Compositions and methods for treating cytotoxic T-cell resistant tumors

Abstract

The present invention provides compositions and methods for treating tumors resistant to checkpoint blockade by activating NK cells. [Solution] A method for treating cancer in a subject is provided, comprising administering to the subject a therapeutically effective amount of a composition comprising one or more activators and a pharmaceutically acceptable carrier, wherein the activators activate NK cells via NKG2D and / or CD16 receptors, thereby causing lysis of one or more cancer cells in the subject, and the cancer is resistant to cytotoxic T cells.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 912,826, filed on 9 October 2019, the entirety of which is incorporated herein by reference.

[0002] All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. To more fully describe the latest art known to those skilled in the art as of the date of the embodiments described herein and claimed herein, the disclosures of these publications are incorporated herein by reference.

[0003] This patent disclosure includes materials that are protected by copyright. The copyright holder reserves all copyrights to the patent document or patent disclosure, except as they appear in the patent files or records of the United States Patent and Trademark Office, and does not object to facsimile reproduction of either the patent document or the patent disclosure.

[0004] Government interests This invention was made possible with government support under grant numbers CA173750, T32 CA207021, and R01 CA238039, awarded by the National Institutes of Health. The government has certain rights to this invention.

[0005] field The embodiments described herein relate to compositions and methods for treating tumors resistant to checkpoint blockade by activating NK cells. [Background technology]

[0006] Checkpoint blockade with antibodies targeting programmed cell death protein 1 (PD-1) or cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitory receptors on T cells can induce persistent anti-tumor immunity even in patients with advanced cancer. However, many patients are unable to benefit from these therapies due to primary or secondary resistance. Cytotoxic T cells play a central role in the effectiveness of checkpoint blockade based on their ability to recognize tumor-derived peptides bound to major histocompatibility complex class I (MHC-I) proteins. When such MHC-I-peptide complexes are recognized by the T cell receptor (TCR), T cells release interferon-γ (IFNγ), inhibiting tumor cell proliferation and enhancing MHC-I protein expression in both tumor cells and dendritic cells. Therefore, resistance to checkpoint blockade is often mediated by the loss of MHC-I expression by tumor cells, either due to mutations in key genes in the MHC-I (B2M, TAP1, TAP2, and other genes) or IFNγ (JAK1, JAK2) pathways, or epigenetic silencing. When the number of newly generated antigens is low or lost, tumor immunity mediated by cytotoxic T cells is also reduced. Currently, there are no alternative immunotherapies for patients with solid tumors resistant to checkpoint blockade and cytotoxic T cells. [Overview of the project]

[0007] Embodiments described herein include methods for treating, preventing, or mitigating the symptoms of cancer in a subject. In embodiments, treating cancer is indicated by stopping or reducing tumor growth and / or metastasis.

[0008] In the embodiments, the cancer is resistant to cytotoxic T cells. In the embodiments, the cancer is MCH class I deficient cancer or cancer resistant to IFN gamma. In the embodiments, the cancer is resistant to immunotherapy, for example, cancer resistant to anti-PD1 and / or anti-PD-L1 antibodies.

[0009] Non-limiting examples of cancers that can be treated by embodiments described herein include melanoma, lung cancer, kidney cancer, bladder cancer, Hodgkin lymphoma, breast cancer, stomach cancer, and pancreatic cancer.

[0010] In one embodiment, the method involves administering a therapeutically effective amount of a composition comprising one or more activators and a pharmaceutically acceptable carrier to a subject. For example, the activator activates NK cells via NKG2D and / or CD16 receptors, thereby causing lysis of one or more cancer cells in the subject, and the cancer is resistant to cytotoxic T cells.

[0011] In embodiments, the activator includes polynucleotides, polypeptides, small molecules, cytokines, or combinations thereof. For example, the activator includes antibodies such as monoclonal antibodies. For example, the antibody includes an anti-MICA antibody, an anti-MICB antibody, or both. In embodiments, the antibody binds to the alpha-3 domain of MICA / B. In embodiments, the antibody includes one or more sequences from Table 1.

[0012] In the embodiment, the activator inhibits tumor-mediated shedding of MICA / MICB, thereby increasing the density of NKG2D receptor ligands on tumor cells. For example, an anti-MICA / MICB antibody inhibits tumor-mediated shedding of MICA / MICB.

[0013] Embodiments may further include the step of administering a therapeutically effective amount of a second composition comprising one or more additional therapeutic or prophylactic agents and a pharmaceutically acceptable carrier to a target. In embodiments, one or more additional therapeutic or prophylactic agents include toxins, radiolabeling, radiotherapy, siRNA, small molecules, peptides, antibodies, genetically modified cells, radiation, or cytokines.

[0014] For example, one or more additional therapeutic or prophylactic medications may include HDAC inhibitors such as panobinostat.

[0015] For example, one or more additional therapeutic or prophylactic drugs may include cytokines such as IL2, IL15, IL12, or IL18.

[0016] For example, one or more additional therapeutic or prophylactic agents include small molecules. In embodiments, the small molecules include proteasome inhibitors.

[0017] For example, one or more additional therapeutic or prophylactic agents may include antibodies such as anti-PD1 antibodies, anti-PDL1 antibodies, and / or anti-CTLA-4 antibodies.

[0018] For example, one or more additional therapeutic or prophylactic drugs may include genetically modified cells such as CAR T cells.

[0019] Embodiments of this specification may further include the step of testing for cancer for Jak1 mutations and / or B2m mutations.

[0020] The embodiments described herein also relate to methods for sensitizing target cancer cells to NK cells.

[0021] In embodiments, the method includes administering a therapeutically effective amount of a composition comprising one or more activators and a pharmaceutically acceptable carrier to a subject. For example, the activator activates NK cells, thereby causing the lysis of one or more cancer cells in the subject, and the cancer is resistant to cytotoxic T cells.

[0022] In this embodiment, activation of the NKG2D receptor and / or CD16 receptor activates NK cells.

[0023] In this embodiment, the activator inhibits MICA / MICB shedding by cancer cells, thereby activating NKG2D and / or CD16 receptors. Thus, MICA / B on the surface of cancer cells activate the NKG2D receptor, the CD16 receptor, or both.

[0024] Other objectives and advantages of the embodiments described herein will be readily apparent from the following description. [Brief explanation of the drawing]

[0025] A patent or application file must contain at least one color drawing. A copy of this patent or patent application publication containing the color drawing will be provided by the Patent Office after the application and payment of the required fees.

[0026] [Figure 1A] This shows the characteristics of NK cells infiltrating human melanoma metastases by single-cell RNA-seq. (A) Isolation of NK cells from blood and melanoma metastases (patient CY158) by flow cytometry. NK cells were identified as single, living lymphocyte-sized cells that were positive for CD45 and CD56 markers but negative for CD3, CD4, CD8a, CD14, CD15, and CD163 markers. The numbers indicate the percentage of NK cells in the overall lymphocyte population (including T and B cells). (B) NK cells in tumors differ from NK cells in blood. Single-cell RNA-seq analysis of blood and tumor-infiltrating NK cells from patient CY158. UMAP plots were used to visualize the blood and tumor-infiltrating NK cell populations, with the percentage of NK cells in each cluster shown for blood and tumor NK cells (left). NK cell clusters are color-coded, and major differentially expressed genes are shown for each cluster (right). (C) Comparison of metastatic and blood NK cell populations by scRNA-seq, integrating data from three patients (CY155, CY158, and CY160). NK cell clusters in blood (left) and metastasis (right) are visualized using UMAP plots. The major differentially expressed genes in each cluster and the percentage of NK cells assigned to a given cluster are shown. (D) mRNA transcripts of selected genes in blood (top) and tumor-infiltrated (bottom) NK cell populations are visualized using UMAP plots. The intensity of the blue color indicates the expression level of the gene shown in individual cells. [Figure 1B]Please refer to the explanation in Figure 1A. [Figure 1C] Please refer to the explanation in Figure 1A. [Figure 1D] Please refer to the explanation in Figure 1A. [Figure 2] This paper identifies NK cell populations and shows the expression of genes related to cytotoxicity and chemokines. (A-D) Identification of NK cell and ILC3 cell populations. Gene expression signatures of NK cells and ILC3s were defined using published single-cell data from human innate lymphocytes isolated from tonsils.31 UMAP plots and violin diagrams show the degree of similarity between these gene expression signatures and the gene expression patterns of sequenced cells. Hematologic and tumor-derived cells were investigated using NK cell signatures (A, B) and ILC3 signatures (C, D). (E) Cytotoxic gene expression signatures (GZMA, GZMB, GZMH, GZMK, GZMM, PRF1, GNLY, and NKG7) of NK cells isolated from blood (top) and melanoma metastases (bottom). UMAP plots and violin diagrams are shown to show the score of this signature across the NK cell clusters. (F, G) UMAP plots showing the expression of chemokines XCL1 and XCL2 (abbreviated as XCL1 / 2) (F) and CCL3, CCL4, CCL4L2 and CCL5 (abbreviated as CCL3 / 4 / 4L2 / 5) (G) in blood (top) and tumor-infiltrating NK cells (bottom). [Figure 3]This paper shows differential expression of inhibitory receptors by NK cells in melanoma metastases compared to circulating NK cells, and identification of NK cell populations by flow cytometry. (A) Expression of inhibitory receptors by NK cells isolated from blood and melanoma metastases. The intensity of blue indicates the gene expression level shown in individual cells. (B) Gene expression used to identify NK cell populations (FGFBP2 and FCGR3A) and effector molecules (GZMA and GZMK). The intensity of blue indicates the gene expression level shown in individual cells. (C) Validation of three NK cell populations identified by scRNA-seq in blood samples by flow cytometry using FGFBP2 and CD16a as markers. NK cells were identified by gating CD45 and CD56-positive cells that were negative for CD3ε, CD19, CD14, CD15, CD163, and dead cell markers. A representative analysis for CY165 patients is shown. (D) Quantification of three NK cell populations in blood and tumor samples based on FGFBP2 and CD16a markers. Granzyme A and K labels are also shown for each of the three populations. MFI = mean fluorescence intensity. Each dot in these graphs represents an individual patient. Statistical analysis was performed by two-way ANOVA, Bonferroni post-hoc test. *p<0.05, **p<0.01, ***p<0.001. [Figure 4]This study demonstrates that inactivation of B2M sensitizes human melanoma cells to MICA / B mAbs. (A) Verification of B2M gene inactivation efficiency. Human A375 melanoma cells were edited with control or B2M gRNA (referred to as control and B2M-KO, respectively). Edited A375 cells were treated with the indicated concentrations of IFNγ for 24 hours, and HLA-A / B / C surface levels were quantified by flow cytometry. MFI = mean fluorescence intensity. (B-C) Human A375 melanoma cells edited with control or B2M gRNA were cultured for 24 hours with MICA (7C6-hIgG1) or the indicated concentrations of isotype control antibodies. Quantification of soluble MICA produced by edited melanoma cells using sandwich ELISA (B). As previously reported, the 7C6 antibody does not interfere with the detection of soluble MICA by ELISA. 20 MICA / B surface protein levels in control and B2M-edited melanoma cells were quantified by flow cytometry using PE-conjugated MICA / B mAb 6D4 (C). As previously reported, this mAb binds to the MICA / B α1-α2 domains and does not compete with 7C6 antibody. 20(D) Effect of human NK cells on A375 melanoma cells dependent on MHC-I expression and MICA mAb treatment. GFP+ A375 melanoma cells edited with control or B2M gRNA were seeded in 96-well plates at a cell density of 5 × 10³ cells per well. After pretreatment of melanoma cells with 7C6-hIgG1 or isotype control mAb (20 μg / ml) for 24 hours, purified human NK cells were added at different effector-to-target ratios (0:1, 0.5:1, or 1:1). IL-2 (300 U / ml) was used to support NK cell survival. The number of GFP+A375 melanoma cells was quantified by imaging cytometry using a Nexcelom Celigo instrument at multiple time points over a 72-hour period. Data (A-D) are representative of three independent experiments. Statistical analysis was performed by two-way analysis of variance (ANOVA) with Bonferroni's multiple comparison test (D). *p<0.05, ***p<0.001. [Figure 5-1]This study demonstrates that MICA / B mAb treatment induces immunity against metastases resistant to cytotoxic T cells. (A) B16F10-MICA cells edited with control, B2m, or Jak1 gRNA were treated with IFNγ (10 ng / ml) or a solvent control (PBS) for 24 hours, and H-2Kb surface levels were analyzed by flow cytometry. (B) MICA mAbs induced immunity against established metastases with inactivating mutations in the B2m or Jak1 gene. B16F10-MICA melanoma cells were edited with control, B2m, or Jak1 gRNA, and 7 × 10⁵ tumor cells were intravenously injected into B-cell-deficient (Ighm- / -) mice. On day 7, a subset of mice was euthanized to quantify metastases, and the remaining mice were treated with 7C6-mIgG2a or a control mAb (200 μg intraperitoneally administered on days 7, 8, and 12). On day 14, lung surface metastases were counted under a stereomicroscope. (C) Effect of MICA mAb treatment on survival of mice with B2m or Jak1-deficient melanoma metastases. WT mice were intravenously inoculated with 2 × 10⁵ B16F10-MICA cells edited with control, B2m, or Jak1 gRNA. Mice were administered 7C6-mIgG2a or isotype control mAbs on days 1 and 2, and mouse survival was recorded. (D) MHC-I expression by LLC1-MICA cells. LLC1-MICA cells were edited with control or B2m gRNA and stimulated with IFNγ (10 ng / ml) or solvent control (PBS) for 24 hours. Surface H-2Kb protein levels were quantified by flow cytometry. (E) MICA mAb treatment of lung metastases formed by LLC1 lung cancer cells. WTC57BL6 / J mice were intravenously inoculated with 1 × 10⁶ (1M) or 1.5 × 10⁶ (1.5M) LLC1-MICA tumor cells edited with control or B2m gRNA. Two days after tumor cell inoculation, mice were treated with the indicated mAb (200 μg administered intraperitoneally). Additional treatment was performed on day 3 and thereafter weekly. Lung metastases were counted on day 14. (F) MICA mAb treatment of LLC1-MICA metastases in mice reconstituted with allogeneic or syngeneic NK cells.Rag2- / -Il2rg- / - double knockout mice were injected with LLC1 cells and NK cells (2 × 10⁵ cells) derived from allogeneic or syngeneic CB6F1 / J or C56BL / 6 mice, respectively. A third group of Rag2- / -Il2rg- / - mice did not receive NK cells. LLC1-MICA tumor cells (7 × 10⁵) were intravenously injected. NK cell transfer was performed 24 hours later. Mice were treated with the indicated antibody (200 μg) on ​​days 2 and 3 after tumor cell inoculation, and weekly thereafter. Metastases were counted on day 14. Data are representative from three independent experiments (A, D), or pooled from three (B, E) or two (C, F) independent experiments. Statistical analysis was performed by two-sided unpaired Student's t-test (B, E-F) and log-rank (Mantel-Cox) test (C). *p<0.05, **p<0.01, ***p<0.001. [Figure 5-2] Please refer to the explanation in Figure 5-1. [Figure 6]This study demonstrates the essential role of NK cells in the treatment of B2m and Jak1-deficient melanoma metastases with MICA / B antibodies. (A) Wild-type (WT) C57BL / 6 mice were intravenously inoculated with 7 × 10⁵ B16F10-MICA cells edited with control, B2m, or Jak1 gRNA. Mice were treated with 7C6-mIgG2a or isotype control mAb (200 μg) on ​​days 1, 2, and 7. Compared to tumor cell inoculation, NK cell depletion was performed by injecting 100 μg of anti-asialoGM1 (anti-asGM1) on days 1, 0, and 7. Control mice were administered isotype control antibodies. Lung surface metastases were quantified 14 days after tumor inoculation. (B) Analysis of NK cell infiltration into lung tissue. Tumor injection and mAb treatment were performed as described in "A" using tumor cells expressing ZsGreen, which allowed for identification by flow cytometry. As previously reported, mice were intravenously injected with APC-conjugated anti-CD45.2 antibody 12 days after inoculation of tumor cells to differentiate between blood and tissue-infiltrating NK cells.20 Lung-infiltrating NK cells were identified as CD3ε-TCRβ-NK1.1+CD49b+EOMES+ live cells, showing low staining for CD45.2-APC (intravenous injection) and high staining for CD45.2-PE-CY7 (added to cell suspension). The ratio of NK cells to ZsGreen+B16F10-MICA cells is shown. (C, D) Number of ZsGreen+B16F10-MICA cells (C) and lung-infiltrating NK cells (D) for the indicated genotypes and treatment groups in the experiments described in (B). EOMES labeling was used to differentiate NK cells from ILC1s. Pooled data from two independent experiments (A-D). Statistical analysis was performed using two-way ANOVA with Bonferroni post-hoc tests (A) or two-tailed unpaired Student t-tests (B-D). *p<0.05, **p, 0.01, ***p<0.001. [Figure 7-1]This study demonstrates that the combination of the HDAC inhibitor panobinostat and a MICA mAb enhances MICA / B surface levels and inhibits metastatic growth in NSG mice reconstituted with human NK cells. (A) Increase in NKG2D ligand mRNA levels after treatment with panobinostat. A375 melanoma cells were treated with panobinostat (50 nM) for 24 hours, and mRNA was extracted for bulk RNA-seq. mRNA levels of NKG2D ligand and MHC class I genes are shown as the ratio (log2 factor change) between the panobinostat and PBS groups. (B) Increase in MICA / B surface protein levels after treatment with panobinostat + MICA / B mAb. A375 melanoma cells were incubated with the indicated mAb (20 μg / ml) and increased concentrations of panobinostat for 24 hours. MICA / B surface levels (left) and A375 cell viability (right) were quantified by flow cytometry. Shedding MICA was quantified by sandwich ELISA (center). (C) Short-term treatment of human melanoma cell lines with panobinostat + MICA / B mAb combination. The indicated melanoma cell lines were treated in vitro for 24 hours with the indicated mAb (20 μg / ml) + increasing concentrations of panobinostat. MICA / B surface levels were quantified by flow cytometry. (D) In ​​vivo synergistic effect of panobinostat + MICA / B mAb treatment on metastatic MICA / B surface protein levels formed by human melanoma cells. NSG mice were intravenously inoculated with 1 × 10⁶ ZsGreen+A375 melanoma cells. Two weeks later, the mice were treated with the indicated mAb (200 μg) + / - panobinostat (10 mg / kg) for the following two days. 24 hours after the final treatment, MICA / B surface levels were analyzed using tumor cells (large, viable ZsGreen+, CD45- cells) in lung metastases. (E-F) NSG mice were reconstituted with purified human NK cells (2 × 10⁶ cells intravenously) grown in vitro. In vivo survival of NK cells was supported by co-administration of IL-2 (7.5 × 10⁴ units) via intraperitoneal injection. On day 1, mice were intravenously inoculated with control or B2M-edited A375 cells (5 × 10⁵).On days 2 and 3, mice were administered a different dose of IL-2, the indicated mAb (200 μg) + / - 10 mg / kg panobinostat. On day 14, the number of lung surface metastases was counted. Demonstrations of experimental design (E) and quantification of lung surface metastases (F). Data representative of three independent experiments (B and C), or pooled data from two independent experiments (D and F). Statistical analysis was performed by two-sided unpaired Student's t-test (D) and two-way ANOVA, Bonferroni's post-hoc test (F). *p<0.05, **p<0.01, ***p<0.001. [Figure 7-2] Please refer to the explanation in Figure 7-1. [Figure 8] Flow cytometry analysis of NK cells from patient blood and tumor samples is shown. NK cells were identified as lymphocyte-sized living cells expressing CD45 and CD56, but not CD3, CD19 (in some samples), CD14, CD15, and CD163. Percentage of NK cells in the total lymphocyte population (including T cells and B cells) for blood NK cells (left) and tumor-infiltrating NK cells (right). [Figure 9] This shows a comparison of circulating and tumor-infiltrating NK cell populations from individual patients using scRNA-seq. (A-B) Single-cell RNA-seq analysis of blood and tumor-infiltrating NK cells from samples of two patients, CY155 (A) and CY160 (B), is shown. UMAP plots were used to visualize the blood and tumor-infiltrating NK cell populations from each patient. The percentage of NK cells in each cluster is also shown for blood and tumor NK cells (left). NK cell clusters are color-coded, and major differentially expressed genes are shown for each cluster (right). [Figure 10]This shows the analysis of ILC1 and ILC2 gene expression signatures and chemokine expression. (A-B) Gene expression signatures for ILC1 and ILC2 cells were defined using published single-cell data from human innate lymphocytes isolated from the tonsils.31 These signatures were used to investigate blood (A) and tumor (B) NK cells. (C) Chemokine expression by NK cells isolated from blood and melanoma metastases. The intensity of the blue color indicates the gene expression level shown in individual cells. [Figure 11-1] Analysis of surface receptors and gene expression signatures is shown. (A-B) Gene expression signatures for activating NK cell receptors (KLRK1, KLRF1, FCGR3A, CD226, CD244, NCR1, NCR2, and NCR3) and inhibitory NK cell receptors (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KLRC1, TIGIT, CD96, HAVCR2, PDCD1, LAG3) were used to compare blood (A) NK cells and tumor-infiltrating (B) NK cells. These signatures were visualized using UMAP and violin diagrams. (C) Single-cell expression of activating and inhibitory receptors by blood (top) and tumor (bottom) NK cells. The intensity of the blue indicates the expression level of the gene shown in individual cells. (D) Expression of NKG2D by NK cells analyzed by flow cytometry. For illustration, a histogram of a CY158 patient is shown. [Figure 11-2] Please refer to the explanation in Figure 11-1. [Figure 12-1]This paper shows the analysis of surface HLA class I and MICA / B proteins on tumor cells from melanoma metastases. Melanoma metastases were surgically resected from patients and analyzed by flow cytometry. Tumor cells were identified as viable, large, single cells that were CD45-negative. (A, B) Expression of conventional MHC-I (HLA-A / B / C) (A) and MICA / B (B) proteins by melanoma cells from lesions, NK cells also analyzed by scRNA-seq. (C-D) Expression of conventional MHC-I protein (C) and MICA / B (D) by metastatic tumor cells from an additional patient group. (E) Quantification of shedding MICA in plasma from indicated melanoma patients (CY156P-CY166) and healthy donors (HD) by sandwich ELISA. Shedding MICB was not detected in these samples. [Figure 12-2] Please refer to the explanation in Figure 12-1. [Figure 13]This study describes the characteristics of B2M-deficient A375 melanoma cells and the inhibition of NK cell-mediated melanoma cell killing by MHC-I recognition. (A) Verification of the efficiency of B2M gene inactivation in A375 melanoma cells. Cells were edited with control or B2M gRNA (referred to as control and B2M-KO, respectively). Cells were treated with IFNγ (1 ng / ml) or PBS at indicated concentrations for 24 hours, and surface HLA-A / B / C proteins were quantified by flow cytometry. (B) Comparison of MICA / B surface levels in control and B2M-edited A375 cells. Cells were treated with 7C6-hIgG1 or isotype control mAb (10 μg / ml) for 24 hours, and MICA / B surface proteins were analyzed by flow cytometry. (C) NK cells isolated from healthy donors were cultured for 24 hours in the presence of 1000 U / ml IL-2. Parental A375 melanoma cells were incubated with 7C6-hIgG1 or isotype control antibody (20 μg / ml) for 24 hours and then used in cytotoxic assays. Killing of A375 cells via NK cells was analyzed using a 4-hour 51Cr release assay. The indicated KIR or isotype control antibody was added to the coculture at 10 μg / ml. Data are representative of three independent experiments (A-C). Statistical analysis was performed by two-way ANOVA, Bonferroni's post-hoc test (C). ***p<0.001. [Figure 14]Characterization of B16F10-MICA cell lines and in vivo activity of MICA mAbs are shown. (A) B16F10-MICA cells edited with control, B2m, or Jak1 gRNA were treated with the indicated concentrations of IFNγ for 24 hours, and surface levels of H-2Kb (left) and MICA (right) were analyzed by flow cytometry. MFI = mean fluorescence intensity. Data representative of three independent experiments. (B) B-cell-deficient (Ighm- / -) mice were intravenously inoculated with 7 × 10⁵ B16F10-MICA cells edited with control, B2m, or Jak1 gRNA. Once metastasis was established (day 7), mice were treated with MICA or isotype control mAbs (200 μg on days 7, 8, and 12). Shedding MICA in plasma samples was quantified using sandwich ELISA. Data were pooled from two independent experiments. Statistical analysis was performed using two-way ANOVA and Bonferroni's post-hoc test. ***p<0.001. [Figure 15-1]This study shows the effects of panobinostat treatment on a panel of human tumor cell lines. (A-B) A375 melanoma cells were treated with panobinostat (50 nM) or a solvent control (PBS) for 24 hours, and gene expression was examined by bulk RNA-seq. Major genes differentially expressed in cells treated with panobinostat or solvent control (A) and major immunological pathways upregulated by panobinostat compared to A375 cells treated with the control (B). FDR: false positive rate; q-val: q value. (C-H) The indicated human tumor cell lines were treated with the indicated antibody (20 μg / ml) and increased concentrations of the HDAC inhibitor panobinostat for 24 hours. MICA / B surface levels were quantified by flow cytometry using PE-labeled 6D4 mAb (graph to the left of CH). Shedding MICA in the supernatant was quantified by sandwich ELISA (graphs to the right of C and D). (E~H)ELISA kits did not detect shedding MICA in these cell lines, possibly due to allele variant specificity or MICB shedding (ELISA was specific for MICA). Depending on the availability of such antibodies at the time of assay, 7C6 antibodies with different Fc regions were used. As previously reported, the Fc region of this antibody does not affect the inhibition of MICA / B shedding. Data are representative of 203 independent experiments. [Figure 15-2] Please refer to the explanation in Figure 15-1. [Figure 16]This study demonstrates that panobinostat did not inhibit the reconstitution of NSG mice with human NK cells. NSG mice were intravenously injected with 2 × 10⁶ in vitro-grown human NK cells from a healthy donor. Immediately after NK cell inoculation, the mice were treated with IL-2 (7.5 × 10⁴ units) to support NK cell survival. Mice were also administered panobinostat (10 mg / kg in PBS) or PBS as a control. After 24 hours, blood NK cells were analyzed by flow cytometry. (A) Number of circulating NK cells identified as CD45+CD56+CD3- viable cells. (B, C) Percentage of blood NK cells labeled with CD16a (B) or NKG2D (C) mAb. Data pooled from two independent experiments (A-C). [Figure 17] A summary of the melanoma metastases investigated is presented. ScRNA-seq analysis was performed on the top three cases (highlighted in red). Using other tumor samples, the tumor cell population was examined by flow cytometry for the expression of MHC class I and MICA / B proteins and NK cells. The locations of surgically resected metastases and prior treatment history are listed. [Figure 18]This study demonstrates that inactivation of the B2M gene enhances NK cell-mediated killing of human melanoma cells in the presence of a MICA / B mAb. (A) Verification of B2M gene inactivation efficiency. Control or B2M-KO human A375 melanoma cells were treated with the indicated concentrations of IFNγ for 24 hours, and HLA-A / B / C surface levels were quantified by flow cytometry. MFI = mean fluorescence intensity. (B-D) Control or B2M-KO human A375 melanoma cells were cultured for 24 hours with the specified concentrations of MICA / B (7C6-hIgG1) or isotype control antibody. Quantification of shedding MICA released by melanoma cells using sandwich ELISA (B). As previously reported (23), the 7C6 antibody did not interfere with the detection of soluble MICA by ELISA. MICA / B surface protein levels on control and B2M-KO melanoma cells were quantified by flow cytometry using PE-conjugated MICA / B mAb 6D4 (C). As previously reported (23), this mAb binds to the MICA / B α1-α2 domains and does not compete with 7C6 antibody. Histogram (D) is representative of the experiment shown in "C". (E) Effect of human NK cells on A375 melanoma cells dependent on MHC-I expression and MICA / B mAb treatment. GFP+ A375 melanoma cells (control or B2M-KO) were seeded in 96-well plates at a density of 5 × 10³ cells per well. After pretreatment of melanoma cells with 7C6-hIgG1 or isotype control mAb (20 μg / ml) for 24 hours, purified human NK cells were added at different effector-to-target ratios (0:1, 0.5:1, or 1:1). IL-2 (300 U / ml) was added to support NK cell survival. The number of GFP+A375 melanoma cells was quantified by imaging cytometry using a Nexcelom Celigo instrument at multiple time points over a 72-hour period. Data (A-E) are representative of three independent experiments. Statistical analysis was performed by two-way analysis of variance (ANOVA) with Bonferroni's multiple comparison test (E). *p<0.05, ***p<0.001. [Figure 19]This study demonstrates that MICA / B mAb treatment induces immunity against melanoma metastases with inactivating mutations in the B2m and Jak1 genes. (A) B16F10-MICA cells (control, B2m-KO, or Jak1-KO) were treated with IFNγ (10 ng / ml) or a solvent control (PBS) for 24 hours, and H-2Kb surface levels were analyzed by flow cytometry. (B) MICA / B mAb treatment against established metastases with inactivating mutations in the B2m or Jak1 genes. B16F10-MICA melanoma cells (7 × 10⁵ control, B2m-KO, or Jak1-KO tumor cells) were intravenously injected into B-cell-deficient (Ighm- / -) mice. On day 7, a subset of mice were euthanized to quantify metastases, and the remaining mice were treated with 7C6-mIgG2a or a control mAb (200 μg intraperitoneally administered on days 7, 8, and 12). On day 14, lung surface metastases were counted under a stereomicroscope. (C) Effect of MICA / B mAb treatment on survival of mice with B2m or Jak1-deficient melanoma metastases. WT mice (Ighm+ / +) were intravenously inoculated with 2 × 10⁵ control, B2m-KO, or Jak1-KO B16F10-MICA cells. Mice were administered 7C6-mIgG2a or isotype control mAbs on days 1 and 2, and mouse survival was recorded. Data (A) are representative of three independent experiments, or data pooled from three (B) or two (C) independent experiments. Statistical analysis was performed by two-sided unpaired Student's t-test (B) and log-rank (Mantel-Cox) test (C). *p<0.05, **p<0.01, ***p<0.001. [Figure 20]This study demonstrates that conventional MHC-I molecules expressed by lung cancer cells inhibit NK cells and reduce the effectiveness of MICA / B antibody treatment. (A) MHC-I expression by LLC1-MICA cells. Control or B2m-KO LLC1-MICA cells were stimulated with IFNγ (10 ng / ml) or solvent control (PBS) for 24 hours. Surface H-2Kb protein levels were quantified by flow cytometry. (B) MICA / B mAb treatment of lung metastases formed by LLC1 lung cancer cells. WTC57BL6 / J mice were intravenously inoculated with 1 × 10⁶ cells (1M) or 1.5 × 10⁶ cells (1.5M) of LLC1-MICA tumor cells (control or B2m-KO). Two days after tumor cell inoculation, mice were treated with the indicated mAb (200 μg administered intraperitoneally). Additional treatment was performed on day 3 and then weekly thereafter. Lung metastases were counted on day 14. (C) MICA / B mAb treatment of LLC1-MICA metastasis in mice reconstituted with allogeneic or syngeneic NK cells. Rag2- / -Il2rg- / - double knockout mice were injected with LLC1 cells and NK cells (2 × 10⁵ cells) derived from allogeneic or syngeneic CB6F1 / J or C56BL / 6 mice, respectively. Group 3 of Rag2- / -Il2rg- / - mice did not receive NK cells. LLC1-MICA tumor cells (7 × 10⁵) were injected intravenously. 24 hours after NK cell transfer. Mice were treated with the indicated antibody (200 μg) on ​​days 2 and 3 after tumor cell inoculation, and weekly thereafter. Metastasis was counted on day 14. Data (A) are representative of three independent experiments, or data pooled from three (B) or two (C) independent experiments. Statistical analysis was performed by two-sided unpaired Student's t-tests (B-C). *p<0.05, **p<0.01, ***p<0.001. [Figure 21]This study demonstrates that NK cells are essential for the treatment of B2m and Jak1-deficient melanoma metastases with MICA / B antibodies. (A) Wild-type (WT) C57BL / 6 mice were intravenously inoculated with 7 × 10⁵ B16F10-MICA cells (control, B2m-KO, or Jak1-KO). Mice were treated with 7C6-mIgG2a or isotype control mAb (200 μg) on ​​days 1, 2, and 7. CD8 T cell depletion was performed by injecting 100 μg of anti-CD8β, while NK cell depletion was performed by injecting 100 μg of anti-asialoGM1 (anti-asGM1) or anti-NK1.1. All depletion antibodies were administered on days 1, 0, and 7 after tumor cell inoculation. Control mice were administered isotype control antibodies. Lung surface metastases were quantified on day 14 after tumor inoculation. (B) Analysis of NK cell infiltration into lung tissue. Tumor injection and mAb treatment were performed as described in "A" using tumor cells expressing ZsGreen to enable identification by flow cytometry. Twelve days after tumor cell inoculation, mice were intravenously injected with APC-conjugated anti-CD45.2 antibody as previously reported (23) to differentiate blood and tissue-infiltrating NK cells. Lung-infiltrating NK cells were identified as CD3ε-TCRβ-NK1.1+CD49b+EOMES+ live cells, showing low staining with CD45.2-APC (intravenous injection) and high staining with CD45.2-PE-CY7 (added to cell suspension). The ratio of NK cells to ZsGreen+B16F10-MICA cells is shown. (C, D) Number of ZsGreen+B16F10-MICA cells (C) and lung-infiltrating NK cells (D) for the genotypes and treatment groups shown in the experiments described in (B). NK cells were differentiated from ILC1s using EOMES labeling. Data pooled from two independent experiments (A-D). Statistical analysis was performed using two-way ANOVA with Bonferroni post-hoc tests (A) or two-tailed unpaired Student t-tests (B-D). *p<0.05, **p, 0.01, ***p<0.001. [Figure 22]This study demonstrates that the combination of the HDAC inhibitor panobinostat and a MICA / B mAb enhances MICA / B surface levels on tumor cells. (A) Increased NKG2D ligand mRNA levels after treatment with panobinostat. A375 melanoma cells were treated with panobinostat (50 nM) for 24 hours, and mRNA was extracted for bulk RNA-seq. mRNA levels of NKG2D ligand and MHC class I genes are shown as the ratio (log2 factor change) between the panobinostat and PBS groups. (B) A375 melanoma cells were treated with panobinostat (50 nM) or a solvent control (PBS) for 24 hours, and the expression of the indicated genes was analyzed by RT-qPCR (3 times per condition). *P<0.05, **p<0.01, and ***p<0.001. Statistical analysis was performed using a two-sided unpaired Student's t-test with Welch's correction. Error bars represent the standard deviation of three technical replications. (C) Increase in MICA / B surface protein levels after treatment with panobinostat + MICA / B mAb. A375 melanoma cells were incubated for 24 hours with the indicated mAb (20 μg / ml) and increasing concentrations of panobinostat. MICA / B surface levels (left) and A375 cell viability (right) were quantified by flow cytometry. Shedding MICA was quantified by sandwich ELISA (center). (D) Representative histogram of the data shown in Figure 22C. (E) Short-term treatment of human melanoma cell lines with panobinostat + MICA / B mAb combination. The indicated melanoma cell lines were treated in vitro for 24 hours with the indicated mAb (20 μg / ml) + increasing concentrations of panobinostat. MICA / B surface levels were quantified by flow cytometry. Cell lines differed in basal and induced levels of MICA / B. These were arranged in order of MICA / B expression from low to high. The data are representative of three independent experiments (B, C, and E). [Figure 23]This study demonstrates that HDAC-MICA / B antibody combination therapy inhibits metastatic proliferation in NSG mice reconstituted with human NK cells. (A) In vivo synergistic effect of panobinostat + MICA / B mAb treatment on MICA / B surface protein levels during metastases formed by human melanoma cells. NSG mice were intravenously inoculated with 1 × 10⁶ ZsGreen+ A375 melanoma cells. Two weeks later, the mice were treated with the indicated mAb (200 μg) + / - panobinostat (10 mg / kg) for the following two days. 24 hours after the final treatment, MICA / B surface levels were analyzed in tumor cells (large, viable ZsGreen+, CD45- cells) during lung metastases. (B~C) NSG mice were reconstituted with purified human NK cells (2 × 10⁶ cells intravenously) grown in vitro. In vivo survival of NK cells was supported by co-administration of IL-2 (7.5 × 10⁴ units) by intraperitoneal injection. On day 1, mice were intravenously inoculated with control or B2M-KO A375 cells (5 × 10⁵). On days 2 and 3, mice were administered different doses of IL-2, the indicated mAb (200 μg) + / - 10 mg / kg panobinostat. On day 3, an additional dose of NK cells was administered. On day 14, the number of lung surface metastases was counted. Demonstration of experimental design (B) and quantification of lung surface metastases (C). Pooled data from two independent experiments (A and C). Statistical analysis was performed by two-sided unpaired Student's t-test (A) and two-way ANOVA, Bonferroni's post-hoc test (C). *p<0.05, **p<0.01, ***p<0.001. [Figure 24]This study describes the characteristics of B2M-deficient A375 melanoma cells and the inhibition of NK cell-mediated melanoma cell killing by MHC-1 recognition. (A) Verification of the efficiency of B2M gene inactivation in A375 melanoma cells. Control and B2M-KO cells were treated with IFNγ (1 ng / ml) or PBS at the indicated concentrations for 24 hours, and surface HLA-A / B / C protein levels were quantified by flow cytometry. (B) Control and B2M-KO A375 melanoma cells were treated with or without IFNγ (50 ng / ml) for 24 hours. Western blots (20 μg of total protein per lane) were probed with antibodies specific to B2M and tubulin. (C) NK cells isolated from healthy donors were cultured for 24 hours in the presence of 1000 U / ml of IL-2. Parental A375 melanoma cells were incubated with 7C6-hIgG1 or isotype control antibody (20 μg / ml) for 24 hours and then used in cytotoxic assays. Killing of A375 cells via NK cells was analyzed using a 4-hour 51Cr release assay. The indicated KIR or isotype control antibody was added to the coculture at 10 μg / ml. Data are representative of three independent experiments (A-C). Statistical analysis was performed by two-way ANOVA, Bonferroni's post-hoc test (C). ***p<0.001. [Figure 25] Characterization of the B16F10-MICA cell line and demonstration of the in vivo activity of MICA / B mAb. Control, B2m-KO, or Jak1-KO B16F10-MICA cells were treated with IFNγ at the indicated concentrations for 24 hours. (B) Surface levels of H2-Db were analyzed by flow cytometry. MFI = mean fluorescence intensity. (C) Control, B2M-KO, or Jak1-KO B16F10 melanoma cells were treated with or without IFNγ (50 ng / ml) for 24 hours. Western blots (20 μg of total protein per lane) were probed with antibodies specific to B2M, JAK1, or GAPDH (loading control). Data representative of three independent experiments. [Figure 26]This study demonstrates that B2m-KO and Jak1-KO B16F10-MICA cells are resistant to CD8 T cell-mediated cytotoxicity. Control, B2m-KO, and Jak1-KO B16F10 melanoma cells were pulsed overnight with Ova peptide (10 nM), washed, and added to 96-well plates (5,000 cells per well). Naive OT-I T cells were added at different effector-to-target ratios (1:1, 2:1, and 5:1; the 0:1 condition did not contain T cells). Cells were co-cultured for 48 hours (8–10 times for each condition). Wells were then washed to remove T cells and tumor dead cells, and the number of attached living tumor cells was counted using a Celigo Image Cytometer. Statistical significance was determined using multiple t-tests. Error bars represent standard deviation. ***p<0.0001. [Figure 27] The following describes the characterization of the control and B2m-KO LLC1-MICA cell lines. (A) Control and B2m-KO LLC1-MICA cell lines were cultured in the indicated concentrations of IFNγ for 24 hours, and H2-Db surface expression was analyzed by flow cytometry. (B) Control or B2m-KO LLC1-MICA cells were treated with or without IFNγ (50 ng / ml) for 24 hours. Western blots (20 μg of total protein per lane) were probed with antibodies specific to B2M or tubulin (loading control). [Figure 28]This study demonstrates the in vivo efficacy of the 7C6 antibody in a B16F10 metastasis model. (A) B-cell-deficient (Ighm- / -) mice were intravenously inoculated with 7 × 10⁵ control, B2m-KO, or Jak1-KO B16F10-MICA cells. Once metastasis was established (day 7), the mice were treated with MICA / B or isotype control mAb (200 μg on days 7, 8, and 12). Shedding MICA in plasma samples was quantified using sandwich ELISA. Data were pooled from two independent experiments. Statistical analysis was performed by two-way ANOVA, Bonferroni's post-hoc test. ***p<0.001. (B) WT mice were intravenously inoculated with control, B2m-KO, or Jak1-KO B16F10-MICA cells and treated with the indicated antibody as shown in Figures 21A and B. Surface expression of NKG2D and CD16 receptors was analyzed in lung-infiltrating NK cells by flow cytometry. Data were pooled from two independent experiments. *p<0.05. Calculated using a two-sided unpaired Student's t-test. [Figure 29] This shows the effect of panobinostat on gene expression in A375 cells. (A-B) A375 cells were treated with panobinostat (50 nM) or PBS for 24 hours and analyzed by bulk RNA-seq as shown in Figure 22A. Major genes differentially expressed in cells treated with panobinostat or solvent control (A), and major immunological pathways upregulated with panobinostat compared to A375 cells treated with control (B). FDR: false positive rate; q-val: q value. [Figure 30] This shows the effect of panobinostat treatment on MICA / B expression in melanoma cells. A representative histogram of data for the primary melanoma cell line shown in Figure 22E. [Figure 31]This study demonstrates the effects of panobinostat and 7C6 antibody on MICA / B expression in a diverse panel of tumor cell lines. (A-F) Human tumor cell lines were treated for 24 hours with the indicated antibody (20 μg / ml) and increased concentrations of the HDAC inhibitor panobinostat. MICA / B surface levels were quantified by flow cytometry using PE-labeled 6D4 mAb (left-hand graphs for A and B, and graphs C-F). Shedding MICA in the supernatant was quantified by sandwich ELISA (right-hand graphs for A and B). Based on the availability of such antibodies at the time of assay, 7C6 antibodies with different Fc regions were used. As previously reported, the Fc region of this antibody does not affect the inhibition of MICA / B shedding (Andrade et al., Science 359, 1537-1542 (2018)). Data are representative of three independent experiments. [Figure 32] This study demonstrates the specificity of the ELISA assay for MICA compared to MICB. Supernatants of B16F10 cell lines transduced with human MICA (allele 009) or MICB (allele 005) cDNA were analyzed using the ELISA assay for MICA (Abcam, Ab59569) used throughout this study. These B16F10 cell lines have been previously described (Andrade et al., Science 359, 1537-1542 (2018)). ELISA detected soluble MICA shed by B16F10-MICA cell lines, but not shed MICB released by B16F10-MICB cells. [Figure 33]This study demonstrates that panobinostat did not inhibit the reconstitution of NSG mice with human NK cells, but rather acted synergistically with 7C6 mAb to enhance surface MICA / B expression on melanoma metastases. NSG mice were intravenously injected with 2 × 10⁶ in vitro-grown human NK cells from a healthy donor. Immediately after NK cell inoculation, the mice were treated with IL-2 (7.5 × 10⁴ units) to support NK cell survival. Mice were also administered panobinostat (10 mg / kg in PBS) or PBS as a control. After 24 hours, blood NK cells were analyzed by flow cytometry. (A) Number of circulating NK cells identified as CD45+CD56+CD3- viable cells. (B, C) Percentage of blood NK cells labeled with CD16a(B) or NKG2D(C) mAb. Data pooled from two independent experiments (A-C). (D) Representative histogram of the data shown in Figure 23A. [Figure 34] This shows the genes and their corresponding species and sequences. [Modes for carrying out the invention]

[0027] Detailed explanation cytotoxic T cells Resistance to cytotoxic T cells can arise from mutations in many pathways. For example, resistance to cytotoxic T cells is often mediated by loss of MHC class I expression or IFNγ signaling in tumor cells, such as mutations in the B2M or JAK1 genes. Activated NK cells can target such resistant tumors, Suitable NK cell-based strategies This has not yet been developed. The embodiments described herein address this limitation. Specifically, it has been shown that B2M and JAK1-deficient metastases become targets of NK cells after treatment with mAbs that block MICA / B shedding, a frequent evasion mechanism in human cancer. An exemplary mAb can be found in WO2018217688A1 (which is incorporated herein by reference in its entirety).

[0028] Furthermore, single-cell analysis of NK cells in human melanoma metastases, including patients whose disease progressed after checkpoint blockade, identified key transcriptional differences between tumor-infiltrating NK cells and circulating NK cells. NK cells are present in most human melanoma metastases, including patients who have failed therapy with PD-1 or CTLA-4 mAbs. These cells possess transcriptional programs that reflect important functions such as cytotoxicity, and secrete chemokines that recruit key immune cell populations required for T-cell-mediated tumor immunity, such as XCL1 and XCL2, which recruit dendritic cells expressing the associated receptor (XCR1).

[0029] Finally, the gene expression programs of the seven tumor-infiltrating NK cell clusters exhibit significant specialization, including cytotoxicity and chemokine secretion. Therefore, NK cell-based immunotherapy offers an opportunity to target tumors with mutations that confer resistance to cytotoxic T cells.

[0030] Abbreviations and definitions A detailed description of one or more embodiments is provided herein. However, these embodiments can be embodied in various forms. Therefore, the specific details disclosed herein should not be construed as limitations, but rather as representative grounds for the claims and for teaching those skilled in the art to use the embodiments described herein in any suitable manner.

[0031] The singular forms “a,” “an,” and “the” include multiple references unless the context explicitly indicates otherwise. The use of the words “a” or “an” in the claims and / or specification with the term “to comprise” may mean “one,” but also coincides with the meanings of “one or more,” “at least one,” and “one or more.”

[0032] Whenever any of the phrases "for example," "etc.," or "including" are used herein, it is understood that they are always accompanied by the phrase "without limitation" unless otherwise explicitly stated. Similarly, "for example," "exemplary," etc., are understood to be non-limiting.

[0033] The term "substantially" allows for deviations from descriptive terms that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term "substantially," even if the term is not explicitly listed.

[0034] Terms such as “comprising,” “including,” “having,” and “involving” (as well as “comprises,” “includes,” “has,” and “involves”) are used interchangeably and have the same meaning. Specifically, each term is defined in accordance with the general U.S. Patent Law definition of “comprising,” and is therefore interpreted as an open term meaning “at least the following,” and also as not excluding any additional features, limitations, aspects, etc. Thus, for example, “a process comprising steps a, b, and c” means that the process comprises at least steps a, b, and c. Whenever the terms “a” or “an” are used, they are understood to mean “one or more,” unless such interpretation is meaningless in the context.

[0035] As used herein, the term “approximately” means roughly, roughly, about, or within that range. When the term “approximately” is used with a numerical range, it modifies that range by extending the upper and lower boundaries of the stated numerical value. Generally, the term “approximately” is used herein to qualify numerical values ​​that are above and below a stated value by a 20 percent above or below (higher or lower) variance.

[0036] Treatment method The embodiments described herein relate to methods for treating cell proliferation disorders such as cancer. More specifically, the embodiments described herein relate to methods for treating checkpoint blockade-resistant cancers such as cancers resistant to cytotoxic T cells.

[0037] The terms “cancer” and “malignant” typically refer to or can describe a physiological condition in mammals characterized by uncontrolled cell growth and one of several characteristic structural and / or molecular features. “Cancerous cells” are understood as cells that possess specific structural properties, lack differentiation, and are often capable of invasion and metastasis. (See DeVita, V. et al. (eds.), 2001, Cancer Principles and Practice of Oncology, 6th Ed., Lippincott Williams & Wilkins, Philadelphia, PA). The term cancer includes, for example, cancers of the female reproductive organs, including ovarian cancer, cervical cancer and uterine cancer; lung cancer; breast cancer; renal cell carcinoma; Hodgkin lymphoma; non-Hodgkin lymphoma; cancers of the genitourinary system, including, for example, kidney cancer, prostate cancer, bladder cancer and urethral cancer; head and neck cancer; liver cancer; cancers of the gastrointestinal system, including, for example, stomach cancer, esophageal cancer, small intestine cancer or colon cancer; cancers of the biliary tract; pancreatic cancer; cancers of the male reproductive system, including, for example, testicular cancer; gestational trophoblastic disease; cancers of the endocrine system, including, for example, thyroid cancer, parathyroid cancer, adrenal cancer, cancerous tumors, insulinoma and PNET tumors; sarcomas, including, for example, Ewing's sarcoma, osteosarcoma, liposarcoma, leiomyosarcoma and rhabdomyosarcoma; mesothelioma; skin cancer; melanoma; cancers of the central nervous system; childhood cancers; and cancers of the hematopoietic system, including, for example, all forms of leukemia, myelodysplastic syndromes, myeloproliferative disorders and multiple myeloma. Cancer also includes urological cancers such as bladder cancer; cancers of the bladder, breast, neck, bile duct, colorectal, esophageal, stomach, head and neck, kidney, liver, and lung; ’This may include nasopharyngeal, ovarian, pancreatic / gallbladder, prostate, and thyroid cancers; musculoskeletal cancers such as osteosarcoma, synovial sarcoma, and rhabdomyosarcoma; soft tissue sarcomas such as MFH / fibrosarcoma, leiomyosarcoma, and Kaposi's sarcoma; hematopoietic malignancies such as multiple myeloma, lymphoma, adult T-cell leukemia, acute myeloid leukemia, and chronic myeloid leukemia; and other neoplasms such as glioblastoma, astrocytoma, melanoma, mesothelioma, and Wilms' tumor (Birchmeier et al., Nat Rev MoI Cell Bio 2003 4(12):912-925).

[0038] The terms "cell proliferation disorder" and "proliferative disorder" can refer to disorders associated with a certain degree of abnormal cell proliferation.

[0039] The term "tumor" can refer to the growth and proliferation of all neoplastic cells, whether malignant or benign, as well as all precancerous and cancerous cells and tissues.

[0040] Unless otherwise indicated by the context, the term “cancer” as used herein may refer to cancer, a cell proliferation disorder, or a tumor. Similarly, unless otherwise indicated by the context, the term “cancer cell” may refer to a cell of cancer, a cell proliferation disorder, or a tumor.

[0041] Unless otherwise indicated by the context, the terms “cancer,” “cell proliferation disorder,” or “tumor” can be used interchangeably.

[0042] The terms “to treat” or “treatment” refer to both therapeutic actions and preventive or protective measures, the desired outcome of which is to prevent, slow (reduce), or reverse undesirable physiological changes or impairments, such as cancer progression. Beneficial or desirable clinical outcomes include, but are not limited to, symptom relief, reduction in disease severity, a stable (i.e., non-worsening) state of the disease, delayed or slowed disease progression, improvement or relief of the condition, and remission (partial or total), whether detectable or not. “Treatment” means extending survival time compared to the survival rate expected without treatment. Those who require treatment include those who already have a condition or impairment, as well as those who are susceptible to a condition or impairment, or those who need to prevent a condition or impairment.

[0043] For example, in the context of cancer, the term “to treat” may include any or all of the following: preventing the growth, proliferation, or metastasis of tumor cells, cancer cells, or tumors; preventing the replication of tumor cells or cancer cells, reducing the overall tumor burden, or reducing the number of cancer cells, or improving one or more symptoms associated with the disease.

[0044] The embodiments described herein provide both prophylactic and therapeutic methods for treating subjects at risk of or susceptible to cancer or other cell proliferation-related diseases or disorders. For example, the methods are used to treat, prevent, or alleviate the signs of cancer. In one embodiment, the methods are used to treat, prevent, or alleviate the signs of solid tumors. Non-limiting examples of other tumors that can be treated by the embodiments described herein include lung cancer, ovarian cancer, prostate cancer, colon cancer, bladder cancer, kidney cancer, breast cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, or stomach cancer. In addition, the methods of the embodiments described herein can be used to treat hematological cancers such as leukemia and lymphomas such as Hodgkin's lymphoma. Alternatively, the methods can be used to treat, prevent, or alleviate the symptoms of metastatic cancer.

[0045] In one embodiment, the embodiments described herein provide a method for treating subjects who have, are at risk of, or are susceptible to cancer that are resistant to cytotoxic T cells and / or T cell-based therapies (such as checkpoint blockade). For example, cytotoxic T cells play a central role in the effectiveness of checkpoint blockade based on their ability to recognize tumor-derived peptides bound to major histocompatibility complex class I (MHC-I) proteins. When such MHC-I-peptide complexes are recognized by the T cell receptor (TCR), T cells release interferon-γ (IFNγ), inhibiting tumor cell proliferation and enhancing MHC-I protein expression in both tumor cells and dendritic cells. Therefore, resistance to checkpoint blockade is often mediated by the loss of MHC-I expression by tumor cells due to either mutations in key genes in the MHC-I (B2M, TAP1, TAP2, and other genes) or IFNγ (JAK1, JAK2) pathways or epigenetic silencing. When the number of newly generated antigens is low or lost, tumor immunity mediated by cytotoxic T cells is also reduced.

[0046] Accordingly, in one embodiment, the embodiments described herein provide a method for preventing, treating, or mitigating a symptomatic cancer or cytoproliferative disorder or impairment of a target by administering a monoclonal antibody or a fragment or derivative thereof (e.g., scFv antibody or bispecific antibody) that activates an antitumor NK cell response to the target. The activated NK cell response can be determined, for example, by analysis of tumor biopsies (e.g., comparison of pre-treatment and post-treatment biopsies). Such analyses include, for example, multicolor immunofluorescence assays for granzyme A and perforin, along with NK cell markers such as NKp46 and CD56.

[0047] In one embodiment, an anti-MICA / B antibody can be administered to the target. Many human cancers express MHC-I polypeptide-associated sequence A (MICA) and MICB (MICA / B) proteins, which function as ligands for the activated NK group 2D (NKG2D) receptor on subpopulations of NK cells and T cells. However, tumors often evade NKG2D receptor-mediated tumor immunity by shedding of MICA / B protein through proteolysis. The α3 domain of MICA / B is essential for shedding, and monoclonal antibodies that bind to this domain can inhibit MICA / B shedding and induce NK cell-mediated tumor immunity. Increased density of MICA / B protein on tumor cells enhanced NKG2D receptor-mediated activation in NK cells, and the Fc segment of the tumor conjugate antibody also activated NK cells via the CD16Fc receptor. Treatment with such MICA / B antibodies significantly altered tumor-infiltrating NK cells into a highly cytotoxic state.

[0048] Non-limiting examples of anti-MICA / B antibodies that may be used in embodiments of this specification include any antibody specific to anti-MICA / B. In one embodiment, the antibody is specific to the α3 domain of MICA / B. See, for example, WO2018217688 (which is incorporated herein by reference in its entirety).

[0049] Accordingly, the embodiments described herein include antibodies such as monoclonal antibodies, such as human monoclonal antibodies, that specifically bind to the MHC class I polypeptide-related sequence A (MICA) and / or B (MICB)a3 domain, which is a shedding site due to proteolysis, and have desirable functional properties. These properties include inhibition of MICA / B shedding by human cancer cells, stabilization of cell surface MICA / B for NK cell recognition, and activation of both the NKG2D receptor and CD16 Fc receptor on NK cells. MICA antibodies having these properties restore immune activation induced by stress molecules that activate cytotoxic lymphocytes.

[0050] In some embodiments, the monoclonal antibody or its antigen-binding moiety that binds to MICA and / or MICB includes a heavy chain and a light chain variable region, and the heavy chain CDR1, CDR2, and CDR3 sequences each include SEQ ID NOs. 1 to 3 as shown in Table 1. In some embodiments, the monoclonal antibody or its antigen-binding moiety includes a heavy chain and a light chain variable region, and the heavy chain CDR1, CDR2, and CDR3 sequences each include SEQ ID NOs. 4 to 6 as shown in Table 1. In some embodiments, the monoclonal antibody or its antigen-binding moiety that binds to MICA and / or MICB includes a heavy chain and a light chain variable region, heavy chain CDR1, CDR2, and CDR3 sequences each including SEQ ID NOs. 1 to 3, and light chain CDR1, CDR2, and CDR3 sequences each including SEQ ID NOs. 4 to 6.

[0051] Provided herein are isolated monoclonal antibodies or their antigen-binding moieties, which are conjugated to MICA and / or MICB and include heavy chain and light chain variable regions, wherein the heavy chain variable region includes an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 7.

[0052] Provided herein are isolated monoclonal antibodies or their antigen-binding moieties, which are conjugated to MICA and / or MICB and include heavy chain and light chain variable regions, wherein the light chain variable region includes an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 8. [Table 1] TIFF2026048806000003.tif230154TIFF2026048806000004.tif42154

[0053] For example, the antibody may include clones 7C6 (e.g., 7C6-hIgG1), 6F11, and / or 1C2.

[0054] In relevant embodiments, embodiments of this specification may include nucleic acids encoding the heavy and / or light chain variable regions of an anti-MICA and / or anti-MICB antibody, or the antigen-binding portion thereof, an expression vector comprising the nucleic acid molecule, and cells converted by the expression vector. Embodiments of this specification may further include a method for preparing an anti-MICA and / or anti-MICB antibody, comprising expressing the anti-MICA and / or anti-MICB antibody in cells and isolating the antibody from the cells.

[0055] Compositions comprising anti-MICA and / or anti-MICB antibodies, or their antigen-binding moieties, and carriers are also provided herein. Immunoconjugates comprising the anti-MICA and / or anti-MICB antibodies described herein, linked to a drug, are also provided herein. Kits comprising anti-MICA and / or anti-MICB antibodies, or their antigen-binding moieties, and instructions for use are also provided herein.

[0056] In embodiments, the antibody may be administered in a therapeutically effective dose, as further described herein.

[0057] The terms “patient” or “subject” are interchangeable. Examples of “patient” or “subject” include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, cats, birds, and poultry. In exemplary embodiments, the patient is a human.

[0058] The term "cancer patient" can refer to an individual diagnosed with cancer. Examples of cancer include solid tumors and tumors of other tissue organs such as breast, ovary, prostate, lung, kidney, stomach, colon, testis, head and neck, pancreas, brain, melanoma, as well as hematological malignancies such as lymphoma and leukemia (including acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma), tumors of the brain and central nervous system (e.g., tumors of the meninges, brain, spinal cord, cranial nerves and other parts of the CNS, e.g., glioblastoma or medulloblastoma); head and / or neck cancer, breast cancer, circulatory cancer. Tumors of the cardinal system (e.g., heart, mediastinum and pleura, and other intrathoracic organs, vascular tumors, and tumor-associated vascular tissue); tumors of the hematological and lymphatic systems (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt lymphoma, AIDS-associated lymphoma, malignant immunoproliferative disorders, multiple myeloma, and malignant plasmacytoplasms, lymphocytic leukemia, myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other leukemias of specific cell types, leukemias of unspecified cell types, lymphoid unspecified malignant neoplasms, hematopoiesis and related tissues (diffuse) Tumors of the excretory system (e.g., large cell lymphoma, T-cell lymphoma, or cutaneous T-cell lymphoma); tumors of the excretory system (e.g., kidneys, renal pelvis, ureters, bladder, and other parts of the urinary tract); tumors of the gastrointestinal tract (e.g., esophagus, stomach, small intestine, colon, colorectum, rectosigmoid junction, rectum, anus, and anal canal); tumors of the liver and intrahepatic bile ducts, gallbladder, and other parts of the biliary tract, pancreas, and other digestive organs; tumors of the oral cavity (e.g., lips, tongue, gums, floor of mouth, palate, parotid gland, salivary gland, tonsils, oropharynx, nasopharynx, pear-shaped fossa, hypopharynx, and other parts of the oral cavity); tumors of the reproductive system (e.g., the vulva, vagina, cervix, uterus, ovaries, and other sites related to the female reproductive organs, the placenta, penis, prostate, testes, and other sites related to the male reproductive organs); tumors of the airway (e.g., the nasal cavity, middle ear, sinuses, larynx, trachea, bronchi, and lungs (such as small cell lung cancer and non-small cell lung cancer)); tumors of the skeletal system (e.g., bones and articular cartilage of the limbs, osteoarticular cartilage, and other sites); tumors of the skin (e.g., malignant melanoma of the skin, non-melanoma skin cancer, basal cell carcinoma of the skin, squamous cell carcinoma of the skin, mesothelioma, Kaposi's sarcoma);Furthermore, these include, but are not limited to, tumors of other tissues including the peripheral and autonomic nervous systems, connective tissue and soft tissue, retroperitoneum and peritoneum, eyes, thyroid gland, adrenal gland, and other endocrine glands and related structures, secondary and unspecified malignant neoplasms of lymph nodes, secondary malignant neoplasms of the respiratory and digestive systems, and secondary malignant neoplasms of other sites. In one embodiment, the cancers are melanoma, lung cancer such as non-small cell lung cancer, prostate cancer, renal cell carcinoma, or colorectal cancer.

[0059] Subjects at risk of or susceptible to cancer or cell proliferation-related disorders or impairments include individuals with a family history of cancer or those exposed to drugs known or suspected to cause cancer. Preventive agents may be administered before the onset of cancerous signs to prevent the disease or, alternatively, delay its progression. Therapeutic agents may be administered after a patient has been diagnosed with cancer to reverse, delay, or halt the progression of the disease.

[0060] In one embodiment, the embodiments described herein provide a method for preventing, treating, or mitigating a symptomatic cancer or cytoproliferative disorder or impairment of a target by administering a monoclonal antibody or a fragment or derivative thereof (e.g., scFv antibody or bispecific antibody) that activates an antitumor NK cell response to the target. Where herein, the term “treating” may include any or all of the following: preventing the growth, proliferation, or metastasis of tumor cells, cancer cells, or tumors; preventing the replication of tumor cells or cancer cells, reducing the overall tumor volume, or reducing the number of cancer cells (e.g., by inducing cytolysis); or improving one or more symptoms associated with the disease.

[0061] As used herein, “metastasis” can refer to the spread of a malignant tumor far beyond its point of origin. Cancer cells can metastasize through the bloodstream, lymphatic system, body cavities, or any combination thereof.

[0062] The term "cell proliferation" can refer to a relative increase in the number of cells, whether, for example, by cell division or by the inhibition of cell death (e.g., necrosis, apoptosis). Inappropriate cell proliferation may result, for example, from improper cell growth, excessive cell division, cell division (i.e., mitosis), and / or improper cell survival.

[0063] The term "cell lysis" can refer to the breakdown of a cell due to the destruction of its wall or membrane. For example, cell destruction (i.e., lysis) may be due to viral, chemical, enzymatic, or osmotic mechanisms that impair the cell's integrity.

[0064] In another aspect, embodiments described herein provide methods for sensitizing cancer or cancer cells to the anti-cancer effects of natural killer cells. Natural killer cells (also known as NK cells, K cells, and / or killer cells) are a type of lymphocyte that plays a role in the host rejection of tumors and virtually infected cells. Natural killer (NK) cells recognize tumor cells by a molecular mechanism substantially different from that required by cytotoxic T cells. NK cell recognition by tumor cells is mediated by ligands associated with malignant transformation, such as DNA damage and cellular stress. While we do not wish to be constrained by theory, tumors resistant to cytotoxic T cells may respond to NK cell-based immunotherapy approaches.

[0065] The activation of natural killer (NK) cells is determined by the balance between negative signals provided by inhibitory receptors during interaction with major histocompatibility complex (MHC) class I molecules and positive signals promoted by various activating receptors. NK cells express a broad range of activating receptors that cooperate in promoting the innate cytotoxic response. These receptors include innate cytotoxic receptors (NCRs), the SLAM family receptor member 2B4, the Ig-like receptor DNAX accessory molecule-1 (DNAM1), and the lectin-like receptor natural killer receptor group 2, member D (NKG2D), NKp46, and CD16a (receptor for IgG).

[0066] NKG2D is a potent activating receptor constitutively expressed on all NK cells, but also on immutable natural killer T (NKT) cells, as well as CD8 + NKG2D is also present on subsets of T cells, such as αβ T cells and γδ T cells. While NKG2D can bind to several ligands that are poorly expressed on healthy cells, it is upregulated upon stimulation in cancer or viral infection situations. Several in vivo models demonstrate the fundamental role of the NKG2D receptor in the NK cell response to abnormal cells.

[0067] The most prominent feature of NKG2D receptors lies in their ability to bind to a large repertoire of self proteins induced by stress pathways, and thus mediate "induced self" recognition. In humans, these ligands include six members of the highly polymorphic MHC class I-related proteins (MIC) A and MICB, as well as UL16-binding protein (ULBP). NKG2D ligands (NKG2DL) are absent on the surface of most healthy tissues but are upregulated under stress conditions such as mitosis, viral infection, and cancer by several pathways acting primarily at the transcriptional and post-transcriptional levels.

[0068] Many human cancers express MHC-I polypeptide-associated sequence A (MICA) and MICB (MICA / B) proteins, which function as ligands for the activated NK group 2D (NKG2D) receptor on subpopulations of NK cells and T cells. However, tumors often evade NKG2D receptor-mediated tumor immunity by shedding of MICA / B proteins through proteolysis. The α3 domain of MICA / B is essential for shedding, and monoclonal antibodies that bind to this domain can inhibit MICA / B shedding and induce NK cell-mediated tumor immunity. Increased density of MICA / B protein on tumor cells enhanced NKG2D receptor-mediated activation in NK cells. Treatment with such MICA / B antibodies significantly altered tumor-infiltrating NK cells into a highly cytotoxic state.

[0069] In addition to NKG2D, the Fc segment of the tumor conjugate antibody also activated NK cells via the CD16Fc receptor. Upon IgG binding, CD16 initiates a signaling cascade, generating diverse responses including antibody-dependent cell-mediated cytotoxicity (ADCC), degranulation, and cytokine secretion. CD16 is expressed on macrophages, natural killer (NK) cells, and neutrophils. In this context, its expression on NK cells is particularly relevant.

[0070] As illustrated by the examples, MHC class I-deficient tumor cells (i.e., tumors resistant to cytotoxic T cells) are efficiently killed in the presence of activators such as MICA antibodies that activate NK cells via NKG2D and CD16. Therefore, tumors resistant to cytotoxic T cells can be targeted by NK cell activation via NKG2D and CD16.

[0071] The embodiments described herein include activators and their use to activate anti-cancer NK cell responses. The term “activator” can refer to any agent that enables the activation of NK cells. For example, an activator can activate NK cells via NKG2D and / or CD16. For example, an activator may be a polynucleotide, polypeptide, biologic, cytokine (e.g., IL-15), or small molecule. Molecular markers of NK cell activation are known in the art and include high levels of expression of cytotoxic proteins (perforin, granzyme A) and cytokines (e.g., IFN-gamma). In one embodiment, the activator is an antibody or fragment thereof, such as an anti-MICA / B antibody. In one embodiment, the activator is an antibody or fragment thereof, such as those listed in Table 1.

[0072] The embodiments described herein are useful for preventing, treating, or mitigating the symptoms of drug-resistant cancer. As used herein, “drug-resistant” or “refractory” cancer, cell proliferation disorder, or tumor may refer to a refractory cancer, cell proliferation disorder, or tumor in which cells exhibit reduced cytotoxicity to a drug compared to equally sensitive cells. For example, tumors and / or cancer cells may exhibit resistance to cytotoxic T cells, such as those induced by checkpoint blockade therapy.

[0073] For example, aspects of the embodiments described herein are useful for preventing, treating, or mitigating symptoms of checkpoint blockade, such as cancers resistant to cytotoxic T cells and cancers resistant to immunotherapies that activate T cells. For example, cancers may be resistant to anti-CTLA4, anti-PD1, and / or anti-PDL1 antibodies. Immune checkpoints are inhibitory pathways that slow or halt the immune response, preventing excessive tissue damage due to uncontrolled activity of immune cells. "Checkpoint blockade (CPB) therapy" refers to therapies that inhibit inhibitory pathways and enable broader immune activity. Such therapies may include treatment with any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof, that inhibits the inhibitory pathway. "Checkpoint blockade therapy" can also refer to stimulation of an existing immune response. In certain embodiments, CPB therapy is therapy with an inhibitor of the programmed death-1 (PD-1) pathway, such as an anti-PD1 antibody, e.g., nivolumab. In other embodiments, CPB therapy is therapy using an anticytotoxic T lymphocyte-associated antigen (CTLA-4) antibody. In additional embodiments, CPB therapy targets another member of the CD28CTLA4 Ig superfamily, such as BTLA, LAG3, ICOS, PDL1, or KIR (Page et al., Annual Review of Medicine 65:27 (2014)). In yet another embodiment, CPB therapy targets a member of the TNFR superfamily, such as CD40, OX40, CD137, GITR, CD27, or TEVI-3. In some cases, targeting of checkpoint inhibitors is achieved using inhibitory antibodies or similar molecules. In other cases, it is achieved with an agonist of the target. Examples of this class include the stimulating targets OX40 and GITR. For example, CPB therapy includes therapy with antibodies selected from anti-CTLA4, anti-PD1, anti-PDL1 antibodies and combinations thereof.

[0074] The nature of the CPB therapy is not important for the embodiments described herein, and examples of suitable agents are described herein. In embodiments, the CPB therapy is a therapy with an antibody selected from anti-CTLA4, anti-PD1, anti-PDL1 antibodies, and combinations thereof. An exemplary anti-CTLA4 antibody is ipilimumab. An exemplary anti-PD1 antibody is nivolumab. A significant number of cancer patients undergoing CPB therapy become resistant to the CPB therapy after an initial period of regression, resulting in tumor progression. Resistance to CPB therapy is associated, among other things, with reduced expression of genes related to antigen processing pathways or their products, such as B2M and Jak1. As described herein, after treatment with an mAb that blocks MICA / B shedding, a frequent avoidance mechanism in human cancer, B2M- and JAK1-deficient metastases became targets for NK cells.

[0075] Aspects of the embodiments described herein are useful for preventing, treating, or alleviating the symptoms of cancer that is resistant to cytotoxic T cells. "T cells" refers to T lymphocytes and includes, but is not limited to, γ:δ + T cells, NK T cells, CD4+ T cells, and CD8+ T cells. Examples of CD4+ T cells include T H 0, T H 1, T H 2 cells, as well as regulatory T cells (T reg ). There are at least three types of regulatory T cells: CD4+CD25+ T reg , CD25 - T H 3 T reg , and CD25 -TR 1 T reg . "Cytotoxic T cells" refers to T cells that can kill other cells. Most cytotoxic T cells are CD8+ MHC class I-restricted T cells, although some cytotoxic T cells are CD4+.

[0076] Most T cell receptors (TCRs) recognize MHC:antigen complexes, which are complexes of peptide antigens (or peptide fragments of antigens) bound to MHC molecules. TCRs are involved in the antigen specificity of each T cell and the limitation of antigen recognition by MHC class I and MHC class II molecules. TCRs derived from CD4+ T cells are restricted to MHC class II; that is, TCRs derived from CD4+ T cells recognize only antigens represented by MHC class II molecules. TCRs derived from CD8+ T cells are restricted to MHC class I and recognize only antigens represented by MHC class I molecules.

[0077] While we do not wish to be constrained by theory, tumors resistant to cytotoxic T cells may respond to NK cell-based immunotherapy approaches. In fact, the loss of MHC-I expression by tumor cells (also known as MHC class I-deficient cancers) increases sensitivity to NK cells because the MHC-I protein functions as a ligand for inhibitory NK cell receptors. Therefore, aspects of the embodiments described herein can also be considered useful for treating, preventing, or mitigating the symptoms of MHC class I-deficient cancers.

[0078] Aspects of the embodiments described herein may also be considered useful for treating, preventing, or mitigating symptoms of cancer resistant to IFN-gamma released by T cells. For example, aspects of the embodiments described herein may include activating NK cells against tumors having one or more loss mutations in the gamma interferon pathway.

[0079] The embodiments described herein may also be considered useful for treating, preventing, or mitigating symptoms of cancer that is MHC class I deficient and resistant to IFN-gamma.

[0080] Furthermore, aspects of the embodiments described herein are useful for preventing, treating, or alleviating symptoms of cancer resistant to cancer immunotherapy. Cancer immunotherapy can refer to a diverse set of therapeutic strategies designed to induce the patient's own immune system to fight tumors.

[0081] Several types of immunotherapy are used to treat cancer. These therapies can either help the immune system directly attack cancer cells or stimulate the immune system in a more general way.

[0082] One type of immunotherapy that helps the immune system act directly against cancer is checkpoint blockers, which are drugs that help the immune system respond more strongly to tumors. These drugs work by releasing inhibitory pathways that prevent T cells from killing cancer cells. These drugs do not directly target the tumor. Instead, they interfere with the ability of cancer cells to evade the immune system's attack. Examples of such immunotherapies include checkpoint blocker inhibitors such as anti-CTLA4, anti-PD1, and / or anti-PDL1. Adoptive cell transfer is a treatment that attempts to enhance the target T cells' natural ability to fight cancer. In this treatment, T cells are taken from the target tumor, then the most cancer-active T cells are grown in large quantities in the laboratory, and then administered back to the target. Adoptive cell transfer may include a therapy called CAR T cell therapy, which uses T cells designed in the laboratory to target specific cancers. Monoclonal antibodies, also known as therapeutic antibodies, are immune system proteins produced in the laboratory. These antibodies are designed to attach to specific targets found on cancer cells. Some monoclonal antibodies mark cancer cells so that they are better found and destroyed by the immune system. These are a type of immunotherapy. Other monoclonal antibodies used in cancer treatment do not evoke a response from the immune system. Such monoclonal antibodies are considered targeted therapy, not immunotherapy. Therapeutic vaccines work against cancer by enhancing the immune system's response to cancer cells.

[0083] Other types of immunotherapy that enhance or stimulate the body's immune response to fight cancer include cytokines, which are proteins produced by the body's cells. They play a crucial role in the body's normal immune response and the immune system's ability to respond to cancer. Two main types of cytokines used to treat cancer are called interferons and interleukins.

[0084] Another aspect of the embodiments described herein relates to compositions and methods for sensitizing cells to anticancer agents. "Sensitizing" can refer to the ability of an activator to increase the sensitivity of a designated system, such as cells or tumors. This can include modifying (i.e., sensitizing) cells to make them more responsive to anticancer compounds or regimens that were previously unresponsive or less responsive. Sensitizing and "more sensitive" can also include increasing the sensitivity of cells or tumors to such an extent that exposure to a substance that was not previously killing results in cell death.

[0085] In embodiments, one or more activators are administered to a subject to treat, prevent, or alleviate the symptoms of cancer. The activators may be provided in a pharmaceutically acceptable composition, which may be in any form that allows the composition to be administered to a patient. For example, the composition may be in liquid or solid form. Typical routes of administration include, but are not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, and intratumoral. Parenteral administration may include subcutaneous, intravenous, intramuscular, intrasternal injection, or infusion techniques. In one embodiment, the composition may be administered by infusion using a minipump infusion system.

[0086] In one embodiment, the pharmaceutical composition can be administered to the subject as an antibody preparation. Antibody preparations, such as those having high specificity and high affinity for their target antigen, can produce effects due to their binding to the target. For example, the α3 domain of MICA / B is an essential domain for shedding, and monoclonal antibodies that bind to this domain can inhibit MICA / B shedding and induce tumor immunity via NK cells. Increased density of MICA / B protein on tumor cells enhanced activation of NK cells via the NKG2D receptor, and the Fc segment of tumor conjugate antibodies also activated NK cells via the CD16Fc receptor. Treatment with such MICA / B antibodies significantly altered tumor-infiltrating NK cells into a highly cytotoxic state.

[0087] The activators of the embodiments described herein, for example, antibodies that specifically bind to the α3 domain of MICA / B, can be administered in the form of pharmaceutical compositions for the treatment of cancer. Principles and precautions related to the preparation of therapeutic pharmaceutical compositions containing antibodies, as well as guidance on component selection, are provided, for example, in Remington: The Science And Practice Of Pharmacy 20th ed. (Alfonso R. Gennaro, et al, editors) Mack Pub. Co., Easton, Pa., 2000, Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994, and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0088] The specific dosage and treatment regimen for any particular patient depends on a variety of factors, including the specific antibody used, its variant or derivative, the patient's age, weight, general health status, sex and diet, administration time, elimination rate, drug combinations, the specific disease being treated, and the severity of the disease. The determination of such factors by healthcare professionals is within the scope of the skills of those skilled in the art. The amount will also depend on the individual patient being treated, the route of administration, the type of formulation, the properties of the compound used, the disease being treated, the severity of the disease, and the desired effect. The amount used can be determined by pharmacokinetic principles well known in the art.

[0089] The terms “effective dose” or “therapeutic effective dose” can refer to the amount of a drug or therapeutic agent (i.e., activator) that is effective in treating (e.g., killing) cancer cells in mammals. In the case of cancer, an effective dose of a drug can reduce the number of cancer cells, shrink the size of a tumor, inhibit (i.e., slow to some extent, and / or stop) the invasion of cancer cells into peripheral organs, inhibit (i.e., slow to some extent, and / or stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more of the symptoms associated with cancer. A drug may be cell proliferation inhibitory and / or cytotoxic to the extent that it can prevent the growth of existing cancer cells and / or kill them. In the case of cancer treatment, effectiveness can be measured, for example, by evaluating the time to disease progression (TTP) and / or determining the response rate (RR).

[0090] The therapeutically effective dose in the embodiments described herein may be the amount necessary to achieve the therapeutic objective. As described above, this may be the binding interaction between an antibody and its target antigen, which in particular interferes with the function of the target. The amount to be administered further depends on the binding affinity of the antibody for its specific antigen, and also on the rate at which the administered antibody is depleted from the free volume of the other target to which it is administered. As another example, this may be the activation of NK cells, such as the anti-cancer activity of NK cells.

[0091] The dosage of the activator administered to the subject (e.g., a patient) is typically 0.1 mg / kg to 100 mg / kg patient body weight, 0.1 mg / kg to 20 mg / kg patient body weight, or 1 mg / kg to 10 mg / kg patient body weight.

[0092] With regard to antibodies, human antibodies have a longer half-life in the human body than antibodies from other species due to their immune response to foreign polypeptides. Therefore, lower doses and less frequent administration of human antibodies can be used. Furthermore, the dose and frequency of antibody administration can be reduced by enhancing antibody uptake and penetration into tissues (e.g., the brain) through modifications such as lipidization. A general range for therapeutically effective administration of the antibodies or antibody fragments of the embodiments described herein may be, in non-limiting examples, about 0.1 mg / kg body weight to about 50 mg / kg body weight. A typical administration frequency may be, for example, twice a day to once a week.

[0093] In embodiments, the smallest inhibitory antibody fragment that specifically binds to the binding domain of the target protein can be used. For example, based on the variable region sequence of the antibody, a peptide molecule that retains the ability to bind to the target protein sequence can be designed. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, for example, Marasco et al, Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)). The formulation may also contain two or more active compounds necessary for the indication being treated, e.g., those having complementary activity that does not adversely affect each other. Alternatively or additionally, the composition may include agents that enhance its function, such as cytotoxic agents, cytokines (e.g., IL-15, IL-12, IL-18), chemotherapeutic agents, or growth inhibitors. Such molecules are appropriately present in combination in amounts effective for the intended purpose.

[0094] The active ingredient can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, respectively.

[0095] Preparations used for in vivo administration must be sterile. This can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0096] Sustained-release preparations can be prepared. Preferred examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, the matrices in the form of molded articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT (an injectable microsphere consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyrate. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow for molecular release over more than 100 days, while certain hydrogels release proteins over shorter periods.

[0097] As described in detail herein, compositions of the embodiments described herein, including those containing one or more activators, can be administered in combination with one or more additional therapeutic or prophylactic regimens. For example, one or more additional therapeutic agents may be chemotherapeutic agents, cytokines (such as IL-15, IL-12, IL-18), radiotherapy, or immunotherapy agents.

[0098] In additional embodiments, the compositions described herein may be administered in combination with other therapeutic or prophylactic regimens, such as radiotherapy.

[0099] Embodiments described herein provide a method for treating a patient's cancer by administering two or more antibodies that bind to the same epitope of an antigen, or to two or more different epitopes of an antigen. Alternatively, cancer can be treated by administering a first antibody that binds to a first antigen and a second antibody that binds to a protein other than the first antigen. For example, the first antibody may bind to MICA / B, and the second antibody may bind to PD1, PDL1, CTLA4, or a combination thereof.

[0100] In other embodiments, cancer can be treated by administering a bispecific antibody that binds to a first antigen and also to proteins other than the first antigen.

[0101] In some embodiments, the embodiments described herein provide administering a first antibody or activator alone, or in combination with a second activator or antibody that recognizes a different protein other than that recognized by the first antibody, along with cells capable of achieving or enhancing an immune response. For example, these cells may be peripheral blood mononuclear cells (PBMCs), or any cell type found in PBMCs, such as cytotoxic T cells, macrophages, and natural killer (NK) cells.

[0102] Furthermore, the present invention provides the administration of one or more activators and other therapeutic agents comprising biomolecules such as small molecules, growth factors, cytokines or peptides, peptide mimes, peptoids, polynucleotides, lipid-derived mediators, low-molecular-weight bioamines, hormones, neuropeptides, and proteases. Small molecules include, but are not limited to, inorganic and small organic molecules. Suitable growth factors or cytokines include IL-2, IL-12, IL-15, IL-18, GM-CSF, and TNF-alpha. Small molecule libraries are known in the art. (See Lam, Anticancer Drug Des., 12:145, 1997.)

[0103] One embodiment also includes (a) evaluating the patient to determine whether the patient has refractory or drug-resistant cancer or cancer resistant to cytotoxic T cells; (b) administering an effective dose of one or more activators to the patient; and (c) monitoring the patient to determine the state of the cancer.

[0104] For example, biological samples derived from a subject can be evaluated for markers of resistance to cytotoxic T cells, such as Jak1 or B2M mutations, or other mutations that disrupt the function of the IFN gamma signaling pathway (e.g., Stat1 mutations) or the MHC class I antigen presentation pathway in tumor cells (e.g., Tap1 or Tap2 mutations). The term “biological sample” may include tissues, cells, and bodily fluids isolated from a subject, as well as tissues, cells, and bodily fluids present within the subject. Therefore, the use of the term “biological sample” includes blood, and fractions or components of blood, including serum, plasma, or lymph. That is, analytes such as mRNA, proteins, or genomic DNA in a biological sample can be detected in vitro and in vivo using the detection methods of the embodiments described herein. For example, in vitro techniques for detecting analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detecting analyte proteins include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include Southern hybridization.

[0105] Steps to evaluate and / or monitor a patient or a patient's cancer may include the use of a probe to detect the presence of cellular markers in a sample. For example, the probe may contain a detectable label. In embodiments, the probe is an antibody, but may be a polynucleotide or a small molecule. The antibody may be polyclonal or monoclonal. A intact antibody, or a fragment thereof (e.g., F ab , scFv, or F (ab)2) can be used. With respect to probes or antibodies, the term “labeled” can encompass direct labeling of the probe or antibody by binding (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include the detection of a primary antibody using a fluorescently labeled secondary antibody, and end-labeling a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin.

[0106] Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, JRCrowther (Ed.), Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christophorus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detecting analyte proteins involve introducing labeled anti-analyte protein antibodies into the target. For example, the antibody may be labeled with a radiomarker whose presence and location in the target can be detected by standard imaging techniques.

[0107] Detection can be facilitated by coupling (e.g., physically linking) a probe or antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Suitable enzyme examples include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dancylcloride, or phycoerythrin; luminescent materials include luminol; bioluminescent materials include luciferase, luciferin, and aequorin; suitable radioactive materials include 125 I, 131 I, 35 S, 32 P or 3 H can be mentioned.

[0108] Compositions for treating cancer The embodiments described herein also relate to compositions for preventing, treating, or alleviating symptoms of cancer or cytoproliferative disorders.

[0109] Furthermore, the embodiments described herein relate to compositions for preventing the growth, proliferation, or metastasis of tumor cells, cancer cells, or tumors; for preventing the replication of tumor cells or cancer cells, reducing the overall tumor mass or the number of cancer cells, and for improving one or more symptoms associated with the disease.

[0110] In embodiments, the composition comprises one or more activators as described herein. For example, the composition may include antibodies or fragments thereof as listed in Table 1. For example, the composition may include cytokines such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, anti-CD40, CD40L, and TNF-α. In embodiments, the cytokine is IL-15.

[0111] The compositions are suitable for veterinary or human administration. The compositions of the embodiments described herein may be in any form that enables administration of the composition to humans or animals. For example, the composition may be in solid, liquid, or gaseous (aerosol) form. Typical routes of administration include, but are not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, and nasal. Parenteral administration includes subcutaneous, intravenous, intramuscular, intrasternal injection, or infusion techniques. For example, the composition is administered parenterally. The pharmaceutical compositions of the embodiments described herein may be formulated so that an activator, such as an anti-MICA / B antibody, is bioavailable when the composition is administered to an animal. The composition may take the form of one or more dose units; for example, a tablet may be a single dose unit, and a container of an activator in aerosol form may hold multiple dose units.

[0112] Materials used in preparing pharmaceutical compositions may be nontoxic in the amounts used. It will be apparent to those skilled in the art that the optimal dose of an active ingredient in a pharmaceutical composition, such as an activator, depends on a variety of factors. These factors include, but are not limited to, the type of animal (e.g., human), the specific form of the activator, the specific disease being treated or prevented, the mode of administration, and the composition used.

[0113] Since pharmaceutically acceptable carriers or vehicles can be particulate, the compositions may be in the form of, for example, tablets or powders. The carrier may be liquid, and the compositions may be, for example, oral syrups or injectable liquids. Furthermore, the carrier may be gaseous, for example, to provide aerosol compositions useful for inhalation administration.

[0114] When intended for oral administration, the composition may be in solid or liquid form, and forms such as semi-solid, semi-liquid, suspension, and gel are included herein as either solid or liquid.

[0115] As a solid composition for oral administration, the composition can be formulated in the form of powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotex; lubricants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavorings such as peppermint, methyl salicylate, or orange flavoring; and colorants.

[0116] If the composition is in the form of a capsule, such as a gelatin capsule, it may contain a liquid carrier such as polyethylene glycol, cyclodextrin, or fatty oil in addition to the above-mentioned types of materials.

[0117] The composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion, or suspension. The liquid may be useful for oral administration or delivery by injection. When intended for oral administration, the composition may contain one or more of the following: sweeteners, preservatives, dyes / colorants, and flavorings. Compositions for injection administration may also contain one or more of the following: surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0118] The liquid compositions of the embodiments described herein, whether in solution, suspension or other similar forms, may also include one or more of the following: a sterile diluent such as water for injection; physiological saline such as physiological saline; Ringer's solution; isotonic saline; fixing oils such as synthetic mono or diglycerides that can function as a solvent or suspension medium; polyethylene glycol, glycerin, cyclodextrin, propylene glycol or other solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering agents such as acetates, citrates or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral compositions may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass, plastic or other materials. Physiological saline may be an adjuvant. Injectable compositions may be sterile.

[0119] The amount of composition effective in treating a disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques. Furthermore, the optimal dosage range can be identified using in vitro or in vivo assays. The precise dose used for a composition also depends on the route of administration and the severity of the disease or disorder, and can be determined according to the practitioner's judgment and the individual patient's situation.

[0120] The composition may contain an effective amount of at least one activator so as to yield a suitable dosage. For example, this amount is at least about 0.01% by weight of the composition. For oral administration, this amount can be varied in the range of about 0.1% to about 80% by weight of the composition. The oral composition may constitute about 4% to about 50% by weight of the composition. The compositions of the embodiments described herein may be prepared so that parenteral dose units contain about 0.01% to about 2% by weight of the activator.

[0121] For intravenous administration, the composition may contain approximately 1 to 250 mg of the activator per kg of animal body weight. For example, the dosage would be in the range of approximately 4 to 25 mg / kg body weight of the activator.

[0122] In the embodiments, the dose of the activator administered to the animal is typically about 0.1 mg / kg of animal body weight to about 1000 mg / kg of animal body weight. For example, the dose of the activator administered to the animal is typically about 0.1 mg / kg to about 250 mg / kg of animal body weight. For example, the dose administered to the animal is about 0.1 mg / kg to about 20 mg / kg of animal body weight, for example, about 1 mg / kg to about 10 mg / kg of animal body weight.

[0123] The composition can be administered by any convenient route, for example, by injection or bolus injection, or by absorption via the epithelium or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa). Administration can be systemic or topical. Various delivery systems, such as encapsulation in liposomes, microparticles, microcapsules, and capsules, are known and can be used to administer the composition. In certain embodiments, two or more activators or compositions are administered to animals. Methods of administration include oral and parenteral administration; parenteral administration including, but not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous; and topical administration including, but not limited to, intranasal, epidural, sublingual, intracerebral, intraventricular, intrathecal, vaginal, percutaneous, rectal, inhalation, or to the ear, nose, eye, or skin. In embodiments, the mode of administration may be left to the discretion of the practitioner and may depend in part on the site of the disease (e.g., site of cancer or within a tumor).

[0124] In one embodiment, the activator or composition is administered parenterally.

[0125] In one embodiment, the activator or composition is administered intravenously by means of an infusion pump or drip infusion.

[0126] In certain embodiments, one or more activators or compositions may be administered topically to an area requiring treatment. This can be achieved, for example, by local injection during surgery; topical application, for example, in combination with postoperative wound dressings; by injection; by catheter; by port; by suppository; or by implant, the implant being made of porous, non-porous, or gelatinous material (including membranes such as Silastic membranes), or fibrous material. In one embodiment, administration may be by direct injection into the site (or prior site) of cancer, tumor, or neoplastic or pre-neoplastic tissue. In another embodiment, administration may be by direct injection into the site (or prior site) of disease manifestation.

[0127] Lung administration can also be used, for example, by the use of an inhaler or nebulizer, and by formulation with an aerosolizing agent, or by perfusion with fluorocarbons or synthetic lung surfactants. In certain embodiments, the activator or composition can be formulated as a suppository using conventional binders and carriers such as triglycerides.

[0128] In another embodiment, the activator can be delivered by vesicles such as liposomes (see Langer, Science 249:1527-1533 (1990), Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989), Lopez-Berestein (ibid.), pp.317-327).

[0129] In yet another embodiment, the activator or composition can be delivered by a controlled release system. In one embodiment, a pump can be used (see Langer (above), Sefton, CRC Crit.Ref.Biomed.Eng.14:201 (1987), Buchwald et al., Surgery 88:507 (1980), Saudek et al., N.Engl.J.Med.321:574 (1989)). In another embodiment, polymer materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974), Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984), Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983). See also Levy et al., Science 228:190 (1985), During et al., Ann. Neurol. 25:351 (1989), and Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, the controlled-release system can be positioned close to the target of the activator or composition, and therefore requires only a small fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release (above), vol.2, pp.115-138 (1984)). Other controlled-release systems discussed in Langer's review (Science 249:1527-1533 (1990)) can be used.

[0130] The term "carrier" can refer to a diluent, adjuvant, or excipient administered with an activator. Such pharmaceutically acceptable carriers can be liquids such as water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Carriers may include saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, and urea. Furthermore, auxiliary agents, stabilizers, thickeners, lubricants, and colorants may be used. In one embodiment, when administered to animals, the activator and pharmaceutically acceptable carrier are sterile. When the activator is administered intravenously, water can serve as the carrier. Saline and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, such as injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. The composition may also contain trace amounts of wetting agents or emulsifiers, or pH buffers.

[0131] The composition may take the form of a solution, suspension, emulsion, tablet, pill, pellet, capsule, liquid-containing capsule, powder, sustained-release formulation, suppository, emulsion, aerosol, spray, suspension, or any other form suitable for use. In one embodiment, the pharmaceutically acceptable carrier is a capsule (see, for example, U.S. Patent No. 5,698,155). Other examples of suitable pharmaceutically acceptable carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0132] In one embodiment, the activator is formulated according to standard procedures as a pharmaceutical composition suitable for intravenous administration to animals such as humans. Typically, the carrier or vehicle for intravenous administration is a sterile isotonic aqueous buffer. Optionally, the composition may also contain a solubilizer. The intravenous administration composition may optionally contain a local anesthetic, such as lignocaine, to alleviate pain at the injection site. In embodiments, the components are supplied separately or mixed together in unit dosage forms, and supplied as a dry, lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachet indicating the amount of activator. When the activator is administered by infusion, it can be dispensed, for example, in an infusion bottle containing sterile pharmaceutical-grade water or saline. When the activator is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0133] Compositions for oral delivery may be in the form of, for example, tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Orally administered compositions may contain one or more optional agents, such as sweeteners like fructose, aspartame, or saccharin, flavorings like peppermint, wintergreen oil, or cherry, coloring agents, and preservatives to provide a pharmaceutically palatable preparation. Furthermore, in tablet or pill form, the composition may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over a longer period. Selectively permeable membranes surrounding osmotically active driving compounds are also suitable for orally administered compounds. In these subsequent platforms, fluids from the environment surrounding the capsule are absorbed by the driving compound, causing it to swell and move the drug or drug composition through the opening. These delivery platforms can provide an essentially zero-order delivery profile, unlike the spike profile of immediate-release formulations. Time-delaying materials such as glycerol monostearate or glycerol stearate can also be used. Oral compositions may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Such carriers may be pharmaceutical grade.

[0134] The composition may be intended for topical administration, in which case the carrier may be in the form of a solution, emulsion, ointment, or gel base. The base may include, for example, diluents such as petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, water, and alcohol, as well as one or more emulsifiers and stabilizers. Thickeners may be present in the composition for topical administration. When intended for transdermal administration, the composition may be in the form of a transdermal patch or an iontophoresis device. The topical formulation may contain an activator at a concentration of about 0.1% to about 10% w / v (weight per unit volume of the composition).

[0135] The composition may be intended for rectal administration, for example, in the form of a suppository that dissolves in the rectum and releases an activator. Compositions for rectal administration may contain an oily base as a suitable non-irritating excipient. Examples of such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0136] The composition may include various materials that alter the physical form of the solid or liquid dosage unit. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is usually inert and can be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0137] The composition may consist of gaseous dosage units, for example, in the form of an aerosol. The term aerosol is used to describe a variety of systems, from colloidal ones to systems consisting of pressurized packages. Delivery can be carried out by liquefaction or compressed gas, or by a suitable pump system for distributing the active ingredient. Aerosols of activators can be delivered in single-phase, two-phase, or three-phase systems for delivering the activator. Aerosol delivery involves necessary containers, activators, valves, sub-containers, spacers, etc., which together can form a kit. The aerosol can be determined by those skilled in the art without excessive experimentation.

[0138] Whether in solid, liquid, or gaseous form, the compositions of the embodiments described herein may include agents used to treat, prevent, or alleviate symptoms of cancer or cytoproliferative disorders.

[0139] Pharmaceutical compositions can be prepared using methodologies well known in the pharmaceutical field. For example, a composition intended for administration by injection can be prepared by combining an activator with water to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. A surfactant is a compound that interacts non-covalently with the activator to promote the dissolution or homogeneous suspension of the activator in an aqueous delivery system.

[0140] In embodiments, the activator is a small molecule. The term “small molecule” can refer to a non-peptidic, non-oligomeric organic compound synthesized in the laboratory or found in nature. A small molecule can refer to a compound that is “natural product-like,” but the term “small molecule” is not limited to “natural product-like” compounds. Rather, a small molecule is typically characterized by having one or more features including having several carbon-carbon bonds, having multiple stereocenters, having multiple functional groups, having at least two different types of functional groups, and having a molecule with a weight of less than 1500, but this characterization is not intended to limit the purpose of the embodiments described herein.

[0141] The term "small molecule scaffold" can refer to a compound having at least one site for functionalization. In one embodiment, a small molecule scaffold may have multiple sites for functionalization. These functionalization sites can be protected or masked, as can be understood by those skilled in the art. These sites may also be found on the underlying ring structure or skeleton.

[0142] In embodiments, the activator is a cytokine. The term "cytokine" can refer to molecules such as proteins released by a population of cells that act as intracellular mediators for the same cell population (autocrine) or another cell population (paracrine). Examples of such cytokines are lymphokines, monokines, and conventional polypeptide hormones. Some cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and beta; Müllerian duct inhibitors; mouse gonadotropin-related peptides; inhibin; activin; vascular endothelial growth factor (VEGF); integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet-derived growth factor (PDGF); transforming growth Other polypeptide factors include factors (TGF), e.g., TG-α and TGF-beta; insulin-like growth factor (IGF), e.g., IGF-I and IGF-II; erythropoietin (EPO); bone induction factor; interferons such as interferon-alpha, -beta and -gamma; colony-stimulating factors (CSF) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL), e.g., IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; LIF and kit ligands (KL, also known as stem cell factors). In embodiments, the activator is a biologic. "Biological" or "biologic" can refer to any pharmaceutically active agent made from an organism and / or its products intended for use as a therapeutic agent.In one embodiment of the embodiments described herein, the biological agent includes, but is not limited to, antibodies, nucleic acid molecules (polynucleotides), such as antisense nucleic acid molecules, polypeptides, or proteins.

[0143] In embodiments, the activator is a polynucleotide. “Polynucleotide” can encompass a single “polynucleotide” as well as multiple “polynucleotides.” “Polynucleotide” can refer to a chain of nucleotides, which may be nucleic acids, nucleic acid sequences, oligonucleotides, nucleotides, or any fragment thereof. It may be genomic DNA, mRNA, cDNA, siRNA, or synthetically derived DNA or RNA, double-stranded or single-stranded, and can be combined with carbohydrates, lipids, proteins, or other materials to perform activities, etc., or to form useful compositions.

[0144] In embodiments, the activator is a polypeptide. As used herein, “polypeptide” can encompass a single “polypeptide” as well as multiple “polypeptides” and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any one or more chains of two or more amino acids and does not refer to a specific length of the product. Thus, any other term used to refer to a peptide, dipeptide, tripeptide, oligopeptide, “protein,” “amino acid chain,” or a chain of two or more amino acids can refer herein to a “polypeptide,” and the term “polypeptide” can be used in place of or interchangeably with any of these terms. “Polypeptide” can also refer to post-expression modification products of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification with amino acids that do not exist in nature. Polypeptides can be derived from natural biological sources or produced by recombinant technology and do not necessarily have to be translated from a specific nucleic acid sequence. Polypeptides can be produced by any method, including chemical synthesis. With respect to amino acid sequences, those skilled in the art will readily recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that modify, add, delete, or substitute a single amino acid or a small percentage of amino acids in the encoded sequence are collectively referred to herein as “conservatively modified variants.” In some embodiments, the modification results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art.

[0145] For example, a "conservative amino acid substitution" is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain. In this art, families of amino acid residues with similar side chains are defined as basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues in immunoglobulin polypeptides can be replaced by other amino acid residues from the same side chain family. In another embodiment, amino acid chains can be replaced by structurally similar chains with different order and / or composition of side chain family members.

[0146] In one embodiment, the activator is an antibody, an antibody fragment, or a derivative thereof. As used herein, “antibody” or “antigen-binding polypeptide” may refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody may be a whole antibody, any antigen-binding fragment, or a single chain thereof. For example, an “antibody” may include any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule having biological activity to bind to an antigen. Non-limiting examples include the complementarity-determining region (CDR) of a heavy or light chain or its ligand-binding portion, the variable region of a heavy or light chain, the constant region of a heavy or light chain, the framework (FR) region, or any portion thereof, or at least a portion of a binding protein. As used herein, the term “antibody” may refer to an immunoglobulin molecule and an immunoglobulin (Ig) molecule, i.e., the immunoactive portion of a molecule containing an antigen-binding site that specifically binds to (immunely reacts with) an antigen. “Specifically binding” or “immunely reacting” means that the antibody reacts with one or more antigenicity-determining sites of a desired antigen and not with other polypeptides.

[0147] As used herein, the terms “antibody fragment” or “antigen-binding fragment” refer to a portion of an antibody, such as F(ab′)2, F(ab)2, Fab′, Fab, Fv, scFv, etc. Regardless of their structure, antibody fragments bind to the same antigens recognized by the complete antibody. The term “antibody fragment” may also include aptamers (such as Spiegelmer), minibodies, and diabodies. The term “antibody fragment” may also include any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen and forming a complex. The antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', F(ab')2, Fd, Fvs, single-chain Fv(scFv), single-chain antibodies, dAb (domain antibodies), minibodies, disulfide-binding Fv(sdFv), fragments containing any of the VL or VH domains, fragments generated by Fab expression libraries, and anti-idiotype (anti-Id) antibodies.

[0148] A "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. A single-chain Fv ("scFv") polypeptide molecule is a covalently linked VH:VL heterodimer, which can be expressed from a gene fusion containing VH and VL coding genes linked by a peptide coding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). In some embodiments, the region is linked by a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility, and serine or threonine for solubility, in which case V H The N-terminus and V LThe C-terminus can be linked to the C-terminus, or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker. Numerous methods have been described for identifying the chemical structures to convert naturally aggregated but chemically separated light and heavy polypeptide chains from the antibody V region into scFv molecules that will fold into a three-dimensional structure substantially similar to the structure of the antigen-binding site. See, for example, U.S. Patents 5,091,513, 5,892,019, 5,132,405, and 4,946,778, which are incorporated in their entirety by reference, respectively.

[0149] Very large naive human scFv libraries have been constructed and can be constructed to provide a large source of antibody genes rearranged for numerous target molecules. Smaller libraries can be constructed from individuals with infectious diseases to isolate disease-specific antibodies. (See Barbas et al., Proc. Natl. Acad. Sci. USA 89:9339-43 (1992), Zebedee et al, Proc. Natl. Acad. Sci. USA 89:3 175-79 (1992)).

[0150] Antibody molecules obtained from humans are classified into five classes of immunoglobulins: IgG, IgM, IgA, IgE, and IgD, which differ from one another in the properties of the heavy chains present in the molecules. Those skilled in the art will understand that the heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and that there are several subclasses within these (e.g., γ1-γ4). Certain classes also have subclasses, such as IgG1, IgG2, IgG3, and IgG4, as well as others. Subclasses (isotypes) of immunoglobulins, such as IgG1, IgG2, IgG3, IgG4, and IgG5, are well-characterized and are known to provide functional specificity. In the case of IgG, a standard immunoglobulin molecule contains two identical light-chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy-chain polypeptides with a molecular weight of 53,000-70,000. The four chains are typically linked by disulfide bonds in a "Y" shape, with the light chains beginning at the mouth of the "Y" and surrounding the heavy chains that continue through the variable region. The immunoglobulin or antibody molecules described herein may be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0151] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be bound to either a kappa or lambda light chain. Generally, light and heavy chains are covalently linked to each other, and when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In heavy chains, the amino acid sequence extends from the N-terminus of the Y-branched ends to the C-terminus at the bottom of each chain.

[0152] Both the light and heavy chains are divided into structural and functional homology regions. The terms “constant” and “variable” are used in a functional sense. The variable domains (VL and VH) of both the light and heavy chain portions determine antigen recognition and specificity. Conversely, the constant domains (CL, as well as CH1, CH2, or CH3) of the light and heavy chains confer important biological characteristics such as secretion, transplacental mobility, Fc receptor binding, and complement binding. The term “antigen-binding site” or “binding region” can refer to the portion of the immunoglobulin molecule involved in antigen binding. Antigen-binding sites are formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly distinct segments within the V regions of the heavy and light chains, called “hypervariable regions,” are inserted between more conserved adjacent segments known as “framework regions” or “FR.” Thus, the term “FR” refers to the naturally occurring amino acid sequences between and adjacent to the hypervariable regions of immunoglobulins. In antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of the heavy chain and the three hypervariable regions of the light chain are called "complementarity-determining regions" or "CDRs".

[0153] The six CDRs present in each antigen-binding domain are short, discontinuous sequences of amino acids that specifically align to form the antigen-binding domain when the antibody takes its three-dimensional configuration in an aqueous environment. The remaining amino acids of the antigen-binding domain, the FR region, exhibit less intermolecular variation. The framework region primarily conforms to a β-sheet structure, with the CDRs linking together to form loops and, in some cases, forming part of the β-sheet structure. The framework region functions to form a scaffold for aligning the CDRs in the correct orientation through non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs provides a complementary surface to the epitope on the antigen in the immune response, facilitating the non-covalent binding of the antibody to the congeneral epitope. The amino acids containing the CDR and framework regions, respectively, can be readily identified by those skilled in the art for the heavy chain or light chain variable region, since they have been previously identified (see “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., USD Department of Health and Human Services, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0154] Where there are two or more definitions for a term used and / or permitted in the art, the definitions of terms used herein are intended to encompass all such meanings unless explicitly stated otherwise. An example is the use of the term “complementarity-determining region” (“CDR”) to describe non-adjacent antigen-binding sites found within the variable regions of both heavy-chain and light-chain polypeptides. These particular regions are described by Kabat et al., USDept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and Chothia et al., J.Mol.Biol.196:901-917 (1987), which are incorporated herein by reference in their entirety. The definitions of CDRs by Kabat and Chothia include overlaps or subsets of amino acid residues when compared to one another. Nevertheless, it is intended that applying either definition to refer to the CDRs of an antibody or its variants is also within the scope of the terms defined and used herein. The exact residue numbers containing a particular CDR vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues constitute a specific CDR simply by knowing the amino acid sequence of the variable region of an antibody.

[0155] Kabat et al. defined a numbering system for variable domain sequences applicable to any antibody. Those skilled in the art can clearly assign this “Kabat numbering” system to any variable domain sequence, without relying on other experimental data of the sequence itself. As used herein, “Kabat numbering” refers to the numbering system described in Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

[0156] As used herein, the term “epitope” may include any protein determinant that can specifically bind to an immunoglobulin, scFv, or T cell receptor. The variable region allows an antibody to selectively recognize and specifically bind to an epitope on an antigen. For example, a combination of the VL and VH domains of an antibody, or a subset of complementarity-determining regions (CDRs), forms a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites located at the ends of each arm of the Y. Epitope determinants typically consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural and specific charge characteristics. For example, antibodies can be produced against the N-terminal or C-terminal peptide of a polypeptide. More specifically, the antigen-binding site is defined by three CDRs on the VH and VL chains, respectively (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3).

[0157] As used herein, the terms “immunological binding” and “immunological binding properties” may refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K) of the interaction. D It can be expressed as (K), and a smaller (K D ) represents greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, whose rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Thus, both "on rate constants" (K) on ) and "off-speed constant" (K off ) can be determined by calculating the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). K off / K onThe ratio of these parameters cancels out all parameters unrelated to affinity, and the dissociation constant K D This is equivalent to (see Davies et al. (1990) Annual Rev Biochem 59:439-473). The antibodies of the present invention can specifically bind to the PD-1 epitope when the equilibrium binding constant (KD), as measured by kinetic assays such as radioligand binding assays or similar assays known to those skilled in the art such as BIAcore, is ≤10 μM, ≤10 nM, ≤10 pM, or ≤100 pM to about 1 pM. "Specifically binds" or "specific to" can mean an antibody that binds to the epitope via its antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. For example, an antibody is said to "specifically bind" to an epitope if it binds to that epitope via its antigen-binding domain more easily than it would to bind to a random, unrelated epitope.

[0158] Various procedures known within the art can be used to produce polyclonal or monoclonal antibodies directed against the proteins of the embodiments described herein, or against their derivatives, fragments, analogs, homologs, or orthologues. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0159] Antibodies can be purified by well-known techniques such as affinity chromatography using protein A or protein G, which primarily provide the IgG fraction of immunoserum. Subsequently, or alternatively, immunospecific antibodies can be purified by immunoaffinity chromatography by immobilizing the specific antigen or epitope that is the target of the desired immunoglobulin onto a column. The purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0160] As used herein, the terms “monoclonal antibody,” “mAb,” “Mab,” or “monoclonal antibody composition” may refer to a group of antibody molecules containing only one species of antibody molecule, consisting of a distinctive light chain gene product and a distinctive heavy chain gene product. In particular, the complementarity-determining region (CDR) of a monoclonal antibody is identical across all molecules in the group. A MAb contains an antigen-binding site that can react immunologically with a specific epitope of an antigen, characterized by a specific binding affinity to it.

[0161] Monoclonal antibodies may include “chimeric” antibodies in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence of an antibody originating from a particular species or belonging to a particular antibody class or subclass, and the remaining chain is identical or homologous to a corresponding sequence of an antibody originating from another species or belonging to another antibody class or subclass, as well as a fragment of such an antibody exhibiting the desired biological activity (see, for example, U.S. Patent No. 4,816,567, Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855). For example, chimeric antibodies can be derived from a variable region from a mouse antibody and a constant region from a human antibody.

[0162] The "humanized" form of a non-human (e.g., rodent) antibody can refer to a chimeric antibody containing the smallest sequence derived from a non-human immunoglobulin. In most cases, the humanized antibody is a human immunoglobulin (recipient antibody), and residues from the recipient's hypervariable region are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, to achieve the desired specificity, affinity, and capability. In some cases, framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the recipient or donor antibody. These modifications are made to further improve the antibody's performance. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are from a human immunoglobulin sequence. Humanized antibodies will also optionally contain at least a portion of the constant region (Fc) of immunoglobulins, typically that of human immunoglobulins. For further details, see Jones et al., 1986, Nature 321:522-525, Riechmann et al., 1988, Nature 332:323-329, and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596.

[0163] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In hybridoma methods, mice, hamsters, or other suitable host animals are typically immunized with an immunizer to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizer. Alternatively, lymphocytes can be immunized in vitro.

[0164] The immunotherapy agent may include protein antigens, their fragments, or fusion proteins. For example, peripheral blood lymphocytes may be used if human-derived cells are desired, or spleen cells or lymph node cells may be used if a non-human mammalian source is desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell line may be transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. For example, rat or mouse myeloma cell lines may be used. The hybridoma cells may be cultured in a suitable medium containing one or more substances that inhibit the proliferation or survival of non-fusioned immortalized cells. For example, if parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium typically contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), and these substances prevent the growth of HGPRT-deficient cells.

[0165] Useful immortalized cell lines are those that efficiently fuse, maintain stable high levels of antibody expression by selected antibody-producing cells, and are sensitive to culture media such as HAT medium. Examples of immortalized cell lines include mouse myeloma lines available from the Salk Institute Cell Distribution Center (San Diego, California) and the American Type Culture Collection (Manassas, Virginia). Human myeloma and mouse-human heterozygous myeloma cell lines have also been described for the production of human monoclonal antibodies. (See Kozbor, J. Immunol, 133:3001 (1984), Brodeur et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

[0166] Next, the culture medium in which hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies against the antigen. For example, the binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosolvent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis as described in Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, for therapeutic applications of monoclonal antibodies, it is important to identify antibodies that have high specificity and high binding affinity to the target antigen.

[0167] After the desired hybridoma cells are identified, clones can be subcloned using limiting dilution procedures and grown using standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986), pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle Medium and RPMI-1640 Medium. Alternatively, hybridoma cells can be grown in vivo as ascites in mammals.

[0168] Monoclonal antibodies secreted by subclones can be isolated or purified from culture media or ascites fluid by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0169] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567 (which is incorporated herein by reference in its entirety). The DNA encoding the monoclonal antibodies of the embodiments described herein can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the mouse antibody). Hybridoma cells of the embodiments described herein can serve as a source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, to obtain the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by substituting the coding sequences of human heavy and light chain constant domains for homologous mouse sequences (see U.S. Patent No. 4,816,567, Morrison, Nature 368,812-13 (1994)) or by covalently bonding all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be used in place of the constant domains of the antibodies in the embodiments described herein, or in place of the variable domain of one antigen-binding site of the antibodies in the embodiments described herein, to produce a chimeric bivalent antibody.

[0170] Fully human antibodies are antibody molecules in which both the light and heavy chain sequences, including, for example, CDRs, are derived from human genes. Such antibodies are referred to herein as “humanized antibodies” or “fully human antibodies.” “Humanized antibodies” can be antibodies derived from non-human species, but their light and heavy chain protein sequences have been modified to increase similarity to antibody variants produced in humans. Humanized antibodies are antibody molecules derived from non-human species antibodies that bind to a desired antigen, having one or more complementarity-determining regions (CDRs) from non-human species and a framework region derived from a human immunoglobulin molecule. Often, framework residues in the human framework region are substituted with corresponding residues from the CDR donor antibody, thereby altering, for example, the antigen binding, and improving it. These framework substitutions are identified by methods well known in the art, for example, by modeling the interaction between CDRs and framework residues to identify framework residues important for antigen binding, and by comparing sequences to identify abnormal framework residues at specific positions. (See, for example, Queen et al., USPat. No. 5, 585, 089 and Riechmann et al., Nature 332:323 (1988), which are incorporated in their entirety herein by reference.) For example, the non-human portion of an antibody (such as the CDR of the light and / or heavy chains) can bind to a target antigen. Humanized monoclonal antibodies are also sometimes referred to herein as “human monoclonal antibodies.”

[0171] Antibodies can be humanized using various techniques known in the art, such as CDR transplantation (EP239,400, PCT International Publication No. 91 / 09967, U.S. Patent Nos. 5,225,539, 5,530,101 and 5,585,089), veneering or resurfacing (EP592,106, EP519,596, Padlan, Molecular Immunology 28(4 / 5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814(1994); Roguska et al., Proc. Natl. Sci. USA 91:969-973(1994)), and chain shuffling (U.S. Patent No. 5,565,332, which is incorporated in its entirety by reference). Humanization (also known as reshaping or CDR grafting) is a well-established technique known to those skilled in the art for reducing the immunogenicity of monoclonal antibodies (mAbs) derived from a different source (usually rodents) and improving the activation of the human immune system (see, for example, Hou S, Li B, Wang L, Qian W, Zhang D, Hong X, Wang H, Guo Y (July 2008) "Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modeling" J Biochem. 144(1):115-20).

[0172] Human monoclonal antibodies, such as fully human and humanized antibodies, can be prepared using trioma technology, human B-cell hybridoma technology (see Kozbor, et al, 1983 Immunol Today 4:72), and EBV hybridoma technology that produces human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies can be used and produced by using human hybridomas (see Cote, et al, 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells with Epstein-Barr virus in vitro (Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96).

[0173] In addition, human antibodies can also be produced using other techniques, such as phage display libraries (see Hoogenboom and Winter, J.Mol.Biol, 227:381 (1991), Marks et al., J.Mol.Biol, 222:581 (1991)). Similarly, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, such as mice in which the endogenous immunoglobulin gene is partially or completely inactivated. After the challenge, human antibody production is observed, which is very similar in all aspects to that seen in humans, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patents No. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as in Marks et al., Bio / Technology 10,779-783 (1992), Lonberg et al., Nature 368,856-859 (1994), Morrison, Nature 368,812-13 (1994), Fishwild et al., Nature Biotechnology 14,845-51 (1996), Neuberger, Nature Biotechnology 14,826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13. It is described in 65-93 (1995).

[0174] Human antibodies can also be produced using transgenic non-human animals that are modified to produce fully human antibodies rather than endogenous antibodies in response to antigenic challenge. (See PCT International Publication 94 / 02602 and U.S. Patent No. 6,673,986). Endogenous genes encoding heavy and light chain immunoglobulin chains in the non-human host are neutralized, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host genome. Human genes are incorporated, for example, using a yeast artificial chromosome containing the required human DNA segment. Animals that provide all the desired modifications are then obtained as offspring by mating intermediate transgenic animals that have fewer complements than the complete complements of the modifications. A non-limiting example of such a non-human animal is the mouse, called Xenomouse™ as disclosed in PCT Publications WO96 / 33735 and WO96 / 34096. This animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained, for example, as preparations of polyclonal antibodies, directly from animals after immunization with the immunogen of interest, or alternatively, from immortalized B cells derived from animals, such as hybridomas that produce monoclonal antibodies. In addition, genes encoding immunoglobulins with human variable regions can be recovered and expressed to directly obtain antibodies, or further modified to obtain antibody analogs, such as single-stranded Fv(scFv) molecules. Thus, therapeutically useful IgG, IgA, IgM, and IgE antibodies can be produced using such techniques. For an overview of this technique for producing human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 73:65-93 (1995).For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies, and protocols for producing such antibodies, see, for example, PCT International Publications 98 / 24893, 96 / 34096, 96 / 33735, U.S. Patents 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporated herein by reference in their entirety. In addition, companies such as Creative BioLabs (Shirley, NY) can offer services to provide human antibodies against selected antigens using technologies similar to those described above.

[0175] An example of a method for producing a non-human host, exemplified as a mouse lacking expression of endogenous immunoglobulin heavy chains, is disclosed in U.S. Patent No. 5,939,598. This can be obtained by a method comprising: deleting a J-segment gene from at least one endogenous heavy chain locus in embryonic stem cells to prevent locus rearrangement and the formation of a transcript of the rearranged immunoglobulin heavy chain locus, wherein the deletion is carried out by a targeted vector containing a gene encoding a selectable marker; and producing a transgenic mouse from embryonic stem cells, wherein its somatic and germ cells contain a gene encoding a selectable marker.

[0176] One method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method involves introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing both a heavy chain and a light chain.

[0177] Further improvements to this procedure include methods for identifying clinically relevant epitopes on an immunogen level, and corresponding methods for selecting antibodies that bind immunospecifically to these relevant epitopes with high affinity, as disclosed in PCT International Publication No. 99 / 53049.

[0178] The antibody of interest can also be expressed by a vector containing a DNA segment encoding the single-strand antibody described above. For example, vectors include, but are not limited to, chemical conjugates containing a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid binding moiety (e.g., polylysine), as described in International Publication No. 93 / 64701, viral vectors (e.g., DNA or RNA viral vectors), fusion proteins such as those described in PCT / US95 / 02140 (WO95 / 22618), i.e., fusion proteins containing a targeting moiety (e.g., an antibody specific to a target cell) and a nucleic acid binding moiety (e.g., protamine), plasmids, phages, and viral vectors. Vectors can be chromosomal, non-chromosomal, or synthetic. Retroviral vectors can also be used, such as Moloney's mouse leukemia virus. DNA viral vectors are also commonly used, including pox vectors such as orthopox or avipox vectors, and herpesvirus vectors such as herpes simplex virus type 1 (HSV) vectors (see Geller, AI et al, J. Neurochem, 64:487 (1995), Lim, F., et al, in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995), Geller, AI et al, Proc Natl. Acad. Sci.: USA 90:7603 (1993), Geller, AI, et al, Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (see LeGal LaSalle et al, Science, 259:988 (1993), Davidson, et al, Nat. Genet See 3:219 (1993), Yang, et al, J. Virol. 69:2004 (1995), and Adeno-associated Virus Vectors (see Kaplitt, MG. et al, Nat. Genet. 8:148 (1994)).

[0179] Poxvirus vectors introduce genes into the cytoplasm of cells. Avipoxvirus vectors result in only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors can be used to introduce nucleic acids into nerve cells. Adenovirus vectors result in shorter-term expression (about 2 months) than adeno-associated virus (about 4 months), and thus shorter than HSV vectors. The particular vector selected will depend on the target cell and the condition being treated. Introduction can be by standard techniques such as infection, transfection, transduction, or transformation. Examples of modes of gene delivery include, for example, naked DNA, CaP04 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0180] Vectors can be used to target essentially any desired target cell. For example, stereotactic injection can be used to direct a vector (e.g., adenovirus, HSV) to a desired location. In addition, particles can be delivered by intracerebroventricular (icv) injection using a minipump infusion system such as the SynchroMed Infusion System. Methods based on bulk flow called convection have also been shown to be effective for delivering large molecules to extended regions of the brain and may be useful for delivering vectors to target cells. (See Bobo et al, Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994), Morrison et al, Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal, and subcutaneous injection, as well as oral or other known routes of administration.

[0181] Using these vectors, large amounts of antibodies can be expressed in various ways and, for example, can be made to bind to tumor cells to prevent shedding of MICA / B.

[0182] The technique can be adapted to produce single-chain antibodies specific to the antigenic protein of the embodiments described herein (see, for example, U.S. Patent No. 4,946,778). In addition, the method is F ab Monoclonal F120 ab This can enable rapid and effective identification of fragments. Antibody fragments containing idiotypes for protein antigens can be produced by techniques known in the art, but are not limited to, (i) F produced by pepsin digestion of antibody molecules. (ab’)2 Fragment, (ii) F (ab’)2 F is generated by reducing the disulfide bridges of the fragment. ab (iii) Fragments, F produced by treatment of antibody molecules with papain and a reducing agent ab Fragments, and (iv)F v Contains fragments.

[0183] Heteroconjugate antibodies are also within the scope of the embodiments described herein. Heteroconjugate antibodies consist of two covalently bound antibodies. Such antibodies enable, for example, the targeting of immune system cells to undesirable cells (see U.S. Patent No. 4,676,980) and enable the treatment of HIV infection (see PCT International Publications 91 / 00360 and 92 / 20373). Antibodies are intended to be prepared in vitro using known methods in the field of protein synthesis chemistry, such as those using crosslinking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of reagents suitable for this purpose include iminothiolates and methyl-4-mercaptobutyrimidates, as well as those disclosed, for example, in U.S. Patent No. 4,676,980.

[0184] The antibodies of the embodiments described herein can be modified with respect to effector function, for example, to enhance the efficacy of the antibody in the treatment of cancer. For example, a cysteine ​​residue can be introduced into the Fc region, thereby enabling the formation of interchain disulfide bonds in this region. The homodimeric antibodies thus produced may have improved internalization ability and / or increased complement-mediated cytotoxicity and antibody-dependent cytotoxicity (ADCC). (See Caron et al, J. Exp Med., 176:1 191-195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, antibodies having a double Fc region, thereby capable of enhanced complement lysis and ADCC ability, can be manipulated. (See Stevenson et al, Anti-Cancer Drug Design, 3:219-230 (1989)).

[0185] In certain embodiments, the antibodies of the embodiments described herein may include Fc variants containing amino acid substitutions that modify the antigen-independent effector function of the antibody, particularly the circulating half-life of the antibody. Such antibodies, when compared to antibodies lacking these substitutions, exhibit either increased or decreased binding to FcRn, and therefore have increased or decreased serum half-lives, respectively. Fc variants having improved affinity for FcRn are expected to have a longer serum half-life, and such molecules have useful applications in methods of treating mammals where a longer half-life of the administered antibody is desirable, for example, to treat chronic diseases or disorders. In contrast, Fc variants having reduced FcRn binding affinity may have a shorter half-life, and such molecules are also useful, for example, for administration to mammals where a shortened circulating time may be advantageous, for example, for in vivo diagnostic imaging, or in situations where the starting antibody has toxic side effects if it remains in circulation for a long period of time. Fc variants with reduced FcRn binding affinity are less likely to cross the placenta and are therefore useful in the treatment of diseases or disorders in pregnant women. In addition, other applications where reduced FcRn binding affinity may be desirable include applications where localization to the brain, kidneys, and / or liver is desirable. In one embodiment, an Fc variant-containing antibody may exhibit reduced transport from the vascular system across the renal glomerular epithelium. In another embodiment, an Fc variant-containing antibody may exhibit reduced transport from the brain across the blood-brain barrier (BBB) ​​into the vascular space. In one embodiment, an antibody with modified FcRn binding includes an Fc domain having one or more amino acid substitutions within the “FcRn binding loop” of the Fc domain. The FcRn binding loop consists of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions that modify FcRn binding activity are disclosed in PCT International Publication 05 / 047327, which is incorporated herein by reference. In certain exemplary embodiments, the antibodies or fragments thereof of the embodiments described herein include an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering).

[0186] In some embodiments, mutations are introduced into the constant region of an mAb so as to alter the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the mAb. For example, the mutation is an LALA mutation in the CH2 domain. In one embodiment, the antibody (e.g., a human mAb, or a bispecific Ab) contains a mutation on one scFv unit of a heterodimeric mAb that reduces ADCC activity. In another embodiment, the mAb contains mutations on both strands of a heterodimeric mAb that completely eliminate ADCC activity. For example, a mutation introduced into one or both scFv units of an mAb is an LALA mutation in the CH2 domain. These mAbs with variable ADCC activity can be optimized so that the mAb exhibits maximum selective killing toward cells expressing one antigen recognized by the mAb, but minimum killing toward a second antigen recognized by the mAb.

[0187] In other embodiments, the antibodies of the embodiments described herein, for use in the diagnostic and therapeutic methods described herein, have a constant region, such as the heavy chain constant region of IgG1 or IgG4, which is modified to reduce or eliminate glycosylation. For example, the antibodies of the embodiments described herein may also include Fc variants that include amino acid substitutions that modify the glycosylation of the antibody. For example, in the Fc variant, glycosylation (e.g., N-linked or O-linked glycosylation) can be reduced. In some embodiments, the Fc variant has reduced glycosylation of an N-linked glycan commonly found at amino acid position 297 (EU numbering). In another embodiment, the antibody has an amino acid substitution near or within a glycosylation motif, such as an N-linked glycosylation motif containing the amino acid sequence NXT or NXS. In a specific embodiment, the antibody includes an Fc variant having an amino acid substitution at amino acid position 228 or 299 (EU numbering). In a more specific embodiment, the antibody comprises an IgG1 or IgG4 constant region containing the S228P and T299A mutations (EU numbering).

[0188] Exemplary amino acid substitutions that reduce or alter glycosylation are disclosed in PCT International Publication No. 05 / 018572, incorporated herein by reference. In some embodiments, the antibodies or fragments of the embodiments described herein are modified to eliminate glycosylation. Such antibodies or fragments may be referred to as “agly” antibodies or fragments (e.g., “agly” antibodies). Not bound by theory, “agly” antibodies or fragments may have improved safety and stability profiles in vivo. Exemplary agly antibodies or fragments include a deglycosylated Fc region of an IgG4 antibody that lacks Fc effector function, thereby eliminating the possibility of Fc-mediated toxicity to normal living tissues and cells expressing PD-1. In yet other embodiments, the antibodies or fragments of the embodiments described herein include a modified glycan. For example, the antibody may have a reduced number of fucose residues on the N-glycan at Asn297 in the Fc region, i.e., it is defucosylated. In another embodiment, the antibody may have a modified number of sialic acid residues on the N-glycan at Asn297 in the Fc region.

[0189] The present invention also covers immunoconjugates, including cytotoxic agents such as toxins (e.g., enzymatically active toxins or fragments thereof of bacterial, fungal, plant, or animal origin) or antibodies conjugated to radioisotopes (i.e., radioconjugates).

[0190] Enzymatically active toxins and their fragments that can be used include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modexin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, geronin, mitogenin, restrictoctocin, phenomycin, enomycin, and trichothecenes. Various radionuclides are available for the production of radioconjugated antibodies. Non-limiting examples include: 212 Bi, 131 I, 131 In, 90 Y, and 186 Re is one example.

[0191] Conjugates of antibodies and cytotoxic agents are prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutarelaldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., triene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al, Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radioactive nucleotides to antibodies. (See PCT International Publication No. 94 / 11026 and U.S. Patent No. 5,736,137).

[0192] Those skilled in the art will recognize that a wide variety of possible parts can be coupled to the resulting antibody or other molecules in the embodiments described herein. (See, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, JMCruse and RELewis, Jr(eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0193] Binding can be achieved by any chemical reaction that will bind the two molecules, insofar as the antibody and the other part retain their respective activities. This binding can include many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordination bonding, and complex formation. In one embodiment, the binding is covalent. Covalent bonding can be achieved by direct condensation of existing side chains or by the incorporation of an external crosslinking molecule. Many divalent or polyvalent linkers are useful for coupling protein molecules, such as antibodies in the embodiments described herein, to other molecules. For example, typical linkers can include organic compounds such as thioesters, carbodiimides, succinimides, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to cover all classes of linkers known in the art, but rather to be an example of more general linkers. (See Killen and Lindstrom, Jour.Immun.133:1335-2549 (1984), Jansen et al., Immunological Reviews 62:185-216 (1982), and Vitetta et al, Science 238:1098 (1987).) Non-limiting examples of linkers are described in the literature. (For example, see Ramakrishnan, S. et al., Cancer Res.44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester).) See also U.S. Patent No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives conjugated to antibodies by oligopeptide linkers.Non-limiting examples of useful linkers that can be used with the antibodies of the embodiments described herein include: (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G), 21558G), (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio)propionamide]hexanoate (Pierce Chem. Co., catalog no. 21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6 [3-(2-pyridyldithio)-propionamide]hexanoate (Pierce Chem. Co.) Examples include (v) Sulfo-NHS (-hydroxysulfosuccinimide: Pierce Chem.Co., catalog number 2165-G) conjugated to EDC.

[0194] The linkers described herein contain components with different attributes, resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylate salts. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, SMPT linkers can contain sterically hindered disulfide bonds and form conjugates with improved stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in fewer available conjugates. For example, sulfo-NHS can increase the stability of carbodimide coupling. Carbodimide bonds (such as EDC), when used in combination with sulfo-NHS, form esters that are more resistant to hydrolysis than carbodimide bond reactions alone.

[0195] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art, such as those described in Epstein et al, Proc. Natl. Acad. Sci. USA, 82:3688 (1985), Hwang et al, Proc. Natl. Acad. Sci. USA, 77:4030 (1980), and U.S. Patents No. 4,485,045 and No. 4,544,545. Liposomes with extended circulation time are disclosed in U.S. Patent No. 5,013,556.

[0196] Useful liposomes, not limited to those described herein, can be produced by reverse-phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a defined pore size to produce liposomes having a desired diameter. The Fab' fragments of the antibodies of the embodiments described herein can be conjugated to liposomes described in Martin et al, J. Biol. Chem., 257:286-288 (1982) via a disulfide exchange reaction.

[0197] The embodiments described herein include isolated monoclonal antibodies, such as those specific to MICA / B. As used herein with respect to nucleic acids such as cells, DNA, or RNA, the term “isolated” refers to molecules isolated from other DNA or RNA present in a natural source of macromolecules. The term “isolated” may also refer to nucleic acids or peptides that are substantially free from cellular material, viral material, or culture media when produced by recombinant DNA technology, or from chemical precursors or other chemicals when chemically synthesized. For example, “isolated nucleic acids” may include nucleic acid fragments that do not exist naturally as fragments and are not found in their natural state. “Isolated” may also refer to cells or polypeptides isolated from other cellular proteins or tissues. Isolated polypeptides may include both purified polypeptides and recombinant polypeptides.

[0198] An "isolated molecule" (e.g., an antibody) is one that has been identified, separated, and / or recovered from components of its natural environment. These contaminants in the natural environment may be substances that could interfere with the molecule's diagnostic or therapeutic use and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the molecule would be purified to (1) a concentration of more than 95% by weight of the molecule, or more than 99% by weight, as determined by the Lowry method; (2) a concentration sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer; or (3) homogenization by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. An "isolated molecule" (e.g., an antibody) includes the molecule in situ within a recombinant cell, since at least one component of the molecule's natural environment is absent. However, typically, an isolated molecule would be prepared by at least one purification step.

[0199] As described herein, the antibodies or drugs of the present invention (also referred herein as “active compounds”), as well as their derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutically appropriate compositions. Such compositions typically comprise the antibody or drug and a pharmaceutically acceptable carrier. Where herein the term “pharmaceutically acceptable carrier” is used, it is intended to include any solvent, dispersion medium, coating agent, antibacterial and antifungal agent, isotonic agent, and absorption retarder, etc., that are compatible with pharmaceutically appropriate administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the art, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer’s solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixative oils may also be used. The use of such media and drugs for pharmaceutically active substances is well known in the art. Unless any conventional medium or agent is incompatible with the active compound, its use in the composition is intended. Auxiliary active compounds may also be incorporated into the composition.

[0200] The pharmaceutical compositions of the embodiments described herein are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in ampoules, disposable syringes, or glass or plastic multi-dose vials.

[0201] A pharmaceutical composition suitable for injectable use can include a sterile aqueous solution (if water-soluble), a dispersion, and a sterile powder for the immediate preparation of a sterile injectable solution or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate-buffered saline (PBS). In an embodiment, the composition is sterile and fluid to the extent that easy syringeability exists. It can be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars, polyhydric alcohols such as mannitol, sorbitol, etc., sodium chloride can be included in the composition. Sustained absorption of the injectable composition can be achieved by including in the composition agents that delay absorption, for example, aluminum monostearate and gelatin.

[0202] Sterile injectable solutions can be prepared, if necessary, by incorporating the active compound in an appropriate solvent having one or a combination of the ingredients described herein in the required amounts and then filtering the solution sterilize. For example, a dispersing agent is prepared by incorporating the active compound in a sterile vehicle containing a basic dispersion medium and the other necessary components derived from those described herein. In the case of a sterile powder for the preparation of a sterile injectable solution, the method of preparation is vacuum drying and lyophilization that yields a powder of the active ingredient and any additional desired components from its previously sterile filtered solution.

[0203] Oral compositions include an inert diluent or an edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, in which case the compound in the fluid carrier is applied orally, swirled in the mouth, and either spat out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds of similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or sterol; flow enhancers such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as peppermint, methyl salicylate, or orange flavor.

[0204] For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas like carbon dioxide, or a nebulizer.

[0205] Systemic administration may also be by mucosal or percutaneous means. For mucosal or percutaneous administration, a penetrating agent suitable for the barrier to penetration is used in the formulation. Such penetrating agents are known in the art and, for example, for mucosal administration, include cleansing agents, bile salts, and fusidic acid derivatives. Mucosal administration can be achieved through the use of nasal sprays or suppositories. For percutaneous administration, the active compound is formulated into ointments, plasters, gels, or creams known in the art.

[0206] The compounds can also be prepared in the form of suppositories (e.g., those having a conventional suppository base such as cocoa butter and other glycerides) or retained enemas for rectal delivery.

[0207] In one embodiment, the active compound is prepared on a carrier that will protect the compound from rapid elimination from the body, such as a controlled-release formulation, which includes implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes targeting cells infected with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0208] For ease of administration and uniformity of dosage, oral or parenteral compositions can be formulated in dose unit forms. As used herein, dose unit forms refer to physically distinct units suitable as a single dose for the subject being treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in conjunction with the required pharmaceutical carrier. The specifications of the unit dosage forms in the embodiments described herein are determined by and directly depend upon the specific properties of the active compound, the therapeutic effect to be achieved, and the limitations inherent in the art to compound such active compounds for the treatment of an individual.

[0209] Pharmaceutical compositions may be included in containers, packs, or dispensers along with administration instructions.

[0210] The compositions described herein, including those containing one or more activators, may be administered in combination with one or more additional prophylactic or therapeutic agents, or one or more additional treatment or prophylactic regimens. Accordingly, the term “combination therapy” may refer to a treatment regimen comprising at least one activator and at least one or more additional prophylactic or therapeutic agents or regimens.

[0211] In one embodiment, the additional agent, such as an additional chemotherapy agent, is an agent that has not been found to make cancer treatment refractory. In another embodiment, the additional agent is an agent that has been found to make cancer treatment refractory. This composition can also be administered to patients who have undergone surgery as a treatment for cancer. In one embodiment, the additional treatment method is radiotherapy.

[0212] In certain embodiments, the activator is administered simultaneously with additional agents, such as chemotherapeutic agents, or simultaneously with radiotherapy. In other specific embodiments, the additional agents or radiotherapy are administered before or after administration of the composition, for at least 1 hour, 5 hours, 12 hours, 1 day, 1 week, or 1 month in one embodiment, and for several months (e.g., up to 3 months) before or after administration of the composition in a further embodiment.

[0213] In embodiments, additional therapeutic and / or prophylactic agents include radiotherapy / radiotherapy, polynucleotides, polypeptides, small molecules, antibodies, genetically modified cells, radiation, or any combination thereof.

[0214] For example, the phrase "radiotherapy" can refer to the use of electromagnetic or particle radiation in the treatment of neoplasms. Examples of radiotherapy are provided in radiotherapy, but are not limited to those known in the art (Hellman, Principles of Radiation Therapy, Cancer, in Principles and Practice of Oncology, 248-75 (Devita et al., ed., 4 edit., volume 1, 1993)).

[0215] For example, additional agents may include small molecules. In embodiments, the small molecules include HDAC inhibitors such as panobinostat. Panobinostat is a type of agent called a histone deacetylase (HDAC) inhibitor. Panobinostat is a non-selective HDAC inhibitor that inhibits several histone deacetylase enzymes that cause apoptosis in malignant cells through multiple pathways. Those skilled in the art will recognize that any one of several HDAC inhibitors, including those FDA approved or in clinical trials, could be used in the embodiments described herein. For example, the HDAC inhibitors vorinostat, romidepsin, and bellinostat are approved for certain T-cell lymphomas, and panobinostat is approved for multiple myeloma. See, for example, Eckschlager, Tomas, et al. "Histone deacetylase inhibitors as anticancer drugs." International Journal of Molecular Sciences 18.7(2017):1414. For example, HDAC inhibitors may be hydroxamic acids, short-chain fatty acids, benzamides, cyclic tetrapeptides, or sirtuin inhibitors.

[0216] In other embodiments, small molecules may include proteasome inhibitors. The proteasome is a protease complex involved in the degradation of endogenous proteins. Proteins to be disrupted are recognized by the proteasome because of the presence of ubiquitin bound to the target protein. The ubiquitin-proteasome pathway plays an essential role in regulating the intracellular concentration of specific proteins, thereby maintaining intracellular homeostasis. Proteasome inhibitors prevent the degradation of these targeted proteins, which can affect multiple intracellular signaling cascades. For example, proteasome inhibitors may be bortezomib (Velcade), carfilzomib (Cyprolis), or ixazomib (Ninlaro).

[0217] In some embodiments, the additional agent may be a polypeptide such as an antibody. In some embodiments, the antibody may be an antibody specific to PD1 (anti-PD1 antibody), PDL1 (anti-PDL1 antibody), or CTLA4 (anti-CTLA4 antibody). In other embodiments, the additional therapeutic agent may be an antibody that binds to an inhibitory receptor on NK cells, such as KIR, TIGIT, NKG2A, and CD161 antibodies. In other embodiments, the antibody may be against CD160, CD96, or TIM-3. Referring to Figure 13, the antibody may be an anti-KIR2DL2 / 3 / 4 antibody.

[0218] In embodiments, additional agents may be chemotherapeutic agents. “Chemotherapeutic agent” can refer to compounds useful in the treatment of cancer. Examples of chemotherapeutic agents that can be administered with the compositions described herein include: antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); anti-estrogen agents (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, phloxuridine, interferon alpha-2b, glutamic acid, pricamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and sulfate). Examples include, but are not limited to, vincristine; hormones (e.g., medroxyprogesterone, estramustine sodium phosphate, ethinylestradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianicene, and testolactone); nitrogen mustard derivatives (e.g., mephalen, colambucil, mechloretamine (nitrogen mustard), and thiotepa); steroids and combinations (e.g., betamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0219] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, pigosulfan, and treosulfan; dacarbazines; aziridines such as benzodopa, carbocon, methuredopa, and uredopa; ethyleneimines and methylamelamamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenthiophosphoramide, and trimethyloromelamamine; TLK 286 (TELCYTA®); acetogenin (especially bratacin and bratacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapacon; lapachol; colchicine; betulinic acid; camptothecin (including synthetic analogues topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; calistatin; CC-1065 (its adze Resin, including karzeresin and bizeresin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycin (such as cryptophycin 1 and cryptophycin 8); drastatin; duocalmycin (including synthetic analogs, KW-2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodictiin; spongitatin; chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobenbitin, fenesterine, prednimustine, trophosphamide or uracil mustard; nitrosoureas such as carmustine, chlorozotosine, fotemustine, lomustine, nimustine, and ranimnustine; bisphosphonates such as clodronate;Antibiotics, such as engine antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega I1 (see, e.g., Agnew, 1994, Chem. Intl. Ed. Engl. 33:183-186)) and anthracyclines, such as anthracycline, AD32, alcarbicin, daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100, idarubicin, KRN5500, menogalil, dynemicin (including dynemicin A), esperamicin, neo Cardinostatin chromophores and related pigment proteins, enediin antibiotic chromophores, acrasinomycin, actinomycin, autoramycin, azaserin, bleomycin (e.g., bleomycin A2, bleomycin B2, and peplomycin), kactinomycin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin) (including biscin, 2-pyrrolinodoxorubicin, liposomal doxorubicin, and deoxydoxorubicin), esorubicin, marcelomycin, mitomycin such as mitomycin C, mycophenolate, thiazophrine, ribavirin, EICAR, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zorubicin; denopterin, pteropterin, and trimethrexate Folic acid analogs such as tetraphosphate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens such as carsterone, drostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal drugs such as aminoglutethimide, mitotane, and trilostane;Folic acid supplements such as folic acid (leucovorin); acegraton; antifolic acid antitumor drugs such as ALIMTA®, LY231514 pemetrexed; dihydrofolate reductase inhibitors such as methotrexate and trimethrexate; antimetabolites such as 5-fluorouracil (5-FU) and UFT, S-1 and capecitabine and their prodrugs; thymidylate synthase inhibitors and glycinamide ribonucleotide formyltransferase inhibitors such as larcitrexed (TOMUDEXRM, TDX); dihydropyrimidine dehydrogenase inhibitors such as enyluracil; aldofosphatidyl Midoglycoside; aminolevulinic acid; amsacrin; bestlabsil; bisanthren; edatraxate; defofamine; demecolsin; diaziquan; elfornithine; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydroxyurea; deferoxamine; lentinan; ronidynin; mytansinoids such as mytansin and ansamitosin; mitogluazone; mitoxantrone; mopidammole; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic Acid; Triadiquan; 2,2',2''-Trichlorotriethylamine; Trichothecene (especially T-2 toxin, Beraclin A, Loridine A, and Angidin); Urethane; Vindesine (ELDISINE®, FILDESIN®); Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Cytosine Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa;Taxoids and taxanes, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® cremophore-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® docetaxel (Rhone-Poulenc). Rorer, Antony, France); Chlorambucil; Gemcitabine (GEMZAR®); 6-Thiogunine; Mercaptopurine; Platinum; Platinum analogs or platinum-based analogs such as cisplatin, oxaliplatin, and carboplatin; Vinblastine (VELBAN®); Epipodophyllines such as etoposide (VP-16), teniposide, tepotecan, 9-aminocamptothecin, camptothecin, and cristinator; Ifosfamide; Mitoxantrone; Vincristine (ONCOVIN®), vindesine, vinca alkaloids, and vinorelbine (NAVELBINE®), etc. Vinca alkaloids; Novantrone; Edatrexate; Daunomycin; Aminopterin; Xeroda; Ibandronate; Topoisomerase inhibitor RFS2000; Difluoromethylhyronitine (DMFO); Retinoids such as retinoic acid; pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for treatment regimens of oxaliplatin (ELOXATIN®) in combination with 5-FU and leucovorin.

[0220] Cytokines that may be administered with the composition include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, anti-CD40, CD40L, and TNF-α.

[0221] In some embodiments, the compositions described herein may be administered in combination with other immunotherapeutic agents. Non-limiting examples of immunotherapeutic agents include simtuzumab, avagovomab, adecatumumab, aftuzumab, alemtuzumab, altumomab, amatsuximab, anatumomab, alsitumomab, bavituximab, bectomomab, bevacizumab, vibatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, sitatuzumab, xixtumumab, cribatuzumab, conatumumab, daratumumab, doroditumab, and zuri. Gotumab, ducizimab, detumomab, desetuzumab, dalotuzumab, eclomeximab, elotuzumab, encituximab, erzmaxomab, etalacizumab, falletuzumab, ficratuzumab, phyditumumab, frambotuzumab, futuximab, ganitumumab, gemtuzumab, girentuzumab, grembatumumab, ibritumomab, igobomab, imagatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab Iratumumab, rabetuzumab, lexatumumab, lintuzumab, rorbotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitsumomab, moxetumomab, nalnatumumab, naptumomab, nesitumumab, nimotuzumab, nofetumomab, okalatuzumab, ofatumumab, oraratumumab, onarutuzumab, oporutuzumab, olegobomab, panitumumab, pulsatuzumab, patrizumab, pemtumomab, Examples include pertuzumab, pintumomab, pritumumab, lacotumomab, radretumumab, rilotumumab, rituximab, lobatumumab, satumomab, sibrotuzumab, siltuximab, solitomab, takatuzumab, tapritumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotzumab, ubrituximab, bertuzumab, borsetuzumab, botumumab, saltumumab, CC49, and 3F8.

[0222] In embodiments, additional agents may be administered over a series of sessions. Any one or a combination of the additional agents described herein may be administered. With respect to radiation, any radiotherapy protocol may be used depending on the type of cancer being treated. For example, X-rays may be irradiated, but are not limited to X-rays. For example, high-energy megavoltage (radiation with energy greater than 1 MeV) may be used for deep tumors, and electron beam and orthovoltage X-ray radiation may be used for skin cancers. Gamma-ray emitting radioisotopes such as radium, cobalt, and other elemental radioisotopes may also be administered.

[0223] Furthermore, the cancer compositions and treatment methods described herein are offered as alternatives to standard anti-cancer therapies, such as chemotherapy or radiotherapy, which have been proven, or may be proven, to be too toxic to the patient undergoing treatment, resulting in unacceptable or unbearable side effects. The patient undergoing treatment may, at their discretion, be treated with another cancer treatment, such as surgery, radiotherapy, or chemotherapy, depending on which treatment is acceptable or tolerable.

[0224] The activators can also be used in vitro or ex vivo for the treatment of certain cancers, including but not limited to leukemia and lymphoma, such as therapies involving autologous stem cell transplantation.

[0225] A method for treating drug-resistant cancer or cancer resistant to cytotoxic T cells comprises administering to a patient in need an effective dose of at least one activator and, optionally, another therapeutic agent that is an anticancer drug. Suitable anticancer agents include, but are not limited to, those described herein, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrouracil, cisplatin, carboplatin, mitomycin C, dacarbazine, procarbidine, topotecan, nitrogen mustard, cytoxan, etoposide, 5-fluorouracil, phloxuridine, doxifluridine, and latitrexate, BCNU, irinotecan, camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel.

[0226] Furthermore, antihormone agents that act to modulate or inhibit the hormonal effects on tumors, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including tamoxifen (including NOLVADEX® tamoxifen), raloxifen, megestrol, droloxifen, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and FARESTON® toremifene; for example, 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestany, fadrozol, RIVISOR® borozole, FEMARA, and other aromatase inhibitors® letrozole that inhibit aromatase, an enzyme that regulates estrogen production in the adrenal gland; and ARIMIDESX® anastozole. Rozole; antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, leuprolide acetate, and goserelin; as well as troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as gene therapy vaccines like ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; vitamin DA analogs such as EB1089, CD1093, and KH1060; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0227] In additional embodiments, additional therapeutic agents may include photodynamic agents such as bertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, and demethoxyhypocrelin A (2BA-2-DMHA); cytokines such as interferon-α, interferon-γ, or tumor necrosis factor; gemcitabine, Velcade® (bortezomib), Revlamid® (lenalidomide), or talamide; lovastatin; 1-methyl-4-phenylpyridinium ion; staurosporine; actinomycins such as actinomycin D or dactinomycin; anthracyclines such as daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zolubicin, and mutoxantrone; MDR inhibitors such as verapamil and Ca2+ATPase inhibitors such as thapsigargin; and any pharmaceutically acceptable salts, acids, or derivatives of the above.

[0228] Other Embodiments The embodiments described herein, along with their detailed description, are intended to illustrate, and not limit, the scope of the embodiments described herein as defined by the appended claims. Other embodiments, advantages, and modifications are within the scope of the following claims.

[0229] Embodiments described herein are further described in the following examples, which do not limit the scope of embodiments described herein as described in the claims. [Examples]

[0230] Examples are provided below to facilitate a more complete understanding of the embodiments described herein. The following examples illustrate exemplary ways of constructing and carrying out the embodiments described herein. However, the scope of the embodiments described herein is not limited to the specific embodiments disclosed in these examples, and similar results can be obtained using alternative methods; therefore, they are for illustrative purposes only.

[0231] Example 1 - Treatment of cancer resistant to checkpoint blockade by loss of MHC class I expression Checkpoint blockade using antibodies that target inhibitory receptors on T cells is widely used in cancer treatment. Antibodies targeting the PD-1 / PD-L1 pathway are approved for the treatment of a variety of cancers, including melanoma, lung cancer, kidney cancer, bladder cancer, and non-limited cases such as Hodgkin lymphoma. Checkpoint blockade requires cytotoxic T cells as effector cells of the immune response, which kill tumor cells after recognizing tumor-derived peptides bound to MHC class I proteins. Tumors that have lost MHC class I expression due to genetic mutations or inactivation of epigenetic mechanisms are resistant to checkpoint blockade. However, MHC class I proteins function as ligands for inhibitory KIR receptors on NK cells. However, the loss of this inhibitory signaling is insufficient to induce tumor immunity because the activation signal of NK cells is absent or weak.

[0232] We demonstrated that MHC class I-deficient tumor cells are efficiently killed in the presence of a MICA antibody that activates NK cells via two major receptors, NKG2D and CD16. Importantly, we demonstrate that the MICA antibody has monotherapy activity against treatment-resistant cancer cells in vivo. While we do not wish to be constrained by theory, MHC class I-deficient tumor cells can be targeted with monoclonal antibodies that induce NK cell activation via major activating receptors such as NKG2D and CD16. For example, such a monoclonal antibody could be a MICA antibody or other tumor-targeting antibody that activates NK cells.

[0233] Currently, there are no immunotherapies for cancers that have lost MHC class I expression. Such cancers are resistant to all immunotherapies that require cytotoxic T cells as an effector mechanism. There is a high clinical need in this area. In our approach, NK cells are activated by signaling via two major NK cell receptors (NKG2D and CD16), but this is not counteracted by inhibitory signals from KIR receptors that bind to MHC class I proteins.

[0234] Aspects of the embodiments described herein include the use of combination therapy with PD-1 or CTLA-4 checkpoint blockade in patients resistant to monotherapy with checkpoint blockade. In most responsive cancers, only 20% of patients respond to PD-1 blockade, so there is much room for improvement.

[0235] Example 2 - Use of natural killer cells for the treatment of tumors resistant to cytotoxic T cells summary Resistance to cytotoxic T cells is often mediated by loss of MHC class I expression or IFNγ signaling in tumor cells, such as mutations in the B2M or JAK1 genes. While NK cells can target such resistant tumors, suitable NK cell-based strategies have yet to be developed. The embodiments described herein address this shortcoming. It has been shown that B2M and JAK1-deficient metastases were targeted by NK cells after treatment with mAbs that blocked MICA / B shedding, a frequent evasion mechanism in human cancers. Single-cell analysis of NK cells in human melanoma metastases was also performed, including patients whose cancer progressed after checkpoint blockade. Major transcriptional differences between tumor-infiltrating and circulating NK cells were identified. Furthermore, the gene expression programs of seven tumor-infiltrating NK cell clusters exhibited important specializations, such as cytotoxicity and chemokine secretion. Thus, NK cell-based immunotherapy offers an opportunity to target tumors with mutations that confer resistance to cytotoxic T cells.

[0236] Prologue Checkpoint blockade using antibodies targeting programmed cell death protein 1 (PD-1) or cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitory receptors on T cells can induce persistent anti-tumor immunity even in patients with advanced cancer. However, many patients are unable to benefit from these therapies due to primary or secondary resistance. 1 Cytotoxic T cells play a central role in the effectiveness of checkpoint blocking based on their ability to recognize tumor-derived peptides bound to major histocompatibility complex class I (MHC-I) proteins. 2 When such MHC-I peptide complexes are recognized by the T cell receptor (TCR), T cells release interferon-γ (IFNγ), inhibiting tumor cell proliferation and enhancing MHC-I protein expression in both tumor cells and dendritic cells. 3 Therefore, resistance to checkpoint blockade is often mediated by the loss of MHC-I expression by tumor cells, either due to mutations in key genes in the MHC-I (B2M, TAP1, TAP2, and other genes) or IFNγ (JAK1, JAK2) pathways, or epigenetic silencing. 4、5、6 When the number of newly generated antigens is low or lost, tumor immunity mediated by cytotoxic T cells is also reduced. 7、8、9、10 Currently, there are no alternative immunotherapies for patients with solid tumors that are resistant to checkpoint blockade and cytotoxic T cells.

[0237] Natural killer (NK) cells recognize tumor cells through molecular mechanisms substantially different from those required by cytotoxic T cells. NK cell recognition by tumor cells is mediated by ligands associated with malignant transformation, such as DNA damage and cellular stress. 12 While we do not wish to be constrained by theory, tumors resistant to cytotoxic T cells may respond to NK cell-based immunotherapy approaches. In fact, loss of MHC-I expression by tumor cells increases sensitivity to NK cells, as the MHC-I protein functions as a ligand for inhibitory NK cell receptors.12 However, inducing tumor immunity via NK cells may also require the effective targeting of immune evasion mechanisms that prevent NK cell-mediated attack on tumor cells. For example, many human cancers express MHC-I polypeptide-associated sequence A (MICA) and MICB (MICA / B) proteins, which function as ligands for activated NK group 2D (NKG2D) receptors on subpopulations of NK cells and T cells. 13、14 However, tumors often evade NKG2D receptor-mediated tumor immunity by shedding through proteolytic degradation of MICA / B proteins. 15、16、17、18、19 Recently, we developed monoclonal antibodies (mAbs) that bind to the α3 domain of MICA / B, a domain essential for shedding. See, for example, Table 1 and WO2018217688A1 (the entirety of which is incorporated herein by reference). These mAbs inhibited MICA / B shedding and induced NK cell-mediated tumor immunity. Increased density of MICA / B protein on tumor cells enhanced NK cell-mediated activation via the NKG2D receptor, and the Fc segment of tumor conjugate antibodies also activated NK cells via the CD16Fc receptor. Treatment with such MICA / B antibodies significantly altered tumor-infiltrating NK cells into a highly cytotoxic state. 20

[0238] Therefore, while we do not wish to be constrained by theory, NK cell-based therapies may be able to treat metastatic lesions resistant to cytotoxic T cells. However, little is known about human NK cells infiltrating solid tumors. We performed a single-cell analysis of NK cells infiltrating human melanoma metastases, including metastases from patients whose tumors progressed after checkpoint blockade. These data identified a novel subpopulation of NK cells and highlighted significant differences between tumor-infiltrating NK cell populations and circulating NK cell populations. Treatment with MICA / Bα3 domain mAbs enabled NK cell-mediated immunization against tumors with inactivating mutations in the MHC-I or IFNγ signaling pathway (B2m and Jak1 mutations, respectively). These results will help guide the development and validation of NK cell-based immunotherapies for human cancers resistant to cytotoxic T cells.

[0239] result Evaluation of single-cell characteristics of NK cells infiltrating human melanoma metastases Increasing evidence demonstrates the crucial role that NK cells play in tumor immunity. 21、22、23、24、25、26、27、28 However, little is known about the NK cell population infiltrating human solid tumors. Therefore, we conducted a single-cell study of NK cells infiltrating human melanoma metastases requiring surgical resection. In the majority of these patients, the tumor progressed after treatment with checkpoint blockade (Figure 17). NK cells were CD45 + CD56 + CD3 - CD4 - CD8a - CD14 - CD15 - CD163 - Lymphocyte-sized viable cells were sorted from tumors and matching blood samples by flow cytometry. NK cells identified by these markers were present at a lower frequency in tumors compared to blood samples. The frequency of NK cells in the overall lymphocyte population varied from 2.47% to 46.10% in blood and from 0.46% to 6.48% in tumors (Figure 8). For example, NK cells were found in the total CD45 of patient CY158 tumors.+ 4.12% of lymphocytes and their total CD45 in the blood + They accounted for 27.7% of lymphocytes (Figure 1).

[0240] The transcriptomes of NK cells isolated from melanoma metastases and matching blood samples from three patients (CY155, CY158, and CY160) were examined by single-cell RNA-seq (scRNA-seq) using the 10X Genomics platform. NK cell clusters were visualized using the nonlinear dimensionality reduction technique, Uniform Manifold Approximation and Projection (UMAP). A pooled analysis of tumor versus blood NK cells from each patient demonstrated significant differences in NK cell distribution across clusters, indicating substantial differences in the transcriptomes of tumor-infiltrating and circulating NK cells. NK cell clusters with similar gene expression profiles were identified in all three patients. The only exception was an NK cell population characterized by differential expression of interferon-inducible genes (ISG15, IFI6), present only in the tumor of patient CY158 (Figures 1 and 9).

[0241] Analysis of blood NK cells from all three patients identified four blood NK cell clusters (bNK.0–bNK.3). Similar analysis of tumor-infiltrating cells from all three patients identified eight NK cell clusters (tNK.0–tNK.7) (Figure 1). The two NK cell clusters identified within the tumor shared transcriptional function with blood NK cells, but were present at significantly different frequencies in these locations. The dominant NK cell cluster within the tumor (tNK.0, 41.1%) shared transcriptional signatures with the minor NK cell population in the blood (bNK.2, 3.3%) (SELL, IL7R, XCL1, and XCL2). The dominant NK cell cluster in the blood (bNK.0) shared expression of many major genes with the tumor cluster tNK.3 (including FGFBP2, FCGR3A, PRF1, and GZMB), but the proportion of tumor-infiltrating NK cells was much lower (10.9% compared to 82.9% in the blood). Furthermore, small proliferating NK cell clusters were present in both blood (bNK.3, 0.9%) and tumors (tNK.7, 1.5%), and these cells shared the expression of cell cycle genes (including PCNA and MKI67). Five other NK cell clusters identified in melanoma metastases (tNK.1, tNK.2, tNK.4, tNK.5, and tNK.6) had entirely different transcriptional programs from the blood NK cell clusters (Figure 1).

[0242] The markers used to isolate NK cells (Figure 1) are also expressed by innate lymphoid cells (ILCs), including the ILC1, ILC2, and ILC3 subpopulations. 29 ILCs are known to be cells found in tissues, and therefore it is possible that ILCs were not present in the blood sample. 30 However, it was important to assess whether some of the NK cell clusters identified in melanoma metastases could be ILCs. Previous scRNA-seq studies identified genes differentially expressed by NK cells, ILC1, ILC2, and ILC3, and these genes were used to assemble the transcriptional signatures of each of these innate lymphoid cell populations. 31All NK cell clusters in the blood had a strong NK cell gene expression signature, but all three ILC signatures had low scores (Figures 2 and 10). Furthermore, seven of the eight NK cell clusters identified within the tumor also had high NK cell gene expression signature scores (Figure 2). The exception was the tNK.5 cluster (gold), which had a high ILC3 gene expression signature score but low NK cell, ILC1, or ILC2 signature scores (Figures 2 and 10). Finally, neither the tumor NK cell population nor the blood NK cell population had a strong ILC1 or ILC2 gene expression signature (Figure 10). Thus, NK cells (7 out of 8 clusters, 93.2% of cells total) and ILC3-like cells (1 cluster, 6.8% of cells) were identified in human melanoma metastases.

[0243] Gene expression programs related to major NK cell functions While research into NK cells in tumor immunity has primarily focused on their cytotoxic function, recent studies have highlighted the crucial role of NK cells in the recruitment of dendritic cells, which are essential for inducing T cell-mediated tumor immunity. 24、26A panel of genes encoding major cytotoxic proteins was used to assemble a cytotoxic gene expression signature (GZMA, GZMB, GZMH, GZMK, GZMM, PRF1, GNLY, and NKG7). This cytotoxic signature was high in most hematopoietic NK cells, particularly in clusters bNK.0, bNK.1, and bNK.3. In tumor NK cells, a gradient of this cytotoxic signature was observed, being highest in tNK.3 and tNK.4 clusters and intermediate in the other five clusters. Consistent with designating these cells as ILC3s, only the tNK.5 cluster was negative for this cytotoxic signature (Figure 2). Different granzyme gene expression patterns were also observed across NK cell clusters. GZMA expression was high in most hematopoietic NK cells, but showed a similar gradient to the cytotoxic signature in tumor NK cells. Interestingly, GZMK expression showed a distinct pattern. While low levels were observed in most blood NK cells, they were high in many tumor NK cell clusters (Figure 3).

[0244] Furthermore, significant differences were observed in chemokine gene expression between tumor NK cells and blood NK cells. Chemokines XCL1 and XCL2 (which bind to the XCR1 chemokine receptor) have recently been shown to play an important role in recruiting cross-presenting DCs (cDC1) to tumors. 26 The expression of these two chemokine genes was substantially higher in tumor NK cells (clusters tNK.0, tNK.1, tNK.2, and tNK.6) compared to hematopoietic NK cells (Figures 2 and 10). High expression of another set of chemokine genes (CCL3, CCL4, CCL4L2, and CCL5) was also observed in many tumor NK cells (clusters tNK.3, tNK.4, and tNK.1), while expression was low in most hematopoietic NK cells (Figures 2 and 10). These chemokines bind to CCR5 and other chemokine receptors and play a crucial role in the recruitment of T cells and other immune cells. CCL5 is also known to contribute to the recruitment of cross-presenting DCs. 26、32Therefore, NK cells present in tumors express many chemokine genes important for the recruitment of DCs, T cells, and other immune cell populations. Note that tumor-infiltrating NK cells expressed substantially higher levels of FOS and JUN, which encode subunits of the AP-1 transcription factor, compared with hematopoietic NK cells (Figure 1). Single-cell data also clearly demonstrated functional specialization among tumor NK cell populations with respect to chemokine gene expression: four clusters of tumor NK cells showed high expression of XCL1 / XCL2, while distinct sets of tumor NK cell clusters showed high expression of CCL3, CCL4, CCL4L2, and CCL5. Thus, these tumor NK cell populations can create distinct microenvironments.

[0245] NK cells integrate signals from the extracellular environment via a series of activating and inhibitory receptors (Figure 11). Among the genes encoding activating receptors, strong signaling for KLRF1 (NKp80 protein) was observed in the majority of hematological and tumor NK cells (Figure 11). The AICL gene, which encodes the ligand for NKp80, may be expressed in both hematological malignancies and solid tumors. 33 While other established activated NK cell receptors showed low signals (NCR1, NCR3, CD226, and NKG2D), it is important to note that some mRNAs tend to produce fairly weak signals by scRNA-seq, even if both mRNA and protein are very abundant within the relevant cell population. For example, KLRK1 mRNA was low in all NK cell populations, including blood NK cells (KLRK1 encodes the NKG2D protein), while HCST mRNA was high (HCST mRNA encodes DAP10, an adapter molecule for NKG2D). Therefore, although we do not wish to be bound by theory, KLRK1 mRNA was difficult to detect by scRNA-seq, while HCST was detected. On the other hand, published reports demonstrate that NKG2D protein can be detected on human blood NK cells from melanoma patients, but at lower levels compared to healthy donors. 34、35NKG2D expression was lower in tumor infiltration compared to blood NK cells (Figure 11). This is likely due to ligand-induced downregulation and TGFβ-induced changes in gene expression. 36

[0246] Furthermore, we observed interesting expression patterns of receptors whose inhibitory function was established within NK cells. Tumor-infiltrating NK cells expressed higher levels of the KLRC1 gene (NKG2A protein) than blood NK cells, and the KLRD1 gene (CD94 protein) was highly expressed by most tumor NK cells and blood NK cells. This indicates that the majority of melanoma-infiltrating NK cells express the inhibitory NKG2A-CD94 receptor that recognizes HLA-E. In addition, strong signaling of the KLRB1 gene (CD161 protein) was observed in both tumor NK cells and blood NK cells, and CD161 is known to inhibit the cytotoxicity of NK cells after binding to CLEC2D on tumor cells and APCs. 37 While signaling for most other inhibitory receptors was weak, interestingly, different expression patterns emerged. CD96 was expressed across the entire NK cell cluster, while the expression of other receptors was limited to one or a small subset of the NK cell cluster (e.g., CD160 and KIR2DL3). Furthermore, KIR2DL3 and KIR2DL4, despite belonging to the same family of inhibitory receptor genes, showed different expression patterns across the entire NK cell cluster (Figures 3 and 11).

[0247] Validation of scRNA-seq data using flow cytometry Using flow cytometry, key findings from scRNA-seq data were validated, and the analysis was extended to a larger population of melanoma patients. Established markers (CD45) + CD56 + CD3 - CD4 - CD8a - CD14 - CD15 - CD163 - CD19 -Live cells of lymphocyte size were used to identify NK cells, and then the major NK cell subpopulations were identified using the CD16a and FGFPB2 markers based on scRNA-seq data. Importantly, the CD16a protein is encoded by the FCGR3A gene. This analysis identified three cell populations: 1) bNK.0 (the most abundant population in the blood) and tNK.3 that expressed the major cytotoxic genes (PRF1 and GZMB), corresponding to FGFBP2 + CD16a + NK cells; 2) FGFPB2 was used as a marker to CD16a + FGFBP2 that could be separated from the population - CD16a + NK cells (these cells corresponded to bNK.1, and tNK.4 and tNK.1 that expressed FCGR3A but not FGFBP2); 3) bNK.2 (a small blood subset) and FGFBP2 corresponding to a rich population of tumor NK cells that did not express FGFBP2 and FCGR3A (not exclusive, mainly tNK.0 and tNK.2) - CD16a - NK cells (Figure 1 and Figure 3). [[ID=I6]]

[0248] Using the FGFBP2 and CD16a markers, these three NK cell populations in melanoma were examined, and blood samples from a total of seven patients were collated. The dominant NK cell population in the blood samples was positive for both FGFBP2 and CD16a, while most of the tumor-infiltrating NK cells were negative for both FGFBP2 and CD16a (Figure 3). This is consistent with the scRNA-seq data (Figure 3). Furthermore, the expression of granzyme A and K in these NK cell populations was examined. The expression of granzyme A and K was higher in the blood compared to tumor NK cells. Also, the granzyme A level was FGFBP2 - CD16a[[ID=ZI]] - Higher in FGFBP2-positive and -negative CD16a compared to blood NK cells + blood NK cells (Figure 3). This is consistent with the scRNA-seq data (Figure 3).

[0249] HLA-A / B / C proteins were detected on the surface of melanoma cells in eight patients, but were low or undetectable in tumor cells in three patients (Figure 12). In particular, one of the tumors studied by scRNA-seq (CY155) had undetectable surface HLA-A / B / C proteins, and this patient had progressed after treatment with PD-1 mAb. Surface MICA / B proteins were detected on the surface of melanoma cells in most cases, albeit at relatively low levels. Shedding MICA was detected in seven of the nine serum samples, which is consistent with the loss of surface MICA from tumor cells due to shedding (Figure 12). Shedding MICA was not detected in the serum of healthy subjects.

[0250] MICA antibodies enhanced the killing of human B2M-deficient melanoma cells via NK cells. Single-cell data demonstrate that NK cells infiltrate human melanoma metastases, including lesions resistant to checkpoint blockade (Figure 17). Therefore, we investigated whether MICA / Bα3 domain-specific antibodies could enhance NK cell-mediated immunity against B2M-deficient tumor cells. 20 The MICA and MICB genes are part of the MHC locus on human chromosome 6, and the encoded proteins share significant structural similarities with MHC proteins. B2M deficiency disables T-cell immunity and responsiveness to T-cell checkpoint blockade, although the MICA / B proteins do not associate with β2-microglobulin or peptides. 5、38、39 Inactivation of the B2M gene in human A375 melanoma cells resulted in complete loss of MHC-I surface protein even after IFNγ stimulation (Figures 4 and 13). B2M deficiency did not interfere with the MICA / B pathway, but treatment with a MICA / Bα3 domain-specific mAb (7C6-hIgG1) inhibited MICA shedding and increased MICA / B surface levels to a level comparable to control and B2M-edited A375 cells (Figures 4 and 13).

[0251] NK cells have inhibitory receptors for MHC-I molecules 12 While not wishing to be bound by theory, B2M-deficient tumor cells may be more sensitive to MICA / B mAb treatment. Using an imaging-based system that allows counting of fluorescent tumor cells in 96-well plates at multiple time points, the kinetics of killing of human A375 melanoma cells by NK cells were studied. The major advantage of this technique is that it allows investigation of the interaction between NK cells and tumor cells in the context of the tumor microenvironment, when the effector-to-target ratio is low. 40 This experiment demonstrated that MICA / B mAb treatment (7C6-hIgG1) was substantially more effective against B2M-KO compared to control A375 melanoma cells. Even at a low effector-to-target ratio (1:1), in the presence of MICA / B mAb, only a few fluorescent B2M-KO melanoma cells remained at late time points (48 - 72 hours) (Figure 4). KIR2DL2, KIR2DL3 and KIR2DL4 are part of the best characterized inhibitory receptors for MHC-I molecules on human NK cells. 12 Blocking of these receptors via antibodies increased killing of 7C6-hIgG1-treated A375 melanoma cells by NK cells (Figure 13). These experiments demonstrated that loss of MHC class I surface expression renders human tumor cells more vulnerable to NK cells in the presence of MICA / B mAb.

[0252] MICA / B antibodies induced NK cell-mediated immunity against metastases resistant to cytotoxic T cells Two mouse models were used to investigate whether MICA / B mAb treatment could induce NK cell-mediated immunity against tumors with inactivating mutations in the MHC-I and IFNγ pathways (B2m and Jak1 mutations respectively). Jak1 mutations were of particular interest since IFNγ is secreted from both T cells and NK cells. IFNγ signaling in tumor cells not only enhances the expression of many genes in the MHC class I pathway but also inhibits tumor cell proliferation. 3Therefore, Jak1 mutations may either adversely affect the ability of NK cells to regulate tumor cell growth or enhance NK cell activation through the loss of MHC class I proteins involved in inhibitory receptors on NK cells. Lentiviral vectors were transduced into B16F10 melanoma and LLC1 lung cancer cell lines to induce the expression of human MICA, which is known to bind to the mouse NKG2D receptor. 20 These mouse models showed significant differences in the patterns of MHC class I expression. B16F10 melanoma cells showed H-2K b Although the basal surface level of the protein was very low, exposure to IFNγ resulted in H-2K b Surface proteins were significantly increased (Figures 5 and 14). In contrast, LLC1 lung tumor cells showed increased H-2K levels upon IFNγ treatment. b The baseline level increased to a detectable level (Figure 5).

[0253] The efficacy of MICA / B mAb treatment was tested in a lung metastasis model by inactivating the B2m or Jak1 gene in B16F10-MICA melanoma cells (Figures 5 and 14). Edited tumor cells were injected intravenously, and treatment was initiated on day 7 when established surface lung metastases were detected (determined by pathological analysis of a subset of mice labeled "pre-treatment group") (Figure 5). As previously reported, B cell-deficient Ighm was used to prevent the development of endogenous antibodies against human MICA. - / - Mice were used as the host. 20Treatment with the MICA mAb (7C6-mIgG2a) inhibited the proliferation of lung metastases in control, B2m-KO, and Jak1-KO B16F10-MICA cells (Figure 5). MICA mAb treatment also reduced plasma levels of shedding MICA (Figure 14). Compared to B16F10-MICA inoculation, MICA mAbs administered on days 1 and 2 significantly increased the survival of wild-type (WT) mice with control, B2m-KO, or Jak1-KO melanoma metastases compared to isotype control mAb treatment (Figure 5). Surprisingly, JAK1-KO mice showed a greater survival benefit compared to B2M-KO mice. This finding was surprising, as it was unknown whether JAK1-KO mice would respond (since NK cells also secrete gamma interferon). As shown herein, NK cells remain active against tumors with loss mutations in the gamma interferon pathway. Furthermore, the effectiveness of MICA / B antibody treatment in an LLC1-MICA tumor model was investigated. Notably, control LLC1 cells expressed MHC-I at baseline, and MHC-I surface protein levels increased upon IFNγ treatment, while B2m-KO LLC1 cells did not express MHC-I even after IFNγ treatment (Figure 5). Tumor cells were intravenously injected into WT mice, and mAb treatment was started on day 2. MICA mAb treatment reduced the number of lung metastases formed by control LLC1-MICA tumor cells. Inactivation of the B2m gene reduced the number of lung metastases to almost undetectable levels compared to control LLC1-MICA cells. Therefore, increasing the number of inoculated tumor cells by 50% resulted in the formation of lung metastases by B2m-KO LLC1-MICA cells. Under these experimental conditions, a significant reduction in the number of B2m-KO LLC1-MICA metastases after treatment with 7C6-mIgG2a was observed compared to isotype control mAbs (Figure 5). Furthermore, we investigated the role of inhibitory receptors for NK cells and MHC-I proteins using an adoptive transfer model. - / - Il2rg - / -KO mice were reconstituted with syngeneic (C57BL / 6 mouse-derived) or allogeneic (CB6F1 mouse-derived) NK cells. Syngeneic and allogeneic NK cells significantly reduced the number of lung metastases formed by LLC1-MICA tumor cells when mice were treated with MICA / B and isotype control mAbs. Furthermore, MICA / B antibody treatment was more effective when allogeneic NK cells were transplanted (Figure 5). As previously reported, allogeneic NK cells are not inhibited by MHC class I proteins on tumor cells (such as LLC1-MICA cells). 41 While we do not wish to be constrained by theory, the data demonstrate that the involvement of MHC-I proteins by inhibitory receptors on NK cells reduces the effectiveness of MICA / B mAb-induced antitumor immunity.

[0254] Next, mechanistic experiments were performed using B16F10-MICA cell lines inoculated into WT mice. When NK cells were depleted, the efficacy of MICA mAbs against both control and B2m-KO B16F10-MICA tumor cells was completely lost, while the efficacy of MICA mAbs against Jak1-KO B16F10-MICA tumor cells was significantly reduced by NK cell depletion (Figure 6). As previously reported, lung infiltrating cells and hematopoietic NK cells were distinguished by intravenous injection of APC-conjugated anti-CD45.2 antibody before euthanasia. 20 MICA mAb treatment increased the degree of NK cell infiltration in control or Jak1-KO B16F10-MICA tumors (Figure 6). In this analysis, NK cell infiltration was normalized to tumor volume because the number of B16F10-MICA tumor cells was substantially reduced in MICA mAb-treated mice (Figure 6). These data demonstrate that MICA mAb treatment inhibits the proliferation of melanoma metastases in an NK cell-dependent manner, even when tumor cells have inactivating mutations in the B2m or Jak1 gene.

[0255] Improvement of MICA / B surface protein levels on human tumor cells treated with a combination of MICA / B mAb and HDAC inhibitor. In the tumor model described above, MICA transcription was regulated by heterologous promoters that induced relatively high levels of MICA, as previously shown. 20 However, in human cancer, MICA / B expression is induced in response to DNA damage and cellular stress. 13 In the human melanoma metastases examined, MICA / B proteins were detectable on the surface of tumor cells in most cases, albeit at low levels, and MICA was present in the serum of 7 out of 9 patients (Figure 12). It is well known that the transcription of MICA and MICB genes is epigenetically regulated by histone deacetylase (HDAC), and that HDAC inhibitors enhance the transcription of these genes. 42 Panobinostat, a pan-HDAC inhibitor, has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of multiple myeloma. 43 Previous studies in multiple cancer mouse models have established that a functionally intact immune system is necessary for panobinostat to be therapeutically active. 44Therefore, we investigated whether the combination of panobinostat and 7C6-hIgG1 mAb could improve MICA / B protein levels by increasing transcription (panobinostat) and stabilizing surface proteins (MICA / B mAb). RNA-seq analysis demonstrated that 24-hour treatment of A375 melanoma cells with panobinostat (50 nM) increased mRNA levels of several genes encoding NKG2D ligands, including MICA, RAET1G, and RAET1L. However, panobinostat did not increase mRNA levels of genes encoding conventional or non-conventional MHC-I molecules (Figure 7). Panobinostat also affected the transcription of many other genes in A375 melanoma cells, some of which were immune-related pathways (Figure 15). The combination of panobinostat and MICA / B mAb significantly increased surface MICA / B protein levels and reduced shedding MICA concentration without reducing cell viability (Figure 7). These conclusions were further supported by analysis of a diverse panel of human tumor cell lines (Figure 15). MICA / B protein levels were also examined using a panel of short-term human melanoma cell lines established from metastatic lesions. 20、45 Treatment with panobinostat and MICA / B mAbs significantly increased the surface density of MICA / B proteins compared to treatment with the individual compounds (Figure 7). These data demonstrate that a combined approach that increases MICA / B gene transcription and stabilizes synthetic proteins significantly increases surface MICA / B proteins on human cancer cells.

[0256] Combining MICA mAbs with panobinostat reduces melanoma metastasis in mice reconstituted with human NK cells. Next, we investigated the in vivo activity of panobinostat against the surface MICA / B protein levels of human melanoma cells. We first established that a selected dose of panobinostat (10 mg / kg) did not adversely affect human NK cells transferred into immunodeficient NSG mice (based on the number of total circulating NK cells, as well as CD16a or NKG2D-positive NK cells, Figure 16). Next, ZsGreen + A375 melanoma cells were intravenously injected into NSG mice, and a two-week waiting period was observed until metastasis was established. Next, the mice were treated twice at 24-hour intervals with panobinostat (or PBS), MICA / B mAb (or isotype control mAb), or a combination of panobinostat and MICA / B mAb (or panobinostat and isotype control mAb). One day later, MICA / B surface protein levels were measured using ZsGreen from lung tissue dissected by flow cytometry. + Quantitative analysis was performed on tumor cells. The selected dose of panobinostat did not significantly increase MICA / B protein levels in melanoma cells, but the combination of panobinostat and MICA / B mAb showed ZsGreen in lung metastases. + The MICA / B surface level of A375 melanoma cells increased (Figure 7).

[0257] Based on previous experience, the survival of transplanted human NK cells was limited in NSG mice, and only a relatively small number of human NK cells infiltrated the lung tissue. Therefore, we initiated treatment one day after inoculation of A375 melanoma cells (Figure 7). Early initiation of treatment may have enabled NK cell recognition of tumor cells that had not yet deeply infiltrated the lung tissue. Only the combination of panobinostat and MICA / B mAb reduced the number of lung metastases formed by control (B2M wild-type) A375 melanoma cells, while monotherapy with either panobinostat or MICA / B mAb was ineffective. In contrast, monotherapy with MICA / B mAb significantly reduced the number of lung metastases formed by B2M-KO A375 melanoma cells (Figure 7). The combination of MICA / B mAb and panobinostat did not enhance this effect against B2M-KO metastases. This is thought to be because NK cell rearrangement was limited in this model. These results demonstrate that MICA / B mAb treatment is more effective against MHC-I-deficient human melanoma metastases in this humanized mouse model, while combination therapy alone is effective against melanoma metastases expressing MHC-I protein.

[0258] Consideration Primary and secondary resistance to checkpoint blockade is a major problem in oncology. Many mechanisms of resistance to checkpoint blockade are related to the MHC-I and IFNγ signaling pathways in tumor cells. These include mutations in B2M or other genes in the MHC-I antigen presentation pathway, transcriptional and epigenetic silencing of nascent antigens or MHC-I expression, and inactivating mutations in the IFNγ signaling pathway. 4、5、6 MICA / B proteins have a similar structure to MHC-I proteins, but they do not assemble with β2-microglobulin. 38 Furthermore, the transcription of the MICA / B gene is regulated not by IFNγ, but by DNA damage and cellular stress. 13Therefore, inactivating mutations in the MHC-I and IFNγ pathways do not adversely affect MICA / B expression.

[0259] It is well known that loss of MHC-I expression eliminates important inhibitory signals for NK cells, but sufficient activation signals are also necessary for the induction of tumor immunity mediated by NK cells. 12 We have shown that metastases with inactivating mutations in the MHC-I (B2M mutation) or IFNγ signaling (JAK1 mutation) pathway can be treated with a MICA / Bα3 domain-specific antibody. This antibody inhibits shedding of MICA / B by proteolysis, which is a common mechanism of evasion from immunity via the NKG2D receptor in human cancers. We have previously shown that treatment with this mAb induces activation of both NKG2D (increased density of MICA / B ligand) and CD16a (Fc region of the mAb) receptors on NK cells. 20 While we do not wish to be constrained by theory, two approaches could be tested to induce NK cell-mediated tumor immunity with such mAbs. First, MICA / Bα3 domain-specific mAbs could be used to treat tumors resistant to checkpoint blockade by inactivating mutations in the MHC-I or IFNγ signaling pathway. Second, co-administration of MICA / B mAbs and PD-1 mAbs could simultaneously induce NK cell and CD8 T cell-mediated immunity against tumor cells, thereby preventing the proliferation of tumor clones resistant to cytotoxic T cells. Such approaches may be particularly interesting for advanced human tumors with extensive heterogeneity. It is important to note that the NKG2D receptor is also expressed by human CD8 T cells, γδ T cells, and ILCs. 14、46、47 While I don't want to be constrained by theory, it is therefore possible that MICA / B mAb treatment can enhance T cell-mediated tumor immunity.

[0260] Many therapeutic approaches used in oncology enhance the expression of MICA / B proteins by tumor cells. For example, it is well known that HDAC inhibitors, such as panobinostat, an FDA-approved drug, enhance the transcription of the MICA / B gene. 43 However, shedding of MICA / B proteins by tumor cells due to proteolysis limits the effectiveness of such drugs on NKG2D receptor activation. The combination of panobinostat and a MICA / B α3 domain antibody has been shown to significantly increase MICA / B surface protein levels and enhance NK cell-mediated immunity in a humanized mouse model of melanoma metastasis. We recognize that this humanized model has significant limitations, particularly the lack of homeostatic cytokine signaling, which restricts the survival of transplanted human NK cells. A similar approach could be used to develop combination therapies with other FDA-approved drugs. While HDAC inhibitors can also induce NKG2D ligand expression in healthy tissue, this aspect could not be evaluated in this study because mice lack the MICA / B gene. It is also known that the DNA damage response induced by radiotherapy potently enhances MICA / B transcription. 48 The combination of local radiotherapy and systemic immunotherapy with MICA / B mAbs is attractive because it limits the immune-related adverse events observed with combinations involving two systemic immunotherapies (such as PD-1 and CTLA-4 mAbs). Furthermore, there is already clinical evidence that combining immunotherapy with radiotherapy (CTLA-4 blockade) can induce systemic tumor immunity against non-irradiated lesions (abscopal effect). 49

[0261] Single-cell data demonstrate the presence of NK cells in human melanoma metastases, including patients whose disease progressed after treatment with PD-1 or CTLA-4 mAbs. Tumor and blood NK cell populations from the same patients demonstrated significant transcriptional differences. Most NK cells in blood samples (82.9%, bNK0 cluster) expressed conventional cytotoxic signatures (including GZMB and PRF1). However, NK cells isolated from melanoma metastases had more diverse gene expression programs. Seven NK cell clusters (and one ILC3 cluster) were detected in tumors, while one dominant NK cell cluster and three smaller NK cell clusters were detected in blood. Most NK cells in tumors were positive for cytotoxic signatures, but there was a gradient between clusters (highest in tNK.3 and tNK.4 clusters, and lower in tNK.0, tNK.1, and tNK.2 clusters). For example, this signature is a global assessment of cytokine function and is more useful than a single marker such as granzyme A or perforin. The most significant difference between tumor-infiltrating NK cells and blood NK cells is related to chemokine gene expression. Two recent publications have shown that NK cells play a crucial role in recruiting cross-presented DCs (cDC1s) to tumors by secreting the chemokines XCL1 and XCL2, which bind to the XCR1 receptor. 24、26 Furthermore, it was found that NK cells and cDC1s frequently interact, indicating that NK cells play a crucial role in the recruitment of DCs, which are important for T cell-mediated tumor immunity. 24Interestingly, we found that tumor-infiltrating NK cells expressed significantly more XCL1 and XCL2 than hematopoietic NK cells. Furthermore, we observed functional specialization between tumor NK cell clusters in terms of chemokine gene expression: NK cells with lower cytotoxic signatures (clusters tNK.0, tNK.1, tNK.2, tNK.6) expressed higher levels of XCL1 and XCL2 than clusters with higher cytotoxic signatures (tNK.3 and tNK.4). Tumor-infiltrating NK cells with higher cytotoxic signatures actually expressed a distinct set of chemokine genes (CCL3, CCL4, CCL4L2, CCL5). The encoded chemokines bind to the CCR5 chemokine receptor and recruit T cells as well as other immune cell populations. 32 Therefore, these data demonstrate significant functional specialization among tumor-infiltrating NK cell populations. NK cells with a low cytotoxic signature tend to express high levels of XCR1-binding chemokines that recruit cDC1s, while NK cells with a higher cytotoxic signature express higher levels of CCR5-binding chemokines that recruit T cells and other immune cells. These data and recent publications suggest that the role of NK cells in tumor immunity needs to be reconsidered in a broader context. NK cells can not only kill tumor cells but also create a favorable microenvironment by recruiting key immune cell populations necessary for protective tumor immunity.

[0262] Recent scRNA-seq studies have analyzed the transcriptome of human NK cells, identifying two NK cell populations in blood and four populations in spleen samples. The blood and spleen NK cell populations were characterized by their distinct transcriptional functions. 50While we do not wish to be constrained by theory, this study and our data verify that the transcriptional state of NK cells is dynamically regulated by the tissue microenvironment. Again, while we do not wish to be constrained by theory, the different NK cell populations identified in tumors are localized to different microenvironments. The apparent functional specialization of NK cell populations within tumors also offers opportunities to enhance distinct aspects of NK cell function, such as increased cytotoxic activity or secretion of chemokines that mobilize cross-presenting DCs.

[0263] In summary, metastases accompanied by mutations that induce resistance to cytotoxic T cells may become targets for NK cells when MICA / B shedding is inhibited by mAbs. Several combination strategies can be tested to further enhance NK cell activity against metastatic lesions: 1. Approaches that enhance MICA / B protein expression by tumor cells (e.g., panobinostat, local radiotherapy). 48 2. Cytokines that enhance NK cell function within tumors and reduce TGFβ-mediated downregulation of NKG2D (such as the IL-15 / IL-15Rα complex). 51 3. Antibodies that target inhibitory receptors on NK cells 52 Single-cell data on metastatic and invasive human NK cells also provide rich information about the gene expression programs of distinct NK cell populations. These single-cell data can inform the future development of strategies to enhance NK cell immunity against tumors resistant to cytotoxic T cells.

[0264] method cell line The B16F10, LLC1, A375, HCT-116, A549, and U937 cell lines were purchased from ATCC (Manassas, Virginia). The RPMI-8226 and U266 cell lines were generously donated (Dana-Farber Cancer Institute, Boston, Massachusetts), and the NCI-H139-Sqc cell line was generously donated. The CY029-S1, CY048-S, CY 21A-S1, CY.119-1A S, and CY36-S1 short-term melanoma cell lines have been previously described. 20、45 All cell lines were tested for mycoplasma negativity using the Universal Mycoplasma Detection Kit (ATCC, catalog no. 30-1012K) or the MycoAlert® Mycoplasma Detection Kit (Lonza, catalog no. LT07-318). All cell lines were obtained from suppliers or collaborators and used after a small number of passages. The A375, HCT-116, A549, U937, RPMI-8226, U266, and NCI-H139-Sqc cell lines were cultured in RPMI-1640 medium, while the B16F10, LLC1, CY029-S1, CY048-S, CY 21A-S1, CY.119-1A S, and CY36-S1 were cultured in DMEM medium. RPMI-1640 and DMEM media were supplemented with 10% FBS, 1× Glutamax, and 1× Penicillin / Streptomycin. All tissue culture reagents were purchased from Gibco (Thermo Fisher Scientific). Cells were cultured at 37°C in 5% CO2.

[0265] Control and B2M-KOA375 cells were generated by transducing parental A375 cells with the lentiCas9-blast vector and then selecting them with blastoscientin. Subsequently, the cells were transduced with the pLKO3G-gRNA-PGK-EGFP vector, which contains a gRNA targeting the human B2M gene inserted between the BsmB1 sites. The control cell line was transduced with the vector backbone. After transduction, the cells were cultured for 24 hours in the presence of recombinant human IFNγ (10 ng / ml) to induce upregulation of MHC-I protein and stained with APC conjugate W6 / 32 antibody (Biolegend, catalog no. 311410). HLA-A / B / C negative B2M-KO cells and HLA-A / B / C positive control cells were sorted by flow cytometry. Parental A375 cells were also transduced with the pHAGE lentiviral vector to enable ZsGreen expression under the control of the EF1α promoter. Zs Green + A375 cells were sorted by flow cytometry and used to examine MICA / B expression in vivo.

[0266] The generation of B16F10 control and B2m-KO cell lines has been reported previously. 53 As described above, MICA expression was achieved by transduction of a pHAGE lentiviral vector carrying a MICA*009-IRES-luciferase expression cassette into control and B2m-KO B16F10 cells under the control of the EF1α promoter. 20 After treating the cells with IFNγ (10 ng / ml) for 24 hours, APC conjugate H-2D b The cells were labeled with an antibody (Biolegend, catalog number 111513) and a PE-conjugated MICA 6D4 antibody (Biolegend, catalog number 320906). Next, MICA + H-2D b- B2M-KO and MICA + H-2D b+ The control sample B16F10 was selected using flow cytometry.

[0267] Jak1-KO B16F10-MICA cells were generated by electroporating a control B16F10-MICA cell line using gRNA targeting the Cas9 protein and the Jak1 gene. Electroporation was performed using an Amaxa® SF Cell Line 96-well Nucleofector® Kit (Lonza, V4SC-2096) in a 4D Nucleofactor (Lonza). The cells were then cultured in recombinant mouse IFNγ (10 ng / ml) for 24 hours to obtain MICA cells. + H-2D b- The cells were isolated by flow cytometry.

[0268] LLC1 cells were first transduced with a pHAGE lentiviral vector carrying a MICA*009 cDNA-IRES-ZsGreen expression cassette under the control of the EF1α promoter. The resulting LLC1-MICA cells were electroporated with Cas9 protein, which was then bound to a gRNA targeting the B2m gene. Control cells were electroporated with Cas9 protein alone. Control and B2m-KO LLC1-MICA cells were treated with IFNγ for 24 hours, followed by H-2K b Cells were sorted by flow cytometry based on H-2K expression. Control LLC1-MICA cells were H-2K b+ However, B2m-KO LLC1 cells were H-2K b- That was the case.

[0269] MICA / B Shedding Assay 5 x 10 4Individual tumor cells were cultured for 24 hours in 96-well plates (flat-bottom for adherent cells, U-bottom for suspension cells) in the presence of varying concentrations of antibody, IFNγ, and / or panobinostat (ApexBio, catalog number A8178), as shown in each figure. After the 24-hour culture period, the plates were centrifuged at 500×g for 5 minutes, and the supernatant was collected for analysis of shedding MICA using the Human MICA ELISA Kit (Abcam, catalog number ab59569). Previously, this ELISA kit was used to demonstrate that 7C6 mAb does not interfere with the detection of shedding MICA. 20

[0270] Adherent cells were detached with Versene (Gibco, catalog no. 15040-066) to maintain the integrity of the MICA / B protein on the cell surface. The Fc receptor was blocked using Human TruStainFcX® (Biolegend, catalog no. 422302), and the cells were stained with PE or APC-conjugated anti-human MICA / B clone 6D4 (Biolegend, catalog no. 320906 or 320908). Importantly, the 6D4 antibody binds to the α1-α2 domains of MICA / B and therefore does not compete with the 7C6 antibody, which targets the α3 domain of MICA / B, as previously mentioned. 20 Cells were also stained with one of the following dead cell markers: 7-AAD (BD Pharmingen®, catalog no. 559925), Zombie UV, Yellow, or Near Infrared (Biolegend, catalog nos. 423108, 423104, and 423106, respectively). Data were acquired using a BD Fortessa X20 or Beckman Coulter CytoFLEX LX, and analysis was performed using FlowJo V10 software.

[0271] Isolation of human and mouse NK cells Human NK cells from healthy individuals (leukopenic color) were isolated by negative selection using the EasySep® Human NK Cell Isolation Kit (Stem Cell Technologies, catalog no. 17955), resulting in an NK cell purity of at least 90%. Leukopenic color was anonymously provided by Brigham and Women's Hospital (Boston, USA). NK cells were grown in vitro in G-Rex 6-well plates (Wilson Wolf, catalog no. 80240M) using RPMI-1640 medium supplemented with 10% FBS, 5% human AB serum, 1,000 U / ml IL-2, and 20 ng / ml IL-15. The medium was replenished weekly until the NK cells were used in the experiment.

[0272] Mouse NK cells were isolated by meshing spleen tissue using a 70 μm cell strainer, followed by erythrocyte lysis (ACK buffer), and staining with PE conjugate anti-mouse CD49b mAb (Biolegend, catalog no. 108908) and APC conjugate anti-mouse CD3ε mAb (Biolegend, catalog no. 100312). NK cells were sorted by flow cytometry, with a typical purity of approximately 99%. These cells were then used in experiments involving allogeneic or syngeneic NK cells in Rag2 - / - Il2rg - / - The KO mice were immediately injected.

[0273] NK cell-mediated killing assay In long-term NK cell-mediated killing assays, GFP + Control and B2M-KOA375 cells were pre-treated for 24 hours with MICA / B or isotype control mAb (20 μg / ml) in tissue culture medium. Subsequently, tumor cells were detached with Versene, washed with PBS, and placed in 5 × 10⁶ wells per well in a black-walled 96-well plate (Corning®, catalog no. 3603). 3The cells were seeded at a cell density of [number]. As shown in the figure, human NK cells were added at different effector-to-target ratios after 1-2 hours, and IL-2 (300 U / ml) was added to support NK cell survival. As previously reported, GFP was measured using a Celigo Image Cytometer (Nexcelom Bioscience, Lawrence, USA). + The number of tumor cells was tracked over time. 40

[0274] In the short-term NK cell killing assay, as described above, A375 melanoma cells were pretreated in tissue culture medium with MICA / B or isotype control mAb (20 μg / ml) for 24 hours, followed by 4 hours of NK cell killing assay. 51 These cells were used as target cells in the Cr release assay. 20 NK cells were isolated by negative selection from the leukocyte apheresis-reduced color and cultured for 24 hours with 1,000 U / ml IL-2 in 96-well U-bottom plates before use in assays. In some experiments, by adding isotype control mAbs or anti-KIR2DL2 / 3 + anti-KIR2DL4 mAbs (BioLegend, catalog numbers 312602 and 347003), 51 In the Cr release assay, KIR receptors on NK cells were blocked.

[0275] Bulk RNA-seq analysis of human A375 melanoma cells Parental A375 cells were cultured for 24 hours in 50 nM panobinostat or a corresponding amount of PBS. Cells were then detached with Versene, and RNA was isolated using the RNeasy Plus MiniKit and the RNase-FreeDNase Set, respectively (both kits from Qiagen, catalog numbers 74134 and 79254, respectively). cDNA generation, sequencing, and analysis were performed as previously reported. 20

[0276] mouse Wild type (WT) C57BL6 / J, Ighm - / -The C57BL6 / J, CB6F1 / J, and NSG mice were purchased from Jackson Laboratories (catalog numbers 000664, 002288, 100007, and 005557, respectively). Rag2 - / - Il2rg - / - The knockout mice were purchased from Taconic (catalog number 4111). The mice were male (except for female NSG mice) and 6-8 weeks old. As previously reported, all mice were housed at the Dana-Farber Cancer Institute's facility. 20 The Institutional Committee for Animal Use approved the procedures used in this study (Animal Protocol Number 08-049).

[0277] Metastasis model in immune-responsive mice B16F10-MICA tumor cells (control, B2m-KO or Jak1-KO) were collected from the tail vein of C57BL / 6 mice (WT or Ighm - / - ) was administered intravenously (as explained in the legend in the figure, 1 to 7 × 10 in 100 μl of PBS depending on the experiment). 5 (individual cells). When mice established metastasis by intraperitoneal injection of isotype control mAb (BioXcell, catalog number BE0085) or 7C6-mIgG2a mAb (200 μg per injection, days 7 and 8, then once a week thereafter) (day 7 after tumor inoculation), Ighm - / - Treatment was initiated in mice. In another protocol, WT mice were injected with antibodies on days 1 and 2, and then weekly thereafter. Antibodies that induced CD8 T cell depletion (100 μg anti-CD8β, BioXcell, catalog BE0223) or NK cell depletion (1:10 dilution anti-asialoGM1, Wako Chemicals, catalog 986-1001) were injected against tumor cells on days -1 and 0, and then weekly thereafter. Mouse IgG1 was used as a control IgG (BioXcell, catalog BE0083). Lung metastases were quantified on day 14 under a stereomicroscope after formalin fixation of tissue. Alternatively, mouse survival was recorded.

[0278] In the LLC1-MICA metastasis model, WT C57BL / 6 mice were given 1.0-1.5 × 10¹⁶ PBS in 0.1 ml. 6 Tumor cells (as shown in the legend in the figure) were inoculated intravenously via the tail vein. 7C6-mIgG2a or isotype control mAb (200 μg) was administered on days 2 and 3 of tumor cell inoculation, and thereafter once weekly. In experiments involving adoptive transfer of NK cells, 2 × 10⁶ cells isolated from WT C57BL / 6 (syngene) or CB6F1 / J (allogeneic) mice were used. 5 Rag2 - / - Il2rg - / - The cells were intravenously injected into knockout mice. These NK cells were identified by flow cytometry as CD3ε - CD49b + The cells were isolated. LLC1-MICA tumor cells (7 × 10) 5 (100 cells) were intravenously injected one day after NK cell transfer. 7C6-mIgG2a or isotype control mAb (200 μg / injection) was administered intraperitoneally on days 2 and 3, and then once a week thereafter. On day 14, mice (WT or Rag2) were administered. - / - Il2rg - / - The knockout patients were euthanized by CO2 inhalation, and as mentioned above, India ink (30%) was injected into their tracheas to enable the counting of lung metastases. 28 Lung tissue was treated with Fekete fixative, and surface metastases were counted using a stereomicroscope.

[0279] Characterization of mouse NK cells in the B16F10-MICA metastasis model. WT C57BL / 6 mice were subjected to a 7×10⁶ experiment with control, B2m-KO, or Jak1-KO genotypes. 510 B16F10-MICA cells were intravenously inoculated. On days 1 and 3 after tumor cell inoculation, mice were treated with 7C6-mIgG2a or isotype control mAb (200 μg / injection). On day 12, mice were intravenously injected with 50 μl of APC conjugate anti-mouse CD45.2 (Biolegend, 109814) to label intravascular immune cells and euthanize them. Lung tissue was finely dissected, resuspended in RPMI-1640 supplemented with 1 mg / ml collagenase type IV, 0.1 mg / ml hyaluronidase, and 20 U / ml DNase, and processed using a gentleMACS instrument (Miltenyi). Next, the cell suspension was treated with mouse TruStain FcX (trademark) (Biolegend, catalog number 101320), as well as PE-Cy7 conjugate anti-mouse CD45.2 (Biolegend, 109830), APC conjugate anti-mouse CD3ε (Biolegend, 100312), APC conjugate anti-mouse TCRβ (Biolegend, 109212), BV785 conjugate anti-mouse NK1.1 (BD Biosciences, 740853), PE-CF594 conjugate anti-mouse CD49b (BD Biosciences, 562453), Alexa488 conjugate anti-mouse EOMES (Invitrogen, 53-4875-82), PE conjugate anti-mouse GZMA (Invitrogen, 12-5831-82), and BV421 conjugate anti-mouse NKG2D (BD Cells were incubated with multiple antibodies, including Biosciences (562800), PERCP-CY5.5 conjugate anti-mouse CD16 / 32 (Biolegend, 101324), BV510 conjugate anti-mouse Ly49C / I (BD Biosciences, 744028), and Zombie UV. Cells were analyzed using a CytoFLEX Flow Cytometer (Beckman Coulter), and data were processed using FlowJo V10.

[0280] Humanized mouse model NSG mice are used to grow human NK cells (1-2 × 10⁻¹⁰) in vitro as described above. 6The cells were reconstituted intravenously (7.5 × 10⁶ cells). As previously reported, IL-2 was injected intraperitoneally. 4 (Individual units) supported in vivo survival of NK cells. 20 A375 melanoma cells (5 x 10 5 Individual cells (control or B2M-KO) were injected on day 1 (day 0). One day after tumor cell inoculation, mice were administered another dose of IL-2, in addition to isotype control (BioXcell, catalog BE0096) or 7C6-hIgG1 mAb (200 μg), and PBS or 10 mg / kg panobinostat (ApexBio, catalog no. A8178). On day 2, mice were again reconstituted with human NK cells from the same donor and administered injections of IL-2, antibody, and PBS or panobinostat. Metastasis was quantified two weeks after the last treatment for the LLC1-MICA metastasis model (injection of India ink into the trachea, treatment of lung tissue with Fekete fixative), as described above.

[0281] 1x10 NSG mouse 6 ZsGreen + The effects of MICA / B mAb and panobinostat treatment on MICA / B surface levels in lung metastases were studied by inoculating A375 melanoma cells. These mice were not administered human NK cells. Once metastasis was established (2 weeks later), mice were treated 2 days later with 7C6-hIgG1 or isotype control mAb (200 μg), as well as panobinostat (10 mg / kg) or PBS as a solvent control. The day after the final treatment, mice were euthanized by CO2 inhalation, and lung tissue was mechanically isolated to maintain MICA / B protein integrity. Tumor cells were identified as large, viable cells that were ZsGreen positive but negative for mouse CD45 antigen. MICA / B surface protein was labeled with 6D4-PEmAb (Biolegend, catalog 320906) and quantified by flow cytometry.

[0282] Characterization of human NK cells in tumor-free NSG mice 2 x 10⁶ NSG mice without tumors 6Human NK cells were intravenously inoculated and proliferated in vitro as described above. Simultaneously, mice were given IL-2 (7.5 × 10⁻¹⁶). 4 As a solvent control, each individual was administered intraperitoneally with either panobinostat (10 mg / kg) or PBS. One day later, blood was collected due to bleeding from the eye, and human NK cells were analyzed by flow cytometry.

[0283] Characterization of human NK cells infiltrating melanoma metastases Melanoma tissue samples were obtained from patients who required surgery for the treatment of unresponsive lesions at Brigham & Women's Hospital. Blood samples were also taken during surgery. Newly excised tumor tissue was isolated using the Tumor Dissociation Kit (Miltenyi Biotec, catalog 130-095-929). Red blood cells in the blood samples were lysed using ACK buffer. NK cells were extracted from the melanoma lesions and corresponding blood samples using a BD Aria flow cytometer (BD Biosciences) to identify CD45 cells. + CD56 + , CD3ε - ,CD4 - CD8a - CD14 - CD15 - and CD163 - They were isolated as single, living lymphocyte-sized cells. These selected NK cells were used for scRNA-seq analysis.

[0284] In follow-up experiments, tumor and hematopoietic NK cells were also stained with anti-FGFPB2, GZMA, GZMK, CD62L, NKG2D, and CD16a antibodies and analyzed by flow cytometry. All fluorescently labeled antibodies were purchased from BioLegend, BD Biosciences, or eBiosciences. For intracellular staining, NK cells were fixed and permeabilized with the True-Nuclear® Transcription Factor Buffer Set (Biolegend, catalog no. 424401) according to the manufacturer's recommendations. Samples were analyzed using a Cytoflex flow cytometer (Beckman Coulter).

[0285] Single-cell RNA-seq Immediately after sorting NK cells (approximately 13,000 NK cells per sample), the cell suspension was washed with 0.05% RNase-free BSA in PBS. The 10X Genomics 3'V2 single-cell assay (10X Genomics) was used to construct the scRNA-seq library. Reverse transcription, cDNA amplification, and library preparation were all performed according to the manufacturer's instructions. The library was sequenced using an Illumina HiSeq 2500 in rapid-run mode, yielding over 25,000 reads per cell.

[0286] Computational analysis of single-cell RNA-seq data The steps of demultiplexing, alignment, filtering, barcode counting, and unique molecular identifier (UMI) counting were performed using 10x Genomics' Cell Ranger software. The analysis was performed using the Seurat 3.0 package. 54 First, each dataset was processed individually, and then data from multiple samples were combined. For each dataset, 1,500 of the most variable genes were selected. Subsequently, principal component analysis (PCA) was performed, and the first 15 principal components (PCs) were used to perform Louvain clustering and uniform manifold approximation and projection (UMAP) embedding. 55、56We checked the most important marker genes in each cluster to identify potential contaminating cell populations such as T cells (CD3D, CD3E, and CD3G), B cells (IGHG1, IGHG2, and JCHAIN), macrophages (LYZ), and melanoma cells (MLANA). These cells were removed before subsequent analysis.

[0287] Next, using Seurat's integration algorithm, we compared paired populations of hematopoietic NK cells and melanoma-infiltrating NK cells from each patient, and further integrated three melanoma-infiltrating NK cell samples and three hematopoietic NK cell samples separately. Using 3,000 of the most variable genes and the first 15 PCs from each sample, we selected 1,000 anchor genes for integration. Subsequently, we repeated PCA, clustering (resolution = 0.3), and UMAP embedding on the integrated dataset. Finally, we performed differential analyses on the integrated dataset to identify genes that were significantly upregulated within each cluster compared to all other cells (adjusted P < 0.05), as well as genes that were differentially expressed between hematopoietic NK cells and melanoma-infiltrating NK cells within each major cluster.

[0288] For gene sets representing specific cellular functions or pathways, the AUCell score for each cell was also calculated to assess variations in gene set activity across the entire cell population. 57 Based on the analysis of the data, we selected gene sets for cytotoxicity and chemokine activity. For NK cell, ILC1, ICL2, and ILC3 identity, we referenced gene signatures defined by previous studies using genes that were significantly upregulated in one of these innate cell types compared to the other three populations (adjusted P<1e-3); in particular, for ILC identity, the same genes were not significantly upregulated in NK cells (adjusted P>1e-2). 31

[0289] statistical analysis All statistical analyses were performed using GraphPad Prism 8 software, and relevant statistical tests are indicated in the legend of each figure.

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[0291] Example 3 - Inhibition of MICA and MICB shedding induces NK cell-mediated immunity against tumors resistant to cytotoxic T cells. summary Resistance to cytotoxic T cells may be mediated by loss of MHC class I expression or IFNγ signaling in tumor cells, such as mutations in the B2M or JAK1 genes. While we do not wish to be constrained by theory, NK cells could target resistant tumors, although NK cell-based strategies have not yet been developed. Again, while we do not wish to be constrained by theory, tumors could be targets for NK cells if an activation signal is provided. Human tumors express MICA and MICB ligands for the activated NKG2D receptor, and shedding of MICA / B by proteolysis is an immune evasion mechanism in many human cancers. B2M and JAK1-deficient metastases have been shown to be targets for NK cells after treatment with mAbs that block MICA / B shedding. Furthermore, we have demonstrated that the FDA-approved HDAC inhibitor panobinostat and a MICA / B antibody synergistically enhance MICA / B surface levels on tumor cells. The HDAC inhibitor enhances MICA / B gene expression, and the MICA / B antibody stabilizes the synthetic protein on the cell surface. The combination of panobinostat and a MICA / B antibody reduces the number of lung metastases formed by human melanoma cell lines in NSG mice reconstituted with human NK cells. Therefore, NK cell-mediated immunity induced by MICA / B-specific mAbs offers an opportunity to target tumors with mutations that confer resistance to cytotoxic T cells.

[0292] Prologue Checkpoint blockade with antibodies targeting programmed cell death protein 1 (PD-1) or cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitory receptors on T cells can induce persistent anti-tumor immunity even in patients with advanced cancer. However, many patients are unable to benefit from these therapies due to primary or secondary resistance (1). Cytotoxic T cells play a role in the effectiveness of checkpoint blockade based on their ability to recognize tumor-derived peptides bound to major histocompatibility complex class I (MHC-I) proteins (2). Recognition of such MHC-I-peptide complexes by the T cell receptor (TCR) leads to T cell-mediated killing via the release of cytotoxic granules containing perforin and granzyme. In addition, interferon-γ (IFNγ) secretion by T cells inhibits tumor cell proliferation and enhances MHC-I protein expression in both tumor cells and dendritic cells (3). Therefore, resistance to checkpoint blockade is mediated by the loss of MHC-I expression by tumor cells, either due to mutations in key genes in the MHC-I (B2M, TAP1, TAP2, and other genes) or IFNγ (JAK1, JAK2) pathways, or epigenetic silencing (4-6). When the number of nascent antigens is low or lost, tumor immunity mediated by cytotoxic T cells is also reduced (7-10). Currently, there are no alternative immunotherapies for patients with solid tumors resistant to checkpoint blockade. While we do not wish to be bound by theory, chimeric antigen receptor (CAR) T cells can target tumor cells lacking MHC-I proteins, but so far, CAR T cells have shown limited efficacy against solid tumors (11).

[0293] Natural killer (NK) cells recognize tumor cells through a different molecular mechanism than that required by cytotoxic T cells. NK cell recognition by tumor cells is mediated by germline-encoded activating receptors that bind to ligands upregulated on tumor cells by cellular processes associated with malignant transformation, such as DNA damage and cellular stress (12). In contrast, T cells recognize MHC-presenting peptides derived from co-existing tumor antigens or nascent antigens produced by somatic mutations (2). Therefore, tumors resistant to cytotoxic T cells may respond to NK cell-based immunotherapy approaches. Indeed, loss of expression by tumor cells increases sensitivity to NK cells because the MHC-I protein functions as a ligand for inhibitory NK cell receptors (12). However, inducing NK cell-mediated tumor immunity may also require effective targeting of immune evasion mechanisms that prevent NK cell-mediated attack by tumor cells. For example, many human cancers express MHC-I chain-related polypeptide A (MICA) and MICB (MICA / B) proteins, which function as ligands for the activated NK group 2D (NKG2D) receptor on subpopulations of NK cells and T cells (13, 14). Tumors evade NKG2D receptor-mediated tumor immunity by shedding of MICA / B proteins through proteolysis (15-22). Monoclonal antibodies (mAbs) that bind to the α3 domain of MICA / B, a domain essential for shedding, were developed. These mAbs inhibited MICA / B shedding and induced NK cell-mediated tumor immunity. Increased density of MICA / B protein on tumor cells enhanced NK cell activation via the NKG2D receptor, and the Fc segment of tumor conjugate antibodies also activated NK cells via the CD16Fc receptor. Treatment with such MICA / B antibodies significantly altered tumor-infiltrating NK cells into a highly cytotoxic state (23).

[0294] The MICA and MICB genes are part of the MHC locus on human chromosome 6, and the encoded proteins share significant structural similarities with MHC proteins. B2M deficiency disables responsiveness to T cell-mediated immunity and T cell checkpoint blockade, while MICA / B proteins do not associate with β2 microglobulin or peptides (5, 24-26). While we do not wish to be bound by theory, inhibition of MICA / B shedding induces NK cell-mediated immunity against metastatic lesions resistant to cytotoxic T cells. Indeed, treatment with mAbs specific to the MICA / B α3 domain has enabled NK cell-mediated immunity against tumors with inactivating mutations in the MHC-I or IFNγ signaling pathway (B2m and Jak1 mutations, respectively). Furthermore, the MICA / B gene is epigenetically regulated by histone deacetylases that inhibit MICA / B expression by tumor cells (27-30). We discovered that HDAC inhibitors act synergistically with MICA / B mAbs in vivo, enhancing MICA / B protein levels on the surface of tumor cells through the enhancement of MICA / B gene transcription (HDAC inhibitors) and the inhibition of MICA / B shedding (MICA / B mAbs). This combination therapy conferred NK cell-mediated immunity against melanoma metastasis in a humanized mouse model. Therefore, NK cell-based immunotherapy that induces activating receptors can be used to treat cancers resistant to cytotoxic T cells.

[0295] material and method cell line The B16F10, LLC1, A375, HCT-116, A549, and U937 cell lines were purchased from ATCC (Manassas, Virginia). The RPMI-8226 and U266 cell lines were donated (Dana-Farber Cancer Institute, Boston, Massachusetts), and the NCI-H139-Sqc cell line was generously donated. The short-term melanoma cell lines CY029-S1, CY048-S, CY21A-S1, CY.119-1A S, and CY36-S1 have been previously reported (23, 31). All cell lines were tested to be negative for mycoplasma before use in experiments using the Universal Mycoplasma Detection Kit (ATCC, catalog no. 30-1012K) or the MycoAlert® Mycoplasma Detection Kit (Lonza, catalog no. LT07-318). All cell lines were obtained from suppliers or collaborators and used after a small number of passages (less than approximately 10 passages). A375, HCT-116, A549, U937, RPMI-8226, U266, and NCI-H139-Sqc cell lines were cultured in RPMI-1640 medium, while B16F10, LLC1, CY029-S1, CY048-S, CY 21A-S1, CY.119-1A S, and CY36-S1 were cultured in DMEM medium. RPMI-1640 and DMEM mediums were supplemented with 10% FBS, 1× Glutamax, and 1× Penicillin / Streptomycin. All tissue culture reagents were purchased from Gibco (Thermo Fisher Scientific). Cells were cultured at 37°C in 5% CO2.

[0296] Control and B2M-KOA375 cells were generated by selecting parental A375 cells with lentiCas9-blast vector (Addgene#52962) followed by blastosidine (Gibco, catalog number R21001). Subsequently, the previously reported (32)pLKO3G-gRNA-PGK-EGFP vector, containing a gRNA targeting the human B2M gene inserted between BsmB1 sites, was transduced into the cells. The control cell line was transduced with the vector backbone. After transduction, cells were cultured for 24 hours in the presence of recombinant human IFNγ (10 ng / ml, BD Biosciences) to induce upregulation of MHC-I proteins. Cells were stained with APC-conjugated W6 / 32 antibody (Biolegend, catalog number 311410), and HLA-A / B / C-negative B2M-KO cells and HLA-A / B / C-positive control cells were sorted by flow cytometry. Parental A375 cells were also transduced with a pHAGE lentiviral vector to enable ZsGreen expression under the control of the EF1α promoter. This vector was generated by inserting the ZsGreen sequence between the NotI and BamHI restriction sites, which were obtained by removing the IRES and ZsGreen sequences from the parental vector. + A375 cells were sorted by flow cytometry and used to examine MICA / B expression in vivo.

[0297] B16F10 control and B2m-KO cell lines have been previously reported (32). MICA expression was achieved by transduction of a pHAGE lentiviral vector carrying a MICA*009-IRES-luciferase expression cassette into control and B2m-KO B16F10 cells under the control of the EF1α promoter. This plasmid has been previously reported (23). B16F10 control and B2m-KO cell lines were labeled with a PE conjugate anti-MICA / B antibody (Biolegend catalog number 320906) and MICA expression was achieved. +Cells were sorted by flow cytometry. Jak1-KO B16F10-MICA cells were generated by electroporating a control B16F10-MICA cell line with gRNA targeting Cas9 protein and the Jak1 gene (Figure 34). Electroporation was performed using an Amaxa® SF Cell Line 96-well Nucleofector® Kit (Lonza, V4SC-2096) in a 4D Nucleofactor (Lonza). Cells were treated with IFNγ (10 ng / ml, BD Biosciences) for 24 hours. Subsequently, cells were treated with a cocktail of PE-conjugated MICA 6D4 antibody (Biolegend, catalog number 320906) and APC-conjugated anti-MHC-I antibody (anti-H-2K). b and anti-H-2D b They were labeled with Biolegend catalog numbers 116518 and 111513, respectively. Next, MICA + MHC-I - B2M-KO and MICA + MHC-I + The control sample B16F10 was classified using flow cytometry.

[0298] LLC1 cells were first transduced with a pHAGE lentiviral vector carrying a MICA*009 cDNA-IRES-ZsGreen expression cassette under the control of the EF1α promoter (Addgene#114007). The resulting LLC1-MICA cells were electroporated with Cas9 protein, which was then bound to a gRNA targeting the B2m gene. Control cells were electroporated with Cas9 protein alone. Control and B2m-KO LLC1-MICA cells were treated with IFNγ (BD Biosciences) for 24 hours, followed by H-2K b Cells were sorted by flow cytometry based on the expression of (clone AF6-88.5 Biolegend). Control LLC1-MICA cells were H-2K b Although positive, B2m-KO LLC1 cells were H-2K b The result was negative.

[0299] Western blotting B16F10, LLC1, and A375 cell lines were treated with or without 50 ng / ml IFNγ (BD Biosciences) for 16 hours. Subsequently, the cells were washed with PBS and lysed in RIPA buffer (Thermo Scientific) supplemented with a protease inhibitor cocktail (Sigma Aldrich). The lysates were refracted at 14,000 rpm. o The samples were centrifuged at 10°C for 10 minutes. Total protein was measured by bicinconinate assay (Thermo Scientific) and normalized before gel loading. Following SDS-PAGE, the samples were transferred to polyvinylidene difluoride membranes, blocked with 5% milk in Tris-buffered saline supplemented with 0.1% Tween, and incubated overnight with appropriate primary antibodies as follows: anti-mouse B2M (R&D Systems), anti-human B2M, JAK1, GAPDH, and tubulin (all from Cell Signaling Technology). After incubation with secondary antibodies conjugated with horseradish peroxidase (Cell Signaling Technology and JacksonImmunoresearch), proteins were visualized by chemiluminescence (Western Lightning and Perkin-Elmer) using a Chemi-Doc instrument (Bio-Rad Laboratories).

[0300] MICA / B Shedding Assay 5 x 10 4Individual tumor cells were cultured for 24 hours in 96-well plates (flat-bottom for adherent cells, U-bottom for suspension cells) in the presence of varying concentrations of antibody, IFNγ, and / or panobinostat (ApexBio, catalog no. A8178), as shown in each figure. After the 24-hour culture period, the plates were centrifuged at 500×g for 5 minutes, and the supernatant was collected for analysis of shedding MICA using the Human MICA ELISA Kit (Abcam, catalog no. ab59569). Importantly, we previously demonstrated that 7C6 mAb does not interfere with the detection of shedding MICA using this ELISA (23).

[0301] Adherent cells were detached with Versene (Gibco, catalog no. 15040-066) to maintain the integrity of the MICA / B protein on the cell surface. The Fc receptor was blocked using Human TruStainFcX® (Biolegend, catalog no. 422302), and the cells were stained with PE or APC-conjugated anti-human MICA / B clone 6D4 (Biolegend, catalog no. 320906 or 320908). Since the 6D4 antibody binds to the α1-α2 domains of MICA / B, it does not compete with the 7C6 antibody that targets the α3 domain of MICA / B, as previously shown (23). Cells were also stained with one of the following dead cell markers: 7-AAD (BD Pharmingen®, catalog no. 559925), Zombie UV, Yellow, or Near Infrared (Biolegend, catalog nos. 423108, 423104, and 423106, respectively). Data were acquired using a BD Fortessa X20 or Beckman Coulter CytoFLEX LX, and analysis was performed using FlowJo V10 software.

[0302] Isolation of human and mouse NK cells Human NK cells from healthy individuals (leukopenic color) were isolated by negative selection using the EasySep® Human NK Cell Isolation Kit (Stem Cell Technologies, catalog no. 17955), resulting in an NK cell purity of at least 90%. Leukopenic color was anonymously provided by Brigham and Women's Hospital (Boston, USA). NK cells were grown in vitro in G-Rex 6-well plates (Wilson Wolf, catalog no. 80240M) using RPMI-1640 medium supplemented with 10% FBS, 5% human AB serum, 1,000 U / ml IL-2, and 20 ng / ml IL-15 (all cytokines were from BD Biosciences). The medium was replenished weekly until the NK cells were used in the experiment.

[0303] Mouse NK cells were isolated by meshing spleen tissue using a 70 μm cell strainer, followed by erythrocyte lysis (ACK buffer), and staining with PE conjugate anti-mouse CD49b mAb (Biolegend, catalog no. 108908) and APC conjugate anti-mouse CD3ε mAb (Biolegend, catalog no. 100312). NK cells were sorted by flow cytometry, with a typical purity of approximately 99%. These cells were then used in experiments involving allogeneic or syngeneic NK cells in Rag2 - / - Il2rg - / - The KO mice were immediately injected.

[0304] NK cell-mediated killing assay In long-term NK cell-mediated killing assays, GFP + Control and B2M-KOA375 cells were pre-treated for 24 hours with MICA / B or isotype control mAb (20 μg / ml) in tissue culture medium. Subsequently, tumor cells were detached with Versene, washed with PBS, and placed in 5 × 10⁶ wells per well in a black-walled 96-well plate (Corning®, catalog no. 3603). 3The cells were seeded at a cell density of 1. As shown in the figure, human NK cells were added at different effector-to-target ratios after 1–2 hours, and IL-2 (300 U / ml, BD Biosciences) was added to support NK cell survival. As previously reported (33), GFP was detected using a Celigo Image Cytometer (Nexcelom Bioscience, Lawrence, USA). + The number of tumor cells was tracked over time.

[0305] In the short-term NK cell killing assay, as described above (23), A375 melanoma cells were pretreated for 24 hours with MICA / B or isotype control mAb (20 μg / ml) in tissue culture medium, followed by 4 hours of NK cell killing assay. 51 These cells were used as target cells in the Cr release assay. NK cells were isolated by negative selection from leukocyte apheresis reduction color, cultured for 24 hours with 1,000 U / ml IL-2 (BD Biosciences) in 96-well U-bottom plates, and then used in the assay. By adding isotype control mAbs or anti-KIR2DL2 / 3 + anti-KIR2DL4 mAbs (BioLegend, catalog numbers 312602 and 347003), 51 In the Cr release assay, KIR receptors on NK cells were blocked.

[0306] CD8 T cell cytotoxicity assay To confirm the resistance of Jak1-KO and B2m-KO B16F10-MICA cell lines to CD8 T cell-mediated cytotoxicity, a Celigo-based imaging cytometry assay was performed (33). Briefly, tumor cells were pulsed overnight with 10 nM Ova peptide, washed with PBS, and added to 96-well plates (5,000 tumor cells per well). Tumor cells were co-cultured with naive OT-I CD8 T cells with different effector-to-target ratios (1:0 no T cells, 1:1, 2:1, and 5:1, 8-10 replications per group). After 48 hours, the supernatant was removed and the wells were washed with PBS to remove dead tumor cells and CD8 T cells. The plates were then analyzed using a Celigo instrument for quantification of living tumor cells.

[0307] Bulk RNA-seq analysis of human A375 melanoma cells Parental A375 cells were treated with panobinostat (50 nM) or a corresponding amount of PBS for 24 hours. Cells were then detached with Versene, and RNA was isolated using the RNeasy Plus MiniKit and the RNase-FreeDNase Set, respectively (both kits from Qiagen, catalog numbers 74134 and 79254, respectively). cDNA generation, sequencing, and analysis were performed as previously reported (23).

[0308] Real-time quantitative PCR (qPCR) A375 cells were treated with panobinostat (50 nM) for 24 hours. Subsequently, the cells were washed twice with PBS, pelletized, and used to extract total RNA using the RNeasy mini kit (#74106, Qiagen) according to the manufacturer's protocol. One microgram of the extracted RNA was used to synthesize cDNA using SuperScript IV VILO Master Mix (ThermoFisher, 11756050). Diluted cDNA was used for qPCR using TaqMan Gene Expression MasterMix (Life Technologies, 4369016), TaqMan probes (MICA-Hs00741286_m1, MICB-Hs00792952_m1, GAPDH-Hs02786624_g1, ULBP2-Hs00607609_mH, and RAET1L-Hs04194671_s1), and the QuantStudio 6 Flex Real-Time PCR System (ThermoFisher). To examine the changes in gene expression between groups, the ΔΔCT value was calculated from the average CT value of three technical replications for each sample in each group. The multiplicative change in gene expression was expressed in comparison to GAPDH (housekeeping gene) for each sample.

[0309] mouse Wild type (WT) C57BL6 / J, Ighm - / - The C57BL6 / J, CB6F1 / J, and NSG mice were purchased from Jackson Laboratories (catalog numbers 000664, 002288, 100007, and 005557, respectively). Rag2 - / - Il2rg - / - Knockout mice were purchased from Taconic (catalog number 4111). The mice were male (except for female NSG mice) and 6–8 weeks old. The mice were housed at the Dana-Farber Cancer Institute and the Icahn School of Medicine at Mount Sinai. The organizing committee for animal use approved the procedures used in this study.

[0310] Metastasis model in immune-responsive mice B16F10-MICA tumor cells (control, B2m-KO or Jak1-KO) were collected from the tail vein of C57BL / 6 mice (WT or Ighm - / - ) was administered intravenously (as explained in the legend in the figure, 1 to 7 × 10 in 100 μl of PBS depending on the experiment). 5 (individual cells). When mice established metastasis by intraperitoneal injection of isotype control mAb (BioXcell, catalog number BE0085) or 7C6-mIgG2a mAb (200 μg per injection, days 7 and 8, then once a week thereafter) (day 7 after tumor inoculation), Ighm - / - Processing was initiated in mice. Another protocol uses WT mice (Ighm + / + Antibodies were injected into the mice on days 1 and 2, and then weekly thereafter. Antibodies that induced CD8 T cell depletion (100 μg anti-CD8β, BioXcell, catalog BE0223) or NK cell depletion (1:10 dilution anti-AsialoGM1, Wako Chemicals, catalog 986-1001, and 100 μg anti-NK1.1, clone PK136, BioXcell) were injected into the tumor cells on days -1 and 0, and then weekly thereafter. Mouse IgG1 was used as the control IgG (BioXcell, catalog BE0083). Lung metastases were quantified on day 14 under a stereomicroscope after formalin fixation of the tissue. Alternatively, mouse survival was recorded.

[0311] In the LLC1-MICA metastasis model, WT C57BL / 6 mice were given 1.0-1.5 × 10¹⁶ PBS in 0.1 ml. 6 Tumor cells (as shown in the legend in the figure) were inoculated intravenously via the tail vein. 7C6-mIgG2a or isotype control mAb (200 μg) was administered on days 2 and 3 of tumor cell inoculation, and thereafter once weekly. In experiments involving adoptive transfer of NK cells, 2 × 10⁶ cells isolated from WT C57BL / 6 (syngene) or CB6F1 / J (allogeneic) mice were used. 5 Rag2 - / - Il2rg - / -The cells were intravenously injected into knockout mice. These NK cells were identified by flow cytometry as CD3ε - CD49b + The cells were isolated. LLC1-MICA tumor cells (7 × 10) 5 (100 cells) were intravenously injected one day after NK cell transfer. 7C6-mIgG2a or isotype control mAb (200 μg / injection) was administered intraperitoneally on days 2 and 3, and then once a week thereafter. On day 14, mice (WT or Rag2) were administered. - / - Il2rg - / - The knockouts were euthanized by CO2 inhalation, and as described above (34), India ink (30%) was injected into the trachea to enable the counting of lung metastases. Lung tissue was treated with Fekete fixative, and surface metastases were counted using a stereomicroscope.

[0312] Characterization of mouse NK cells in the B16F10-MICA metastasis model. WTC57BL / 6 mice were subjected to a control, and 7 × 10⁶ mice with either the B2m-KO or Jak1-KO genotype. 510 B16F10-MICA cells were intravenously inoculated. On days 1 and 3 after tumor cell inoculation, mice were treated with 7C6-mIgG2a or isotype control mAb (200 μg / injection). On day 12, mice were intravenously injected with 50 μl of APC conjugate anti-mouse CD45.2 (Biolegend, 109814) to label intravascular immune cells and euthanize them. Lung tissue was finely dissected, resuspended in RPMI-1640 supplemented with 1 mg / ml collagenase type IV, 0.1 mg / ml hyaluronidase, and 20 U / ml DNase, and processed using a gentleMACS instrument (Miltenyi). Next, the cell suspension was treated with mouse TruStain FcX (trademark) (Biolegend, catalog number 101320), as well as PE-Cy7 conjugate anti-mouse CD45.2 (Biolegend, 109830), APC conjugate anti-mouse CD3ε (Biolegend, 100312), APC conjugate anti-mouse TCRβ (Biolegend, 109212), BV785 conjugate anti-mouse NK1.1 (BD Biosciences, 740853), PE-CF594 conjugate anti-mouse CD49b (BD Biosciences, 562453), Alexa488 conjugate anti-mouse EOMES (Invitrogen, 53-4875-82), PE conjugate anti-mouse GZMA (Invitrogen, 12-5831-82), and BV421 conjugate anti-mouse NKG2D (BD Cells were incubated with multiple antibodies, including Biosciences (562800), PERCP-CY5.5 conjugate anti-mouse CD16 / 32 (Biolegend, 101324), BV510 conjugate anti-mouse Ly49C / I (BD Biosciences, 744028), and Zombie UV. Cells were analyzed using a CytoFLEX Flow Cytometer (Beckman Coulter), and data were processed using FlowJo V10.

[0313] Humanized mouse model Human NK cells (1-2 × 10⁻¹) were grown in vitro from NSG mice as described above. 6The cells were reconstituted intravenously (individual cells). As previously reported (23), IL-2 (Peprotech, catalog number 200-02) was administered by intraperitoneal injection (7.5 × 10⁶). 4 (in units of individual cells) supported the in vivo survival of NK cells. A375 melanoma cells (5 × 10) 5 Individual cells (control or B2M-KO) were injected on day 1 (day 0). One day after tumor cell inoculation, mice were administered another dose of IL-2, in addition to isotype control (BioXcell, catalog BE0096) or 7C6-hIgG1 mAb (200 μg), and PBS or 10 mg / kg panobinostat (ApexBio, catalog no. A8178). On day 2, mice were again reconstituted with human NK cells from the same donor and administered injections of IL-2, antibody, and PBS or panobinostat. Metastasis was quantified two weeks after the last treatment for the LLC1-MICA metastasis model (injection of India ink into the trachea, treatment of lung tissue with Fekete fixative), as described above.

[0314] 1x10 NSG mouse 6 ZsGreen + The effects of MICA / B mAb and panobinostat treatment on MICA / B surface levels in lung metastases were studied by inoculating A375 melanoma cells. These mice were not administered human NK cells. Once metastasis was established (2 weeks later), mice were treated 2 days later with 7C6-hIgG1 or isotype control mAb (200 μg), as well as panobinostat (10 mg / kg) or PBS as a solvent control. The day after the final treatment, mice were euthanized by CO2 inhalation, and lung tissue was mechanically isolated to maintain MICA / B protein integrity. Tumor cells were identified as large, viable cells that were ZsGreen positive but negative for mouse CD45 antigen. MICA / B surface protein was labeled with 6D4-PEmAb (Biolegend, catalog 320906) and quantified by flow cytometry.

[0315] Characterization of human NK cells in tumor-free NSG mice 2 x 10⁶ NSG mice without tumors 6 Human NK cells were intravenously inoculated and proliferated in vitro as described above. Simultaneously, mice were given IL-2 (7.5 × 10⁻¹⁶). 4 Each patient was administered intraperitoneally with Peprotech, and either panobinostat (10 mg / kg) or PBS as a solvent control. One day later, blood was collected due to hemorrhage in the eye, and human NK cells were analyzed by flow cytometry.

[0316] statistical analysis All statistical analyses were performed using GraphPad Prism 8 software, and relevant statistical tests are indicated in the legend of each figure.

[0317] result Killing of human B2M-deficient melanoma cells via NK cells is enhanced by MICA mAbs. The effect of MICA / Bα3 domain-specific antibodies on NK cell-mediated immunization against human B2M-deficient tumor cells was investigated. Inactivation of the B2M gene in human A375 melanoma cells resulted in complete loss of MHC-I surface protein even after IFNγ...

Claims

1. A method for treating cancer in the subject, Administering a therapeutically effective amount of a composition containing one or more activators and a pharmaceutically acceptable carrier to the subject. The method comprising the activator activating NK cells via NKG2D and / or CD16 receptors, thereby causing lysis of one or more cancer cells in the subject, wherein the cancer is resistant to cytotoxic T cells.

2. The method according to claim 1, wherein the activator comprises a polynucleotide, a polypeptide, a small molecule, or a combination thereof.

3. The method according to claim 1, wherein the activator comprises an anti-MICA antibody, an anti-MICB antibody, or both.

4. The method according to claim 3, wherein the antibody comprises a monoclonal antibody.

5. The method according to claim 3, wherein the antibody binds to the alpha-3 domain of MICA / B.

6. The method according to claim 3, wherein the antibody comprises one or more sequences from Table 1.

7. The method according to claim 1, wherein the activator inhibits MICA / MICB shedding by the tumor, thereby increasing the density of NKG2D receptor ligand on tumor cells.

8. The method according to claim 1, further comprising administering to the subject a therapeutically effective amount of a second composition comprising one or more therapeutic agents and a pharmaceutically acceptable carrier.

9. The method according to claim 8, wherein the one or more therapeutic agents include small molecules, toxins, radiolabeling, radiotherapy, siRNA, peptides, antibodies, genetically modified cells, radiation, or cytokines.

10. The method according to claim 8, wherein the one or more therapeutic agents include an HDAC inhibitor.

11. The method according to claim 10, wherein the HDAC inhibitor is panobinostat.

12. The method according to claim 9, wherein the cytokine includes IL2, IL15, IL12, or IL18.

13. The method according to claim 9, wherein the small molecule comprises a proteasome inhibitor.

14. The method according to claim 9, wherein the antibody comprises an anti-PD1 antibody and / or an anti-CTLA-4 antibody.

15. The method according to claim 9, wherein the genetically modified cells are CAR T cells.

16. The method according to claim 1, wherein the cancer is MCH class I deficiency cancer or IFN gamma-resistant cancer.

17. The method according to claim 1, wherein the cancer is resistant to immunotherapy.

18. The method according to claim 1, wherein the cancer is resistant to the anti-PD1 / PD-L1 antibody.

19. The method according to claim 1, wherein the cancer includes melanoma, lung cancer, kidney cancer, bladder cancer, Hodgkin lymphoma, breast cancer, stomach cancer, and pancreatic cancer.

20. The method according to claim 1, wherein treating cancer is demonstrated by stopping or reducing tumor growth and / or metastasis.

21. The method according to claim 1, further comprising the step of testing the cancer for Jak1 mutations and / or B2m mutations.

22. A method for sensitizing target cancer cells to NK cells, Administering a therapeutically effective amount of a composition containing one or more activators and a pharmaceutically acceptable carrier to the subject. The method comprising the activator activating NK cells, thereby causing the lysis of one or more cancer cells in the subject, and the cancer being resistant to cytotoxic T cells.

23. The method according to claim 22, wherein the activation of the NKG2D receptor and / or CD16 receptor activates NK cells.

24. The method according to claim 22, wherein the activator inhibits the shedding of MICA / MICB by the cancer cells, thereby activating the NKG2D and / or CD16 receptor.

25. The method according to claim 22, wherein MICA / B on the surface of the cancer cells activates the NKG2D receptor, the CD16 receptor, or both.

26. The method according to claim 22, wherein the cancer is MCH class I deficiency cancer or IFN gamma-resistant cancer.

27. The method according to claim 22, further comprising the step of testing the cancer cells for Jak1 mutations and / or B2m mutations.

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