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WO2020165402A1 - Régulation positive du gd2 par inhibition d'ezh2 dans le traitement du cancer - Google Patents

Régulation positive du gd2 par inhibition d'ezh2 dans le traitement du cancer Download PDF

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WO2020165402A1
WO2020165402A1 PCT/EP2020/053874 EP2020053874W WO2020165402A1 WO 2020165402 A1 WO2020165402 A1 WO 2020165402A1 EP 2020053874 W EP2020053874 W EP 2020053874W WO 2020165402 A1 WO2020165402 A1 WO 2020165402A1
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cell
car
specific
cells
ezh2 inhibitor
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Claudia Roessig
Bianca ALTVATER
Sareetha KAILAYANGIRI
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Westfaelische Wilhelms Universitaet Muenster
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Westfaelische Wilhelms Universitaet Muenster
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    • A61K40/42—Cancer antigens
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    • A61K40/4258—Gangliosides, e.g. GM2, GD2 or GD3
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    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
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Definitions

  • the present invention relates an Enhancer of Zeste Homolog 2 (EZH2) inhibitor for use in a method of treating a solid tumor in a subject, wherein the method further comprises the administration of a G D2 -specific CAR-T cell or CAR-NK cell or a G D2 -specific antibody to the subject. Further, the present invention relates to a G D2 -specific chimeric antigen receptor (CAR-) T cell or CAR-NK cell or a G D2 -specific antibody for use in a method of treating a solid tumor in a subject, wherein (i) the method further comprises the administration of an EZH2 inhibitor to the subject; and/or (ii) the subject has been and/or is treated with an EZH2 inhibitor.
  • EZH2 Enhancer of Zeste Homolog 2
  • the present invention relates to methods for increasing the surface expression of G D2 in solid tumor cells and kits and kits-of-part comprising an EZH2 inhibitor and a G D2 -specific CAR-T cell or a G D2 -specific CAR-NK cell or a G D2 -specific antibody.
  • Chimeric antigen receptors are recombinant proteins that link antibody-derived antigen-binding domains to stimulatory T cell signaling pathways. Expressed in immune effector cells, they induce activation responses to surface-expressed tumor target antigens, resulting in antigen-specific tumor cytolysis (Eshhar et al, 1993). T cells engineered to express CARs against the B-lineage antigen CD19 are active to induce and maintain remissions in refractory B lineage leukemias (Gardner et al, 2017; Lee et al, 2015; Maude et al, 2018) and lymphomas (Neelapu et al, 2017; Schuster et al, 2018). The development of CAR T cells for solid tumors is limited by the paucity of target antigens reliably expressed at high densities on cancer cells but not normal cells.
  • Ewing sarcoma as one example of a solid tumor, is an aggressive solid mesenchymal malignancy arising in bone and soft tissues (Ladenstein et al, 2010). EwS are characterized by a specific chromosomal translocation, most commonly of chromosomes 22 and 11 [t(1 1 ,22)(q24; 12)], resulting in the aberrant chimeric transcription factor EWSR1-FLI1 (Delattre et al, 1992). The inventors and others have shown that EwS can express the disialoganglioside G D2 on the cell surface (Kailayangiri et al, 2017; Long et al, 2016).
  • G D2 expression normally characterizes immature neuroectodermal cells, with restricted and low-level tissue expression after birth on neuronal cells in the CNS, peripheral nerves and mesenchymal stroma cells (Martinez et al, 2007; Suzuki, 1965), reviewed in (Rossig et al, 2018). G D2 was first evaluated as a therapeutic target in neuroblastoma, a cancer with abundant G D2 surface expression due to its tissue origin from neuroectoderm (Ladenstein et al, 2018; Yu et al, 2010).
  • the present invention relates to an Enhancer of Zeste Homolog 2 (EZH2) inhibitor for use in a method of treating a solid tumor in a subject, wherein the method further comprises the administration of a G D2 -specific CAR-T or CAR-NK cell or a G D2 -specific antibody cell to the subject.
  • EZH2 Enhancer of Zeste Homolog 2
  • the present invention additionally relates to a G D2 -specific chimeric antigen receptor (CAR)-T cell or CAR-NK cell or a G D2 -specific antibody for use in a method of treating a solid tumor in a subject, wherein (i) the method further comprises the administration of an EZH2 inhibitor to the subject; and/or (ii) the subject has been and/or is treated with an EZH2 inhibitor.
  • CAR chimeric antigen receptor
  • the EZH2 inhibitor may be selected from the group consisting of GSK126, EPZ-6438 (tazemetostat), 3-deazaneplanocin A (DZNep), EPZ005687, EI 1 , and UNC1999.
  • the solid tumor may be selected from the group consisting of Ewing sarcoma, breast cancer, preferably an adenocarcinoma of the breast, neuroblastoma, melanoma, small cell lung carcinoma, lung carcinoma, brain tumors, retinoblastoma and osteosarcoma.
  • the solid tumor is Ewing sarcoma.
  • the solid tumor is neuroblastoma.
  • the solid tumor is osteosarcoma.
  • the solid tumor is breast cancer.
  • the solid tumor is lung cancer.
  • the CAR-T cell or the CAR-NK cell preferably comprises a chimeric antigen receptor specific (CAR) for G D2 and wherein the CAR is Q ⁇ 2-BBz or GD2-CD28 z.
  • the CAR ⁇ 02-BBz has an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to the amino acid sequence shown in SEQ ID NO: 3.
  • the CAR Q ⁇ 2-28z has an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to the amino acid sequence shown in SEQ ID NO: 4.
  • ⁇ 02-BBz or GD2-CD28C may comprise the CDR-H1 as depicted in SEQ ID NO: 15, the CDR-H2 as depicted in SEQ ID NO: 16, the CDR-H3 as depicted in SEQ ID NO: 17, the CDR-L1 as depicted in SEQ ID NO: 18, the CDR-L2 as depicted in SEQ ID NO: 19 and the CDR-L3 as depicted in SEQ ID NO: 20.
  • the G D2 -specific antibody preferably is dinutuximab or dinutuximab beta or an antibody having the same CDR sequences as is dinutuximab (produced in mouse cells) or dinutuximab beta (ch 14.18/produced in CHO cells) or having the same VH and VL regions as dinutuximab or dinutuximab beta.
  • Dinutuximab or dinutuximab beta may comprise the CDR-H1 as depicted in SEQ ID NO: 15, the CDR-H2 as depicted in SEQ ID NO: 16, the CDR-H3 as depicted in SEQ ID NO: 17, the CDR-L1 as depicted in SEQ ID NO: 18, the CDR-L2 as depicted in SEQ ID NO: 19 and the CDR-L3 as depicted in SEQ ID NO: 20.
  • the EZH2 inhibitor may be capable of increasing the surface expression of G D2 on G D2 - low and/or G D2 -negative cancer cells.
  • the EZH2 inhibitor does not increase the surface expression of G D2 in non cancer cells.
  • the EZH2 inhibitor may be administered before and/or simultaneously to administration of the CAR-T cell or CAR-NK cell or a G D2 -specific antibody to the subject.
  • the EZH2 inhibitor is for use of the invention or the G D2 -specific CAR-T cell or CAR-NK cell for use of the invention, wherein the method comprises a further anti-cancer treatment.
  • the further anti-cancer treatment is chemotherapy, radiotherapy, immunotherapy or surgery.
  • the subject may be human.
  • the present invention relates to a combination of an EZH2 inhibitor, and a G D2 -specific CAR-T cell or CAR-NK cell or a G D2 -specific antibody for use in a method of treating a solid tumor in a subject.
  • the EZH2 inhibitor is selected from the group consisting of GSK126, EPZ-6438 (tazemetostat), 3-deazaneplanocin A (DZNep), EPZ005687, EI1 , and UNC1999.
  • the present invention also relates to a method of enhancing susceptibility of a tumor or a tumor cell to G D2 -specific CAR-T cell or CAR-NK cell or GG D2 -specific antibody treatment comprising contacting the tumor or tumor cell with an EZH2 inhibitor.
  • the method of enhancing susceptibility of a tumor or a tumor cell to G D2 -specific CAR-T cell or CAR-NK cell or G D2 -specific antibody treatment comprises administering the EZH2 inhibitor to a subject.
  • the tumor or tumor cell may be in the subject.
  • the method of enhancing susceptibility of a tumor or a tumor cell to G D2 -specific CAR-T cell or CAR-NK cell or G D2 -specific antibody treatment may be an in vitro method.
  • the tumor or tumor cell is in a subject and the EZH2 inhibitor is administered to the subject or in other words, the method of enhancing susceptibility of a tumor or a tumor cell to G D2 -specific CAR-T cell or CAR-NK cell or G D2 -specific antibody treatment may be an in vivo method.
  • the present invention relates to a kit comprising an EZH2 inhibitor and a Go 2 -specific CAR-T cell or CAR-NK cell or a G D2 -specific antibody.
  • the present invention further relates to a kit-of-parts comprising an EZH2 inhibitor and a G D2 -specific CAR-T cell or CAR-NK cell or a G D2 -specific antibody.
  • (A) Go surface expression by flow cytometry in 19 EwS cell lines. According to RFI, cell lines were categorized as G D2 pos (RFI 32) or G D2 neg (RFI ⁇ 2). Representative experiments of two or three.
  • FIG. 1 Upregulation of G D 2 expression by EZH2 inhibition is reversible and limited to EwS cell lines.
  • A G D2 surface expression by flow cytometry in 8 G D2 neg EwS cell lines cultured with 4 pM GSK126 or equivalent volumes of DMSO (control) for 7 days (upper panel) and for 28 days (lower panel).
  • RD RFI after incubation with DMSO
  • RG RFI after incubation with GSK126.
  • FIG. 1 Expression of EZH2 and ganglioside synthases GD3S and GD2S in EwS cells.
  • A EZH2 gene expression in 9 G D2 neg and 10 G D2 pos EwS cell lines by quantitative RT- PCR.
  • B GD3S and GD2S gene expression in 9 G D2 neg and 10 G D2 pos EwS cell lines by quantitative RT-PCR.
  • C GD3S and GD2S gene expression in 9 G D2 neg EwS cell lines following 14 days of incubation in the presence of 4 pM GSK126 or DMSO (control) by quantitative RT-PCR.
  • D Screen shots of ChIP-seq data analysis of SK-N-MC EwS cells using UCSC genome Browser.
  • FIG. 4 High G D2 surface expression in EwS cells does not affect in vitro proliferation colony formation, chemosensitivity, and in vivo tumorigenicity.
  • A FACS selection strategy of tumor cell subpopulations with the highest (Go 2 hi, 30% or 1 %) and lowest (G D2 IOW, 30% or 1 %) G D2 surface expression, exemplified by cell line VH-64.
  • B G D2 surface expression on sorted G D2 hi and G D2 low subpopulations (30%) determined by flow cytometry every 6 days during subsequent cell culture over a period of 3 weeks. The median RFIs of G D2 expression in the two subpopulations are shown in relation to bulk cells from the same cell lines. *p ⁇ 0.02 (t-test).
  • C In vitro expansion of tumor cells from the sorted subpopulations (30%) for 6 days of standard monolayer cultures, quantified by trypan blue exclusion and cell counting.
  • D Colony formation of tumor cells from sorted subpopulations (30%) in semisolid media after 7 or 8 days of culture.
  • E Viabilities of tumor cells from the sorted subpopulations (30%) following incubation with the indicated concentrations of doxorubicin by luminometry.
  • F Tumor formation of G D2 hi and G D2 low subpopulations selected from VH-64 and MS-EwS-16 cells after subcutaneous or intravenous transplantation into NSG mice at the indicated cell numbers.
  • FIG. 5 Disruption of G D2 surface expression in EwS cells by GD3S gene editing does not affect colony formation, tumorigenicity and chemosensitivity.
  • A G D2 expression in 3 EwS cell lines following disruption of the GD3S gene by CRISPR/Cas9 genome editing (GD3S KO) or in wild-type cells (mock control) by flow cytometry.
  • B Proliferation of GD3S KO or wild-type EwS cells after 3-day in vitro culture by luminometry.
  • C Colony formation of GD3S KO or wild-type EwS cells in semisolid media.
  • D Viabilities of GD3S KO and wild-type cells following incubation with the indicated concentrations of doxorubicin by luminometry.
  • FIG. 1 Figure 6. EZH2 inhibitor pretreatment sensitizes G D 2-negative EwS cell lines to in vitro cytolysis by G D2 -specific CAR-T cells.
  • A Exemplary gating strategies for quantification of intracellular cytokine expression and CD107a expression in CAR (GD2-BBz)-transduced T cells.
  • B, C Quantification of intracellular expression of IFN-y (B) and TNF-a (C) in non- transduced (NT) or ⁇ 02-BBz transduced T cells co-incubated for 6 h with EwS cell lines pretreated with 4 mM GSK126 or DMSO alone for 14 days. Shown is one representative experiment of two.
  • FIG. 7 In vitro expansion and colony formation of Go 2 neg EwS cells cultured in the presence of GSK126.
  • A In vitro expansion rates of 9 G D2 neg EwS cell lines cultured in the presence of 4 pM GSK126 or equivalent volumes of DMSO (control) for 14 days by trypan blue exclusion and cell counting.
  • B Colony formation of tumor cells cultured in semisolid medium in the presence of 4 pM GSK126 or equivalent volumes of DMSO (control) for 14 days. Statistical analysis for both assays was done by t-test.
  • FIG. 8 Proliferation, colony formation and viability of tumor cells from G D2 pos and G D2 neg EwS cell lines.
  • A Proliferation of tumor cells from 10 G D2 pos and 9 G D2 neg EwS cell lines (defined as in Figure 1A) after 3 days of in vitro culture by luminometry. RLU, relative luminescence unit. Statistical analysis by t-test.
  • B Colony formation of tumor cells from 10 G D2 pos and 9 G D2 neg EwS cell lines in semisolid agar. Statistical analysis by t-test.
  • C Viabilities of tumor cells from 10 G D2 pos and 9 G D2 neg EwS cell lines following incubation with the indicated concentrations of doxorubicin by luminometry. Statistical analysis by Rank sum test for all concentrations.
  • FIG. 11 Pretreatment of G D2 -negative Ewing sarcoma cell lines with the EZH2 inhibitor renders the tumor cells susceptible to G D2 -specific antibody dependent cytolysis.
  • PBMCs were pre-activated with 500 U/ml IL-2 for 48 h and then analyzed for 4 h in the presence and absence of the G D2 -specific antibody Dinutuximab (100 pg/ml) at effector-to- target (E:T) ratios of 40:1 , 20: 1 and 10: 1.
  • the tumor cells RD-ES were treated with 10 mM DMSO (control) or GSK126 for 7 days prior the co-incubation. % cytolysis was quantified by luminometry.
  • Figure 12 In vitro EZH2 inhibition induces G D 2 upregulation also in lung cancer.
  • Tumor cells from the lung cancer cell line A549 were cultured in the presence of 1 mM tazemetostat dissolved in DSMO (dark grey) or DMSO (control, light grey) for 14 days, and then G D2 surface expression (PE) was quantified by flow cytometry. Isotype controls of the DMSO- treated cells (white area, black dashed lines) and of the GSK126-treated cells (white area, solid black line) were also performed.
  • G D2 is a very interesting target for tumor therapy because of its almost exclusive expression on tumor cells.
  • therapies making use of this tumor-specific expression are hampered by the inhomogeneous expression of G D2 on tumor cells.
  • G D2 -low or even -negative populations that can escape such a therapy.
  • previous tries to develop a therapy targeting G D2 -expressing tumor cells were not successful.
  • G D2 -specific CAR- T cell or CAR-NK cell therapies.
  • EZH2 enhancer of Zeste Homolog 2
  • EZH2 Enhanccer of Zeste Homolog 2
  • EZH2 a histone- lysine N-methyltransferase enzyme
  • Histone-lysine N- methyltransferase EZH2 ENX-1 or Lysine N-methyltransferase 6.
  • EZH2 participates in histone methylation and, ultimately, transcriptional repression.
  • EZH2 acts as a histone methyltransferase and silences genes involved in cell differentiation in a highly context- dependent manner by depositing repressive histone marks at histone 3 lysine 27 (H3K27me3) (Comet et al, 2016) by using the cofactor S-adenosyl-L-methionine. Methylation activity of EZH2 facilitates heterochromatin formation thereby silences gene function.
  • An exemplary EZH2 can be found in the UniProt database using the accession number Q15910-v2, whose sequence is also shown in SEQ ID NO: 11. [036]
  • An ⁇ ZH2 inhibitor” as used herein relates to an inhibitor, which reduces or inhibits the activity of EZH2.
  • Reducing or inhibiting the activity of EZH2 may relate to a reduced methylation of lysine 27 of histone H3 (H3K27).
  • the activity of EZH2 may be determined by an assay as essentially described in Example 1 and Fig. 1C.
  • the rate of lysine 27 of histone H3 trimethylation (H3K27me3) is analyzed by Western Blot using an antibody that detects the trimethylated form of H3K27.
  • the EZH2 inhibitor GSK126 almost completely blocks the trimethylation of H3K27 in comparison to a mock control.
  • An exemplary anti-H3K27me3 antibody is available from abeam as ab6002.
  • EZH2 and histone H3 are prepared in reaction buffer (20 mM Hepes pH 7.5, 10 mM MgCI2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na 3 V0 4 , 2 mM DTT, 1 % DMSO).
  • reaction buffer (20 mM Hepes pH 7.5, 10 mM MgCI2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na 3 V0 4 , 2 mM DTT, 1 % DMSO).
  • a putative EZH2 inhibitor is added to the reaction.
  • the reaction may then be started by the addition of 3 H-SAM (S-adenosyl methionine) and incubated, e.g. for 1 h at 37 °C. After washing away the unbound or not-reacted 3 H-SAM, the radioactivity of the histone H3 may be measured and compared to a control, in which no EZH2 inhibitor has been added.
  • An EZH2 inhibitor may reduce the activity of EZH2 by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% compared to a mock control.
  • EZH2 inhibitors [037] Most of the EZH2 inhibitors known so far target the interaction of EZH2 with its co-factor S-adenosyl methionine (SAM), i.e. compete for SAM binding to EZH2.
  • SAM S-adenosyl methionine
  • GSK126 is a SAM- competitive EZH2 inhibitor.
  • the EZH2 inhibitors of the invention preferably are EZH2 specific and do not inhibit any other histone methylases such as EZH1. Examples of EZH2 inhibitors include, but are not limited to, GSK126, EPZ-6438 (tazemetostat), 3-deazaneplanocin A (DZNep), EPZ005687, EI 1 , and UNC1999.
  • Table 1 shows the structure of different EZH2 inhibitors:
  • G D2 expression marks in breast cancer a cell population with higher capacity to self-renew and reinitiate tumor growth (Battula et al, 2012; Liang et al, 2013).
  • GD 2 expression in EwS cells was surprisingly not associated with distinct functional properties (see also Examples 3 and 4).
  • the administration of an EZH2 inhibitor does not induce a more dangerous phenotype.
  • the inventors After having found that the administration of an EZH2 inhibitor increases the surface expression of G D2 in tumor cells, the inventors further analyzed whether this increase is limited to solid tumor cells. As shown in Fig. 2C, healthy and non-solid tumor cells do not express G D2 even after stimulation with an EZH2 inhibitor. This feature underlines the safety of increasing GD2 expression on solid tumor cells. Thus, increasing the surface expression of G D2 is safe. Accordingly, the EZH2 inhibitor preferably does not increase the surface expression of G D2 in non-cancer cells, more preferably in non-solid tumor cells.
  • a therapeutic strategy based on the surprising findings of the inventors would include the administration of an EZH2 inhibitor to a subject to increase the expression of G D2 specifically on the solid tumor.
  • the increased surface expression of G D2 on the solid tumor may then be used for targeting G D2 -specific CAR-T cells or CAR-NK cells to the solid tumor cells.
  • those solid tumor cells may then be specifically killed, thereby treating the solid tumor.
  • a G D2 -specific antibody may be administered to the subject.
  • the present invention relates to an Enhancer of Zeste Homolog 2 (EZH2) inhibitor for use in a method of treating a solid tumor in a subject, wherein the method further comprises the administration of a G D2 -specific CAR-T cell or CAR-NK cell or a G D2 -specific antibody to the subject.
  • EZH2 Enhancer of Zeste Homolog 2
  • the present invention relates to an Enhancer of Zeste Homolog 2 (EZH2) inhibitor for use in a method of treating a solid tumor in a subject, wherein the method further comprises the administration of a G D2 -specific CAR-T cell to the subject.
  • EZH2 Enhancer of Zeste Homolog 2
  • the present invention relates to an Enhancer of Zeste Homolog 2 (EZH2) inhibitor for use in a method of treating a solid tumor in a subject, wherein the method further comprises the administration of a G D2 -specific CAR-NK cell to the subject.
  • EZH2 Enhancer of Zeste Homolog 2
  • the present invention relates to an Enhancer of Zeste Homolog 2 (EZH2) inhibitor for use in a method of treating a solid tumor in a subject, wherein the method further comprises the administration of G D2 -specific antibody to the subject.
  • EZH2 Enhancer of Zeste Homolog 2
  • the present invention also relates to a G D2 -specific chimeric antigen receptor (CAR-) T cell or CAR-NK cell or a G D2 -specific antibody for use in a method of treating a solid tumor in a subject, wherein (i) the method further comprises the administration of an EZH2 inhibitor to the subject; and/or (ii) the subject has been and/or is treated with an EZH2 inhibitor.
  • CAR- G D2 -specific chimeric antigen receptor
  • the present invention also relates to a G D2 -specific chimeric antigen receptor (CAR-) T cell for use in a method of treating a solid tumor in a subject, wherein (i) the method further comprises the administration of an EZH2 inhibitor to the subject; and/or (ii) the subject has been and/or is treated with an EZH2 inhibitor.
  • CAR- G D2 -specific chimeric antigen receptor
  • the present invention also relates to a G D2 -specific chimeric antigen receptor (CAR-) NK cell for use in a method of treating a solid tumor in a subject, wherein (i) the method further comprises the administration of an EZH2 inhibitor to the subject; and/or (ii) the subject has been and/or is treated with an EZH2 inhibitor.
  • CAR- G D2 -specific chimeric antigen receptor
  • the present invention also relates to a G D2 -specific antibody for use in a method of treating a solid tumor in a subject, wherein (i) the method further comprises the administration of an EZH2 inhibitor to the subject; and/or (ii) the subject has been and/or is treated with an EZH2 inhibitor.
  • an EZH2 inhibitor it is possible to begin with the administration of an EZH2 inhibitor to the subject prior to, i.e. before, treatment with G D2 -specific CAR-T or CAR-NK cells or the G D2 - specific antibody. It is also possible to first start the treatment with G D2 -specific CAR-T or CAR- NK cells or the G D2 -specific antibody and then to support this treatment by the administration of an EZH2 inhibitor to the subject, i.e. to simultaneously administer the EZH2 inhibitor and the CAR-T or CAR-NK cell or G D2 -specific antibody to the subject.
  • the EZH2 inhibitor may be administered to the subject at any time before or simultaneously to the administration of the CAR-T cell or the CAR-NK cell or the G D2 -specific antibody to the subject.
  • a “chimeric antigen receptors” is a membrane-bound artificial receptor that is designed to bind to a target cell-specific antigen such as G D2 and then induce a signal cascade in an effector cell such as a T cell or a NK cell that expresses the CAR.
  • CARs usually may comprise an extracellular domain, transmembrane domain and intracellular domain.
  • the extracellular domain is the region of the CAR that is exposed to the extracellular fluid and comprises an antigen recognition region.
  • the antigen recognition domain is designed to bind GD 2 .
  • Examples for such an antigen recognition domain include, but are not limited to, an antibody or an antibody fragment or a proteinaceous binding molecule with antibody-like properties.
  • the antibody fragment thereof may be selected from the group consisting of an scFv, a di-scFv, a bi-scFv, a Fab, an Fc, an F(ab’)2, a pFc’, a nanobody, an affibody, a DARPin, a diabody, a camelid, an engineered T cell receptor and a monobody.
  • the antibody may be selected from the group consisting of an lgA1 , an lgA2, an IgD, an IgM, an IgE, an lgG1 , an lgG2, an lgG3, and an lgG4.
  • the CAR may comprise at least a portion of a single chain variable fragment (scFv).
  • the antibody may be human, fully human, humanized, human engineered, non-human, and/or chimeric antibody.
  • the transmembrane domain and/or the intracellular domain of the CAR may comprise at least a portion of a cytoplasmic signaling domain.
  • the intracellular domain may comprise at least a portion of a signaling molecule selected from the group comprising CD3zeta, CD28, and 4-1 BB, 2B4, ICOS, OX-40, or other.
  • the signaling molecule may comprise CD3zeta.
  • the signaling molecule may comprise CD28.
  • the signaling molecule may comprise 4-1 BB.
  • the intracellular domain may comprise at least a portion of CD3zeta.
  • the intracellular domain may comprise at least a portion of CD28.
  • the intracellular domain may comprise at least a portion of 4-1 BB.
  • the intracellular domain may comprise at least a portion of OX-40.
  • the intracellular domain may comprise at least a portion of 2B4.
  • the intracellular domain may comprise at least a portion of ICOS.
  • the intracellular domain may comprise at least a portion of a cytoplasmic signaling domain from one or more signaling molecules.
  • the intracellular domain may comprise at least a portion of two or more cytoplasmic signaling domains.
  • the two or more cytoplasmic signaling domains may be from two or more different signaling molecules.
  • the intracellular domain may comprise at least a portion of three or more cytoplasmic signaling domains.
  • the intracellular domain may comprise at least a portion of four or more cytoplasmic signaling domains.
  • the intracellular domain may comprise at least a portion of a ligand that binds to one or more signaling molecules.
  • a “G D2 -specific CAR” comprises an extracellular domain that binds G D2 , i.e. a G D2 - specific extracellular domain, preferably when G D2 is expressed on the solid tumor.
  • G D2 -specific CAR comprises an extracellular domain that binds G D2 , i.e. a G D2 - specific extracellular domain, preferably when G D2 is expressed on the solid tumor.
  • a person skilled in the art can readily determine whether an extracellular domain of a CAR binds G D2 .
  • a G D2 -specific extracellular domain of a CAR may bind G D2 with a binding affinity of less than about 1 nM, about 2 nM, about 2.5 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 12nM, about 14 nM, about 16 nM, about 18 nM, about 20 nM, about 25 nM, about 50 nM, about 100 nM, about 125 nM or about 250 nM.
  • G D2 -specific CARs include, but are not limited to, ⁇ 02-BBz as depicted in the nucleic acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 3, or GD2-28C, as depicted in the nucleic acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 4.
  • the G D2 -specific CAR-T cells or CAR-NK cells described herein may comprise or express a G D2 -specific CAR as defined herein.
  • ⁇ 02-BBz and GD2-28 z for example, both comprise a single-chain antibody domain (scFv) of the monoclonal antibody 14.G2a.
  • a scFv derived from monoclonal antibody 3F8 or any other G D2 - specific monoclonal antibody may be used in a G D2 -specific CAR.
  • Antibody 3F8 or a scFv derived from 3F8 may comprise 3 heavy chain CDRs as depicted in SEQ ID NOs: 5-7 and 3 light chain CDRs as depicted in SEQ ID NOs: 8-10, which may be comprised in a G D2 -specific CAR comprised in a G D2 -specific CAR-T cell or CAR-NK cell.
  • Antibody 14.G2a or an scFv derived from 14.G2a may comprise the CDR-H1 as depicted in SEQ ID NO: 15, the CDR-H2 as depicted in SEQ ID NO: 16, the CDR-H3 as depicted in SEQ ID NO: 17, the CDR-L1 as depicted in SEQ ID NO: 18, the CDR-L2 as depicted in SEQ ID NO: 19 and the CDR-L3 as depicted in SEQ ID NO: 20, which may be comprised in a G D2 -specific CAR comprised in a Goa- specific CAR-T cell or CAR-NK cell.
  • Antibody 14.G2a may comprise a heavy chain as depicted in SEQ ID NO: 21 and a light chain as depicted in SEQ ID NO: 22, which may be comprised in a G D 2-specific CAR comprised in a G D 2-specific CAR-T cell or CAR-NK cell. Further G D 2-specific CARs are also described in WO 2013/040371 and WO 2016/134284, which are hereby incorporated by reference.
  • CDRs sequences of the disclosure follow the definition according to Kabat, as described in Sequences of Proteins of immunological Interest, US Department of Health and Human Services (1991), eds. Kabat et al. Other standards for defining CDRs exist as well, such as the definition according to Maass 2007 (Journal of Immunological Methods 324 (2007) 13-25).
  • Another standard for characterizing the antigen binding site is to refer to the hypervariable loops as described by Chothia (see, e.g., Chothia, et al. (1992); J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995) EMBO J.
  • CDRs A further standard for defining CDRs is the definition according to AbM, used by Oxford Molecular's AbM antibody modelling software. See, generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). It is understood that embodiments described with respect to the CDR definition of Kabat, can alternatively be implemented using similar described relationships such as with respect to Maass, AbM, or Chothia definition.
  • the CAR ⁇ 02-BBz has an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to the amino acid sequence shown in SEQ ID NO: 3.
  • the CAR ⁇ 02-BBz may comprise the CDR-H1 as depicted in SEQ ID NO: 15, the CDR-H2 as depicted in SEQ ID NO: 16, the CDR-H3 as depicted in SEQ ID NO: 17, the CDR-L1 as depicted in SEQ ID NO: 18, the CDR-L2 as depicted in SEQ ID NO: 19 and the CDR-L3 as depicted in SEQ ID NO: 20.
  • the CAR GD2-28C has an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity to the amino acid sequence shown in SEQ ID NO: 4.
  • the CAR Q ⁇ 2-28z may comprise the CDR-H1 as depicted in SEQ ID NO: 15, the CDR-H2 as depicted in SEQ ID NO: 16, the CDR-H3 as depicted in SEQ ID NO: 17, the CDR-L1 as depicted in SEQ ID NO: 18, the CDR-L2 as depicted in SEQ ID NO: 19 and the CDR-L3 as depicted in SEQ ID NO: 20.
  • identity or“sequence identity” is meant a property of sequences that measures their similarity or relationship.
  • sequence identity means the percentage of pair-wise identical residues - following (homology) alignment of a sequence of a polypeptide of the invention with a sequence in question - with respect to the number of residues in the longer of these two sequences. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100.
  • the percentage of sequence identity can, for example, be determined herein using the program BLASTP, version blastp 2.2.5 (November 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res. 25, 3389-3402).
  • the percentage of homology is based on the alignment of the entire polypeptide sequences (matrix: BLOSUM 62; gap costs: 11.1 ; cutoff value set to 10 3 ) optionally including the propeptide sequences, using the SEQ ID NO: 3 or 4 as reference in a pairwise comparison. It is calculated as the percentage of numbers of "positives" (homologous amino acids) indicated as result in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.
  • A“G D2 -specific antibody” as used herein relates to any antibody that specifically binds to GD2, preferably on the surface of a solid tumor or solid tumor cell, and stimulates the subject’s immune system to attack the solid tumor or solid tumor cell. It is understood that the Goa- specific antibody is preferably a therapeutic antibody.
  • the G D 2-specific antibody may induce an antibody-dependent cell-mediated cytotoxicity (ADCC) thereby stimulating the lysis of the solid tumor or the solid tumor cells by immune cells such as natural killer cells (see also Example 6).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the G D 2-specific antibody may be conjugated to a radioactive moiety or may be in the form of antibody-drug conjugates (ADC) or to an immune-activating agent in the form of a bispecific T cell engager.
  • a "bispecific T cell engager" (BiTE) preferably refers to a class of artificial bispecific monoclonal antibodies that are capable of directing a host's immune system, more specifically the T cells' cytotoxic activity, against cancer cells.
  • a bispecific T cell engager is preferably a fusion protein comprising or consisting of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain.
  • One of the scFvs binds to T cells, e.g. via the CD3 receptor, and the other to a tumor cell, e.g. via a tumor specific molecule such as a tumor specific antigen, which preferably is in the context of the invention G D2 .
  • exemplary G D2 -specific antibodies include, but are not limited to, dinutuximab and dinutuximab beta or antibodies having the same CDRs as dinutuximab or dinutuximab beta or antibodies having the same VH and VL region as dinutuximab or dinutuximab beta.
  • Such an antibody may have a VH and/or VL region that has an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or even higher sequence identity with the VH and/or VL region of dinutuximab or dinutuximab beta.
  • the VH domain of dinutuximab or dinutuximab beta is depicted in SEQ ID NO: 21 and the VL domain of dinutuximab or dinutuximab beta is depicted in SEQ ID NO: 22.
  • Dinutuximab or dinutuximab beta may comprise may comprise the CDR-H1 as depicted in SEQ ID NO: 15, the CDR-H2 as depicted in SEQ ID NO: 16, the CDR-H3 as depicted in SEQ ID NO: 17, the CDR-L1 as depicted in SEQ ID NO: 18, the CDR-L2 as depicted in SEQ ID NO: 19 and the CDR-L3 as depicted in SEQ ID NO: 20.
  • Dinutuximab is described in WO 2013/189516, which is hereby incorporated by reference.
  • G D2 -specific antibodies are preferably administered by parenteral administration such as an intravenous injection.
  • G D2 relates to a disialoganglioside that is also known as ganglioside G D2 . It may be represented by the following Formula I, which shows its structure.
  • G D2 may be expressed on tumors of neuroectodermal origin, including human neuroblastoma and melanoma or on tumor cells from sarcomas and other solid cancers, with highly restricted expression on normal tissues.
  • the relatively tumor specific expression of G D2 makes it a suitable target for immunotherapy with monoclonal antibodies or with artificial T cell receptors.
  • a CAR-T cell or CAR-NK immunotherapy makes use of an effector cell, i.e. a T cell or a NK cell, which has been genetically engineered with the CAR.
  • the effector cell may be a T cell.
  • the effector cell may be a cell of a T cell lineage.
  • the effector cell may be a mature T cell.
  • the effector cell may be a cytotoxic T cell.
  • the effector cell may be a naive T cell.
  • the effector cell may be a memory stem cell T cell (TMSC).
  • the effector cell may be a central memory T cell (TCM).
  • TCM central memory T cell
  • the effector cell may be an effector T cell (TE).
  • the effector cell may be a CD4+ T cell.
  • the T cell may be a CD8+ T cell.
  • the effector cell may be a CD4+ and CD8+ cell.
  • the effector cell may be an alpha-beta T cell.
  • the effector cell may be a gamma-delta T cell.
  • the effector cell may be a natural killer T cell.
  • the effector cell may be a natural killer (NK) cell.
  • the effector cell may be a cytokine-induced killer (CIK) cell.
  • the premise of CAR-T or CAR-NK immunotherapy in general is to modify effector cells such as T cells or NK cells to recognize cancer cells in order to more effectively target and destroy them.
  • the T cells or NK cells are genetically engineered to target solid tumor cells expressing G D2 on their surface, i.e. with a G D2 -specific CAR.
  • T cells or NK cells may be obtained from people, genetically altered ex vivo to express a CAR, and the resulting CAR-T cells or CAR-NK cells may then be infused into patients to attack their tumors such as solid tumors described herein.
  • Effector cells may be derived from a patient's own blood (autologous) or derived from the T cells or NK cells of another healthy donor (allogenic). Once isolated from a person, these T cells or NK cells are genetically engineered to express a specific G D2 -specific CAR, which programs them to target G D2 that is present on the surface of solid tumors described herein.
  • Methods for producing a CAR-T cell are e.g. disclosed in WO 2017/049166, which is hereby incorporated by reference in its entirety. These methods may analogously be applied to CAR-NK cells.
  • T cells or NK cells are isolated from the subject. Ex vivo, they are genetically modified to express the CAR, e.g. by viral transduction or transfection of a vector that comprises the CAR as well as regulatory elements such as a promoter. After verification of the genetic modification, the resulting CAR-T cells or CAR-NK cells may be administered to the subject. Suitable modes of administration for CAR-T cells or CAR-NK cells include intravenous, subcutaneous, intracavitary (for example by reservoir-access device), intraperitoneal, and direct injection into a tumor mass.
  • A“solid tumor” as used herein relates to an abnormal mass of tissue that usually does not contain cysts or liquid areas.
  • solid tumors are sarcomas, carcinomas, and lymphomas.
  • a solid tumor include, but are not limited to, Ewing sarcoma, breast cancer, preferably an adenocarcinoma of the breast, neuroblastoma, melanoma, small cell lung carcinoma, lung carcinoma, brain tumors, retinoblastoma and osteosarcoma.
  • the solid tumor is a Ewing sarcoma (EwS).
  • the solid tumor is osteosarcoma.
  • the tumor is neuroblastoma.
  • a “subject” as used herein relates to any mammal, including a human.
  • the subject is an adult, an adolescent or an infant.
  • terms “individuaf or " atient" are used and are intended to be interchangeable with “subject.”
  • the subject may suffer from a disease such as a solid tumor described herein.
  • the administration of an EZH2 inhibitor increases the surface expression in solid tumors such as Ewing sarcoma (EwS) but also in osteosarcoma, breast cancer and neuroblastoma.
  • EwS Ewing sarcoma
  • the EZH2 inhibitor of the invention preferably is capable of increasing the surface expression of G D2 on G D2 -low and/or G D2 -negative cancer cells.
  • “Surface expression of G D2 ” within this context relates to the amount or level of G D2 that is expressed and can be determined on the cell surface of a cell, e.g. a cancer cell or a cell of a solid tumor.
  • an “increase” of the surface expression of G D2 may mean an increase by at least 1.1 -fold, 1.25-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 25-fold, 50-fold or even 100-fold.
  • the surface expression of G D2 may be classified by using flow cytometry. One exemplary method is described in the materials and methods section of the examples. Here, the surface expression of G D2 was determined and the RFI (relative fluorescence intensity) of each cell line or groups of cell has been calculated. G D2 -negative cells may have a RFI of 2 or lower while G D2 -positive cells may have an RFI of more than 2. RFI was calculated by dividing median fluorescence intensities of mAb-stained cells by those obtained with isotype antibodies
  • the EZH2 inhibitor of the invention preferably does not increase the surface expression of G D2 in non-cancer cells.
  • “Non-cancer cells” as used herein relate to cell present in healthy tissues of the subject. These cells are characterized by stopping cell division when no cell division or the like is needed for tissue growth, repair or function and are unlike cancer cells, which continue cell division even in instances, where cell division is not needed.
  • the EZH2 inhibitor may be administered in combination or separated from the administration of the CAR-T cells or the CAR-NK cells or the G D2 -specific antibody.
  • administration means administering of a therapeutically effective dose of the EZH2 inhibitor to a subject.
  • administration also relates to a method of incorporating the EZH2 inhibitor into tissues of an organism. Different routes of administration are possible.
  • the EZH2 inhibitor of the present invention can, for example, be administered via different ways such as any parenteral or non-parenteral (enteral or topical) route that is therapeutically effective for (preferably proteinaceous) drugs.
  • Parenteral application methods include, for example, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intrathecal, intranasal, intra- atrial, intraperitoneal or intravenous injection and infusion techniques, e.g. in the form of injection solutions, infusion solutions or tinctures.
  • Non-parenteral delivery modes are, for instance, enteral delivery modes such as oral delivery, e.g. in the form of pills, tablets, capsules, solutions or suspensions, or rectally, e.g. in the form of suppositories.
  • Topical application routes include epicutaneous or inhalational applications.
  • EZH2 inhibitors and pharmaceutical compositions of the present invention can be administered in formulations containing conventional non-toxic pharmaceutically acceptable excipients or carriers, additives and vehicles as desired and described herein.
  • EZH2 inhibitors are often small molecules that are suitable for an oral administration.
  • the EZH2 inhibitor is administered orally.
  • a simultaneous parenteral administration e.g. an intravenous injection, together with the CAR-T cell or the CAR-NK cell or the Goa- specific antibody is envisioned.
  • a composition comprising the EZH2 inhibitor and the CAR-T cell or CAR-NK cell or the G D 2-specific antibody is administered to the subject, preferably by an intravenous injection.
  • Cancer treatment very often does not only rely on only one agent or medicament for treating. Very often, there is a combination of different medicaments to provide a better treatment of the tumor.
  • the treatment of the solid tumor comprising the administration of an EZH2 inhibitor and a G D2 -specific CAR-T cell or CAR-NK cell or the G D2 -specific antibody as described herein may be supported by a further anti-cancer treatment.
  • the method of treating a solid tumor of the invention may comprise a further anti-cancer treatment.
  • Exemplary further anti-cancer treatments include, but are not limited to, chemotherapy, radiotherapy, immunotherapy or surgery.
  • the present invention also relates to a combination of an EZH2 inhibitor and a G D2 - specific CAR-T cell or a combination of an EZH2 inhibitor and a Go 2 -specific CAR-NK cell or a combination of an EZH2 inhibitor and the G D2 -specific antibody for use in a method of treating a solid tumor in a subject.
  • the combination may be in the form of a pharmaceutical composition.
  • the combination or the pharmaceutical composition of the invention may further comprise a suitable buffer.
  • Suitable buffers include buffers, in which the fusion proteins or pharmaceutical compositions of the present invention can be stored or in which they can be directly administered to the subject. In the latter case, such buffer is a non-toxic, physiologically acceptable buffer.
  • All embodiments described herein within the context of the EZH2 inhibitor for use in a method of treating a solid tumor or a G D2 -specific CAR-T cell or CAR-NK cell or the G D2 -specific antibody for use in a method of treating a solid tumor also apply for the combination of an EZH2 inhibitor and a G D2 -specific CAR-T cell or a combination of an EZH2 inhibitor and a G D2 -specific CAR NK-cell or the G D2 -specific antibody.
  • Example 5 shows that the upregulation of G D2 by an EZH2 inhibitor increases the susceptibility of Ewing Sarcoma (EwS), a solid tumor, to a G D2 -specific CAR-T cell treatment.
  • EwS Ewing Sarcoma
  • This feature of EZH2 inhibitors may be used to make solid tumors more susceptible to a G D2 - specific CAR-T cell or NK cell treatment but also for G D2 -specific antibody treatments. Therefore, the present invention additionally relates to a method of enhancing susceptibility of a tumor or a tumor cell to a G D2 -specific CAR-T cell or CAR-NK cell treatment or the G D2 -specific antibody comprising contacting the tumor or tumor cell with an EZH2 inhibitor.
  • the method of enhancing susceptibility of a tumor or a tumor cell to G D2 -specific CAR-T cell or CAR-NK cell treatment or G D2 -specific antibody treatment of the invention is an in vitro method.
  • the tumor or tumor cell is in a subject and the EZH2 inhibitor is administered to the subject.
  • All embodiments described herein within the context of the EZH2 inhibitor for use in a method of treating a solid tumor or a G D2 -specific CAR-T cell or CAR-NK cell or the G D2 -specific antibody for use in a method of treating a solid tumor also apply for the method of enhancing susceptibility of a tumor or a tumor cell to a G D2 -specific CAR-T cell or CAR-NK cell or the G D2 -specific antibody treatment.
  • “Susceptibility of a tumor or a tumor cell to G D2 -specific CAR-T cell or NK cell or G D2 - specific antibody treatment” as used herein relates to the efficacy of a G D2 -specific CAR-T cell or NK cell or G D2 -specific antibody treatment against a specific tumor or tumor cell of a solid tumor as described herein.
  • a G D2 -specific CAR-T cell or CAR-NK cell therapy or a G D2 - specific antibody therapy shows a higher efficacy after treatment with one or more EZH2 inhibitor(s), e.g.
  • the solid tumor or tumor cells shows a higher susceptibility to G D2 -specific CAR-T cell or CAR-NK cell treatment or a G D2 -specific antibody treatment.
  • Assays for determining the amount of solid tumor cells killed by a CAR-T cell or CAR-NK cell treatment or the cytotoxicity of a CAR-T cell or CAR-NK cell are known to a person skilled in the art.
  • One example for such an assay is a calcein-AM release assay as described in the“Materials and Methods” section of the Examples or published in Neri et al.
  • “Higher susceptibility” of the solid tumor or tumor cell to a G D2 -specific CAR-T cell or CAR-NK cell therapy may relate to an increase of killed G D2 -treated tumor or tumor cells by at least 1.1 -fold, 1.25-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 25-fold, 50-fold or even 100-fold in comparison to untreated or mock-treated tumors or tumor cells.
  • the present invention further relates to a kit comprising an EZH2 inhibitor and a G D2 - specific CAR-T cell or CAR-NK cell or a G D2 -specific antibody.
  • the present invention further relates to a kit-of-parts comprising an EZH2 inhibitor and a G D2 -specific CAR-T cell or CAR-NK cell or a G D2 -specific antibody.
  • the kit or the kit-of-parts may further comprise a pharmaceutically acceptable vehicle.
  • the kit or the kit-of-parts may further comprise a buffer, preservative agents, coloring agents and the like.
  • less than 20 means less than the number indicated.
  • more than or greater than means more than or greater than the indicated number, e.g. more than 80% means more than or greater than the indicated number of 80%.
  • MS-EwS-1 previously described as MS-PES-1 (Kailayangiri et al, 2012)
  • MS-EwS-4 originally described as MS-PES-4 (Kailayangiri et al, 2017)
  • MS-EwS-6 originally described as MS-PES-6 (Spurny et al, 2018)
  • MS-EwS-16 originally described as DC-ES-6 (Leuchte et al, 2014)
  • MS-EwS-15 originally described as DC-ES-15 (Unland et al, 2015)
  • MS-EwS-34 were established from biopsy material of individual patients obtained at metastatic relapse, as reported previously (Kailayangiri et al, 2012; Leuchte et al, 2014; Spurny et
  • A4573, 5838, TTC-466 and TC-32 were a gift from the Children's Hospital Los Angeles.
  • A673, TC-71 , SK-ES-1 , RD-ES, and CADO-ES-1 were purchased from DSMZ (Braunschweig, Germany).
  • A204 is a rhabdoid tumor cell line purchased from DSMZ (Braunschweig, Germany).
  • Normal human fibroblasts generated from skin biopsies were obtained from Cliona Rooney (Houston, TX, USA).
  • Mesenchymal stem cells of two donors were cultured as described (Gieseke et al, 2013).
  • the B-cell precursor leukemia cell line SupB15 and the T cell leukemia cell line Jurkat were purchased from ATCC.
  • T cell cultures and B-lymphoblastoid cell lines were generated from healthy donors as described (Altvater et al, 2006). The identity of the cell lines was confirmed by short tandem repeat (STR) profiling.
  • STR short tandem repeat
  • tumor cells were cultured in collagen-coated 25 or 75 cm 2 tissue culture flasks (MS-EwS-1 , MS-EwS-4, MS- EwS-6, MS-EwS-15, MS-EwS-16, MS-EwS-34, TC-71 , TC-32, VH-64, CADO-ES-1 , SK-ES-1 , SK-N-MC, RD-ES and WE-68) or in uncoated flasks (all others) in RPMI 1640 medium (Invitrogen, Germany), supplemented with 10% heat-inactivated fetal calf serum (FCS; Thermo Fisher) and 2 mM L-glutamine (Sigma-Aldrich, Germany) and maintained at
  • EwS cell lines were sorted into G D2 low (30%) and G D2 hi (30%) subpopulations as described above and were seeded with 1x10 6 cells in 25 mm 2 culture flasks. Every six days, cells were harvested and stained with PE-conjugated G D2 antibody 14.G2a over a period of three weeks. The RFI of G D2 expression in each subpopulation was calculated in relation to control cells having undergone the sorting procedure without selection of subpopulations.
  • EwS tumor cells were harvested, counted and seeded in collagen-coated 6-well plates (Sarstedt, Germany) at 0.1 to 0.3x10 6 cells/well in a total volume of 2 ml. After 2-4 h, the EZH2 inhibitor GSK126 (Active Biochemicals, USA) or tazemetostat (Cayman Chemicals, USA) dissolved in DMSO or DMSO alone as control were added at concentration of 10 to 12 mM, 4 pM or 1 pM as indicated in the figures. After 4 days of incubation at 37 °C and 5% C02, the medium was changed and the EZH2 inhibitor was added again at the same concentrations. Every 7 days, cells were pooled, harvested, counted and analyzed for the G D2 expression described as above or lysates for western blot analysis were generated as detailed below.
  • EZH2 inhibitor or DMSO treated tumor cells were homogenized in 30-100 pi ice-cold RIPA-buffer (0.1 % DTT, Sigma-Aldrich) with fresh protease inhibitor cocktail (Roche, Germany), shortly fractured in liquid nitrogen, thawed on ice, and then clarified by spinning for 15 min at 4°C and 20,000 g. After measuring the protein concentration with Bradford reagent, 50 pg of the sample was separated by electrophoresis on an SDS/15% polyacrylamide gel and then electro blotted onto a nitrocellulose membrane (Bio-Rad).
  • Blocking was done in TBST buffer containing 5% BSA for 1 h, followed by incubation with anti-H3K27me3 antibody (Abeam, Germany) diluted 1 : 1 ,000 in TBST containing 5% BSA for 12 h at 4 °C.
  • the membrane was incubated with HRP-linked anti-mouse IgG whole Ab (GE Healthcare, Germany) at 1 :2,000 in TBST 5% BSA for 1 h at RT, followed by treatment with enhanced chemo luminescence reagent (ECL, Plus Western Blotting Detection System, GE Healthcare) and either exposed to Hyperfilm ECL film (GE Healthcare) for 1 min or directly analyzed by the Imager (ECL Chemostar, INTAS Science Imaging, Germany). Equal protein loading was determined by Ponceau staining. Equal protein loading was also determined by shortly washing the membrane in TBST buffer, followed by incubation with 7 ml RestorTM Plus stripping buffer (Thermo Scientific) for 15 min at RT.
  • the membrane was then washed twice in 20 ml TBST buffer for 10 min at RT, followed by blocking with TBST buffer with 5% nonfat dry milk for 1 h and detection with a p-actin-specific antibody (Cell Signaling, Germany) diluted 1 :5,000 in TBST with 5% BSA for 12 h at 4°C. After washing, the membrane was incubated with HRP-linked anti-rabbit antibody (GE Healthcare) 1 :2,000 in TBST 5% milk for 1 h, followed by detection as described above.
  • HRP-linked anti-rabbit antibody GE Healthcare
  • the relative luminescence units (RLUs) measured after 96 h of incubation were divided by the means of the RLUs on day 0 for each cell line. The results represent the rises of ATP levels indicating proliferation.
  • RLUs relative luminescence units
  • tumor cells were sorted into G D2 low (30%) and G D2 hi (30%) subpopulations as described above and seeded at 1x10 6 cells in 25 mm 2 culture flasks. On day six, cells were harvested, resuspended in RPMI media and counted using trypan blue staining and a microscope. Fold expansion was calculated by dividing the absolute cell numbers on day six by the seeded cell numbers.
  • EwS cells were plated in triplicate in 35 mm tissue culture dishes (Thermo Scientific, USA) in methylcellulose enriched media (1.9% methylcellulose, 15% fetal bovine serum, 0.23% bovine serum albumin, 1 % Penicillin/Streptomycin (Pen/Strep) and 82% Iscove's Modified Dulbecco's Media) in a total volume of 4 ml.
  • Cells were plated at 450 cells/ml in 1 ml volumes and incubated for 7 to 8 days at 37°C and 5% C02 to allow for anchorage-independent clonal growth. Numbers of colonies from each culture dish were calculated using an inverted microscope and a scoring grid. The mean colony numbers (colony-forming units, CFU) of the triplicate dishes were used for the graphic analyses.
  • the chemosensitivity of EwS cells was determined by quantification of the numbers of viable cells using the CellTiter Glo® Luminescent Cell Viability Assay (Promega, Mannheim, Germany). Tumor cells were seeded in opaque-walled 96-well plates at 5,000 cells per well in 50 pi of growth medium and incubated with doxorubicin at dilutions of 0.02, 0.1 , 0.2, 0.5 and 1 pmol/l at 37°C and 5% C02. After 72 h of incubation, 100 pi of CellTiter Glo® reagent were added to each well. The plates were placed on an orbital shaker for 2 min, and then incubated for 10 min at RT in the dark. The amount of metabolized ATP was quantified by determining the relative light units with GloMax Discover (Promega, Mannheim, Germany). Percent viability was determined by dividing the RLU of doxorubicin-treated cells with untreated cells x 100.
  • the biological scaffold SISmuc was prepared from porcine gut as previously described (Gottlich et al, 2016) followed by removal of mesentery and vascular tree from completely decellularized explants.
  • the matrix is registered under the trade mark BioVaSc-TERM® and after seeding with tumor cells under OncVaSc-TERM®. All explantations were in compliance with the German Animal Protection Laws ( ⁇ 4 Abs. 3 TierSchG) and the institute's animal protection officer regularly informed the responsible authorities.
  • the animals received proper attention and care in compliance with the Guide for Care and Use of Laboratory Animals published by the National Institute of Health (NIH publication no. 85e23, revised 1996) and was approved by the institutional animal protection board.
  • SISmuc For the treatment with EZH2 inhibitor, 1x10 5 EwS cells were seeded on the luminal side of the SISmuc.
  • static culture was performed for 3 days by administering 2.5 ml of cell specific media (RPMI-1640 + 10% FCS + Na-Pyruvate + Pen/Strep) to the cell crowns.
  • cell specific media RPMI-1640 + 10% FCS + Na-Pyruvate + Pen/Strep
  • the reseeded SISmuc scaffolds were placed into the chambers of customized flow bioreactors and attached to a tubing system containing 45 ml of cell specific media connected to a peristaltic pump which provides a slow media flow of 3-4 ml/min.
  • 3D tumors on the scaffolds were treated with the EZH2 inhibitor or control for 14 days, then extracted from the bioreactor and cut in half to fix one part in 4% formaldehyde for further paraffin embedding and to extract the cells from the other half for flow cytometry analysis using PBS/EDTA and Trypsin. G D2 staining was performed as described above.
  • H&E Hematoxylin and eosin
  • NSG mice NOD Scid gamma mice were used from own breeding in the central animal experimental facility Muenster (ZTE), originally purchased from Charles River (Germany) and were housed in pathogen-free rooms in type-2L (long) individually ventilated cages (Charles River) with a maximum of six animals per cage. They were allowed access to sterile food and water ad libitum. The RT is held constantly at 21 °C. Eight to 12-week old NSG mice of both genders were used for the experiments.
  • VH-64 or MS-EwS-16 cells were sorted into G D2 low (30%) and G D2 hi (30%) subpopulations as described above and were injected at 1x10 4 to 25x10 4 into the right and left flanks of NSG mice. Tumor growth was monitored at regular intervals and measured using a caliper. Mice were anesthetized with isoflurane and sacrificed when the tumor volumes reached the experimental endpoint. If no tumor was detectable, mice were sacrificed after 12 weeks. Additionally, VH-64 cells sorted into the 1 % lowest and highest G D2 - expressing cells were injected at 1x10 3 cells each into the right and left flanks of NSG mice. Tumor volume measurement was done as above.
  • VH-64 cells transduced with luciferase were sorted into the 30% lowest and highest G D2 -expressing cells and injected at 1x10 4 to 25x10 4 cells into the tail veins of NSG mice. EwS engraftment and growth were monitored weekly starting 2 weeks after transplantation by BLI using IVIS Spectrum Imaging System (PerkinElmer). Mice were injected intraperitoneally (i.p.) with D- luciferin (150 mg/kg; Synchem OHG, s039).
  • mice were anesthetized with isoflurane (2% isoflurane, 0.5 L/min oxygen), and images were acquired after 6 min in a dorsal and ventral position (f/stp: 1 , bining: 4, exposure time: automatic). Mice were sacrificed after tumor volumes reached the experimental endpoints. If no tumor was detectable, mice were sacrificed after 12 weeks.
  • isoflurane 2% isoflurane, 0.5 L/min oxygen
  • images were acquired after 6 min in a dorsal and ventral position (f/stp: 1 , bining: 4, exposure time: automatic). Mice were sacrificed after tumor volumes reached the experimental endpoints. If no tumor was detectable, mice were sacrificed after 12 weeks.
  • Primers for EZH2 (QT00054614), ST8SIA1 (G D3 synthase, QT00054159) and B4GALNT1 (GD 2 synthase, QT02564009) were purchased from Qiagen.
  • Primers for the reference gene HPRT1 forward primer 5'-TGAGGATTTGGAAAGGGTGT (SEQ ID NO: 12), reverse primer 5’-GAGCACACAGAGGGCTACAA (SEQ ID NO: 13)
  • HPRT1 forward primer 5'-TGAGGATTTGGAAAGGGTGT (SEQ ID NO: 12), reverse primer 5’-GAGCACACAGAGGGCTACAA (SEQ ID NO: 13)
  • Amplification was performed in triplicate reactions in two different runs at 95° C for 15 min, followed by 94° C for 15 sec and 40 cycles of 55° C (30 sec) and 12 C (30 sec) on a CFX96 Thermal Cycler (BioRad).
  • Cq threshold cycle
  • Cq values were determined using CFX Manager (BioRad). For the analysis the triplicates of each runs were taken together. Relative gene expression levels were calculated by 2A-AACq-method compared to HPRT1 and untreated or G D2 neg cells used as reference.
  • the sgRNA used for ST8SIA1 (NCBI Gene ID: 6489; G D3 synthase) knock-out was designed using the website http://crispr.mit.edu/.
  • the sgRNA (CACCGCCATTGAAGAAATGCGCGG (SEQ ID NO: 14)) was cloned into the BsmBI sites of the lentiviral vector lentiCRISPR_v2 (Addgene 52961) (Sanjana et al, 2014). Production of lentiviral supernatant was performed as described in Brabetz et al, 2017.
  • HEK293T cells were co-transfected with lentiCRISPR_v2 and the helper plasmids pMD2.G (Addgene plasmid #12259) and psPAX2 (Addgene plasmid #12260) containing gagpol and VSV-G sequences. Lentiviral supernatant was harvested after 48 h and used to transduce the tumor cells in 10 cm plates using 6 pg/ml Sequabrene (Sigma) over night. After puromycin selection for one week, G D3 S gene knockout was analyzed by staining for G D2 surface expression as described above.
  • the T cells were harvested and transferred to 24-well non-tissue culture-treated plates coated with Retronectin and co incubated with viral supernatant for 48 h, as described (Altvater et al, 2009).
  • 5x10 6 transduced or non-transduced T cells were then transferred to gas-permeable culture devices with 50 ml capacity (Wilson Wolf Manufacturing, 80040S) in 35 ml of culture medium for 14-16 days.
  • the G D2 -specific CAR GD2-BBZ as shown in SEQ ID NO: 1 was previously described (Kailayangiri et al, 2012). Briefly, it contains the single-chain antibody domain (scFv) of the monoclonal antibody (mAb) 14.G2a (Rossig et al, 2001), the hinge domain of human lgG1 followed by the transmembrane domain of CD28 and the signaling domains derived from 4-1 BB and CD3Z.
  • the CAR gene was codon-optimized and then sub cloned into the Agel and Xhol sites of the retroviral vector SFG (Altvater et al, 2009). Generation of stable retroviral producer cell lines and production of recombinant retrovirus for transduction of T cells was performed as described (Altvater et al, 2009).
  • T cells (3x10 5 /well) on days 13-14 after transduction were incubated with an equal number of target cells and with the EZH2 inhibitor for 14 days in a total volume of 200 pi in Eppendorf tubes in the presence of Monensin (eBioscience, Germany) (1 pl/ml) and CD107a- PE antibody (Biolegend, Germany) (100 ng/ml) for 3 h at 37°C and 5% C02.
  • the cells were washed, then incubated with CF488-conjugated G D2 anti-idiotype antibody ganglidiomab and a CD3-specific antibody for 15 min, fixed in 1 % PFA and analyzed by flow cytometry.
  • T cells incubated with medium alone or with DMSO-treated EwS cells served as controls.
  • CAR T cells The lytic activity of CAR T cells was tested using a calcein-AM release assay (Neri et al, 2001). EwS target cells treated with 4 pM GSK126 for 14 days were washed twice with PBS and then resuspended in PBS at a final concentration of 2x10 6 cells/ml and incubated with 10 pM calcein-AM (Thermo Fisher, Germany) for 30 min at 37°C with occasional shaking. After two washes in medium (RPMI, 10% FCS) cells were adjusted to 10 5 cells/ml. The test was performed in flat bottom 96-well microtiter plates (Thermo Scientific, Germany).
  • CAR-T cells at effector-to-target (E:T) cell ratios from 40: 1 to 10: 1 were seeded in triplicates together with 1x10 4 calcein AM-labeled target cells for spontaneous (only target cells in complete medium) and maximum release (only target cells in medium plus 9% Triton X-100). After 4 h at 37°C in 5% C02, the plates were centrifuged for 5 min and then 75 pi supernatant was harvested and transferred into new black-walled 96-well microtiter plates (Greiner Bio-One, Germany). Samples were measured using a GloMax® Discover multi-mode microplate reader (Promega, Germany) (Excitation 475 nm/Emission 500-550 nm).
  • Example 1 Pharmacological inhibition of EZH2 selectively upregulates surface GD2 expression in EwS cells
  • RFI relative fluorescence intensity
  • EwS cells were seeded onto a biological tissue matrix consisting of decellularized small-intestine submucosa and mucosa (SISmuc) and cultured in a dynamic bioreactor system in the presence or absence of 4 mM (SK-ES-1) or 12 mM (MS-EwS- 4) GSK126 for 14 days.
  • Immunohistochemistry analysis confirmed formation of multilayered tumor tissue on the matrix ( Figure 2D).
  • GSK126 effectively upregulated cell surface expression of G D2 on EwS cells also in the 3D model ( Figure 2D).
  • Figure 9 additionally provides evidence, that an EZH2 inhibitor such as tazemetostat can increase the surface expression of G D2 in osteosarcoma and neuroblastoma.
  • Figure 10 also shows that GSK126 enhances the surface expression of G D2 in the breast cancer cell line MDA-MB-231.
  • EZH2 inhibition selectively and reliably upregulates the ganglioside G D2 on the cell surface of EwS cells, also in a complex 3D tumor model and using chemically different pharmacological inhibitors.
  • Example 2 EZH2 modulates G D 2 expression in EwS cells by regulating expression of genes involved in G D 2 biosynthesis.
  • G D2 expression of G D2 during development is regulated through stage- and tissue- specific expression of glycosyltransferases, G D3 synthase (GD3S) and G D2 synthase (GD2S), which synthesize G D3 from its precursor G M 3 and convert G D3 to G D2 , respectively (Ngamukote et al, 2007).
  • G D3 synthase GD3S
  • G D2 synthase GD2 synthase
  • the inventors quantified transcripts of EZH2 and of GD3S and GD2S in 9 G D2 neg and 10 G D2 pos EwS cell lines by RT-PCR. EZH2 expression was not different in G D2 neg versus G D2 pos cell lines ( Figure 3A).
  • Example 3 G D2 pos and G D 2neg EwS cell lines and subpopulations of EwS cells have comparable in vitro proliferation rates, clonogenicity, tumorigenicity and chemosensitivity
  • G D2 Safe therapeutic upregulation of G D2 requires knowledge of the functional significance of G D2 expression in EwS.
  • G D2 was found to define a malignant population with a higher capacity to self-renew and reinitiate tumor growth than G D2 -negative cells (Battula et al, 2012; Liang et al, 2017). Inducing G D2 expression by epigenetic regulation could thus promote a cell population with high aggressiveness and metastatic properties.
  • the inventors compared the in vitro expansion and colony-forming capacities of EwS cell lines expressing high or low densities of G D2 identified by flow cytometry (Figure 1A).
  • G D2 hi expression within individual EwS cell lines does not confer to enhanced tumor-initiating capacities.
  • Example 4 Loss of G D 2 by genetic disruption of G D 3 synthase in EwS cells does not affect in vitro growth, clonogenicity and chemosensitivity
  • Example 5 Upregulation of surface G D 2 in EwS cells by pharmacological inhibition of EZH2 enables effective targeting by G D2 -specific CAR T cells.
  • the capacity of the tumor cells to induce antigen-specific, CAR-mediated T cell activation was assessed by quantification of the intracellular cytokines IFN-y and TNF-a and of CD107a, a marker of degranulation and prerequisite for perforin-mediated toxicity.
  • GSK126 pretreatment significantly enhanced cytolysis of initially G D2 neg EwS cells by ⁇ 02-BBz transduced T cells ( Figure 6E).
  • EZH2 inhibition sensitizes G D2 neg EwS cells to antigen-specific functional interactions with G D2 -redirected CAR T cells and to CAR T cell mediated in vitro cytolysis.
  • Example 6 Pretreatment of G D 2-negative Ewing sarcoma cell lines with the EZH2 inhibitor renders the tumor cells susceptible to G D2 -specific antibody dependent cytolysis
  • PBMCs were pre-activated with 500 U/ml IL-2 for 48 h and then analysed for 4 h in the presence and absence of the G D2 -specific antibody Dinutuximab (100pg/ml) as described herein, i.e. a light chain having an amino acid sequence as depicted in SEQ ID NO: 22 and a heavy chain having an amino acid sequence as depicted in SEQ ID NO: 21 , at effector-to-target (E:T) ratios of 40: 1 , 20: 1 and 10: 1.
  • the tumor cells RD-ES were treated with 10mM DMSO (control) or GSK126 for 7 days prior the co-incubation.
  • % cytolysis was quantified by luminometry. As apparent from Fig. 1 1 , Ewing sarcoma cells pretreated with the EZH2 inhibitor but not untreated cells from the same cell line are sensitive to Dinutuximab-dependent cytolysis.
  • Example 7 In vitro EZH2 inhibition induces G D2 upregulation also in lung cancer cells
  • Tumor cells from the lung cancer cell line A549 were cultured in the presence of 1 mM tazemetostat dissolved in DSMO (dark grey) or DMSO (control, light grey) for 14 days, and then G D2 surface expression was quantified by flow cytometry. As apparent from Fig. 12, pharmaceutical EZH2 inhibition induces a 4.7-fold induction of G D2 surface expression in a lung cancer cell line.
  • CD22-targeted CAR T cells induce remission in B- ALL that is naive or resistant to CD19-targeted CAR immunotherapy. Nat Med 24: 20-28.
  • EWS-FLI 1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma. Cancer Cell 26: 668-681.
  • Neural ganglioside GD2 identifies a subpopulation of mesenchymal stem cells in umbilical cord. Cell Physiol Biochem 23: 415-424.

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Abstract

La présente invention concerne un activateur de l'inhibiteur de séquence activatrice de l'homologue zeste 2 (EZH2) destiné à être utilisé dans un procédé de traitement d'une tumeur solide chez un sujet, le procédé comprenant en outre l'administration d'une cellule CAR-T spécifique du GD2 ou d'une cellule CAR-NK ou d'un anticorps ou d'un dérivé d'anticorps spécifique du GD2 au sujet. En outre, la présente invention concerne une cellule T (CAR) à récepteur antigénique chimérique spécifique du GD2 ou une cellule CAR-NK destinée à être utilisée dans un procédé de traitement d'une tumeur solide chez un sujet, (i) le procédé comprenant en outre l'administration d'un inhibiteur d'EZH2 au sujet ; et/ou (ii) le sujet a été et/ou est traité avec un inhibiteur d'EZH2. De plus, la présente invention concerne des procédés pour augmenter l'expression de surface du GD2 dans des cellules tumorales solides et des trousses et des trousses de composant comprenant un inhibiteur d'EZH 2 et une cellule CAR-T spécifique du GD2 ou une cellule CAR-NK spécifique du GD2.
PCT/EP2020/053874 2019-02-14 2020-02-14 Régulation positive du gd2 par inhibition d'ezh2 dans le traitement du cancer Ceased WO2020165402A1 (fr)

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