Methods and compositions for the treatment of pancreatic cancer

Abstract

To provide various compositions and methods useful for the treatment of pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC), and methods for activating pancreatic tissue-specific anti-tumor T cell immunity.SOLUTION: In certain embodiments, such methods comprise administration of IL33. In certain embodiments, such methods comprise administration of PD-1 and / or PD - L1 inhibitors.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 868,976, filed June 30, 2019, and U.S. Provisional Patent Application No. 62 / 937,219, filed November 18, 2019, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on June 30, 2020, is named MSKCC_042_WO1_SL.txt and is 15,983 bytes in size.

[0003] Inclusion by Citation Under statutes permitting incorporation by reference, all references cited herein are incorporated by reference in their entirety. Also incorporated by reference are any manufacturer's manuals or catalogs of products cited or referred to herein. References incorporated by reference herein, or any teachings therein, may be used in the practice of the present invention. Superscript numbers following the text of this specification refer to numbered references identified in the "Reference List" section of this patent application. [Background technology]

[0004] background Antibodies targeting the PD-1 / PD-L1 pathway and the CTLA-4 pathway are useful as cancer immunotherapies. However, most cancers lack pre-existing anti-tumor T cells and are unresponsive. Pancreatic ductal adenocarcinoma (PDAC) is one of the most immunotherapy-resistant and lethal cancers. Therefore, there is an urgent need for new and improved treatments for PDAC, including immunotherapy-resistant PDAC. The present invention fulfills this need. Summary of the Invention

[0005] Summary of the Invention The present invention is based in part on a series of important discoveries, which are described in more detail in the Examples section of this specification. Briefly, using a unique cohort of long-term survivors of pancreatic ductal adenocarcinoma (PDAC), we found that tumor expression of group 2 innate lymphocytes (ILC2s) and the ILC2-activating ligand interleukin (IL)-33 positively correlated with tumor immune cytolytic activity and long-term patient survival. Using a PDAC mouse model, we found that the IL33-ILC2 axis activates pancreatic tissue-specific antitumor T cell immunity. Surprisingly, recombinant IL33 (rIL33) significantly inhibited the expression of PDAC TILC2s and CD8s. + It was found to activate T cells and cure over 70% of tested mice. Furthermore, the combination of rIL33 treatment and PD-1 / PD-L1 pathway blockade (using αPD-1) was shown to synergistically expand pancreatic tissue-specific ILC2s and enhance the antitumor effects of αPD-1 in both PD-1 partially sensitive and PD-1 resistant models. Importantly, in a highly aggressive PD-1-resistant tumor model, treatment with rIL33 or αPD-1 alone had limited efficacy, whereas the combination of rIL33 and αPD-1 significantly reduced tumor size compared with all other treatments, reducing tumor volume by approximately 40% and improving survival by 50%. Based on these and other discoveries described herein, the present invention provides a variety of new and improved compositions and methods for the treatment of pancreatic cancer.

[0006] For example, in one aspect, the present invention provides a method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, the method comprising administering to a subject having PDAC an effective amount of IL33, thereby treating PDAC in the subject.

[0007] In another aspect, the present invention provides a method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, comprising administering to a subject having PDAC effective amounts of: (a) IL33 and (b) a PD-1 and / or PD-L1 inhibitor, thereby treating PDAC in the subject.

[0008] In yet another embodiment, the present invention provides a method of activating pancreatic tissue-specific anti-tumor T cell immunity in a subject in need thereof, comprising administering to a subject having PDAC an effective amount of IL33, thereby activating tissue-specific anti-tumor T cell immunity in the subject. In some such embodiments, the activation of pancreatic tissue-specific anti-tumor T cell immunity is achieved by activation / proliferation of pancreatic ILC2 cells and / or CD8 + Including T cell activation.

[0009] In another embodiment, the invention provides a method of activating pancreatic tissue-specific anti-tumor T cell immunity in a subject in need thereof, comprising administering to a subject with PDAC an effective amount of (a) IL33 and (b) a PD-1 and / or PD-L1 inhibitor, thereby activating pancreatic tissue-specific anti-tumor T cell immunity in the subject. In some such embodiments, the activation of pancreatic tissue-specific anti-tumor T cell immunity is achieved by activation / proliferation of pancreatic ILC2 cells and / or CD8 + Including T cell activation.

[0010] In another aspect, the present invention provides a method of activating pancreatic ILC2 cells, the method comprising contacting pancreatic ILC2 cells with an effective amount of IL33, thereby activating the pancreatic ILC2 cells.

[0011] In another aspect, the invention provides a method of activating pancreatic ILC2 cells, the method comprising contacting pancreatic ILC2 cells with an effective amount of (a) IL33 and (b) a PD-1 and / or PD-L1 inhibitor, thereby activating the pancreatic ILC2 cells.

[0012] In another aspect, the invention provides a method of sensitizing PDAC tumor and / or pancreatic ILC2 cells to a PD-1 and / or PD-L1 inhibitor, comprising contacting the PDAC tumor and / or pancreatic ILC2 cells with an effective amount of IL33, thereby sensitizing the PDAC tumor and / or pancreatic ILC2 cells to the PD-1 and / or PD-L1 inhibitor.

[0013] In some embodiments, the present invention provides a composition comprising (a) IL33 and (b) a PD-1 and / or PD-L1 inhibitor. For example, in some embodiments, the present invention provides a composition for use in treating PDAC comprising (a) IL33 and (b) a PD-1 and / or PD-L1 inhibitor.

[0014] In some embodiments, the methods summarized above or described elsewhere herein also include the preliminary step of determining whether the subject's tumor and / or pancreas contains ILC2 cells that express the IL-33 receptor, or determining whether the ILC2 cells express the IL-33 receptor. In some such embodiments, an IL33 and / or PD-1 / PD-L1 inhibitor is administered to the subject only if the subject's tumor and / or pancreas contains ILC2 cells that express the IL-33 receptor. Similarly, in some such embodiments, ILC2 cells are contacted with an IL33 and / or PD-1 / PD-L1 inhibitor only if the ILC2 cells express the IL-33 receptor.

[0015] In some embodiments, the present invention provides various cell therapy methods. For example, in one embodiment, the present invention provides a method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, comprising administering to a recipient subject having PDAC an effective amount of activated donor pancreatic ILC2 cells, wherein the donor pancreatic ILC2 cells are obtained from the donor subject and activated ex vivo / in vitro by contact with IL33, and wherein the donor subject and recipient subject are allogeneic, thereby treating PDAC in the recipient subject.

[0016] Similarly, in another aspect, the present invention provides a method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, comprising: (a) contacting donor pancreatic ILC2 cells obtained from a donor subject with IL33 ex vivo / in vitro to generate activated donor pancreatic ILC2 cells; and (b) administering the donor activated pancreatic ILC2 cells to a recipient subject having pancreatic ductal adenocarcinoma (PDAC), wherein the donor subject and recipient subject are allogeneic, thereby treating PDAC in the recipient subject.

[0017] Similarly, in yet another aspect, the present invention provides a method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, comprising: (a) obtaining donor pancreatic ILC2 cells from a donor subject; (b) contacting the donor pancreatic ILC2 cells with IL33 ex vivo / in vitro to generate activated donor pancreatic ILC2 cells; and (c) administering the activated donor pancreatic ILC2 cells to a recipient subject having pancreatic ductal adenocarcinoma (PDAC), wherein the donor subject and recipient subject are allogeneic, thereby treating PDAC in the recipient subject.

[0018] In some embodiments, the cell therapy methods described above or elsewhere herein also include a step of ex vivo / in vitro expansion of the donor pancreatic ILC2 cells and / or activated donor pancreatic ILC2 cells prior to administering the activated donor pancreatic ILC2 cells to the recipient subject. In some of the cell therapy methods described above or elsewhere herein, the donor pancreatic ILC2 cells administered to the recipient subject are a substantially pure population of ILC2 cells. Such a substantially pure population of ILC2 cells can be obtained by any suitable cell isolation / purification method, for example, cell sorting based on the presence of one or more pancreatic ILC2 markers (such as those described in the Examples section of this patent application). In some of the cell therapy methods described above or elsewhere herein, the donor and recipient subjects are the same individual, such that the methods are autologous cell therapy methods. Similarly, in some of the cell therapy methods described above or elsewhere herein, the donor and recipient subjects have the same MHC / HLA type.

[0019] In some embodiments summarized above or described elsewhere herein, the subject (including the donor subject and / or recipient subject, in the case of cell therapy methods) is a human. In some embodiments summarized above or described elsewhere herein, the subject (including the donor subject and / or recipient subject, in the case of cell therapy methods) is a non-human mammal. In some embodiments summarized above or described elsewhere herein, the subject (including the donor subject and / or recipient subject, in the case of cell therapy methods) is a mouse. In some embodiments summarized above or described elsewhere herein, the subject (including the donor subject and / or recipient subject, in the case of cell therapy methods) has PDAC that is partially or completely resistant to PD-1 and / or PD-L1 inhibitor treatment.

[0020] In some embodiments summarized above or described elsewhere herein, the IL33 is recombinant IL33. In some embodiments summarized above or described elsewhere herein, the IL33 is human IL33. In some embodiments summarized above or described elsewhere herein, the IL33 is recombinant human IL33. In some embodiments summarized above or described elsewhere herein, the IL33 is murine IL33. In some embodiments summarized above or described elsewhere herein, the IL33 is recombinant murine IL33.

[0021] In those embodiments summarized above or described elsewhere herein that include a PD-1 inhibitor, in some such embodiments, the PD-1 inhibitor is an antibody. In some embodiments summarized above or described elsewhere herein, the PD-1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, AMP-224, AMP-514, and PDR001.

[0022] In some of the embodiments summarized above or described elsewhere herein that include a PD-L1 inhibitor, in some such embodiments, the PD-L1 inhibitor is an antibody. In some embodiments, the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, durvalumab, BMS-936559, and CK-301.

[0023] These and other aspects of the present invention are further described in the Detailed Description, Figures, and Examples sections of this patent application. Furthermore, one skilled in the art will recognize that the various aspects of the present invention described throughout this patent application can be combined in a variety of different ways, and that such combinations are within the scope of the present invention. [Brief explanation of the drawings]

[0024] [Figure 1]Figures 1a-f: IL33-dependent ILC2s infiltrate human and mouse pancreatic cancers. (Figure 1a) ILC gating, frequency, and phenotype in unselected human PDAC patients. (Figure 1b) ILC2 frequency (top) and correlation with survival (bottom) in tumor tissue microarrays from short- and long-term PDAC survivors. (Figure 1c) Association of bulk tumor IL33 mRNA with survival and correlation with tumor cytolytic index (CYT) in short- and long-term PDAC survivors. (Figure 1d) ILC gating and frequency in PDAC mice. (Figure 1e) Intratumoral ILC frequency and number in Rag2- / - PDAC mice treated with αCD90.2 or isotype (Iso) antibody. (Figure 1f) ILC gating, frequency, and number in Il33+ / + and Il33- / - PDAC mice. High and Low in Figure 1b and Low in Figure 1c are defined as values ​​higher or lower than the median of the cohort, respectively. Data were collected 14 days after tumor implantation (Fig. 1d-f). n, number of tumors from individual patients or mice. Horizontal bars indicate median values. Data in Fig. 1d-f were pooled from ≥2 independent experiments with n≥3 / group; each point represents one mouse analyzed separately. P values ​​were determined by Tukey's one-way ANOVA (Fig. 1a) and Kruskal-Wallis multiple comparisons post-hoc test (Fig. 1d), two-tailed Mann-Whitney test (Fig. 1b, e, f), two-tailed log-rank (Fig. 1b, c, survival curves), and linear regression (Fig. 1c). [Figure 2]Figure 2a-g: The IL33-ILC2 axis activates tissue-specific cancer immunity. Tumor weight, volume, and survival rate of Il33+ / + and Il33- / - orthotopic (Figure 2a) or subcutaneous (Figure 2b) PDAC mice. (Figure 2c) Frequency of total CD8+ T cells (left) and IFN-producing (right) cells in orthotopic Il33+ / + and Il33- / - PDAC tumors. (Figure 2d) Tumor weight of T cell-depleted Il33+ / + and Il33- / - orthotopic PDAC mice. (Figure 2e) Frequency of tumor rejection and tumor weight in Il33+ / + and Il33- / - orthotopic and subcutaneous KPC-OVA PDAC mice. (Figure 2f) Study design (left), frequency of tumor rejection (middle), and tumor weight (right) of KPC-OVA PDAC tumors in iCOS-T mice with intact or depleted ILC2s. (Figure 2g) Frequency of OVA-specific CD8+ T cells in the draining lymph nodes of orthotopic KPC-OVA PDAC iCOS-T mice with intact or depleted ILC2s. Data were collected 14 days (Figure 2a, c, d), 28 days (Figure 2b), 42 days (Figure 2e), and 8 days (Figure 2f, g) after transplantation. Horizontal bars indicate median values, and error bars indicate s.e.m. Data were pooled from two or more independent studies with n ≥ 4 / group. n and data points represent individual mice analyzed separately. P values ​​were determined by two-sided Mann-Whitney test (Fig. 2a-g), two-sided log-rank test (Fig. 2a, b, survival curves), two-way ANOVA with Sidak's multiple comparison test (Fig. 2a, b, tumor volume) and chi-square test (Fig. 2e, f, % rejection). [Figure 3]Figures 3a-h: ILC2s stimulate tissue-specific cancer immunity by recruiting intratumoral dendritic cells. (Figure 3a) Tumor weight, volume, and survival rate of orthotopic and subcutaneous PDAC mice treated with vehicle or recombinant IL33 (rIL33). (Figure 3b) Tumor weight and volume of orthotopic and subcutaneous PDAC mice treated with vehicle or recombinant IL18 (rIL18). (Figure 3c) Gating, frequency, and number of ILC2s in orthotopic PDAC mice treated with rIL33 (DLN vehicle, n=13; tumor vehicle, n=12). (Figure 3d) Gating and frequency of CD103+ dendritic cells (DCs) in tumors of orthotopic PDAC mice treated with rIL33. (Figure 3e) Tumor weight, volume, and (Figure 3f) frequency of CD103+ DCs in tumors of wild-type (WT) and ILC2-deficient orthotopic PDAC mice treated with rIL33. (Figure 3g) Tumor volume in WT and CD103+ DC-deficient Batf3- / - orthotopic PDAC mice treated with rIL33. (Figure 3h) Migration of purified DCs to Ccl5. Data were collected at weeks 5 (Figure 3c, d) and 7 (Figure 3e, f) after tumor implantation. Data were pooled from two or more independent experiments, with n ≥ 3 / group. n and data points represent individual mice analyzed separately or (Figure 3h) individual replicates. P values ​​were determined by two-tailed log-rank test (Figure 3a, survival curves), two-way ANOVA (Figure 3a, b, e, g, tumor volumes), and two-tailed Mann-Whitney test (Figure 3a-f, h).

[0025] [Figure 4]Figures 4a-i: PD-1 blockade activates TILC2s. (Figure 4a) Tumor volume and survival, (Figure 4b) gating, frequency, and count, and (Figure 4c) scRNA-seq (n=7,022 ILC2 single cells) in treated PDAC mice, as nonlinear representations of the top 15 principal components. Cells are color-coded by cluster (left) or treatment and tissue (right). (Figures 4a, b) n=n for left and right graphs, respectively. (Figure 4d) Tumor volume in wild-type (WT) or ILC2-deficient PDAC mice treated with αPD-1 + rIL33. (Figure 4e) TILC2s were sorted and purified from WT or Pdcd1- / - PDAC mice treated with rIL33 and transplanted into ILC2-deficient PDAC recipients, and tumor volume was measured. (Figures 4f-h) TILC2s were purified from rIL33-treated PDAC CD45.1 donor mice and transplanted into ILC2-deficient CD45.2 PDAC recipient mice, which were then treated with αPD-1 after cell transplantation. Tumor volume and weight in recipient mice 10 weeks after cell transplantation (Figure 4f), frequency of CD45.1 and CD45.2 cells (Figure 4g), and frequency of T cells (Figure 4) are shown (TILC2s-: all groups, n = 8; TILC2+: spleen, n = 9; DLN, n = 7; tumor, n = 7). Frequency in Figure 4g = percentage of immune cells derived from living donors or recipients. (Figure 4i) Tumor volume (vehicle, n = 13; other groups, n = 10) and survival rate (vehicle and αPD-1, n = 15; rIL33, n = 24; rIL33 + PD-1, n = 26) of treated PDAC mice (KPC 52 cells). DLN, draining lymph node. Data were collected 5 weeks (Figure 4b), 10 days (Figure 4c), and 6 weeks (Figure 4d) after orthotopic tumor cell implantation. Horizontal bars indicate median values, and error bars indicate s.e.m. Data were pooled from two or more independent experiments with n ≥ 3 per group. n and data points indicate individual mice analyzed separately. scRNA-seq data represent purified single cells pooled from biological replicates (vehicle n = 10, rIL33 n = 5, αPD-1 + rIL33 n = 5).P values ​​were determined by two-way ANOVA with Tukey's multiple comparison post hoc (Fig. 4a, df, i, tumor volume), two-tailed Mann-Whitney (Fig. 4b, d, g, h), and two-tailed log-rank (Fig. 4a, i, survival curve) tests. [Figure 5]Figures 5a–h: Identification of IL33-dependent ILCs in pancreatic cancer. (Figure 5a) Gating strategy for identifying human ILCs. The first plot was pre-gated on live cells (DRAQ7-) and singlets. Lineage (Lin) 1 cocktail: CD5, CD11b, CD11c, CD16, FcεR1. Lin 2 cocktail: CD3, CD19, TCRα / β. ILCs were identified as Lin- CD56- CD25+ CD127+ cells. FMO, fluorescence minus one control. (Figure 5b) Representative images of immunofluorescence of ILC2s in tumor tissue microarrays from short- and long-term PDAC survivors (n=96). Arrows, putative ILC2s. (Figure 5c) Top row: Overall survival rates for patients with intratumoral mRNA levels of ILC-stimulating cytokines greater than (high) or less than (low) the median. Lower panel: Correlation between ILC-activating cytokine expression and immune cytolytic index (CYT) in long-term and short-term human PDAC survivors. Curves were fitted by linear regression. (Figure 5d) Gating strategy for identifying mouse ILCs. The first plot was pre-gated on live cells (DRAQ7-) and singlets. Lineage (Lin) 1 cocktail: CD5, CD11b, CD11c, FcεR1. Lin 2 cocktail: CD3, CD19. ILCs were identified as Lin- NK1.1- CD25+ CD127+ cells, and ILC2s as Lin- NK1.1- CD25+ St2+ cells. Gating in orthotopic PDAC mice is shown. (Figure 5e) Intratumoral ILC frequency is shown in orthotopic PDAC mice established with KPC cell lines 8-1 and 18-3, and in autologous KPC mice (KPCSpont) with spontaneous PDAC. For comparison, the combined ILC frequency (KPC 4662) is included as in Figure 1d. (Figure 5f) ILC phenotype in PDAC mice. Gray curve, isotype control; numbers, mean fluorescence intensity. (Figure 5g) ILC proliferation rate in PDAC mouse tissues. (Figure 5h) Changes in non-ILC cell frequency in Rag2- / - PDAC mice treated with αCD90.2 or isotype antibodies. Data were analyzed at 14 days (Figure 5d-f), 10 days (Figure 5h), or the indicated time points after tumor implantation.n indicates individual mice analyzed separately in at least two independent experiments, with n ≥ 2 / group. P values ​​were determined by two-tailed log-rank (Figure 5c, upper), linear regression (Figure 5c, lower), or two-tailed Mann-Whitney test (Figure 5g). P values ​​in Figure 5g indicate comparison of tumors to all other organs.

[0026] [Figure 6]Figures 6a-o: Host-derived IL33 activates pancreatic ILC2s. (Figure 6a) mRNA expression of ILC1 (IL12, IL15, IL18), ILC2 (IL25, IL33, TSLP), ILC3-inducing cytokines (IL23), and IL33 receptor (ST2) was calculated from previously reported mRNA microarrays in orthotopic PDAC tumors (left) and autologous PDAC tumors from KPC mice (right). (Figure 6b) Representative IL33 immunohistochemistry (IHC) results for IL33Low and IL33High human (tissue microarray, n = 96) and mouse PDAC (n = 3 / group). (Figure 6c) Frequency of human PDAC patients showing IL33 positivity by IHC on human PDAC tumor microarrays. (Figure 6d) Multiplex immunofluorescence of IL33, tubular adenocarcinoma marker CK19, and myeloid cell carcinoma markers CD11b and Iba in mouse PDAC (top row). Arrows indicate IL33-expressing cells. IL33 mean fluorescence intensity (MFI) of non-immune (CD45-), immune (CD45+), macrophage (TAM), and monocytic and granulocytic myeloid-derived suppressor cell (M-MDSC and G-MDSC) populations in tumors of IL33Cit reporter PDAC mice (bottom row). (Figure 6e) Representative IL33 protein expression by IHC in orthotopic PDAC tumors of Il33+ / + (WT) mice and non-tumor-bearing pancreas of Il33- / - mice (n = 3 / group). (Figure 6f) ILC frequency (top row) and cell number (bottom row) in organs and draining lymph nodes (DLN) of Il33+ / + and Il33- / - orthotopic PDAC mice. (Figure 6g) Gating and frequency of IL4 and IL5 expression in intratumoral ILCs in Il33+ / + and Il33- / - orthotopic PDAC mice. (Figure 6h) ILC2 and immune cell frequencies in orthotopic Rag2- / - and Rag2- / -γc- / - PDAC mice treated with or without recombinant IL33 (rIL33) (Figure 6i). (Figure 6j) Frequency of ST2+ tumor ILCs in mice with subcutaneous (SQ) and orthotopic PDAC. (Figure 6k) Tumors in orthotopic and subcutaneous PDAC mice. (Figure 6l) Tumor weights in Il33+ / + and Il33- / - littermate PDAC mice. (Figure 6m) Study scheme of bone marrow chimeras to evaluate the contribution of hematopoietic cell-derived IL33 to tumor control.(Figure 6n) Hematopoietic cell reconstitution and tumor weight (Figure 6o) in irradiated CD45.1 congenic mice reconstituted with either CD45.2 Il33+ / + or CD45.2 Il33- / - bone marrow. Data were collected on days 14 (Figure 6a, d, f, g, j, o) and 10 (Figure 6h, i) after tumor implantation. n indicates individual mice analyzed separately in at least two independent experiments; n ≥ 2 / group. P values ​​were determined by one-way ANOVA (Figure 6a) or two-tailed Mann-Whitney test (Figure 6d, fh, j, l, o). [Figure 7] Figures 7a-e: Host-derived IL33 activates pancreatic T cell immunity. (Figure 7a) Gene set enrichment analysis of bulk RNA-seq from CD45+ immune cells purified from Il33+ / + and Il33- / - PDAC mice. Enrichment plots and enrichment scores for three gene sets comparing expression in Il33- / - versus Il33+ / + (n=3 mice / group). FDR, false discovery rate. (Figure 7b) Gating of CD8+ T cells and (Figure 7c) the frequency of various immune cell types (left) and CD4+ T cell lineages (right) in Il33+ / + and Il33- / - orthotopic PDAC mice. (Figure 7d) Frequency of T cell central memory (Tcm) cells (CD45+CD3+CD8+CD44+CD62L+) in tumor-draining lymph nodes and non-tumor-draining distant lymphoid organs (inguinal lymph nodes and spleen) in Il33+ / + and Il33- / - orthotopic PDAC mice. (Figure 7e) Frequency of CD8+ T cells in subcutaneous PDAC tumors. DC, dendritic cell; MDSC, myeloid-derived suppressor cell; NK, natural killer cell; NKT, natural killer T cell; Treg, regulatory T cell. Data were analyzed at 14 days after tumor implantation or at the indicated time points. Horizontal bars represent median values, and error bars represent s.e.m. n indicates individual mice analyzed separately in at least two independent experiments; n≥2 / group. P values ​​were determined by one-way ANOVA (Figure 7d). [Figure 8]Figures 8a-g: IL33 and ILCs do not directly induce tumor cell death. (Figure 8a) Tumor weight in Rag2- / - and Rag2- / -γc- / - PDAC mice treated with vehicle or recombinant murine IL33 (rIL33). (Figure 8b) Histological tumor cell differentiation status (right) and representative hematoxylin and eosin-stained sections (left) in Il33+ / + and Il33- / - PDAC mice. (Figure 8c) Trichrome staining in tumors from Il33+ / + and Il33- / - PDAC mice (n=3 / group). (Figure 8d) Immunohistochemistry for smooth muscle actin in tumors from Il33+ / + and Il33- / - PDAC mice (n=3 / group). (Figure 8e) Intratumoral ST2 expression on KPC cells in Il33+ / + and Il33- / - orthotopic PDAC mice. (Figure 8f) ST2 expression on live KPC cells (DRAQ7 stains dead cells) after in vitro rIL33 treatment (n = 3 / group). (Figure 8g) KPC cell number, viability, proliferation (Ki-67), and apoptosis (annexin) after in vitro rIL33 treatment (n = 6 / group). n in Figures 8a-e indicates individual mice analyzed separately in at least two independent experiments, n ≥ 3 / group. n in Figures 8f,g indicates technical replicates and is representative of at least two independent experiments. P values ​​were determined by two-tailed Mann-Whitney test (Figure 8a).

[0027] [Figure 9]Figures 9a-d: ILC2s induce the priming of antigen-specific CD8+ T cells. (Figure 9a) Gating and frequency of intratumoral ILC2s in ILC2-inactive mice (diphtheria toxin [DT]-treated Icos+ / +; CD4Cre / +) and ILC2-deficient mice (DT-treated Icosfl.DTR / +; CD4Cre / +). (Figure 9b) Gating and frequency of OVA-specific CD8+ T cells in the spleens of ILC-intact and ILC-deficient mice. OVA-specific T cells were detected as SIINFEKL (SEQ ID NO: 15)-tetramer+ cells. (Figure 9c) Gating and frequency of central memory CD8+ T (TCM) cells (CD45+CD3+CD8+CD44+CD62L+) in the tumor-draining lymph nodes and spleens of ILC-intact and ILC-deficient mice. (Figure 9d) ST2 expression in CD45+CD3+CD8+ T cells after tumor implantation in PDAC mice. Data were collected 14 days after tumor implantation or at the indicated time points. DLN, draining lymph node; MFI, mean fluorescence intensity. Horizontal bars indicate median values, and error bars indicate s.e.m. n indicates individual mice analyzed separately in at least two independent experiments, with n≥2 / group. P values ​​were determined by two-tailed Mann-Whitney test (Figure 9a-c) and two-way ANOVA with Tukey's multiple comparison post-test (Figure 9d, showing comparison of tumor ILCs with all other groups). [Figure 10]Figures 10a-i: Immunophenotyping in rIL33-treated PDAC mice. (Figure 10a) Tumor establishment rates of orthotopic and subcutaneous KPC-OVA PDAC tumors in vehicle (veh) and rIL33-treated mice. (Figure 10b) Gating (left) and frequency (right) of IL18R1 expression on tumor ILCs in subcutaneous (SQ) and orthotopic PDAC mice. (Figure 10c) Gating (left) and frequency (right) of splenic ILC2s after rIL33 treatment in orthotopic PDAC mice. (Figure 10d) Gating (left) and frequency (right) of tumor ILC2s after rIL33 treatment in subcutaneous PDAC mice. (Figure 10e) Gating (left) and frequency (right) of cytokine and PD-1 expression on tumor CD8+ T cells after rIL33 treatment in orthotopic PDAC mice. (Fig. 10f) Immune cell frequency in orthotopic PDAC mice treated with vehicle and rIL33. (Fig. 10g) Gating strategy for identifying CD103+ dendritic cells. (Fig. 10h) Gating (left; tumor) and frequency (right) of ILC2s in tumors and draining lymph nodes (DLNs) of wild-type (WT) or Rorafl / fl IL7rCre (ILC2-deficient) PDAC mice after rIL33 treatment. (Fig. 10i) Gating (left) and frequency (right) of PD-1+ CD8+ T cells in tumors of rIL33-treated WT and Batf3- / - mice. Data were collected 6 weeks (Fig. 10a), 5 weeks (Fig. 10b), and 3 weeks (Fig. 10i) after tumor implantation. n indicates individual mice analyzed separately in at least two independent experiments, n≧2 / group. P values ​​were determined by two-tailed Mann-Whitney test (Fig. 10a, f, i). [Figure 11]Figures 11a-c: Single-cell RNA sequencing of tumor and draining lymph node ILC2s from PDAC mice. (Figure 11a) Study design for in vivo processing, purification, and single-cell analysis of ILC2s. (Figures 11b, c) Quality assessment metrics. (Figure 11b) Scatter plot showing the relationship between the number of unique molecular identifiers (# of UMIs) and the number of genes (# of genes) for each cell. (Figure 11c) Violin plot showing the distribution of gene counts (left), UMI counts (center), and the percentage of normalized reads from mitochondrial genes (right) in each treatment group (columns) and tissue (rows). Each dot represents a single cell. For each treatment group and organ, data represent purified single cells collected from biological replicates of n = 10 (vehicle), n = 5 (rIL33), and n = 5 (αPD-1 + rIL33) PDAC mice. [Figure 12] Figures 12a-f: Activated ILC2s from tumors and draining lymph nodes have distinct transcriptional signatures. (Figure 12a) Single-cell analysis of 1,634 rIL33-activated tumor and draining lymph node (DLN) ILC2s (study design outlined in Figure 11a). UMAP plots show single cells (points) in a nonlinear representation of the top 15 principal components. (Figure 12a) Expression of transcription factors (TFs) in ILC2s (Gata3, Id2, Rora) and ILC3s (gene, Rorc; protein, Rorγt), (Figure 12b) expression of ILC2 surface markers, and (Figure 12c) cluster and organ expression. Expression of the ILC-1 TF Tbx21 (T-bet) was undetectable. (Figure 12d, e) Differentially expressed genes by cluster and (Figure 12e) organ (TILC2 and DLN ILC2). (Figure 12f) Distribution of Ccl5 expression from tumor and DLN ILC2s. Violin plots show distributions with circles indicating minimum, maximum, and median values. Each point in Figures 12a and 12b represents a single cell. For each treatment group and organ, data represent purified single cells pooled from n = 5 biological replicates of rIL33-treated PDAC mice. P values ​​are from a two-tailed pairwise Wilcoxon signed-rank test.

[0028] [Figure 13]Figure 13a-i: Combinatorial treatment of αPD-1 and rIL33 induces a unique transcriptional profile in tumor ILC2s. (Figure 13a) Expression of co-inhibitory immune checkpoints in tumor ILC2s from vehicle-treated PDAC mice by single-cell RNA sequencing (scRNA-seq). (Figure 13b) Gating and frequency of PD-1+ ILC2s in vehicle- and rIL33-treated PDAC mice. DLN, draining lymph node. (Figure 13c) ILC2 frequency in treated PDAC mice. Corresponding tumor volume, weight, cell number, and scRNA-seq are shown in Figures 4a-c. (Figure 13d) scRNA-seq of ILC2s from treated PDAC mice. Expression of ILC1 (gene, Tbx21; protein, Tbet), ILC2 (Gata3, Id2, Rora), and ILC3 (gene, Rorc; protein, Rorγt) transcription factors (TFs) in purified tumor and draining lymph node (DLN) ILC2s. Corresponding UMAP plots by cluster and treatment are shown in Figure 4c. The top differentially expressed genes by treatment and tissue (Figure 13e), clusters (Figure 13f), and expression distribution of selected differentially expressed genes by treatment and tissue (Figure 13g) (tumor: vehicle n=28, rIL33 n=752, rIL33+PD-1 n=2,635; DLN rIL33 n=882, rIL33+PD-1 n=2,725). (Figure 13h) UMAP plot of 3,387 single-tumor ILC2s in a nonlinear representation of the top 15 principal components. (Fig. 13i) Differentially expressed genes by treatment in tumor ILC2s. Each point in Fig. 13d and Fig. 13h represents a single cell. For each treatment group and organ, data represent purified single cells pooled from biological replicates of n=10 (vehicle), n=5 (rIL33), and n=5 (αPD-1+rIL33) PDAC mice. Violin plots show distribution with circles indicating minimum, maximum, and median values. Horizontal bars in Fig. 13b and Fig. 13c indicate median values. P values ​​were determined by two-tailed Mann-Whitney test (Fig. 13b, c) and two-tailed Wilcoxon rank-sum test (Fig. 13g). [Figure 14]Figure 14a-g: Activated tumor ILC2s express PD-1 and colocalize with PD-1+ T cells. Orthotopic PDAC mice (C57Bl / 6 WT, Pdcd1- / -, CD45.1) were administered 500 ng of carrier-free recombinant murine IL33 daily for 10 days (study design shown in Figure 4e, f). Live CD45+, Lineage-, CD90+, CD25+, ST2+ tumor ILC2s (TILC2s) were sorted and purified to 98% purity on day 10 post-implantation. 5x105 TILC2s were immediately transferred intraperitoneally (ip) into IL7rCre / +Rorafl / fl (ILC2-deficient) CD45.2 mice bearing orthotopic PDAC tumors on days 7 and 14 post-tumor implantation. Control mice received an equivalent volume of PBS via ip injection. (Figure 14a) Representative plot of TILC2 sorting (top) and post-sorting purity (bottom). (Figure 14b) Representative plot showing PD-1 expression on sorted TILC2 from WT and CD45.1 mice in the study design outlined in Figures 4e and 4f. (Figure 14c) Survival and intratumoral CD8+ T cell frequency in orthotopic KPC 4662-GFP and KPC 52 PDAC tumors; horizontal bars in c represent median values. (Figure 14d) Frequency of PD-1+ ILC2 (left) and correlation with PD-1+ T cells in human PDAC (right). (Figure 14e) Linear regression analysis of IL33 and PD-1 mRNA in the bulk tumor transcriptome of short- and long-term PDAC survivors (left) and survival association of PD-1+ cells in tumor tissue microarrays of short- and long-term PDAC survivors (right). High and low are defined as higher or lower than the cohort median. (Fig. 14f) A model linking the IL33-TILC2 axis to T cell immunity in pancreatic cancer. (Fig. 14g) Expression distribution of costimulatory molecules in untreated tumor ILC2s by single-cell RNA sequencing. Study design shown in Fig. 11a; data represent purified single cells pooled from biological replicates of n=10 (vehicle). Data are representative of purity and PD-1 expression in sorted TILC2s in two independent studies with n≧4 / group (Fig. 14a, b). n and data points indicate individual mice and patients analyzed separately.P values ​​were determined by two-sided Mann-Whitney (Fig. 14c) and two-sided log rank (Fig. 14c, e, survival curves) tests, and linear regression (Fig. 14d, e). DETAILED DESCRIPTION OF THE INVENTION

[0029] Detailed Description While some of the key aspects of the present invention have been described above in the Summary of the Invention and Examples and in the Claims of this patent application, this Detailed Description section provides certain additional details regarding the compositions and methods of the present invention and is intended to be read in conjunction with all other sections of this patent application.

[0030] Definitions and Abbreviations As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The term "a" (or "an") and the terms "one or more" and "at least one" can be used interchangeably.

[0031] Furthermore, "and / or" is to be taken as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" used in phrases such as "A and / or B" is intended to include A and B, A or B, A alone and B alone. Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to include A, B and C; A, B or C; A or B; A or C; B or C; A and B; A and C; B and C; A alone; B alone; and C alone, etc.

[0032] Units, prefixes, and symbols are written in the form accepted by the International System of Units (SI). Numeric ranges are inclusive of the numbers defining the range.

[0033] When a numerical value is preceded by "about" or "approximately," it includes that numerical value and values ​​up to and including 10% of that numerical value.

[0034] Numbers in parentheses or superscripts herein refer to numbered references listed in the "Reference List" at the end of this specification.

[0035] Where an embodiment is described with the phrase "comprising," similar embodiments that are otherwise described with the terms "consisting of" and / or "consisting essentially of" are included.

[0036] As used herein, the abbreviation "IL33" means interleukin 33. As used herein, the abbreviation "rIL33" means recombinant interleukin 33. As used herein, the abbreviation "PDAC" means pancreatic ductal adenocarcinoma. As used herein, the abbreviation "ILC2" refers to group 2 innate lymphoid cells. As used herein, the abbreviation "TILC2" refers to tumor ILC2. It should be noted that all embodiments described herein that refer to ILC2 are also intended to encompass TILC2, and for all methods described herein as involving ILC2, ILC2 alternatives that are specifically directed against TILCs are also contemplated by the present invention.

[0037] As used herein, the abbreviation "PDX" means patient-derived xenograft.

[0038] As used herein, the abbreviation "PD-1" refers to programmed death 1, also known as programmed death protein 1 or programmed cell death protein 1.

[0039] As used herein, the abbreviation PD-L1 stands for programmed cell death ligand 1, which is the ligand for PD-1.

[0040] As used herein, the abbreviation "IP" or "ip" refers to intraperitoneal administration. Drugs are typically administered to mice via the IP route, which is thought to be analogous to the IV route used by humans.

[0041] As used herein, the abbreviation "IT" refers to intratumoral administration, e.g., a drug injected directly into a tumor is delivered intratumorally.

[0042] As used herein, the abbreviation "IV" means intravenous administration.

[0043] As used herein, the terms "inhibiting" and "blocking" are used interchangeably with the terms "inhibit" or "block" and the terms "inhibitor" or "blocker." The terms "inhibiting" and "blocking" refer to any detectable and statistically significant decrease in a given biological activity.

[0044] As used herein, the term "allogeneic" means deriving from, originating in, or being members of the same species, where the members are genetically related or unrelated but genetically similar. "Allogeneic transplantation" or "allogeneic cell therapy" refers to the administration of cells obtained from a donor (or cells derived from cells obtained from a donor) to a recipient, where the recipient is of the same species as the donor. In embodiments of the invention involving the administration of allogeneic pancreatic ILC2 cells to a subject, the allogeneic cells are obtained from a donor of the same species as the subject to whom the cells are administered (i.e., the recipient). In some embodiments, the allogeneic cells are obtained from a donor with the same MHC / HLA type as the subject to whom the cells are administered (i.e., the recipient), i.e., the donor of the cells and the recipient of the cells are MHC- or HLA-matched. In some embodiments, cells (e.g., pancreatic ILC2 cells) are (a) obtained from a donor, (b) maintained and / or cultured and / or expanded and / or activated (e.g., with IL33) ex vivo / in vitro, and (c) then administered to a subject of the same species as the donor. For example, in some embodiments, pancreatic ILC2 cells are obtained from a donor, activated ex vivo / in vitro with IL33, and then administered to a recipient subject of the same species as the donor. Such methods can be referred to as allogeneic transplants or transplantation or cell therapy, and the donor or cells obtained from the donor can be referred to as allogeneic with respect to the recipient. Similarly, in some embodiments, ILC2 cells are obtained from a donor, activated ex vivo / in vitro with IL-33, and then administered to a recipient subject of the same species and MHC / HLA type as the donor.

[0045] As used herein, the term "autologous" means derived from or originating from the same subject (i.e., the same individual). "Autologous transplantation" or "autologous cell therapy" refers to the administration of cells obtained from a donor (or cells derived from cells obtained from a donor) to a recipient, wherein the donor and recipient are the same individual. In certain embodiments, the methods of the present invention comprise administering autologous pancreatic ILC2s to a subject, wherein the pancreatic ILC2s are obtained from the same individual / subject to whom the pancreatic ILC2s are administered (i.e., the donor and recipient are the same person). In certain embodiments, the pancreatic ILC2s are (a) obtained from a subject, (b) maintained and / or cultured and / or expanded and / or activated ex vivo / in vitro, and (c) then administered to the same subject. For example, in some such embodiments, the pancreatic ILC2s are obtained from a subject, activated with IL33 ex vivo / in vitro, and then administered to the same subject. Such methods may be referred to as autologous transplants or transplants or cell therapies, and the donor or cells obtained from the donor may be referred to as autologous with respect to the recipient.

[0046] As used herein, the term "substantially pure," when used in reference to a cell population, refers to a population of cells in which at least about 50%, preferably at least about 75-80%, more preferably at least about 85-90%, and most preferably at least about 95% of the cells comprising the total cell population have a particular cell marker characteristic / profile. Thus, a "substantially pure" cell population contains less than about 50%, preferably less than about 20-25%, more preferably less than about 10-15%, and most preferably less than about 5% of cells that do not exhibit the given marker characteristic / profile.

[0047] As used herein, the term "sorting" with respect to cells refers to the separation of cells based on physical properties or the presence of markers (e.g., sorting using side scatter (SSC) and / or forward scatter (FSC), or fluorescence-activated cell sorting (FACS), e.g., sorting using labeled antibodies).

[0048] As used herein, the term "isolated" with respect to a cell means a specified cell type that is separated from at least one other cell type, e.g., another cell type with which the specified cell type coexists in nature, e.g., in the body.

[0049] Other abbreviations and definitions are described elsewhere herein or are well known in the art.

[0050] activator The methods and compositions provided by the present invention include a variety of different active agents, including, but not limited to, IL33 and PD-1 inhibitors.

[0051] In those embodiments of the present invention involving IL33, any suitable IL33 molecule can be used. In preferred embodiments, the IL33 molecule used is an IL33 molecule from the species to which the IL33 will be administered. For example, for administration to humans, human IL33 is used, while for administration to mice, mouse IL33 is used. For example, for administration to mice, recombinant mouse IL33 (commercially available from R&D Systems, in a carrier-containing or carrier-free form) can be used. Mouse IL33 having the amino acid sequence set forth in UniProtKB / Swiss-Prot.:Q8BVZ5.1 can be used. Similarly, for administration to humans, recombinant mouse IL33 (commercially available from R&D Systems, in a carrier-containing or carrier-free form) can be used. Human IL33 having the amino acid sequence set forth in UniProtKB / Swiss-Prot.:O95760.1 can be used. Other examples of suitable human IL33 sequences that can be used include, but are not limited to, those consisting of or comprising SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, IL33 is isolated / purified from an animal that produces it (e.g., mouse, human). In some embodiments, IL33 is recombinantly produced—i.e., it is expressed in any suitable expression system from recombinant DNA encoding IL33. In some embodiments, IL33 is synthetically produced. In some embodiments, IL33 is modified to increase or improve its half-life, stability, bioavailability, or to improve any other desired biological property. In some embodiments, IL33 comprises a half-life-increasing moiety. Any such variant known in the art can be used, so long as the modified IL33 retains the ability to bind to and activate the IL33 receptor on ILC2 cells. Examples of modifications that can be used include, but are not limited to, pegylation, conjugation to an Fc immunoglobulin domain (e.g., SEQ ID NO: 9), conjugation to an albumin-binding domain, and hexadecenoic acid modification.Modifications useful for the purification, secretion, or production of IL33 may be used, including, but not limited to, expression / purification tags (e.g., His tags such as SEQ ID NO: 3 or SEQ ID NO: 4), protease recognition sites (e.g., TEV protease recognition sites such as SEQ ID NO: 5), secretion signals (e.g., SEQ ID NO: 6), and linker sequences (e.g., SEQ ID NO: 7 or 8). Examples of modified forms of human IL33 that can be used include, but are not limited to, those consisting of or generated from SEQ ID NO: 10 (including a His tag and a TEV protease recognition site), SEQ ID NO: 11 (including a secretion signal, a His tag, and a linker), or SEQ ID NO: 12 (including a secretion signal, an IgG4-Fc sequence, and a linker). For all examples herein referring to IL33, the present invention contemplates and encompasses embodiments in which the IL33 consists of or comprises any one of SEQ ID NOs: 1, 2, 10, 11, or 12.

[0052] [Table 1-1] [Table 1-2] [Table 1-3]

[0053] In those embodiments of the invention involving a PD-1 inhibitor, any suitable PD-1 inhibitor known in the art can be used. In some embodiments, the PD-1 inhibitor is an antibody. In some embodiments, the PD-1 inhibitor is selected from the group consisting of pembrolizumab (Keytruda, Merck), nivolumab (Opdivo, Bristol-Myers Squibb), cemiplimab (Libtayo, Regeneron), AMP-224 (GlaxoSmithKline), AMP-514 (GlaxoSmithKline), and PDR001 (Novartis).

[0054] In those embodiments of the invention involving a PD-L1 inhibitor, any suitable PD-L1 inhibitor known in the art can be used. In some embodiments, the PD-L1 inhibitor is an antibody. In some embodiments, the PD-L1 inhibitor is selected from the group consisting of atezolizumab (Tecentriq, Roche Genentech), avelumab (Bavencio, Merck Serono and Pfizer), durvalumab (Imfinzi, AstraZeneca), BMS-936559 (Bristol-Myers Squibb), and CK-301 (Checkpoint Therapeutics).

[0055] Many of the embodiments of the invention described above and / or elsewhere herein involve the use of a PD-1 and / or PD-L1 inhibitor. It should be noted that in all cases, the invention also includes the same embodiments with the variation that only a PD-1 inhibitor is used.

[0056] In certain embodiments, the methods of the present invention can be practiced using equivalent analogs, homologs, variants, or derivatives of any of the specific active agents described herein. Such analogs, homologs, variants, or derivatives should retain the primary functional properties of the specific molecules described herein. For example, in the case of PD-1 and / or PD-L1 inhibitors, any suitable analogs, homologs, variants, or derivatives of such agents can be used, provided that they retain PD-1 and / or PD-L1 inhibitory activity. In the case of IL33, any suitable analogs, homologs, variants, or derivatives of such agents can be used, provided that they retain the ability to bind to and activate the IL33 receptor on ILC2 cells.

[0057] In certain embodiments, the present invention provides compositions comprising at least one active agent described herein and one or more other components useful in formulating the composition for delivery to a subject, such as a diluent, buffer, carrier, stabilizer, dispersant, suspending agent, thickener, excipient, preservative, etc.

[0058] Treatment method The present invention provides various methods of treatment. For example, in certain embodiments, the present invention provides a method of treatment comprising administering to a subject in need thereof an effective amount of one or more active agents described herein (e.g., an IL33 and / or PD-1 and / or PD-L1 inhibitor). The present invention also provides various methods of activating ILC2 cells in pancreatic tumors (e.g., PDAC tumors). These methods also generally comprise administering to a subject in need thereof an effective amount of one or more active agents described herein (e.g., an IL33 and / or PD-1 and / or PD-L1 inhibitor). In certain embodiments, the present invention also provides various cell therapies. These cell therapies comprise administering to a recipient subject with PDAC an effective amount of activated donor pancreatic ILC2 cells obtained from a donor subject and activated ex vivo / in vitro by contact with IL33.

[0059] As used herein, the terms "treat," "treating," and "treatment" encompass achieving and / or carrying out a method of achieving a detectable improvement in one or more clinical indicators or symptoms associated with pancreatic cancer (e.g., PDAC). For example, such terms include, but are not limited to, reducing the growth rate of a pancreatic tumor (or pancreatic tumor cells), halting the growth of a pancreatic tumor (or pancreatic tumor cells), causing regression of a pancreatic tumor (or pancreatic tumor cells), reducing the size of a pancreatic tumor (e.g., measured in terms of tumor volume or tumor mass), downgrading a pancreatic tumor, removing a pancreatic tumor (or pancreatic tumor cells), preventing, delaying, or slowing the recurrence (rebound) of a pancreatic tumor, improving symptoms associated with a pancreatic tumor, improving survival from a pancreatic tumor, inhibiting or reducing the spread (e.g., metastasis) of a pancreatic tumor, and the like. In each embodiment described herein that refers to a method of treatment, methods of achieving any one or more of the specific parameters listed above are also contemplated. For example, for each embodiment described herein that refers to a method of treating pancreatic cancer (e.g., PDAC), the following methods are also contemplated, intended, and within the scope of the present invention: (a) a method of reducing the growth rate of a pancreatic tumor (or pancreatic tumor cells), (b) a method of stopping the growth of a pancreatic tumor (or pancreatic tumor cells), (c) a method of causing regression of a pancreatic tumor (or pancreatic tumor cells), (d) a method of reducing the size (e.g., as measured by tumor volume or tumor mass) of a pancreatic tumor, (e) a method of downgrading a pancreatic tumor, (f) a method of removing a pancreatic tumor (or pancreatic tumor cells), (g) a method of preventing, delaying, or slowing the recurrence (rebound) of a pancreatic tumor, (h) a method of ameliorating symptoms associated with a pancreatic tumor, (i) a method of improving survival from a pancreatic tumor, and (j) a method of inhibiting or reducing the spread (e.g., metastasis) of a pancreatic tumor.

[0060] As used herein, the term "subject" includes all mammals, including, but not limited to, humans, non-human primates, dogs, cats, rodents (e.g., rats, mice, guinea pigs), cows, pigs, sheep, goats, horses, etc. - all mammalian species used in animal husbandry, as well as animals kept as pets, in zoos, etc. In some embodiments, the subject is a human.

[0061] In some embodiments, the methods and compositions of the present invention can be used to treat any pancreatic tumor in a subject in need thereof (i.e., a subject with pancreatic cancer). In preferred embodiments, the methods and compositions are used for pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof (i.e., a subject with PDAC). In some embodiments, the methods and compositions are used to treat PD-1 / PD-L1 inhibitor-resistant PDAC in a subject in need thereof (i.e., a subject with PD-1 and / or PD-L1 inhibitor-resistant PDAC).

[0062] In some embodiments, the subject has a tumor that is resistant to treatment with other methods and / or compositions. As used herein, the terms "resistance" and "resistance" are used consistent with their normal usage in the art and consistent with the understanding of these terms by physicians (e.g., oncologists) who treat cancer. For example, consistent with normal meaning in the art, a tumor or subject may be considered "resistant" to a treatment method or treatment with a drug (or drug combination) if the subject's tumor (or tumor cells) grow and / or progress and / or metastasize and / or recur despite the use of the method or administration of the drug (or drug combination). In some instances, a tumor may initially be sensitive to treatment with a particular method or drug (or drug combination), but later become resistant to such treatment.

[0063] In some embodiments, the subject has a pancreatic tumor (e.g., PDAC) that has recurred after prior treatment with other compositions or methods, including, but not limited to, chemotherapy, radiation therapy, or surgical resection, or any combination thereof. In some embodiments, the subject has a pancreatic tumor that has not been previously treated.

[0064] As used herein, the term "effective amount" refers to an amount of an active agent or cells described herein sufficient to achieve or contribute toward achieving one or more desired clinical results, as described above in the description of "treatment," and / or to activate ILC2 cells in pancreatic tumors (e.g., PDAC tumors). An appropriate "effective" amount in any individual case may be determined using standard techniques known in the art, such as dose escalation studies, and may take into account factors such as the desired route of administration (e.g., systemic administration vs. intratumoral administration), the desired frequency of administration, and the like. In certain embodiments, an "effective amount" may be an amount previously shown to be effective in clinical trials and / or approved for administration to human subjects. For example, in certain embodiments, an effective amount of IL33, a PD-1 inhibitor, or a PD-L1 inhibitor may be an amount previously shown to be effective in clinical trials and / or approved for administration to human subjects. Furthermore, an "effective amount" may be determined for any co-administration method used. Those skilled in the art can readily conduct such dosing studies, for example, using the assays described in the Examples of this patent, to determine appropriate dosages to use - which involve administering an agent described herein to a subject (such as an animal subject routinely used in the pharmaceutical arts to conduct dosing studies).

[0065] For example, in some embodiments, the dosage of the active agent of the present invention may be calculated based on studies in humans or other mammals conducted to determine the efficacy and / or effective amount of the active agent. The dosage may be determined by methods known in the art and may vary depending on factors such as the pharmaceutical form of the active agent, the route of administration, whether only one active agent is used or multiple active agents are used (e.g., when a first active agent is used in combination with a second active agent, a lower dosage of the first active agent may be required), and patient characteristics, including age, weight, or the presence of any medical conditions that affect drug metabolism.

[0066] In embodiments described herein that refer to a particular dose of an agent to be administered based on mouse studies, one of skill in the art can readily determine the equivalent dose for human studies based on the mouse dose, e.g., using dosing studies and calculations of the type known in the art and / or described herein.

[0067] In certain embodiments, appropriate doses of the various active agents described herein can be determined by conducting dose studies of the type standard in the art, such as dose escalation studies, starting with, for example, doses shown to be effective in mice in the Examples of this patent application.

[0068] In some embodiments, one or more active agents are used at about their maximum tolerated dose, as determined, for example, in a Phase I clinical trial and / or a dose escalation study. In some embodiments, one or more active agents are used at about 90% of their maximum tolerated dose. In some embodiments, one or more active agents are used at about 80% of their maximum tolerated dose. In some embodiments, one or more active agents are used at about 70% of their maximum tolerated dose. In some embodiments, one or more active agents are used at about 60% of their maximum tolerated dose. In some embodiments, one or more active agents are used at about 50% of their maximum tolerated dose. In some embodiments, one or more active agents are used at about 40% of their maximum tolerated dose. In some embodiments, one or more active agents are used at about 30% of their maximum tolerated dose.

[0069] In practicing the treatment methods described herein, any suitable method or route of administration can be used to deliver the active agents and / or cells described herein, or a combination thereof. In some embodiments, systemic administration can be employed, such as oral administration or intravenous (IV) administration, or any other suitable method or route of systemic administration known in the art. In some embodiments, intratumoral (IT) administration can be employed. In some embodiments, intraperitoneal (IP) delivery can be employed. For example, the active agents described herein can be administered systemically or locally by injection, by infusion via a catheter, using an implantable drug delivery device, or by any other means known in the art. Those skilled in the art can select an appropriate delivery method or route depending on the situation, e.g., whether an active agent or cells are being administered, and, in the case of an active agent, the properties of the active agent (e.g., its stability, half-life, etc.).

[0070] In certain embodiments, the compositions and treatment methods provided herein may be used in conjunction with other compositions and treatment methods known to be useful in tumor therapy, including, but not limited to, surgical methods (e.g., for tumor resection), radiation therapy methods, treatment with chemotherapeutic agents, treatment with angiogenic agents, treatment with tyrosine kinase inhibitors, or treatment with immune checkpoint inhibitors, etc. Similarly, in certain embodiments, the treatment methods provided herein may be used in conjunction with methods used to monitor disease status / progression, such as biopsy methods and diagnostic methods (e.g., MRI or other imaging diagnostic methods).

[0071] For example, in some embodiments, the methods described herein may be performed prior to surgical resection of a tumor, e.g., to shrink the tumor prior to surgical resection, hi other embodiments, the methods described herein may be performed both before and after surgical resection of a tumor.

[0072] In some embodiments, the treatment methods described herein can be used in conjunction with performing diagnostic tests to determine whether a subject has a tumor that is likely to respond to treatment. For example, in some embodiments, before treatment is initiated, a diagnostic assay is performed to determine whether the subject has PDAC and / or pancreatic cancer (e.g., PDAC) that includes cells that express PD-1 and / or PD-L1 and, among them, express an IL33 receptor, such as ST2.

[0073] In the cell therapy-based methods described herein, protocols used for other types of cell therapy for tumors (e.g., other types of autologous cell therapy for tumors) can be easily adapted for use with ILC2 cells. For example, modified methods of TIL therapy and CAR-T cell therapy can be used. Each of these methods involves obtaining cells from a donor, manipulating those cells in some way in vitro / ex vivo, and then administering those cells to a recipient. For example, methods for obtaining lymphocytes from a donor, isolating / purifying those cells, culturing / maintaining / expanding those cells in vitro / ex vivo, and administering those cells to a recipient are well known in the art, and modifications of such methods can be used in combination with the ILC2-based cell therapies described herein. Regarding the purification of pancreatic ILC2 cells obtained from a subject, this can be performed using standard cell separation / purification methods known in the art, such as FAC-based methods. Several ILC2 markers that can be used in such cell separation / purification methods are described in the Examples herein. Other suitable markers are known in the art. [Example]

[0074] Example The present invention is further illustrated by the following non-limiting "Examples" and the figures referred to herein. Superscript numbers in the Examples refer to numbered references in the literature list herein.

[0075] Example 1 Tissue-specific activation of innate lymphoid cells enhances PD-1 checkpoint blockade immunotherapy in pancreatic cancer overview Group 2 innate lymphoid cells (ILC2s) regulate inflammation and immunity in tissues 1 ILC2s have been detected in cancers of these tissues. 2 However, their role in cancer immunity and immunotherapy remains unclear. Here, we found that ILC2s infiltrate pancreatic ductal adenocarcinoma (PDAC) and activate tissue-specific tumor immunity. Interleukin-33 (IL33) promotes tumor ILC2s (TILC2s) and CD8+ in orthotopic pancreatic tumors, but not in ectopic skin tumors. + It activates T cells and restricts the growth of pancreatic-specific tumors. Resting and activated TILC2 express the inhibitory checkpoint receptor PD-1, and TILC2 is further expanded by PD-1 blockade (αPD-1), enhancing tumor control. PD-1 blockade acts directly on TILC2 to enhance antitumor immunity and the effects of αPD-1 immunotherapy, revealing that activated TILC2 is a novel target of αPD-1. Finally, PD-1 + TILC2 and PD-1 + Both ILC2s and T cells were confirmed to be present in the majority of human PDACs. Thus, we have identified ILC2s as novel anti-cancer immune cells for PDAC immunotherapy. More broadly, ILC2s emerge as tissue-specific cancer immune enhancers that amplify the effects of αPD-1 immunotherapy. Because ILC2s and T cells coexist in human cancers, sharing activation and inhibitory pathways, cotargeting anti-cancer ILC2s and T cells may be a broadly applicable immunotherapeutic approach.

[0076] TILC2 is involved in the invasion of pancreatic cancer In unselected human primary PDAC, we analyzed immune cell lineage markers (Lin - ), but lack ILCs (CD25, CD127) 1We found intratumoral cells expressing IL-33 receptors ST2 / IL1RL1 / IL33R and GATA3 (Fig. 1a, Fig. 5a). These putative IL-33Cs were more abundant in "hot" tumors (activated CD8 + T cell-rich) 3 TILC2 was enriched in rare long-term PDAC survivors with IL33, and high TILC2 frequency correlated with longer survival (Figures 1b and 5b). Consistently, high bulk tumor RNA expression of the ILC2-activating cytokine IL33, but not other ILC-activating cytokines, was associated with longer survival (Figures 1c and 5c). Furthermore, IL33, but not other ILC-activating cytokines, correlated with higher intratumoral immune cytolytic activity (Figures 1c and 5c). Although these data assess RNA abundance rather than protein abundance, they suggest that IL33 and TILC2 activate antitumor immunity in human PDAC.

[0077] Next, we developed autochthonous "KPC" mice with Kras and p53 mutations. 4 and orthotopic PDAC mouse models (PDAC mice 5、6 In both models, the presence of ILCs was investigated in human PDAC tumors and in mouse ILC2s. 1,7 Mouse TILC2 cells proliferated in tumors but not in adjacent organs (Fig. 1d, Fig. 5g), consistent with their tissue resident nature. 8 , Rag2 - / - In mice, the lymphocyte antigen CD90.2 was targeted and reduced (Figure 1e, Figure 5h). Thus, ILC2s are conserved cells that proliferate locally in mouse and human PDAC.

[0078] Identification of signals inducing TILC2 proliferation in PDAC and KPC mice 11 In both studies, IL33 was most highly expressed in tumors compared with other ILC-induced cytokines (Figure 6a). 6, which was heterogeneously expressed in both human and mouse PDAC (Fig. 6b-c), and in intratumoral myeloid cells. 12、13 To understand the role of IL33 and TILC2 in PDAC immunity, we investigated the expression of IL33 in long-term human PDAC survivors. High , IL33 reflecting ILC2-enriched inflammatory tumors High We tested TILC2 dependency in PDAC mice. - / - PDAC mice are Il33 + / + Compared to PDAC mice, TILC2 frequency, number (Fig. 1f, Fig. 6f), and cytokine production (Fig. 6g) were reduced, indicating that TILC2 proliferation and function were IL33-dependent. Consistently, recombinant IL33 (rIL33) inhibited ILC-proficient Rag2 cells. - / - We expanded ILCs in PDAC mice, but not in ILC-deficient Rag2 - / - γc - / - These studies demonstrated that IL33 promotes the proliferation of TILC2 in PDAC mice (Fig. 6h, i).

[0079] TILC2 enhances tumor immunity in tissues ILC2s have tissue-specific phenotypes 14 Therefore, we hypothesized that the effect of TILC2 on PDAC immunity is tissue-specific. To test this, we contrasted the effect of IL33 deletion on tumor growth in the pancreas and skin (pancreatic TILC2 express ST2, whereas skin TILC2 do not; Figure 6j). 14、15 ). Il33 + / + Il33 with orthotopic PDAC compared with animals - / - Mice developed larger tumors, accelerated tumor growth, and poorer survival (Figure 6a), in contrast to subcutaneous PDAC mice, which do not display an IL33-dependent phenotype (Figure 2b, Figure 6k). These mice were fully backcrossed to the same genetic background, but lacked IL33 expression. - / - Against IL33 + / +The observation of larger tumors in littermates confirmed that these differences were not due to potential minor genetic mismatches (Figure 6l). - / - These antitumor effects were dependent on IL33 derived from host hematopoietic cells, as the tumors in chimeric mice that received bone marrow transplants were larger than those in control mice (Fig. 6m-o). + / + and Il33 - / - CD45 purified from orthotopic PDAC mice + RNA sequencing (RNA-seq) of intratumoral immune cells revealed Il33 - / - PDAC immune cells exhibited reduced transcriptional signals related to T cell activation and MHC-I antigen processing, suggesting a defect in T cell priming (Fig. 7a). Consistently, Il33 - / - Orthotopic PDAC mice express tumor-infiltrating CD8, but not subcutaneous + There was a low frequency of T cells, with no consistent changes in the frequencies of other immune cells, and central memory CD8 + T cells (TCM) were reduced, but not observed in distant lymph nodes (Fig. 2c, Fig. 7c-e). + / + IL33 compared to mice - / - The increase in tumor size in mice was abolished by pan-T cell depletion (Fig. 2d), and rIL33-treated Rag2 - / - There was no difference in tumor weight in PDAC mice (Fig. 8a), confirming that the antitumor effect of IL33 is T cell-mediated. - / - and Il33 + / + Orthotopic tumors in PDAC mice also had similar histology, collagen, and fibroblast content (Figure 8b-d), and rIL33 had no effect on tumor cells in vitro (Figure 8e-g), indicating that IL33 does not directly affect tumor or stromal cells. These data suggest that IL33 activates TILC2 to upregulate CD8 + It has been revealed that tissue-specific cancer immunity is activated by inducing T cells.

[0080] We next investigated CD8 +To determine whether the effects of IL33 on T cells are tissue-specific, we investigated CD8 + We investigated the rejection phenotype of KPC cells (KPC-OVA) expressing the T cell rejection antigen ovalbumin by comparing them in different tissue sites. + / + 70% of mice rejected orthotopic KPC-OVA tumors, whereas Il33 - / - The percentage of Il33 mice was 0%. + / + and Il33 - / - 100% of mice rejected subcutaneous KPC-OVA tumors (Fig. 2e). This phenotype is consistent with the ILC2 deficiency and ineffective CD8 + To assess whether this is due to T cell priming, ICOS + CD4 + T cells 16 Using iCOS-T mice, which allow ILC2 depletion with diphtheria toxin while sparing ILCs, we investigated the expression of antigen-specific CD8+ / CD8+ ILCs in the DLN. + Acute depletion of T cells and testing were performed (Fig. 2f, Fig. 9a). ILC2 depletion resulted in Il33 - / - Recapitulating the phenotype, orthotopic KPC-OVA tumors were associated with higher tumor rejection rates and larger tumor sizes, whereas subcutaneous tumors showed no difference (Fig. 2f). However, given the differences in rejection assessment time and depletion efficacy, Il33 - / - Tetramer analysis in ILC2-depleted orthotopic KPC-OVA mice revealed that OVA-specific CD8 + Decrease in T cells and CD8 in DLN + Decrease in TCM (IL33 - / - As seen in mice (Fig. 2g, Fig. 9B, C). Therefore, ILC2 deficiency partially reflects IL33 deficiency. + Although a direct effect on T cells cannot be ruled out, intratumoral CD8 + We found that ST2 was not expressed on T cells (Fig. 9d). Collectively, these loss-of-function studies suggest that the IL33-TILC2 axis primes tissue-specific CD8+ T cell immunity in PDAC.

[0081] Next, to investigate whether rIL33 treatment has similar tissue-specific antitumor effects, we found that rIL33 inhibited tumor formation and prolonged survival in orthotopic PDAC mice, but had no effect on subcutaneous PDAC mice, which resulted in progressive tumor growth and ulceration requiring euthanasia (Fig. 3a), and had similar tissue-specific antitumor effects in KPC-OVA PDAC mice (Fig. 10a). + skin ILC2s 14 rIL18, a cytokine that preferentially activates IL18R + Although ILCs restricted the growth of infiltrated subcutaneous PDAC, IL18R + Orthotopic PDAC cells lacking ILCs were not restricted (Fig. 3b, Fig. 10b). rIL33 selectively expanded ILC2s in the DLN and tumors of orthotopic PDAC mice (Fig. 3c), but not in the spleen or subcutaneous PDAC (Fig. 10c, d). ILC2 expansion was significantly enhanced by intratumoral CD8 + This was accompanied by enhanced T cell cytokine potency and upregulation of PD-1 (Fig. 10e), and no consistent changes were observed in other intratumoral immune cells (Fig. 10f), although we cannot rule out the possibility that ILC2s indirectly regulate the function of antitumor CD8 + Consistent with T cell priming, rIL33 treatment increased intratumoral CD103 expression. + The number of dendritic cells (DCs) doubled (Fig. 3d, Fig. 10g), and CD8 + T cell priming and recruitment to PDAC 6 To investigate whether the effects of rIL33 depend on ILC2s, we investigated the effects of rIL33 on Rora cells with PDAC. fl / fl Il7r Cre / + Mice were administered rIL33 to constitutively delete ILC2s 16 ILC2 deletion (Fig. 1h) impaired the efficacy of rIL33 (Fig. 3e) and reduced intratumoral CD103 (Fig. 3f). + The proliferation of DCs was attenuated. + DC deletion loss Batf3 - / - In mice, no antitumor effect was observed (Fig. 3g), and intratumoral CD8 +The expression of PD-1 on T cells was also not induced (Fig. 10i), suggesting that rIL33-mediated tumor control is not related to CD103. + We were able to establish that DCs are essential. To confirm whether TILC2s produce chemokines to recruit DCs to tumors, we used single-cell RNA-seq (scRNA-seq) (Fig. S11a-c). We found that activated TILC2s and DLN ILC2s retained markers of ILC2 identity but displayed distinct transcriptional profiles (Fig. S12a-e). rIL33-activated TILC2s upregulated CD103. + They selectively express Ccl5 (Fig. 12f), which encodes a chemokine that recruits DCs to tumors. 17 , and was found to induce efficient DC migration in vitro (Fig. 3h). Collectively, these data suggest that rIL33 promotes TILC2 proliferation and CD103 uptake, potentially via Ccl5 production. + Recruiting DCs to tumors and CD8 + These results suggest that T cells may be activated to induce therapeutic tumor immunity.

[0082] Activation of TILC2 by PD-1 blockade Since stimulating ILC2 with rIL33 has an antitumor effect, we sought to further enhance ILC2 activation. Recent data suggest that, like T cells, ILC2s also act as co-inhibitors. 2,18 It has been shown that the activity of ILC2 is regulated through immune checkpoint pathways. Specifically, the immune checkpoint PD-1 regulates the development of ILC2 in mice. 19 , marking effector ILCs 19 When genetically deleted or inhibited with a blocking antibody (PD-1), IL33-activated ILC2s exhibit greater proliferation and effector function in mice and humans. 20 PD-1 + ILC2s are also present in human tumors 2 However, the simultaneous activation and inhibition of ILC2s for cancer therapy remains a relatively unexplored area.

[0083] Using scRNA-seq (Fig. 11a-c), we found that PD-1 was the only detectable co-inhibitory molecule expressed by TILC2 at baseline (Fig. 13a). rIL33 treatment upregulated PD-1 in a subset of TILC2 but not in DLN ILC2 (Fig. 13b), suggesting that PD-1 may functionally suppress activated TILC2. We therefore investigated whether combining rIL33 and αPD-1 could cooperatively activate TILC2 and enhance the antitumor effect. Consistent with PD-1 expression exclusively on rIL33-activated TILC2, αPD-1 alone induced a partial response, as previously reported in PDAC6 (Fig. 4a), but did not significantly alter TILC2 frequency (Fig. 4b, Fig. 13c). Combining rIL33 and αPD-1 maximized ILC2 expansion in tumors and DLNs (Figure 4b) and enhanced tumor control compared with PD-1 alone (Figure 4a). To investigate whether αPD-1 activates ILC2s by blocking cell-intrinsic PD-1, we compared the single-cell transcriptional profiles of TILC2s and DLN ILC2s after in vivo treatment. While TILC2s retained ILC2 transcriptional and cellular identity regardless of treatment (Figure 13d), TILC2s from rIL33- and αPD-1-treated PDAC mice had a unique transcriptional phenotype compared with all other conditions (Figure 4c), revealing increased expression of the ILC2-specific marker, canonical (amphiregulin [Areg]). 14 and non-canonical (CXCL2) 21 Increased expression of effector molecules, cell activation machinery (Junb, Fosl2, Ybx1), and co-inhibitory immune checkpoints was observed (Figure 13e-i). Finally, the antitumor effect of the dual therapy was abolished in ILC2-deficient mice (Figure 4d), demonstrating that ILC2s are required for the efficacy of αPD-1 and rIL33 dual therapy. These results suggest that αPD-1 preferentially amplifies activated TIL32, possibly by inhibiting the PD-1 pathway on ILC2s rather than on T cells alone.

[0084] PD-1 TILC2 inhibition is cell-intrinsic To confirm whether blocking the intracellular PD-1 pathway in activated TILC2 contributes to the antitumor effect of dual therapy, we investigated the effects of rIL33-activated PD-1-proficient (wild-type [WT]) or PD-1-deficient (Pdcd1 - / - ) TILC2s were transplanted into tumor-bearing ILC2-deficient mice (Fig. 4e, Fig. 14a). WT TILC2 delivery did not show antitumor effects in established tumors, whereas Pdcd1 - / - TILC2 restricted tumor growth, indicating that disrupting PD-1 signaling on TILC2 could enhance tumor control (Figure 4e). + To test whether TILC2 could directly amplify the effects of PD-1 therapy in established tumors, we used a sorted and purified rIL33-activated congenic line, CD45.1 + TILC2 was identified as a CD45.2 + ILC2-deficient mice were transplanted with PD-1 after transplantation (Fig. 4f). More than 97% of the transplanted TILC2s expressed PD-1. + (Fig. 10b) and accumulated in the tumors and DLN, but not in the spleen, of αPD-1-treated recipient mice, persisting for up to 9 weeks after transplantation (Fig. 4g). + Transplantation of TILC2 cells enhanced the efficacy of αPD-1, restricted tumor growth, and increased T cell frequencies in the tumors and DLN (but not the spleen) of recipient mice (Figure 4h). Together, these data demonstrate that blocking PD-1 signaling in rIL33-activated TILC2 cells has a direct antitumor effect and amplifies PD-1 effects.

[0085] IL33 Low To investigate rIL33 and PD-1 efficacy in αPD-1-resistant tumors, we performed IL33 Low IL33 to mimic the immunological and survival characteristics of short-term human PDAC survivors Low Tumors (Figure 6b) were generated and CD8 +We selected an aggressive "non-inflammatory" PDAC model (KPC52) with 50% fewer T cells and a median survival of only 2 weeks (Fig. 14c). KPC52 PDAC mice do not display the sequential stages of PDAC tumorigenesis from preinvasive neoplasia to invasive PDAC seen in spontaneous KPC mice, but they recapitulate the αPD-1 resistance seen in spontaneous KPC mice and human PDAC (Fig. 4i). We found that the combination of rIL33 and αPD-1 reduced tumor volume by nearly 40% and improved mouse survival by nearly 50% (Fig. 4i). Finally, to assess the potential for treating PDAC patients with the combination of rIL33 and αPD-1, we investigated the PD-1 resistance observed in nearly 60% of human PDAC. + TILC2 and PD-1 + The frequency of T cells was low, and there was a significant correlation between these two cell types (Fig. 14d), suggesting that they frequently co-occur in human PDAC. Furthermore, IL33 mRNA was significantly correlated with PD-1 mRNA, which is associated with prolonged survival (Fig. 14e), suggesting that the IL33-PD-1 axis may positively influence human PDAC survival. 22 Taken together, activating ILC2s with rIL33 can amplify responses to αPD-1 in both PD-1 partially sensitive and PD-1 resistant tumors.

[0086] Consideration Our results suggest that activating ILC2s may be a broader strategy for promoting T cell priming in ILC2-infiltrating cancers (Figure 14f). However, given the tissue-specific phenotype of ILC2s, further testing is needed to determine whether activating ILC2s has similar effects across various cancer types.

[0087] Although ILC2s express immune checkpoints, it was unclear whether they could be effectively treated with immune checkpoint inhibitors. We confirmed that blocking PD-1 on activated ILC2s has an antitumor effect, suggesting that ILC2s may partially contribute to the efficacy of PD-1 pathway blockade in human cancers and more broadly highlighting that differences in checkpoint blockade responses may depend on tissue-specific factors.

[0088] The expression of several immune regulatory molecules by activated ILC2s (Figure 14g) suggests that a broader range of checkpoints may be targeted in tumor ILC2s and T cells. Because ILC2s and T cells coexist in human cancers with common costimulatory and co-inhibitory pathways, strategies that jointly target ILC2s and T cells are needed for cancer immunotherapy.

[0089] The data presented in this example were published by the present inventors in the journal "Nature" (see Moral et al., "ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity" Nature, 2020, March, Vol. 579(7797), pp. 130-135. doi: 10.1038 / s41586-020-2015-4, Epub 2020 Feb 19). The entire contents of this publication, including supplementary materials, are incorporated herein by reference.

[0090] Example 2 material and method This example describes the materials and methods used in carrying out the experiments described in Example 1. mouse C57BL / 6 (wild type, WT, CD45.2), C57BL / 6 CD45.1, Rag2 - / - , Rag2 - / - c - / - , Batf3 - / - and Pdcd1 - / -Mice were purchased from Jackson Labs. Il33 - / - , Il33 Cit / + was a gift from MJ Rosen. Cd4 Cre / + Icos fl-Dtr / + and Il7r Cre / + Rorα fl / fl was a gift from ANJ McKenzie and has been previously reported. 16,23 For all experiments, 6-12 week old mice were age- and sex-matched and randomly assigned to specific treatment groups, with at least two independent experiments performed overall. G12D / + ;LSL-Trp53 R172H / + (KPC mouse) has been reported 4 The experimental sample size was determined without a formal power calculation. Animals were housed and maintained in a specific pathogen-free animal facility, and all experiments were conducted in accordance with Memorial Sloan Kettering Cancer Center (MSKCC) Institutional Animal Care and Use Committee (IACUC)-approved protocols and in compliance with all relevant ethical regulations.

[0091] Cell lines and animal studies All tumor cell lines were derived from KPC mice. Pdx1-Cre;LSL-Kras G12D / + ;LSL-Trp53 R172H / + KPC4662 cells (gift from RH Vonderheide) were transfected with GFP and used for all experiments unless otherwise indicated. Ptf1a-Cre;LSL-Kras G12D / + ;LSL-Trp53 R172H / + Mouse-derived KPC8-1, 18-3, and 52 cells were kindly provided by C. Iacobuzio-Donahue. KPC4662 cells engineered to express OVA were previously described. 24(A gift from RH Vonderheide). All cell lines were recognized as bonafide PDAC cell lines based on histopathological verification by an expert pancreatic cancer pathologist. Orthotopic tumors established with KPC4662 cells were characterized by IL33 High The size of the orthotopic tumors established with KPC52 cells was transiently reduced by PD-1 therapy initiated at the time of transplantation (PD-1 partial sensitivity). Low The tumors were resistant to PD-1 therapy initiated at the time of transplantation and did not decrease in size (PD-1 resistance). All cell lines were routinely tested using the MycoAlert Mycoplasma Detection Kit (Lonza). Orthotopic PDAC tumors were established as previously reported. 5 Briefly, mice were anesthetized with a ketamine / xylazine cocktail, and a small (7 mm) left flank incision was made. Tumor cells (10 6 KPC cells / mouse: 1.25 × 10 5 KPC-OVA cells (10 per mouse) were suspended in Matrigel (Becton Dickinson), diluted 1:1 with cold phosphate-buffered saline (PBS) (total volume 50 μl), and injected into the tail of the pancreas with a 26-gauge needle. Successful injection was confirmed by the appearance of a fluid bubble without intraperitoneal leakage. The abdominal wall was closed with absorbable Vicryl RAPIDE sutures (Ethicon) and the skin with wound clips (Roboz). For subcutaneous PDAC tumors, tumor cells (10 6 KPC cells / mouse: 1.25 × 10 5 KPC-OVA cells (per mouse) were resuspended in sterile PBS (Fisher Scientific) and implanted subcutaneously. Mice were sacrificed at the indicated time points and processed for histology or flow cytometry. Autochthonous KPC mice were sacrificed when tumors were detectable by ultrasound. Tumor volume was measured for orthotopic tumors using continuous ultrasound (Vevo 2100 Linear Array Imaging and Vivo LAB Version 3.1.1, Fuji Film Visual Sonics) as previously reported. 25For subcutaneous tumors, the length and width of the tumor were measured every 2-3 days with a vernier caliper, and the tumor volume was calculated as follows: Volume = 1 / 2 length × width 2 For survival analysis, survival was calculated as the number of patients with a tumor volume of 500 mm 3 Tumors were euthanized when they reached a maximum IACUC-defined tumor volume of 2 cm, as determined by the health status of the mice, or required euthanasia as defined by institutional IACUC guidelines. 3 No randomization was performed in the handling of the experimental mice, as the treatment groups had to be known.

[0092] T cell depletion CD4 and CD8 cells were depleted by intraperitoneal (ip) injection of 250 μg of anti-mouse CD4 antibody (clone GK1.5, BioXcell, InVivoPlus) and 250 μg of anti-mouse CD8a antibody (clone 2.43, BioXcell, InVivoPlus). Control mice received a rat IgG2b isotype control (clone LTF-2, BioXcell, InVivoPlus). Mice were treated daily for 3 days prior to tumor implantation and then every 3 days for the duration of the experiment. CD4 + and CD8 + T cell depletion was confirmed by flow cytometry analysis of tumors and secondary lymphoid organs (>85% depletion).

[0093] ILC depletion Rag2 - / - Mouse ILCs were depleted by intraperitoneal injection of 300 μg of anti-mouse CD90.2 (clone 30-H12, BioXCell) on days 0, 1, 3, 6, 9, and 13 after tumor implantation, as previously reported. 26 CD4 Cre / + Icos fl-DTR / + Experimental mice and Cd4 Cre / + Icos + / +Control mice were treated with diphtheria toxin (Sigma Aldrich) at a dose of 25 ng / g mouse body weight via ip injection to deplete ILC2s. Mice were treated the day before tumor implantation and then every other day for a total of five doses, as previously reported. 16 ILC2 depletion was confirmed by flow cytometry analysis of tumors (Figure 9a).

[0094] Bone marrow chimera Bone marrow was collected from CD45.2 congenitally labeled donor mice, filtered through a 70 mm filter, centrifuged, resuspended in sterile PBS, and diluted with 10 per 200 μl. 8 The concentration of viable cells was 10 per recipient mouse. CD45.1 congenitally labeled C57BL / 6J recipient mice were irradiated (5.5 Gy × 2, 6-hour intervals) 24 hours before bone marrow transplantation and maintained with endofloxacin water for 4 weeks after irradiation. Single-cell suspensions of CD45.2 bone marrow chimeras (10 per recipient mouse) in sterile PBS were prepared. 8 Viable cells (1000 cells) were transplanted into each recipient mouse via retro-orbital injection. Reconstitution was confirmed by flow cytometry of peripheral blood 4 and 8 weeks after transplantation. Tumor transplantation experiments were performed 12 weeks after transplantation.

[0095] Recombinant IL33, IL18 and PD-1 blockade For rIL33, mice were injected intraperitoneally with 500 ng of carrier-free recombinant mouse IL33 (R&D Systems) in sterile PBS daily for 7 days, and then every 2 days thereafter, and treated as previously described. 15 For rIL18, mice were treated with ip injections of 2 μg of carrier-free recombinant murine IL-18 (R&D Systems) in sterile PBS on days 3, 7, 11, and 15 after tumor inoculation. 27The chimeric anti-mouse PD-1 antibody (4H2) used in this study, designed as a mouse IgG1 isotype monoclonal antibody (mAb), was shown to bind to PD-1-expressing CHO transfectants and block the binding of PD-L1 and PD-L2 to these cells. The affinity of 4H2 for mouse PD-1, as measured by surface plasmon resonance using PD-1-Fc, was 4.68 × 10 -9 M. Each batch was certified to contain <0.5 EU / mg endotoxin and >95% purity. All dosing solutions were prepared in PBS. Mice were treated with 250 μg of anti-PD1 antibody by i.p. injection every 2 days. A partial response was defined as a transient reduction in tumor size during continued αPD-1 administration, followed by subsequent regrowth. A lack of tumor reduction during continued αPD-1 administration was defined as resistance.

[0096] Human samples All tissues were collected at MSKCC after approval of the study protocol by the MSKCC Institutional Review Board. Informed consent was obtained from all patients. This study was conducted in strict compliance with all institutional ethical guidelines. All tumor samples were surgically resected primary PDAC.

[0097] Tissue microarrays (TMAs) were constructed as previously described from tumor and adjacent non-tumor cores of formalin-fixed, paraffin-embedded tissue blocks from short-term (n = 45 tumors, 5 normal tissues) and long-term (n = 51 tumors, 5 normal tissues) survivors of PDAC. 3 A subset of patients was randomly selected for tissue microarray construction. Patients who received neoadjuvant therapy were excluded. All tumors underwent pathological review and histological confirmation by two PDAC pathologists before analysis. To exclude perioperative deaths, long-term survivors were defined as patients with an overall survival of more than 3 years from surgery, and short-term survivors as patients with an overall survival of more than 3 months but less than 1 year from surgery. ILC2 High and ILC2 Lowwas defined as greater than or less than the median ILC2 frequency of the entire TMA cohort, respectively.

[0098] Tumor transcriptome profiling. A random subset of patients was randomly selected and subjected to transcriptome profiling as previously described. 3 Patients within the TMA cohort with tumor tissue available for transcriptome assessment were included in the analysis shown in Figure 1b to allow for protein confirmation of RNA expression. Extracted RNA was characterized using an Agilent BioAnalyzer and quantified by fluorometry (Ribogreen). RNA preparation for whole-transcriptome expression analysis was performed using the WT Pico Reagent Kit (Affymetrix). To capture both coding and multiple noncoding RNAs, reverse transcription was initiated at the poly(A) tail as well as the full-length RNA. RNA amplification was achieved by low-cycle PCR and linear amplification using the T7 in vitro transcription technique. cRNA was then converted to a biotinylated sense-strand DNA hybridization target. The prepared target was hybridized to the GeneChip Human Transcriptome Array 2.0 (Affymetrix). Cleaning was performed using the GeneChip Hybridization, Wash, and Stain kit on a Fluidics Station 450 / 250. Array scanning was performed using a GeneChip Scanner 3000. Array data analysis was performed using the Affymetrix Expression Console software (SST-RMA algorithm to summarize the signal from array probesets). Immune cytolytic activity was determined as previously described. 28 .

[0099] Cell isolation Mouse and human PDAC tumors and adjacent pancreas were mechanically dissociated and incubated in collagenase (5 mg / ml collagenase II for mouse tumors and 5 mg / ml collagenase IV for human tumors; Worthington Biochemical Corp., Fisher Scientific), DNAse I (0.5 mg / ml; Roche Diagnostics), and Hank's balanced salt solution (Gibco, Fisher Scientific) at 37°C for 30 minutes. The digestion was then stopped with fetal bovine serum (FBS; Life Technologies), and the cells were filtered sequentially through 100 mm and 40 mm nylon cell strainers (Falcon, Fisher Scientific). The tumors, adjacent pancreas, and lymph nodes were then mechanically dissociated and filtered through 100 mm and 40 mm nylon cell strainers (Falcon, Fisher Scientific) using PBS containing 1% FBS (Life Technologies). Spleens were mechanically dissociated and filtered through 70 mm and 40 mm nylon cell strainers (Falcon, Fisher Scientific) with 1% FBS in PBS, followed by red blood cell lysis (RBC lysis buffer, ThermoFisher Scientific). Mouse Fc receptors were lysed using FcεRIII / II-specific antibodies (1 × 10 6 and blocked with 1 μg per cell; clone 2.4G2, Bio X Cell).

[0100] ILC2 adoptive cell transfer CD45.1 C57Bl / 6 or Pdcd1 - / - Orthotopic PDAC mice were administered 500 ng of carrier-free recombinant murine IL33 (R&D Systems) in sterile PBS for 10 days. Live cells, CD45 + ,system - , CD90 + , CD25 + , ST2 + TILC2s were selected and purified to 98% purity on day 10 after transplantation using an Aria Cell sorter (BD Biosciences). 5Tumor ILC2s were injected i.p. into orthotopic PDAC tumor-bearing IL7r mice on days 7 and 14 after tumor implantation. Cre / + Ror fl / fl The recipient mice were immediately transplanted into CD45.2 mice. Control mice received the same volume of PBS via i.p. injection. αPD-1 treatment in recipient mice was initiated on the day of ILC2 cell transplantation. Tissues were harvested at the indicated time points.

[0101] Flow cytometry Single-cell suspensions were stained with antibody cocktails at 4°C in the dark, washed, and analyzed using a FACS LSR Fortessa (BD Biosciences). Mouse ILCs were analyzed as live cells, CD45 + ,system - (CD3, CD5, NK1.1, CD11b, CD11c, CD19, FcεR1), CD25 + , CD127 + Defined as cells 7、1 Mouse immune cells were defined as follows: ILC2 = live cells, CD45 + ,lineage - ,CD25 + ,ST2 + Cells; Central memory T cells = live cells, CD45 + ,CD3 + ,NK1.1 - ,CD8 + ,CD62l + ,CD44 + ; Dendritic cells = live cells, CD45 + ,CD3 - ,NK1.1 - ,Gr1 - , F4 / 80 - , CD11c + , MHC-II + ;B cells = living cells, CD45 + , CD3 - , CD19 + ;T cells = live cells, CD45 + , CD3 + ;CD4 + T cells = live cells, CD45 + , CD3 + , CD4+ ;CD8 + T cells = live cells, CD45 + , CD3 + , CD8 + ; Regulatory T cells = live cells, CD45 + , CD3 + , CD4 + FoxP3 + ;Tumor-associated macrophages = live cells, CD45 + , CD11b + , F4 / 80 + , GR1 - Myeloid-derived suppressor cells (MDSCs) = live cells, CD45 + , CD3 - , CD11b + , F4 / 80 - , GR1 +Mouse cells were stained with the following antibodies: CD45 (clone 30-F11, Pacific Blue), CD45.1 (clone A20, BV711), NK1.1 (clone PK136, APC), Gr-1 (clone RB6-8C5, BV605), and CD103 (clone 2E7, BV711) from Biolegend; and CD5 (clone 53-7.3, APC), CD11c (clone HL3, APC), NK1.1 (clone PK136, BV605), CD4 (clone RM4-5, BV786), CD62L (clone MEL-14, APC), CD19 (clone 1D3, BV510), Ly6C (clone AL-21, PerCP-Cy5.5), Ly6G (clone 1A8, AF700), and PD1 (clone J) from BD Biosciences. 43, BV605), TNF-α (clone MP6-XT22, BV510), IFN-γ (clone XMG1.2, APC-Cy7), CD90.2 (clone 53-2.1, BV786), Tbet (clone Q4-46, BV711), Ror-t (clone Q31-378, BV786), Gata3 (clone L50-823, PE-Cy7), and IL4 (clone 11B11, BV650); ThermoFisher Scientific: CD3 (clone 17A2, Alexa Fluor 700), CD11b (clone M1 / 70, APC), CD11b (clone M1 / 70, PerCP-Cy5.5), CD8 (clone 53-6.7, Alexa Fluor 700), CD19 (clone 1D3, Alexa Fluor 700), FcR1 (clone MAR-1, APC), F4 / 80 (clone BM8, PE-Cy5), CD3 (clone 145-2C11, PE-Cy7), MHC-II (clone M5 / 114.15.2, Alexa Fluor 700), CD44 (clone IM7, PerCP-Cy5.5), CD127 (clone A7R34, FITC), and CD25 (clone PC61.5, PerCP-Cy5).5), IL5 (clone TRFK5, PE), CD86 (clone GL1, PE), CD11c (clone N418, FITC), ST2 (clone RMST2-2, PE-Cy7), and FoxP3 (clone FJK-16S, APC); and SINFEKL tetramer (catalog number TB-5001-1, PE) from MBL International.

[0102] Human ILCs were cultured using live CD45 + ,system - (CD3, CD5, CD56, CD11b, CD11c, CD16, CD19, TCRα / β, FcεR1), CD25 + , CD127 + Defined as a cell 7 Human cells were stained with the following antibodies: BD Biosciences GATA3 (clone L50-823, BV711), TBET (clone O4-46, BV650), and RORγ-T (clone Q21-559, PE); Biolegend CRTH2 (clone BM16, PE-Cy7), CD11b (clone ICRF44, APC), CD56 (clone NCAM16.2, BV650), CD25 (clone BC96, PerCP-Cy5.5), CD45 (clone HI30, Pacific Blue), and TCRα / β (clone IP26, APC); ThermoFisher Scientific CD16 (clone CB16, APC), CD11c (clone 3.9, APC), CD127 (clone RDR5, FITC), and CD3 (clone OKT3, Alexa Fluor). 700), ST2 (clone hIL33Rcap, PE), CD5 (clone L17F12, APC), CD19 (clone HIB19, AF700), and FcεR1 (clone AER-37, APC). A human-specific antibody against IL33 (clone 390412, PE) was purchased from R&D Systems. All samples for flow cytometry were from prospectively collected, unselected PDAC patients.

[0103] To examine intracellular cytokine production, single-cell suspensions of tumors were stimulated ex vivo with phorbol 12-myristate (PMA, 100 ng / ml) and ionomycin (1 ng / ml) in the presence of brefeldin A (10 μg / ml) (all from Sigma-Aldrich) for 6 hours at 37°C. Cells were then surface stained, fixed, permeabilized, and stained for cytokine production using the Fixation and Permeabilization Buffer Kit (Invitrogen, ThermoFisher Scientific) according to the manufacturer's recommendations. Appropriate isotype controls were used as directed. Analysis was performed using FlowJo (versions 9 and 10, Tree Star).

[0104] immunohistochemistry Tissues were fixed in paraformaldehyde (Fisher Scientific) for 24 hours and embedded in paraffin. Tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems) and then antigen retrieval was performed with CC1 buffer (Ventana Medical Systems). Sections were blocked with Background Buster solution (Innovex) for 30 minutes, followed by avidin-biotin blocking for 8 minutes (Ventana Medical Systems). Antibodies for mouse IL33 (AF3626, R&D Systems), mouse smooth muscle actin (Abcam), and human IL33 (AF3625, R&D Systems) were applied, and sections were incubated for 4 hours. Subsequently, sections were incubated with a 1:200 dilution of biotinylated rabbit anti-goat IgG (Vector Labs) or biotinylated goat anti-rabbit IgG (Vector Labs) for 60 minutes. Detection was performed using a DAB detection kit (Ventana Medical Systems) according to the manufacturer's instructions. All sections containing cells showing cytoplasmic or nuclear positivity for IL33 were designated as having positive staining. Slides were counterstained with Masson's trichrome or hematoxylin and eosin and coverslipped with Permount (Fisher Scientific). All tissue sections were evaluated by an independent PDAC pathologist.

[0105] Immunofluorescence mouse IL33 / CD11b / CK19 / Iba1 immunofluorescence: Multiplex immunofluorescence staining was performed using a Discovery XT processor (Ventana Medical Systems) as described. 29 . IL33: Sections were first incubated with anti-mIL33 (R&D Systems, Cat. No. AF3626, 1 μg / ml) for 4 hours, followed by incubation with biotinylated horse anti-goat IgG (Vector Laboratories) at a 1:200 dilution for 60 minutes. Detection was performed with streptavidin-HRP D (part of the DABMap kit, Ventana Medical Systems), followed by incubation with Tyramide Alexa Fluor 488 (Invitrogen) at the indicated dilutions according to the manufacturer's instructions.

[0106] CD11b: Sections were then incubated with anti-CD11b (Abcam, clone EPR1544) for 5 hours, followed by incubation with biotinylated goat anti-rabbit IgG (Vector Laboratories) at a 1:200 dilution for 60 minutes. Detection was performed with streptavidin-HRP D (part of the DABMap kit, Ventana Medical Systems), followed by incubation with Tyramide Alexa 594 (Invitrogen) at the indicated dilutions according to the manufacturer's instructions.

[0107] CK19: Slides were then incubated with anti-CK19 (Abcam, clone EP1580Y) for 5 hours, followed by a 60-minute incubation with biotinylated goat anti-rabbit (Vector Laboratories) diluted 1:200. Detection was performed with streptavidin-HRP D (part of the DABMap kit, Ventana Medical Systems), followed by incubation with Tyramide Alexa Fluor 546 (Invitrogen) at the indicated dilutions according to the manufacturer's instructions.

[0108] Iba1: Finally, sections were incubated with anti-Iba1 (Wako, catalog no. 019-19741) for 5 hours, followed by a 1:200 dilution of biotinylated goat anti-rabbit IgG (Vector Laboratories) for 60 minutes. Detection was performed using streptavidin-HRP D (part of the DABMap kit, Ventana Medical Systems), followed by incubation with Tyramide Alexa 647 (Invitrogen) prepared at the indicated dilution according to the manufacturer's instructions. After staining, slides were counterstained with DAPI (Sigma-Aldrich) for 10 minutes and coverslipped with Mowiol.

[0109] Human Tissue sections were deparaffinized with Leica Bond buffer (Leica Biosystems) and subjected to antigen retrieval with Leica Bond ER2 buffer (Leica Biosystems). First, sections were incubated with anti-PD-1 antibody (Cell Marque, clone NAT105) for 1 hour and detected using the Bond Polymer Refine Detection kit (Leica Biosystems) and Tyramide Alexa Fluor 488 (Invitrogen). Next, sections were incubated with anti-CD3 antibody (DAKO, catalog number A0452) for 1 hour and detected using the Bond Polymer Refine Detection kit (Leica Biosystems) and Tyramide CF594 (Biotum). Next, sections were incubated with anti-GATA3 antibody (Cell Marque, clone L50-823) for 1 hour and detected using the Bond Polymer Refine Detection kit (Leica Biosystems) and CF 543 (Biotum). Finally, sections were incubated with anti-CD45 antibody (DAKO, clone 2B11 + PD7 / 26) for 1 hour and detected with the Bond Polymer Refine Detection kit (Leica Biosystems) and Tyramide Alexa Fluor 647 (Invitrogen). All detections were prepared at the specified dilutions according to the manufacturer's instructions. After staining, slides were counterstained with DAPI (Sigma-Aldrich) for 10 minutes and coverslipped with Mowiol.

[0110] Digital Image Processing and Analysis Slides were digitized using a Panoramic Flash 250 (3Dhistech, Budapest, Hungary) with a Zeiss 20x / 0.8NA objective and custom filters for A488, A546, A594, and A647. Each core was exported to a multichannel TIFF file and analyzed using a custom macro written in FIJI / ImageJ. For quantification, each nucleus was segmented using the DAPI channel after appropriate processing and background subtraction. Each nucleated cell was then assessed for the presence or absence of other markers after setting appropriate thresholds for each marker. The number of cells bearing a particular marker combination was tallied. ILC2s were identified as CD45. + CD3 - GATA3 + Nucleated cells, PD-1-expressing ILC2s, express CD45 + CD3 - GATA3 + PD-1 + Nucleated cells, PD-1 expressing T cells, CD45 + CD3 + PD-1 + Nucleated cells were defined as nucleated cells. For each patient, the frequency of each cell type as a percentage of total nucleated cells was calculated in triplicate cores, and then the mean frequency of the triplicate cores was determined to calculate the final cell frequency per patient.

[0111] RNA sequencing Mice: Tissues from orthotopic PDAC mice (n = 6) were harvested and dispersed into single-cell suspensions as described above. Tumor-infiltrating leukocytes were positively selected by magnetic-activated cell sorting using mouse CD45 MicroBeads (Miltenyi Biotec). Purification of magnetically sorted cells was confirmed by flow cytometry and was greater than 95%. RNA was isolated from sorted cells using the RNeasy Plus Mini Kit (Qiagen). Poly(A) capture and paired-end RNA-seq were performed by the MSKCC Integrated Genomics Core Facility. Specifically, after RiboGreen quantification and quality control using an Agilent BioAnalyzer, 500 ng of total RNA was subjected to poly(A) selection and TruSeq library preparation (TruSeq Stranded mRNA LT Kit, catalog number RS-122-2102) according to Illumina's instructions, followed by eight cycles of PCR. Samples were barcoded and run on a HiSeq 4000 using the HiSeq 3000 / 4000 SBS Kit (Illumina) with 100bp / 100bp paired-end migration. An average of 83 million paired reads were generated per sample. Ribosomal reads accounted for a maximum of 0.03% of all reads generated, and the proportion of mRNA bases averaged 76.6%. Expression datasets were loaded into Gene Set Enrichment Analysis (GSEA) 3.0. Gene set databases for antigen presentation and T cell-mediated immunity were selected from MSIGDB v6.1, and the false discovery rate was set to 0.25 or less to facilitate exploratory discovery. GSEA was run with 1000 permutations. Three gene set databases met this threshold: GO 0002474 Antigen Processing and Presentation of Peptide Antigen Via MHC Class I, GO 0002711 Positive Regulation of T Cell Mediated Immunity, and GSE19825 Naive vs Day 3 Effector CD8 T Cell Up.

[0112] Single-cell RNA sequencing Library preparation, sequencing, and raw data post-processing for single-cell immune profiling were performed at the Epigenomics Core at Weill Cornell Medicine.

[0113] Single-cell RNA library preparation and sequencing Vehicle, IL33 alone, IL33 +Single-cell suspensions of fluorescence-activated cell sorted ILC2 cells from PD-1-treated pancreatic KPC tumors and mesenteric DLN were prepared as described above. scRNA-seq libraries were generated according to 10X Genomics specifications (Chromium Single Cell V(D)J User Guide PN-1000006, 10X Genomics, Pleasanton, CA, USA). Four independent cell suspensions (85-90% viable) at a concentration of 90-200 cells / µl were loaded onto the 10X Genomics Chromium platform to generate gel beads-in-emulsion (GEMs), targeting approximately 2000 single cells per sample. After GEM generation, the sample was incubated at 53°C for 45 minutes in a C1000 Touch Thermal cycler with a 96-Deep Well Reaction Module (Bio-Rad, Hercules). Cell barcodes and Template Switch Oligos (TSOs) conjugated to Unique Molecular Identifiers (UMIs) were added to generate 5'-barcoded poly(A) cDNA. The GEMs were excised, and the single-stranded cDNA was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, Waltham, MA). The cDNA was amplified for 16 cycles (98°C for 45 seconds, 98°C for 20 seconds, 67°C for 30 seconds, and 72°C for 1 hour). The quality of the cDNA was assessed using an Agilent Bioanalyzer 2100 (Santa Clara, CA), yielding a product of approximately 1,200 bp. 50 ng of cDNA was enzymatically fragmented, end-repaired to A-tails, subjected to double-sided size selection with SPRIselect beads (Beckman Coulter, Indianapolis, IN), and ligated to the adapters provided in the kit.Each library was subjected to 14 cycles of PCR amplification using the index provided in the kit (98°C for 45 seconds, 98°C for 20 seconds, 54°C for 30 seconds, 72°C for 20 seconds x 14 cycles, 72°C for 1 minute, 4°C hold) to introduce a unique sample index. The indexed libraries underwent a second round of double-sided size selection, after which the libraries were quantified using Qubit fluorometry (Thermo Fisher Scientific, Waltham, MA). Quality was assessed using an Agilent Bioanalyzer 2100, yielding an average library size of 450 bp. None of the treated samples had concentrations below the limit of detection, and 18 cycles of cDNA amplification and 16 cycles of sample indexing were performed. The libraries were diluted to 10 nM and clustered using a NovaSeq600 with a paired-end read flow cell. R1 (10x barcode and UMI) was sequenced for 28 cycles, I7 Index (sample index) for 8 cycles, and R2 (transcript) for 89 bases. Approximately 100 million clusters were obtained per sample (except for tumors from vehicle-treated mice, which yielded approximately 10 million clusters). Primary processing of the sequence images was performed using Illumina Real Time Analysis software (RTA). 10x Genomics Cell Ranger Single Cell Software suite v3.0.2 (https: / / support.10xgenomics.com / single-cell-gene-expression / software / pipelines / latest / what-is-cell-ranger) was used for sample demultiplexing, alignment to the mouse genome reference mm10, filtering, UMI counting, single-cell 5'-end gene counting, and quality control using manufacturer parameters. Approximately 11,000 single cells passed quality control, yielding an average of approximately 41,000 reads per cell (sequence saturation of 48%).

[0114] scRNA-seq data processing To identify clusters in the combined dataset, the Seurat R package version 3.1 pipeline was used. 30 First, individual datasets were loaded into R as count matrices, converted to Seurat objects, and selected for genes expressed in 3 or more cells and for cells with at least 200 genes detected. Next, using a standard preprocessing workflow, cells were filtered based on excluding cells with more than 2,500 or fewer than 200 expressed unique genes, and cells with mitochondrial gene content greater than 5%.

[0115] After filtering, samples were integrated, and gene expression measurements from retained cells were log-transformed, normalized by total expression per cell, and scaled by 10,000 molecules per cell. Next, the top 2,000 genes with the highest variability across single cells were identified and subjected to principal component (PC) analysis. After examining jackstraw and elbow plots, the top 15 PCs were selected using K-nearest neighbor (KNN) clustering with a cluster resolution of 0.4. Six to eight clusters were identified across all sample- and tumor-combined merged datasets. The top 15 PCs were then visualized using nonlinear dimensionality reduction with UMAP. Gene expression differences between clusters for gene marker discovery were determined using the Wilcoxon rank-sum test, as used in the Seurat package. To compare gene expression between samples, pairwise comparisons using the Wilcoxon rank-sum test were performed with Holm P-value adjustment.

[0116] In vitro assay KPC 4662-GFP cells were cultured in 96-well flat-bottom plates (Falcon) in complete medium: RPMI-1640 (Gibco, ThermoFisher) containing L-glutamine, supplemented with 10% fetal bovine serum (Life Technologies), 100 units / ml penicillin, 100 μg / ml streptomycin, and recombinant IL-33 at concentrations of 0, 10, 100, and 500 ng / ml for 1 week. Medium and cytokines were replenished every 48 hours. Viability was measured using a colorimetric tetrazolium salt assay (Cell Counting Kit, Dojindo Molecular Technologies) according to the manufacturer's instructions, and cells were read on a Synergy HT Multi-Detection Microplate Reader (Biotek). Cells were harvested and stained for Annexin V (ThermoFisher Scientific), Ki-67 (clone SolA15, ThermoFisher Scientific), and ST2 (clone RMST2, ThermoFisher Scientific). For all in vitro studies, 2–3 technical replicates were performed per independent experiment.

[0117] In vitro dendritic cell migration assay Mouse splenic DCs were isolated and enriched using a mouse pan-DC isolation kit (Miltenyi Biotech) according to the manufacturer's protocol. DC purity was assessed by flow cytometry (>70% CD11c expression in live cells). + The cells were cultured at 5x10 cells / well with 50 ng / ml recombinant mouse GM-CSF (Biolegend). 5Splenic DCs were seeded overnight at 1x10 cells / ml in complete RPMI medium. Chemotaxis of splenic DCs was then analyzed by transwell migration assay. 600 μl of RPMI with or without 100 ng / ml recombinant mouse Ccl5 (Biolegend) was added to the lower chamber of a 6.5 mm transwell plate with a 5.0-μm pore polycarbonate membrane insert (Sigma-Aldrich). 200 μl of RPMI was also added to the upper chamber, and the plate was equilibrated at 37°C with 5% CO2 for 15 minutes. Then, 1x10 DCs were cultured in 100 μl of RPMI. 5 Splenic DCs were added to the upper chamber and incubated for 2 hours at 37°C in 5% CO2. After incubation, the membrane insert was carefully removed and cells were harvested from the lower chamber. Migrated DCs were incubated with DAPI and CD11c antibody for 20 minutes at 4°C, and Precision Count Beads™ (Biolegend) were added and counted for live, migrated, and CD11c cells using flow cytometry according to the manufacturer's protocol. + The number of cells was quantified.

[0118] statistical processing Data are presented as medians. Because many statistically significant effects were observed in the data without calculating a preanalysis sample size, no statistical methods were used to determine sample size. Comparisons between two groups were performed using a two-tailed unpaired Mann-Whitney test with the Benjamini-Krieger-Yekutieli false discovery approach for comparisons across multiple time points. Comparisons between multiple groups were performed using a one-way ANOVA test followed by a Kruskal-Wallis post hoc test. Comparisons between multiple groups across multiple time points were performed using a two-way ANOVA test. Correlations between two variables were calculated using linear regression. Survival curves were compared using a two-tailed log-rank test. Tumor incidence rates were compared using a chi-square test. All alpha levels were 0.05, and P < 0.05 was considered significant. Statistical analysis was performed using Prism 7.0 (GraphPad Software).

[0119] The information presented in this example was published by the present inventors in the journal "Nature" (see Moral et al., "ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity," Nature, 2020, March, Vol. 579(7797), pp. 130-135. doi: 10.1038 / s41586-020-2015-4, Epub 2020 Feb 19). The entire contents of this publication, including supplementary materials, are incorporated herein by reference.

[0120] Example 3 Effective treatment of PDAC in mice using IL33-activated ILC2 cell therapy We conducted a study to evaluate the efficacy of IL33-activated ILC2 cell therapy using a mouse model of PDAC. Unless otherwise specified, the mice, PDAC model, activators (IL33, αPD-1), and protocol were as described in Examples 1 and 2 above. IL33 was administered to "donor" mice to activate pancreatic tumor ILC2 cells. Pancreatic tumor ILC2 cells were then isolated and purified from the donor mice and administered to "recipient" ILC2-deficient mice with established PDAC tumors. Some recipient mice were also administered an anti-PD-1 antibody. While no significant effect on PDAC tumors was observed in recipient mice administered only donor ILC2 cells, recipient mice administered donor ILC2 cells and anti-PD-1 antibody showed a significant reduction in PDAC tumor size.

[0121] Example 4 Human clinical trials Clinical trials are conducted to demonstrate the safety and / or efficacy of treating PDAC. Clinical trials are conducted to demonstrate the safety and / or efficacy of treating PDAC with IL33 and / or PD-1 / PD-L1 inhibitors in adult human subjects. PDAC patients are identified and enrolled. Patients are assigned to specific groups. Different groups receive either: (a) IL33 alone, (b) an approved PD-1 and / or PD-L1 inhibitor alone, or (c) IL33 and an approved PD-1 and / or PD-L1 inhibitor. Within each group, there may be multiple different subgroups that use different agents (e.g., different PD-1 / PD-L1 inhibitors), different doses, and different administration schedules. IL33 is administered intravenously (IV). In some subgroups, IL33 may be in its native form. In some subgroups, IL33 may be modified by including one or more half-life-improving moieties. Different subgroups may receive different doses of IL33 (within a range of possible doses). Different subgroups can receive IL33 at different frequencies (e.g., daily, or every 2, 3, 4, 5, 6, or 7 days). Approved PD-1 and / or PD-L1 inhibitors are administered at approved doses, by approved dosing schedules, and by approved routes of administration (e.g., based on criteria such as those approved for the treatment of certain cancers).

[0122] Reference list 1. Vivier, E. et al. Innate Lymphoid Cells: 10 Years On. Cell 174, 1054-1066 (2018). 2. Salimi, M. et al. Activated innate lymphoid cell populations accumulate in human tumor tissues. BMC Cancer 18, 341 (2018). 3. Balachandran, V. P. et al. Identification of unique neoantigen qualities in long-term survivors of pancreatic cancer. Nature 551, 512-516 (2017). 4. Hingorani, S. R. et al. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell 7, 469-483 (2005). 5. Pylayeva-Gupta, Y., Lee, K. E., Hajdu, C. H., Miller, G. & Bar-Sagi, D. Oncogenic Kras-induced GM-CSF production promotes the development of pancreatic neoplasia. Cancer Cell 21, 836-847 (2012). 6. Li, J. et al. Tumor Cell-Intrinsic Factors Underlie Heterogeneity of Immune Cell Infiltration and Response to Immunotherapy. Immunity 49, 178-193.e7 (2018). 7. Brestoff, J. R. et al. Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit obesity. Nature 519, 242-246 (2015). 8. Gasteiger, G., Fan, X., Dikiy, S., Lee, S. Y. & Rudensky, A. Y. Tissue residency of innate lymphoid cells in lymphoid and nonlymphoid organs. Science 350, 981-985 (2015). 9. Monticelli, L. A. et al. Arginase 1 is an innate lymphoid-cell-intrinsic metabolic checkpoint controlling type 2 inflammation. Nat Immunol 17, 656-665 (2016). 10. Kirchberger, S. et al. Innate lymphoid cells sustain colon cancer through production of interleukin-22 in a mouse model. Journal of Experimental Medicine 210, 917-931 (2013). 11. Neesse, A. et al. CTGF antagonism with mAb FG-3019 enhances chemotherapy response without increasing drug delivery in murine ductal pancreas cancer. Proceedings of the National Academy of Sciences 110, 12325-12330 (2013). 12. Hardman, C. S., Panova, V. & McKenzie, A. N. J. IL‐33 citrine reporter mice reveal the temporal and spatial expression of IL‐33 during allergic lung inflammation. Eur J Immunol 43, 488-498 (2013). 13. Talabot-Ayer, D. et al. The mouse interleukin (Il)33 gene is expressed in a cell type- and stimulus-dependent manner from two alternative promoters. J. Leukoc. Biol. 91, 119-125 (2012). 14. Ricardo-Gonzalez, R. R. et al. Tissue signals imprint ILC2 identity with anticipatory function. Nat Immunol 19, 1093-1099 (2018). 15. Dalmas, E. et al. Interleukin-33-Activated Islet-Resident Innate Lymphoid Cells Promote Insulin Secretion through Myeloid Cell Retinoic Acid Production. Immunity 47, 928-942.e7 (2017). 16. Oliphant, C. J. et al. MHCII-mediated dialog between group 2 innate lymphoid cells and CD4(+) T cells potentiates type 2 immunity and promotes parasitic helminth expulsion. Immunity 41, 283-295 (2014). 17. Boettcher, J. P. et al. NK Cells Stimulate Recruitment of cDC1 into the Tumor Microenvironment Promoting Cancer Immune Control. Cell 172, 1022-1037.e14 (2018). 18. Miyamoto, C. et al. Runx / Cbfβ complexes protect group 2 innate lymphoid cells from exhausted-like hyporesponsiveness during allergic airway inflammation. Nat Commun 10, 447 (2019). 19. Yu, Y. et al. Single-cell RNA-seq identifies a PD-1hi ILC progenitor and defines its development pathway. Nature 539, 102-106 (2016). 20. Taylor, S. et al. PD-1 regulates KLRG1(+) group 2 innate lymphoid cells. Journal of Experimental Medicine 214, 1663-1678 (2017). 21. Kim, J. et al. Intratumorally Establishing Type 2 Innate Lymphoid Cells Blocks Tumor Growth. The Journal of Immunology 196, 2410-2423 (2016). 22. Diana, A. et al. Prognostic value, localization and correlation of PD-1 / PD-L1, CD8 and FOXP3 with the desmoplastic stroma in pancreatic ductal adenocarcinoma. Oncotarget 7, 40992-41004 (2016). 23. Donovan, C. et al. Roles for T / B lymphocytes and ILC2s in experimental chronic obstructive pulmonary disease. J. Leukoc. Biol. 105, 143-150 (2019). 24. Evans, R. A. et al. Lack of immunoediting in murine pancreatic cancer reversed with neoantigen. JCI Insight 1, (2016). 25. Sastra, S. A. & Olive, K. P. Quantification of murine pancreatic tumors by high-resolution ultrasound. Methods Mol. Biol. 980, 249-266 (2013). 26. Monticelli, L. A. et al. Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus. Nat Immunol 12, 1045-1054 (2011). 27. Ma, Z. et al. Augmentation of Immune Checkpoint Cancer Immunotherapy with IL18. Clin Cancer Res 22, 2969-2980 (2016). 28. Rooney, M. S., Shukla, S. A., Wu, C. J., Getz, G. & Hacohen, N. Molecular and genetic properties of tumors associated with local immune cytolytic activity. Cell 160, 48-61 (2015). 29. Yarilin, D. et al. Machine-based method for multiplex in situ molecular characterization of tissues by immunofluorescence detection. Sci Rep 5, 9534 (2015). 30. Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 36, 411-420 (2018).

Claims

1. 1. A method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, comprising administering to a subject having PDAC an effective amount of IL33, thereby treating the PDAC in the subject.

2. 1. A method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, comprising administering to a subject having PDAC effective amounts of (a) IL33 and (b) a PD-1 and / or PD-L1 inhibitor, thereby treating the PDAC in the subject.

3. 1. A method for activating pancreatic tissue-specific anti-tumor T cell immunity in a subject in need thereof, comprising administering an effective amount of IL33 to a subject having PDAC, thereby activating pancreatic tissue-specific anti-tumor T cell immunity in the subject.

4. 1. A method for activating pancreatic tissue-specific anti-tumor T cell immunity in a subject in need thereof, comprising administering to a subject having PDAC effective amounts of (a) IL33 and (b) a PD-1 and / or PD-L1 inhibitor, thereby activating pancreatic tissue-specific anti-tumor T cell immunity in the subject.

5. A method for activating pancreatic ILC2 cells, comprising contacting pancreatic ILC2 cells with an effective amount of IL33, thereby activating the pancreatic ILC2 cells.

6. A method for activating pancreatic ILC2 cells, comprising contacting the pancreatic ILC2 cells with effective amounts of (a) IL33 and (b) a PD-1 and / or PD-L1 inhibitor, thereby activating the pancreatic ILC2 cells.

7. The method of claim 5 or 6, wherein the ILC2 cells are contacted with IL33, a PD-1 inhibitor, or a PD-L1 inhibitor in vitro / ex vivo.

8. The method of claim 5 or 6, wherein the ILC2 cells are contacted with IL33, a PD-1 inhibitor, or a PD-L1 inhibitor in vivo.

9. A method for sensitizing PDAC tumor and / or pancreatic ILC2 cells to a PD-1 inhibitor and / or a PD-L1 inhibitor, comprising contacting the PDAC tumor and / or pancreatic ILC2 cells with an effective amount of IL33, thereby sensitizing the PDAC tumor and / or pancreatic ILC2 cells to the PD-1 inhibitor and / or the PD-L1 inhibitor.

10. The method of any one of claims 1 to 4, wherein the subject is a human.

11. 5. The method of any one of claims 1 to 4, wherein the subject is a non-human mammal.

12. The method of any one of claims 1 to 4, wherein the subject is a mouse.

13. 5. The method of any one of claims 1 to 4, wherein the subject has PDAC that is partially or totally resistant to PD-1 and / or PD-L1 inhibitor treatment.

14. The method of any one of claims 1 to 9, wherein the IL33 is recombinant IL33.

15. The method of claim 10, wherein the IL33 is human IL33.

16. The method of claim 10, wherein the IL33 is recombinant human IL33.

17. The method of claim 12, wherein the IL33 is mouse IL33.

18. The method of claim 12, wherein the IL33 is recombinant mouse IL33.

19. The method of any one of claims 2, 4, 6 and 9, wherein the PD-1 inhibitor is an antibody.

20. 10. The method of any one of claims 2, 4, 6 and 9, wherein the PD-1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, AMP-224, AMP-514 and PDR001.

21. The method of any one of claims 2, 4, 6 and 9, wherein the PD-L1 inhibitor is an antibody.

22. 10. The method of any one of claims 2, 4, 6 and 9, wherein the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, durvalumab, BMS-936559 and CK-301.

23. A pharmaceutical composition comprising (a) IL33 and (b) a PD-1 and / or PD-L1 inhibitor.

24. A pharmaceutical composition for use in treating PDAC, comprising: (a) IL33 and (b) a PD-1 and / or PD-L1 inhibitor.

25. A method for treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, comprising: (a) administering to a recipient subject having PDAC an effective amount of activated donor pancreatic ILC2 cells, wherein the donor pancreatic ILC2 cells are obtained from the donor subject and activated ex vivo / in vitro by contact with IL33, and wherein the donor subject and recipient subject are allogeneic, thereby treating PDAC in the recipient subject.

26. A method for treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, comprising: (a) contacting donor pancreatic ILC2 cells obtained from a donor subject with IL33 ex vivo / in vitro to produce activated donor pancreatic ILC2 cells; and (b) administering the donor activated pancreatic ILC2 cells to a recipient subject having pancreatic ductal adenocarcinoma (PDAC), wherein the donor subject and recipient subject are allogeneic, thereby treating PDAC in the recipient subject.

27. A method for treating pancreatic ductal adenocarcinoma (PDAC) in a subject in need thereof, comprising: (a) obtaining donor pancreatic ILC2 cells from a donor subject; (b) contacting the donor pancreatic ILC2 cells with IL33 ex vivo / in vitro to generate activated donor pancreatic ILC2 cells; and (c) administering the activated donor pancreatic ILC2 cells to a recipient subject having pancreatic ductal adenocarcinoma (PDAC), wherein the donor subject and the recipient subject are allogeneic, thereby treating the PDAC in the recipient subject.

28. 28. The method of any one of claims 25 to 27, further comprising expanding the donor pancreatic ILC2 cells and / or the activated donor pancreatic ILC2 cells ex vivo / in vitro prior to administering the activated donor pancreatic ILC2 cells to the recipient subject.

29. 29. The method of any one of claims 25 to 28, wherein the donor subject and the recipient subject are the same person, and wherein the method is an autologous cell therapy.

30. 29. The method of any one of claims 25 to 28, wherein the donor subject and the recipient subject have the same MHC / HLA type.

31. (none)

32. 31. The method of any one of claims 25 to 30, wherein the donor subject and the recipient subject are human.

33. 31. The method of any one of claims 25 to 30, wherein the donor subject and the recipient subject are non-human mammals.

34. 31. The method of any one of claims 25 to 30, wherein the donor subject and the recipient subject are mice.

35. 35. The method of any one of claims 25 to 34, wherein the recipient subject has PDAC that is partially or totally resistant to PD-1 and / or PD-L1 inhibitor treatment.

36. 36. The method of any one of claims 25 to 35, wherein the IL33 is recombinant IL33.

37. 36. The method of any one of claims 25 to 35, wherein the donor subject and the recipient subject are human and the IL33 is human IL33.

38. 36. The method of any one of claims 25 to 35, wherein the donor subject and the recipient subject are human and the IL33 is recombinant human IL33.

39. 36. The method of any one of claims 25 to 35, wherein the donor subject and the recipient subject are mice and the IL33 is mouse IL33.

40. 36. The method of any one of claims 25 to 35, wherein the donor subject and the recipient subject are mice and the IL33 is recombinant mouse IL33.

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