INTRACELLULAR KINASE ASSOCIATED WITH RESISTANCE AGAINST T-CELL MEDIATED CYTOTOXICITY, AND USES THEREOF
20230228760 · 2023-07-20
Assignee
Inventors
- Valentina VOLPIN (Regensburg, DE)
- Philipp BECKHOVE (Regensburg, DE)
- Antonio SORRENTINO (Regensburg, DE)
- Michael BOUTROS (Heidelberg, DE)
- Nisit KHANDELWAL (Martinsried, DE)
- Tillmann MICHELS (Martinsried, DE)
- Peter SENNHENN (Martinsried, DE)
Cpc classification
G01N33/57492
PHYSICS
International classification
G01N33/50
PHYSICS
Abstract
The invention is based on the identification of the intracellular kinase calcium/calmodulin-dependent protein kinase 1D (CAMK1D) as a key checkpoint inhibitor in tumour cells mediating resistance against cytotoxic T lymphocytes (CTL). CAMK1D was identified in PD-L1 refractory tumours to impair CTL-induced death receptor signalling and apoptosis via caspase inhibition. The invention offers therapeutic approaches involving impairing CAMK1D immune checkpoint function by various CAMK1D inhibitors, especially nucleic acid or small molecule inhibitors of CAMK1D and/or treatments involving CAMK1D inhibitors with death receptor agonists. The medical approaches of the invention are useful for treating subjects suffering from various proliferative disorders; preferably such proliferative disorders that are characterized by a resistance to CTL mediated immune responses, or which are refractory or resistant to treatments with other immune checkpoint therapies, such as PD1-PDL1 antagonistic treatments. Provided are the medical applications and corresponding diagnostic approaches, kits, CAMK1D inhibitors and screening methods for the identification of new therapeutic agents for the treatment of proliferative disorders.
Claims
1. A method for identifying and/or characterising a compound suitable for a treatment of a disease, disorder or condition that is characterised by a resistance against death receptor signalling, in particular a resistance against a cell-mediated immune response, and that is characterised by expression or activity of CAMK1D, the method comprising the steps of: (a) bringing into contact a first cell or cell-free system which comprises and/or expresses CAMK1D mRNA or protein and (i) a candidate compound, or (ii) a candidate compound and a cell-dependent or cell-independent cytotoxic stimulus; and (b) determining (i) the expression, activity, function and/or stability of the (eg protein or mRNA of) CAMK1D, in the first cell or cell-free system; and/or (ii) the cytotoxicity of the cell-dependent or cell-independent cytotoxic stimulus against the first cell or cell-free system; wherein: (i) a reduced expression, activity function and/or stability of the (eg protein or mRNA of) CAMK1D, in said first cell or cell-free system contacted with the candidate compound compared to said first cell or cell-free system not contacted with said candidate compound; and/or (ii) an enhanced cytotoxicity of the cell-dependent or cell-independent cytotoxic stimulus against the first cell or cell-free system contacted with the candidate compound compared to the cytotoxicity of the cell-dependent or cell-independent cytotoxic stimulus against the first cell or cell-free system not contacted with the candidate compound; indicates that the candidate compound is a compound suitable for the treatment of the disease, disorder or condition that is characterised by resistance against death receptor signalling, in particular resistance against a cell-mediated immune response, and that is characterised by expression or activity of CAMK1D.
2. A method for identifying and/or characterising a compound suitable for a treatment of a disease, disorder or condition that is characterised by resistance against death receptor signalling, in particular resistant against a cell-mediated immune response, and that is characterised by expression or activity of CAMK1D, the method comprising the steps of: (a) bringing into contact a first cell or cell free system which comprises and/or expresses CAMK1D mRNA or protein and one or more effector caspase(s)—in particular caspase-3, -6 and/or -7—mRNA or protein, and (i) a candidate compound, or (ii) a candidate compound, and a cell-dependent or cell-independent cytotoxic stimulus; and (b) determining (i) the expression, activity, function and/or stability of the (eg protein or mRNA of) of the one or more effector caspase(s) or of one or more phosphorylated effector caspase(s)—in particular caspase-3, -6 and/or -7—in the first cell or cell-free system; and/or (ii) the cytotoxicity of the cell-dependent or cell-independent cytotoxic stimulus against the first cell or cell-free system; wherein: (i) an increased expression, activity, function and/or stability of the (eg protein or mRNA of) one or more effector caspase(s) or of one or more phosphorylated effector caspase(s)—in particular caspase-3, -6 and/or -7—in the first cell or cell-free system, in said first cell or cell-free system contacted with the candidate compound compared to said first cell or cell-free system not contacted with said candidate compound; and/or (ii) an enhanced cytotoxicity of the cell-dependent or cell-independent cytotoxic stimulus against the first cell or cell-free system contacted with the candidate compound compared to the cytotoxicity of the cell-dependent or cell-independent cytotoxic stimulus against the first cell or cell-free system not contacted with the candidate compound; indicates that the candidate compound is a compound suitable for the treatment of the disease, disorder or condition that is characterised by resistance against death receptor signalling, in particular resistance against a cell-mediated immune response, and that is characterised by expression or activity of CAMK1D.
3. A method for identifying and/or characterising a compound suitable for a treatment of a disease, disorder or condition that is characterised by resistance against death receptor signalling, in particular resistant against a cell-mediated immune response, and that is characterised by expression or activity of CAMK1D, the method comprising the steps of: (a) bringing into contact a first cell or cell free system which comprises and/or expresses CAMK1D mRNA or protein and calmodulin mRNA or protein, and (i) a candidate compound, or (ii) a candidate compound, and a cell-dependent or cell-independent cytotoxic stimulus; and (b) determining or detecting (i) the expression, activity, function and/or stability of the (eg protein or mRNA of) of calmodulin in the first cell or cell-free system; and/or (ii) the specific binding of a Ca2+/calmodulin protein complex to CAMK1D protein; wherein: (i) an increased expression, activity function and/or stability of the (eg protein or mRNA of) calmodulin in the first cell or cell-free system, in said first cell or cell-free system contacted with the candidate compound compared to said first cell or cell-free system not contacted with said candidate compound; and/or (ii) a reduced specific binding of a Ca2+/calmodulin protein complex to CAMK1D protein in the presence of the candidate compound compared to the absence of the candidate; indicates that the candidate compound is a compound suitable for the treatment of the disease, disorder or condition that is characterised by resistance against death receptor signalling, in particular resistance against a cell-mediated immune response, and that is characterised by expression or activity of CAMK1D.
4. The method of any one of claim 1 to 3, wherein the cell-dependent or cell-independent cytotoxic stimulus is a substance or composition capable of binding to, and activating or increasing activity of, a death receptor signalling pathway, or a downstream component of death receptor signalling, in the cell or cell-free, and preferably is an agonist of TNR6 signalling, such as a membrane bound or soluble TNR6 ligand (FAS ligand), or is an agonist of TRAIL receptor signalling, such as TRAIL.
5. A Calcium/calmodulin-dependent protein kinase type 1D (CAMK1D) inhibitor for use in a treatment of a proliferative disorder in a subject, wherein the treatment involves inhibiting an activity, function, expression and/or stability of CAMK1D, and thereby sensitising cells involved with the proliferative disorder to a cell-dependent or cell-independent cytotoxic stimulus; wherein the treatment comprises administering the CAMK1D inhibitor to the subject.
6. A CAMK1D inhibitor for use in a treatment of a proliferative disorder in a subject, the treatment comprising exposing cells involved with the proliferative disorder in the subject to: (i) a cell-dependent or cell-independent cytotoxic stimulus; and (ii) the CAMK1D inhibitor.
7. The CAMK1D inhibitor for use of claim 6, wherein in (i) the cells involved with the proliferative disorder are exposed to the cell-dependent or cell-independent cytotoxic stimulus by (a) a cell-mediated immune response, such as CTL response, wherein the immune cells express and/or secrete a cell-dependent or cell-independent cytotoxic stimulus, in particular wherein the cells involved with the proliferative disorder are exposed to the immune cells; (b) an administration of immune cells which express and/or secrete a cell-dependent or cell-independent cytotoxic stimulus; and/or (c) an administration of a substance or composition eliciting the cell-dependent or cell-independent cytotoxic stimulus to the subject.
8. The CAMK1D inhibitor for use of claim 6 or 7, wherein in (ii), exposing a cell involved with the proliferative disorder in the subject to the CAMK1D inhibitor is sensitising cells involved with the proliferative disorder to a pro apoptotic stimulus, and wherein the treatment comprises administering the CAMK1D inhibitor to the subject
9. A CAMK1D inhibitor for use in a treatment of a proliferative disorder in a subject, wherein the treatment is for sensitizing a cell involved with the proliferative disorder to a cell-dependent or cell-independent cytotoxic stimulus, the treatment comprising administering the CAMK1D inhibitor to the subject.
10. A CAMK1D inhibitor for use in a treatment for the sensitisation of a subject suffering from a proliferative disorder to a therapy involving the administration of a cell-dependent or cell-independent cytotoxic stimulus to the subject, the treatment comprising administering the CAMK1D inhibitor to the subject
11. The CAMK1D inhibitor for use of any one of claims 5 to 10, wherein the cell-dependent or cell-independent cytotoxic stimulus is selected from a substance or composition capable of binding to, and activating or increasing an activity of, a death receptor signalling pathway in the cells involved with the proliferative disorder, for example selected from (i) an agonist of TNR6 signalling (such as an agonistic anti-TNR6 antibody, a membrane bound or soluble TNR6 ligand (FAS ligand), or (ii) an agonist of TRAIL receptor signalling, such as a TRAIL or an agonistic anti-TRAIL receptor (anti-“DR4” or anti-“DR5”) antibody.
12. The CAMK1D inhibitor for use of any one of item 5 to 11, wherein the cell-dependent or cell-independent cytotoxic stimulus is capable of inducing apoptosis in the cells involved with the proliferative disorder via activation of one or more caspases.
13. The CAMK1D inhibitor for use of any one of claims 5 to 12, wherein the proliferative disorder is a tumour, such as a solid or a liquid tumour.
14. The CAMK1D inhibitor for use of any one of claims 5 to 13, wherein the proliferative disorder is characterized by expression of (i) mRNA and/or protein of CAMK1D, or (ii) expression of mRNA and/or protein CAMK1D and expression of a death receptor, in particular such as TNR6 (Fas) or a TRAIL receptor (DR4/DR5); in the cells involved with the proliferative disorder, and thus preferably tumour cells.
15. The CAMK1D inhibitor for use of any one of claims 5 to 14, wherein the CAMK1D inhibitor is (i) a small molecule, in particular, a small molecule ligand or a small cell-permeable molecule; or is (ii) selected from a polypeptide, peptide, glycoprotein, a peptidomimetic, an antibody or antibody-like molecule (such as an intra-body); a nucleic acid such as a DNA or RNA, for example an antisense DNA or RNA, a ribozyme, an RNA or DNA aptamer, siRNA, shRNA and the like, including variants or derivatives thereof such as a peptide nucleic acid (PNA); a genetic construct for targeted gene editing, such as a CRISPR/Cas9 construct and/or guide RNA/DNA (gRNA/gDNA) and/or tracrRNA; a hetero-bi-functional compound (such as a PROTAC or a HyT molecule); a carbohydrate such as a polysaccharide or oligosaccharide and the like, including variants or derivatives thereof; a lipid such as a fatty acid and the like, including variants or derivatives thereof.
16. An in vitro method for determining whether a subject has, or is at risk of, developing a proliferative disorder, such as a tumour, that is associated with cellular resistance against a cell-dependent or cell-independent cytotoxic stimulus, such as of a cell-mediated immune response, the method comprising the step of: (a) detecting an applicable biomarker in a biological sample from said subject; wherein the detection of the applicable biomarker in the sample indicates the proliferative disorder, or a risk of developing the proliferative disorder, in the subject; and wherein the applicable biomarker is one or more selected from the group consisting of: (i) CAMK1D, in particular the presence (or an amount) of or expression and/or activity of CAMK1D, preferably of phosphorylated CAMK1D; (ii) death receptor, in particular the presence (or an amount) of or expression and/or activity of a death receptor; (iii) A death receptor ligand or a cell expressing a death receptor ligand, such as a FAS ligand, in particular the presence (or an amount) of or expression and/or activity of a death receptor ligand.
17. An in vitro method for determining whether a subject has, or has a risk of developing, a disease, disorder or condition that is associated with resistance against pro apoptotic stimuli, such as pro apoptotic stimuli elicited by cell-mediated immune responses, and wherein the proliferative disorder is associated with expression or activity of CAMK1D, the method comprising the steps of: (a) contacting cells of the subject suspected to be involved with the disease, disorder or condition with a CAMK1D inhibitor in the presence of a pro apoptotic signal: (i) immune cells capable of eliciting or eliciting pro apoptotic stimuli towards cells involved with the proliferative disease, disorder or condition, such as lymphocytes, T-cells, CTLs and TILs; or (ii) a pro apoptotic stimulus such as a soluble or substrate bound death receptor agonist or ligand; and (b) determining initiation of apoptosis in the cells involved with the proliferative disorder of the subject, wherein an enhancement of the initiation of apoptosis in the cells of the subject indicates that the subject has or has a risk of developing such disease, disorder or condition.
18. An in vitro method for stratifying a subject that suffers from a proliferative disorder into a patient group that is distinguished by having a poor prognosis or into a patient group that does not have a poor prognosis, the method comprising the steps of: (a) detecting an applicable biomarker in a biological sample from said subject, in particular wherein the biological sample comprises cells involved with the proliferative disorder; wherein the detection of the applicable biomarker in the sample indicates that the subject is stratified into the group of patients having a poor prognosis, and wherein no detection of the applicable biomarker in the sample indicates that the subject is stratified into the group of patients not having a poor prognosis; and wherein the applicable biomarker is one, preferably both, selected from the group consisting of: (i) CAMK1D, in particular the presence (or an amount) of or expression and/or activity of CAMK1D, preferably of phosphorylated CAMK1D; and (ii) a death receptor, such as a member of tumour necrosis factor (TNF) receptor superfamily (e.g., TNFR1, TNR6 (Fas), DR3, DR4, DR5, DR6, and LTpR) in particular the presence (or an amount) of or expression and/or activity of a death receptor;
19. The in vitro method of claim 18, wherein the applicable biomarker is detected in cells involved with the proliferative disorder contained in the biological sample.
20. A use of an antigen binding protein (ABP) capable of binding specifically to CAMK1D or phosphorylated CAMK1D in an in-vitro diagnosis of a proliferative disease, disorder or condition in a subject; wherein the proliferative disease, disorder or condition is associated with resistance against a pro apoptotic signal, such as pro apoptotic stimuli elicited by cell-mediated immune responses, and wherein the proliferative disease, disorder or condition is associated with expression or activity of CAMK1D.
21. A kit for use in a diagnostic method for determining whether a subject has, or has a risk of developing, a disease, disorder or condition that is associated with resistance against a pro apoptotic stimulus, such as a cell-mediated response mediated by a TNR6 ligand positive immune cell, and that is associated with expression or activity of CAMK1D; wherein: the diagnostic method comprises a step of surgically obtaining a sample from the subject; and the kit comprises: (a) either (x) a nucleic acid capable of binding specifically to CAMK1D, or (y) an ABP binding specifically to CAMK1D; and (b) optionally (i) instructions describing how to use the ABP or a nucleic acid or kit for detecting CAMK1D activity in the sample; and/or (ii) one or more other item, component, reagent or other means useful for the use of the kit or the detection of CAMK1D activity in the sample.
Description
BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCES
[0297] The figures show:
[0298]
[0299]
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[0302]
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[0304]
[0305] The sequences show:
TABLE-US-00003 SEQ ID NO: 1 (CAMK1D; UniProt identifier: Q81U85-1, database entry of May 13, 2020): 10 20 30 40 50 MARENGESSS SWKKQAEDIK KIFEFKETLG TGAFSEVVLA EEKATGKLFA 60 70 80 90 100 VKCIPKKALK GKESSIENEI AVLRKIKHEN IVALEDIYES PNHLYLVMQL 110 120 130 140 150 VSGGELFDRI VEKGFYTEKD ASTLIRQVLD AVYYLHRMGI VHRDLKPENL 160 170 180 190 200 LYYSQDEESK IMISDFGLSK MEGKGDVMST ACGTPGYVAP EVLAQKPYSK 210 220 230 240 250 AVDCWSIGVI AYILLCGYPP FYDENDSKLF EQILKAEYEF DSPYWDDISD 260 270 280 290 300 SAKDFIRNLM EKDPNKRYTC EQAARHPWIA GDTALNKNIH ESVSAQIRKN 310 320 330 340 350 FAKSKWRQAF NATAVVRHMR KLHLGSSLDS SNASVSSSLS LASQKDCLAP 360 370 380 STLCSFISSS SGVSGVGAER RPRPTTVTAV HSGSK SEQ ID NO: 2 (CAMK1D; UniProt identifier: Q81U85-1, database entry of May 13, 2020): 10 20 30 40 50 MARENGESSS SWKKQAEDIK KIFEFKETLG TGAFSEVVLA EEKATGKLFA 60 70 80 90 100 VKCIPKKALK GKESSIENEI AVLRKIKHEN IVALEDIYES PNHLYLVMQL 110 120 130 140 150 VSGGELFDRI VEKGFYTEKD ASTLIRQVLD AVYYLHRMGI VHRDLKPENL 160 170 180 190 200 LYYSQDEESK IMISDFGLSK MEGKGDVMST ACGTPGYVAP EVLAQKPYSK 210 220 230 240 250 AVDCWSIGVI AYILLCGYPP FYDENDSKLF EQILKAEYEF DSPYWDDISD 260 270 280 290 300 SAKDFIRNLM EKDPNKRYTC EQAARHPWIA GDTALNKNIH ESVSAQIRKN 310 320 330 340 350 FAKSKWRQAF NATAVVRHMR KLHLGSSLDS SNASVSSSLS LASQKDCAYV AKPESLS SEQ ID NO: 3 (siRNA sequence targeting CAMK1D) UGAAGUGUAUCCCUAAGAA SEQ ID NO: 4 (siRNA sequence targeting CAMK1D) CAAAUCACCUGUACUUGGU SEQ ID NO: 5 (siRNA sequence targeting CAMK1D) CCGAAAAUCUCUUGUACUA SEQ ID NO: 6 (siRNA sequence targeting CAMK1D) GAGAAGGACCCGAAUAAAA SEQ ID NO: 7 (gRNA sequence for targeting CAMK1D by gene editing) TCGATCGGATAGTGGAGAAG SEQ ID NO: 8 (gRNA sequence for targeting CAMK1D by gene editing) GGAGATAGTATACGGCATCC SEQ ID NO: 9 (gRNA sequence for targeting CAMK1D by gene editing) TAGCCGAGGAGAAAGCTACT SEQ ID NO: 10 (gene editing target sequence at locus: Chr.2: 5362021-5362043 on GRCm38) GGAGATAGTATACGGCATCC SEQ ID NO: 11 (Calmodulin (CaM); UniProt identifier: P0DP23, database entry of May 15, 2020): 10 20 30 40 50 MADQLTEEQI AEFKEAFSLF DKDGDGTITT KELGTVMRSL GQNPTEAELQ 60 70 80 90 100 DMINEVDADG NGTIDFPEFL TMMARKMKDT DSEEEIREAF RVFDKDGNGY 110 120 130 140 ISAAELRHVM TNLGEKLTDE EVDEMIREAD IDGDGQVNYE EFVQMMTAK SEQ ID NO: 12 (human Calcium/calmodulin- dependent protein kinase kinase 1 isoform 1; UniProt identifier: Q8N5S9-1, database entry of May 15, 2020): 10 20 30 40 50 MEGGPAVCCQ DPRAELVERV AAIDVTHLEE ADGGPEPTRN GVDPPPRARA 60 70 80 90 100 ASVIPGSTSR LLPARPSLSA RKLSLQERPA GSYLEAQAGP YATGPASHIS 110 120 130 140 150 PRAWRRPTIE SHHVAISDAE DCVQLNQYKL QSEIGKGAYG VVRLAYNESE 160 170 180 190 200 DRHYAMKVLS KKKLLKQYGF PRRPPPRGSQ AAQGGPAKQL LPLERVYQEI 210 220 230 240 250 AILKKLDHVN VVKLIEVLDD PAEDNLYLVF DLLRKGPVME VPCDKPFSEE 260 270 280 290 300 QARLYLRDVI LGLEYLHCQK IVHRDIKPSN LLLGDDGHVK IADFGVSNQF 310 320 330 340 350 EGNDAQLSST AGTPAFMAPE AISDSGQSFS GKALDVWATG VTLYCFVYGK 360 370 380 390 400 CPFIDDFILA LHRKIKNEPV VFPEEPEISE ELKDLILKML DKNPETRIGV 410 420 430 440 450 PDIKLHPWVT KNGEEPLPSE EEHCSVVEVT EEEVKNSVRL IPSWTTVILV 460 470 480 490 500 KSMLRKRSFG NPFEPQARRE ERSMSAPGNL LVKEGFGEGG KSPELPGVQE DEAAS SEQ ID NO: 13 (human Calcium/calmodulin- dependent protein kinase kinase 1 isoform 2; UniProt identifier: Q8N5S9-2, database entry of May 15, 2020): 10 20 30 40 50 MEGGPAVCCQ DPRAELVERV AAIDVTHLEE ADGGPEPTRN GVDPPPRARA 60 70 80 90 100 ASVIPGSTSR LLPARPSLSA RKLSLQERPA GSYLEAQAGP YATGPASHIS 110 120 130 140 150 PRAWRRPTIE SHHVAISDAE DCVQLNQYKL QSEIGKGAYG VVRLAYNESE 160 170 180 190 200 DRHYAMKVLS KKKLLKQYGF PRRPPPRGSQ AAQGGPAKQL LPLERVYQEI 210 220 230 240 250 AILKKLDHVN VVKLIEVLDD PAEDNLYLAL QNQAQNIQLD STNIAKPHSL 260 270 280 290 300 LPSEQQDSGS TWAARSVFDL LRKGPVMEVP CDKPFSEEQA RLYLRDVILG 310 320 330 340 350 LEYLHCQKIV HRDIKPSNLL LGDDGHVKIA DFGVSNQFEG NDAQLSSTAG 360 370 380 390 400 TPAFMAPEAI SDSGQSFSGK ALDVWATGVT LYCFVYGKCP FIDDFILALH 410 420 430 440 450 RKIKNEPWF PEEPEISEEL KDLILKMLDK NPETRIGVPD IKLHPVVVTKN 460 470 480 490 500 GEEPLPSEEE HCSVVEVTEE EVKNSVRLIP SWTTVILVKS MLRKRSFGNP 510 520 FEPQARREER SMSAPGNLLV
EXAMPLES
[0306] Certain aspects and embodiments of the invention will now be illustrated byway of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.
[0307] The examples show:
Example 1: CAMK1D Protects PD-L1+ Tumour Cells Against Death Receptor Signalling by Cytotoxic T Cells
[0308] In order to identify novel genes involved in immune escape mechanisms of cancer cells, a high-throughput screening approach recently developed (45) was adapted. The HLA-A2 positive human multiple myeloma cell line KMM-1 was used as a tumour model in this study because KMM-1 cells express high levels of PD-L1 and also lower levels of another recently characterized immune-checkpoint molecule, CCR9 (45). As a reporter system for tumour cell survival the inventors stably transfected KMM-1 cells with e-GFP-firefly luciferase, allowing to apply luminescence imaging as a reliable parameter for immune mediated tumour cell destruction in a HTP format. As a source of tumour-reactive T cells marrow-infiltrating, PD-1 positive T cells (MILs) from an HLA-A2-matched patient were used. These MILs were not terminally exhausted as they showed strong IFN-gamma secretion after polyclonal stimulation, which even exceeded that of a well-established tumour antigen specific CD8.sup.+ cytotoxic T cell clone, SK-1 (Survivin TC) (45). Moreover, they recognized and reacted by substantial IFN-gamma secretion also against KMM-1 tumour cells, despite high levels of PD-L1 expression on KMM-1. However, they exerted only limited capacity to kill KMM-1 cells (20% killing at 10:1 E:T ratio with 5000 KMM-1-luc cells; not shown) suggesting the presence of resistance mechanisms against T cell attack in KMM-1 cells. Silencing of firefly-luciferase (siFLuc) was used as positive control for siRNA transfection efficacy, while silencing of genes essential for tumour cell survival, such as ubiquitin C (UBC) or transfection with a mixture of siRNAs inducing cell death (siCD) resulted in strong reduction of luciferase expression, indicating appropriate gene silencing and sensitivity of the luciferase-based readout. The strongest immune modulatory effect (high impact on T cell killing and no viability impact) was elicited by the serine/threonine calcium/calmodulin-dependent protein kinase 1D (CAMK1D)
[0309] Based on the strong immune resistance phenotype associated with CAMK1D expression in the screens for immune checkpoint inhibitors, the inventors focused on validation and characterization of the immune regulatory role of the intracellular kinase CAMK1D. So far, an immune-related function of CAMK1D in cancer evasion has not been studied. As a first experiment, the inventors de-convoluted the pool of CAMK1D targeting siRNAs from used in the HTP-screen to exclude potential dominant off-target effects of single siRNAs within the pool. Three out of four CAMK1D targeting siRNAs (s1, s2 and s3) and the pool of all siRNAs increased T cell mediated cytotoxicity, while no viability impact of the individual or pooled siRNAs per se was detected (
[0310] Next, CAMK1D expression was studied in a large cohort of CD138-purified malignant plasma cells from multiple myeloma patients with monoclonal gammopathy of unknown significance (MGUS), human myeloma cell lines (HMCL), memory B cells (MBC), plasmablasts (PPC) and normal bone marrow plasma cells (BMPC). CAMK1D expression was highest in MBC but it was also expressed in all MM, MGUS, PPC, and in 30/32 HMCL samples and these showed higher expression than normal bone marrow plasma cells (BMPCs) (Figure id). Thus, these data indicate that CAMK1D is consistently expressed in human multiple myelomas and confers resistance against cytotoxic T cell attack. Classical immune-checkpoint molecules expressed by tumour cells regulate the activity of cytotoxic T cells mostly through engagement of inhibitory receptors on T cells. Since CAMK1D is an intracellular kinase, it may indirectly regulate T cell activity. Parameters of T cell effector function upon contact with CAMK1D proficient or deficient KMM-1 cells were studied next, including the secretion of the T cell effector cytokines INF-γ, Granzyme B, IL-2 or TNF-α. Although consistently increased T cell-mediated tumour cell killing was detected after CAMK1D knockdown in KMM-1 cells, functional analysis of T cells did not reveal any increased T cell function after interaction with CAMK1D-deficient compared to wt tumour cells (
Example 2: CAMK1D Immune Checkpoint Function Blocks FasL and TRAIL Induced T Cell Cytotoxicity
[0311] KMM-1 cells were then exposed to the cytotoxic agents FasL (rHuFasL), TRAIL (rHuTRAIL) or TNF (rHuTNF) commonly used by T cells to kill their target cells. The respective cell death-mediating receptors for FasL and TRAIL, Fas, DR4 and DR5 were strongly expressed on KMM-1 cells while the TNF receptors TNFR1 and TNFR2 were not expressed (
Example 3: CAMK1D has Immune Checkpoint Function in Multiple Tumour Entities and Allows for Patient Stratification
[0312] Since Fas-FasL interactions represent a major cytotoxic principle in tumour immunology, we wondered whether CAMK1D might protect not only multiple myeloma but also solid tumour cells against immune rejection. Therefore Fas expression was analysed on several human cancer cell lines. Fas expression was low in the pancreatic cancer cell line PANC-1 and in the breast cancer cell line MCF-7. However, strong Fas and CAMK1D expression was found in Mel270, which is a PD-L1.sup.+ human uveal melanoma (UVM) cell line (
[0313] The clinical outcome of a cohort of uveal melanoma patients together with genome-wide RNA expression data from their tumour tissue is available at the TCGA database and allows an analysis of the prognostic impact of CAMK1D in this highly immunotherapy-refractory patient population. CAMK1D expression in UVM might protect those tumours with strong Fas receptor expression against immune rejection. Therefore the inventors stratified patients in this cohort according to expression levels of CAMK1D and Fas (above/below median). Kaplan-Meier analyses show that overexpression of CAMK1D in Fas receptor.sup.high tumours but not in Fas receptor.sup.low tumours correlate with poor patient prognosis (
[0314] Using the TCGA database CAMK1D and PD-L1 co-regulation was studied in other tumour entities that are largely unresponsive to anti-PD-1 treatment, specifically in ovarian, pancreatic, colorectal, stomach and esophageal cancer and in glioblastoma. Among them, CAMK1D and PD-L1 were co-expressed in ovarian, pancreatic, stomach and esophageal cancer. As observed in UVM, significant correlations of CAMK1D and Fas receptor expression with poor outcome in these cancers was detected (
Example 4: CAMK1D Regulates the Activity of Effector Caspases-3, -6 and -7 after Fas Receptor Activation
[0315] FasL binding to Fas receptor results in complex signalling events. This induces on the one hand the caspase cascade that finally activates endonucleases to initiate apoptosis by DNA fragmentation and on the other hand stimulates Ca.sup.2+ influx into the cytoplasm, which ultimately triggers CAMK1D activation. CAMK1D might thus interfere with the cellular apoptotic cascade to mediate its tumour protective effect. To clarify this assumption, the impact of CAMK1D expression on tumour cell killing in the absence of effector caspases was analysed. Indeed, silencing of each of the individual downstream effector caspase (caspase-3, -6 and -7) completely abrogated the increased lysis of CAMK1D-deficient tumour cells after FasL exposure (
[0316] CAMK1D activation depends on binding to calmodulin (CaM) which upon Ca.sup.2+ influx induces a conformational change allowing the CAMK-kinase (CAMKK) to phosphorylate and fully activate CAMK1D (62, 63). FasL-expressing MILs thus might trigger Ca.sup.2+ release in KMM-1 cells sufficient for CAMK1D activation. Hence, the intracellular Ca.sup.2+ in KMM-1 cells on single cell level was compared after exposure to MILs or rHuFasL and it was found that both procedures induced a similar, robust increase of intracellular Ca.sup.2+ shortly after treatment (
[0317] Activation of CAMK1D requires binding to Ca.sup.2+/calmodulin complexes which can be inhibited by W-7 hydrochloride (64). Treatment with 5 μM W-7 hydrochloride is not toxic to KMM-1 cells (
##STR00004##
QPP is also known under CAS ID #CAS-404828-08-6 and has a IUPAC name (5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine.
[0318] QPP was identified as CAMK1D inhibitor in a radiometric protein kinase assay (PanQinase® Activity Assay) used for measuring the kinase activity of a recombinantly expressed CAMK1D protein kinase. All assays were performed with a BeckmanCoulter/SAGIAN™ Core System. Using varying concentrations an IC50 of 2.94E-06 was determined for QPP (shown in
[0319] The additional treatment with recombinant FasL induced a significant tumour cell viability, confirming that CAMK1D plays a substantial role in conferring resistance towards tumour cell apoptosis (
Example 5: CAMK1D Knock-Out Reduces Tumour Growth In-Vivo
[0320] To further confirm the role of CAMK1D in mediating cancer resistance against immune attack in vivo, the inventors knocked out Camk1d in the murine colon adenocarcinoma cell line MC38 using the CRISPR/Cas9 technique. In vitro analysis of MC38 Camk1d-deficient tumour cells already revealed sensitivity towards FasL as well as TRAIL mediated apoptosis. Thus, MC38 Camk1d KO as well as MC38 NTS (non-targeting sequence) cells were injected into the left and right flank of the same mouse of both immunodeficient NSG and immunocompetent C57BL6 mice. MC38 Camk1d KO and MC38 NTS tumours outgrew in a similar manner in NSG mice, while a significant difference was observed in the immunocompetent C57BL6 mice, where Camk1d-deficient tumours grew as in the NSG mice, while the growth of Camk1d-deficient tumours was significantly retarded (
Example 6: CAMK1D Binds and Phosphorylates Effector Caspases
[0321] To elucidate mechanistic aspects of CAMK1D involvement in the Fas-signalling cascade, activation of caspase-8 and -9, the prototypic initiator caspases of the extrinsic and intrinsic apoptotic pathway, respectively (65). FasL-induced activation of caspase-8 and -9 was comparably effective in CAMK1D proficient and -deficient KMM-1 cells (
[0322] Notably, the inhibitory Ser150 phosphorylation site of caspase-3 (67) and the corresponding Ser257 of caspase-6 (68) are located in the kinase-function critical distance of up to 4 amino acids apart from the predicted binding site for CAMK1D. CAMK1D might thus be able to phosphorylate Ser150 and Ser257 of caspases-3 and -6. Indeed, CAMK1D deficient KMM-1 cells showed a strongly reduced phosphorylation level of inhibitory serine residues of both caspase-3 and -6 already at steady-state conditions (
[0323] Taken together, these results demonstrate that in tumours, CAMK1D upon its activation through FasL regulates activation and activity of all effector caspases after cytotoxic T cell encounter. These results further suggest that this is at least partially achieved by the inhibitory phosphorylation of the effector caspases.
[0324] Materials and Methods:
[0325] Experimental Model and Subject Details: Patients, Healthy Donors, and Samples:
[0326] Patients presenting with previously untreated multiple myeloma (n=332) or monoclonal gammopathy of unknown significance (MGUS; n=22) at the University Hospitals of Heidelberg and Montpellier as well as 10 healthy normal donors have been included in the study approved by the ethics committee (#229/2003 and S-152/2010) after written informed consent. Patients were diagnosed, staged and response to treatment assessed according to standard criteria (33-35).
[0327] Samples: Normal bone marrow plasma cells and myeloma cells were purified using anti-CD138 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) (36-40). Peripheral CD27.sup.+ memory B-cells (n=11) were FACS-sorted as described (41). The human myeloma cell lines U266, RPMI-8226, LP-1, OPM-2, SK-MM-2, AMO-1, JJN-3, NCI-H929, KMS-12-BM, KMS-11, KMS-12-PE, KMS-18, MM1.S, JIM3, KARPAS-620, L363 and ANBL6 were purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and the American Type Cell Culture (Wesel, Germany), the XG-lines were generated at INSERM U1040 (Montpellier, France) (42). KMM-1 cells were obtained from the National Institutes of Biomedical Innovation, Health and Nutrition (Osaka, Japan). Cell line identity was assessed for proprietary cell lines by DNA-fingerprinting, mycoplasma-contamination excluded by PCR-based assays, and EBV-infection status by clinical routine PCR-based diagnostics. Polyclonal plasmablastic cells (n=10) were generated as published (38, 43, 44). The human uveal melanoma cell line Mel270 was established, characterized and provided by Prof. Griewank (University Hospital Essen) (45). KMM-1-luc cells were generated after transfection with a pEGFP-luc plasmid (provided by Dr. Rudolf Haase, LMU Munich, Germany) and selected for the G418-resistance gene. Lipofectamine LTX was used as transfection reagent according to the manufacturer's instructions. Transfected cells were selected for 14 days with G418-containing medium (0.6 mg/mL). KMM-1-luc cells were sorted twice for the expression of GFP by flow cytometry and cultured in the presence of 0.6 mg/mL G418. Cell sorting was conducted in collaboration with the DKFZ sorting core facility, using the FACSARIA II cell sorter (BD). KMM-1, U266 and Mel270 were cultured under standard conditions in RPMI media supplemented with 10% fetal calf serum, 100 U/mL penicillin G and 100 μg/ml streptomycin at 37° C. in a humidified atmosphere under 5% CO.sub.2.
[0328] MILs Isolation
[0329] Marrow-infiltrating lymphocytes were isolated from the bone marrow of a multiple myeloma patient. Briefly, T cells were isolated from the negative fraction of CD138-sorted bone marrow cells using untouched Human T cells Dynabeads (Invitrogen). Cells were stained for anti-CD3 (Pacific Blue™ anti-human CD3 (Clone OKT3), Biolegend), anti-CD4 (APC/Cy7 mouse anti-human CD4 (Clone RPA-T4), BD Biosciences) and anti-CD8 (Pacific Blue™ mouse anti-human CD8 (Clone RPA-T8), BD Biosciences), tested for HLA-A2 positivity (APC mouse anti-human HLA-A2 Clone BB7.2 (RUO), BD Biosciences) and subsequently expanded using the rapid expansion protocol.
[0330] MILs Expansion
[0331] MILs cultures were ex-vivo expanded using a modified version of the Rapid Expansion Protocol (REP) (46, 47). 2×10.sup.6 of freshly isolated MILs were diluted to 6×10.sup.5 cell/mL in CLM supplemented with 3000 U/mL rHuIL2 (Novartis Pharma). Cells were incubated in 25 cm.sup.2 tissue culture flask for 48h at 37° C. and 5% CO.sub.2. PBMCs from three different buffy coats (at a ratio of 1:1:1) were irradiated with 60 Gy (Gammacell 1000) and used as feeder cells to support MILs expansion. 2×10.sup.6 MILs were co-incubated with 2×10.sup.8 feeder cells (in a ratio 1:100) in 400 mL of MIL expansion medium (CLM/AIM-V) with 30 ng/mL OKT3 antibody (Thermo Scientific) and 3000 IU/mL IL-2 for 5 days in a G-Rex 100 cell culture flask. Afterwards, 250 mL of supernatant was changed with 150 mL of fresh media and IL-2 was replenished to keep the concentration at 3000 IU/mL. On day 7, MILs were divided into three G-Rex 100 flasks in a final volume of 250 mL medium each and media was again replenished on day 11. On day 14 of the expansion, MILs were counted and frozen in aliquots of 40×10.sup.6 cells/mL in freezing media A (60% AB serum and 40% RPMI1640) and B (80% AB serum and 20% DMSO).
[0332] Generation of Flu-Antigen Specific CD8.sup.+ T Cells
[0333] For the generation of flu-specific CD8.sup.+ T cells (flu TC), PBMCs from HLA-A2 healthy donors were isolated. Total CD8.sup.+ T cells were sorted from PBMCs by magnetic separation (Miltenyi) (day 0) and expanded in the presence of A2-matched flu peptide (GILGFVFTL) for 14 days. Irradiated autologous CD8.sup.− fraction was used as feeder cells during the first 7 days of expansion. Afterwards, irradiated T2 cells were used as fresh feeder cells. On day 1 and day 8, 100 IU/mL IL2 (Novartis Pharma) and 5 ng/μL IL15 (R&D Systems) were added to the expansion. The percentage of flu-antigen specific T cells was determined by pentamer staining (GILGFVFTL-APC, ProImmune) on day 7 and 14 via flow cytometry analysis. After antigen-specific expansion, flu TC were sorted by FACS and expanded further for 14 days by using rapid expansion protocol.
[0334] PCR and qPCR
[0335] Gene expression was measured using end-point PCR. Briefly, total RNA was isolated from cell pellets using the RNeasy Mini kit (Qiagen) according to the manufacturer's guidelines. 1 μg of RNA was reverse transcribed to complementary DNA (cDNA) using the QuantiTect reverse transcription kit (Qiagen) according to the manufacturer's protocol. Synthesized cDNA was amplified using conventional PCR. PCR samples were set up in a 25 μL volume using 2×MyTaq HS Red Mix (Bioline), 500 nM of gene-specific primer mix and 100 ng of template cDNA. The PCR program was set as the following: 95° C. for 3 min, 35 cycles of 3 repetitive steps of denaturation (95° C. for 30 s), annealing (60° C. for 30 s) and extension (72° C. for 30 s), and a final step at 72° C. for 5 min. PCR products were run on a 2% agarose gel in TAE buffer using a gel electrophoresis system (Thermo Scientific) and DNA bands were visualized using UV light of myECL Imager (Thermo Scientific). Knockdown efficiency of siRNA sequences was measured by quantitative PCR (qPCR). For qPCR, 10 ng of template cDNA, 2× QuantiFast SYBR Green PCR mix (Qiagen) and 300 nM of gene-specific primer mix was used per 20 μL reaction and each sample was prepared in triplicates. Reactions were run using the QuantStudio 3 (Applied Biosystems). Expression of several genes was normalized to the expression of β-actin gene and the analysis was performed using comparative Ct method.
[0336] Gene expression profiling using U133 2.0 plus arrays (Affymetrix, Santa Clara, Calif., USA) was performed as published (36, 37, 48). Expression data are deposited in ArrayExpress under accession numbers E-MTAB-317.
[0337] Survival and Correlation Analysis Using the Cancer Genome Atlas (TCGA)
[0338] Transcriptomic normalized RNA-Seq by Expectation-Maximization (RSEM) and clinical data from different tumor entities was downloaded using the TCGA2STAT package for R (49). Log 2-normalized expression values were correlated (Person's r) using the ggpubr package for R. Survival curves were generated using survminer package for R. FAS expression was cut at the median to generate Fas high and low sets. Similarly, CAMK1D expression was cut at the median for the Kaplan-Meier survival curves. Significance was calculated using the log-rank test.
[0339] Reverse siRNA Transfection
[0340] Gene knockdown in tumor cells was induced using reverse siRNA transfection with Lipofectamine RNAiMAX (Thermo Scientific). Briefly, 200 μL of 250 nM siRNA solution was added to each well of a 6-well plate. 4 μl of RNAiMAX transfection reagent was diluted in 196 μL of RPMI (Sigma-Aldrich) and incubated for 10 min at room temperature (RT). 400 μL of additional RPMI was added and 600 μL of RNAiMAX mix was given to the siRNA coated wells and incubated for 30 min at RT. 3.5×10.sup.5 KMM-1 (WT or luc) cells were resuspended in 1.2 mL of antibiotic-free RPMI culture medium supplemented with 10% FCS, seeded in the siRNA-RNAiMAX containing wells and incubated for 48 h at 37° C., 5% CO.sub.2. Final siRNA concentration was 25 nM in all cases.
[0341] Phospho-Protein Isolation
[0342] To isolate phosphorylated proteins from cells, tumor cells were pelleted at 0.5×g for 5 min and washed once with PBS at 4° C. The cell pellets were lysed with one pellet volume of Phosphoplex Lysis Buffer (Merck Millipore) containing protease inhibitor cocktail (Cabliochem, 1:100) and phosphatase inhibitor cocktail (Sigma-Aldrich, 1:100) at 4° C. for 15 min on a rotator. Samples were centrifuged at 17000 g at 4° C. for 15 min. Supernatants containing the protein lysates were collected into fresh tubes and quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer's protocol. Proteins were stored at −20° C.
[0343] SDS-PAGE
[0344] 30 μg of protein lysates were denaturated in 4×NuPAGE LDS Sample Buffer (Thermo Scientific) containing 100% ß-mercaptoethanol (PAN) at 70° C. for 10 min. Samples were spun down and separated on NuPAGE 4-12% Bis-Tris Gels (Thermo Scientific) along with PageRuler Prestained Protein Ladder (Thermo Scientific) and run at 115-150 V for 90 min.
[0345] Semi-Dry Western Blot
[0346] Proteins were transferred from the gel to a PVDF membrane (Millipore) using a semi-dry western blot method. The PVDF blotting membrane (Merck Millipore) was activated in 100% methanol (Merck Millipore) for 1 min and afterwards placed in Transfer Buffer (Thermo Science) until use. Blots were assembled from anode to cathode into the Pierce Power Blot cassette (Thermo Scientific) and run at 24 V for 10 min. Membranes were washed in ix TBS and then placed in blocking solution (5% BSA/0.05% TBST) for 2 h. Primary antibodies (anti-CAMK1D (Abcam) 1:20000, anti-caspase-3 (Abcam) 1:750, anti-caspase-6 (Abcam) 1:2000, anti-caspase-7 (Thermo Scientific) 1:1000, anti-caspase-3 (phospho S150) (Abcam) 1:850, anti-caspase-6 (phospho S257) (Abcam) 1:250 and sodium potassium ATPase (Abcam) 1:20000) were diluted in 5% BSA/0.05% TBST and kept on the membrane overnight at 4° C. on a rotator. Membranes were then washed three times for 10 min with 1% BSA/0.05% TBST. Afterwards, HRP-conjugated secondary antibodies (anti-rabbit 1:4000, Santa Cruz or anti-mouse 1:4000, Santa Cruz) were added to 1% BSA/TBST and kept on the membrane at room temperature for 1h on a shaker. Thereafter, the membranes were washed for 10 min with 1% BSA/TBST, then TBST and lastly with TBS. The blots were incubated with the ECL Detection Reagent (Reagent A and Reagent B, 1:1, GE Healthcare) for 4 min and the chemiluminescence was detected with myECL Imager (Thermo Scientific).
[0347] Co-Immunoprecipitation Assay
[0348] For detection of direct protein-protein interaction, co-immunoprecipitation was performed. Briefly, 10×10.sup.6 tumor cells were seeded in 10 cm.sup.2 petri dishes. The next day, cells were stimulated for 4 h with 100 ng/mL rHuFasL (Biolegend). Unstimulated cells were used as negative control. Afterwards, tumor cells were detached, resuspended in ice cold TBS and centrifuged at 400 g for 6 min at 4° C. Supernatant was discarded, cell pellet was resuspended in 1.5 mL TBS and centrifuged at 500 g for 8 min at 4° C. Cell pellet was lysed with 1.5 mL lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% NP40 or Triton-X) containing protease inhibitor (Roche complete 25×) and kept on a rotator for 1 h at 4° C. Afterwards, cells were centrifuged for 20 min at 20000 g at 4° C. Supernatant was collected and centrifuged for further 5 min at 20000 g at 4° C. Meanwhile, protein-G agarose (Sigma-Aldrich) was washed with 1 mL TBS and centrifuged for 1 min at 12000 g. 1 mL of cell supernatant containing cytoplasmatic proteins was added to 60 μL protein-G agarose, incubated with anti-caspase-3 (1:50) (Cell Signaling), anti-caspase-6 (1:50) (Abcam) or anti-caspase-7 (1:100) (Cell Signaling) antibodies and incubated overnight on a rotator at 4° C. 90 μL of cell lysates were frozen at −20° C. The next day, the immunoprecipitated samples were centrifuged at 12000 g at 4° C. for 1 min. Supernatant was discarded and protein-G agarose was washed three times with lyses buffer and centrifuged at 12000 g at 4° C. for 1 min. 2×LDS containing 10% β-mercaptoethanol was added to the immunoprecipitated samples, while 4×LDS containing 10% β-mercaptoethanol was added to the lysates. Samples were denaturated for 10 min at 95° C. on a thermocycler. Samples were spun down and separated on NuPAGE 4-12% Bis-Tris Gels (Thermo Scientific) along with PageRuler Prestained Protein Ladder (Thermo Scientific) and run at 115-150 V for 90 min. After electrophoresis, proteins were transferred on a PVDF membrane (Millipore). Anti-CAMK1D antibody (1:10000) was diluted in 5% BSA/0.05% TBST and kept on the membrane overnight at 4° C. on a rotator. Membranes were then washed three times for 10 min with 1% BSA/0.05% TBST. Afterwards, HRP-conjugated secondary antibodies (anti-rabbit 1:3000) (Santa-Cruz) was added to 1% BSA/TBST and kept on the membrane at room temperature for 1 h on a shaker. The membrane was washed. The blot was incubated with the ECL Detection Reagent (Reagent A and Reagent B, 1:1, GE Healthcare) for 4 min and the chemiluminescence was detected with myECL Imager (Thermo Scientific).
[0349] Plasmid Transfection
[0350] To generate KMM-1-luc cells, 3.5×10.sup.5 KMM-1 WT cells were seeded in a 6 well plate and incubated at 37° C. overnight. 15 μL Lipofectamine LTX reagent were diluted in 150 μL Opti-MEM medium (Gibco). Simultaneously, 3.5 μg of pEGFP-Luc plasmid was diluted in 175 μL Opti-MEM medium and 3.5 μL of PLUS Reagent was added. 150 μL of diluted DNA was added to 150 μL diluted Lipofectamine LTX (Life Technologies) reagent and incubated for 5 min at RT. DNA-lipid complex was then added to the growth medium of the myeloma cells. Cells were incubated at 37° C. for 48 h before investigation of transfection efficacy by flow cytometry.
[0351] Luciferase-Based Cytotoxicity Assay
[0352] KMM-1-luc cells were reverse transfected with the desired siRNA sequences in white 96-well-plate (Perkin Elmer) and incubated for 48 h at 37° C., 5% CO.sub.2. At the same day of transfection MILs were thawn and treated with benzonase (too IU/mL) (Merck). Cell density was adjusted to 0.6×10.sup.6 cells/mL in CLM supplemented with 3000 IU/mL rhuIL-2 (Novartis) for 48 h. IL-2 was depleted 24 h before the co-culture. Flu TC were thawn 6 h before co-culture. For the cytotoxicity setting, MILs, flu TC, the supernatant of activated MILs or rHuFasL were added to transfected tumor cells at desired E:T ratio/concentration, and incubated for 20 h at 37° C., 5% CO.sub.2. For the viability setting, only CLM was added to the tumor cells. After co-culture, supernatant was removed, remaining tumor cells were lysed using 40 μL/well of cell lysis buffer for 10 min. After tumor cell lysis, 60 μL/well of luciferase assay buffer was added and luciferase intensity was measured by using the Spark 20M plate reader (Tecan) with a counting time of 100 msec. Luciferase activities (relative luminescence units=RLUs) were either represented as raw luciferase values or as normalized data to scramble or unstimulated controls.
[0353] Real-Time Live-Cell Imaging Assay
[0354] Target genes in KMM-1 or U266 tumor cells were knocked down with reverse siRNA transfection for 48 h. The reverse siRNA transfection was performed using transparent 96 well microplates (TPP). In parallel, MILs were thawn and prepared as previously described. After 48 h, MILs (E:T 10:1) or rHuFasL (100 ng/mL) were added to the target cells in CLM with YOYO-1 (final concentration 1:5000) and co-cultured at 37° C. For viability controls the according amount of CLM with YOYO-1 (final concentration 1:5000) was added. MILs or rHuFasL-mediated tumor lysis was imaged on the green channel using an IncuCyte ZOOM live cell imager (ESSEN BioScience) for the indicated time points at a 10× magnification. Data were analyzed with the Incucyte ZOOM 2016A software by creating a top-hat filter-based mask for the calculation of the area of YOYO-1 incorporating cells (indicating dead cells).
[0355] ELISA
[0356] Tumor cells were transfected with the indicated siRNAs in a 96-well plate. Afterwards, T cells were added at the indicated E:T ratio for 20 h and 100 μL of supernatants were harvested for the detection of IFN-γ (Human IFN-γ ELISA Set; BD OptEIA), IL-2 (Human IL-2 ELISA Set; BD OptEIA), Granzyme B (Human Granzyme B ELISA development kit; Mabtech) and TNF (Human TNF ELISA Set; BD OptEIA). Experiments were performed according to the manufacturer's instructions. Polyclonal stimulation (Dynabeads Human T-Activator CD3/CD28, Invitrogen) was used as positive control. Absorbance was measured at λ=450 nm, taking λ=570 nm as reference wavelength using the Spark microplate reader (TECAN).
[0357] Flow Cytometry (FACS)
[0358] Flow cytometry was used for the detection of proteins expressed on the plasma membrane of tumor and T cells. Intracellular staining was performed for the detection of caspase-3 (FITC Active Caspase-3 Apoptosis Kit, BD Bioscience) according to manufacturer's instruction. Tumor cells were detached from plates using PBS-EDTA, centrifuged at 500×g for 5 min and resuspended in FACS buffer (5×10.sup.5 cells/tube). Live T cell and tumor cells were distinguished by using Live/Dead Fixable Yellow dead Cell Stain (Life Technologies) followed by blocking with kiovig (human plasma-derived immunoglobulin, Baxter, Deerfield, Ill., USA) at a concentration of 100 μg/mL in FACS buffer (PBS, 2% FCS) for 15 min in the dark on ice. Samples were washed two times in FACS buffer and incubated with either fluorophore-conjugated primary antibodies or isotype control (APC anti-human CD274 (PD-L1) (Clone 29E.2A3), Biolegend; Alexa Fluor 647 Mouse anti-human CCR9 (Clone 112509 (RUO), BD Biosciences; Brilliant Violet 421 anti-human CD95 (Fas) (Clone DX2), Biolegend; PE anti-human CD95 (Fas) (Clone DX2), Biolegend; APC anti-human CD261 (DR4, TRAIL-R1) (Clone DJR1), Biolegend; PE anti-human CD262 (DR5, TRAIL-R2) (Clone DJR2), Biolegend; Biotin anti-human CD120a (TNFR1) (Clone W15099A), Biolegend; PE/Cy7 anti-human CD120b (TNFR2) (Clone 3G7A02), Biolegend; PE/Cy7 anti-human CD279 (PD-1) Antibody, Biolegend); APC mouse anti-human CD178 (Clone NOK-1), BD Biosciences; PE anti-human CD253 (TRAIL) (Clone RIK2), Biolegend; APC anti-human TNF-α (Clone Mab11), Biolegend for 20 min on ice in the dark. Afterwards, cells were washed twice and acquired with the FACS Canto II cell analyzer machine (BD Bioscience) or FACSLyrics Flow cytometer and data were analyzed using FlowJo (Tree Star).
[0359] Calcium Imaging
[0360] KMM-1 cells grown on coverslips were washed with Ringer solution (118 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 1.2 mM Na.sub.2HPO.sub.4, 2 mM NaH.sub.2PO.sub.4, 1.8 mM CaCl2), 5 mM glucose, 9.1 mM HEPES, pH 7.4, with NaOH) and loaded with Fura-2-AM ester (Thermo Fisher Scientific, Waltham, USA) for 45 min. After 15 min, MILs or rHuFasL (50 ng/ml) was added to scr siRNA transfected cells and recording of the intracellular free Ca.sup.2+ was continued for further 30 minutes. Experiments were performed using a ZEISS live cell imaging setup based on an inverse microscope (Axio Observer Z.1) equipped with Fluar 40×/1.3 objective lens (ZEISS, Germany). Fura 2-AM-loaded KMM-1 cells were illuminated with light of 340 nm or 380 nm (BP 340/30 HE, BP 387/15 HE) using a fast wavelength switching and excitation device (Lambda DG-4, Sutter Instrument), and fluorescence was detected at 510 nm (BP 510/90 HE and FT 409) using an AxioCam MRm LCD camera (ZEISS). Data were recorded and analyzed with ZEN 2012 software (ZEISS, Jena, Germany).
[0361] Generation of Supernatants of Activated MILs
[0362] For the generation of the supernatant of polyclonally activated MILs, 1×10.sup.6 MILs were suspended in 1 mL of CLM collected in a 15 mL tube and stimulated with 25 μL of Dynabeads Human T-Activator CD3/CD28 (Thermo Scientific). Afterwards, only the supernatant (100 μL/well) of activated T cells was added to knocked down tumor cells and incubated overnight at 37° C., 5% CO.sub.2. Luciferase-based cytotoxicity assay was performed. Alternatively, MILs were stimulated with tumor cells at an E:T ratio of 10:1. After 20 h co-culture, plates were centrifuged at 450 g for 5 min and 100 μL/well of the supernatant was collected for cytokines detection (ELISA).
[0363] Functional Neutralization
[0364] For the functional neutralization experiment, anti-FasL (Biolegend) or isotype control (Biolegend) were pre-incubated with MILs for 1 h at 37° C., 5% CO.sub.2. As negative control, antibodies were cultivated in the absence of T cells. Afterwards, antibody-containing supernatants were used to stimulate KMM-1-luc cells, which were reverse transfected with the indicated siRNAs. The final concentration of the neutralizing antibodies was 100 ng/mL for anti-FasL and isotype control. As positive control recombinant FasL protein (too ng/mL, Biolegend) was added to the tumor cells instead of T cells. 20 h after co-culture, luciferase intensity was measured.
[0365] Blocking Assays
[0366] For the experiments using the anti-Calmodulin (W7) (Tocris) inhibitor, 1×10.sup.4 KMM-1-luc (scr or CAMK1D-transfected) cells/well were seeded in white 96 well plates (Perkin Elmer) in 100 μL of RPMI 10% FCS. The small molecule inhibitor was added at the indicated concentrations for 1 h at 37° C., before 100 ng/mL rHuFasL or medium control was added. DMSO treatment served as negative control. After 20 h stimulation, luciferase-based cytotoxicity assay was performed. For CAMK1D inhibition, 1×10.sup.4 KMM-1-luc or 1×10.sup.4 Mel270 cells/well were incubated overnight in a 96 well plate. OMX2001 was added at the indicated concentrations 1 h before rHuFasL stimulation (100 ng/mL) or medium control. DMSO treatment served as negative control. After 20h stimulation, luciferase-based cytotoxicity assay was performed.
[0367] Luminex Assays
[0368] Tumor cells were stimulated with rHuFasL (too ng/mL) for 15 min, 30 min, 1 h, 2 h, 4 h and 8 h. Unstimulated cells served as control. For the detection of intracellular phosphorylated analytes, a general pathway (MILLIPLEX MAP Multi-Pathway Magnetic Bead 9-Plex kit, Millipore) was used. For the detection of proteins involved in the activation of apoptosis the MILLIPLEX MAP Early Phase Apoptosis 7-plex-kit (Millipore) together with active caspase-3 Magnetic Bead MAPmate (Millipore) was used. Beads specific for GAPDH served as normalization control. 20 μg of protein lysates were used for the detection of ERK/MAP kinase 1/2 (Thr185/Tyr187), Akt (Ser473), STAT3 (Ser727), JNK (Thr183/Tyr185), P70 S6 kinase (Thr412), NF-kB (Ser536), STAT5A/B (Tyr694/699), CREB (Ser133), and p38 (Thr180/Tyr182) phosphorylated Akt (Ser473), JNK (Thr183/Tyr185), Bad (Ser112), Bel-2 (Ser70), p53 (Ser46), cleaved caspase-8 (Asp384), cleaved caspase-9 (Asp315) and active caspase-3 (Asp175). The assay was performed according to the manufacturer's instructions and samples were measured using the MAGPIX Luminex instrument (Merck Millipore).
[0369] In Vivo Experiment
[0370] Experiments were performed in two cohorts of mice: C57BL6 (n=12) and NOD/SCID gamma chain (NSG) mice (n=12) were subcutaneously injected with 1×10.sup.5 MC38 Camk1d KO (g3 clone 11) or 1×10.sup.5 MC38 NTS (clone 12) cells each into the right and left flank of one mouse, respectively. Tumor growth was measured twice a week and the volume was determined using the following formula: Tumor volume (mm.sup.3)=(Width.sup.2×Length)×(π/6). Mice were sacrificed when tumors exceeded 1.5 cm in diameter.
[0371] Statistics
[0372] For statistical analysis, GraphPad Prism software v6.0 (GraphPad Software, La Jolla, Calif., USA was used. If not differently stated, statistical differences between the control and the test groups were determined by using two-tailed unpaired Student's t-test. In all statistical tests, a p-value s 0.05 was considered significant with *=p≤0.05, **=p≤0.01, ***=p≤0.001 and ****=p≤0.0001.
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