WO2024251981A1 - Detecting and/or modulating susceptibility to btn2a1 and/or btn3a1 targeting therapeutics - Google Patents

Detecting and/or modulating susceptibility to btn2a1 and/or btn3a1 targeting therapeutics 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

The present invention relates to an ex vivo method for determining susceptibility to a BTN2A1 and/or BTN3A1 targeting 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 butyrophilin subfamily 3 member A1 (BTN3A1). The present invention further relates to a BTN2A1 and/or BTN3A1 targeting therapeutic for use in the treatment of cancer, infectious disease or auto-immune disease, wherein the BTN2A1 and/or BTN3A1 targeting therapeutic is administered separately, sequentially or simultaneously to at least one PI3K-AKT1-mTOR pathway activator and/or at least one phosphorylation-inducing agent.

Description

Detecting and/or modulating susceptibility to BTN2A1 and/or BTN3A1 targeting therapeutics
Technical field
The present invention relates to treatment of cancer, infectious disease or auto-immune disease, in particular treatments that target BTN2A1 and/or BTN3A1 expressing cells, for example Vy9V52 TCR based treatments.
Background of the invention
Increasing evidence show that human gdT cells have an essential role in cellular stress sensing and immune surveillance for both microbial and autologous stress (e.g. tumorigenesis) (1). Infiltration of gdT 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 Vg9Vd2T cells most likely at the first line of defense during transformational processes of a healthy to a cancer cell, however it remains unclear which process triggers gdT cells during early transformation even though the anti-tumor role of gdT cells has been implicated in various tumor models with established tumors (4-7). 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). Because of the broad-, but tumor-specific recognition of malignantly-transformed cells, and the lack of MHC-restriction, Vg9Vd2T cells harbor great clinical potential as an immunotherapy for cancer (10-12). Even though it is well-established that the gdTCR itself is essential for recognition of pAgs, a clear ligand-TCR interaction has not been found yet. Recently however, butyrophilin-3 isoform A1 (BTN3A1) and BTN2A1 have emerged as key proteins for recognition of tumor cells by Vy9V52-T cells (15) where BTN2A1 is directly bound by the gamma chain of Vg9Vd2TCR. The role of the small GTPase RHOB, was previously demonstrated to relocalize to the membrane and interact with BTN3A1 upon pAg build-up (16). This is associated with cytoskeletal changes and reduced mobility of BTN3A1 in the membrane, implying a sort of “membrane trapping” mechanism. It is thought that direct binding of pAgs to the intracellular B30.2 domain subsequently induces joint conformational and spatial changes (referred as CD277J), rendering the BTN3A1 molecule ‘activated’ for interaction with the y6-TCR (16, 17). Even with the identification of BTN2A1 (11, 14, 15) and the understanding of BTN3A1 regulation through RHOB (11 , 16), it is not fully elucidated how this mechanism is regulated and even more important at which stage of transformation from a healthy cell to a cancer cell this mechanism is turned on and would allow in principle recognition of a cancer cell through a Vg9Vd2TCR. This understanding is however crucial to better understand early immune cancer surveillance and allow a better development of Vg9Vd2TCR based immune therapies. Importantly, it may allow detecting and/or modulating susceptibility of cancer patients for BTN2A1 and/or BTN3A1 targeting therapy, in particular through a Vg9Vd2TCR.
It is an objective of the present invention to overcome one or more of the above-mentioned or other problems in the art, preferably to provide for a method for detecting and/or modulating susceptibility of patients for BTN2A1 and/or BTN3A1 targeting therapy, in particular Vg9Vd2TCR based therapy.
Summary of the invention
The present inventors unraveled the regulation of expression of butyrophilin subfamily 2 member A1 (BTN2A1) and BTN3A1 on cancer cells (or infected cells I cells affected by autoimmune disease) and surprisingly found that
(1) a single oncogenic mutation (in particular ErbB2V777E);
(2) activating PI3KCA-AKT1-mTOR pathway, and/or
(3) 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.
Accordingly, 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
- at least one PI3K-AKT1-mTOR pathway activator; and/or
- at least one phosphorylation-inducing agent.
Detailed description of the invention
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.
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.
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. Recent studies have reported that the BTN2A1 binding peptide of the Vy9 domain of the Vy9V52 TCR interacts with BTN2A1 using germline encoded residues, independent of the CDR3y residues, and that this interaction is important for the activation of Vy9V<52 T cells.
Accordingly, the present method allows for determining susceptibility to a BTN2A1 and/or BTN3A1 targeting agent/therapeutic of a (cancer) patient and/or of cancer cells/tissue, such as a tumour, or alternatively, of an infectious disease patient and/or of infected cells/tissue, or of an auto-immune disease patient and/or of cells affected by auto-immune disease.
In this regard, “susceptibility” means whether the respective (cancer or infectious/auto- immune disease) patient (likely) will respond positively to a BTN2A1 and/or BTN3A1 targeting agent/therapeutic, e.g. the treatment (likely) will lead to tumour shrinkage, tumour stabilization, reduced cancer associated pain, and/or prolonged survival of the patient (or eradicating infection/infected cells). In addition or alternatively, “susceptibility” in this regard means whether the respective (cancer) cells/tissue (e.g. tumour) or (infected) cells will respond positively to a BTN2A1 and/or BTN3A1 targeting agent/therapeutic, i.e. the treatment (likely) will lead to killing of said (cancer) cells/tissue (e.g. tumour), reduce growth of said (cancer) cells/tissue (e.g. tumour) and/or tumour shrinkage, tumour stabilization, or alternatively, killing of (infected cells) (or recognition of cells affected by auto-immune disease and subsequent inhibiting/blocking of T cell activation).
In other words, determining susceptibility to a BTN2A1 and/or BTN3A1 targeting agent/therapeutic can be interchangeable with predicting response to a BTN2A1 and/or BTN3A1 targeting agent/therapeutic, or interchangeable with predicting efficacy/effectiveness of a BTN2A1 and/or BTN3A1 targeting agent/therapeutic. In some embodiments, susceptibility to a BTN2A1 and/or BTN3A1 targeting agent/therapeutic may refer to eligibility to Vy9V<52 T cell receptor recognition.
Conversely, in the case no or reduced susceptibility is determined, the present method can be used to predict and/or prevent toxicity (and/or side effects) of a BTN2A1 and/or BTN3A1 targeting agent/therapeutic.
The sample as used in the present method may be or comprise blood, blood serum, urine, lacrimal fluid, or saliva. Preferably the sample comprises cells which are not healthy cells, preferably diseased cells. The cells preferably (surface) express BTN2A1 and/or BTN3A1. More preferably the sample comprises cancer cells and/or extracellular vesicles from cancer cells, or cells potentially affected/targeted by any auto immune process. Alternatively, the sample comprises infected cells and/or extracellular vesicles from infected cells (in case of a patient with infectious disease). Typically, the sample is or comprises a cancer tissue biopsy. 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.
As said, in order to determine susceptibility to a BTN2A1 and/or BTN3A1 targeting agent/therapeutic, 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.
The Erb-B2 receptor tyrosine kinase 2, also known as HER2 (human epidermal growth factor receptor 2), is well known to a skilled person and refers to a protein that plays a significant role in cell growth and division. It is a member of the human epidermal growth factor receptor (EGFR) family of proteins. HER2 is encoded by the ERBB2 gene and is found on the surface of certain cells, including cancer cells. It functions as a receptor that receives signals from growth factors, leading to the activation of intracellular signalling pathways that regulate cell proliferation, survival, and differentiation.
In accordance with the present disclosure, it was found that presence, 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 , is indicative of susceptibility to a BNT2A1 and/or BTN3A1 targeting agent/therapeutic and that 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 , is indicative of reduced susceptibility to a BNT2A1 and/or BTN3A1 targeting agent/therapeutic (relative to said presence). The said amino acid other than valine may be glutamic acid.
In addition or alternatively, in order to determine susceptibility to a BTN2A1 and/or BTN3A1 targeting agent/therapeutic, the method may comprise determining ii) level of PI3K-AKT1- mTOR pathway activity.
The PI3K-AKT1-mTOR pathway is well known to a skilled person and may refer to a cellular signalling pathway involved in regulating various cellular processes, including cell growth, proliferation, survival, and metabolism. It is an important pathway implicated in many physiological functions as well as in the development and progression of various diseases, including cancer. The pathway can be seen as comprising of three main components:
- Phosphoinositide 3-kinase (PI3K): PI3K is an enzyme that phosphorylates certain lipids, known as phosphoinositides, present in the cell membrane. Activation of PI3K leads to the production of phosphatidylinositol-3,4,5-trisphosphate (PIP3), a lipid second messenger.
- Protein Kinase B (also known as AKT): AKT, or protein kinase B, is a serine/threonine kinase that is recruited to the cell membrane when PIP3 is produced. Once at the membrane, AKT is activated through phosphorylation by other enzymes. Activated AKT then phosphorylates various downstream targets involved in cell growth, proliferation, and survival.
- Mammalian Target of Rapamycin (mTOR): mTOR is a protein kinase that integrates signals from various pathways, including the PI3K-AKT1 pathway, to regulate protein synthesis, cell growth, and metabolism. mTOR exists in two distinct complexes, mTORCI and mTORC2, each with different functions. mTORCI is particularly important in regulating protein synthesis and cell growth in response to nutrient availability and growth factors.
The present inventors found that upregulation of PI3K-AKT1-mTOR pathway activity, e.g. relative to healthy cells/tissue, is indicative of susceptibility to a BNT2A1 and/or BTN3A1 targeting agent/therapeutic. Conversely, no upregulation of PI3K-AKT1-mTOR activity, e.g. relative to healthy cells/tissue, is indicative of reduced susceptibility to a BNT2A1 and/or BTN3A1 targeting agent/therapeutic (relative to said upregulation).
In addition or alternatively, in order to determine susceptibility to a BTN2A1 and/or BTN3A1 targeting agent/therapeutic, the present method may comprise determining iii) phosphorylation status of BTN3A1.
The present inventors found that phosphorylation of a serine at a position corresponding to position 296 in SEQ ID NO:4 and/or phosphorylation of a threonine at a position corresponding to position 297 in SEQ ID NO:4 is indicative of susceptibility to a BNT2A1 and/or BTN3A1 targeting agent/therapeutic. Conversely, no phosphorylation of a serine at a position corresponding to position 296 in SEQ ID NO:4 and/or no phosphorylation of a threonine at a position corresponding to position 297 in SEQ ID NO:4 is indicative of reduced susceptibility to a BNT2A1 and/or BTN3A1 targeting agent/therapeutic (relative to said phosphorylation at position 296 and/or 297).
Said positions 296 and/or 297 may be within the juxtamembrane region of BTN3A1.
As mentioned earlier herein, the present method may determine: 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.
It should be noted that in case only one of i), ii), iii) is indicative of susceptibility to a BNT2A1 and/or BTN3A1 targeting agent/therapeutic, the method already determines susceptibility to BNT2A1 and/or BTN3A1 targeting agent/therapeutic, but if two or even three of i), ii), iii) are indicative, the respective patient or (cancer) cells may have increased or even more increased susceptibility to a BNT2A1 and/or BTN3A1 targeting agent/therapeutic, relative to if only one of i), ii), iii) is indicative. 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 sequencing a nucleic acid sequence encoding ErbB2.
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. There are different methods available for DNA sequencing, including Sanger sequencing (also known as chaintermination sequencing) and Next-Generation Sequencing (NGS) technologies, all well known to a skilled person.
In addition or alternatively, 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).
The skilled person is aware of how such antibody may be obtained using well known methods, for example by 1) animal immunization by injecting the peptide into an animal, typically rabbits or mice. This process triggers an immune response, leading to the production of antibodies specific to the peptide. And subsequent 2) antibody screening to identify antibodies that specifically bind to the mutant peptide but not the non-mutated peptide (or vice versa). Techniques like ELISA (enzyme-linked immunosorbent assay) or Western blotting can be used for this purpose.
In addition to the mutation in ErbB2, also the below KRAS mutations were found to be (also) associated with activation of the PI3K-AKT1-mTOR pathway.
Accordingly, in a preferred embodiment, step i) of the present method additionally (or alternatively) determines presence or absence in Kirsten rat sarcoma viral oncogene homolog (KRAS) of
- an amino acid other than Lys at a position corresponding to position 5 in SEQ ID NO:2;
- an amino acid other than Vai at a position corresponding to position 7 in SEQ ID NO:2;
- an amino acid other than Vai at a position corresponding to position 8 in SEQ ID NO:2;
- an amino acid other than Vai at a position corresponding to position 9 in SEQ ID NO:2;
- an amino acid other than Gly at a position corresponding to position 10 in SEQ ID NO:2;
- an amino acid other than Ala at a position corresponding to position 11 in SEQ ID NO:2; - an amino acid other than Gly at a position corresponding to position 12 in SEQ ID NO:2;
- an amino acid other than Gly at a position corresponding to position 13 in SEQ ID NO:2;
- an amino acid other than Vai at a position corresponding to position 14 in SEQ ID NO:2;
- an amino acid other than Ser at a position corresponding to position 17 in SEQ ID NO:2;
- an amino acid other than Ala at a position corresponding to position 18 in SEQ ID NO:2;
- an amino acid other than Leu at a position corresponding to position 19 in SEQ ID NO:2;
- an amino acid other than Gin at a position corresponding to position 22 in SEQ ID NO:2;
- an amino acid other than Leu at a position corresponding to position 23 in SEQ ID NO:2;
- an amino acid other than His at a position corresponding to position 27 in SEQ ID NO:2;
- an amino acid other than Glu at a position corresponding to position 31 in SEQ ID NO:2;
- an amino acid other than Pro at a position corresponding to position 34 in SEQ ID NO:2;
- an amino acid other than Thr at a position corresponding to position 35 in SEQ ID NO:2;
- an amino acid other than lie at a position corresponding to position 36 in SEQ ID NO:2;
- an amino acid other than Vai at a position corresponding to position 45 in SEQ ID NO:2;
- an amino acid other than Thr at a position corresponding to position 58 in SEQ ID NO:2;
- an amino acid other than Ala at a position corresponding to position 59 in SEQ ID NO:2;
- an amino acid other than Gly at a position corresponding to position 60 in SEQ ID NO:2;
- an amino acid other than Gly at a position corresponding to position 61 in SEQ ID NO:2;
- an amino acid other than Glu at a position corresponding to position 62 in SEQ ID NO:2;
- an amino acid other than Glu at a position corresponding to position 63 in SEQ ID NO:2;
- an amino acid other than Asp at a position corresponding to position 69 in SEQ ID NO:2;
- an amino acid other than Tyr at a position corresponding to position 71 in SEQ ID NO:2;
- an amino acid other than Met at a position corresponding to position 72 in SEQ ID NO:2;
- an amino acid other than Thr at a position corresponding to position 74 in SEQ ID NO:2;
- an amino acid other than Cys at a position corresponding to position 80 in SEQ ID NO:2;
- an amino acid other than Asp at a position corresponding to position 92 in SEQ ID NO:2;
- an amino acid other than Arg at a position corresponding to position 97 in SEQ ID NO:2;
- an amino acid other than Lys at a position corresponding to position 117 in SEQ ID NO:2;
- an amino acid other than Ala at a position corresponding to position 146 in SEQ ID NO:2;
- an amino acid other than Lys at a position corresponding to position 147 in SEQ ID NO:2;
- an amino acid other than Vai at a position corresponding to position 152 in SEQ ID NO:2;
- an amino acid other than Asp at a position corresponding to position 154 in SEQ ID NO:2;
- an amino acid other than Phe at a position corresponding to position 156 in SEQ ID NO:2;
- an amino acid other than Arg at a position corresponding to position 164 in SEQ ID NO:2;
- an amino acid other than Cys at a position corresponding to position 180 in SEQ ID NO:2;
- an amino acid other than Lys at a position corresponding to position 182 in SEQ ID NO:2;
- an amino acid other than lie at a position corresponding to position 183 in SEQ ID NO:2; - an amino acid other than Lys at a position corresponding to position 184 in SEQ ID NO:2; and/or
- an amino acid other than Lys at a position corresponding to position 185 in SEQ ID NO:2, preferably wherein presence is indicative of susceptibility to a BNT2A1 and/or BTN3A1 targeting agent/therapeutic and wherein absence is indicative of reduced susceptibility to a BNT2A1 and/or BTN3A1 targeting agent/therapeutic (relative to said presence).
As known by a skilled person, 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.
It should be noted that determining presence or absence of one or more of the above indicated amino acids in KRAS 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) and 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).
In addition, the below P53 mutations were found to be (also) associated with activation of the PI3K-AKT1-mTOR pathway.
Accordingly, in addition (or alternatively), step i) of the present method determines presence or absence in Protein 53 (P53) of
- an amino acid other than Pro at a position corresponding to position 82 in SEQ ID NO:3;
- an amino acid other than Vai at a position corresponding to position 97 in SEQ ID NO:3;
- an amino acid other than Gly at a position corresponding to position 105 in SEQ ID NO:3;
- an amino acid other than Ser at a position corresponding to position 106 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 110 in SEQ ID NO:3;
- an amino acid other than Tyr at a position corresponding to position 126 in SEQ ID NO:3;
- an amino acid other than Lys at a position corresponding to position 132 in SEQ ID NO:3;
- an amino acid other than Met at a position corresponding to position 133 in SEQ ID NO:3;
- an amino acid other than Ala at a position corresponding to position 138 in SEQ ID NO:3;
- an amino acid other than Cys at a position corresponding to position 141 in SEQ ID NO:3;
- an amino acid other than Gin at a position corresponding to position 144 in SEQ ID NO:3; - an amino acid other than Pro at a position corresponding to position 151 in SEQ ID NO:3;
- an amino acid other than Pro at a position corresponding to position 152 in SEQ ID NO:3;
- an amino acid other than Thr at a position corresponding to position 155 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 156 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 158 in SEQ ID NO:3;
- an amino acid other than Tyr at a position corresponding to position 163 in SEQ ID NO:3;
- an amino acid other than Gin at a position corresponding to position 167 in SEQ ID NO:3;
- an amino acid other than Vai at a position corresponding to position 172 in SEQ ID NO:3;
- an amino acid other than Vai at a position corresponding to position 173 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 174 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 175 in SEQ ID NO:3;
- an amino acid other than His at a position corresponding to position 179 in SEQ ID NO:3;
- an amino acid other than Glu at a position corresponding to position 180 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 181 in SEQ ID NO:3;
- an amino acid other than Ala at a position corresponding to position 189 in SEQ ID NO:3;
- an amino acid other than His at a position corresponding to position 193 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 196 in SEQ ID NO:3;
- an amino acid other than Vai at a position corresponding to position 197 in SEQ ID NO:3;
- an amino acid other than Asn at a position corresponding to position 210 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 213 in SEQ ID NO:3;
- an amino acid other than Pro at a position corresponding to position 219 in SEQ ID NO:3;
- an amino acid other than Tyr at a position corresponding to position 220 in SEQ ID NO:3;
- an amino acid other than Ser at a position corresponding to position 227 in SEQ ID NO:3;
- an amino acid other than His at a position corresponding to position 233 in SEQ ID NO:3;
- an amino acid other than Tyr at a position corresponding to position 234 in SEQ ID NO:3;
- an amino acid other than Asn at a position corresponding to position 235 in SEQ ID NO:3;
- an amino acid other than Tyr at a position corresponding to position 236 in SEQ ID NO:3;
- an amino acid other than Met at a position corresponding to position 237 in SEQ ID NO:3;
- an amino acid other than Cys at a position corresponding to position 238 in SEQ ID NO:3;
- an amino acid other than Ser at a position corresponding to position 241 in SEQ ID NO:3;
- an amino acid other than Cys at a position corresponding to position 242 in SEQ ID NO:3;
- an amino acid other than Gly at a position corresponding to position 244 in SEQ ID NO:3;
- an amino acid other than Gly at a position corresponding to position 245 in SEQ ID NO:3;
- an amino acid other than Met at a position corresponding to position 246 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 248 in SEQ ID NO:3;
- an amino acid other than lie at a position corresponding to position 251 in SEQ ID NO:3;
- an amino acid other than Leu at a position corresponding to position 252 in SEQ ID NO:3;
- an amino acid other than Leu at a position corresponding to position 257 in SEQ ID NO:3; - an amino acid other than Glu at a position corresponding to position 258 in SEQ ID NO:3;
- an amino acid other than Leu at a position corresponding to position 265 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 267 in SEQ ID NO:3;
- an amino acid other than Vai at a position corresponding to position 272 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 273 in SEQ ID NO:3;
- an amino acid other than Cys at a position corresponding to position 275 in SEQ ID NO:3;
- an amino acid other than Pro at a position corresponding to position 278 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 280 in SEQ ID NO:3;
- an amino acid other than Asp at a position corresponding to position 281 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 282 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 283 in SEQ ID NO:3;
- an amino acid other than Glu at a position corresponding to position 285 in SEQ ID NO:3;
- an amino acid other than Glu at a position corresponding to position 286 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 290 in SEQ ID NO:3;
- an amino acid other than Lys at a position corresponding to position 292 in SEQ ID NO:3;
- an amino acid other than Lys at a position corresponding to position 305 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 306 in SEQ ID NO:3;
- an amino acid other than Pro at a position corresponding to position 309 in SEQ ID NO:3;
- an amino acid other than Gly at a position corresponding to position 325 in SEQ ID NO:3;
- an amino acid other than Arg at a position corresponding to position 337 in SEQ ID NO:3;
- an amino acid other than Leu at a position corresponding to position 344 in SEQ ID NO:3;
- an amino acid other than His at a position corresponding to position 365 in SEQ ID NO:3; and/or
- an amino acid other than Ser at a position corresponding to position 366 in SEQ ID NO:3, preferably wherein presence is indicative of susceptibility to a BNT2A1 and/or BTN3A1 targeting agent/therapeutic and wherein absence is indicative of reduced susceptibility to a BNT2A1 and/or BTN3A1 targeting agent/therapeutic (relative to said presence).
Protein 53 (p53), 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.
It will be understood that 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. by more than 1 , 2, 3, 4, 5, 10, 15, 20, 25% in number of mRNA molecules) of 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, MAP2K3, MAP2K6, MAP3K7, MAPK1, MAPK10, MAPK8, MAPK9, MAPKAP1, MKNK1 , MKNK2, MYD88, NCK1 , NFKBIB, NGF, NOD1 , PAK4, PDK1 , PFN1 , PIK3R3, PIKFYVE, PIN1, PITX2, PLA2G12A, PLCB1 , PLCG1, PPP1CA, PPP2R1B, PRKAA2, PRKAG1 , PRKAR2A, PRKCB, PTEN, PTPN11, RAC1, RAF1, RALB, RIPK1, RIT1, RPS6KA1, RPS6KA3, RPTOR, SFN, SLA, SLC2A1 , SMAD2, SQSTM1, STAT2, TBK1, THEM4, TIAM1 , TNFRSF1A, TRAF2, TRIB3, TSC2, UBE2D3, LIBE2N, VAV3, YWHAB (and/or determining downregulation of expression of one or more of PTEN and/or MAP2K1);
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. 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.
In addition or alternatively, 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: RPS6KB1, PDPK1, PIK3CA, TSC1, PTEN, EIF4B, PRKCA, PAK1, AKT2, GRB2 and/or downregulation by more than 10% of PIK3R1 , MTOR, TSC2, PRKCZ, AKT1 , GRB2, EIF4A, HSPB1 , and RHEB.
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).
Genes up-regulated by activation of the PI3K/AKT/mTOR pathway. (From: Liberzon et al; Cell Syst, 2015 Dec 23; 1(6):417-425. doi: 10.1016/j.cels.2015.12.004).
Figure imgf000014_0001
ACACA 31 ACACA acetyl-CoA carboxylase alpha
ACTR2 10097 ACTR2 actin related protein 2
ACTR3 10096 ACTR3 actin related protein 3
ADCY2 108 ADCY2 adenylate cyclase 2
ADRBK1 156 GRK2 G protein-coupled receptor kinase 2
AKT1 207 AKT1 AKT serine/threonine kinase 1
AKT1S1 84335 AKT1S1 AKT1 substrate 1
AP2M1 1173 AP2M1 adaptor related protein complex 2 subunit mu 1
ARF1 375 ARF1 ADP ribosylation factor 1
ARHGDIA 396 ARHGDIA Rho GDP dissociation inhibitor alpha
ARPC3 10094 ARPC3 actin related protein 2/3 complex subunit 3
ATF1 466 ATF1 activating transcription factor 1
CAB39 51719 CAB39 calcium binding protein 39 CAB39L 81617 CAB39L calcium binding protein 39 like
CALR 811 CALR calreticulin
CAMK4 814 CAMK4 calcium/calmodulin dependent protein kinase IV
CDK1 983 CDK1 cyclin dependent kinase 1
CDK2 1017 CDK2 cyclin dependent kinase 2
CDK4 1019 CDK4 cyclin dependent kinase 4
CDKN1A 1026 CDKN1A cyclin dependent kinase inhibitor 1 A
CDKN1 B 1027 CDKN1 B cyclin dependent kinase inhibitor 1 B
CFL1 1072 CFL1 cofilin 1
CLTC 1213 CLTC clathrin heavy chain
CSNK2B 1460 CSNK2B casein kinase 2 beta
CXCR4 7852 CXCR4 C-X-C motif chemokine receptor 4
DAPP1 27071 DAPP1 dual adaptor of phosphotyrosine and 3-
Phosphoinositides 1
DDIT3 1649 DDIT3 DNA damage inducible transcript 3
DUSP3 1845 DUSP3 dual specificity phosphatase 3
E2F1 1869 E2F1 E2F transcription factor 1
ECSIT 51295 ECSIT ECSIT signaling integrator
EGFR 1956 EGFR epidermal growth factor receptor
EIF4E 1977 EIF4E eukaryotic translation initiation factor 4E
FASLG 356 FASLG Fas ligand
FGF17 8822 FGF17 fibroblast growth factor 17
FGF22 27006 FGF22 fibroblast growth factor 22
FGF6 2251 FGF6 fibroblast growth factor 6
GNA14 9630 GNA14 G protein subunit alpha 14
GNGT1 2792 GNGT1 G protein subunit gamma transducin 1
GRB2 2885 GRB2 growth factor receptor bound protein 2
GSK3B 2932 GSK3B glycogen synthase kinase 3 beta
HRAS 3265 HRAS HRas proto-oncogene, GTPase
HSP90B1 7184 HSP90B1 heat shock protein 90 beta family member 1
IL2RG 3561 IL2RG interleukin 2 receptor subunit gamma
IL4 3565 IL4 interleukin 4
IRAK4 51135 IRAK4 interleukin 1 receptor associated kinase 4
ITPR2 3709 ITPR2 inositol 1 ,4, 5-trisphosphate receptor type 2
LCK 3932 LCK LCK proto-oncogene, Src family tyrosine kinase
MAP2K3 5606 MAP2K3 mitogen-activated protein kinase kinase 3
MAP2K6 5608 MAP2K6 mitogen-activated protein kinase kinase 6
MAP3K7 6885 MAP3K7 mitogen-activated protein kinase kinase 7
MAPK1 5594 MAPK1 mitogen-activated protein kinase 1
MAPK10 5602 MAPK10 mitogen-activated protein kinase 10
MAPK8 5599 MAPK8 mitogen-activated protein kinase 8 MAPK9 5601 MAPK9 mitogen-activated protein kinase 9
MAPKAP1 79109 MAPKAP1 MAPK associated protein 1
MKNK1 8569 MKNK1 MAPK interacting serine/threonine kinase 1
MKNK2 2872 MKNK2 MAPK interacting serine/threonine kinase 2
MYD88 4615 MYD88 MYD88 innate immune signal transduction adaptor
NCK1 4690 NCK1 NCK adaptor protein 1
NFKBIB 4793 NFKBIB NFKB inhibitor beta
NGF 4803 NGF nerve growth factor
NOD1 10392 NOD1 nucleotide binding oligomerization domain 1
PAK4 10298 PAK4 p21 (RAC1) activated kinase 4
PDK1 5163 PDK1 pyruvate dehydrogenase kinase 1
PFN1 5216 PFN1 profilin 1
PIK3R3 8503 PIK3R3 phosphoinositide-3-kinase regulatory subunit 3
PIKFYVE 200576 PIKFYVE phosphoinositide kinase, FYVE-type zinc finger
PIN1 5300 PIN1 peptidylprolyl cis/trans isomerase, NIMA-lnteracting 1
PITX2 5308 PITX2 paired like homeodomain 2
PLA2G12A 81579 PLA2G12A phospholipase A2 group XIIA
PLCB1 23236 PLCB1 phospholipase C beta 1
PLCG1 5335 PLCG1 phospholipase C gamma 1
PPP1CA 5499 PPP1CA protein phosphatase 1 catalytic subunit Alpha
PPP2R1 B 5519 PPP2R1 B protein phosphatase 2 scaffold subunit Abeta
PRKAA2 5563 PRKAA2 protein kinase AMP-activated catalytic subunit Alpha
2
PRKAG1 5571 PRKAG1 protein kinase AMP-activated non-catalytic subunit
Gamma 1
PRKAR2A 5576 PRKAR2A protein kinase cAMP-dependent type II regulatory
Subunit Alpha
PRKCB 5579 PRKCB protein kinase C beta
PTEN 5728 PTEN phosphatase and tensin homolog
PTPN11 5781 PTPN11 protein tyrosine phosphatase non-receptor Type 11
RAC1 5879 RAC1 Rac family small GTPase 1
RAF1 5894 RAF1 Raf-1 proto-oncogene, serine/threonine kinase
RALB 5899 RALB RAS like proto-oncogene B
RIPK1 8737 RIPK1 receptor interacting serine/threonine kinase 1
RIT1 6016 RIT1 Ras like without CAAX 1
RPS6KA1 6195 RPS6KA1 ribosomal protein S6 kinase A1
RPS6KA3 6197 RPS6KA3 ribosomal protein S6 kinase A3
RPTOR 57521 RPTOR regulatory associated protein of MTOR complex 1
SFN 2810 SFN stratifin
SLA 6503 SLA Src like adaptor
SLC2A1 6513 SLC2A1 solute carrier family 2 member 1 SMAD2 4087 SMAD2 SMAD family member 2
SQSTM1 8878 SQSTM1 sequestosome 1
STAT2 6773 STAT2 signal transducer and activator of transcription 2
TBK1 29110 TBK1 TANK binding kinase 1
THEM4 117145 THEM4 thioesterase superfamily member 4
TIAM1 7074 TIAM1 TIAM Rac1 associated GEF 1
TNFRSF1A 7132 TNFRSF1A TNF receptor superfamily member 1 A
TRAF2 7186 TRAF2 TNF receptor associated factor 2
TRIB3 57761 TRIB3 tribbles pseudokinase 3
TSC2 7249 TSC2 TSC complex subunit 2
UBE2D3 7323 UBE2D3 ubiquitin conjugating enzyme E2 D3
UBE2N 7334 UBE2N ubiquitin conjugating enzyme E2 N
VAV3 10451 VAV3 vav guanine nucleotide exchange factor 3
YWHAB 7529 YWHAB tyrosine 3-monooxygenase/tryptophan 5- monooxygenase Activation Protein Beta
Below additional genes are shown that are differentially regulated by activation of the PI3K/AKT/mTOR pathway. (From: Song et al, J Cell Mol Med 9:59-71 (2005); Hers et al Cell Signal 23:1515-27 (2011); Hennessy et al Nat Rev Drug Discov 4:988-1004 (2005); Laplante et al J Cell Sci 122:3589-94 (2009); Kim et al Mol Cells 35:463-73 (2013); Meng et al Development 145:dev152595 (2018); Lamming et al Springerplus 3:735 (2014); Yap et al Curr Opin Pharmacol 8:393-412 (2008); Li et al Arch Toxicol 89:711-31 (2015); Georgescu et al Genes Cancer 1 :1170-7 (2010); Song et al Nat Rev Mol Cell Biol 13:283-96 (2012); Hollander et al Nat Rev Cancer 11:289-301 (2011) Lasarge et al Front Mol Neurosci 7:18 (2014); Cantley et al Science 296:1655-7 (2002); Bertotti et al Clin Cancer Res 21 :3377-83
(2015); Makker et al J Mol Endocrinol 53:R103-18 (2014); Wymann et al Biochim Biophys Acta 1436:127-50 (1998)).
Figure imgf000017_0001
EGF 1950 EGF phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha
EGFR 1956 EGFR mechanistic target of rapamycin kinase
PIK3CA 5290 PI3K3CA KRAS proto-oncogene, GTPase
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
AKT2 208 AKT2 AKT serine/threonine kinase 2
AKT3 10000 AKT3 AKT serine/threonine kinase 3 BAD 572 BAD BCL2 associated agonist of cell death
IGF1 3479 IGF1 insulin like growth factor 1
IGFR 3480 IGFR insulin like growth factor 1 receptor
TSC1 7248 TSC1 TSC complex subunit 1
TSC2 7249 TSC2 TSC complex subunit 2
RHEB 6009 RHEB Ras homolog, mTORCI binding
MTOR 2475 MTOR mechanistic target of rapamycin kinase
EIF4EBP1 1978 EIF4EBP1 eukaryotic translation initiation factor 4E binding protein
1
CHUK 1147 CHUK conserved helix-loop-helix ubiquitous kinase
IKBKB 3551 IKBKB inhibitor of nuclear factor kappa B kinase subunit beta
IKBKG 8517 IKBKG inhibitor of nuclear factor kappa B kinase subunit gamma
NFKBIA 4792 NFKBIA NFKB inhibitor alpha
NFKB1 4790 NFKB1 nuclear factor kappa B subunit 1
RELA 5970 RELA RELA proto-oncogene, NF-kB subunit
GRB2 2885 TSC2 growth factor receptor bound protein 2
SOS1 6654 RHEB SOS Ras/Rac guanine nucleotide exchange factor 1
SOS2 6655 MTOR SOS Ras/Rac guanine nucleotide exchange factor 2
HRAS 3265 TSC2 HRas proto-oncogene, GTPase
KRAS 3845 RHEB KRas proto-oncogene, GTPase
NRAS 4893 MTOR NRas proto-oncogene, GTPase
Figure imgf000018_0001
BRAF 673 BRAF B-Raf proto-oncogene, serine/threonine kinase
MAP2K1 5640 MAP2K1 mitogen-activated protein kinase kinase 1
MAP2K2 5605 MAP2K2 mitogen-activated protein kinase kinase 2
Genes found to be involved in orchestrating BTN3A1 expression, ultimately leading to increased BTN2A1 surface expression
Figure imgf000018_0002
RHOB 388 RHOB Ras Homolog Gene Family, Member B
SYNJ2 8871 SYNJ2 Synaptic Inositol 1 ,4, 5-Trisphosphate 5-Phosphatase 2
CARMIL1 55604 LRRC16A Capping Protein Regulator And Myosin 1 Linker 1
PHLDB2 90102 PHLDB2 Pleckstrin Homology Like Domain Family B Member 2
In addition or alternatively, determining level of PI3K-AKT1-mT0R pathway activity, as under ii) of the present method, 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. Downregulation 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.
Furthermore, determining phosphorylation status of BTN3A1 , as in iii) of the present method, 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. In addition or alternatively, iii) may be performed by detecting upregulation of kinase (PRKCQ, Protein kinase C theta (PKC-0)) expression, relative to healthy tissue (see Figure 3G).
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. First, the respective protein may be extracted from the sample, and transferred to a membrane, which facilitates antibody binding and detection. Then, 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). Depending on the labelling of the secondary antibody, the appropriate detection method may be applied.
It will be clear that 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. Accordingly, the method may comprise a further step of administering a BTN2A1 and/or BTN3A1 targeting therapeutic to selected patients.
Hence, also foreseen is a BTN2A1 and/or BTN3A1 targeting therapeutic (or agent) for use in the treatment of cancer (or infectious disease or auto-immune disease), the use comprising a) 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; iii) phosphorylation status of BTN3A1 ; and/or iv) regulation of BTN3A1 expression and/or formation of BTN3A1 and BTN2A1 heterodimers, b) selecting patients for who i), ii), iii) and/or iv) indicate susceptibility to a BTN2A1 and/or BTN3A1 targeting therapeutic (as set out herein), c) administering a BTN2A1 and/or BTN3A1 targeting therapeutic to selected patients.
In a preferred embodiment, 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
- separately, sequentially or simultaneously to at least one PI3K-AKT1-mTOR pathway activator;
- separately, sequentially or simultaneously to at least one phosphorylation-inducing agent; and/or separately, sequentially or simultaneously to a modulator of one or more of RHOB, PHLDB2, SYNJ2 and CARMIL-1. For example, C3 transferase inhibits RhoB, and Rhotekin, CN01 (Rho activator) activate RhoB. See e.g. Cell Rep. 2016 May 31 ; 15(9): 1973-1985.
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.
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.
It was found that recognition of cancer cells (or infected cells or cells affected by auto-immune process), by the 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). Accordingly, 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. In other words, (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).
In addition or alternatively, 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 , MAPK10, MAPK8, MAPK9, MAPKAP1, MKNK1, MKNK2, MYD88, NCK1 , NFKBIB, NGF, NOD1 , PAK4, PDK1 , PFN1, PIK3R3, PIKFYVE, PIN1 , PITX2, PLA2G12A, PLCB1 , PLCG1, PPP1CA, PPP2R1 B, PRKAA2, PRKAG1, PRKAR2A, PRKCB, PTPN11 , RAC1, RAF1, RALB, RIPK1, RIT1, RPS6KA1 , RPS6KA3, RPTOR, SFN, SLA, SLC2A1 , SMAD2, SQSTM1 , STAT2, TBK1 , THEM4, TIAM1, TNFRSF1A, TRAF2, TRIB3, TSC2, UBE2D3, UBE2N, VAV3, YWHAB (and/or at least one agent that downregulates expression of one or more of PTEN and/or MAP2K1);
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, such as pamidronate or zoledronate, 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).
In particular, 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. In addition or alternatively, 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.
Preferably, 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). In addition or alternatively, 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.
Most natural T cell receptors (TCR) comprise complete alpha (a) and beta (P) chains, but a minority of natural immune cells express an alternate receptor, formed by complete gamma (Y) and 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. The Variable domains of both the TCR a-chain and p- chain, or both the TCR y-chain and 5-chain, each may have three hypervariable or complementarity determining regions (CDRs). An exogenous immune receptor according to the disclosure is preferably defined as not being an endogenous T cell receptor. For example, 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. expressed from a transgene construct and not from endogenous loci. An exogenous immune receptor according to the disclosure 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 (CARs) 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.
In one aspect of the disclosure, 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.
The T-cell receptor or extracellular domain thereof preferably is a Y9<52 T-cell receptor or extracellular domain thereof. As described herein, the term “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 allows first the bispecific construct to bind to a T and/or NK cell and then later to recruit this cell to a tumor. 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. The small size of a construct according to the disclosure, together with the non-cellular nature, render said construct as an ideal treatment tool for infectious diseases and cancer. These constructs provide great promise for treatment of cancer and infectious diseases.
Fusion of the extracellular domain of a ybTCR as tumor binding domain to, for example, an anti-CD3 scFv can effectively target T cells to tumor cells without the need for engineering TEGs. ybTCR anti-CD3 bispecific molecules (abbreviated as GABs) can redirect CD3+ effector cells towards several tumor cell lines of both hematologic and solid origin and preserve the mode of action of tumor recognition, thereby opening a new universe of antigens to the bispecific format.
Accordingly, in the construct as described above:
- 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.
In addition or alternatively, in said construct, the binding domain might be modified so as to inhibit T cell activation. For example, the construct may bind inhibitory domain(s) of the T cell. Specifically, in the construct described above, 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).
Accordingly, in the construct as described above:
- 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 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. Some preferred examples of such amino acid sequences include 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. For example, 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. In addition or alternatively, the y<5 T-cell receptor or extracellular domain thereof is fused to the T-cell- and/or Natural Killer (NK) cell-binding domain.
The construct of the present disclosure can combine tumor targeting with immune cell recruitment and thus can avoid a major drawback of engineered immune cells such as CAR-T and TEGs which relates to the rather challenging logistic for such advanced therapy medicinal products (ATMPs). Generating ATMPs is an individualized, cumbersome, and costly process that in most cases takes week and can associate with production failures. 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). In addition or alternatively, 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. Preferably, 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.
Preferably in one embodiment, the nucleic acid encoding the immune receptor (or extracellular domain thereof) 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 nucleic acid or nucleic acids that encode the immune receptor (or extracellular domain thereof) according to the disclosure 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. Preferably, 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. Introduction of the nucleic acid or nucleic acids may be via transfection or transduction methods depending on what type of nucleic acid or nucleic acids are used. It is understood that depending on what type of genetic construct or constructs are used, the genetic construct may consist of DNA or RNA. For example, 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. the genetic construct, being transformed from RNA into double stranded DNA thereby allowing expression therefrom. Integration is advantageous as it allows for proliferation of transduced cells while maintaining the viral vector genome comprising the genetic construct. 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. As said, when 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. Alternatively, 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.
Accordingly, 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 HEK293F™ 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 HEK293F™ 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, e.g. T cells or NK cells of a subject, 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. In cellular therapy 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).
Also envisaged are op T-cells with y<5 TCRs (TEGs) according to the disclosure 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.
Also provided is 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. The term "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).
In general, formulations may be administered that comprise between about 1 x 104 and about
1 x 1O10 immune cells, or between 0.1-10, or 1 -100, 10-1000 mg construct. In addition or alternatively, formulations may be administered that comprise between 0.1-10, or 1 -100, 10- 1000 mg EGF. In addition or alternatively, formulations may be administered that comprise between 0.1-10, or 1 -100, 10-1000 mg phosphorylation inducing agent. In most cases, the formulation may comprise between about 1 x 105 and about 1 x 109 immune cells, from about 5 x 105 to about 5 x 108 immune cells, or from about 1 x 106 to about 1 x 107 immune cells. A physician may ultimately determine appropriate dosages to be used.
The BTN2A1 and/or BTN3A1 targeting therapeutic according to the present disclosure (e.g. a y<5 TCR or extracellular domain thereof comprising BTN2A1 binding peptide), the construct according to the present disclosure, or the cell 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. Examples of non-aqueous solvents are 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.
Accordingly, there is provided for a method for treatment of a cancer and/or an infection (such as infectious disease), 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. Preferably, the subject is human.
As will be clear, 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.
Therapy in the present disclosure may involve prophylactic administration or therapeutic administration in humans that are (suspected to be) suffering from e.g. a cancer or an infectious disease (or autoimmune disease). Thus, the BTN2A1 and/or BTN3A1 targeting therapeutic according to the disclosure, any one of the constructs according to the present disclosure, or the cell according to the present disclosure, 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.
For example, in the treatment of leukemia, 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. 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 , NFKBIB, NGF, NOD1 , PAK4, PDK1 , PFN1 , PIK3R3, PIKFYVE, PIN1, PITX2, PLA2G12A, PLCB1 , PLCG1, PPP1CA, PPP2R1B, PRKAA2, PRKAG1 , PRKAR2A, PRKCB, PTPN11, RAC1 , RAF1 , RALB, RIPK1 , RIT1, RPS6KA1, RPS6KA3, RPTOR, SFN, SLA, SLC2A1 , SMAD2, SQSTM1, STAT2, TBK1, THEM4, TIAM1 , TNFRSF1A, TRAF2, TRIB3, TSC2, UBE2D3, UBE2N, VAV3, YWHAB; (and/or at least one agent that upregulates expression of PTEN); and
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. In the context of the present disclosure, 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. During T-cell development, 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" T cells that successfully complete the y gene rearrangement before the p gene rearrangement express a y6TCR and remain CD4"CD8" [47], The T cell receptor associates with the CD3 protein to form a T cell receptor complex. T cells, i.e. expressing an apTCR or a yQTCR, express the T cell receptor complex on the cell surface. 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. Similarly, 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. However, various subsets may also be considered part of the innate immunity where a restricted TCR is used as a pattern recognition receptor. For example, 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.
In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".
Methods of carrying out the conventional techniques used in methods of the present invention will be evident to the skilled worker, and are disclosed for example in Molecular Cloning: A Laboratory Manual (eds. Sambrook, J. & Russell, D.W.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2001).
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Vy9V52 TCR T cells recognize early transformed CRC organoids. (A) Patient derived CRC organoids (PDO) were co-cultured with VY9V52TCR T cells in the presence of PAM and 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. (I) IFNy production by VY9V52TCR T cells after co-culture with either healthy colon organoids (normal) or CRC organoids single-mutated for APC, p53 and KRAS in the presence of 100uM PAM. (D) Cluster heatmap displaying Gene Set Variation Analysis (GS A) enrichment scores (ES) for 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 .(F) 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. (I) Healthy colon organoids (WT), CRC organoids mutated for APCKO 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. (J) Expression of BTNx genes in epithelial cells from adenocarcinoma patients, represented as per sample pseudobulk profiles from a publicliy available single-cell RNAseq dataset. Scaled from -1 to 1 with white representing negative, (grey representing zero) and black representing positive Z- scores.
Figure 2. Transformed cells upregulate BTN2A1 surface expression via PI3K kinase activity. (A) 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. (F) Multiple tumor cell lines were pre-treated with either PI3K kinase inhibitor, AKT inhibitor, mTOR inhibitor Rapamycin or mTOR inhibitor Torinl and subsequently co-cultured with Vy9V52TCR T cells. After an overnight co-culture, supernatant was used to determine IFNy production by the Vy9V52TCR T cells. (G) Heatmap illustrating the expression of genes from the 'PI3K_and_AKT_family’ gene list, in the WT-AKPS model, with or without PAM. Scaled from -1 to 1 with white representing negative, (grey representing zero) and black representing positive Z-scores. (H) Heatmap of Pearson's correlation of the expression of genes from the 'PI3K_and_AKT_family’ and BTNx genes in the WT-AKPS model, with or without PAM. Scaled from -1 to 1 with white representing negative, (grey representing zero) and black representing positive correlation scores.
Figure 3. Phopshosites of juxtamembrane region of BTN3A1 affect Vy9V52TCR T cells target recognition. (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. (E) CD107a degranulation of Vy9V52TCR T cells was measured upon co-culture with either the Phospho-deficient or the phosphor-mimic mutated HEK293FT cells in the presence of PAM. (F) Flow cytometry FRET measurement was performed between phosphomutated variants of BTN3A1 and both, RHOB and BTN2A1-HA. (G) Heatmap illustrating the expression of kinases predicted to be active targeting the S297 and T297 depicted by Piero Giansanti et.al. , in the WT-AKPS model, with or without PAM. Scaled from -1 to 1 with white representing negative, (grey representing zero) and black representing positive Z-scores.
Figure 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. To dissect the differences between each gene and the 'Scrambled' control, estimated marginal means (EMMs) were calculated for each gene using the emmeansO function from the emmeans R package. Subsequently, pairwise comparisons were conducted employing Dunnett's test. The results from these pairwise comparisons were summarized to include confidence intervals and adjusted p-values:
Each comparison was annotated with significance markers to visually denote the level of statistical significance: **** for p < 0.001, *** for p < 0.01, ** for p < 0.05, * for p < 0.1 , empty (ns) for non-significant results.
Figure 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.
Subsequently, cells were fixed and permeabilized. A DuoLink™ proximity ligation assay (PLA) 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. Scaled from -1 to 1 with white representing negative and grey representing positive Z-scores. (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.
SEQUENCES REFERRED TO:
ERBB2 mutation V777E
SEQ ID NO:1 :
MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQWQGNLE LTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLN NTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNR SRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGP KHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDV GSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCK KIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIR GRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALL HTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPRE YVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPI WKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLWVLGWFGILIKR RQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIW I PDGEN VKI PVAI KVLRENTSPKAN KEI LDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLM PY GCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDF GLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGI PAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFWI
QNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRS
SSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPL
QRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERP
KTLSPGKNGWKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPER
GAPPSTFKGTPTAENPEYLGLDVPV
V777E - indicated V may be mutated to E
KRAS amino acid sequence
SEQ ID NO:2:
MTEYKLVWGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQ
EEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLP
SRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIK
KOI IM
From Prior et al; A comprehensive survey of Ras mutations in cancer; Cancer Res. 2012 May 15; 72(10): 2457-2467. Doi: 10.1158/0008-5472.CAN-11-2612
P53 amino acid sequence
SEQ ID NO:3:
MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPD
EAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKS
VTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEWRRCPHH
ERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCN
SSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHE
LPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEP
GGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD
From Nivlin et al; Mutations in the p53 Tumor Suppressor Gene; Genes Cancer. 2011 Apr;
2(4): 466-474. doi: 10.1177/1947601911408889
BTN3A1 amino acid sequence
SEQ ID NO:4: MKMASFLAFLLLNFRVCLLLLQLLMPHSAQFSVLGPSGPILAMVGEDADLPCHLFPTMSAET
MELKWVSSSLRQVVNVYADGKEVEDRQSAPYRGRTSILRDGITAGKAALRIHNVTASDSGK YLCYFQDGDFYEKALVELKVAALGSDLHVDVKGYKDGGIHLECRSTGWYPQPQIQWSNNK GENIPTVEAPVVADGVGLYAVAASVIMRGSSGEGVSCTIRSSLLGLEKTASISIADPFFRSAQ RWIAALAGTLPVLLLLLGGAGYFLWQQQEEKKTQFRKKKREQELREMAWSTMKQEQSTRV
KLLEELRWRSIQYASRGERHSAYNEWKKALFKPADVILDPKTANPILLVSEDQRSVQRAKEP QDLPDNPERFNWHYCVLGCESFISGRHYWEVEVGDRKEWHIGVCSKNVQRKGWVKMTPE NGFWTMGLTDGNKYRTLTEPRTNLKLPKPPKKVGVFLDYETGDISFYNAVDGSHIHTFLDVS FSEALYPVFRILTLEPTALTICPA
Amino acid Three letter code One letter code alanine ala A arginine arg R asparagine asn N aspartic acid asp D asparagine or aspartic acid asx B cysteine cys C glutamic acid glu E glutamine gin Q glutamine or glutamic acid glx Z glycine gly G histidine his H isoleucine ile I leucine leu L lysine lys K methionine met M phenylalanine phe F proline pro P serine ser S threonine thr T tryptophan trp W tyrosine tyr Y valine val V any amino acid any N
In case of inconsistency between the above sequences and the corresponding sequences in the ST.26 sequence listing, the above sequences may be used. Alternatively, the sequences in the ST.26 sequence listing may be used. EXPERIMENTAL SECTION
In this experimental section, the present inventors demonstrate that a single oncogenic mutation (in particular ErbB2V777E) is associated with enhanced PI3K-AKT1-mTOR pathway activity which is sufficient to upregulate surface expressed BTN2A1 , a known ligand of Vg9Vd2TCR on tumor cells. Full activation of T cells through a Vy9V52TCR is then catered by phosphorylation of JTM amino acids of BTN3A1 leading to the activating heterodimerization of BTN2A1 and BTN3A1.
Accordingly, the present inventors propose a method for determining susceptibility, of a cancer or infectious disease patient, to BTN2A1 and/or BTN3A1 targeting therapy, wherein the method comprises 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-mOR pathway activity; and/or iii) phosphorylation status of butyrophilin subfamily 3 member A1 (BTN3A1).
The present inventors also propose use of a BTN2A1 and/or BTN3A1 targeting therapeutic in the treatment of cancer, wherein the BTN2A1 and/or BTN3A1 targeting therapeutic is administered separately, sequentially or simultaneously to
- at least one PI3K-AKT1-mTOR pathway activator; and/or
- at least one phosphorylation-inducing agent.
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. In addition, PI3K activity leads to upregulation of BTN3A1 expression upon PAM treatment (PI3K activity sensitizes cells for PAM-induced BTN3A1 up regulation). 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. Using a protein interactome mapping pipeline, the inventors identified 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. Increasing evidence show that human y<5T cells have an essential role in cellular stress sensing and immune surveillance for both microbial and autologous stress (e.g. tumorigenesis)1. Infiltration of ybT cells in various tumors has been shown to have a favorable prognostic value2 and play an important role in the immunosurveillance of early tumor development in mice3, 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 tumors4-7. Vy9V52T cells, which are considered the most innate-like subset of gamma delta T cells in general1, 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 pathway9. Because of the broad-, but tumor-specific recognition of malignantly transformed cells, and the lack of MHC-restriction, Vy9V52T cells harbor great clinical potential as an immunotherapy for cancer10-12. Even though it is well-established that the ybTCR itself is essential for recognition of pAgs, 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 BTN2A111’13-15. BTN2A1 has emerged as key protein for recognition of tumor cells by Vy9V52-T cells14 where it is directly bound by the gamma chain of Vy9V52TCR. The role of the small GTPase RHOB, was previously demonstrated by the inventor’s group to relocalize to the membrane and interact with BTN3A1 upon pAg build-up16. This is associated with cytoskeletal changes and reduced mobility of BTN3A1 in the membrane, implying a sort of “membrane trapping” mechanism. It is thought that direct binding of pAgs to the intracellular B30.2 domain enables heterodimerization of BTN3A1 with BTN2A1. The subsequently induced joint conformational and spatial changes stabilize BTN2A1 -homodimers, which are then available for interaction with the yb-TCR16-18.
Even with the identification of BTN2A111’13’14, the understanding of BTN3A1 regulation through RHOB11 16, and the most recent observation that pAgs glue BTN3A1 and BTN2A118, it is not fully elucidated how this mechanism is regulated. Recently, it has been shown to be partially controlled by the AMP-activated protein kinase (AMPK) pathway, and activation of this pathway led to increased transcription of both molecules, BTN3A and BTN2A1 , followed by enhanced activation of Vy9V52T cells19. Although, these new insights defined a signature which is predictive for recognition of tumors in cancer patients19, 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. 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 formats11 12 as well as apT cells expressing a high affinity Vy9V52 TCR20'22. 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.
RESULTS
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 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). In screenings to assess tumor recognition in the absence and presence of PAM, 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. (Figure 1A). 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 (APCKO), KRAS (KRASG12D), TP53 (p53KO) and SMAD (SMADKO), 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). 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 mice23.
To delineate whether sensitivity to Vy9V52TCR after PAM-treatment is already imprinted in organoids after malignant transformation, and whether PAM treatment further differently acts on healthy versus malignant tissues, the inventors conducted 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). The test comparing normal and AKPS without PAM treatment resulted in a p-value of 0.02312, indicating a statistically significant difference in pathway enrichment between WT and AKPS under 'no PAM' conditions, implying that malignant transformation indeed creates a different molecular imprint on cells. The major changes associated with the AKPS mutation covered biological terms related to signaling. The inventors noticed that the highest enrichment score was calculated for the 'CHOLESTEROL_HOMEOSTASIS' term in the mutated samples, representing genes of the mevalonate pathway (ACAT, HMGCS1 , MVK, PMVK, MVD, IDI1 , FDPS). Comparing either healthy or AKPS-mutants with or without PAM showed significant differences in enrichment scores in the normal (pval=0.0005891) and the AKPS conditions (pval= 7.761 e-09), mainly associated with cell cycle, and implied that PAM acts on gene expression of both healthy and malignant tissues. However, when comparing the PAM-treated normal and Pam-treated AKPS-mutant organoids, there was no significant change in enrichment scores (p-value of 0.8822), suggesting that PAM's effect on the gene expression landscape was consistent across both normal and AKPS conditions, as far as the HALLMARK scores are concerned and that the different underlying molecular profiles in healthy and transformed tissues are the primary reason for PAM-responsiveness and not a selective effect of PAM on tumor cells only. Within this context differential expression analysis comparing the effects of PAM treatment on gene activity demonstrated that normal organoids had a larger array of PAM- responsive genes, both induced and repressed (404 and 578 genes, respectively), than AKPS mutant organoids, which showed changes in 131 and 466 genes, respectively (Figure 1E). BTN genes, which are pivotal in the recognition of target cells by y8T cells13’14, demonstrated the most pronounced expression in AKPS mutants (Figure 1G). The 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). However, 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.
To assess at which stage of malignant transformation can be made susceptible to Vy9V02TCRs the inventors made use of a step-wise mutagenesis colon organoid model, where the introduction of single mutations of APC (APCKO), KRAS (KRASG12D), TP53 (p53KO) and SMAD (SMADKO) into normal (healthy) colon organoids represents models for pre- cancerous lesions while AKPS mutant served as a fully developed CRC23 model. After coincubation of Vy9V62TCR T cells with organoids treated with/without PAM, a single gene mutation introduced in the normal colon was already able to induce IFNy production after addition of PAM (Figure 1 H). The inventors chose APCKO and AKPS mutant CRC organoids to represent very early and late stages of CRC development, respectively, to further characterize the capacity of a Vy9V62TCR to potentially sense early or late-stage cancer development. Already APCKO showed as AKPS mutants an enhanced expression of BTN2A1 protein at the cell membrane. Importantly, the inventors found BTN2A1 protein to be present already in the early, pre-cancerous APC-mutant stage in the absence of PAM (Figure 11). Also, the by the inventors previously described PAM-induced redistribution of intracellular RhoB11’16 was already detected in single APC-mutant organoids.
To validate our observation that early malignant transformation primarily impacts BTN2A1 expression in pre-cancerous lesions in humans, the inventors consulted existing single cell RNA-sequencing (scRNAseq) datasets from patients at various disease progression stages of CRC. 24 Analysing pseudo bulk profiles of the epithelial cell population revealed that always BTN2A1 expression levels were heightened in pre-cancerous (polyp) stages when compared to the “unaffected” baseline in both studied patients (Figure 1 J), which parallels the increased BTN2A1 expression seen at comparable stages in our model. The inventors could also observe that one pre-cancerous lesion BTN2A1 , BTN3A1 and BTN3A2 were increased.
Phosphoinositide 3-kinase/AKT1 activity during early transformation is important for Vy9V52TCR activation
To confirm that single oncogenic mutations generally mediate susceptibility to Vy9V62TCR recognition, the inventors tested the effect of single ErbB2 mutations and overexpression in breast tissue25"27. MCF10a benign breast tissue cell line was engineered to mimic various oncogenic ErbB2 gene related mutations such as overexpression of HER2 (ErbB2AMP), a single mutation in the extracellular domain (ErbB2S310F) or amplified kinase activity mutant (ErbB2V777E). Only MCF10a cells expressing kinase mutant HER2 variant (ErbB2V777E) triggered IFNy production of Vy9V02TCR T cells, again exclusively in the presence of PAM (Figure 2A) while benign cells and the two other oncogenic variants remained unrecognized, suggesting that it is not the transformation event in general that promotes susceptibility to recognition by a Vy9V62TCR, but rather very specific mutations that also differ depending on the tissue type of tumor. Since 44hosphons in ErbB2 often result in PI3K pathway activation 28 the inventors pre-treated MFC10a ErbB2V777E tumor cells with PI3K inhibitor (Pictilisib) which led to significant reduction of IFNy production of Vy9V62TCR T cells upon co-culture while HER2 CAR-T cell reactivity remained unaffected. The surface expression of Vy9V62TCR ligand, BTN2A1 was significantly increased upon ErbB2V777E mutation already in the absence of PAM, and for this increase PI3 kinase activity in MCF10a cells is important, since pan PI3K inhibition depleted BTN2A1 surface expression (Figure 2B). BTN3A surface expression however did not change either upon ErbB2V777E 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. When analysing phosphorylation of AKT in MCF10a HER2 mutants, 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. To further clarify the signalling cascade downstream of PI3K necessary for PAM-dependent activation of Vy9V02TCR T cells, the inventors blocked various key kinases that might be activated by PI3K in the subsequent experiments. In all tumor cell lines inhibition of both, PI3K and AKT1 significantly reduced PAM-dependent Vy9V52TCR activity, while specific MEK inhibitors did not alter recognition of the tumor cells (Figure 2E). Pre-treatment of solid (HT-29, SKBR3) and haematological (Daudi, K562, RPMI- 8226) tumor cell lines with blocking reagents of mTOR reduced recognition significantly, in a similar or even higher potency as inhibiting AKT activity (Figure 2F). Inhibition of both mTOR- involved complexes, mTORCI and mT0RC2, by inhibitor Torinl was superior in reducing activation of Vy9V52TCR T cells compared to inhibition of only mTOR complex 1 (mTORCI) by Rapamycin (Figure 2F). Since BTN2A1 surface expression was solely dependent on PI3K activity in tumor cell lines, while Vy9V02T cell activation was triggered by the combination of increased PI3K pathway activity and the presence of PAM, the inventors wondered whether the activity of this pathway is exclusively transformation dependent. The expression patterns of genes of the PI3K-AKT-mTOR pathway over our AKPS-models displayed varied trends in terms of being induced by PAM or transformation. However, the key components of the pathway (PRKCA/B, PIK3CA, PAK1 , AKT2) were predominantly influenced by the transformation process itself (Figure 2G) and thus highly correlated with BTN2A1 expression (Figure 2H), suggesting that as the disease progresses, these key pathway components and BTN2A1 expression are linked.
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, GNGT1 , GRB2, GSK3B, HRAS, HSP90B1 , IL2RG, IL4, IRAK4, ITPR2, LCK, MKNK1 , MKNK2, MYD88, NCK1 , NFKBIB, NGF, NOD1 , PAK4, PDK1 , PFN1 , PIK3R3, PIKFYVE, PIN1 , PITX2, PLA2G12A, PLCB1 , PLCG1 , PPP1CA, PPP2R1 B, PRKAA2, PRKAG1 , PRKAR2A, PRKCB, PTEN, PTPN11 , RAC1 , RAF1 , RALB, RIPK1 , RIT1 , RPS6KA1 , RPS6KA3, RPTOR, SFN, SLA, SLC2A1 , SMAD2, SQSTM1 , STAT2, TBK1 , THEM4, TIAM1 , TNFRSF1A, TRAF2, TRIB3, TSC2, UBE2D3, UBE2N, VAV3, YWHAB; and 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.
These data together indicate that PI3K/AKT1/mTOR activity in the early onset of malignant transformation drives BTN2A1 surface upregulation on tumor cells and therefore makes them susceptible for Vy9V52TCR targeting.
BTN3A1 phosphorylation affects surface expression of BTN3A1 and BTN2A1
To this end the inventors revealed that transformation induced AKT/mTOR activity promotes cell surface expression of BTN2A1 which is important for binding of the Vy9 TCR chain. However, in all models full activation of Vy9V52TCR T cells required PAM treatment. Although the inventors have not been able to identify the driver of PAM-mediated BTN3A1 upregulated the inventors could further characterize molecular mechanisms once BTN3A1 is upregulated by revisiting previously generated mass spectrometry data generated to assess PAM-induced changes in surface proteins 11. This analysis identified two potential 46hosphor- sites on the juxtamembrane (JTM) region of the BTN3A1 molecule; the residues S296 and T297 that were phosphorylated upon PAM (Figure 3A). To dissect functional impact of PAM-induced phosphorylation on BTN3A1 from phosphorylation events induced by endogenous phosphoantigen (pAg) levels, the inventors constructed modified versions of the BTN3A1 sequence that mimic protein conformation of either a phosphorylated (46hosphor- mimic variant) or an unphosphorylated state (46hosphor-deficient variant). These 46hosphor- variants were developed by substituting residues S296 and T297 with aspartic acid which is known to function similarly to phosphorylated serine and threonine (D, 46hosphor-mimic) or alanine which is employed regularly to inhibit residue phosphorylation (A, 46hosphor- deficient) To understand whether phosphorylation mimicking of here-investigated residues is required for BTN3A1 membrane orchestration, the inventors performed FRET measurements that showed that 62hosphor-deficient BTN3A1 is unable to interact with both BTN2A1 and RhoB, another important player in the orchestration of BTN3A11629. (Figure 3F) suggesting an important role of JTM phosphorylation of BTN3A1 in enabling Vy9V52TCR full activation., Functionally introduction of 62hosphor-mimic BTN3A1 variant resulted in higher activation of Vy9V52TCR T cells upon PAM (Figure 3D and 3E). To assess potential regulators of this phosphorylation the inventors used gene expression profiles of the normal and AKPS- mutated colorectal organoids, and identified protein kinase C theta (PRCKQ) as the only kinase that follow PAM-treatment and upregulates by PAM regardless of the mutational status of the tissue (Figure 3G). This data let hypothesize that PAM-induced kinase activation results in phosphorylation of the S296 and T297 motifs of BTN3A1. BTN3A1 phosphorylation then alters interaction of BTN3A1 with BTN2A1, and RhoB.
Interactome platform identifies potential BTN3A1 -proximate proteins involved in Vv9V52TCR-induced tumor targeting
To study now in more detail which additional protein complexes are in the close proximity of BTN3A1 after addition of PAM, the inventors set up an interactome pipeline in the proximity of BTN3A1 that are exclusively related to Vy9V52TCR activation (Figure 4A). As first step, the inventors made use of Proximity-dependent biotin identification (BiolD) approach30 by overexpressing BTN3A1 fused to bacterial biotin ligase BirA* to I C-terminus in HEK293F cells. Cells were treated with 100 pM PAM and 50 pM biotin for 24h, which led to biotinylation of BTN3A1-close proteins, after which cells were lysed and biotinylated proteins were pulled down using streptavidin beads. Identification of proteins was done by mass spectrometry analyzing quadruplicate samples, which resulted in a list of 60 candidate proteins including 31 hits under no treatment, 19 upon PAM treatment and 10 hits that occurred upon both conditions. To determine if candidate proteins play a physiological role in tumor targeting by Vy9V52TCR T cells, 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 summary of these knock-down experiments provided evidence that eight candidate proteins, namely PHLDB2, PKP2, SYNJ2, CARMIL1, PA2G4, TANC1, SDK2, HNRPC and have a functional impact on PAM-dependent activation of Vy9V52TCR T cells, as possible additional modulators of the BTN3A1-RhoB axis.
Novel protein interactome linked to BTN3A1 and RHOB in PAM-treated tumor cells
The inventors wondered if PAM-induced spatial rearrangements of BTN3A1 -related proteins as observed with BiolD-tagging could partially be the result of post-transcriptional or post- translational regulation of proteins in tumor cells. Therefore, the inventors assessed 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. In addition to BTN3 and RhoB 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). Next, 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. To further map how BTN3A interaction dynamics occur with our new five candidate proteins in a PAM-dependent manner, the inventors analyzed co-localization between BTN3A membrane clusters and each of the candidate proteins using confocal microscopy in MZ1851Rccells. Examples of the region of interest selections and representative images are shown. The inventors found that while PHLDB2 moves out of while SYNJ2 and CARMIL1 moves into BTN3A membrane clusters upon PAM treatment within the range of approximately 200 nm (Figure 5B). To confirm if PHLDB2, SYNJ2 and CARMIL1 truly interact with BTN3A1 in tumor cell membranes the inventors studied interaction at higher resolution measured by Proximity ligation assay (PLA) which can visualize protein interactions within approximately 40 nm. Similarly to the previously described small GTPase RHOB 16, SYNJ2 and CARMIL1 both showed a significant PAM-dependent interaction with BTN3A1 while PHLDB2 showed no significant interaction with BTN3A1 (Figure 5C. To understand whether next to spatial regulation at the protein level the expression of the here-investigated genes are regulated similarly to BTNs. While CARMIL1 and RHOB gene expression strongly correlated with BTN3A1 gene expression both in our normal colon and AKPS CRC models, PHLDB2 and SYNJ2 expression did not seem to be linked to these changes suggesting the involvement of these molecules in BTN3A1 expression is regulated entirely post-transcriptionally (Figure 5D). To demonstrate that the here-identified BTN3A1 -network is significant for the immunosurveillance of Vy9Vb2TCR T cells in a clinically more relevant setting, the inventors knocked out the individual candidate genes in AKPS mutant organoids using CRISPR/Cas system and tested Vy9V52TCR T cells reactivity against them. KO of each individual genes similarly reduced both IFNy production and direct tumor killing of Vy9V52TCR T cells in response to AKPS mutant organoids (Figure 5E and F).
The inventors conclude that PHLDB2, SYNJ2 and CARMIL1 are next to RHOB involved in orchestrating PAM-induced cytoskeletal rearrangements in tumor cells that is a prerequisite for spatial and conformational changes in BTN3A1 leading to Vy9Vb2TCR T cell activation.
IMPLICATIONS
Despite the growing interest in Vy9V52T cells and their anti-tumor potential and the research aiming to resolve the unknown mechanisms of induction and regulation of this anti-tumor effect, there are still many mysteries to resolve. One of them being how tumor recognition is being triggered, and another how it is exactly regulated.
Here, the inventors have demonstrated that potential sensitivity to that 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 Vy9V52TCR14’29, and thereby making early transformed cells susceptible for Vy9V52TCR recognition. However, for recognition by Vy9V52TCR T cells this step is preferably accompanied by additional upregulation of BTN3A1. The inventors observed that in primary pre-cancerous lesions, both can be observed, cells expressing BTN2A1 only or BTN2A1 and additional molecules of the BTN3 complex, implying earlier tumor control by Vy9V52T cells in some but not all pre-cancerous lesions. This conclusion was implied by our observation that full activation of T cells via the Vy9V52TCR, typically involve additional parallel steps.
Additional steps include increased levels of intracellular phosphoantigens (pAg), which can be mimicked by aminobisphosphonates, like Pamidronate (PAM). Although the inventors could not characterize the natural trigger of BTN3A upregulation, the PAM-model system implied that phosphorylation of the juxtamembrane (JTM) region of BTN3A1 is important in achieving full recognition by Vy9V52T cells, and provided a group of kinases most likely responsible for this step. These steps together result in a tightly regulated composition of membrane expressed BTN2A1 and BTN3A1 which is fed by a constant intracellular protein pool. 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.
Our data, in line with previous findings11 13’29 imply that BTN2A1 and BTN3A1 membrane expression dynamics are tightly regulated together in human cells. It has been previously suggested that various pathways including AMPK19 or EBV-induced activation of JNK31 can influence BTN surface expression by enhancing transcription of BTN2A1 and BTN3A. In contrary, the inventors found the total BTN2A1 protein pool does not differ between normal (healthy) and transformed cells, however, in normal cells the inventors found no or low surface expression. And despite the contribution of these studies to better understand regulation of Vy9V52T cell targeting, it is still unknown at what stage of tumorigenesis exactly Vy9V52T cells begin to recognize and eliminate transformed cells. In this study, the inventors show that single mutations that induces higher PI3K activity were sufficient to allow expression of BTN2A1 at the cell membrane and to enable binding of the gamma chain of the VY9V52TCR and was also not lost during later cancer stages. This observation implies that 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. Since the inventors show that depletion of PI3 kinase activity and in particular AKT phosphorylation and mTOR activity in tumor cells impairs Vy9V52TCR reactivity implies that targeted therapies acting on the PI3K pathway should be carefully designed as they might interfere with endogenous innate responses against the tumor. On the other hand, PI3K activation in cancer patients, next to PAM treatment, might provide a new avenue to possibly sensitize tumors for Vy9V52TCR- driven attack. When combining the knowledge from these studies with next generation engineering strategies such as TEGs20’22 and Gamma delta TCR anti-CD3 bispecific molecules (GABs)12 might employ full potential of how to maximize targeting potential via Vy9V52TCRs.
Interestingly, in contrast to the previously mentioned JNK pathway, which is a downstream signaling pathway of PI3K/AKT32, AMPK and AKT are often seen to act as antagonists in regulating autophagy and apoptosis3334, however, it has been shown that under certain conditions, e.g. cancer, activation of AMPK, especially via AMPK-activator AICAR, can also activate AKT3536 and mTORC236, and vice versa, inhibition of AMPK via compound C reduced AKT activity35. 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.
While PI3K activity directly affects re-localization of BTN2A1 to the cell membrane, BTN3A1 surface expression remained unaffected by this oncogenic offset. Although 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 transformation37 and leads to this accumulation of pAgs. The inventors used aminobisphosphonates (ABP), like Pamidronate (PAM) as model system to characterize the next molecular steps. Therefore, the oncogenic-regulated translocation of BTN2A1 to the cell surface and subsequent binding of pAg to the B30.2 domain of BTN3A1 upon ABP treatment, eventually leads to heterodimerization of BTN2A1 and BTN3A1 and ultimately to TCR binding and activation.
Recently, pAg were shown to act as glue between BTN molecules BTN2A1 and BTN3A1 to activate Vy9V52T cells18, and previously, the juxtamembrane (JTM) region have been identified as being sensitive for translating pAg-induced inside-out-signaling17’38. The inventors have now identified two amino acids that are likely to be phosphorylated specifically upon PAM treatment and phosphorylation of these sites led was associated with higher IFNy production by Vy9V52TCR T cells upon co-culture.
Furthermore, the data suggests that phosphorylation of JTM BTN3A1 sites promotes interaction with RhoB and heterodimerization of BTN3A1 and BTN2A1 which will ultimately stabilize BTN2A1 at the cell surface and further increase in membrane surface clusters that altogether will form immunological synapses that enable proper T cell activation. These events are not only present in very early transformation events but likely also preserved in late stages of tumors, since colorectal organoids mutated for all four colon cancer associated oncogenes are equally recognized by Vy9V52TCR T cells as single APC mutated ones.
A closer look at downstream mechanisms after BTN2A1 and BTN3A1 are expressed at the cell membrane surface allowed to reveal that BTN2A1/BTN3A1 surface dynamics are tightly regulated by spatial rearrangements of so-far unknown intracellular modulators which are functionally essential to allow Vy9V52TCR targeting. In this study the inventors established a detailed ABP-dependent interactome of BTN3A1 -related proteins using various protein proximity techniques that resolved spatial interaction hierarchies among the studied proteins. PHLDB2 was found, as the only protein of all candidates to occupy BTN3A1 -proximity membrane clusters without showing detectable direct close interaction with BTN3A1 , implying its role in tissues when yet no functional recognition by Vy9V52T cells can occur. PHLDB2 dissociates from these clusters either once PAM is present or in tumor lesions with increased pAg levels, suggesting a complementary role in preventing the accumulation of BTN protein membrane clusters as also suggested by others3940 (Figure x).
The inventors have shown in the past that active 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 cells16. 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 RhoB41. Our data let hypothesize that 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 dynamics4243 and furthermore has also been implicated in vesicle trafficking44, emphasizing and a role in supporting RhoB-mediated BTN3A1 recycling11 and enable recognition of tumor cells by Vy9V52T cells.
Like SYNJ2 and PHLDB2, also CARMIL1 most likely accommodates the formation of cytoskeletal rearrangement45 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. Like AKT1 , PHLDB2 contains a PH-domain, which has a high affinity to membrane-bound PIP339, an oncogenic product by PI3K46. Therefore, a role of PHLDB2 in recruitment of BTN proteins to the cell membrane is not excluded and needs to be further investigated.
In summary, 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. However, 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. Within the context 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+L), anti-LRRC16A/CARMIL1 Rabbit polyclonal IgG (NBP1- 91221), anti-PHLDB2 Rabbit polyclonal IgG (NBP2-38238), anti-CASKIN1 Rabbit polyclonal IgG (HPA055990), anti-HNRNPC mouse monoclonal IgG (AMAb91010), anti-PKP2 Rabbit polyclonal IgG (PA553144), anti-ANKRD26 Rabbit polyclonal IgG (PA559240), anti-VPS13A Rabbit polyclonal IgG (PA554483), anti-TANC1 Rabbit polyclonal IgG (PA557797), anti-SDK2 Rabbit polyclonal IgG (ABIN 1386438), anti-NBEA rabbit Polyclonal IgG (HPA040385), anti- PA2G4 Mouse monoclonal IgG (ABIN518579), anti-SYNJ2 Rabbit polyclonal IgG (PA556784). 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).
Cell and patient derived organoid culture. 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. MCF10A cells were cultured in DMEM/Ham’s F-12 (Gibco) supplemented with, 1% Penicillin/Streptomycin (Gibco), Ala-Gin (Ultra-Glutamine) (Gibco), 5% heat-inactivated horse serum (Lonza), 0.01 mg/ml insulin (Gibco), 500 ng/ml hydrocortisone (Sigma), 100ng/ml cholera toxin (Sigma), 20 ng/ml epidermal growth factor (EGF) (Peprotech). Cell lines were routinely tested for mycoplasma and STR type verified by - 53 -hosphor. PBMCs were isolated from buffy coats using ficoll-paque obtained from the Blood bank Sanquin. Colon organoids were established and cultured as previously described47. Before co-cultures, organoids were recovered from the BME using TrypLE express.
Generation of engineered T-cells. ybTCR and HER2 CAR T-cells were generated as previously published22. In short, Phoenix-ampho cells were transfected with env (COLT- GALV), gag-pol (HIT60) and pBullet retrovirus constructs containing either both TCR chains or the HER2 CAR sequence. Pre-activated T-cells were subsequently transduced twice with the viral supernatant of these cells with polybrene. Transduced T-cells were isolated and expanded using a rapid expansion protocol.
Viral transduction MCF10a. pBABE-Puro retroviral vector (EV) and pBABE-Puro vectors carrying ERBB2 wild-type and mutants were co-transfected independently with plIMVC (Addgene #8449) and VSV-G (Addgene #8454) retroviral packaging plasmids into HEK-293T cells using PEI-transfection. Medium was refreshed after 24hrs and viral supernatant was collected 48hrs and 72hrs after transfection. Viral particles were added to low passage MCF10A cells and incubated overnight. Twenty-four hours following transduction, MCF10A cells were selected using 2 pg/ml Puromycin (Gibco) for two days. Gene transduction and protein expression were validated using western analysis. Established cell lines were immediately expanded and cryopreserved in low passage aliquots.
Plasmids. Retroviral pBabe-Puro plasmids were purchased from Addgene: ERBB2 (#40978), ERBB2::S310F (#40991), and ERBB2:: A775_G775ins , G776C (V777E) (#40979). All plasmids and mutations were verified by Sanger Sequencing.
BiolD. pcDNA3.1 MCS-BirA(R118G)-HA was a gift from Kyle Roux (Addgene plasmid # 36047; http://n2t.net/addgene:36047; RRID:Addgene_36047). The DNA sequence of BTN3A1-BirA(R118G)-HA has been subcloned into pBullet retrovirus constructs. Phoenix- ampho cells were transfected with env (COLT-GALV), gag-pol (HIT60) and pBullet retrovirus constructs containing BTN3A1-BirA(R118G)-HA constructs. HEK293FT cells were subsequently transduced twice with the viral supernatant of these cells with polybrene. Transduced cells were selected using 2,5 mg/ml G418. Expression was confirmed by confocal microscopy. The preparation of the samples for analysis by LS-MS followed the protocol by Roux et al 30’48. In short, HEK293FT WT or BTN3A1-BirA(R118G)-HA expressing cells were 50 pM Biotin alone or 50 pM Biotin and 100 pM Pamidronate. Cells were lysed and sonicated to generate whole cell lysates. Biotinylated proteins were pulled down using streptavidin-magnetic beads and samples were then analyzed by LS-MS. Each condition was prepared as quadruplicate.
Western Blot. Cell lines were seeded in a 10 cm dish overnight to confluency. Cells were rinsed with ice cold PBS and lysed with lysis buffer containing 1% NP-40, 150 mM NaCI, 20 mM Tris HCI pH 7,6 and protease inhibitors (complete, Roche, #11873580001). Protein concentration was determined using Pierce BCA Protein Assay Kit - Rapid Gold (Fisher Scientific, #15776178) and 5 pg total protein was loaded on each lane of a Mini Protean TGX Gel (BioRad, #4561093) together with a standard (WesternC, BioRad, #1610376) and run at 140V. The protein was then transferred to a 0.2 pm nitrocellulose membrane (BioRad, #1704158) using Trans-Blot Turbo System. The membrane was incubated in blocking buffer (PBS-T + 5% BSA for 1h at RT, washed three times with PBS-T (0,1% Tween) and incubated overnight on a rotator with the respective primary antibodies as indicated. After three times washing with PBS-T at RT, membranes were incubated with the respective secondary antibodies for 1h at RT on a rotator. After final washings, membranes were developed using the ECL Kit and measured.
Inhibitors. The following inhibitors were used in this study; PI3K inhibitor GDC-0941 (Pictilisib) (SelleckChem, #S1065), pan-AKT inhibitor MK2206 (SelleckChem, #S1078), MEK inhibitor AZD6244 (Selumetinib) (SelleckChem, #S1008), mTORC 1 inhibitor Rapamycin (Sirolimus) (SelleckChem, #S1039), mTORC 1 / 2 inhibitor Torin 1 (SelleckChem, #S2827).
Stimulation assays. 50.000 tumor cells were co-cultured overnight at 37°C in a roundbottom 96-well plate (Nunc, Thermo-Fisher Scientific) with 50.000 Vy9V52-expressing T cells (TEGs) or HER2 CAR expressing T cells in RPMI-GlutaMAX medium, containing pamidronate as indicated. Furthermore, for the inhibitor experiments tumor cells were pretreated overnight with either PI3K (Pictilisib, GDC-0941), AKT (MK2206), MEK (Selumetinib, AZD6244) inhibitors, Rapamycin or Torin. Before co-culture cells were washed twice with fresh RPMI medium and afterwards adjusted to 50.000 cells in 100 pL RPMI culture medium. After co-culture, 100pL supernatant was used for INF-y detection using the Invitrogen™ eBioscience™ human IFN-y ELISA kit ready-set-go by following the manufactures protocol42.
Flow Cytometry. Cells were counted and 200.000 per condition were put in a FACS tube, after which 1 mL FACS buffer (PBS, 1% Na-azide) was added and tubes spun down at 1500RPM for 5 min. Supernatant was discarded and, for the tetramer and bead stainings with soluble TCRs, cells were incubated with tetramer/bead solution for 30 minutes at RT. Cells are washed with PBS and subsequently, 20pL of the antibody mixture in FACS buffer was added to each well, after which cells were resuspended and incubated for 30min at RT. 1mL FACS buffer was added to each well and cells were spun down at 1500RPM for 5 min, supernatant removed and resuspended in 1mL FACS buffer and spun down again at 1500RPM for 5 min. Finally, supernatant was removed and 150pL 1 % PFA was added. Samples were measured on a FACSCanto 2 (BD bioscience) using FACSDiva software.
Intracellular staining and colocalization analysis. MZ1851rc cells were seeded in a 16- well Nunc Lab-Tek™ chamber at 1500 cells per well in 200pL cDMEM and cultured for 76h at 37°C, 5% CO2 in a wet chamber (petri dish + wet paper cloth). Medium was removed and 200pL cDMEM with 100pM pamidronate (PAM) (where indicated) was added to the wells. After 24h incubation, cells were washed with PBS and subsequently fixed with 4% paraformaldehyde (PFA) for 15min at RT. Cells were permeabilized for 10min using either 100% ice-cold methanol, 0.3% Triton X100 in PBS, or 0.1% saponin in PBS (changed protocol over time, saponin seems to work best) at RT. Sample blocking was performed by incubating wells for 1h at RT with blocking solution (PBS, 5% Pooled human serum (HuS), 1% BSA, 1% Na-azide + either 0.3% Triton X-100 or 0.1% saponin). Primary antibodies diluted in antibody dilution buffer (PBS, 1% BSA, 1% Na-azide + either 0.3% Triton X-100 or 0.1% saponin) were added to the wells and were incubated either 1h at RT, or overnight at 4°C. Cells were washed 3x with PBS and secondary antibody diluted in antibody dilution buffer was added and incubated either 1 h at RT, or overnight at 4°C. Finally, the chamber gasket and any glue residue was removed from the well chamber using a sterile tweezer and surgical blade, some Prolong Diamond Antifade mountant with DAPI was added and a coverglass applied. Before imaging, any leftover residue was removed with a Kimwipe and slide was kept in a dark place for at least 30min. Imaging was performed on a Zeiss LSM710 confocal laser scanning microscope using a 63x 1.40 oil immersion objective. Analysis of pictures was done blinded to conditions using Velocity™ image analysis software (PerkinElmer) by selecting areas of interest on membrane clusters of BTN3A, and determining the Pearson’s correlation coefficient. Representative images were made using Imaged software (NiH).
Animal models. NSG mice were administered total body irradiation of 1.65 Gy on day -1. AKPS mutated PDOs were injected subcutaneously in the right flank on day 0. On day 1 and 7, 107TEG-001 or TEG-LM1 cells were administered intravenously in pamidronate (10 mg/kg body weight). Next to T-cell administration, 0.6 x 106 III of IL-2 in incomplete Freund’s adjuvant was injected subcutaneously at day 1 as described in49. Bioluminescence was used to weekly monitor PDO outgrowth over time.
Proximity ligation assays. Duolink™ PLA Fluorescence protocol was followed.41 Duolink Plus and minus probes and detection reagents orange were obtained from Merck. MZ1851rc cells were seeded in a 16-well Nunc Lab-Tek™ chamber (Thermo-Fisher Scientific, Nunc) at 15000 per well per 200pL cDMEM and cultured for 76h. afterwards, medium was gently removed and 100pM pamidronate in cDMEM was added where indicated and incubated overnight. Wells were washed 1x with PBS and fixed with 4% PFA for 15min at RT. Wells were washed 3x with PBS and permeabilized using 0.1% saponin in PBS for 15min at RT. Wells were blocked using Duolink blocking buffer and primary antibodies diluted in Duolink PLA diluent were added to the wells and incubated for 1h at RT in a humidity chamber (empty pipette tip box with elevated plateau covered in parafilm for slide to sit on, water around it). Wells were washed 2x with 1x wash buffer A (Merck) and incubated for 1 hour at 37°C in a wet chamber with 35pL/well of Duolink min/plus PLA probe mix (anti-mouse-plus, anti-rabbit- minus). Wells were washed 2x with 1x wash buffer A and incubated with 35pL/well Duolink ligation buffer with 1 :40 Ligase enzyme added from freezing block and incubated for 30 min at 37°C in a wet chamber. Wells were washed 2x with 1x wash buffer A again and incubated with Duolink Orange amplification buffer with 1 :80 polymerase enzyme added from freezing block and incubated for 100min at 37°C. Finally, wells were washed 2x with 1x wash buffer B (Merck) for 10min and 1x with O.OIx wash buffer B for 1 min. All remaining buffer was discarded and further preparation of the slide was done in the same way as described for intracellular staining. Imaging was performed on a Zeiss LSM710 confocal laser scanning microscope using both a 20x objective or a 63x 1.40 oil immersion objective. PLA clusters per image and number of nuclei were determined using Velocity™ image analysis software (PelkinElmer).
Fluorescence Resonance Energy Transfer (FRET). To study direct interaction between proteins upon phosphoantigen accumulation, HEK293T cells expressing - 57 -hosphor- variants of BTN3A1 were first dissociated using Trypsin-EDTA, transferred into a FACS tube and resuspended in complete DM EM. The cells were then treated with 100 pM PAM for 1 hour at 37°C and washed with FACS buffer (PBS, 1% Na-azide). After that, the cells were blocked with PBS containing 5% BSA for 15 minutes and then incubated for 30 minutes at room temperature with an antibody conjugated with the donor fluorochrome at 10OpI staining volume. Cells were washed once and then split into two samples: donor and donor+acceptor. Donor samples were incubated with FACS buffer and donor+acceptor samples were incubated for 30 minutes at room temperature with an antibody conjugated with the acceptor fluorochrome at 50pl staining volume. After washing with FACS buffer twice, samples were fixed with 1% PFA. The donor fluorescence was measured using a FACS Canto-ll flow cytometer (BD Biosciences) where donor fluorescence of the donor+acceptor (double stained) samples was compared with the one of the samples labeled only with donor antibody. FRET efficiency was calculated from the fractional decrease of the donor fluorescence in the presence of the acceptor. Background noise in the donor fluorescent channel due to spectral overlap with different fluorescence channels was excluded from the calculations, by subtracting the measured mean fluorescence intensity (MFI) on unlabeled and single stained samples from the MFI of the donor and donor+acceptor samples respectively. The centrifugation steps during washes were done at 1500RPM for 5 minutes and the antibodies were diluted in FACS buffer. The samples stained with antibodies conjugated to Alexa Fluor 594 were validated for proper staining by remeasurement using an LSR Fortessa cell analyzer (BD Biosciences). Generation of HEK-293FT - 58 -hosphor-variants. For the generation of the cell lines stably expressing BTN3A1 phosphovariants and BTN3A2, the BTN3A1-Flag as well as the BTN3A2 sequence were codon optimized, custom synthesized and cloned in pBullet-IRES-puro plasmid vectors. The retroviral particles produced by transfection in Phoenix ampho cells were generated with the same procedure used to generate TEGs. The viral supernatant from the Phoenix ampho cells was used to transduce the HEK 293T BTN3KO cell line. 48 hours post-transduction 1.5 pg/mL of puromycin was supplemented to the culture medium for antibiotic selection and it was carried out until the control (untransduced) cell line died completely.
Software used. Office 2016 (Microsoft), Illustrator CS61 Illustrator 2019 (Adobe systems), Zen 2009 / 2012 (Zeiss), Imaged (NiH), Volocity image analysis software (PerkinElmer), GraphPad Prism 8, Microplate Manager (Bio-Rad), FACSDiva (BD Biosciences).
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Claims

1. Ex vivo method for determining susceptibility of cells to a butyrophilin subfamily 2 member A1 (BTN2A1) binding peptide and/or butyrophilin subfamily 3 member A1 (BTN3A1) binding peptide, 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 , wherein presence, 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 , is indicative of susceptibility to a BNT2A1 binding peptide and/or BTN3A1 binding peptide and wherein 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 , is indicative of reduced susceptibility to a BTN2A1 and/or BTN3A1 ; ii) level of PI3K-AKT1-mTOR pathway activity, 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, 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, MKNK1 , MKNK2, MYD88, NCK1, NFKBIB, NGF, NOD1, PAK4, PDK1, PFN1, PIK3R3, PIKFYVE, PIN1, PITX2, PLA2G12A, PLCB1, PLCG1, PPP1CA, PPP2R1 B, PRKAA2, PRKAG1 , PRKAR2A, PRKCB, PTEN, PTPN11, RAC1 , RAF1, RALB, RIPK1 , RIT1, RPS6KA1, RPS6KA3, RPTOR, SFN, SLA, SLC2A1 , SMAD2, SQSTM1, STAT2, TBK1, THEM4, TIAM1 , TNFRSF1A, TRAF2, TRIB3, TSC2, UBE2D3, UBE2N, VAV3, YWHAB, EGF, PIK3CA, PIK3CB, PIK3CD, AKT2, AKT3, BAD, IGF1, IGFR, TSC1, RHEB, mTOR, EIF4EBP1, CHUK, IKBKB, IKBKG, NFKBIA, NFKB1 , RELA, SOS1 , SOS2, HRAS, KRAS, NRAS and BRAF, 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; and/or iii) phosphorylation status of BTN3A1 , wherein phosphorylation of a serine at a position corresponding to position 296 in SEQ ID NO:4 and/or phosphorylation of a threonine at a position corresponding to position 297 in SEQ ID NO:4 is indicative of susceptibility to a BNT2A1 binding peptide and/or BTN3A1 binding peptide and wherein no phosphorylation of a serine at a position corresponding to position 296 in SEQ ID NO:4 and/or no phosphorylation of a threonine at a position corresponding to position 297 in SEQ ID NO:4 is indicative of reduced susceptibility to a BNT2A1 binding peptide and/or BTN3A1 binding peptide, wherein susceptibility means that treatment of the cells with to a BNT2A1 binding peptide and/or BTN3A1 binding peptide will lead to killing and/or reduced growth of the cells.
2. Ex vivo method according to claim 1, wherein the BTN2A1 binding peptide is a T-cell receptor y-chain domain, preferably a T-cell receptor yQ-chain domain and/or which is comprised in an (exogeneous) immune receptor or extracellular domain thereof, preferably a yb T-cell receptor or extracellular domain thereof, more preferably a y9b2 T-cell receptor or extracellular domain thereof; and/or the BTN3A1 binding peptide is a T-cell receptor b-chain domain, preferably a T-cell receptor b2-chain domain and/or which is comprised in an (exogeneous) immune receptor or extracellular domain thereof, preferably a yb T-cell receptor or extracellular domain thereof, more preferably a y9b2 T-cell receptor or extracellular domain thereof.
3. Ex vivo method according to claim 2, wherein the BTN2A1 binding peptide and/or BTN3A1 binding peptide is 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 yb T-cell.
4. Ex vivo method according to any one of the previous claims, wherein i) is performed by sequencing a nucleic acid sequence encoding ERBB2 and/or by detecting 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; iii) is performed by detecting 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 and/or iii) is performed by detecting upregulation of kinase (PRKCQ) expression, relative to healthy tissue; and/or the method additionally comprises determining in a sample that has been obtained from a patient of iv) regulation of BTN3A1 expression, by transcriptomic analysis, preferably by determining, relative to healthy tissue, downregulation or upregulation of expression of one or more of the following genes: RHOB, PHLDB2, and CARMIL-1.
5. Ex vivo method according to any one of the previous claims, wherein in i) the amino acid other than valine is glutamic acid and wherein ii) is 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, PIK3R1, MTOR, TSC2, PRKCZ, AKT1, GRB2, EIF4A, HSPB1 , and RHEB, 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.
6. Ex vivo method according to any one of the previous claims, the method further comprising selecting patients that are susceptible to a BTN2A1 binding peptide and/or BTN3A1 binding peptide and/or excluding patients that are not susceptible to a BTN2A1 binding peptide and/or BTN3A1 binding peptide.
7. Ex vivo method according to any one of the previous claims, wherein the sample
- comprises blood, serum, urine, lacrimal fluid, or saliva;
- comprises not healthy cells and/or diseased cells;
- comprises cancer cells and/or extracellular vesicles from cancer cells; and/or
- has been obtained from a human patient.
8. Ex vivo method according to any one of the previous claims, wherein the cells are cancer cells 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.
9. BTN2A1 binding peptide and/or BTN3A1 binding peptide for use in the treatment of a disease chosen from cancer, infectious disease and auto-immune disease, wherein the BTN2A1 binding peptide and/or BTN3A1 binding peptide is administered separately, sequentially or simultaneously to
- at least one PI3K-AKT1-mTOR pathway activator which is at least one growth factor ligand, preferably epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), or transforming growth factor (TGF), more preferably Epidermal Growth Factor (EGF); and - preferably at least one phosphorylation-inducing agent which is at least one (bi)phosphonate, preferably pamidronate.
10. BTN2A1 binding peptide and/or BTN3A1 binding peptide for use according to claim 9, wherein the BTN2A1 binding peptide and/or BTN3A1 binding peptide is administered separately, sequentially or simultaneously to a modulator of one or more of RHOB, PHLDB2, and CARMIL-1.
11. BTN2A1 and/or BTN3A1 targeting therapeutic for use according to any one of claim 9-10, wherein the BTN2A1 binding peptide is a T-cell receptor y-chain domain, preferably a T-cell receptor y9-chain domain and/or which is comprised in an (exogeneous) immune receptor or extracellular domain thereof, preferably a yb T-cell receptor or extracellular domain thereof, more preferably a y9b2 T-cell receptor or extracellular domain thereof; and/or the BTN3A1 binding peptide is a T-cell receptor b-chain domain, preferably a T-cell receptor b2-chain domain and/or which is comprised in an (exogeneous) immune receptor or extracellular domain thereof, preferably a yb T-cell receptor or extracellular domain thereof, more preferably a y9b2 T-cell receptor or extracellular domain thereof.
12. BTN2A1 binding peptide and/or BTN3A1 binding peptide for use according to any one of claims 9-11 , wherein the BTN2A1 binding peptide and/or BTN3A1 binding peptide is 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 yb T-cell.
13. BTN2A1 binding peptide and/or BTN3A1 binding peptide for use according to any one of claims 9-12, wherein the cancer is chosen 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) tumor, stomach cancer, esophageal cancer; wherein the infectious disease is chosen from bacterial infection, fungal infection, viral infection, COVID-19, Hanta virus infection, sepsis, pneumonia, meningitis, acute respiratory distress syndrome, necrotizing fasciitis; and/or wherein the auto-immune disease is chosen 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.
14. BTN2A1 and/or BTN3A1 targeting therapeutic for use according to any one of claims 9- 13, wherein the use
- comprises intravenous administration of said BTN2A1 binding peptide and/or BTN3A1 binding peptide; and/or
- is for use in the treatment of a human patient.
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