined in the absence and presence of PAM by Western Blot, expression differences (PAM vs no PAM) as ratio are depicted in the figure.
  • B MZ1851rc cells were treated overnight with either OpM or 100pM PAM. Afterwards, cells were fixed, permeabilized, and stained for all of the indicated candidate interacting proteins and BTN3A1. Bars indicate mean +/-SEM. Statistical significance of differences between no PAM and PAM conditions were determined using unpaired parametric T-tests. All analyses were performed blinded to sample conditions.
  • C MZ1851rc cells were either treated overnight with 100pM PAM or left untreated.
  • a DuoLinkTM proximity ligation assay was performed to assess interaction between CD277 and either PHLDB2, SYNJ2, or CARMIL1 respectively.
  • Each condition was paired with a technical control (C) constituted by leaving out one of the primary antibodies. All the technical control samples were pulled together to form the control condition.
  • Multiplicity adjusted P-values were calculated using a two-way ANOVA with Tukey’s multiple comparison test. Bars indicate mean +/-SEM.
  • D Heatmap of Pearson's correlation of the expression of genes from the ‘Biol D candidates’ and the BTNx genes in the WT-AKPS model, with or without PAM.
  • E IFNy production of Vy9V52TCR T cells after co-culture with AKPS CRC organoids knocked out for PHLDB2, SYNJ2 and CARMIL1 , respectively in the presence of 100 pM PAM.
  • F Granzyme B production of Vy9V52TCR T cells after co-culture with KO-variants of AKPS mutant organoids knocked out for PHLDB2, SYNJ2 and CARMIL1 with the presence of 100 pM PAM.
  • V777E - indicated V may be mutated to E
  • Vy9V52T cells Although tumor infiltrating Vy9V52T cells often have a good prognostic value, their role in cancer immune surveillance remains to be defined.
  • the inventors employed two independent, genetically engineered step-wise mutagenesis models of human colorectal and breast cancer to demonstrate that a single oncogenic mutation which led to enhanced PI3K activity introduced into healthy cells or organoids is sufficient to upregulate surface expressed BTN2A1 , a known ligand of Vy9V52TCR on tumor cells.
  • PI3K activity leads to upregulation of BTN3A1 expression upon PAM treatment (PI3K activity sensitizes cells for PAM-induced BTN3A1 up regulation).
  • Vy9V52TCR For full activation of T cells through a Vy9V52TCR, phosphorylation of juxtamembrane (JTM) amino acids of BTN3A1 is important leading to the activating heterodimerization of BTN2A1 and 3A1.
  • JTM juxtamembrane
  • PHLDB2, SYNJ2 and CARMIL1 As key players in controlling surface dynamics of BTN2A1 and 3A1 during early transformation. This mode of action allowed Vy9V52TCR T cells to control tumors in vitro and in vivo emphasizing the crucial role of these molecules from early mutagenesis to advanced cancer stages and the therapeutic potential of a Vy9V52TCR.
  • y ⁇ 5T cells have an essential role in cellular stress sensing and immune surveillance for both microbial and autologous stress (e.g. tumorigenesis) 1 .
  • microbial and autologous stress e.g. tumorigenesis 1 .
  • Infiltration of ybT cells in various tumors has been shown to have a favorable prognostic value 2 and play an important role in the immunosurveillance of early tumor development in mice 3 , putting Vy9V52T cells most likely at the first line of defense during transformational processes of a healthy to a cancer cell, however it was unclear which process triggers y ⁇ 5T cells during early transformation even though the anti-tumor role of y ⁇ 5T cells has been implicated in various tumor models with established tumors 4-7 .
  • Vy9V52T cells which are considered the most innate-like subset of gamma delta T cells in general 1 , are activated by intermediate metabolites of the isoprenoid/mevalonate pathway, such as isopentenyl-5-pyrophosphate (IPP) 8 , also referred to as phosphoantigens (pAgs) which can build up in cancerous- or virally-infected cells due to disruption of the mevalonate pathway.
  • Aminobiphosphonate (ABP) drugs such as pamidronate (PAM) can also further increase cellular pAg levels, by inhibiting farnesyl diphosphate synthase (FPPS), an essential enzyme in this pathway 9 .
  • FPPS farnesyl diphosphate synthase
  • Vy9V52T cells harbor great clinical potential as an immunotherapy for cancer 10-12 .
  • the exact mechanism of ligand-receptor interaction has not been found yet, intracellular pAgs are bound to the B30.2 domain of butyrophilin-3 isoform A1 (BTN3A1) which leads to complex formation with BTN2A1 11 ’ 13-15 .
  • BTN2A1 has emerged as key protein for recognition of tumor cells by Vy9V52-T cells 14 where it is directly bound by the gamma chain of Vy9V52TCR.
  • these new insights defined a signature which is predictive for recognition of tumors in cancer patients 19 , the signature could not elucidate at which stage of transformation from and healthy cell to a cancer cell the BTN- pathway is turned on.
  • the inventors used therefore a step wise mutagenesis model for colon and breast cancer, which remodels different steps during mutagenesis to characterize expression patterns of BTN2A1 , BTN3A1 and RHOB during transformation which identified PI3K activity as essential step to upregulate BTN2A1.
  • This model allowed also to hunt for novel players regulating in particular the heavily orchestrated BTN3A1 molecule by an innovative proximity proteomics approach.
  • Vy9V52TCR To properly analyze recognition of a tumor cell through Vy9V52TCR and overcome diversity in innate receptor expression and diversity in function of natural Vy9V52T cells the inventors used soluble Vy9V52TCR formats 11 12 as well as apT cells expressing a high affinity Vy9V52 TCR 20 ' 22 .
  • This strategy allowed to characterize the orchestration of BTN2A1 and BTN3A1 during early transformation and late-stage cancers where BTN2A1 traffics early during mutagenesis to the cell membrane in close proximity to BTN3A1 , a process heavily regulated by PHLDB2, SYNJ2 and CARMIL1.
  • Vy9V52TCR T cells target early transformation events in colorectal cancer (CRC) which features are preserved in late CRC stages to enable targeting by Vy9V52TCR T cells
  • CRC colorectal cancer
  • the inventors investigated both the a priori sensitivity of tumors to recognition by a Vy9V52TCR as well as the presence of an altered mevalonate pathway in healthy versus diseased tissues by adding Pamidronate (PAM).
  • PAM Pamidronate
  • a cells expressing Vy9V52TCR exhibited the production of IFNy when co-cultured with a variety of colorectal cancer (CRC) cell lines and patient derived tumor organoids selectively in the presence of PAM.
  • Vy9V52TCR To discern whether recognition by a Vy9V52TCR is a general mechanism of also healthy colon tissues or distinct feature of malignant transformation , the inventors utilized a colon organoid model derived from healthy tissues and then altered to carry APC (APC KO ), KRAS (KRAS G12D ), TP53 (p53 KO ) and SMAD (SMAD KO ), referred to as AKPS mutant, simulating a fully developed CRC 23 . IFNy production after co-incubation of Vy9V ⁇ 52TCR T cells was exclusively observed in AKPS mutants in the presence of pamidronate (PAM), implying that recognition of tumors by a Vy9V52TCR is a hallmark of malignant transformation ( Figure 1 B).
  • APC APC
  • KRAS G12D KRAS G12D
  • TP53 p53 KO
  • SMAD KO SMAD KO
  • Vy9V02TCR T cells were also able to control CRC outgrowth for at least 43 days after tumor injection (Figure 1 C) in vivo and improved overall survival when NSG-mice were engrafted with APKS mutant CRC organoids which is the only mutant that engrafts in mice 23 .
  • RNA-sequencing from the normal (healthy) and AKPS organoids, with and without PAM.
  • the inventors applied a Wilcoxon rank sum tests over the enrichment scores of a gene set variation analysis (GSVA) focusing on HALLMARK gene sets ( Figure 1D).
  • GSVA gene set variation analysis
  • BTN2A1 gene encoding for the main interacting protein with the Vy9 chain but not BTN3A1/2 genes were significantly elevated already after malignant transformation in the absence of PAM and was also not further increased by PAM (Figure 1G, ), PAM addition significantly increased BTN3A1 gene expression levels in AKPS-mutants ( Figure 1G).
  • patterns of other gene expression had little overlap with the ‘killer signature,’ a set of genes previously found in an extensive CRISPR-screening in the context of immune-mediated tumor cell destruction upon PAM treatment 19 , implying that the recently published killer signature does not fully explain the general sensitivity upon malignant transformation to Vy9V52TCR-mediated recognition.
  • APC KO showed as AKPS mutants an enhanced expression of BTN2A1 protein at the cell membrane.
  • BTN2A1 protein found already in the early, pre-cancerous APC-mutant stage in the absence of PAM ( Figure 11).
  • the by the inventors previously described PAM-induced redistribution of intracellular RhoB 11 ’ 16 was already detected in single APC-mutant organoids.
  • Phosphoinositide 3-kinase/AKT1 activity during early transformation is important for Vy9V52TCR activation
  • MCF10a benign breast tissue cell line was engineered to mimic various oncogenic ErbB2 gene related mutations such as overexpression of HER2 (ErbB2 AMP ), a single mutation in the extracellular domain (ErbB2 S310F ) or amplified kinase activity mutant (ErbB2 V777E ).
  • BTN3A surface expression however did not change either upon ErbB2 V777E mutation or PI3K inhibition but it significantly decreased upon any oncogenic mutations induced into benign cells (Figure 2C).
  • the inventors could confirm the dependency of BTN2A1 and independency of BTN3A1 expression, respectively, on PI3K signalling in multiple targeted cell lines in the absence of PAM.
  • the inventors found that while AKT was phosphorylated on position Ser473 in all lines in the presence of full culture medium containing growth factors, only a mutation in position V777E showed phosphorylation already in the absence of growth factors (Figure 2D), confirming the special role of this mutation.
  • Upregulation of PI3K- AKT1-mT0R activity can be detected or modulated by upregulation (or downregulation) of the pathway regulators, for example PI3K(CA), PTEN, AKT1 , mTOR, KRAS, BRAF, MAP2K1 , MAP2K2 as listed above, or via one or more other regulators such as ACACA, ACTR2, ACTR3, ADCY2, ADRBK1 , AKT1 , AKT1S1 , AP2M1 , ARF1 , ARHGDIA, ARPC3, ATF1 , CAB39, CAB39L, CALR, CAMK4, CDK1 , CDK2, CDK4, CDKN1A, CDKN1 B, CFL1 , CLTC, CSNK2B, CXCR4, DAPP1 , DDIT3, DUSP3, E2F1 , ECSIT, EGFR, EIF4E, FASLG, FGF17, FGF22, FGF6, GNA14
  • BTN3A1 phosphorylation affects surface expression of BTN3A1 and BTN2A1
  • Interactome platform identifies potential BTN3A1 -proximate proteins involved in Vv9V52TCR-induced tumor targeting
  • the inventors knocked down each candidate protein in HEK293F and MZ1851RC cells using siRNAs and used these cells as target against Vy9V52TCR T cells in the presence of PAM and calculated to what level KD influenced IFNy production by Vy9V52TCR T cells (Figure 4B).
  • the inventors used the same target cells also against Wilms tumor 1 specific (WT-1) apTCR T cells after loading with WT-1 peptide to exclude that candidate proteins are not involved in general T cell-target cell interactions.
  • the inventors investigated changes of expression of the candidate proteins upon PAM treatment in a panel of Vy9V52TCR- activating tumor cell lines including breast cancer line MDA-MB 231 , HEK293FT, renal cell carcinoma cell line MZ1851rc and head and neck cancer cell line SCC9 by performing Western blot analysis.
  • the inventors focused our further analysis on five out of eight new candidate proteins against which reliable antibodies were available (PHLDB2, PKP2, SYNJ2, CARMIL1, PA2G4) that allowed both western blot and cellular expression analysis.
  • the inventors found that PAM treatment induced a significant increase of RhoB and SYNJ2 protein and decrease of PHLDB2 protein expression in all tested cell lines in the presence of PAM ( Figure 5A).
  • the inventors compared protein expression in HEK293FT wt and HEK BTN3 KO cells in the presence and absence of PAM of the newly identified proteins and observed partial loss of 48hosphor48nn of SYNJ2, suggesting its expression is partially co-regulated with BTN3A1.
  • Vy9V52TCR T cells is imprinted as early as after earliest single mutations of a variety of oncogenes.
  • the inventors show that AKT phosphorylation and mTOR activity via enhanced PI3K activity in transformed cells results in enhanced BTN2A1 surface expression, the ligand for the gamma chain of Vy9V52TCR 14 ’ 29 , and thereby making early transformed cells susceptible for Vy9V52TCR recognition.
  • this step is preferably accompanied by additional upregulation of BTN3A1.
  • Additional steps include increased levels of intracellular phosphoantigens (pAg), which can be mimicked by aminobisphosphonates, like Pamidronate (PAM).
  • PAM aminobisphosphonates
  • JTM juxtamembrane
  • BTN3A1 While intracellular pAg-induced changes in total expression of BTN proteins were minimal, the inventors found additional series of spatial rearrangements directly related to BTN3A1, and identified three novel functionally relevant proteins that showed PAM-dependent spatial dynamics, CARMIL1 , SYNJ2, and PHLDB2.
  • Vv9V52TCR-based therapies 10 targeting tumors with oncogenic mutations promoting PI3K activity can be used at very early stages of malignant transformation or many tumors with low mutational load.
  • the fact that not only artificial organoids mutated for all tumor associated genes, but also patient derived colorectal organoids are recognized by Vy9V52TCR cells shows that these molecular rearrangements are preserved during tumor development and will allow targeting of primary but most likely also metastatic lesions.
  • PI3K activation in cancer patients might provide a new avenue to possibly sensitize tumors for Vy9V52TCR- driven attack.
  • next generation engineering strategies such as TEGs 20 ’ 22 and Gamma delta TCR anti-CD3 bispecific molecules (GABs) 12 might employ full potential of how to maximize targeting potential via Vy9V52TCRs.
  • AMPK and AKT are often seen to act as antagonists in regulating autophagy and apoptosis 3334 , however, it has been shown that under certain conditions, e.g. cancer, activation of AMPK, especially via AMPK-activator AICAR, can also activate AKT 3536 and mTORC2 36 , and vice versa, inhibition of AMPK via compound C reduced AKT activity 35 . It is also possible, that for the initial trigger, BTN2A1 surface expression, an oncogenic PI3K-activating mutation is necessary, but that further regulation and enhancement can be achieved by other pathways, such as AMPK.
  • BTN3A1 surface expression remained unaffected by this oncogenic offset.
  • the inventors have not been able to identify the precise molecular steps needed for upregulation of BTN the inventors showed that the ability to be susceptible to Vy9V52T cells is very early imprinted in pre-cancerous lesions.
  • the inventors show that accumulation of intracellular pAg are important and dysregulation of the mevalonate pathway is a frequent hallmark of malignant transformation 37 and leads to this accumulation of pAgs.
  • the inventors used aminobisphosphonates (ABP), like Pamidronate (PAM) as model system to characterize the next molecular steps.
  • pAg were shown to act as glue between BTN molecules BTN2A1 and BTN3A1 to activate Vy9V52T cells 18 , and previously, the juxtamembrane (JTM) region have been identified as being sensitive for translating pAg-induced inside-out-signaling 17 ’ 38 .
  • JTM juxtamembrane
  • RhoB relocates to the cell membrane in the vicinity of BTN3A1 dimers and plays a crucial role in the high turn-over of BTN3A1 in cancer cells 16 .
  • Our new data imply that active RhoB might cause relocalization of SYNJ2 from the cytosol to the cell membrane upon PAM stimulation and support Rac1, another small GTPase closely linked to RhoB 41 .
  • RhoB 41 Another small GTPase closely linked to RhoB 41 .
  • SYNJ2 being enriched in larger size clusters but also in direct interaction with BTN3A1 in the presence of PAM could lead to a PAM-induced protein aggregation close to BTN3A1.
  • the phosphoinositode-5 phosphatase SYNJ2 has been shown to be involved in actin-based cytoskeleton dynamics 4243 and furthermore has also been implicated in vesicle trafficking 44 , emphasizing and a role in supporting RhoB-mediated BTN3A1 recycling 11 and enable recognition of tumor cells by Vy9V52T cells.
  • CARMIL1 most likely accommodates the formation of cytoskeletal rearrangement 45 and might altogether the final step to stabilize BTN2A1-BTN3A1 dimers in the immunological synapse on the tumor cell surface. While the inventors found prove on multiple levels for co-localization or interaction with BTN3A1 in a PAM-dependent manner with CARMIL1, SYNJ2 and PHLDB2, their relationship to BTN2A1 remains unclear.
  • PHLDB2 contains a PH-domain, which has a high affinity to membrane-bound PIP3 39 , an oncogenic product by PI3K 46 . Therefore, a role of PHLDB2 in recruitment of BTN proteins to the cell membrane is not excluded and needs to be further investigated.
  • the inventors show that during early mutagenesis hallmarks for the recognition of a cell through Vy9V52TCRare induced such as BTN2A1 surface upregulation and RhoB relocalization.
  • the inventors found activated PI3K pathway in single oncogene mutated tumors creates the basis for the susceptibility of cancer cells to Vy9V52TCR.
  • increased levels of pAgs either induced by PAM or through endogously increased levels frequently increased in cancer cells are also important to allow recognition by Vy9V52TCR.
  • the inventors identified phosphorylation of BTN3A1 JTM region as additional independent mechanism important to achieve a complex recognized by a Vy9V52TCR.
  • the inventors identified three novel key players, namely CARMIL1 , SYNJ2 and PHLDB2, that directly regulates BTN3A1 surface expression and therefore controls BTN2A1- BTN3A1 dimer dynamics on the cell surface. These findings do not only shed light in the role of Vy9V52T cells during early cancer immune surveillance but have also great implication for all Vy9V52T cell based immune therapies.
  • Antibodies The following antibodies were used: Anti-CD277/BTN3A Alexa Fluor 647 (FAB7316R, Clone 849203), Anti-CD277/BTN3A PE Mab (FAB7316P, Clone 849203), anti- CD277/BTN3A (clone 20.1 , LSC106569), pan-ybTCR PE (IMMU510, B49176), Granzyme B APC (QA16A02, 372204), CD107a PE (H4A3), CD8a PerCP-Cy5.5 (RPA-T8, 301032), anti- RhoB mouse monoclonal (C-5, sc-8048), anti-RhoB rabbit polyclonal (abeam, ab170611), anti-CD3 (clone: OKT3), Goat-anti-Rabbit AF488 IgG (H+L), anti-Rabbit AF488 Fab fragment, Goat-anti-Mouse AF488 IgG (H
  • Rabbit mAb ERBB2 (29D8) (1:1 ,000; #2165; Cell Signaling Technology), rabbit mAb AKT (1 :1 ,000; #9272; Cell Signaling Technology), rabbit Phospho-AKT Ser473 (1:1,000; #9272; Cell Signaling Technology), rabbit mAb Phospho-AKT Thr308 (D25E6) (1 :1,000; #13038; Cell Signaling Technology), Rabbit mAb P44/42 MAPK (Erk 1 / 2 ) (137F5) (1 :1,000; #4695; Cell Signaling Technology), rabbit mAb Phospho-MAPK (Erk 1 Thr202/Tyr204) (D13.14.4E) (1 :1 ,000; #4370; Cell Signaling Technology, mouse mAb GAPDH (1;5000, G8795, MERCK/Sigma-Aldrich).
  • HEK293FT, MDA-MB-231 , MZ1851rc, HT29, SKBR-3, Caco-2, HL-60, Phoenix-ampho and MCF10a cells lines were cultured in DMEM+GlutaMAX with 10% fetal calf serum (FCS) and 1% Penicillin-Streptomycin. Daudi cells were cultured in in RPMI-GlutaMAX with 1% Pen/Strep and 10% FCS.

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Abstract

La présente invention concerne un procédé ex vivo pour déterminer la sensibilité à un agent thérapeutique ciblant BTN2A1 et/ou BTN3A1, le procédé consistant à déterminer dans un échantillon prélevé sur un patient : i) la présence ou l'absence, dans le récepteur Erb-B2 tyrosine kinase 2 (ErbB2), d'un acide aminé autre que la valine à une position correspondant à la position 777 dans SEQ ID NO : 1 ; ii) le niveau d'activité de la voie PI3K-AKT1-mTOR ; et/ou iii) l'état de phosphorylation du membre A1 de la sous-famille 3 de la butyrophiline (BTN3A1). La présente invention concerne en outre un agent thérapeutique ciblant BTN2A1 et/ou BTN3A1 destiné à être utilisé dans le traitement du cancer, d'une maladie infectieuse ou d'une maladie auto-immune, l'agent thérapeutique ciblant BTN2A1 et/ou BTN3A1 étant administré séparément, successivement ou simultanément à au moins un activateur de la voie PI3K-AKT1-mTOR et/ou au moins un agent induisant la phosphorylation.
PCT/EP2024/065794 2023-06-09 2024-06-07 Détection et/ou modulation de la sensibilité à des agents thérapeutiques ciblant btn2a1 et/ou btn3a1 Pending WO2024251981A1 (fr)

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