Diagnostic, therapeutic and other immunomodulatory and Anti-tumor uses of tap protein modulation
Patent Information
- Application Number
- PCT/US2026/019608
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-04-28
- Filing Date
- 2026-03-17
- Publication Date
- 2026-09-24
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Abstract
Description
[0001] ATTORNEY DOCKET NO. YU 8874 PCT
[0002] DIAGNOSTIC, THERAPEUTIC AND OTHER IMMUNOMODULATORY AND ANTI TUMOR USES OF TAP PROTEIN MODULATION
[0003] CROSS-REFERENCE TO RELATED APPLICATIONS
[0004] This application claims the benefit of and priority to U. S. Provisional Application No.
[0005] 63 / 773,372 filed March 17, 2025, U. S. Provisional Application No. 63 / 794,188 filed April 24, 2025, and U. S. Provisional Application No. 63 / 796,234 filed April 28, 2025, each of which is herein incorporated by reference in its entirety.
[0006] REFERENCE TO SEQUENCE LISTING
[0007] The Sequence Listing XML submitted as a filed named “YU8874PCT.xml”, created on March 17, 2026, and having a file size of 32,548 bytes is hereby incorporated by reference pursuant to 37 C. F. R. § 1.834(c)(1).
[0008] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0009] This invention was made with government support under CA219603 awarded by the National Institutes of Health. The government has certain rights in the invention.
[0010] FIELD OF THE INVENTION
[0011] The disclosed invention is generally in the field of immune modulation and specifically in the area of increasing immune response against cancer and reducing immune response in autoimmunity and inflammation.
[0012] BACKGROUND OF THE INVENTION
[0013] Therapies targeting the inhibitory checkpoints CTLA-4 and PD-1 axis induce prominent responses and lasting clinical benefit in a subset of patients with different tumor types, including non-small cell lung cancer (NSCLC) (Doroshow and Herbst, 2018;
[0014] Jhunjhunwala et al., 2021; Lopez de Rodas et al., 2022; Rizvi et al., 2018; Schalper et al., 2016). The recognition of cancer cells by T-cells and the therapeutic efficacy of immune checkpoint blockers depend on a fully functional HLA class-I antigen processing machinery (APM) (Mpakali and Stratikos, 2021; Sadagopan et al., 2022; Thompson et al., 2020).
[0015] Consistent with this notion, deleterious genomic alterations in β2M and HLA class-I genes occur in a subset of patients with acquired resistance to immune checkpoint blockers (Beck and Trowsdale, 2000; Gettinger et al., 2017; Sade- Feldman et al., 2017; Zaretsky et al., 2016). Moreover, the silencing of β2M in malignant lung cancer cells is sufficient to confer resistance to PD-1 axis blockers in a syngeneic carcinogen- induced mouse model (Gettinger et al., 2017). A high frequency of cancer-cell selective downregulation of β2M, HLA class-I and HLA class-II proteins in immunotherapy naive human NSCLCs associated 1
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[0017] with distinct tumor microenvironment composition and outcomes (Datar et al., 2021). The reduced expression of HLA class-I APM proteins in malignant cells was not explained by deleterious mutations in the genes encoding these proteins, indicating alternative, non-genomic mechanisms mediating these responses (Datar et al., 2021; Sadagopan et al., 2022).
[0018] Adequate processing and presentation of immunogenic peptides involve additional APM components beyond HLA class-I proteins, such as immuno-proteasome subunits, transporters associated with antigen processing (TAP) which transport peptides to the endoplasmic reticulum (ER), and key ER chaperones which assist the peptide loading on the HLA class-I molecules (Datar et al., 2021; Mpakali and Stratikos, 2021; Sadagopan et al., 2022). To date, the possible role of these APM components in immune evasion and immunotherapy resistance remains poorly understood. The species-specific nature of the HLA class-I system is a prominent barrier for the use of animal models to study mechanistic and therapeutic aspects, and therefore studies using human samples or model systems are required to assess the role of APM in this context. In addition, the HLA class-I APM components can be expressed both in tumor and stromal cells, which limits the capacity of bulk molecular analysis methods such as mRNA sequencing or mass spectrometry-based protein measurements to identify cancer-cell selective alterations. Moreover, the levels of mRNA transcripts using expression-based analysis such as RT-PCR, RNA- sequencing or single-cell RNA sequencing may not strictly mirror the protein levels. Therefore, selective identification of alterations in APM markers in cancer cells is better supported by localized or in situ strategies to characterize the signal present in each cell type compartment (Datar et al., 2021). To date, the mechanisms mediating defective antigen presentation in cancercells remain poorly understood and have not been exploited diagnostically and / or therapeutically in cancer patients.
[0019] Thus, there remains a need for improved means of detecting, diagnosing, and treating subjects with cancer.
[0020] It is object of the invention to identify a mechanism for adaptive immune evasion and immunotherapy resistance in cancer cells and to provide new diagnostic methods and therapeutic avenues to treat cancer subjects, particularly those that currently do not benefit from standard immunotherapy using immune checkpoint blockade (ICBs).
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[0023] BRIEF SUMMARY OF THE INVENTION
[0024] The majority of patients with NSCLC do not have effective therapeutic options. Immunotherapy using ICBs can achieve prominent and durable clinical benefit in a fraction of patients, but the majority show both primary and acquired resistance to treatment. The mechanism mediating immunotherapy resistance is poorly understood and this limits the capacity to overcome it. Identified herein is a new and dominant mechanism for adaptive immune evasion and immunotherapy resistance mediated by TAP2 downregulation in lung cancer cells. Using detailed spatial molecular analysis of human NSCLC cohorts and functional in vitro studies, the dominant effect of cancer-cell TAP2 downregulation in tumor immune evasion and immunotherapy resistance is demonstrated. Further, the data illustrates the mechanism by which cancer- cell TAP2 downregulation mediates adaptive immune escape and identifies IL-4 produced in the tumor microenvironment as the causative signal mediating the epigenetic TAP2 silencing. Finally, strategies to revert TAP2 suppression in lung cancer cells and re-sensitize them to PD-1 blocking antibodies are exemplified.
[0025] Thus, this mechanism, although sophisticated / complex, is also reversible. As such, also provided are diagnostic methods, and therapeutic compositions and methods for treating cancer patients including but not limited to those that currently do not benefit from standard immunotherapy using ICBs. For example, disclosed herein are multiple approved and nonapproved compounds able to modulate TAP2 downregulation. Results presented herein show this pathway to be a potent regulator of immunity in multiple cancers beyond NSCLC, and therefore has implications for immunostimulatory therapies for cancer patients more generally, but also activating the pathway to limit immune responses in the context of autoimmune diseases.
[0026] Thus provided are methods and compositions for modulating a TAP2 signaling axis to increase or decrease immune and / or inflammatory responses.
[0027] The data presented in the experiments below also supports a conclusion that in addition to dysfunctional antigen presentation, reduced or absent TAP transport creates retrograde accumulation of charged peptides in the nucleus, altering the chromatin accessibility and leading to IFNy resistance and reduced T-cell chemoattraction. More specifically, and without being bound by theory, it is believed that the cellular state and acquisition of immunosuppressive properties can be modulated based on the retrograde accumulation of short intranuclear peptides and chromatin changes secondary to and / or
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[0030] independent from reduced ER transport under TAP2 loss. This mechanism may reflect a general feedback loop that is corrupted by cancer cells to resist chronic IFNy exposure. It is believed both cancer and / or non-cancer cells can be subject to these mechanisms.
[0031] Thus, also provided are methods and compositions for increasing and decreasing immune and inflammatory responses by modulating the accumulation of short intranuclear peptides and / or chromatin changes and / or gene expression associated with intranuclear peptide changes.
[0032] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or can be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.
[0033] BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The accompanying drawings illustrate several forms of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.
[0035] Figures 1A-1K: Cancer-cell selective downregulation of TAPI and TAP2 in NSCLC and association with CD8+ TILs and treatment-specific outcomes. (Figs. 1A-1B) Representative multicolor immunofluorescence microphotographs showing the simultaneous detection of cytokeratin (CK) positive tumor epithelial cells (green), CD8+ TILs (white), TAPI (red), and TAP2 (yellow) proteins in human NSCLC. Cell nuclei were stained with DAPI (blue). (Figs. 1C-1D) Charts showing the frequency of cancer-cell TAPI and TAP2 downregulation in baseline samples from 4 cohorts of NSCLC patients treated with chemotherapy (Cohorts #1 and #2) or using PD-1 axis blockers (Cohorts #3 and #4). (Fig. IE) Mean frequency of cancer-cell TAPI and TAP2 downregulation across 7 NSCLC cohorts (Fig. 9C shows cohorts #5, 6, and 7). (Figs. 1F-1G) Levels of CD8+ TILs across tumors with and without TAPI and TAP2 downregulation in NSCLC cohorts #1-4. CD8 QIF scores are shown as thousands of fluorescence units. (Figs. 1H-1K) Kaplan-Meier graphical analysis of the overall survival of patients with low (red line) and high (blue line) cancer-cell TAPI or TAP2 levels. Cases were stratified using a TAPI or TAP2 protein tumor / stromal ratio of 1.4. Statistical significance was calculated using the Mann- Whitney test for marker
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[0038] levels and the log-rank test for survival analysis *, p<0.05; **, p<0.01. ns, not significant; HR, hazard ratio; OS, overall survival; QIF, quantitative immunofluorescence.
[0039] Figures 2A-2P: TAP2 downregulation reduces the levels of surface peptide-HLA complexes in lung cancer cells. (Fig.2A) Schematic of expected tumor antigen presentation machinery (APM) modulation in cancer cells. (Fig. 2B) Outline of the experimental strategy for the measurement of surface HLA-A2-HER2369-377 and HLA-A2-MAGE3271-279 complexes by flow cytometry in lung cancer cells. (Figs. 2C-2N) Levels of surface peptide-HLA complexes in human A549 or PC9 lung cancer cells transfected with a scrambled / control siRNA or with siRNAs targeting TAPI and / or TAP2, and left untreated (gray histograms) or stimulated with IFNy (blue histograms) or IFNy with TNFa (red histograms). Panels C-E and I-K show the surface levels of HLA-A2-HER2369-377 and panels F-H and L-N show the levels of HLA-A2-MAGE3271-279. (Figs. 2O-2P) Levels of surface peptide-HLA complexes in A549 cells after TAP2 elimination (KO) left untreated (gray histograms) or stimulated with IFNy (blue histograms) or IFNy with TNFa (red histograms). Panel O shows the surface levels of HLA-A2-HER2369-377 and panel P the levels of HLA-A2-MAGE3271-279. An isotype control antibody (IgG) was used as a background signal reference. The statistical significance was calculated using t test. *, p<0.05; **, p<0.01; ***, pcO. OOl. MFI, mean fluorescent intensity; si, siRNA; scr, scrambled; Tx, treatment; ns, not significant; WT, wild type.
[0040] Figures 3A-3Y: TAP2 reduction protects cancer-cells from tumor antigen-specific T-cell killing. (Fig. 3A) Schematic showing the expected tumor antigen-specific recognition by cognate CD8+ T cells. (Fig. 3B) Outline of the experimental strategy to measure changes in the cell death and viability in cancer-cell / T-cell co-cultures by flow cytometry, LDH release and MTT assay. (Figs. 3C-3V) Levels of cancer-cell Annexin V positivity, LDH release or MTT staining of A549 or PC9 lung cancer cells transfected with scrambled siRNA or siRNAs targeting TAPI and / or TAP2; and left untreated (black) or stimulated with IFNy (blue) or IFNy with TNFa (red). Different effector (CD8+ T cells) to target (tumor cell) ratios including 0:1, 2:1 and 5:1 were analyzed. Panels C and M show representative flow cytometry plots for the identification of apoptotic malignant cells using EpCAM+ Annexin V+ events. Panels D-F and N-P show the levels of EpCAM+ / Annexin V+ cells under different experimental conditions. Panels G-I and Q-S show the LDH levels and panels J-L and T-V display the cellular viability using MTT staining. (Figs. 3W-3Y) Levels of cancercell apoptosis and viability of A549 cells with TAP2 elimination (KO) left untreated (black)
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[0043] or stimulated with IFNy (blue) or IFNy with TNFa (red); and after exposure to different effectortarget cell ratios. An isotype control antibody (IgG) was used as a background signal reference. The statistical significance after treatments was calculated using a t test. *, p<0.05; **, p<0.01; ***, p<0.001. si, siRNA; scr, scrambled; Tx, treatment; ns, not significant; WT, wild type.
[0044] Figures 4A-4I: Downregulation of TAP2 alters intracellular immunomodulatory pathways and reduces the sensitivity to proinflammatory cytokines in lung cancer-cells. (Fig.
[0045] 4A) Impact of TAP2 downregulation in multiple immunomodulatory transcripts after targeted transcriptomic analysis of A549 cells. The transcripts level changes in TAP2 deficient cells were normalized relative to those of the scrambled transfected cells. (Fig.4B) Graph showing the levels of SOCS1 protein expression by flow cytometry in A549 cells transfected with a scrambled / control siRNA (black bars) or with TAP2 siRNA (white bars). (Fig.4C) Levels of SOCS1 protein expression by flow cytometry in A549 cells transfected with an empty vector (black bars) or with a vector encoding FLAG-TAP2 (white bars). (Fig. 4D - Fig.4G) Volcano plots showing the change in the levels of multiple immunomodulatory transcripts in A549 cells transfected with scr siRNA (siScr) or with TAP2 siRNAs (siTAP2) and stimulated with IFNy (D-E) or IFNy with TNFa (F-G). (Fig. 4H) Impact of TAP2 downregulation on multiple phosphorylated intracellular proteins using phosphoprotein arrays of cell lysates obtained from A549 cells transfected with siScr or with siTAP2 and stimulated with IFNy with TNFa. The phosphoprotein level changes were normalized relative to those of the scrambled transfected cells. (Fig. 41) Schematic of the TAP2-SOCS1 modulation and its impact on the of IFNy and TNFa pathways in cancer-cells. Statistical significance was calculated using t test. *, p<0.05; **, p<0.01. FC, fold change; MFI, mean fluorescent intensity; si, siRNA; scr, scrambled.
[0046] Figures 5A-5O: Myeloid-cell derived IL-4 reduces TAP2 expression in lung cancercells. (Fig. 5A) TAP2 mRNA expression levels in primary NSCLCs from the TCGA cohort stratified with the median IL-4 mRNA expression. (Fig.5B) TAP2 protein levels measured using flow cytometry in permeabilized A549 cells treated with IL-4 for different time periods. (Figs. 5C-5D) Levels of TAP2 or SOCS1 protein in A549 cells treated with IL-4, IL-4 with IFNy or IL-4 with IFNy and TNFa. (Figs. 5E-5F) Surface levels of HLA-A2-HER2369-377 or HLA-A2-MAGE3271-279 complexes measured by flow cytometry in in A549 cells treated with IL-4, IFNy and TNFa or IL-4 plus IFNy and TNFa. (Figs. 5G-5I) levels of cancer-cell apoptosis measured by Annexin V positivity or cell viability using LDH
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[0049] release and MTT assay of A549 cells treated with IL-4, IL-4 with IFNy or IL-4 with IFNy and TNFa followed by co-incubation with tumor-antigen specific effector CD8+ T-cells. (Fig. 5J) Representative multicolor fluorescence images showing the expression and spatial location of CK protein (green), TAP2 protein (yellow), IL-4 mRNA (red) and CD1 lb mRNA (white) in primary human NSCLC using multiplexed protein / RNA staining. Nuclei were stained using DAPI (blue). (Figs. 5K-5L) Levels of IL-4 mRNA (Fig. 5K) or TAP2 protein (Fig. 5L) measured in selected tumor tissue compartments or stratified by the median IL-4 levels in samples from Cohorts #1 and #2. (Figs. 5M-5O) Levels of CD8+ / CD25+ T-cells (M) or EpCAM+ / Annexin V+ apoptotic cancer-cells (Figs. 5N-5O) measured by flow cytometry in single-cell suspensions obtained from primary human NSCLCs pre-incubated with IFNy and TNFa followed by treatment with an anti-IL-4R antibody (aIL-4r) and / or the PD-1 blocking antibody Pembrolizumab (aPD-1). The analysis of cancer-cell apoptosis was also conducted in tumor-cell suspensions with selective T-cell elimination (TECS). An isotype control antibody (IgG) was used as a background signal reference. For A statistical significance was calculated using unpaired t test and for B-I statistical significance was calculated using paired t test. *, p<0.05; **, p<0.01; ***, pcO. OOl; ****, pcO. OOOl. ns, not significant; MFI, mean fluorescent intensity; T, tumor; S, stroma.
[0050] Figures 6A-6J: IL-4 reduces TAP2 expression via epigenetic remodeling in lung cancer. (Fig. 6A) Schematic of the strategy for obtaining and analyzing single cell preparations from paired surgically resected morphologically normal adjacent to tumor (NAT) and tumor tissues from NSCLC patients with low TAP2 protein levels. (Fig. 6B) Volcano plot showing the differential expression of multiple mRNA transcripts between nontumor lung tissue and NSCLC analyzed using bulk RNA sequencing. (Fig. 6C) Differential expression of TAP2 and SOCS1 mRNA transcripts in paired preparations from non-tumor lung tissue and NSCLC. The results show the mean from 3 independent cases. (Fig. 6D) Heatmap representation of the chromatin accessibility peaks located within the TAP2 gene promoter region using ATAC-seq (Fig. 6E) Predicted transcription factor (TF) binding sites (SEQ ID NOS:23-30 respectively) with the highest affinity scores for the TAP2 promoter region. (Figs. 6F-6H) Graphs and heatmap showing the chromatin accessibility of the promoter region of the TAP2 gene measured using ATACseq in control A549 cells or after treatment with IL-4 or IFNy and TNFa. (Figs. 6I-6J) Graphs and heatmap showing the chromatin accessibility of the promoter region of the SOCS1 gene measured using ATACseq in control A549 cells or after treatment with IL-4 or IFNy and TNFa. Promoter regions were
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[0053] considered as the DNA sequences between the gene Transcription Start Site (TSS, +3kb) and Transcription End Site (TES, -3kb). FC, fold change.
[0054] Figures 7A-7O: Upregulation and downregulation of TAP2 protein expression. (Fig.
[0055] 7 A) Schematic showing the strategy for high throughput screening of pharmacologic compounds using TAP2 and PD-L1 staining in A549 cells. (Figs. 7B-7D) Representative histograms showing the TAP2 protein upregulation, down regulation, PD-L1 protein increase, and PD-L1 protein decrease measured by immunofluorescence in A549 cells treated with individual compounds from the Pharmakon 1600 library (Fig. 7B), Enzo 640 FDA library (Fig. 7C) and Enzo epigenetic library (44 compounds) (Fig.7D). The protein scores were normalized relative to those of the samples treated with the vehicle control (DMSO) alone. (Fig. 7E) Table showing 6 compounds from the 3 libraries, selected for additional experiments to determine their EC50. (Figs. 7F-7G) Concentration-response curves showing the TAP2 protein level changes in A549 cells after exposure to different concentrations of the compounds indicated in (Fig. 7E). (Fig. 7H) Outline of experiments to study changes in TAP2 protein and surface peptide-HLA complex levels using flow cytometry in A549 cells treated with different concentrations of S AHA / Vorinostat. 7I-7K) Levels of TAP2 protein (Fig. 71), surface HLA-A2-HER2369-377 (Fig. 7J) or HLA-A2-MAGE3271-279 complexes (Fig. 7K) measured by flow cytometry in A549 cells with or without treatment for 24 hours with 1 or 3.5 LI of SAHA / Vorinostat. An isotype control antibody (IgG) was used as a background signal reference. Statistical significance was calculated using paired t-test. p<0.01; ****, p<0.0001. FC, fold change; ns, non- significant; No-Tx, no treatment; MFI, mean fluorescent intensity. (Fig. 7L) is a dot plot showing the compounds identified as up and down regulating TAP2, and gating the 10% strongest TAP2 reducers and illustrating selected drugs including Aminopterin, Pemetrexed, Dinitolmide, and Amprolium. (Fig. 7M) is a flow-diagram of bioinformatical processing of hits from the screen. (Figs. 7N and 70) are a histogram of “pathways” enriched based on signature genes and representative drugs associated with modulation thereof (upper panel), and a pie chart showing drug classification by function (lower panel) for the strongest 10% of compounds identified in the screen for Pharmakon 1600 (7N) and Enzo 640 FDA (70) libraries.
[0056] Figure 8: Graphical model summarizing the IL-4 mediated epigenetic regulation of TAP2 in lung cancer. IL-4 signaling through IL-4R epigenetically suppresses TAP2 expression leading to cancer cell adaptive immune evasion. Downregulation of TAP2 induces SOCS1 expression, suppresses IFN gamma pathway activation, limits sensitivity to
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[0059] proinflammatory cytokines and reduces the levels of antigenic HLA-peptide complexes in malignant cells.
[0060] Figures 9A-9D: Cancer-cell selective downregulation of TAPI and TAP2 in NSCLC.
[0061] (Figs. 9A-9B) Representative multicolor immunofluorescence microphotographs showing the simultaneous detection of cytokeratin (CK) positive tumor epithelial cells (green), CD8+ TILs (white), TAP1 (red) and TAP2 (yellow) proteins in human NSCLC. Cell nuclei were stained with DAPI (blue). (Fig. 9C) Charts showing the frequency of cancer-cell TAPI and TAP2 downregulation in baseline samples from (Cohort #5, Cohort #6 and Cohort #7. (Fig.
[0062] 9D) Levels of CD8+ T-cells across tumors with and without TAPI and TAP2 downregulation in NSCLC cohorts #5-7. CD8 QIF scores are shown as thousands of fluorescence units. Statistical significance was calculated using the Mann-Whitney test for marker levels *, p<0.05. ns, not significant; HR, hazard ratio; QIF, quantitative immunofluorescence.
[0063] Figures 10A-10B: Cancer-cell selective downregulation of TAPI and TAP2 and association with survival outcomes in NSCLC. (Figs. 10A-10B) Kaplan-Meier graphical analysis of the overall survival of patients with low (red line) and high (blue line) cancer-cell TAP2 levels. Cases with available clinical annotation were stratified using a TAP2 tumor / stromal ratio of 1.4. Statistical significance was calculated using log-rank test for survival analysis. HR, hazard ratio; OS, overall survival.
[0064] Figures 11A-11D: Validation of HLA- A2-HER2 and HLA-A2-MAGE3 antibodies.
[0065] (Figs. 11A-11D) Cells were incubated with HER2 peptides or MAGE3 peptides followed by surface HLA-A2-HER2369-377 and HLA-A2-MAGE3271-279 levels by flow cytometry respectively. (Fig. 11A) contour plots (Fig. 11B) A549 cells (Fig. 11C) H1975 cells (Fig. 11D) H520 cells Bar graphs represent percent (%) positive cell counts. Statistical significance was calculated using paired t-test. *, p<0.05; **, p<0.01. ns, non-significant.
[0066] Figures 12A-12D: TAPI and TAP2 knockdown validation. (Fig. 12A) mRNA levels of TAPI and TAP2 in A549 cells left untreated (gray bar) or treated with IFNy (blue bars) or IFNy with TNFa (red bars). (Figs. 12B-12C) A549 cells were incubated with scrambled siRNA (siScr) or TAPI siRNA (siTAPl) or TAP2 siRNA (siTAP2) and knockdown efficacy by flow. (Fig. 12D) TAP2 CRISPR knockout validated by flow cytometry. An isotype control antibody (IgG) was used as a background signal reference. Statistical significance was calculated using paired t-test. *, p<0.05; **, p<0.01; ***, p<0.001. ns, non-significant.
[0067] Figure 13: Transient expression of TAP2. A549 cells were transfected with FLAG tagged TAP2 and FLAG staining analysis by flow cytometry. An isotype control antibody
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[0070] (IgG) was used as a background signal reference. Statistical significance was calculated using paired t-test. **, p<0.01. MFI, mean fluorescent intensity.
[0071] Figures 14A-14J: IL-4 regulates expression of TAP2. (Fig. 14A) Frequency of TAP2 gene mutations (including missense, indels, deep deletion) in NSCLC samples from the MSKCC collection. The frequency of wild type and mutation indicated with white-colored text within the pie chart. (Fig. 14B) TAP2 mRNA expression stratified with the median cutoff of IFNy and IL-8 mRNA expression in NSCLC samples from TCGA cohort. (Fig. 14C) A549 cells were treated with IL-4 or IFNy or IFNy with TNFa and TAPI expression using flow cytometry. (Figs. 14D-14H) PC9 cells treated with IL-4, IL-4 with IFNy or IL-4 with IFNy and TNFa and (Fig. 14D) TAP2 expression (Fig. 14E) SOCS1 expression (Fig. 14F) TAP2 expression (Fig. 14G) surface HLA-A2-HER2369-377 and (Fig. 14H) surface HLA-A2-MAGE3271-279 levels analyzed by flow cytometry. (Figs. 141- 14J) Spatially resolved expression of IL-4 and CD lib mRNAs expression in 2 retrospective NSCLC cohorts related to Figures 5K-5L. An isotype control antibody (IgG) was used as a background signal reference. Statistical significance was calculated using the paired t test. *, p<0.05; **, p<0.01; ***, pcO. OOl; ****, p<0.0001. h, hours; MFI, mean fluorescent intensity; ns, not significant.
[0072] Figures 15A-15B: Preparation of TEST from human NSCLC tissues. (Figs. 15A-15B) NSLCL tissue suspension after T cell eliminated from cell suspension (TECS) and surface levels of annexin V positive on EpCAM-i- cells. Contour plots.
[0073] Figure 16A-16D are plots showing TAP2 loss reduces the surface HLA-peptide complexes and protects lung cancer cells from tumor-antigen specific killing only after IFNy treatment.
[0074] Figures 17A-17E show modulation of inflammatory signals by TAP2 downregulation or loss in human lung cancer.
[0075] Figures 18A-18C are plots showing an altered response to IFNy in TAP2 deficient lung cancer cells.
[0076] Figures 19A-19E are plots showing immunopeptidome changes in TAP2 deficient lung cancer cells.
[0077] Figures 20A-20B are plots showing changes in chromatin accessibility in TAP2 deficient lung cancer cells.
[0078] Figures 21A and 21B are plots showing alteration in intracellular transport of immunogenic peptides in TAP2 deficient lung cancer cells.
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[0081] Figures 22A and 22B are plots showing the impact of peptide loading on IFNy response in TAP2 deficient lung cancer cells.
[0082] DETAILED DESCRIPTION OF THE INVENTION
[0083] The disclosed method and compositions can be understood more readily by reference to the following detailed description of particular forms and the Example included therein and to the Figures and their previous and following description.
[0084] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular forms only and is not intended to be limiting.
[0085] I. Definitions
[0086] It is to be understood that the disclosed compounds, compositions, and methods are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular forms and forms only and is not intended to be limiting.
[0087] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a composition or method is disclosed and discussed and a number of modifications that can be made to the composition or method are discussed, each and every combination and permutation of the composition and method and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination
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[0090] of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials. These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific form or combination of forms of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
[0091] The term “hit” refers to a test compound that shows desired properties in an assay. The term “test compound” refers to a chemical to be tested by one or more screening method(s) as a putative modulator. A test compound can be any chemical, such as an inorganic chemical, an organic chemical, a protein, a peptide, a carbohydrate, a lipid, or a combination thereof. Usually, various predetermined concentrations of test compounds are used for screening, such as 0.01 micromolar, 1 micromolar and 10 micromolar. Test compound controls can include the measurement of a signal in the absence of the test compound or comparison to a compound known to modulate the target.
[0092] The terms “high,” “higher,” “increases,” “elevates,” or “elevation” refer to increases above basal levels, e.g., as compared to a control. The terms “low,” “lower,” “reduces,” or “reduction” refer to decreases below basal levels, e.g., as compared to a control.
[0093] The term “modulate” as used herein refers to the ability of a compound to change an activity in some measurable way as compared to an appropriate control. As a result of the presence of compounds in the assays, activities can increase or decrease as compared to controls in the absence of these compounds. Preferably, an increase in activity is at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound. Similarly, a decrease in activity is preferably at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound. A compound that increases a known activity is an “agonist”. One that decreases, or prevents, a known activity is an “antagonist”.
[0094] The term “inhibit” means to reduce or decrease in activity or expression. This can be a complete inhibition or activity or expression, or a partial inhibition. Inhibition can be
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[0097] compared to a control or to a standard level. Inhibition can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96. 97, 98, 99, or 100%.
[0098] The term “monitoring” as used herein refers to any method in the art by which an activity can be measured.
[0099] The term “providing” as used herein refers to any means of adding a compound or molecule to something known in the art. Examples of providing can include the use of pipettes, pipettemen, syringes, needles, tubing, guns, etc. This can be manual or automated. It can include transfection by any mean or any other means of providing nucleic acids to dishes, cells, tissue, cell-free systems and can be in vitro or in vivo.
[0100] The term “preventing” as used herein refers to administering a compound prior to the onset of clinical symptoms of a disease or conditions so as to prevent a physical manifestation of aberrations associated with the disease or condition.
[0101] The term “in need of treatment” as used herein refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals) that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a care giver's expertise, but that include the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the compounds of the invention.
[0102] As used herein, “subject” includes, but is not limited to, animals, plants, bacteria, viruses, parasites and any other organism or entity. The subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian. The subject can be an invertebrate, more specifically an arthropod (e.g., insects and crustaceans). The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.
[0103] By “treatment” and “treating” is meant the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the
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[0106] improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. It is understood that treatment, while intended to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, need not actually result in the cure, amelioration, stabilization or prevention. The effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and / or quantitiative terms. Thus, for example, characteristics or features of a disease, pathological condition, or disorder and / or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.
[0107] A cell can be in vitro. Alternatively, a cell can be in vivo and can be found in a subject. A “cell” can be a cell from any organism including, but not limited to, a bacterium.
[0108] In one aspect, the compounds described herein can be administered to a subject comprising a human or an animal including, but not limited to, a mouse, dog, cat, horse, bovine or ovine and the like, that is in need of alleviation or amelioration from a recognized medical condition.
[0109] By the term “effective amount” of a compound as provided herein is meant a nontoxic but sufficient amount of the compound to provide the desired result. As will be pointed out below, the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount.” However, an appropriate effective amount can be determined by one of ordinary skill in the art using only routine experimentation.
[0110] The dosages or amounts of the compounds described herein are large enough to produce the desired effect in the method by which delivery occurs. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent
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[0113] of the disease in the subject and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician based on the clinical condition of the subject involved. The dose, schedule of doses and route of administration can be varied.
[0114] The efficacy of administration of a particular dose of the compounds or compositions according to the methods described herein can be determined by evaluating the particular aspects of the medical history, signs, symptoms, and objective laboratory tests that are known to be useful in evaluating the status of a subject in need of treatment for diseases and / or conditions such as those disclosed herein. These signs, symptoms, and objective laboratory tests will vary, depending upon the particular disease or condition being treated or prevented, as will be known to any clinician who treats such patients or a researcher conducting experimentation in this field. For example, if, based on a comparison with an appropriate control group and / or knowledge of the normal progression of the disease in the general population or the particular individual: (1) a subject’s physical condition is shown to be improved (e.g., a tumor has partially or fully regressed), (2) the progression of the disease or condition is shown to be stabilized, or slowed, or reversed, or (3) the need for other medications for treating the disease or condition is lessened or obviated, then a particular treatment regimen will be considered efficacious.
[0115] By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
[0116] Any of the disclosed compounds can be used therapeutically in combination with a pharmaceutically acceptable carrier. The compounds described herein can be conveniently formulated into pharmaceutical compositions composed of one or more of the compounds in association with a pharmaceutically acceptable carrier. See, e.g., Remington's Pharmaceutical Sciences, latest edition, by E. W. Martin Mack Pub. Co., Easton, PA, which discloses typical carriers and conventional methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the compounds described herein. These most typically would be standard carriers for administration of compositions to humans. In one aspect, humans and non-humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Other compounds will be administered according to standard procedures used by those skilled in the art.
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[0119] The pharmaceutical compositions described herein can include, but are not limited to, carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions can also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
[0120] The compounds and pharmaceutical compositions described herein can be administered to the subject in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Thus, for example, a pharmaceutical composition described herein can be administered to the subject, e.g., as described herein. Moreover, a pharmaceutical composition can be administered to a subject vaginally, rectally, intranasally, orally, by inhalation, or parenterally, for example, by intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intraarterial, intralymphatic, intravenous, intrathecal and intratracheal routes. Parenteral administration, if used, is generally characterized by injection and includes intravenous (IV), subcutaneous (SC), intramuscular (IM), epidural and intra-articular injection, as well as surgical insertion of depots in the organ or tissue of interest (Bittner, et al., BioDrugs., 32:425-440 (2018); Lee et al., J. Pharm.
[0121] Investig., 49: 459-476 (2019); Chaudhary et al., Crit. Rev. Ther. Drug Carrier Syst., 36:137-181 (2019)). Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions (Park et al., J Control Release, 342:53-65 (2022); Nkanga, et al., Advanced Drug Delivery Reviews, 167:19-46, (2020); Sheikh, et al., Asian Journal of Pharmaceutics, 10(4): S465-S471 (2016); Rhee et al.. Pharmaceutical Technology Drug Delivery, p. S6 (2010)). An exemplary approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See for example, U. S. Patent No. 9,700,630, WO 2006 / 125620, and KR 101898816.
[0122] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions which can also contain buffers, diluents and other suitable additives. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based
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[0124] 45836485.1ATTORNEY DOCKET NO. YU 8874 PCT
[0125] on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
[0126] Formulations for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable.
[0127] Compositions for oral administration can include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders can be desirable.
[0128] The materials described herein as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method.
[0129] Disclosed are mixtures formed by performing or preparing to perform the disclosed method.
[0130] Whenever the method involves mixing or bringing into contact compositions or components or reagents, performing the method creates a number of different mixtures. For example, if the method includes 3 mixing steps, after each one of these steps a unique mixture is formed if the steps are performed separately. In addition, a mixture is formed at the completion of all of the steps regardless of how the steps were performed. The present disclosure contemplates these mixtures, obtained by the performance of the disclosed methods as well as mixtures containing any disclosed reagent, composition, or component, for example, disclosed herein.
[0131] Disclosed are systems useful for performing, or aiding in the performance of, the disclosed method. Systems generally comprise combinations of articles of manufacture such as structures, machines, devices, and the like, and compositions, compounds, materials, and the like. Such combinations that are disclosed or that are apparent from the disclosure are contemplated.
[0132] The disclosed methods include the determination, identification, indication, correlation, diagnosis, prognosis, etc. (which can be referred to collectively as “identifications”) of subjects, diseases, conditions, states, etc. based on measurements, detections, comparisons, analyses, assays, screenings, etc. Such identifications are useful for many reasons. For example, and in particular, such identifications allow specific actions to be taken based on, and relevant to, the particular identification made. For example, diagnosis of
[0133] 17
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[0135] a particular disease or condition in particular subjects (and the lack of diagnosis of that disease or condition in other subjects) has the very useful effect of identifying subjects that would benefit from treatment, actions, behaviors, etc. based on the diagnosis. For example, treatment for a particular disease or condition in subjects identified is significantly different from treatment of all subjects without making such an identification (or without regard to the identification). Subjects needing or that could benefit from the treatment will receive it and subjects that do not need or would not benefit from the treatment will not receive it.
[0136] Accordingly, also disclosed herein are methods comprising taking particular actions following and based on the disclosed identifications. For example, disclosed are methods comprising creating a record of an identification (in physical — such as paper, electronic, or other — form, for example). Thus, for example, creating a record of an identification based on the disclosed methods differs physically and tangibly from merely performing a measurement, detection, comparison, analysis, assay, screen, etc. Such a record is particularly substantial and significant in that it allows the identification to be fixed in a tangible form that can be, for example, communicated to others (such as those who could treat, monitor, followup, advise, etc. the subject based on the identification); retained for later use or review; used as data to assess sets of subjects, treatment efficacy, accuracy of identifications based on different measurements, detections, comparisons, analyses, assays, screenings, etc., and the like. For example, such uses of records of identifications can be made, for example, by the same individual or entity as, by a different individual or entity than, or a combination of the same individual or entity as and a different individual or entity than, the individual or entity that made the record of the identification. The disclosed methods of creating a record can be combined with any one or more other methods disclosed herein, and in particular, with any one or more steps of the disclosed methods of identification.
[0137] As another example, disclosed are methods comprising making one or more further identifications based on one or more other identifications. For example, particular treatments, monitorings, follow-ups, advice, etc. can be identified based on the other identification. For example, identification of a subject as having a disease or condition with a high level of a particular component or characteristic can be further identified as a subject that could or should be treated with a therapy based on or directed to the high level component or characteristic. A record of such further identifications can be created (as described above, for example) and can be used in any suitable way. Such further identifications can be based, for example, directly on the other identifications, a record of such other identifications, or a
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[0140] combination. Such further identifications can be made, for example, by the same individual or entity as, by a different individual or entity than, or a combination of the same individual or entity as and a different individual or entity than, the individual or entity that made the other identifications. The disclosed methods of making a further identification can be combined with any one or more other methods disclosed herein, and in particular, with any one or more steps of the disclosed methods of identification.
[0141] As another example, disclosed are methods comprising treating, monitoring, following-up with, advising, etc. a subject identified in any of the disclosed methods. Also disclosed are methods comprising treating, monitoring, following-up with, advising, etc. a subject for which a record of an identification from any of the disclosed methods has been made. For example, particular treatments, monitorings, follow-ups, advice, etc. can be used based on an identification and / or based on a record of an identification. For example, a subject identified as having a disease or condition with a high level of a particular component or characteristic (and / or a subject for which a record has been made of such an identification) can be treated with a therapy based on or directed to the high level component or characteristic. Such treatments, monitorings, follow-ups, advice, etc. can be based, for example, directly on identifications, a record of such identifications, or a combination. Such treatments, monitorings, follow-ups, advice, etc. can be performed, for example, by the same individual or entity as, by a different individual or entity than, or a combination of the same individual or entity as and a different individual or entity than, the individual or entity that made the identifications and / or record of the identifications. The disclosed methods of treating, monitoring, following-up with, advising, etc. can be combined with any one or more other methods disclosed herein, and in particular, with any one or more steps of the disclosed methods of identification.
[0142] The disclosed measurements, detections, comparisons, analyses, assays, screenings, etc. can be used in other ways and for other purposes than those disclosed. Thus, the disclosed measurements, detections, comparisons, analyses, assays, screenings, etc. do not encompass all uses of such measurements, detections, comparisons, analyses, assays, screenings, etc.
[0143] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field
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[0146] relevant to the present disclosure as it existed before the priority date of each claim of this application.
[0147] Throughout this specification the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated clement, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
[0148] II. Biomarkers for Immune Modulation
[0149] As described herein, TAP2 has a negative predictive role in cancer-cell immunotherapy. TAP1 downregulation alone had a lower impact on the surface antigen levels after cytokine stimulation that was inconsistent across the different peptide-HLA complexes analyzed, and the concurrent downregulation of both TAP1 and TAP2 produced an effect comparable to TAP2 reduction alone supporting a dominant effect this protein. These results demonstrate that TAP2 reduction can limit the levels of surface HLA class-I antigens in lung cancer cells, particularly under proinflammatory conditions. TAP2 downregulation also induced a more prominent reduction in the apoptosis of unstimulated A549 cells and almost totally prevented the tumor-antigen specific T-cell mediated killing induced by proinflammatory cytokines (Figures 3E-3F). In the provided experiments, the concurrent downregulation of both TAP 1 and TAP2 produced an effect comparable to TAP2 downregulation alone. Together, these results demonstrate that TAP2 reduction mediates, and is sufficient to induce tumor antigen-specific T-cell immune evasion in cancer cells.
[0150] As shown in Figures 4C and 13, TAP2 overexpression significantly reduced SOCS1 protein levels supporting the bi-directional modulation of both proteins in cancer cells. These results show that TAP2 downregulation alters the immune transcriptome of cancer cells and suppresses proinflammatory signals via SOCS1 increase. Together the results support the existence of a previously unrecognized feed-forward mechanism where TAP2 expression, that is normally induced by proinflammatory cytokines, can limit proinflammatory responses of cancer cells via transcriptional SOCS1 control. Results also show that IL-4 reduces TAP2 expression, mimics the effects of TAP2 downregulation and mediates adaptive immune evasion of cancer cells.
[0151] Thus, TAP2, TAP1, SOCS1, and IL-4 are biomarkers of the disclosure.
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[0154] In some forms, subjects are identified and / or selected for treatment of cancer and infections based on reduced expression of TAP2 and / or TAPI, and / or increased expression of SOCS1 and / or IL-4.
[0155] In some forms, subjects are identified and / or selected for treatment of autoimmune disease and / or inflammation based on increased expression of TAP2 and / or TAPI, and / or reduced expression of SOCS1 and / or IL-4.
[0156] Provided herein are methods of detection of one or two of the disclosed biomarkers, and use thereof in the diagnosis, prognosis, and / or treatment of subjects in need thereof.
[0157] A. Source of the Biomarkers
[0158] The disclosed biomarkers are proteins and / or their encoding mRNA. Some forms provide these biomolecules in isolated form such as in a biological sample. The preferred biological source for detection of the biomarkers is tissue (e.g., tissue suspected of being cancerous) including biopsy material from a tumor, or microenvironment and / or cells thereof.
[0159] In some forms, intact cells are subjected to biomarker detection. For example, a sample may be obtained and processed using well-known and routine clinical methods. In some aspects, the biological sample includes a plurality of cells. In certain aspects, the biological sample includes fresh or frozen tissue. In specific aspects, the biological sample includes formalin fixed, paraffin embedded tissue.
[0160] In some forms, the cells are permeabilized. In some forms, a cell lysate or homogenate is subjected to biomarker detection.
[0161] B. Methods of Detecting Biomarkers
[0162] Generally, the level of the expression biomarker is determined by measuring the amount of nucleic acids and / or protein expressed from a corresponding gene encoding the expression biomarker to be detected. As discussed in more detail below, detection and measurement of nucleic acids can be accomplished using techniques such as quantitative polymerase chain reaction (qPCR), reverse transcription quantitative PCR (RT-qPCR), or next-generation sequencing (NGS). These methods detect and quantify messenger RNA (mRNA) or other nucleic acid fragments associated with the target biomarkers, providing a measure of gene expression levels. Alternatively, detection of protein expression may be performed using immunoassays such as enzyme-linked immunosorbent assays (ELISA), immunohistochemistry (IHC), or multiplex bead-based assays. These methods utilize antibodies specific to the target biomarkers to measure protein levels in the sample.
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[0165] For example, the sample can be subjected to one or more forms of molecular analysis to detect the presence or absence of biomarkers. These analyses may quantify specific gene transcripts, gene products, or other molecular markers. These various readouts can be obtained using any suitable molecular means, including but not limited to polymerase chain reaction (PCR), arrays, sequencing, and combinations thereof. The substrate of the molecular analysis is typically nucleic acids. Depending on the particular technique(s) employed, DNA and / or RNA can serve as the substrate for analysis.
[0166] Suitable molecular techniques for use in the disclosed methods are well-established in the art. See, e.g., U. S. Patent No. 11,807,909 and U. S. Publication Nos. 2022 / 0017946, 2021 / 0371940, 2021 / 0308351, and 2021 / 0171898, each of which is specifically incorporated by reference in its entirety.
[0167] The methods can include amplification and / or sequencing of nucleic acids in collected subject samples. In preferred forms, the target polynucleotide is mRNA. The mRNA can be collected from the cells themselves, or be cell-free at the time of collection (i.e., liquid biopsy). These nucleic acids are typically derived from target cells (e.g., cancer cells) present in the sample.
[0168] Prior to amplification, the methods can include assessing the quality of the isolated nucleic acids, such as mRNA, to ensure suitability for downstream processing. This assessment may involve evaluating both the integrity and concentration of the nucleic acids.
[0169] The molecular analysis can include the use of primers and / or probes that are specific to the biomarkers. Such amplification and / or detection can be used to detect and determine the identity of one or more expression biomarkers in the sample. Such amplification and / or detection can be performed without the need for sequencing, using methods such as qPCR or droplet digital PCR (ddPCR). Alternatively, amplification can be performed using nonspecific or random primers for broader analysis, followed by sequencing for detailed characterization.
[0170] In some forms, RNA is analyzed directly. In other forms, RNA is converted to DNA for subsequent molecular analysis. Most typically, this is accomplished by a reverse transcription (RT) reaction. Typically, the RT primer is a single-stranded oligonucleotide (e.g., containing DNA) that can act as a point of initiation for nucleic acid synthesis by an RT polymerase under suitable conditions. Typically, the RT primer is able to hybridize to a target RNA (e.g., miRNA, mRNA) to facilitate cDNA synthesis by a reverse transcriptase. The primer can be specific, random, or semi-random.
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[0173] The method of detecting can also include detecting the level of at least one control biomarker for normalization.
[0174] As described, the methods provided herein can include subjecting the RNA isolated from the sample to polymerase chain reaction (PCR) for nucleic acid amplification. Any PCR technique known in the art can be used to amplify the nucleic acids. In various aspects, the polymerase chain reaction includes Real-Time PCR (quantitative PCR or qPCR), Reverse-Transcriptase (RT-PCR), Multiplex PCR, Nested PCR, High Fidelity PCR, Fast PCR, Hot Start PCR, Long-range PCR, Arbitrary Primed PCR, Digital PCR, Droplet Digital PCR (ddPCR), isothermal amplification PCR, Endpoint PCR (Qualitative PCR), or a combination of any thereof. In various aspects, the polymerase chain reaction includes qPCR or ddPCR.
[0175] Quantitative PCR (qPCR) can be used to quantify the presence of bimarkers.
[0176] Conventional PCR followed by gel electrophoresis can also be used for size-based confirmation of the amplified products. Additionally, loop-mediated isothermal amplification (LAMP) PCR can be employed as a rapid, highly specific, and sensitive method for detecting biomarkers that could be performed without the need for thermal cyclers.
[0177] In some forms, the analysis can include quantitative detection of the nucleic acid, such as using an oligonucleotide probe or nucleic acid dye. In some forms, the quantitative detection occurs as part of the polymerase chain reaction performed (e.g., using real-time PCR or quantitative PCR (qPCR)). In some forms, the quantitative detection occurs after the polymerase chain reaction is performed. For example, in some forms, the quantitation can use a Nanopore or similar nanotechnology to detect the nucleic acids. See, for example, Kang et al., “Ready-to-use nanopore platform for the detection of any DNA / RNA oligo at attomole range using an Osmium tagged complementary probe” Scientific Reports 10, 19790 (2020), which is incorporated herein by reference in its entirety.
[0178] qPCR is a well-established method for the detection, quantification, and analysis of biomarkers in clinical samples. Exemplary qPCR methods relevant to these applications include: (1) non-specific fluorescent dye intercalation with double- stranded DNA and (2) sequence-specific oligonucleotide DNA probes labeled with fluorescent reporters, which detect target sequences only after hybridization with their complementary sequences.
[0179] Each of the PCR reactions described above can be performed according to standard methods in the art, including those provided in laboratory manuals such as Sambrook, J., et al. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001; Spector, D. L. et al., Cells: A Laboratory Manual, Cold
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[0182] Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1998: Carruthers, W., and Coldham, I., Modern Methods of Organic Synthesis (4th Edition), Cambridge University Press, Cambridge, U. K., 2004, herein incorporated by reference in its entirety.
[0183] The disclosed methods can also include a step of sequencing nucleic acids. The nucleic acid substrate for sequencing can be the nucleic acids from the original sample and / or an amplified nucleic acid product thereof, e.g., produced by a PCR protocol. In various aspects, the analysis can include sequencing the nucleic acids using, but not limited to, Sanger sequencing, single molecule real-time (SMRT) sequencing, nanopore DNA sequencing, massively parallel signature sequencing (MPSS), colony sequencing, 454 pyrosequencing, Illumina sequencing, combinatorial probe anchor synthesis (cPAS), SOLiD sequencing, ion torrent semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, or a combination of any thereof, optionally using a microfluidic system. In some forms, the sequencing is or includes Illumina, Ion Torrent, PacBio, or Oxford Nanopore technology, or any similar next-generation sequencing platforms.
[0184] For example, oxford nanopore sequencing technology used in Nanopore sequencing is the third generation single-molecule sequencing technology, which has the advantages of simple sample handling, fast and long sequencing length (>10 kbp) compared with NGS second-generation technology that utilizes amplified signals. Oxford Nanopore sequencing is an emerging third-generation sequencing technology that can generate ultra-long reads exceeding 800 kb (Iain et al., Nat Biotechnol 36, 338-345, doi:10.1038 / nbt.4060 (2018)) in a portable device called MinlON. These long reads come without much compromise on reads consensus accuracy since the sequencing errors are mostly random (Loman et al., Nat Methods 12, 733-U751, doi:10.1038 / Nmeth.3444 (2015)). They hold great promise in calling and phasing variants, assembling scaffolds, and prospectively detecting epigenetic marks (Cretu et al., Nat Commun 8, 1326, doi:10.1038 / s41467-017-01343-4 (2017), Simpson et al., Nat Methods 14, 407-410, doi:10.1038 / nmeth.4184 (2017)).
[0185] In still further forms, the analysis can include qualitative detection, such as using an agarose gel, polyacrylamide electrophoresis, restriction endonuclease digestion, dot blots, liquid chromatography, electrochemoluminescence, or a combination of any thereof. In general, any method known in the art to sequence, quantify, or detect nucleic acid may be used in the methods provided herein. In any aspect provided herein, identifying the nucleic acid as indicative of the presence of the biomarkers is contemplated.
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[0188] Alternatively, the level of the expression biomarker is determined by measuring the amount of protein expressed from a corresponding gene encoding the biomarker. Assays for qualitatively and / or quantitatively detecting proteins are described as follows.
[0189] Traditional immunoassays including, for example, sandwich immunoassays including ELISA or fluorescence-based immunoassays, as well as other enzyme immunoassays can be used for detecting protein biomarkers. In other forms, the detection of the protein biomarker is carried out on slides of test material (e.g., immunohistochemistry), Western blotting, surface plasmon resonance (e.g. Biacore), or by flow cytometry (FACS) analysis). Other specific examples include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA) and counting immunoassay (CIA), homogeneous enzyme-multiplied immunoassays (“EMIT”), apoenzyme reactivation immunoassay (“ARIS”), dipstick immunoassays, and immuno-chromatography assays. Most assays now use nonradioactive labels. Enzyme immunoassays (enzyme-linked immunosorbent assays, or ELISA; immunometric assays) can use enzymes as labels, such as, for example, horseradish peroxidase or alkaline phosphatase. Chemiluminescent immunoassays (CIA) can use luminol. Fluorimetric immunoassays (FIA) use fluorescent compounds (e.g., fluorescein) as labels.
[0190] The protein detection assays can be homogenous or heterogeneous assays, competitive and non-competitive assays. There are four main kinds of ELISA: sandwich, competitive, direct, and indirect assays. These methods differ in how the antibody or antigen is attached to the solid plate, and how the signal is detected. For example, in a sandwich ELISA, for example, an antibody is immobilized on a plate. The sample containing the target antigen is added, which binds to the antibody and so is immobilized on the plate. Next, a second type of antibody is added, which also binds to the target antigen on the plate, forming a ‘sandwich’ with the target antigen in the middle. The second antibody is linked to an enzyme, called a reporter enzyme, which allows the binding reaction to be measured by creating a color signal. To create this signal, first any unbound antibody is washed away, and a colorimetric substrate is added. The enzyme catalyzes a reaction of the substrate, creating a color change. A stronger color signal indicates more target antigen is present. An example of this is a home pregnancy test.
[0191] In some forms, the assay is in the form of a sandwich assay, which is a noncompetitive immunoassay, wherein the molecule to be detected and / or quantified is bound to a first antibody and to a second antibody. The first antibody may be bound to a solid
[0192] 25
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[0194] phase, e.g., a bead, a surface of a well or other container, a chip or a strip, and the second antibody is an antibody which is labeled, e.g. with a dye, with a radioisotope, or a reactive or catalytically active moiety. The amount of labeled antibody bound to the analyte is then measured by an appropriate method. The general composition and procedures involved with “sandwich assays” are well-established and known to the skilled person.
[0195] Immunohistochemistry (IHC) is a process of localizing antigens (e.g., proteins) in tissue utilizing antigen-specific antibodies. The antigen-binding antibody can be conjugated or fused to a tag that allows its detection, e.g., via visualization. In some forms, the tag is an enzyme that can catalyze a color-producing reaction, such as alkaline phosphatase or horseradish peroxidase. The enzyme can be fused to the antibody or non-covalently bound, e.g., using a biotin-avidin system. Alternatively, the antibody can be tagged with a fluorophore, such as fluorescein, rhodamine, DyLight Fluor or Alexa Fluor. The antigenbinding antibody can be directly tagged or it can itself be recognized by a detection antibody that carries the tag.
[0196] Western blotting can be used and can be quantitative or qualitative. A typical Western blotting procedure includes the steps of immunoprecipitating a target protein from a lysate of cells expressing the protein, performing an SDS-PAGE with said protein, transferring the protein to a nitrocellulose membrane, incubating the nitrocellulose membrane with said antibody, detecting said antibody with a secondary antibody conjugated to a fluorescent or chromogenic compound (e.g. peroxidases such as horseradish peroxidase (HRP), alkaline phosphatase (AP), IRDye near-infrared (NIR) fluorescent dyes), and quantifying the respective signal of said compound (e.g. fluorescence, luminescence, chromogenic enzyme substrate).
[0197] The ratio of two signals generated by Western blotting employing the same antibody but two different samples can be calculated, thereby determining how much more / less (foldchange) of the biomarker is present in one sample compared to another.
[0198] In some forms, the corresponding protein can be detected in the supernatant separated from the sample following centrifugation. In some forms, exosomes can be collected by an additional centrifugation step, and then be processed and evaluated for proteins corresponding to the expression biomarkers. Extracellular vesicles (EVs), e.g., exosomes, typically refer to lipid vesicles formed by cells or tissue. Extracellular vesicles, including exosomes, can be isolated using differential centrifugation, flotation density gradient
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[0201] centrifugation, filtration, high performance liquid chromatography, and immunoaffinitycapture.
[0202] For example, one of the most common isolation technique for isolating exosomes from a sample is differential centrifugation, whereby large particles and cell debris in the sample are separated using centrifugal force between 200-100, OOOxg and the exosomes are separated from supernatant by the sedimenting exosomes at about 100, OOOxg. Purity can be improved, however, by centrifuging the samples using flotation density gradient centrifugation with sucrose or Optiprep. Tangential flow filtration combined with deuterium / sucrose-based density gradient ultracentrifugation was employed to isolate therapeutic exosomes for clinical trials.
[0203] Ultrafiltration and high performance liquid chromatography (HPLC) are additional methods of isolating EVs based on their size differences. EVs prepared by HPLC are highly purified.
[0204] Other common techniques for EV collection involve positive and / or negative selection using affinity-based methodology. Antibodies can be immobilized in different media conditions and combined with magnetic beads, chromatographic matrix, plates, and microfluidic devices for separation. For example, antibodies against exosome-associated antigens — such as cluster of differentiation (CD) molecules CD74, CD3, CD 19, CD68 — can be used for affinity-based separation of exosomes. Non-exosome vesicles that carry these or different antigens can also be isolated in a similar way.
[0205] C. Diagnosis
[0206] 1. Single Markers
[0207] The biomarkers can be used in diagnostic tests to assess cancer and / or other biomarker-related disease and disorder status in a subject, e.g., to distinguish between normal cells and diseased cells, and disease status. For example, disease status includes, without limitation, the presence or absence of disease (e.g., cancer v. non-cancer), characterization of cells including cancer or other cells (e.g., level of aberrant biomarker activity), the risk of developing disease, the stage of the disease (e.g., non-invasive or early-stage cancer v. invasive or metastatic cancer), the progress of disease (e.g., progress of disease or remission of disease over time) and the effectiveness or response to treatment of disease. Based on this status, further procedures may be indicated, including additional diagnostic tests or therapeutic procedures or regimens. Representative cancers and therapies are discussed in more detail below.
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[0209] 45836485.1ATTORNEY DOCKET NO. YU 8874 PCT
[0210] The biomarkers discussed herein can be present and / or expressed in cancer including but not limited to lung cancer, and, therefore, each is individually useful in aiding in the determination of cancer and / or other TAP pathway related diseases and disorders. The method involves, first, measuring the selected biomarker in a subject sample using the methods described herein, and, second, comparing the measurement with a diagnostic amount or cut-off that distinguishes a positive cancer and / or other biomarker-related disease and disorder status from a negative cancer and / or other biomarker-related disease and disorder status. The diagnostic amount represents a measured amount of a biomarker above which a subject is classified as having a particular status.
[0211] For example, because the TAPI and TAP2 are down-regulated compared to normal during cancer and / or other TAP signaling-related diseases and disorders in which the immune system is suppressed (e.g., infection), then a measured amount below the diagnostic cutoff provides a diagnosis or status of the cancer and / or other related diseases and disorders.
[0212] Because the SOCS1 and / or IL-4 may be up-regulated compared to normal during cancer and / or other TAP signaling-related diseases and disorders in which the immune system is suppressed (e.g., infection), then a measured amount above the diagnostic cutoff provides a diagnosis or status of the cancer and / or other related diseases and disorders.
[0213] Because the TAPI and TAP2 may be up-regulated compared to normal during autoimmune, inflammation, and other related diseases and disorders in which the immune system is overactive, then a measured amount above the diagnostic cutoff provides a diagnosis or status of the diseases and disorders.
[0214] Because the SOCS1 and / or IL-4 may be down- regulated compared to normal during autoimmune, inflammation, and other related diseases and disorders in which the immune system is overactive, then a measured amount below the diagnostic cutoff provides a diagnosis or status of the diseases and disorders.
[0215] As is well understood in the art, by adjusting the particular diagnostic cut-off used in an assay, one can increase sensitivity or specificity of the diagnostic assay depending on the preference of the diagnostician. The particular diagnostic cut-off can be determined, for example, by measuring the amount of the biomarker in a statistically significant number of samples from subjects with the different cancer statuses and drawing the cut-off to suit the diagnostician's desired levels of specificity and sensitivity.
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[0218] 2. Combinations of Markers
[0219] While individual biomarkers are useful diagnostic biomarkers, a combination of biomarkers may provide greater predictive value of a particular status than single biomarkers alone. Specifically, the detection of a plurality of biomarkers in a sample can increase the sensitivity and / or specificity of the test. Thus, in one form, two or more, three or more, or even all four of the biomarkers can be detected and used to assess the status of cancer and / or other biomarker-related disease and disorder in a subject.
[0220] D. Determining Risk of Developing Disease
[0221] Methods for determining the risk of developing disease in a subject are also provided. Biomarker amounts or patterns can be characteristic of various risk states, e.g., high, medium, or low. The risk of developing a disease is determined by measuring the relevant biomarker or biomarkers and then either submitting them to a classification algorithm or comparing them with a reference amount and / or pattern of biomarkers that is associated with the particular risk level.
[0222] E. Determining Stage of Disease
[0223] Another form provides methods for determining the stage of disease in a subject. Each stage of the disease can have a characteristic amount of a biomarker or relative amounts of a set of biomarkers (a pattern). The stage of a disease is determined by measuring the relevant biomarker or biomarkers and then either submitting them to a classification algorithm or comparing them with a reference amount and / or pattern of biomarkers that is associated with the particular stage.
[0224] F. Determining Course (Progression / Remission) of Disease
[0225] Still another form provides methods for determining the course of disease in a subject. Disease course refers to changes in disease status over time, including disease progression (worsening) and disease regression (improvement). Overtime, the amounts or relative amounts (e.g., the pattern) of the biomarker(s) changes. This method involves measuring one or more biomarkers in a subject at least two different time points, e.g., a first time and a second time, and comparing the change in amounts, if any. The course of disease is determined based on these comparisons. Similarly, this method is useful for determining the response to treatment. If a treatment is effective, then the biomarkers will trend toward normal, while if treatment is ineffective, the biomarkers will trend toward disease indications.
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[0228] G. Subject Management
[0229] In certain forms of the method including the detection and / or analysis of one or more biomarkers further include managing subject treatment based on the status. Such management includes the actions of the physician or clinician subsequent to determining cancer and / or other biomarker- related disease and disorder status. For example, if a physician makes a diagnosis of cancer and / or other biomarker-related disease and disorder, then a certain regime of treatment, such as prescription or administration of chemotherapy, radiation, immunotherapy, including, but not limited to administration of the compositions discussed in more detail below, might follow. Alternatively, a diagnosis of non-cancer or benign tumor might be followed with further testing to determine a specific disease that the patient might be suffering from. Also, if the diagnostic test gives an inconclusive result on cancer and / or other biomarker-related disease and disorder, further tests may be required.
[0230] One form provides a method for selecting a subject for treatment for cancer and / or other biomarker-related disease and disorder by detecting the presence or quantity of one or more biomarkers provided herein in a sample from a subject suspected of having cancer and / or other biomarker-related disease and disorder, comparing the levels of biomarker in the sample to a predetermined standard, wherein the patient is selected for treatment for cancer and / or other biomarker-related disease and disorder if certain biomarkers or levels of biomarkers are detected in the sample. Such treatments can be those known to be effective and / or preferred for treating subjects with aberrant biomarker-positive conditions.
[0231] In some forms, the methods additionally or alternatively include identifying the subject as not having a biomarker-related disease and disorder, when the test is negative. Thus, although the subject may have a cancer or another disease or disorder, the subject can be identified as negative for aberrant biomarker-related cancer and other diseases and disorder. Such forms may lead to selection of alternative treatments and may avoid treatments known to be effective or preferred for treating subjects with aberrant biomarkerpositive conditions, and / or may include treatments that are known not to be effective and / or preferred for treating subjects with aberrant biomarker-positive conditions.
[0232] Additional forms relate to the communication of assay results or diagnoses or both to technicians, physicians or patients, for example. In certain forms, computers will be used to communicate assay results or diagnoses or both to interested parties, e.g.: physicians and their patients. In some forms, the assays will be performed or the assay results analyzed in a
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[0235] country or jurisdiction which differs from the country or jurisdiction to which the results or diagnoses are communicated.
[0236] In a preferred form a diagnosis based on the presence or absence in a test subject of any of the disclosed biomarkers is communicated to the subject as soon as possible after the diagnosis is obtained. The diagnosis may be communicated to the subject by the subject's treating physician. Alternatively, the diagnosis may be sent to a test subject by email or communicated to the subject by phone. A computer may be used to communicate the diagnosis by email or phone. In certain forms, the message containing results of a diagnostic test may be generated and delivered automatically to the subject using a combination of computer hardware and software which will be familiar to artisans skilled in telecommunications. In certain forms all or some of the method steps, including the assaying of samples, diagnosing of diseases, and communicating of assay results or diagnoses, may be carried out in diverse (e.g., foreign) jurisdictions.
[0237] H. Biomarkers in Screening Assays
[0238] The biomarkers can be used to screen for compounds that modulate the expression of the biomarkers in vitro or in vivo, which compounds in turn may be useful in treating or preventing cancer and / or other biomarker-related disease or disorder in patients. Compounds suitable for therapeutic testing may be screened initially by identifying compounds which reduce the presence of one or more biomarkers in the cancer and / or other biomarker-related disease or disorder cells.
[0239] Test compounds capable of modulating the presence and / or expression of any of the biomarkers in cancer cells or other biomarker-related diseases and disorders may be administered to patients who are suffering from or are at risk of developing cancer or other disease or disorder having the biomarkers. For example, the administration of a test compound that decreases the activity of a particular biomarker may decrease the risk of cancer and / or other biomarker-related disease and disorder in a patient if the increased activity of the biomarker is responsible or indicative, at least in part, for the onset of the cancer and / or other biomarker-related disease and disorder.
[0240] At the clinical level, screening a test compound includes obtaining samples from test subjects before and after the subjects have been exposed to a test compound. The levels in the samples of one or more of the biomarkers can be measured and analyzed to determine whether the levels of the biomarkers change after exposure to a test compound. The samples can be analyzed by any appropriate means known to one of skill in the art including e.g., by
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[0243] the means described herein. In a further form, the changes in the level of expression of one or more of the biomarkers can be measured using in vitro methods and materials. For example, human tissue cultured cells which express, or are capable of expressing, one or more of the biomarkers can be contacted with test compounds. Subjects who have been treated with test compounds can be routinely examined for any physiological effects which may result from the treatment. In particular, the test compounds can be evaluated for their ability to decrease disease likelihood in a subject. Alternatively, if the test compounds are administered to subjects who have previously been diagnosed with cancer and / or other biomarker- related disease and disorder, test compounds will be screened for their ability to slow or stop the progression of the disease.
[0244] I. Assessing the Effectiveness of Treatment or Risk for Developing Cancer and / or other Biomarker-related Disease and Disorder
[0245] Methods for determining the course of cancer and / or other biomarker-related diseases and disorders in a subject are also provided. Disease course refers to changes in disease status over time, including disease progression (worsening) and disease regression (improvement). Over time, the amounts or relative amounts (e.g., the pattern) of the biomarkers changes. Accordingly, this method involves measuring one or more biomarkers in a subject at least two different time points, e.g., a first time and a second time, and comparing the change in amounts, if any. The course of disease is determined based on these comparisons. Similarly, this method is useful for determining the response to treatment. If a treatment is effective, then the biomarkers will trend toward normal, while if treatment is ineffective, the biomarkers will trend toward disease indications.
[0246] In yet another example, the biomarkers can be used in studies to determine if the subject is at risk for developing cancer and / or another biomarker-related disease or disorder.
[0247] III. Methods of Treatment
[0248] Methods of treating a subject in need thereof are also provided.
[0249] The methods can stand alone or be carried out in combination with any of the other methods provided herein, including, but not limited to the diagnostic and prognostic methods discussed above.
[0250] Results presented herein show that the epigenetically-mediated cancer cell downregulation of TAP2 in -42% of primary NSCLCs from large patient cohorts was strongly associated with reduced CD8+ tumor infiltrating lymphocytes (TILs) and worse outcomes after immunotherapy. The underlying mechanism involves both antigen display and antigen
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[0253] independent transcriptional adaptations, SOCS1 upregulation, accumulation of intranuclear peptides, IFNy resistance and reduced chemoattraction. Thus provided is an overarching mechanism that TAP2 downregulation or its loss or absence is a common and dominant mechanism of adaptive immune evasion in human cells acting through antigen-dependent and antigen-independent routes to support malignant cell survival and progression. Because the levels of TAP2 vary across different cell types and tissues, and in response to microenvironmental cues, it is believed that both cancer and / or non-cancer cells can be subject to these mechanisms.
[0254] Thus provided are methods and compositions for modulating a TAP2 signaling axis to increase or decrease immune and / or inflammatory responses.
[0255] The data presented in the experiments below also supports a conclusion that in addition to dysfunctional antigen presentation, reduced or absent TAP transport creates retrograde accumulation of charged peptides in the nucleus, altering the chromatin accessibility and leading to IFNy resistance and reduced T-cell chemoattraction. More specifically, and without being bound by theory, it is believed that the cellular state and acquisition of immunosuppressive properties can be modulated based on the retrograde accumulation of short intranuclear peptides and chromatin changes secondary to reduced ER transport under TAP2 downregulation or loss. This mechanism may reflect a general feedback loop that is corrupted by cancer cells to resist chronic IFNy exposure. More specifically, immune evasion is accomplished in a TAP2 deficient cancer cells by one, two, or all three of (1) presentation of fewer surface antigens, (2) INF gamma resistance, and (3) reduced chemoattraction of immune cells via downregulation of chemokines. It is believed both cancer and / or non-cancer cells can be subject to these mechanisms.
[0256] Thus, also provided are methods and compositions for modulating chromatin and / or gene expression, optionally for increasing and decreasing immune and inflammatory responses. Such methods typically include modulating the accumulation of short intranuclear peptides and / or chromatin changes, which may be secondary to and / or independent of loss of TAP2. Thus, target cells of these methods include not only those that express TAP2, but also those that are not expressing TAP2. In some forms, the compositions and methods change chromatin structure and / or gene expression, such as those involved in pro- and / or antiinflammatory pathways.
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[0259] For example, in some forms, reducing the number or bioavailability of basic peptides in the nucleus induces an immune or inflammatory response, optionally by increasing IFNy sensitivity and / or inducing expression of genes associated therewith.
[0260] Additionally or alternatively, in some forms increasing the presence or bioavailability of acidic peptides in the nucleus induces an immune or inflammatory response, optionally by increasing IFNy sensitivity and / or inducing expression of genes associated therewith.
[0261] In other forms, increasing the number or bioavailability of basic peptides in the nucleus reduces an immune or inflammatory response, optionally by increasing IFNy resistance and / or reducing expression of genes associated therewith.
[0262] Additionally or alternatively, in some forms reducing the presence or bioavailability of acidic peptides in the nucleus reduces an immune or inflammatory response, optionally by increasing IFNy resistance and / or reducing expression of genes associate therewith.
[0263] Without being bound by theory, it is believed that basic peptides in the nucleus can bind to chromatin, particularly in negatively charged regions such as the acidic patch of histones, and thereby repress gene expression. Similarly, acidic peptides may serve as a “molecular sink”, altering the local physico-chemical properties and shielding basic peptides in the nucleus from binding chromatin and / or may directly target other aspects of gene expression.
[0264] A. Treating Patients
[0265] 1. Increasing an Immune and / or Inflammatory Response In some forms, the subject is in need of an increase in expression of a TAP protein such as TAP2 and / or TAPI, and / or a decrease in expression of SOCS1 and / or IL-4, and / or a decrease in level or bioavailability of intranuclear basic peptides and / or an increase in the level or bioavailability of intracellular acidic peptides. Such forms include, e.g., a subject where an increased immune and / or inflammatory response is desired such as cancer or infection. Such subject can be administered an effective amount of a compound or composition that increases expression of a TAP protein such as TAP2 and / or TAPI, and / or decreases expression of SOCS1 and / or IL-4 and / or decreases the intranuclear level or bioavailability of basic peptides and / or increases the level or bioavailability of intranuclear acidic peptides in cancer or infected cells or other target cells in which an increased sensitivity to an immune and / or inflammatory response thereto is desired.
[0266] In some forms, the subject has cancer or an infection.
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[0269] a. Cancer
[0270] Thus, the disclosed methods can be used to treat, reduce, and / or prevent cancer in a subject. Therefore, the disclosed compounds or compositions can be administered in an effective amount to treat, reduce, and / or prevent cancer in a subject. The effective amount or therapeutically effective amount of the compound or composition to treat cancer or a tumor thereof is typically a dosage sufficient to reduce or prevent a least one symptom of the cancer, or to otherwise provide a desired pharmacologic and / or physiologic effect. The symptom may be physical, such as tumor burden, or biological such as reducing proliferation or increasing death of cancer cells. In some forms, the amount is effective to kill tumor cells or reduce or inhibit proliferation or metastasis of the tumor cells. In some forms, the amount is effective to reduce tumor burden. In some forms, the amount is effective to reduce or prevent at least one comorbidity of the cancer. Typically the compound or composition is effective to induce or increase an immune response against the cancer or a tumor thereof.
[0271] In a mature animal, a balance usually is maintained between cell renewal and cell death in most organs and tissues. The various types of mature cells in the body have a given life span; as these cells die, new cells are generated by the proliferation and differentiation of various types of stem cells. Under normal circumstances, the production of new cells is so regulated that the numbers of any particular type of cell remain constant. Occasionally, though, cells arise that are no longer responsive to normal growth-control mechanisms. These cells give rise to clones of cells that can expand to a considerable size, producing a tumor or neoplasm. A tumor that is not capable of indefinite growth and does not invade the healthy surrounding tissue extensively is benign. A tumor that continues to grow and becomes progressively invasive is malignant. The term cancer typically refers to a malignant tumor. In addition to uncontrolled growth, malignant tumors exhibit metastasis. In this process, small clusters of cancerous cells dislodge from a tumor, invade the blood or lymphatic vessels, and are carried to other tissues, where they continue to proliferate. In this way a primary tumor at one site can give rise to a secondary tumor at another site.
[0272] The compositions and methods described herein are useful for treating subjects having benign or malignant tumors by delaying or inhibiting the growth of a tumor in a subject, reducing the growth or size of the tumor, inhibiting or reducing metastasis of the tumor, and / or inhibiting or reducing symptoms associated with tumor development or growth.
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[0275] Malignant tumors which may be treated can be classified according to the embryonic origin of the tissue from which the tumor is derived. Carcinomas are tumors arising from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs and glands. The disclosed compositions are particularly effective in treating carcinomas.
[0276] Sarcomas, which arise less frequently, are derived from mesodermal connective tissues such as bone, fat, and cartilage. The leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone marrow. Leukemias proliferate as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors may show up at numerous organs or tissues of the body to establish a cancer.
[0277] The disclosed compounds and compositions can be used to treat cells undergoing unregulated growth, invasion, or metastasis.
[0278] A representative but non-limiting list of cancers that the compounds and compositions can be used to treat include cancers of the blood and lymphatic system (including leukemias, Hodgkin’s lymphomas, non-Hodgkin’s lymphomas, solitary plasmacytoma, multiple myeloma), cancers of the genitourinary system (including prostate cancer, bladder cancer, renal cancer, urethral cancer, penile cancer, testicular cancer,), cancers of the nervous system (including mengiomas, gliomas, glioblastomas, ependymomas) cancers of the head and neck (including squamous cell carcinomas of the oral cavity, nasal cavity, nasopharyngeal cavity, oropharyngeal cavity, larynx, and paranasal sinuses), lung cancers (including small cell and non-small cell lung cancer), gynecologic cancers (including cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer ovarian and fallopian tube cancer), gastrointestinal cancers (including gastric, small bowel, colorectal, liver, hepatobiliary, and pancreatic cancers), skin cancers (including melanoma, squamous cell carcinomas, and basal cell carcinomas), breast cancer (including ductal and lobular cancer and triple negative breast cancers), and pediatric cancers (including neuroblastoma, Ewing’s sarcoma, Wilms tumor, medulloblastoma). Accordingly, in some forms, the present disclosure relates to a method of treating breast, ovarian, colon, prostate, lung, brain, skin, liver, stomach, pancreatic or blood based cancer.
[0279] In some forms, the lung cancer is Non-Small Cell Lung Cancer (NSCLC).
[0280] b. Infections
[0281] The disclosed methods can also be used to treat, reduce, and / or prevent an infection in a subject. Therefore, the disclosed compounds or compositions can be administered in an effective amount to treat, reduce, and / or prevent an infection in a subject. The effective
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[0284] amount or therapeutically effective amount of the compound or composition to treat an infection is typically a dosage sufficient to reduce or prevent a least one symptom of the infection, or to otherwise provide a desired pharmacologic and / or physiologic effect.
[0285] Typically the compound or composition is effective to induce or increase an immune response against the infection or causative agent thereof.
[0286] Stimulating an immune response in a host is desirable, for example, when the host suffers from a viral infection, bacterial infection, fungal, or protozoa infection.
[0287] Representative infections that can be treated, include but are not limited to infections cause by microoganisms including, but not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type B (HIB), Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and Treponema, Vibrio, Yersinia, Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosoma mansoni.
[0288] Representative viruses that can be treated include, but are not limited to, Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., Coronavirus, such as severe acute respiratory syndrome (SARS) virus, and SARS-CoV-2), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strain)), Flaviviridae, e.g., Hepatitis C virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, and Dengue virus 4), Hepadnaviridae, Herpesviridae (e.g., Human herpesvirus 1, 3, 4, 5, and 6, and Cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g., Influenzavirus A and B and C), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), Parvoviridae, Picornaviridae
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[0291] (e.g., poliovirus, rhinovirus, hepatovirus, and aphthovirus), Poxviridae (e.g., vaccinia and smallpox virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentivirus, such as human immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae (for example, rabies virus, measles virus, respiratory syncytial virus, etc.), Togaviridae (for example, rubella virus, dengue virus, etc.), and Totiviridae.
[0292] Representative parasites that can be treated include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosoma mansoni.
[0293] 2. Reducing an Immune and / or Inflammatory Response In some forms, the subject is in need of a decrease in expression and / or levels of a TAP protein such as TAP2 and / or TAPI, and / or an increase in expression and / or levels of SOCS1 and / or IL-4, and / or an increase in the level or bioavailability of intranuclear basic peptides and / or decrease in the levels or bioavailability of intranuclear acidic peptides. Such forms include, e.g., subject wherein a reduced immune response is desired, such where the subject as autoimmunity or an inflammatory disease or disorder, or a transplant. Such subjects can be administered an effective amount of a compound or composition that reduces expression and / or levels of a TAP protein such as TAP2 and / or TAPI, and / or increases expression and / or levels of SOCS1 and / or IL-4 and / or increases the level or bioavailability of intranuclear basic peptides and / or decreases the level or bioavailability of intranuclear acidic peptides in autoimmune and / or transplanted cells / tissue or other target cells in which an reduced sensitivity to an immune and / or inflammatory response thereto is desired.
[0294] a. Inflammatory and Autoimmune Diseases Representative inflammatory responses or autoimmune diseases that can be treated according to the disclosed methods include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, anklosing spondylitis, antiphospholipid syndrome, autoimmune Addison’ s disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (alps), autoimmune thrombocytopenic purpura (ATP), Behcet’s disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome immune deficiency, syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial
[0295] 38
[0296] 45836485.1ATTORNEY DOCKET NO. YU 8874 PCT
[0297] pemphigoid, cold agglutinin disease, Crest syndrome, Crohn’s disease, Dego’s disease, dermatomyositis, dermatomyositis - juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia - fibromyositis, grave’s disease, guillain-barre, hashimoto’s thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), Iga nephropathy, insulin dependent diabetes (Type I), juvenile arthritis, Meniere’s disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglancular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud’s phenomenon, Reiter’s syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren’s syndrome, stiff-man syndrome, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener’s granulomatosis.
[0298] b. Transplant Rejection
[0299] In some forms, the compounds or compositions are administered in an effective amount of reduce or prevent transplant rejection. The transplanted material can be cells, tissues, organs, limbs, digits or a portion of the body, preferably the human body. The transplants are typically allogenic or xenogenic. The disclosed compounds and compositions are administered to a subject in an effective amount to reduce or inhibit transplant rejection. Compounds and compositions can be administered systemically or locally by any acceptable route of administration. In some forms, the compounds and compositions are administered to a site of transplantation prior to, at the time of, or following transplantation. In one form, the compound or composition is administered to a site of transplantation parenterally, such as by subcutaneous injection.
[0300] In other forms, the compound or composition is administered directly to cells, tissue or organ to be transplanted ex vivo. In one form, the transplant material is contacted with a compound or composition prior to transplantation, after transplantation, or both.
[0301] In other forms, the compound or composition is administered to immune tissues or organs, such as lymph nodes or the spleen.
[0302] The transplant material can be modified prior to transplant. For example, the transplant material can be genetically modified to express a protein that aids in the inhibition or reduction of transplant rejection. In some forms, the transplant material is genetically modified to a disclosed compound or composition in an amount effective to inhibit or reduce transplant rejection in a transplant recipient.
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[0304] 45836485.1ATTORNEY DOCKET NO. YU 8874 PCT
[0305] The transplant material can be treated with enzymes or other materials that remove cell surface proteins, carbohydrates, or lipids that are known or suspected in being involved with immune responses such as transplant rejection.
[0306] In some forms, the transplant material is cells, tissue, and / or organs. Populations of any types of cells can be transplanted into a subject. The cells can be homogenous or heterogenous. Heterogeneous means the cell population contains more than one type of cell. Exemplary cells include progenitor cells such as stem cells and pluripotent cells which can be harvested from a donor and transplanted into a subject. The cells are optionally treated prior to transplantation as mention above. Such treatment includes transfecting the cells ex vivo with a nucleic acid construct enabling the cells to express a disclosed compound or composition in vitro and in vivo. Methods for transfecting cells are well known in the art.
[0307] Ex vivo methods of nucleic acid delivery can include, for example, the steps of harvesting cells from a subject, culturing the cells, transducing them with an expression vector, and maintaining the cells under conditions suitable for expression of the encoded polypeptides. These methods are known in the art of molecular biology. An exemplary nucleic acid vector includes but is not limited to an adenoviral vector. The transduction step can be accomplished by any standard means used for ex vivo gene therapy, including, for example, calcium phosphate, lipofection, electroporation, viral infection, and biolistic gene transfer. Alternatively, liposomes or polymeric microparticles can be used. Cells that have been successfully transduced then can be selected, for example, for expression of the coding sequence or of a drug resistance gene. The cells then can be lethally irradiated (if desired) and injected or implanted into the subject. Other exemplary cells that can be transplanted include, but are not limited to, islet cells, hematopoietic cells, muscle cells, cardiac cells, neural cells, embryonic stem cells, adult stem cells, T cells, lymphocytes, dermal cells, mesoderm, endoderm, and ectoderm cells.
[0308] Any tissue can be used as a transplant. Exemplary tissues include skin, adipose tissue, cardiovascular tissue such as veins, arteries, capillaries, valves: neural tissue, bone marrow, pulmonary tissue, ocular tissue such as corneas and lens, cartilage, bone, and mucosal tissue. The tissue can be modified as discussed above.
[0309] Exemplary organs that can be used for transplant include, but are not limited to kidney, liver, heart, spleen, bladder, lung, stomach, eye, tongue, pancreas, intestine, etc. The organ to be transplanted can also be modified prior to transplantation as discussed above.
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[0312] One form provides a method of inhibiting or reducing chronic transplant rejection in a subject by administering an effective amount of a disclosed compound or composition to inhibit or reduce chronic transplant rejection relative to a control.
[0313] c. Graft- versus-host disease (GVHD)
[0314] The disclosed compounds and compositions also be used to treat graft- versus-host disease (GVHD) by administering an effective amount of the compound or composition to alleviate one or more symptoms associated with GVHD. GVHD is a major complication associated with allogeneic hematopoietic stem cell transplantation in which functional immune cells in the transplanted marrow recognize the recipient as “foreign” and mount an immunologic attack. It can also take place in a blood transfusion under certain circumstances. Symptoms of GVD include skin rash or change in skin color or texture, diarrhea, nausea, abnormal liver function, yellowing of the skin, increased susceptibility to infection, dry, irritated eyes, and sensitive or dry mouth.
[0315] d. Diabetes
[0316] The disclosed compounds and compositions can also be used to treat diabetes. The method includes transplanting insulin producing cells in a subject and administering to the subject an effective amount of a disclosed compound or composition to reduce or inhibit transplant rejection. Preferably the insulin producing cells are beta cells or islet cells. In certain forms, the insulin producing cells are recombinant cells engineered to produce insulin. The insulin producing cells may also be genetically modified to produce the compound or composition, as described herein.
[0317] The insulin producing cells can be encapsulated within a matrix, such as a polymeric matrix, using suitable polymers, including, but not limited to alginate, agarose, hyaluronic acid, collagen, synthetic monomers, albumin, fibrinogen, fibronectin, vitronectin, laminin, dextran, dextran sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, chitin, chitosan, heparan, heparan sulfate, or a combination thereof.
[0318] B. Compositions
[0319] Exemplary compounds that can modulate (i.e., increase or decrease) expression or bioactivity of TAP2, TAPI, SOCS1, and / or IL-4 provided and discussed below and exemplified in the working examples. Also provided are compounds that can modulate the levels and / or biophysical properties, e.g., basic and / or acidic, of peptides in target cells. See, e.g., Figures 7A-7O. Any of the compounds discussed anyway herein including in the
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[0321] 45836485.1ATTORNEY DOCKET NO. YU 8874 PCT
[0322] experiments below and their associate figures, including but not limited to Figures 7A-7O, can be used in the disclosed methods.
[0323] 1. Increased Expression
[0324] In some forms, a compound that increases expression and / or levels of a protein such as TAP2, TAPI, SOCS1, or IL-4 is the protein itself (e.g., protein therapy) or a nucleic acid encoding the protein. In some forms, the compound is a transcription factor that increases expression of the TAP2, TAPI, SOCS 1 or IL-4. Such compounds can be, e.g., a small molecule, a nucleic acid, a protein, or a nucleic acid encoding the protein.
[0325] Sequences for TAP2, TAPI, SOCSL and IL-4 are known in the art. Exemplary, nonlimiting sequences are provided as SEQ ID NOS:31-34, respectively. Thus, in some forms, protein therapy includes delivery and / or expression of one or more of the following proteins, or functional fragments or variants thereof with at least 70, 75, 80, 85, 90, or 95 sequence identity thereto, or a nucleic acid encoding any of the foregoing:
[0326] MRLPDLRPWTSLLLVDAALLWLLQGPLGTLLPQGLPGLWLEGTLRLGGLWGLLKLRGLLGFV GTLLLPLCLATPLTVSLRALVAGASRAPPARVASAPWSWLLVGYGAAGLSWSLWAVLSPPGA QEKEQDQVNNKVLMWRLLKLSRPDLPLLVAAFFFLVLAVLGETLIPHYSGRVIDILGGDFDP HAFASAIFFMCLFSFGSSLSAGCRGGCFTYTMSRINLRIREQLFSSLLRQDLGFFQETKTGE LNSRLSSDTTLMSNWLPLNANVLLRSLVKVVGLYGFMLSISPRLTLLSLLHMPFTIAAEKVY NTRHQEVLRE IQDAVARAGQWREAVGGLQTVRSFGAEEHEVCRYKEALEQCRQLYWRRDLE RALYLLVRRVLHLGVQMLMLSCGLQQMQDGELTQGSLLSFMIYQESVGSYVQTLVYIYGDML SNVGAAEKVFSYMDRQPNLPSPGTLAPTTLQGWKFQDVSFAYPNRPDRPVLKGLTFTLRPG EVTALVGPNGSGKSTVAALLQNLYQPTGGQVLLDEKPISQYEHCYLHSQWSVGQEPVLFSG SVRNNIAYGLQSCEDDKVMAAAQAAHADDF IQEMEHGI YTDVGEKGSQLAAGQKQRLAIARA LVRDPRVLILDEATSALDVQCEQALQDWNSRGDRTVLVIAHRLQTVQRAHQILVLQEGKLQK LAQL (SEQ ID NO:31, Q03519 · TAP2_HUMAN)
[0327] MASSRCPAPRGCRCLPGASLAWLGTVLLLLADWVLLRTALPRIFSLLVPTALPLLRVWAVGL SRWAVLWLGACGVLRATVGSKSENAGAQGWLAALKPLAAALGLALPGLALFRELISWGAPGS ADSTRLLHWGSHPTAFWSYAAALPAAALWHKLGSLWVPGGQGGSGNPVRRLLGCLGSETRR LSLFLVLWLSSLGEMAIPFFTGRLTDWILQDGSADTFTRNLTLMS ILTIASAVLEFVGDGI YNNTMGHVHSHLQGEVFGAVLRQETEFFQQNQTGNIMSRVTEDTSTLSDSLSENLSLFLWYL VRGLCLLGIMLWGSVSLTMVTLITLPLLFLLPKKVGKWYQLLEVQVRESLAKSSQVAIEALS AMPTVRSFANEEGEAQKFREKLQEIKTLNQKEAVAYAVNSWTTSISGMLLKVGILYIGGQLV
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[0330] TSGAVSSGNLVTFVLYQMQFTQAVEVLLSIYPRVQKAVGSSEKIFEYLDRTPRCPPSGLLTP LHLEGLVQFQDVSFAYPNRPDVLVLQGLTFTLRPGEVTALVGPNGSGKSTVAALLQNLYQPT GGQLLLDGKPLPQYEHRYLHRQVAAVGQEPQVFGRSLQENIAYGLTQKPTMEEITAAAVKSG AHSFISGLPQGYDTEVDEAGSQLSGGQRQAVALARALIRKPCVLILDDATSALDANSQLQVE QLLYESPERYSRSVLLITQHLSLVEQADHILFLEGGAIREGGTHQQLMEKKGCYWAMVQAPA DAPE (SEQ ID NO:32, Q03518 · TAP1_HUMAN)
[0331] MVAHNQVAADNAVSTAAEPRRRPEPSSSSSSSPAAPARPRPCPAVPAPAPGDTHFRTFRSHA DYRRITRASALLDACGFYWGPLSVHGAHERLRAEPVGTFLVRDSRQRNCFFALSVKMASGPT SIRVHFQAGRFHLDGSRESFDCLFELLEHYVAAPRRMLGAPLRQRRVRPLQELCRQRIVATV GRENLARIPLNPVLRDYLSSFPFQI (SEQ ID NO:33, O15524 · SOCS1_HUMAN)
[0332] MGLTSQLLPPLFFLLACAGNFVHGHKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKN TTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCP VKEANQSTLENFLERLKTIMREKYSKCSS (SEQ ID NO:34, P05112 · IL4_HUMAN)
[0333] In some forms, a compound that increases TAP2 expression and / or levels is a small molecule. Exemplary small molecules that increase expression of TAP2 are discussed in the experiments below and their associate figures, and any such compounds can be used in the disclosed methods. Exemplary preferred compounds include, but are not limited to, Dasatinib, Vinorelbine, Docetaxel, Vinblastine, Vindesine and Mitomycin; the anti-inflammatory / anti-gout agent Colchicine, the cardiac glycosides Digoxin and Ouabain; as well as the anti-parasitic / anti-fungal agents Pyrithione Zinc, Oxibendazole, Albendazole, Fenbendazole, Podofilox, Gentian and Emetine; the non-selective HDAC inhibitor Oxamflatin, the HDAC6 inhibitor BML-281, and the class-I / IIDAC6 inhibitor SAHA / Vorinostat.
[0334] 2. Decreased Expression
[0335] In some forms, a compound that decreases expression and / or levels of a protein such as TAP2, TAPI, SOCS1, or IL-4 is an inhibitory polypeptide; a small molecule or peptidomimedic, or an inhibitory nucleic acid that targets genomic or expressed nucleic acids (e.g., TAP2, TAPI, SOCS1, IL-4 mRNA), or a vector that encodes an inhibitory nucleic acid. The compound can reduce the expression or bioavailability of TAP2, TAPI, SOCS1 or IL-4. TAP2, TAPI, SOCS1, or IL-4 inhibition can be competitive, non-competitive, uncompetitive,
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[0338] or product inhibition. Thus, an TAP2, TAPI, SOCS 1, or IL-4 inhibitor can directly inhibit TAP2, TAPI, SOCS1, or IL-4; a TAP2, TAPI, SOCS1, or IL-4 inhibitor can inhibit another factor in a pathway that leads to induction, persistence, or amplification of TAP2, TAPI, SOCS1, or IL-4 expression; or a combination thereof.
[0339] In some forms, a compound that reduces TAP2 expression and / or levels is a small molecule. Exemplary compounds that reduce expression of TAP2 are discussed in the experiments below and their associate figures, and any such compounds can be used in the disclosed methods. Exemplary preferred compounds include, but are not limited to, aminopterin, pemetrexed, dinitolmide, and amprolium. See, e.g., Figure 7L. Also provided are the results of a bioinformatics analysis including the identification of pathway enrichment based on signature genes of the proteins targeted the positive hits, drugs associated with modulation thereof, and drug classifications by function. See, e.g., Figures 7M-7O.
[0340] The inhibitor can be a functional nucleic acid. Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction. As discussed in more detail below, functional nucleic acid molecules can be divided into the following non-limiting categories: antisense molecules, siRNA, miRNA, aptamers, ribozymes, triplex forming molecules, RNAi, and external guide sequences. The functional nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.
[0341] Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functional nucleic acids can interact with the mRNA or the genomic DNA of a target polypeptide or they can interact with the polypeptide itself. Often functional nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other situations, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place.
[0342] Therefore, the compositions can include one or more functional nucleic acids designed to reduce expression of the TAP2, TAPI, SOCS1, or IL-4 gene, or a gene product thereof. For example, the functional nucleic acid or polypeptide can be designed to target and reduce or inhibit expression or translation of TAP2, TAPI, SOCS 1, or IL-4 mRNA; or to
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[0344] 45836485.1ATTORNEY DOCKET NO. YU 8874 PCT
[0345] reduce or inhibit expression, reduce activity, or increase degradation of TAP2, TAPI, SOCS1, or IL-4 protein. In some forms, the composition includes a vector suitable for in vivo expression of the functional nucleic acid.
[0346] In some forms, the protein(s) targeted for decreased expression includes the amino acid sequence of any one of SEQ ID NOS:31-34, or functional fragments or variants thereof with at least 70, 75, 80, 85, 90, or 95 sequence identity thereto.
[0347] a. Antisense
[0348] The functional nucleic acids can be antisense molecules. Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAse H mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. There are numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule. Exemplary methods include in vitro selection experiments and DNA modification studies using DMS and DEPC. It is preferred that antisense molecules bind the target molecule with a dissociation constant (Kd) less than or equal to 10’6, 10‘8, 10’10, or 10‘12.
[0349] b. Aptamers
[0350] The functional nucleic acids can be aptamers. Aptamers are molecules that interact with a target molecule, preferably in a specific way. Typically aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind small molecules, such as ATP and theophiline, as well as large molecules, such as reverse transcriptase and thrombin. Aptamers can bind very tightly with Kd’s from the target molecule of less than 10’12M. It is preferred that the aptamers bind the target molecule with a Kd less than 1 O’6,
[0351] 1 O’8, 1 O’10, or 10’12. Aptamers can bind the target molecule with a very high degree of specificity. For example, aptamers have been isolated that have greater than a 10,000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule. It is preferred that the aptamer have a Kd with the target molecule at least 10, 100, 1000, 10,000, or 100,000 fold lower than the Kd with a
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[0354] background binding molecule. It is preferred when doing the comparison for a molecule such as a polypeptide, that the background molecule be a different polypeptide.
[0355] c. Ribozymes
[0356] The functional nucleic acids can be ribozymes. Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. It is preferred that the ribozymes catalyze intermolecular reactions. There are a number of different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes. There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo. Preferred ribozymes cleave RNA or DNA substrates, and more preferably cleave RNA substrates. Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions. This property makes ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence.
[0357] d. Triplex Forming Oligonucleotides
[0358] The functional nucleic acids can be triplex forming molecules. Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed in which there are three strands of DNA forming a complex dependent on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a Kd less than 10-6, 10-8, 10-10, or 10-12.
[0359] e. External Guide Sequences
[0360] The functional nucleic acids can be external guide sequences. External guide sequences (EGSs) are molecules that bind a target nucleic acid molecule forming a complex, which is recognized by RNase P, which then cleaves the target molecule. EGSs can be designed to specifically target a RNA molecule of choice. RNAse P aids in processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA: EGS complex to mimic the natural tRNA substrate. Similarly, eukaryotic EGS / RNAse P-directed cleavage of RNA can
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[0363] be utilized to cleave desired targets within eukarotic cells. Representative examples of how to make and use EGS molecules to facilitate cleavage of a variety of different target molecules are known in the art.
[0364] f. RNA Interference
[0365] In some forms, the functional nucleic acids induce gene silencing through RNA interference. Gene expression can also be effectively silenced in a highly specific manner through RNA interference (RNAi). This silencing was originally observed with the addition of double stranded RNA (dsRNA) (Fire, et al. (1998) Nature, 391:806-11; Napoli, etal. (1990) Plant Cell 2:279-89; Hannon, (2002) Nature, 418:244-51). Once dsRNA enters a cell, it is cleaved by an RNase III -like enzyme, Dicer, into double stranded small interfering RNAs (siRNA) 21-23 nucleotides in length that contains 2 nucleotide overhangs on the 3’ ends (Elbashir, etal. (2001) Genes Dev., 15:188-200; Bernstein, et al. (2001) Nature, 409:363-6; Hammond, et al. (2000) Nature, 404:293-6). In an ATP dependent step, the siRNAs become integrated into a multi-subunit protein complex, commonly known as the RNAi induced silencing complex (RISC), which guides the siRNAs to the target RNA sequence (Nykanen, et al. (2001) Cell, 107:309-21). At some point the siRNA duplex unwinds, and it appears that the antisense strand remains bound to RISC and directs degradation of the complementary mRNA sequence by a combination of endo and exonucleases (Martinez, et al. (2002) Cell, 110:563-74). However, the effect of iRNA or siRNA or their use is not limited to any type of mechanism.
[0366] Short Interfering RNA (siRNA) is a double-stranded RNA that can induce sequencespecific post-transcriptional gene silencing, thereby decreasing or even inhibiting gene expression. In one example, a siRNA triggers the specific degradation of homologous RNA molecules, such as mRNAs, within the region of sequence identity between both the siRNA and the target RNA. For example, WO 02 / 44321 discloses siRNAs capable of sequencespecific degradation of target mRNAs when base-paired with 3’ overhanging ends, herein incorporated by reference for the method of making these siRNAs.
[0367] Sequence specific gene silencing can be achieved in mammalian cells using synthetic, short double-stranded RNAs that mimic the siRNAs produced by the enzyme dicer (Elbashir, et al. (2001) Nature, 411:494498) (Ui-Tei, et al. (2000) FEBS Lett 479:79-82). siRNA can be chemically or in vz'tro-synthesized or can be the result of short double- stranded hairpinlike RNAs (shRNAs) that are processed into siRNAs inside the cell. Synthetic siRNAs are generally designed using algorithms and a conventional DNA / RNA synthesizer. Suppliers
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[0370] include Ambion (Austin, Texas), ChemGenes (Ashland, Massachusetts), Dharmacon (Lafayette, Colorado), Glen Research (Sterling, Virginia), MWB Biotech (Esbersberg, Germany), Proligo (Boulder, Colorado), and Qiagen (Vento, The Netherlands). siRNA can also be synthesized in vitro using kits such as Ambion’ s SILENCER® siRNA Construction Kit.
[0371] The production of siRNA from a vector is more commonly done through the transcription of a short hairpin RNAse (shRNAs). Kits for the production of vectors having shRNA are available, such as, for example, Imgenex’s GENESUPPRESSOR™ Construction Kits and Invitrogen’s BLOCK- IT™ inducible RNAi plasmid and lenti virus vectors.
[0372] In some form, the functional nucleic acid is siRNA, shRNA, miRNA. In some forms, the composition includes a vector expressing the functional nucleic acid. Methods of making and using vectors for in vivo expression of functional nucleic acids such as antisense oligonucleotides, siRNA, shRNA, miRNA, EGSs, ribozymes, and aptamers are known in the art.
[0373] g. Other Gene Editing Compositions
[0374] In some forms the functional nucleic acids are gene editing compositions. Gene editing compositions can include nucleic acids that encode an element or elements that induce a single or a double strand break in the target cell’s genome, and optionally a polynucleotide. The compositions can be used, for example, to reduce or otherwise modify expression of TAP2, TAPI, SOCS1, or IL-4.
[0375] Strand Break Inducing Elements
[0376] CRISPR / Cas
[0377] In some forms, the element that induces a single or a double strand break in the target cell’s genome is a CRISPR / Cas system. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an acronym for DNA loci that contain multiple, short, direct repetitions of base sequences. The prokaryotic CRISPR / Cas system has been adapted for use as gene editing (silencing, enhancing or changing specific genes) for use in eukaryotes (see, for example, Cong, Science, 15:339(6121):819-823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012)). By transfecting a cell with the required elements including a Cas gene and specifically designed CRISPRs, the organism's genome can be cut and modified at any desired location. Methods of preparing compositions for use in genome editing using the CRISPR / Cas systems are described in detail in WO 2013 / 176772 and WO 2014 / 018423, which are specifically incorporated by reference herein in their entireties.
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[0380] In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-matc sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. One or more tracr mate sequences operably linked to a guide sequence (e.g., direct repeat-spacer-direct repeat) can also be referred to as pre-crRNA (pre-CRISPR RNA) before processing or crRNA after processing by a nuclease.
[0381] In some forms, a tracrRNA and crRNA are linked and form a chimeric crRNA-tracrRNA hybrid where a mature crRNA is fused to a partial tracrRNA via a synthetic stem loop to mimic the natural crRNA:tracrRNA duplex as described in Cong, Science, 15:339(6121):819-823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012)). A single fused crRNA-tracrRNA construct can also be referred to as a guide RNA or gRNA (or singleguide RNA (sgRNA)). Within an sgRNA, the crRNA portion can be identified as the ‘target sequence’ and the tracrRNA is often referred to as the ‘scaffold’.
[0382] There are many resources available for helping practitioners determine suitable target sites once a desired DNA target sequence is identified. For example, numerous public resources, including a bioinformatically generated list of about 190,000 potential sgRNAs, targeting more than 40% of human exons, are available to aid practitioners in selecting target sites and designing the associate sgRNA to affect a nick or double strand break at the site. See also, crispr.u-psud.fr / , a tool designed to help scientists find CRISPR targeting sites in a wide range of species and generate the appropriate crRNA sequences.
[0383] In some forms, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a target cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. While the specifics can be varied in different engineered CRISPR systems, the overall methodology is similar. A practitioner interested in using CRISPR technology to target a DNA sequence can insert a short DNA fragment containing the target sequence into a guide RNA expression plasmid. The sgRNA expression plasmid contains the target sequence (about 20 nucleotides), a form of the tracrRNA sequence (the scaffold) as well as a suitable promoter and necessary elements for proper processing in eukaryotic cells. Such vectors are commercially available
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[0386] (see, for example, Addgene). Many of the systems rely on custom, complementary oligos that are annealed to form a double stranded DNA and then cloned into the sgRNA expression plasmid. Co-expression of the sgRNA and the appropriate Cas enzyme from the same or separate plasmids in transfected cells results in a single or double strand break (depending of the activity of the Cas enzyme) at the desired target site.
[0387] Zinc Finger Nucleases
[0388] In some forms, the element that induces a single or a double strand break in the target cell’s genome is a nucleic acid construct or constructs encoding a zinc finger nucleases (ZFNs). ZFNs are typically fusion proteins that include a DNA-binding domain derived from a zinc-finger protein linked to a cleavage domain.
[0389] The most common cleavage domain is the Type IIS enzyme Fokl. Fokl catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, U. S. Pat. Nos. 5,356,802; 5,436, 150 and 5,487,994; as well as Li et al. Proc., Natl. Acad. Set. USA 89 (1992):4275-4279; Li et al. Proc. Natl. Acad. Sci. USA, 90:2764-2768 (1993); Kim et al. Proc. Natl. Acad. Sci. USA. 91:883-887 (1994a); Kim et al. J. Biol. Chem. 269:31,978-31,982 (1994b). One or more of these enzymes (or enzymatically functional fragments thereof) can be used as a source of cleavage domains.
[0390] The DNA-binding domain, which can, in principle, be designed to target any genomic location of interest, can be a tandem array of CyS2His2 zinc fingers, each of which generally recognizes three to four nucleotides in the target DNA sequence. The Cys2His2 domain has a general structure: Phe (sometimes Tyr)-Cys-(2 to 4 amino acids)-Cys-(3 amino acids)-Phe(sometimes Tyr)-(5 amino acids)-Leu-(2 amino acids)-His-(3 amino acids)-His. By linking together multiple fingers (the number varies: three to six fingers have been used per monomer in published studies), ZFN pairs can be designed to bind to genomic sequences 18-36 nucleotides long.
[0391] Engineering methods include, but are not limited to, rational design and various types of empirical selection methods. Rational design includes, for example, using databases including triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U. S. Pat. Nos. 6, 140,081; 6,453,242; 6,534,261; 6,610,512; 6,746,838; 6,866,997; 7,067,617; U. S. Published Application Nos. 2002 / 0165356;
[0392] 50
[0393] 45836485.1ATTORNEY DOCKET NO. YU 8874 PCT
[0394] 2004 / 0197892; 2007 / 0154989; 2007 / 0213269; and International Patent Application Publication Nos. WO 98 / 53059 and WO 2003 / 016496.
[0395] Transcription Activator-Like Effector Nucleases In some forms, the clement that induces a single or a double strand break in the target cell’s genome is a nucleic acid construct or constructs encoding a transcription activator-like effector nuclease (TALEN). TALENs have an overall architecture similar to that of ZPNs, with the main difference that the DNA-binding domain comes from TAL effector proteins, transcription factors from plant pathogenic bacteria. The DNA-binding domain of a TALEN is a tandem array of amino acid repeats, each about 34 residues long. The repeats are very similar to each other; typically they differ principally at two positions (amino acids 12 and 13, called the repeat variable diresidue, or RVD). Each RVD specifies preferential binding to one of the four possible nucleotides, meaning that each TALEN repeat binds to a single base pair, though the NN RVD is known to bind adenines in addition to guanine. TAL effector DNA binding is mechanistically less well understood than that of zinc-finger proteins, but their seemingly simpler code could prove very beneficial for engineered-nuclease design. TALENs also cleave as dimers, have relatively long target sequences (the shortest reported so far binds 13 nucleotides per monomer) and appear to have less stringent requirements than ZFNs for the length of the spacer between binding sites. Monomeric and dimeric TALENs can include more than 10, more than 14, more than 20, or more than 24 repeats.
[0396] Methods of engineering TAL to bind to specific nucleic acids are described in Cermak, et al, Nucl. Acids Res. 1-11 (2011). US Published Application No. 2011 / 0145940, which discloses TAL effectors and methods of using them to modify DNA. Miller et al. Nature Biotechnol 29: 143 (2011) reported making TALENs for site-specific nuclease architecture by linking TAL truncation variants to the catalytic domain of Fokl nuclease. The resulting TALENs were shown to induce gene modification in immortalized human cells. General design principles for TALE binding domains can be found in, for example, WO 2011 / 072246.
[0397] 3. Modulation of Intracellular Peptides
[0398] a. Compounds for Targeting Microproteins and Defective Ribosomal Products (DRiPs)
[0399] A fraction of the cytosolic peptides forming the repertoire loaded onto HLAs for antigen presentation are derived from degradation of microproteins and DRiPs. Microproteins are usually <100 amino acid long peptides that can originate in traditional coding and
[0400] 51
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[0402] presumed non-coding regions of the genome, and may have roles in diseases such as cancer (PMID: 39379206). DRiPs originate from misfolded or mistranslated protein sequences, are expected to be rapidly degraded under physiological conditions and have roles in immunogenicity and disease (PMID: 21962745). Small molecules and antibiotics modulating protein synthesis such as Puromycin cause premature chain termination during translation, creating rapid increases in truncated, defective proteins that are immediately processed into peptides, and thus can be used for this purpose in the disclosed methods.
[0403] b. Minigene Constructs
[0404] Transient mRNA or DNA minigenes encoding peptide flanking regions can be used to preferentially generate and intracellularly increase the levels of antigenic peptides while avoiding broader, unwanted antigen complexity. These peptides could then accumulate in the nucleus and produce immunomodulatory effects.
[0405] Rationally designed basic and / or acid peptides and mimetics (discussed in more detail below) can also be used.
[0406] Thus, provided expression constructs for expressing basic and / or acid peptides for nuclear modulation. In some forms, the peptides are in range of 7-12, 7-11, 7-10, 7-9, 7-8, 8-12, 8-11, 8-10, 8-9, 9-12, 9-11, 9-10, 10-12, 10-11, or 11-12 amino acids, c.g., 7, 8, 9, 10, 11, or 12. The peptides can be a plurality of a single peptide (i.e., homogeneous), or can be mixture of different sizes and / or structures (i.e., heterogenous). Suitable constructs and formats for peptide expression are discussed below. In some forms, the compositions are designed to selectively produce 9-mer peptides with specific structures, polarities and charges to favor their intracellular transport and location / accumulation, particularly in the nucleus. In some forms, this strategy provides a means of bypassing proteasomal processing and TAP transport by delivering the specific peptide directly into the ER for MHC loading and / or nuclear accumulation.
[0407] c. Pharmacological Modulation of Trimming
[0408] Once intracellular peptides are inside the endoplasmic reticulum (ER), they are "trimmed" to the correct 9-amino acid length by enzymes like ERAP1 and ERAP2. Small molecule inhibitors or activators of ERAP can shift the peptide repertoire and modify their probability of being loaded and transported. Inhibiting ERAP1 can lead to the accumulation of longer precursors (10-15 mers), while overexpressing it can "over-trim" peptides, potentially revealing or destroying specific epitopes. In some forms, this strategy is used to modulate the size, type and localization of peptides within cell compartments. Thus provided
[0409] 52
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[0411] are ERAP1 and ERAP2 inhibitors and activators. The same or similar molecular strategies discussed above for modulating TAP2, TAPI, SOCS1 and IL-4 can be used, and thus are hereby incorporated by reference for modulating ERAP1 and / or ERAP2.
[0412] d. Enhancing Endogenous Processing (UPS Activation) Since the Ubiquitin-Proteasome System (UPS) is the primary source and engine for peptide generation, increasing its activity can boost production of peptides and enhance their nuclear accumulation. The use of strategies to accelerate protein degradation such as proinflammatory cytokines could increase the proteasomal activity to surpass antigen presentation gradient and favor the accumulation of nuclear peptides. This strategy could also be achieved by enhancing protein degradation of specific sequences using PROTACs, Degrons, and / or other alternative approaches, to ultimately create high localized concentrations of specific peptides with desired properties derived from specific proteins targeted for degradation.
[0413] e. Nanoparticles and Nanocarriers
[0414] Biodegradable nanoparticles, such as PLGA-based carriers, loaded with peptides could be used to modulate the intracellular and nuclear accumulation of desired peptides. Such nanoparticlcs could also contain additional compounds or signals to modulate proteosomal function and / or to direct them to the nucleus (or other compartments). Other suitable nanoparticles and nanocarriers are discussed in more detail below.
[0415] f. Mimetic peptides
[0416] Mimetic peptide therapy uses short chains of amino acids designed to mimic natural biological molecules, acting as targeted signaling agents to treat diseases. Both natural, recombinant, and synthetic mimetic peptides with desired physio-chemical properties (e.g. size, hydrophobicity, charge, pH etc.) (such as those discussed above with respect to minigene constructs and below with respect to exemplary peptides, and incorporated here by reference thereto) can be synthesized and delivered to cells to accumulate within specific cellular compartments such as the ER and / or nucleus (Zaman et al., Therapeutic peptides targeting intracellular molecules; European Polymer Journal, Volume 219, 16 October 2024, 113386), which is specifically incorporated by reference herein in its entirety.
[0417] g. Peptide scavengers
[0418] pH-responsive peptide scavengers can be designed to bind peptides preferentially within the acidic intracellular compartments and / or milieu of inflamed, uninflamed or normal tissues while remaining relatively inactive elsewhere. Although many pathogenic peptides are
[0419] 53
[0420] 45836485.1ATTORNEY DOCKET NO. YU 8874 PCT
[0421] generated intracellularly, this concept may be also be useful in settings of ongoing tissue injury, where antigenic fragments are released into local inflammatory or lymphatic compartments.
[0422] h. Exemplary Peptides
[0423] One of skill in the art will understand the difference between basic, neutral, and acid peptides for use in the disclosed compositions and methods. Typically, as used herein, basic peptides have a positive charge a physiological pH (e.g., 7.4), acidic peptides have a negative charge at physiological pH. Basic peptides (also referred to herein as positively charged peptides) can be characterized by a high content of basic amino acids, e.g., — lysine (Lys), arginine (Arg), or histidine (His) — which are protonated and positively charged at this pH. Basic peptides have a high pl, meaning they remain positively charged unless the surrounding environment is highly alkaline (pH > pl). Acidic peptides (also referred to herein as negatively charged peptides) can contain a high proportion of acidic amino acids, such as aspartic acid (Asp, D) and glutamic acid (Glu, E), which deprotonate and become negatively charged carboxylate groups in neutral or basic environments. For acidic peptides, the pl is low (usually below pH 7). The isoelectric point (pl) of a peptide is the specific pH at which it carries a net charge of zero, determining its solubility and electrophoretic mobility. It is calculated by averaging the two pKa values that bracket the neutral state, and is highly dependent on amino acid sequence.
[0424] In some forms, basic peptides have a pH of greater than 7, or greater than or equal to 7.5, 8, 8.5., 9, 9.5, 10, 10.5, 11, 11.5. 12, or more, e.g., under physiological conditions. In some forms, acidic peptides have a pH of less 7, or less than or equal to 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, or 2, e.g., under physiological conditions.
[0425] For illustrative purposes only, provided in the Table below are not limiting examples of peptides that basic, acid, or neutral according to the criteria above. Although these peptides can be used in the disclosed compositions and methods, they are non-limiting. One of skill in the art can design alternative peptides with similar structural and functional features.
[0426] 9-mer peptides HLA Binding | ~
[0427] Acirficfbasic
[0428] (Gene name) affinity |
[0429] pH“1t t Baric
[0430] SAf PEVRSL (i HALVq U strong pH-7 Ffenira! FAYPAPL£V(PSM01) OWsfiwg | pM~3.3 Acidic
[0431] Aekiic
[0432] - - -
[0433]
[0434] VrRSSSF'GVfA'fcSt) 0. weak i pH- 11.1 Basie
[0435] 54
[0436] 45836485.1ATTORNEY DOCKET NO. YU 8874 PCT
[0437] C. Isolated Nucleic Acid Molecules
[0438] Isolated nucleic acid sequences encoding TAP2, TAPI, SOCS1, IL-4, ERAP1, or ERAP2 proteins, polypeptides, fusions fragments and variants thereof, as well as inhibitor nucleic acids, and vectors and other expression constructs encoding the foregoing and peptides for modulation of intracellular peptide levels and / or types are also disclosed herein. As used herein, “isolated nucleic acid” refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a mammalian genome, including nucleic acids that normally flank one or both sides of the nucleic acid in a mammalian genome (e.g., nucleic acids that encode non- TAP2, TAPI, SOCS1, IL-4, and ERAP proteins). The term “isolated” as used herein with respect to nucleic acids also includes the combination with any non-naturally-occurring nucleic acid sequence, since such non-naturally-occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome.
[0439] An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, a cDNA library or a genomic library, or a gel slice containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid.
[0440] The nucleic acid sequences encoding the polypeptides include genomic sequences. Also disclosed are mRNA sequence wherein the exons have been deleted. Other nucleic acid sequences encoding the polypeptides, such polypeptides that include the aboveidentified amino acid sequences and fragments and variants thereof, are also disclosed.
[0441] Nucleic acids encoding the polypeptides may be optimized for expression in the expression host of choice. Codons may be substituted with alternative codons encoding the same amino acid to account for differences in codon usage between the organism from which the nucleic
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[0443] 45836485.1ATTORNEY DOCKET NO. YU 8874 PCT
[0444] acid sequence is derived and the expression host. In this manner, the nucleic acids may be synthesized using expression host-preferred codons.
[0445] Nucleic acids can be in sense or antisense orientation, or can be complementary to a reference sequence encoding a polypeptide. Nucleic acids can be DNA, RNA, or nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modification can improve, for example, stability, hybridization, or solubility of the nucleic acid. Common modifications are discussed in more detail below.
[0446] Nucleic acids encoding polypeptides can be administered to subjects in need thereof. Nucleic delivery involves introduction of “foreign” nucleic acids into a cell and ultimately, into a live animal. Compositions and methods for delivering nucleic acids to a subject are known in the art (see Understanding Gene Therapy, Lemoine, N. R., ed., BIOS Scientific Publishers, Oxford, 2008).
[0447] 1. Vectors and Host Cells
[0448] Vectors encoding polypeptides, fusion, fragments, and variants and inhibitor nucleic acids thereof are also provided. Nucleic acids, such as those described above, can be inserted into vectors for expression in cells. As used herein, a “vector” is a replicon, such as a plasmid, phage, virus or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Vectors can be expression vectors. An “expression vector” is a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.
[0449] Nucleic acids in vectors can be operably linked to one or more expression control sequences. For example, the control sequence can be incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream
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[0452] from the transcription initiation site. A coding sequence is “operably linked” and “under the control” of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence.
[0453] Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalo vims, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).
[0454] An expression vector can include a tag sequence. Tag sequences are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, Flag™ tag (Kodak, New Haven, CT), maltose E binding protein and protein A.
[0455] Vectors containing nucleic acids to be expressed can be transferred into host cells. The temi “host cell” is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques. Although not limited to a particular technique, a number of these techniques are well established within the art. Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride mediated transformation. Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection. Ilost cells (e.g., a prokaryotic cell or a eukaryotic cell such as a CHO cell) can be used to, for example, produce the polypeptides or fusion polypeptides described herein.
[0456] The vectors can be used to express TAP2, TAPI, SOCS1, or IL-4 or nucleic acids inhibitory thereof in cells. An exemplary vector includes, but is not limited to, an adenoviral vector. One approach includes nucleic acid transfer into primary cells in culture followed by autologous transplantation of the ex vivo transformed cells into the host, either systemically or into a particular organ or tissue. Ex vivo methods can include, for example, the steps of
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[0459] harvesting cells from a subject, culturing the cells, transducing them with an expression vector, and maintaining the cells under conditions suitable for expression of the encoded polypeptides. These methods are known in the art of molecular biology. The transduction step can be accomplished by any standard means used for ex vivo gene therapy, including, for example, calcium phosphate, lipofection, electroporation, viral infection, and biolistic gene transfer. Alternatively, liposomes or polymeric microparticles can be used. Cells that have been successfully transduced then can be selected, for example, for expression of the coding sequence or of a drug resistance gene. The cells then can be lethally irradiated (if desired) and injected or implanted into the subject. In one form, expression vectors containing nucleic acids encoding fusion proteins are transfected into cells that are administered to a subject in need thereof.
[0460] In vivo nucleic acid therapy can be accomplished by direct transfer of a functionally active DNA into mammalian somatic tissue or organ in vivo. Nucleic acids may also be administered in vivo by viral means. Nucleic acid molecules encoding polypeptides or fusion proteins may be packaged into retrovirus vectors using packaging cell lines that produce replication-defective retroviruses, as is well-known in the art. Other vims vectors may also be used, including recombinant adenoviruses and vaccinia virus, which can be rendered nonreplicating. In addition to naked DNA or RNA, or viral vectors, engineered bacteria may be used as vectors.
[0461] Nucleic acids may also be delivered by other carriers, including liposomes, polymeric micro- and nanoparticles and polycations such as asialoglycoprotein / polylysine.
[0462] In addition to virus- and carrier-mediated gene transfer in vivo, physical means well-known in the art can be used for direct transfer of DNA, including administration of plasmid DNA and particle-bombardment mediated gene transfer.
[0463] 2. Oligonucleotide Composition
[0464] The disclosed nucleic acids nucleic acids can be DNA or RNA nucleotides which typically include a heterocyclic base (nucleic acid base), a sugar moiety attached to the heterocyclic base, and a phosphate moiety which esterifies a hydroxyl function of the sugar moiety. The principal naturally-occurring nucleotides include uracil, thymine, cytosine, adenine and guanine as the heterocyclic bases, and ribose or deoxyribose sugar linked by phosphodiester bonds.
[0465] In some forms, the oligonucleotides are composed of nucleotide analogs that have been chemically modified to improve stability, half-life, or specificity or affinity for a target
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[0467] 45836485.1ATTORNEY DOCKET NO. YU 8874 PCT
[0468] receptor, relative to a DNA or RNA counterpart. The chemical modifications include chemical modification of nucleobases, sugar moieties, nucleotide linkages, or combinations thereof. As used herein ‘modified nucleotide” or “chemically modified nucleotide” defines a nucleotide that has a chemical modification of one or more of the heterocyclic base, sugar moiety or phosphate moiety constituents. In some forms, the charge of the modified nucleotide is reduced compared to DNA or RNA oligonucleotides of the same nucleobase sequence. For example, the oligonucleotide can have low negative charge, no charge, or positive charge.
[0469] Typically, nucleoside analogs support bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, where the analog backbone presents the bases in a manner to permit such hydrogen bonding in a sequence-specific fashion between the oligonucleotide analog molecule and bases in a standard polynucleotide (e.g., singlestranded RNA or single-stranded DNA). In some forms, the analogs have a substantially uncharged, phosphorus containing backbone.
[0470] a. Heterocyclic Bases
[0471] The principal naturally-occurring nucleotides include uracil, thymine, cytosine, adenine and guanine as the heterocyclic bases. The oligonucleotides can include chemical modifications to their nucleobase constituents. Chemical modifications of heterocyclic bases or heterocyclic base analogs may be effective to increase the binding affinity or stability in binding a target sequence. Chemically-modified heterocyclic bases include, but are not limited to, inosine, 5-(l-propynyl) uracil (pU), 5-(l-propynyl) cytosine (pC), 5-methylcytosine, 8-oxo-adenine, pseudocytosine, pseudoisocytosine, 5 and 2-amino-5-(2'-deoxy-.beta.-D-ribofuranosyl)pyridine (2-aminopyridine), and various pyrrolo- and pyrazolopyrimidine derivatives.
[0472] b. Sugar Modifications
[0473] Oligonucleotides can also contain nucleotides with modified sugar moieties or sugar moiety analogs. Sugar moiety modifications include, but are not limited to, 2'-O-aminoetoxy, 2'-O-amonioethyl (2'-OAE), 2'-O-methoxy, 2'-O-methyl, 2-guanidoethyl (2’-OGE), 2’-O,4'-C-methylene (LNA), 2’-O-(methoxyethyl) (2’-0ME) and 2’-O-(N-(methyl)acetamido) (2’-OMA). 2’-O-aminoethyl sugar moiety substitutions are especially preferred because they are protonated at neutral pH and thus suppress the charge repulsion between the TFO and the target duplex. This modification stabilizes the C3’-endo conformation of the ribose or dexyribose and also forms a bridge with the i-1 phosphate in the purine strand of the duplex.
[0474] 59
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[0476] In some forms, the nucleic acid is a morpholino oligonucleotide. Morpholino oligonucleotides are typically composed of two more morpholino monomers containing purine or pyrimidine base-pairing moieties effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, which arc linked together by phosphorus-containing linkages, one to three atoms long, joining the morpholino nitrogen of one monomer to the 5' exocyclic carbon of an adjacent monomer. The purine or pyrimidine base-pairing moiety is typically adenine, cytosine, guanine, uracil or thymine. The synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U. S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337.
[0477] Important properties of the morpholino-based subunits typically include: the ability to be linked in a oligomeric form by stable, uncharged backbone linkages; the ability to support a nucleotide base (e.g. adenine, cytosine, guanine, thymidine, uracil or inosine) such that the polymer formed can hybridize with a complementary-base target nucleic acid, including target RNA, with high Tm, even with oligomers as short as 10-14 bases: the ability of the oligomer to be actively transported into mammalian cells; and the ability of an oligomer: RNA heteroduplex to resist RNAse degradation.
[0478] In some forms, oligonucleotides employ moipholino-bascd subunits bearing basepairing moieties, joined by uncharged linkages, as described above.
[0479] c. Internucleotide Linkages
[0480] Oligonucleotides connected by an intemucleotide bond that refers to a chemical linkage between two nucleoside moieties. Modifications to the phosphate backbone of DNA or RNA oligonucleotides may increase the binding affinity or stability oligonucleotides, or reduce the susceptibility of oligonucleotides nuclease digestion. Cationic modifications, including, but not limited to, diethyl-ethylenediamide (DEED) or dimethylaminopropylamine (DMAP) may be especially useful due to decrease electrostatic repulsion between the oligonucleotide and a target. Modifications of the phosphate backbone may also include the substitution of a sulfur atom for one of the non-bridging oxygens in the phosphodiester linkage. This substitution creates a phosphorothioate intemucleoside linkage in place of the phosphodiester linkage. Oligonucleotides containing phosphorothioate internucleoside linkages have been shown to be more stable in vivo.
[0481] Examples of modified nucleotides with reduced charge include modified intemucleotide linkages such as phosphate analogs having achiral and uncharged intersubunit linkages (e.g., Sterchak, E. P. et al., Organic. Chem., 52:4202, (1987)), and uncharged
[0482] 60
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[0484] morpholino-based polymers having achiral intersubunit linkages (see, e.g., U. S. Pat. No. 5,034,506), as discussed above. Some internucleotide linkage analogs include morpholidate, acetal, and polyamide-linked heterocycles.
[0485] In another form, the oligonucleotides arc composed of locked nucleic acids. Locked nucleic acids (LNA) are modified RNA nucleotides (see, for example, Braasch, et al., Chem. Biol., 8(1 ): 1 -7 (2001)). LNAs form hybrids with DNA which are more stable than DNA / DNA hybrids, a property similar to that of peptide nucleic acid (PNA) / DNA hybrids. Therefore, LNA can be used just as PNA molecules would be. LNA binding efficiency can be increased in some forms by adding positive charges to it. Commercial nucleic acid synthesizers and standard phosphoramidite chemistry are used to make LNAs.
[0486] In some forms, the oligonucleotides are composed of peptide nucleic acids. Peptide nucleic acids (PNAs) are synthetic DNA mimics in which the phosphate backbone of the oligonucleotide is replaced in its entirety by repeating N-(2-aminoethyl)-glycine units and phosphodiester bonds are typically replaced by peptide bonds. The various heterocyclic bases are linked to the backbone by methylene carbonyl bonds. PNAs maintain spacing of heterocyclic bases that is similar to conventional DNA oligonucleotides, but are achiral and neutrally charged molecules. Peptide nucleic acids arc composed of peptide nucleic acid monomers.
[0487] Other backbone modifications include peptide and amino acid variations and modifications. Thus, the backbone constituents of oligonucleotides such as PNA may be peptide linkages, or alternatively, they may be non-peptide peptide linkages. Examples include acetyl caps, amino spacers such as 8-amino-3,6-dioxaoctanoic acid (referred to herein as O-linkers), amino acids such as lysine are particularly useful if positive charges are desired in the PNA, and the like. Methods for the chemical assembly of PNAs are well known. See, for example, U. S. Patent Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 and 5,786,571.
[0488] Oligonucleotides optionally include one or more temrinal residues or modifications at either or both termini to increase stability, and / or affinity of the oligonucleotide for its target. Commonly used positively charged moieties include the amino acids lysine and arginine, although other positively charged moieties may also be useful. Oligonucleotides may further be modified to be end capped to prevent degradation using a propylamine group. Procedures for 3' or 5' capping oligonucleotides are well known in the art.
[0489] In some forms, the nucleic acid can be single stranded or double stranded.
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[0491] 45836485.1ATTORNEY DOCKET NO. YU 8874 PCT
[0492] D. Delivery Vehicles
[0493] The disclose compounds can be administered and taken up into the cells of a subject with or without the aid of a delivery vehicle. Appropriate delivery vehicles for the disclosed inhibitors arc known in the art and can be selected to suit the particular inhibitor. For example, if the compound is a nucleic acid or vector, the delivery vehicle can be a viral vector, for example a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). The viral vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome (see e.g., Pastan et al., (1988) Proc. Natl. Acad. Sci. U. S. A. 85:4486;
[0494] Miller et al., (1986) Mol. Cell. Biol. 6:2895). The recombinant retrovirus can then be used to infect and thereby deliver to the infected cells nucleic acid encoding the compound inhibitor. The exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors (Mitani et al., Hum. Gene Then 5:941-948 (1994)), adeno-associated viral (AAV) vectors (Goodman et al., Blood 84:1492-1500 (1994)), lentiviral vectors (Naidini et al., Science 272:263-267 (1996)), pseudotyped retroviral vectors (Agrawal et al., Exper. Hematol. 24:738-747 (1996)).
[0495] Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, Schwartzenberger et al., Blood 87:472-478 (1996)). For example in some forms, the CTPS1 inhibitor is delivered via a liposome. Commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as well as other liposomes developed according to procedures standard in the art are well known. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.). This disclosed compounds and compositions and methods can be used in conjunction with any of these or other commonly used gene transfer methods.
[0496] In some forms, the delivery vehicle is incorporated into or encapsulated by a nanoparticle, microparticle, micelle, synthetic lipoprotein particle, or carbon nanotube. For example, the compounds and compositions can be incorporated into a vehicle such as polymeric microparticles which provide controlled release of the compound. In some forms,
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[0498] 45836485.1ATTORNEY DOCKET NO. YU 8874 PCT
[0499] release of the drug(s) is controlled by diffusion of the compound out of the microparticles and / or degradation of the polymeric particles by hydrolysis and / or enzymatic degradation. Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives. Polymers which arc slowly soluble and form a gel in an aqueous environment, such as hydroxypropyl methylcellulose or polyethylene oxide may also be suitable as materials for drug containing microparticles. Other polymers include, but are not limited to, polyanhydrides, poly (ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3 -hydroxybut rate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof.
[0500] E. Protein Transduction Domains and Targeting Moieties
[0501] 1. Protein Transduction Domains
[0502] Any of the compounds disclosed herein, including TAP2, TAPI, SOCS1, IL-4, ERAP1, and ERAP2, inhibitors and compounds that increase the bioactivity of TAP2, TAPI, SOCS1, IL-4, ERAP1, and ERAP2, and other peptides and mimics disclose herein e.g., for modulating intracellular peptide levels and / or types and delivery vehicles including the compounds, can be modified to one or more domains for enhancing delivery of the compound across the plasma membrane in into the interior of cells. The compounds can be modified to include a protein transduction domain (PTD), also known as cell penetrating peptides (GPPS). PTDs are known in the art, and include, but are not limited to, small regions of proteins that are able to cross a cell membrane in a receptor-independent mechanism (Kabouridis, P., Trends in Biotechnology (11):498-503 (2003)). Although several of PTDs have been documented, the two most commonly employed PTDs are derived from TAT (Frankel and Pabo, Cell, 55(6): 1189-93(1988)) protein of HIV and Antennapedia transcription factor from Drosophila, whose PTD is known as Penetratin (Derossi et al., J Biol Chem., 269(14):10444-50 (1994)).
[0503] The Antennapedia homeodomain is 68 amino acid residues long and contains four alpha helices. Penetratin is an active domain of this protein which consists of a 16 amino acid sequence derived from the third helix of Antennapedia. TAT protein consists of 86 amino acids and is involved in the replication of HIV-1. The TAT PTD consists of an 11 amino acid sequence domain (residues 47 to 57; YGRKKRRQRRR (SEQ ID NO:1)) of the parent protein that appears to be important for uptake. Additionally, the basic domain Tat(49-57) or RKKRRQRRR (SEQ ID NO:2) has been shown to be a PTD. TAT has been
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[0506] favored for fusion to proteins of interest for cellular import. Several modifications to TAT, including substitutions of Glutatmine to Alanine, i.e., Q- A, have demonstrated an increase in cellular uptake anywhere from 90% (Wender et al., Proc Natl Acad Sci USA., 97(24):13003-8 (2000)) to up to 33 fold in mammalian cells. (Ho et al., Cancer Res., 61(2):474-7 (2001)) The most efficient uptake of modified proteins was revealed by mutagenesis experiments of TAT-PTD, showing that an 11 arginine stretch was several orders of magnitude more efficient as an intercellular delivery vehicle. Thus, some forms include PTDs that are cationic or amphipathic. Additionally, exemplary PTDs include, but are not limited to, poly-Arg - RRRRRRR (SEQ ID NO:3); PTD-5 - RRQRRTSKLMKR (SEQ ID NO:4); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:5); KALA - WEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:6); and RQIKIWFQNRRMKWKK (SEQ ID NO:7).
[0507] In some forms, the compounds includes an endosomal escape sequence that improves delivery of the compound to the interior of the cell. Endosomal escape sequences are known in the art, see for example, Barka, et al., Histochem. Cytochem., 48(11): 1453-60 (2000) and Wadia and Stan, Nat. Med., 10(3):310— 5 (2004).
[0508] 2. Targeting Signal or Domain
[0509] Any of the compounds disclosed herein, including both TAP2, TAPI, SOCS1, IL-4, ERAP1, and ERAP2 inhibitors and compounds that increase the bioactivity of TAP2, TAPI, SOCS1, IL-4, ERAP1, and ERAP2, and other peptides and mimics disclose herein e.g., for modulating intracellular peptide levels and / or types and delivery vehicles including the compounds can be modified to include one or targeting signals or domains. The targeting signal or sequence can be specific for a host, tissue, organ, cell, organelle, an organelle such as the nucleus, or cellular compartment. Moreover, the compounds and compositions disclosed here can be targeted to other specific intercellular regions, compartments, or cell types.
[0510] In some forms, the targeting signal binds to a ligand or receptor which is located on the surface of a target cell such as to bring the compound and cell membranes sufficiently close to each other to allow penetration of the compound into the cell. Additional forms are directed to specifically delivering the compound to specific tissue or cell types.
[0511] Preferably, the targeting moiety enhances targeting to muscle, most preferably muscle satellite cells.
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[0514] In a preferred form, the targeting molecule is selected from the group consisting of an antibody or antigen binding fragment thereof, an antibody domain, an antigen, a cell surface receptor, a cell surface adhesion molecule, a viral envelope protein and a peptide selected by phage display that binds specifically to a defined cell.
[0515] Targeting domains to specific cells can be accomplished by modifying the disclosed compounds to include specific cell and tissue targeting signals. These sequences target specific cells and tissues, but in some forms the interaction of the targeting signal with the cell does not occur through a traditional receptor:ligand interaction. The eukaryotic cell includes a number of distinct cell surface molecules. The structure and function of each molecule can be specific to the origin, expression, character and structure of the cell.
[0516] Determining the unique cell surface complement of molecules of a specific cell type can be determined using techniques well known in the art.
[0517] One skilled in the art will appreciate that the tropism of the compound can be altered by changing the targeting signal.
[0518] It is known in the art that nearly every cell type in a tissue in a mammalian organism possesses some unique cell surface receptor or antigen. Thus, it is possible to incorporate nearly any ligand for the cell surface receptor or antigen as a targeting signal. For example, peptidyl hormones can be used a targeting moieties to target delivery to those cells which possess receptors for such hormones. Chemokines and cytokines can similarly be employed as targeting signals to target delivery of the complex to their target cells. A variety of technologies have been developed to identify genes that are preferentially expressed in certain cells or cell states and one of skill in the art can employ such technology to identify targeting signals which are preferentially or uniquely expressed on the target tissue of interest Another form provides an antibody or antigen binding fragment thereof bound to the disclosed recombinant polypeptides acting as the targeting signal. The antibodies or antigen binding fragment thereof are useful for directing the fusion protein to a cell type or cell state. In one form, the fusion protein possesses an antibody binding domain, for example from proteins known to bind antibodies such as Protein A and Protein G from Staphylococcus aureus. Other domains known to bind antibodies are known in the art and can be substituted. In certain forms, the antibody is polyclonal, monoclonal, linear, humanized, chimeric or a fragment thereof. Representative antibody fragments are those fragments that bind the antibody binding portion of the non- viral vector and include Fab, Fab', F(ab’), Fv diabodies, linear antibodies, single chain antibodies and bispecific antibodies known in the art.
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[0521] In some forms, the targeting domain includes all or part of an antibody that directs the compound to the desired target cell type or cell state. Antibodies can be monoclonal or polyclonal, but are preferably monoclonal. For human gene therapy purposes, antibodies are derived from human genes and arc specific for cell surface markers, and are produced to reduce potential immunogenicity to a human host as is known in the art. For example, transgenic mice which contain the entire human immunoglobulin gene cluster are capable of producing "human" antibodies can be utilized. In one form, fragments of such human antibodies are employed as targeting signals. In a preferred form, single chain antibodies modeled on human antibodies are prepared in prokaryotic culture.
[0522] Additional forms are directed to specifically delivering the compound to intracellular compartments or organelles. Eukaryotic cells contain membrane bound structures or organelles.
[0523] For example, in some forms, the compounds include a nuclear localization signal. Most proteins transported across the nuclear envelope contain a nuclear localization signal (NLS). The NLS is recognized by a nuclear import complex, enabling active transport to the nucleus. Even the transport of small proteins that can diffuse through the nuclear pore is increased by an NLS. NLS domains arc known in the art and include for example, SV 40 T antigen or a fragment thereof, such as PKKKRKV (SEQ ID NO:8). The NLS can be simple cationic sequences of about 4 to about 8 amino acids, or can be bipartite having two interdependent positively charged clusters separated by a mutation resistant linker region of about 10-12 amino acids. The cauliflower mosaic virus (CMV) major capsid protein, CP, possesses an amino- terminal NLS. Additional representative NLS include but are not limited to GKKRSKV (SEQ ID NO:9); KSRKRKL (SEQ ID NOTO);
[0524] KRPAATKKAGQAKKKKLDK (SEQ ID NO: 11); RKKRKTEEESPLKDKAKKSK (SEQ ID NO: 12); KDCVMNKHHRNRCQYCRLQR (SEQ ID NO:13); PAAKRVKLD (SEQ ID NO:14); and KKYENVVIKRSPRKRGRPRK (SEQ ID NO:15).
[0525] F. Formulations
[0526] The disclosed compounds can be formulated in a pharmaceutical composition.
[0527] Pharmaceutical compositions can be for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), enteral, transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, pulmonary, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.
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[0530] The compositions can be administered systemically.
[0531] Drugs can be formulated for immediate release, extended release, or modified release. A delayed release dosage form is one that releases a drug (or drugs) at a time other than promptly after administration. An extended release dosage form is one that allows at least a twofold reduction in dosing frequency as compared to that drug presented as a conventional dosage form (e.g. as a solution or prompt drug-releasing, conventional solid dosage form). A modified release dosage form is one for which the drug release characteristics of time course and / or location are chosen to accomplish therapeutic or convenience objectives not offered by conventional dosage forms such as solutions, ointments, or promptly dissolving dosage forms. Delayed release and extended release dosage forms and their combinations are types of modified release dosage forms.
[0532] Formulations are typically prepared using a pharmaceutically acceptable “carrier” composed of materials that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. The “carrier” is all components present in the pharmaceutical formulation other than the active ingredient or ingredients. The term “carrier” includes, but is not limited to, diluents, binders, lubricants, disintegrators, fillers, and coating compositions.
[0533] “Carrier” also includes all components of the coating composition which may include plasticizers, pigments, colorants, stabilizing agents, and glidants. The delayed release dosage formulations may be prepared as described in references such as “Pharmaceutical dosage form tablets”, eds. Liberman et al. (New York, Marcel Dekker, Inc., 1989), “Remington - The science and practice of pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6thEdition, Ansel et.al., (Media, PA: Williams and Wilkins, 1995) which provides information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.
[0534] The compound can be administered to a subject with or without the aid of a delivery vehicle. Appropriate delivery vehicles for the compounds are known in the art and can be selected to suit the particular active agent. For example, in some forms, the active agent(s) is incorporated into or encapsulated by, or bound to, a nanoparticle, microparticle, micelle, synthetic lipoprotein particle, or carbon nanotube. For example, the compositions can be incorporated into a vehicle such as polymeric particles which provide controlled release of the active agent(s). In some forms, release of the drug(s) is controlled by diffusion of the
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[0537] active agent(s) out of the particles and / or degradation of the polymeric particles by hydrolysis and / or enzymatic degradation.
[0538] Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives. Polymers which arc slowly soluble and form a gel in an aqueous environment, such as hydroxypropyl methylcellulose or polyethylene oxide, may also be suitable as materials for drug containing particles or particles. Other polymers include, but are not limited to, polyanhydrides, poly (ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3 -hydroxybut rate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof. In some forms, both agents are incorporated into the same particles and are formulated for release at different times and / or over different time periods. For example, in some forms, one of the agents is released entirely from the particles before release of the second agent begins. In other forms, release of the first agent begins followed by release of the second agent before the all of the first agent is released. In still other forms, both agents are released at the same time over the same period of time or over different periods of time.
[0539] 1. Formulations for Parenteral Administration
[0540] Compounds and pharmaceutical compositions thereof can be administered in an aqueous solution, by parenteral injection. The formulation may also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of the active agent(s) and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions include diluents sterile water, buffered saline of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and optionally, additives such as detergents and solubilizing agents (e.g., TWEEN® 20, TWEEN® 80 also referred to as POLYSORBATE® 20 or 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and com oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be lyophilized and redissolved / resuspended immediately before use. The formulation may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.
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[0543] 2. Oral Immediate Release Formulations
[0544] Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gclatin capsules can prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art.
[0545] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name Eudragit® (Roth Pharma, Westerstadt, Germany), Zein, shellac, and polysaccharides.
[0546] Additionally, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.
[0547] Optional pharmaceutically acceptable excipients present in the drug-containing tablets, beads, granules or particles include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants. Diluents, also termed "fillers," arc typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to,, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powder sugar.
[0548] Binders are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydorxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid and polyvinylpyrrolidone.
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[0551] Lubricants are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.
[0552] Disintegrants are used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as crosslinked PVP (Polyplasdone XL from GAF Chemical Corp).
[0553] Stabilizers are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions.
[0554] Surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but arc not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, POLOXAMER® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
[0555] If desired, the tablets, beads granules or particles may also contain minor amount of nontoxic auxiliary substances such as wetting or emulsifying agents, dyes, pH buffering agents, and preservatives.
[0556] 3. Extended release dosage forms
[0557] The extended release formulations are generally prepared as diffusion or osmotic systems, for example, as described in “Remington - The science and practice of pharmacy”
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[0560] (20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000). A diffusion system typically consists of two types of devices, reservoir and matrix, and is well known and described in the art. The matrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form. The three major types of materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include, but not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and carbopol 934, polyethylene oxides. Fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate. Alternatively, extended release formulations can be prepared using osmotic systems or by applying a semi-permeable coating to the dosage form. In the latter case, the desired drug release profile can be achieved by combining low permeable and high permeable coating materials in suitable proportion.
[0561] The devices with different drug release mechanisms described above could be combined in a final dosage form having single or multiple units. Examples of multiple units include multilayer tablets, capsules containing tablets, beads, granules, etc.
[0562] An immediate release portion can be added to the extended release system by means of either applying an immediate release layer on top of the extended release core using coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads.
[0563] Extended release tablets containing hydrophilic polymers are prepared by techniques commonly known in the art such as direct compression, wet granulation, or dry granulation processes. Their formulations usually incorporate polymers, diluents, binders, and lubricants as well as the active pharmaceutical ingredient. The usual diluents include inert powdered substances such as any of many different kinds of starch, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidine can also be used. Polyethylene glycol, hydrophilic polymers,
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[0566] ethylcellulose and waxes can also serve as binders. A lubricant is necessary in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.
[0567] Extended release tablets containing wax materials are generally prepared using methods known in the art such as a direct blend method, a congealing method, and an aqueous dispersion method. In a congealing method, the drug is mixed with a wax material and either spray- congealed or congealed and screened and processed.
[0568] 4. Delayed release dosage forms
[0569] Delayed release formulations are created by coating a solid dosage form with a film of a polymer which is insoluble in the acid environment of the stomach, and soluble in the neutral environment of small intestines.
[0570] The delayed release dosage units can be prepared, for example, by coating a drug or a drug-containing composition with a selected coating material. The drug-containing composition may be, e.g., a tablet for incorporation into a capsule, a tablet for use as an inner core in a "coated core" dosage form, or a plurality of drug-containing beads, particles or granules, for incorporation into cither a tablet or capsule. Preferred coating materials include bioerodible, gradually hydrolyzable, gradually water-soluble, and / or enzymatically degradable polymers, and may be conventional "enteric" polymers. Enteric polymers, as will be appreciated by those skilled in the art, become soluble in the higher pH environment of the lower gastrointestinal tract or slowly erode as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly in the colon. Suitable coating materials for effecting delayed release include, but are not limited to, cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and / or ethyl methacrylate, and other methacrylic resins that are commercially available under the tradename EUDRAGIT®.
[0571] (Rohm Pharma; Westerstadt, Germany), including EUDRAGIT®. L30D-55 and L100-55 (soluble at pH 5.5 and above), EUDRAGIT®. E-100 (soluble at pH 6.0 and above),
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[0574] EUDRAGIT®. S (soluble at pH 7.0 and above, as a result of a higher degree of esterification), and EUDRAGITS®. NE, RL and RS (water-insoluble polymers having different degrees of permeability and expandability); vinyl polymers and copolymers such as polyvinyl pyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene- vinyl acetate copolymer; enzymatically degradable polymers such as azo polymers, pectin, chitosan, amylose and guar gum; zein and shellac. Combinations of different coating materials may also be used. Multi-layer coatings using different polymers may also be applied.
[0575] The preferred coating weights for particular coating materials may be readily determined by those skilled in the art by evaluating individual release profiles for tablets, beads and granules prepared with different quantities of various coating materials. It is the combination of materials, method and form of application that produce the desired release characteristics, which one can determine only from the clinical studies.
[0576] The coating composition may include conventional additives, such as plasticizers, pigments, colorants, stabilizing agents, glidants, etc. A plasticizer is normally present to reduce the fragility of the coating, and will generally represent about 10 wt. % to 50 wt. % relative to the dry weight of the polymer. Examples of typical plasticizers include polyethylene glycol, propylene glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate, castor oil and acetylated monoglycerides. A stabilizing agent is preferably used to stabilize particles in the dispersion. Typical stabilizing agents are nonionic emulsifiers such as sorbitan esters, polysorbates and polyvinylpyrrolidone. Glidants are recommended to reduce sticking effects during film formation and drying, and will generally represent approximately 25 wt. % to 100 wt. % of the polymer weight in the coating solution. One effective glidant is talc. Other glidants such as magnesium stearate and glycerol monostearates may also be used. Pigments such as titanium dioxide may also be used. Small quantities of an anti-foaming agent, such as a silicone (e.g., simethicone), may also be added to the coating composition.
[0577] Methods of manufacturing
[0578] As will be appreciated by those skilled in the art and as described in the pertinent texts and literature, a number of methods are available for preparing drug-containing tablets, beads, granules or particles that provide a variety of drug release profiles. Such methods include, but are not limited to, the following: coating a drug or drug-containing composition with an appropriate coating material, typically although not necessarily incorporating a
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[0581] polymeric material, increasing drug particle size, placing the drug within a matrix, and forming complexes of the drug with a suitable complexing agent.
[0582] The delayed release dosage units may be coated with the delayed release polymer coating using conventional techniques, c.g., using a conventional coating pan, an airless spray technique, fluidized bed coating equipment (with or without a Wurster insert). For detailed information concerning materials, equipment and processes for preparing tablets and delayed release dosage forms, see Pharmaceutical Dosage Forms: Tablets, eds. Lieberman et al. (New York: Marcel Dekker, Inc., 1989), and Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 6.sup.th Ed. (Media, PA: Williams & Wilkins, 1995).
[0583] A preferred method for preparing extended release tablets is by compressing a drugcontaining blend, e.g., blend of granules, prepared using a direct blend, wet-granulation, or dry-granulation process. Extended release tablets may also be molded rather than compressed, starting with a moist material containing a suitable water-soluble lubricant. However, tablets are preferably manufactured using compression rather than molding. A preferred method for forming extended release drug-containing blend is to mix drug particles directly with one or more excipients such as diluents (or fillers), binders, disintegrants, lubricants, glidants, and colorants. As an alternative to direct blending, a drug-containing blend may be prepared by using wet-granulation or dry-granulation processes. Beads containing the active agent may also be prepared by any one of a number of conventional techniques, typically starting from a fluid dispersion. For example, a typical method for preparing drug-containing beads involves dispersing or dissolving the active agent in a coating suspension or solution containing pharmaceutical excipients such as polyvinylpyrrolidone, methylcellulose, talc, metallic stearates, silicone dioxide, plasticizers or the like. The admixture is used to coat a bead core such as a sugar sphere (or so-called "non-pareil") having a size of approximately 60 to 20 mesh.
[0584] An alternative procedure for preparing drug beads is by blending drug with one or more pharmaceutically acceptable excipients, such as microcrystalline cellulose, lactose, cellulose, polyvinyl pyrrolidone, talc, magnesium stearate, a disintegrant, etc., extruding the blend, spheronizing the extrudate, drying and optionally coating to form the immediate release beads.
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[0587] 5. Formulations for Mucosal and Pulmonary
[0588] Administration
[0589] Active agent(s) and compositions thereof can be formulated for pulmonary or mucosal administration. The administration can include delivery of the composition to the lungs, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa. In a particular form, the composition is formulated for and delivered to the subject sublingually.
[0590] In one form, the compounds are formulated for pulmonary delivery, such as intranasal administration or oral inhalation. The respiratory tract is the structure involved in the exchange of gases between the atmosphere and the blood stream. The lungs are branching structures ultimately ending with the alveoli where the exchange of gases occurs. The alveolar surface area is the largest in the respiratory system and is where drug absorption occurs. The alveoli are covered by a thin epithelium without cilia or a mucus blanket and secrete surfactant phospholipids. The respiratory tract encompasses the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli. The upper and lower airways are called the conducting airways. The terminal bronchioli then divide into respiratory bronchiole, which then lead to the ultimate respiratory zone, the alveoli, or deep lung. The deep lung, or alveoli, is the primary target of inhaled therapeutic aerosols for systemic drug delivery.
[0591] Pulmonary administration of therapeutic compositions composed of low molecular weight drugs has been observed, for example, beta-androgenic antagonists to treat asthma. Other therapeutic agents that are active in the lungs have been administered systemically and targeted via pulmonary absorption. Nasal delivery is considered to be a promising technique for administration of therapeutics for the following reasons: the nose has a large surface area available for drug absorption due to the coverage of the epithelial surface by numerous microvilli, the subepithelial layer is highly vascularized, the venous blood from the nose passes directly into the systemic circulation and therefore avoids the loss of drug by first-pass metabolism in the liver, it offers lower doses, more rapid attainment of therapeutic blood levels, quicker onset of pharmacological activity, fewer side effects, high total blood flow per cm3, porous endothelial basement membrane, and it is easily accessible.
[0592] The term aerosol as used herein refers to any preparation of a fine mist of particles, which can be in solution or a suspension, whether or not it is produced using a propellant.
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[0595] Aerosols can be produced using standard techniques, such as ultrasonication or high-pressure treatment.
[0596] Carriers for pulmonary formulations can be divided into those for dry powder formulations and for administration as solutions. Aerosols for the delivery of therapeutic agents to the respiratory tract are known in the art. For administration via the upper respiratory tract, the formulation can be formulated into a solution, e.g., water or isotonic saline, buffered or un-buffered, or as a suspension, for intranasal administration as drops or as a spray. Preferably, such solutions or suspensions are isotonic relative to nasal secretions and of about the same pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0. Buffers should be physiologically compatible and include, simply by way of example, phosphate buffers. For example, a representative nasal decongestant is described as being buffered to a pH of about 6.2. One skilled in the art can readily determine a suitable saline content and pH for an innocuous aqueous solution for nasal and / or upper respiratory administration.
[0597] Preferably, the aqueous solution is water, physiologically acceptable aqueous solutions containing salts and / or buffers, such as phosphate buffered saline (PBS), or any other aqueous solution acceptable for administration to an animal or human. Such solutions are well known to a person skilled in the art and include, but are not limited to, distilled water, de-ionized water, pure or ultrapure water, saline, phosphate-buffered saline (PBS). Other suitable aqueous vehicles include, but are not limited to, Ringer's solution and isotonic sodium chloride. Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.
[0598] In another form, solvents that are low toxicity organic (i.e. nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydrofuran, ethyl ether, and propanol may be used for the formulations. The solvent is selected based on its ability to readily aerosolize the formulation. The solvent should not detrimentally react with the compounds. An appropriate solvent should be used that dissolves the compounds or forms a suspension of the compounds. The solvent should be sufficiently volatile to enable formation of an aerosol of the solution or suspension. Additional solvents or aerosolizing agents, such as freons, can be added as desired to increase the volatility of the solution or suspension.
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[0601] In one form, compositions may contain minor amounts of polymers, surfactants, or other excipients well known to those of the art. In this context, “minor amounts” means no excipients are present that might affect or mediate uptake of the compounds in the lungs and that the excipients that are present are present in amount that do not adversely affect uptake of compounds in the lungs.
[0602] Dry lipid powders can be directly dispersed in ethanol because of their hydrophobic character. For lipids stored in organic solvents such as chloroform, the desired quantity of solution is placed in a vial, and the chloroform is evaporated under a stream of nitrogen to form a dry thin film on the surface of a glass vial. The film swells easily when reconstituted with ethanol. To fully disperse the lipid molecules in the organic solvent, the suspension is sonicated. Nonaqueous suspensions of lipids can also be prepared in absolute ethanol using a reusable PARI LC Jet+ nebulizer (PARI Respiratory Equipment, Monterey, CA).
[0603] Dry powder formulations (“DPFs”) with large particle size have improved flowability characteristics, such as less aggregation, easier aerosolization, and potentially less phagocytosis. Dry powder aerosols for inhalation therapy are generally produced with mean diameters primarily in the range of less than 5 microns, although a preferred range is between one and ten microns in aerodynamic diameter. Large “carrier” particles (containing no drug) have been co-delivered with therapeutic aerosols to aid in achieving efficient aerosolization among other possible benefits.
[0604] Polymeric particles may be prepared using single and double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation, simple and complex coacervation, interfacial polymerization, and other methods well known to those of ordinary skill in the art. Particles may be made using methods for making microspheres or microcapsules known in the art. The preferred methods of manufacture are by spray drying and freeze drying, which entails using a solution containing the surfactant, spraying to form droplets of the desired size, and removing the solvent.
[0605] The particles may be fabricated with the appropriate material, surface roughness, diameter and tap density for localized delivery to selected regions of the respiratory tract such as the deep lung or upper airways. For example, higher density or larger particles may be used for upper airway delivery. Similarly, a mixture of different sized particles, provided with the same or different active agents may be administered to target different regions of the lung in one administration.
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[0608] 6. Topical and Transdermal Formulations
[0609] Transdermal formulations may also be prepared. These will typically be gels, ointments, lotions, sprays, or patches, all of which can be prepared using standard technology. Transdermal formulations can include penetration enhancers.
[0610] A “gel” is a colloid in which the dispersed phase has combined with the continuous phase to produce a semisolid material, such as jelly.
[0611] An “oil” is a composition containing at least 95% wt of a lipophilic substance.
[0612] Examples of lipophilic substances include but are not limited to naturally occurring and synthetic oils, fats, fatty acids, lecithins, triglycerides and combinations thereof.
[0613] A “continuous phase” refers to the liquid in which solids are suspended or droplets of another liquid are dispersed, and is sometimes called the external phase. This also refers to the fluid phase of a colloid within which solid or fluid particles are distributed. If the continuous phase is water (or another hydrophilic solvent), water-soluble or hydrophilic drugs will dissolve in the continuous phase (as opposed to being dispersed). In a multiphase formulation (e.g., an emulsion), the discreet phase is suspended or dispersed in the continuous phase.
[0614] An “emulsion” is a composition containing a mixture of non-miscible components homogenously blended together. In particular forms, the non-miscible components include a lipophilic component and an aqueous component. An emulsion is a preparation of one liquid distributed in small globules throughout the body of a second liquid. The dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase. When oil is the dispersed liquid and an aqueous solution is the continuous phase, it is known as an oil-in-water emulsion, whereas when water or aqueous solution is the dispersed phase and oil or oleaginous substance is the continuous phase, it is known as a water-in-oil emulsion. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, especially non-ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non-aqueous materials, particularly glycols such as propylene glycol. The oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers.
[0615] “Emollients” are an externally applied agent that softens or soothes skin and are generally known in the art and listed in compendia, such as the “Handbook of Pharmaceutical
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[0618] Excipients”, 4thEd., Pharmaceutical Press, 2003. These include, without limitation, almond oil, castor oil, ceratonia extract, cetostearoyl alcohol, cetyl alcohol, cetyl esters wax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycol palmitostearate, glycerin, glycerin monostearate, glyceryl monooleate, isopropyl myristate, isopropyl palmitate, lanolin, lecithin, light mineral oil, medium-chain triglycerides, mineral oil and lanolin alcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil, starch, stearyl alcohol, sunflower oil, xylitol and combinations thereof. In one form, the emollients are ethylhexyl stearate and ethylhexyl palmitate.
[0619] “Surfactants” are surface-active agents that lower surface tension and thereby increase the emulsifying, foaming, dispersing, spreading and wetting properties of a product. Suitable non-ionic surfactants include emulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate, poloxamer, povidone and combinations thereof. In one form, the non-ionic surfactant is stearyl alcohol.
[0620] “Emulsifiers” are surface active substances which promote the suspension of one liquid in another and promote the formation of a stable mixture, or emulsion, of oil and water. Common emulsifiers are: metallic soaps, certain animal and vegetable oils, and various polar compounds. Suitable emulsifiers include acacia, anionic emulsifying wax, calcium stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolin alcohols, lecithin, medium-chain triglycerides, methylcellulose, mineral oil and lanolin alcohols, monobasic sodium phosphate, monoethanolamine, nonionic emulsifying wax, oleic acid, poloxamer, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, propylene glycol alginate, selfemulsifying glyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulfate, sorbitan esters, stearic acid, sunflower oil, tragacanth, triethanolamine, xanthan gum and combinations thereof. In one form, the emulsifier is glycerol stearate.
[0621] A “lotion” is a low- to medium-viscosity liquid formulation. A lotion can contain finely powdered substances that are insoluble in the dispersion medium through the use of suspending agents and dispersing agents. Alternatively, lotions can have as the dispersed phase liquid substances that are immiscible with the vehicle and are usually dispersed by means of emulsifying agents or other suitable stabilizers. In one form, the lotion is in the
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[0624] form of an emulsion having a viscosity of between 100 and 1000 centistokes. The fluidity of lotions permits rapid and uniform application over a wide surface area. Lotions are typically intended to dry on the skin leaving a thin coat of their medicinal components on the skin’s surface.
[0625] A “cream” is a viscous liquid or semi-solid emulsion of either the “oil-in-water” or “water-in-oil type”. Creams may contain emulsifying agents and / or other stabilizing agents. In one form, the formulation is in the form of a cream having a viscosity of greater than 1000 centistokes, typically in the range of 20,000-50,000 centistokes. Creams are often time preferred over ointments as they are generally easier to spread and easier to remove.
[0626] An emulsion is a preparation of one liquid distributed in small globules throughout the body of a second liquid. The dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase. When oil is the dispersed liquid and an aqueous solution is the continuous phase, it is known as an oil-in-water emulsion, whereas when water or aqueous solution is the dispersed phase and oil or oleaginous substance is the continuous phase, it is known as a water-in-oil emulsion. The oil phase may consist at least in part of a propellant, such as an HFA propellant. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, especially non-ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non-aqueous materials, particularly glycols such as propylene glycol. The oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers.
[0627] A sub-set of emulsions are the self-emulsifying systems. These drug delivery systems are typically capsules (hard shell or soft shell) composed of the drug dispersed or dissolved in a mixture of surfactant(s) and lipophilic liquids such as oils or other water immiscible liquids. When the capsule is exposed to an aqueous environment and the outer gelatin shell dissolves, contact between the aqueous medium and the capsule contents instantly generates very small emulsion droplets. These typically are in the size range of micelles or nanoparticles. No mixing force is required to generate the emulsion as is typically the case in emulsion formulation processes.
[0628] The basic difference between a cream and a lotion is the viscosity, which is dependent on the amount / use of various oils and the percentage of water used to prepare the formulations. Creams are typically thicker than lotions, may have various uses and often one
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[0631] uses more varied oils / butters, depending upon the desired effect upon the skin. In a cream formulation, the water-base percentage is about 60-75 % and the oil-base is about 20-30 % of the total, with the other percentages being the emulsifier agent, preservatives and additives for a total of 100 %.
[0632] An “ointment” is a semisolid preparation containing an ointment base and optionally one or more active agents. Examples of suitable ointment bases include hydrocarbon bases (e.g., petrolatum, white petrolatum, yellow ointment, and mineral oil); absorption bases (hydrophilic petrolatum, anhydrous lanolin, lanolin, and cold cream); water- removable bases (e.g., hydrophilic ointment), and water-soluble bases (e.g., polyethylene glycol ointments). Pastes typically differ from ointments in that they contain a larger percentage of solids. Pastes are typically more absorptive and less greasy that ointments prepared with the same components.
[0633] A “gel” is a semisolid system containing dispersions of small or large molecules in a liquid vehicle that is rendered semisolid by the action of a thickening agent or polymeric material dissolved or suspended in the liquid vehicle. The liquid may include a lipophilic component, an aqueous component or both. Some emulsions may be gels or otherwise include a gel component. Some gels, however, are not emulsions because they do not contain a homogenized blend of immiscible components.
[0634] Suitable gelling agents include, but are not limited to, modified celluloses, such as hydroxypropyl cellulose and hydroxyethyl cellulose; Carbopol homopolymers and copolymers; and combinations thereof. Suitable solvents in the liquid vehicle include, but are not limited to, diglycol monoethyl ether; alklene glycols, such as propylene glycol; dimethyl isosorbide; alcohols, such as isopropyl alcohol and ethanol. The solvents are typically selected for their ability to dissolve the drug. Other additives, which improve the skin feel and / or emolliency of the formulation, may also be incorporated. Examples of such additives include, but are not limited, isopropyl myristate, ethyl acetate, C12-C15 alkyl benzoates, mineral oil, squalane, cyclomethicone, capric / caprylic triglycerides, and combinations thereof.
[0635] Foams consist of an emulsion in combination with a gaseous propellant. The gaseous propellant consists primarily of hydrofluoroalkanes (HFAs). Suitable propellants include HFAs such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoropropane (HFA 227), but mixtures and admixtures of these and other HFAs that are currently approved or may become approved for medical use are suitable. The propellants preferably are not
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[0638] hydrocarbon propellant gases which can produce flammable or explosive vapors during spraying. Furthermore, the compositions preferably contain no volatile alcohols, which can produce flammable or explosive vapors during use.
[0639] Buffers are used to control pH of a composition. Preferably, the buffers buffer the composition from a pH of about 4 to a pH of about 7.5, more preferably from a pH of about 4 to a pH of about 7, and most preferably from a pH of about 5 to a pH of about 7. In a preferred form, the buffer is triethanolamine.
[0640] Preservatives can be used to prevent the growth of fungi and microorganisms.
[0641] Suitable antifungal and antimicrobial agents include, but are not limited to, benzoic acid, butylparaben, ethyl paraben, methyl paraben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, and thimerosal.
[0642] Additional agents that can be added to the formulation include penetration enhancers. In some forms, the penetration enhancer increases the solubility of the drug, improves transdermal delivery of the drug across the skin, in particular across the stratum corneum, or a combination thereof. Some penetration enhancers cause dermal irritation, dermal toxicity and dermal allergies. However, the more commonly used ones include urea, (carbonyldiamide), imidurea, N, N-diethylformamide, N-methyl-2-pyrrolidone, 1-dodecal-azacyclopheptane-2-one, calcium thioglycate, 2-pyrrolidone, N, N-diethyl-m-toluamide, oleic acid and its ester derivatives, such as methyl, ethyl, propyl, isopropyl, butyl, vinyl and glycerylmonooleate, sorbitan esters, such as sorbitan monolaurate and sorbitan monooleate, other fatty acid esters such as isopropyl laurate, isopropyl myristate, isopropyl palmitate, diisopropyl adipate, propylene glycol monolaurate, propylene glycol monooleatea and nonionic detergents such as BRIJ® 76 (stearyl poly(10 oxyethylene ether), BRIJ® 78 (stearyl poly(20)oxyethylene ether), BRIJ® 96 (oleyl poly(10)oxyethylene ether), and BRIJ® 721 (stearyl poly (21) oxyethylene ether) (ICI Americas Inc. Corp.). Chemical penetrations and methods of increasing transdermal drug delivery are described in Inayat, et al., Tropical Journal of Pharmaceutical Research, 8(2): 173-179 (2009) and Fox, et al., Molecules, 16:10507-10540 (2011). In some forms, the penetration enhancer is, or includes, an alcohol such ethanol, or others disclosed herein or known in the art.
[0643] Delivery of drugs by the transdermal route has been known for many years.
[0644] Advantages of a transdermal drug delivery compared to other types of medication delivery such as oral, intravenous, intramuscular, etc., include avoidance of hepatic first pass
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[0647] metabolism, ability to discontinue administration by removal of the system, the ability to control drug delivery for a longer time than the usual gastrointestinal transit of oral dosage form, and the ability to modify the properties of the biological barrier to absorption.
[0648] Controlled release transdermal devices rely for their effect on delivery of a known flux of drug to the skin for a prolonged period of time, generally a day, several days, or a week. Two mechanisms are used to regulate the drug flux: either the drug is contained within a drug reservoir, which is separated from the skin of the wearer by a synthetic membrane, through which the drug diffuses; or the drug is held dissolved or suspended in a polymer matrix, through which the drug diffuses to the skin. Devices incorporating a reservoir will deliver a steady drug flux across the membrane as long as excess undissolved drug remains in the reservoir; matrix or monolithic devices are typically characterized by a falling drug flux with time, as the matrix layers closer to the skin are depleted of drug. Usually, reservoir patches include a porous membrane covering the reservoir of medication which can control release, while heat melting thin layers of medication embedded in the polymer matrix (e.g., the adhesive layer), can control release of drug from matrix or monolithic devices.
[0649] Accordingly, the active agent can be released from a patch in a controlled fashion without necessarily being in a controlled release formulation.
[0650] Patches can include a liner which protects the patch during storage and is removed prior to use; drug or drug solution in direct contact with release liner; adhesive which serves to adhere the components of the patch together along with adhering the patch to the skin; one or more membranes, which can separate other layers, control the release of the drug from the reservoir and multi-layer patches, etc., and backing which protects the patch from the outer environment.
[0651] Common types of transdermal patches include, but are not limited to, single-layer drug-in-adhesive patches, wherein the adhesive layer contains the drug and serves to adhere the various layers of the patch together, along with the entire system to the skin, but is also responsible for the releasing of the drug; multi-layer drug-in-adhesive, wherein which is similar to a single-layer drug-in-adhesive patch, but contains multiple layers, for example, a layer for immediate release of the drug and another layer for control release of drug from the reservoir; reservoir patches wherein the drug layer is a liquid compartment containing a drug solution or suspension separated by the adhesive layer; matrix patches, wherein a drug layer of a semisolid matrix containing a drug solution or suspension which is surrounded and partially overlaid by the adhesive layer; and vapor patches, wherein an adhesive layer not
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[0654] only serves to adhere the various layers together but also to release vapor. Methods for making transdermal patches are described in U. S. Patent Nos. 6,461,644, 6,676,961, 5,985,311, and 5,948,433.
[0655] G. Immune Checkpoint Modulators
[0656] The methods can include administering an immune checkpoint modulator to the subject, particular subjects with cancer. Immune checkpoints can be stimulatory or inhibitory, and tumors can use these checkpoints to protect themselves from immune system attacks. Currently approved checkpoint therapies block inhibitory checkpoint receptors, but investigations into therapies that activate stimulatory checkpoints are also underway. Thus, the immune checkpoint modulator can be one that blocks an inhibitory checkpoint, or activates a stimulatory checkpoint. Typically, the immune checkpoint modulator is one that induces or otherwise activates or increases an immune response against target cells for example cancer cells or infected cells. Accordingly, in some forms, the immune checkpoint modulator can be a chimeric antigen receptor (CAR) directed cell such as a CAR-T cell. In another form, the immune checkpoint modulator can be an oncolytic virus.
[0657] In preferred forms, the immune checkpoint modulator blocks an inhibitory checkpoint. Blockade of negative feedback signaling to immune cells thus results in an enhanced immune response against tumors. Thus, in some forms the immune checkpoint modulator is administered to the subject in an effective amount to block an inhibitory checkpoint. Exemplary compounds are those that block or otherwise inhibit, for example, PD-1, PD-L1, or CTLA4.
[0658] Dosage regimens or cycles of the agents can be completely or partially overlapping, or can be sequential. Thus, the immune checkpoint modulator can be administered before, concurrently with, after or any combination thereof with a disclosed compound or composition.
[0659] 1. PD-1 antagonists
[0660] In some fomrs, the active agents are PD-1 antagonists. Activation of T cells normally depends on an antigen-specific signal following contact of the T cell receptor (TCR) with an antigenic peptide presented via the major histocompatibility complex (MHC) while the extent of this reaction is controlled by positive and negative antigen-independent signals emanating from a variety of co-stimulatory molecules. The latter are commonly members of the CD28 / B7 family. Conversely, Programmed Death- 1 (PD-1) is a member of the CD28 family of receptors that delivers a negative immune response when induced on T cells. Contact
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[0663] between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that decreases T cell multiplication and / or the strength and / or duration of a T cell response.
[0664] Suitable PD-1 antagonists are described in U. S. Patent Nos. 8,114,845, 8,609,089, and 8,709,416, and include
[0665] compounds or agents that either bind to and block a ligand of PD-1 to interfere with or inhibit the binding of the ligand to the PD-1 receptor, or bind directly to and block the PD-1 receptor without inducing inhibitory signal transduction through the PD- 1 receptor.
[0666] In some forms, the PD-1 receptor antagonist binds directly to the PD-1 receptor without triggering inhibitory signal transduction and also binds to a ligand of the PD-1 receptor to reduce or inhibit the ligand from triggering signal transduction through the PD-1 receptor. By reducing the number and / or amount of ligands that bind to PD-1 receptor and trigger the transduction of an inhibitory signal, fewer cells are attenuated by the negative signal delivered by PD- 1 signal transduction and a more robust immune response can be achieved.
[0667] It is believed that PD-1 signaling is driven by binding to a PD-1 ligand (such as B7-H1 or B7-DC) in close proximity to a peptide antigen presented by major histocompatibility complex (MHC) (sec, for example, Freeman, Proc. Natl. Acad. Sci. U. S. A, 105:10275-10276 (2008)). Therefore, proteins, antibodies or small molecules that prevent co-ligation of PD-1 and TCR on the T cell membrane are also useful PD-1 antagonists.
[0668] In preferred forms, the PD-1 receptor antagonists are small molecule antagonists or antibodies that reduce or interfere with PD-1 receptor signal transduction by binding to ligands of PD-1 or to PD-1 itself, especially where co-ligation of PD-1 with TCR does not follow such binding, thereby not triggering inhibitory signal transduction through the PD-1 receptor.
[0669] Other PD-1 antagonists include antibodies that bind to PD-1 or ligands of PD-1 such as PD-L1 (also known as B7-H1) and PD-L2 (also known as B7-DC), and other antibodies.
[0670] Suitable anti-PD- 1 antibodies include, but are not limited to, those described in the following publications:
[0671] PCT / IL03 / 00425 (Hardy et al., WO / 2003 / 099196)
[0672] PCT / JP2006 / 309606 (Korman et al., WO / 2006 / 121168)
[0673] PCT / US 2008 / 008925 (Li et al., WO / 2009 / 014708)
[0674] PCT / JP03 / 08420 (Honjo et al., WO / 2004 / 004771)
[0675] PCT / JP04 / 00549 (Honjo et al., WO / 2004 / 072286)
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[0678] PCT / IB 2003 / 006304 (Collins et al., WO / 2004 / 056875) PCT / US2007 / 088851 (Ahmed et al., WO / 2008 / 083174)
[0679] PCT / US 2006 / 026046 (Korman et al., WO / 2007 / 005874)
[0680] PCT / US 2008 / 084923 (Terrett et al., WO / 2009 / 073533)
[0681] Berger et al., Clin. Cancer Res., 14:3044-3051 (2008).
[0682] A specific example of an anti-PD-1 antibody is MDX-1106 (see Kosak, US 20070166281 (pub. 19 July 2007) at par. 42), a human anti-PD-1 antibody, preferably administered at a dose of 3 mg / kg.
[0683] Exemplary anti-B7-Hl antibodies include, but are not limited to, those described in the following publications:
[0684] PCT / US06 / 022423 (WO / 2006 / 133396, pub. 14 December 2006) PCT / US07 / 088851 (WO / 2008 / 083174, pub. 10 July 2008)
[0685] US 2006 / 0110383 (pub. 25 May 2006)
[0686] A specific example of an anti-B7-Hl antibody is MDX-1105 (WO / 2007 / 005874, published 11 January 2007)), a human anti-B7-Hl antibody.
[0687] For anti-B7-DC antibodies see 7,411,051, 7,052,694, 7,390,888, and U. S. Published Application No. 2006 / 0099203.
[0688] The antibody can be a bi-specific antibody that includes an antibody that binds to the PD-1 receptor bridged to an antibody that binds to a ligand of PD-1, such as B7-H1. In some forms, the PD-1 binding portion reduces or inhibits signal transduction through the PD-1 receptor.
[0689] Other exemplary PD-1 receptor antagonists include, but are not limited to B7-DC polypeptides, including homologs and variants of these, as well as active fragments of any of the foregoing, and fusion proteins that incorporate any of these. In a preferred form, the fusion protein includes the soluble portion of B7-DC coupled to the Fc portion of an antibody, such as human IgG, and does not incorporate all or part of the transmembrane portion of human B7-DC.
[0690] The PD-1 antagonist can also be a fragment of a mammalian B7-H1, preferably from mouse or primate, preferably human, wherein the fragment binds to and blocks PD-1 but does not result in inhibitory signal transduction through PD-1. The fragments can also be part of a fusion protein, for example an Ig fusion protein.
[0691] Other useful polypeptides PD-1 antagonists include those that bind to the ligands of the PD-1 receptor. These include the PD-1 receptor protein, or soluble fragments thereof,
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[0694] which can bind to the PD-1 ligands, such as B7-H1 or B7-DC, and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction. B7-H1 has also been shown to bind the protein B7.1 (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). Such fragments also include the soluble ECD portion of the PD-1 protein that includes mutations, such as the A99L mutation, that increases binding to the natural ligands (Molnar et al., PNAS, 105:10483-10488 (2008)). B7-1 or soluble fragments thereof, which can bind to the B7-H1 ligand and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction, are also useful.
[0695] PD-1 and B7-H1 anti-sense nucleic acids, both DNA and RNA, as well as siRNA molecules can also be PD-1 antagonists. Such anti-sense molecules prevent expression of PD-1 on T cells as well as production of T cell ligands, such as B7-H1, PD-L1 and / or PD-L2. For example, siRNA (for example, of about 21 nucleotides in length, which is specific for the gene encoding PD-1, or encoding a PD-1 ligand, and which oligonucleotides can be readily purchased commercially) complexed with carriers, such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119(8): 2231-2244 (2009), are readily taken up by cells that express PD-1 as well as ligands of PD-1 and reduce expression of these receptors and ligands to achieve a decrease in inhibitory signal transduction in T cells, thereby activating T cells.
[0696] Exemplary PD- 1 inhibitors include, but are not limited to,
[0697] • Pembrolizumab (formerly MK-3475 or lambrolizumab, Keytruda) was developed by Merck and first approved by the Food and Drug Administration in 2014 for the treatment of melanoma.
[0698] • Nivolumab (Opdivo) was developed by Bristol-Myers Squibb and first approved by the FDA in 2014 for the treatment of melanoma.
[0699] • pidilizumab, by CureTech
[0700] • AMP-224, by GlaxoSmithKline and Medlmmune
[0701] • AMP-514, by GlaxoSmithKline and Medlmmune
[0702] • PDR001, by Novartis
[0703] • cemiplimab, by Regeneron and Sanofi
[0704] Exemplary PD-L1 inhibitors include, but are not limited to,
[0705] • Atezolizumab (Tecentriq) is a fully humanised IgGl (immunoglobulin 1 antibody developed by Roche Genentech. In 2016, the FDA approved atezolizumab for urothelial carcinoma and non-small cell lung cancer.
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[0708] • Avelumab (Bavencio) is a fully human IgGl antibody developed by Merck Serono and Pfizer. Avelumab is FDA approved for the treatment of metastatic merkel-cell carcinoma. It failed phase III clinical trials for gastric cancer.
[0709] • Durvalumab (Imfinzi) is a fully human IgGl antibody developed by AstraZeneca.
[0710] Durvalumab is FDA approved for the treatment of urothelial carcinoma and unresectable non-small cell lung cancer after chemoradiation.
[0711] • BMS-936559, by Bristol-Myers Squibb
[0712] • CK-301, by Checkpoint Therapeutics
[0713] See, e.g., Iwai, et al.. Journal of Biomedical Science, (2017) 24:26, DOI
[0714] 10.1186 / S12929-017-0329-9.
[0715] 2. CTLA4 antagonists
[0716] Other molecules useful in mediating the effects of T cells in an immune response are also contemplated as active agents. For example, in some forms, the molecule is an agent binds to an immune response mediating molecule that is not PD-1. In a preferred form, the molecule is an antagonist of CTLA4, for example an antagonistic anti-CTLA4 antibody. An example of an anti-CTLA4 antibody is described in PCT / US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).
[0717] Dosages for anti-PD-1, anti-B7-Hl, and anti-CTLA4 antibody, are known in the art and can be in the range of 0.1 to 100 mg / kg, with shorter ranges of 1 to 50 mg / kg preferred and ranges of 10 to 20 mg / kg being more referred. An appropriate dose for a human subject is between 5 and 15 mg / kg, with 10 mg / kg of antibody (for example, human anti-PD-1 antibody, like MDX-1106) most preferred.
[0718] Specific examples of CTLA antagonists include Ipilimumab, also known as MDX-010 or MDX-101, a human anti-CTLA4 antibody, preferably administered at a dose of about 10 mg / kg, and Tremelimumab a human anri-CTLA4 antibody, preferably administered at a dose of about 15 mg / kg. See also Sammartino, et al., Clinical Kidney Journal, 3(2): 135- 137 (2010), published online December 2009.
[0719] In other forms, the antagonist is a small molecule. A series of small organic compounds have been shown to bind to the B7-1 ligand to prevent binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277:7363-7368 (2002). Such small organics could be administered alone or together with an anti-CTLA4 antibody to reduce inhibitory signal transduction of T cells.
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[0722] 3. Chimeric Antigen Receptor directed cells
[0723] The modulator can be a chimeric antigen receptor directed cell. The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers to a set of polypeptides, typically two in the simplest forms, which when in an immune effector cell, provides the cell with specificity for a cancer cell, and with intracellular signal generation. In some forms, a CARincludes at least an antigen binding domain such as an extracellular binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to as "an intracellular signaling domain") including a functional signaling domain derived from a stimulatory molecule and / or costimulatory molecule as defined below. In one form, the stimulatory molecule is a zeta chain (“zeta stimulatory domain”) associated with a T cell receptor complex. In one form, the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one costimulatory molecule (e.g., 4-1BB (i.e., CD137), CD27 and / or CD28). In some forms, the CAR includes a chimeric fusion protein including an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain including a functional signaling domain derived from a stimulatory molecule. In various forms, CARs are fusion proteins of single-chain variable fragments (scFv) fused to a CD3-zeta transmembrane domain. However, other intracellular signaling domains such as CD28, 41 -BB and 0x40 may be used in various combinations to give the desired intracellular signal. In some forms, CARs disclosed herein include an extracellular binding domain.
[0724] The term “antigen binding domain” is used in the context of the present disclosure to refer to the portion of the CAR that specifically recognizes and binds to the antigen of interest. The “antigen binding domain” may be derived from a binding protein disclosed herein such as an antibody or fragment thereof. In some forms, the “binding domain” is a single-chain variable fragment (scFv). In certain forms, the “binding domain” includes the complementarity determining regions of a binding protein disclosed herein. In this form, the CAR directed cell can represent the combination of a cell-penetrating antibody (assuming it penetrates a cancer cell) that induces or increase DNA damage or reduces or impairs DNA damage repair, or a combination thereof and an immune checkpoint modulator that induces, increases, or enhances an immune response. For example, the binding domain can represent the cell-penetrating antibody and the modified T-cell can represent the immune cell modulator. In another example, a CAR-directed cell disclosed herein is administered with a cell-penetrating antibody disclosed herein.
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[0727] The terms “zeta” or “CD3-zeta” are used herein to define the protein provided as GenBank Acc. No. BAG36664.1, or the equivalent residues from a non- human species and a “zeta stimulatory domain” or alternatively a “CD3-zeta stimulatory domain” is defined as the amino acid residues from the cytoplasmic domain of the zeta chain, or functional derivatives thereof, that are sufficient to functionally transmit an initial signal necessary for T cell activation.
[0728] The term “immune effector cell,” is used herein to refer to a cell that is involved in an immune response (e.g. promotion of an immune effector response). Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloic-derived phagocytes. In some forms, the immune effector cell(s) is allogenic. In some forms, the immune effector cell(s) is autologous. In some forms, the immune checkpoint modulator is a CAR directed T cell (CAR-T cell). Exemplary CAR-T cells include Axicabtagene ciloleucel (KTE-C19, Axi-cel), Tisagenlecleucel, Lisocabtagene Maraleucel (liso-cel; JCAR017).
[0729] Immune effector cells such as T cells may be activated and expanded generally using methods previously described, such as for example, as described in U. S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041. As a general example, a population of immune effector cells e.g., T regulatory cell depleted cells, may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3 complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells.
[0730] 4. Oncolytic virus
[0731] The modulator can be an oncolytic virus. The term “oncolytic virus” is used in the context of the present disclosure to refer to viruses that are able to infect and reduce growth of cancer cells. For example, oncolytic viruses can inhibit cell proliferation. In some forms, oncolytic viruses can kill cancer cells. In some forms, the oncolytic virus preferentially infects and inhibits growth of cancer cells compared with corresponding normal cells. In another form, the oncolytic virus preferentially replicates in and inhibits growth of cancer cells compared with corresponding normal cells.
[0732] In some forms, the oncolytic virus is able to naturally infect and reduce growth of cancer cells. Examples of such viruses include Newcastle disease virus, vesicular stomatitis, myxoma, reovirus, sindbis, measles and coxsackievirus. Oncolytic viruses able to naturally
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[0735] infect and reduce growth of cancer cells generally target cancer cells by exploiting the cellular aberrations that occur in these cells. For example, oncolytic viruses may exploit surface attachment receptors, activated oncogenes such as Ras, Akt, p53 and / or interferon (IFN) pathway defects.
[0736] In another form, oncolytic viruses encompassed by the present disclosure are engineered to infect and reduce growth of cancer cells. Exemplary viruses suitable for such engineering include oncolytic DNA viruses, such as adenovirus, herpes simplex virus (HSV) and Vaccinia virus; and oncolytic RNA viruses such as Lentivirus, Reovirus, Coxsackievirus, Seneca Valley Virus, Poliovirus, Measles virus, Newcastle disease virus, Vesicular stomatitis virus (VSV) and parvovirus such as rodent protoparvoviruses H-1PV. In some forms, the oncolytic virus includes a backbone of an above referenced virus.
[0737] In some forms, tumor specificity of an oncolytic virus can be engineered to mutate or delete gene(s) required for survival of the vims in normal cells but expendable in cancer cells. For example, the oncolytic vims can be engineered by mutating or deleting a gene that encodes thymidine kinase, an enzyme needed for nucleic acid metabolism. In this example, vimses are dependent on cellular thymidine kinase expression, which is high in proliferating cancer cells but repressed in normal cells. In another example, the oncolytic vims is engineered to include a capsid protein that binds a tumor specific cell surface molecule. In some fomrs, the capsid protein is a fibre, a penton or hexon protein. In another example, the oncolytic vims is engineered to include a tumor specific cell surface molecule for transductionally targeting a cancer cell. Exemplary tumor specific cell surface molecules can include an integrin, an EGF receptor family member, a proteoglycan, a disialoganglioside, B7-H3, CA-125, EpCAM, ICAM-1, DAF, A21, integrin-α2β1, vascular endothelial growth factor receptor 1, vascular endothelial growth factor receptor 2, CEA, a tumour associated glycoprotein, CD19, CD20, CD22, CD30, CD33, CD40, CD44, CD52, CD74, CD152, CD 155, MUC1, a tumor necrosis factor receptor, an insulin-like growth factor receptor, folate receptor a, transmembrane glycoprotein NMB, a C-C chemokine receptor, PSMA, RON-receptor, and cytotoxic T-lymphocyte antigen 4.
[0738] The oncolytic vims can be replication-competent. In some forms, the oncolytic vimses selectively replicate in cancer cells when compared with corresponding normal cells.
[0739] Conditional replication can be achieved by, for example, the insertion of a tumorspecific promoter driving the expression of a critical gene(s). Such promoters can be identified based on differences in gene expression between tumor and corresponding
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[0742] surrounding tissue. Exemplary native promoters include AFP, CCKAR, CEA, erbB2, Cerb2, COX2, CXCR4, E2F1, HE4, LP, MUC1, PSA, Survivin, TRP1, STAT3, hTERT and Tyr. Exemplary composite promoters include AFP / hAFP, SV40 / AFP, CEA / CEA, PSA / PSA, SV40 / Tyr and Tyr / Tyr.
[0743] Various viruses may be engineered as outlined in the above referenced examples. The oncolytic virus can be, for example, a modified HSV, Lentivirus, Baculovirus, Retrovirus, Adenovirus (AdV), Adeno-associated virus (AAV) or a recombinant form such as recombinant adeno-associated virus (rAAV) or a derivative thereof such as a self-complementary AAV (scAAV) or non-integrating AV. The oncolytic virus can be a modified HSVThe oncolytic virus can be a modified lentivirus. Other exemplary viruses include vaccina virus, vesicular stomatitis virus (VSV), measles virus and maraba virus.
[0744] In other examples, the oncolytic virus may be one of various AV or AAV serotypes. In some forms, the oncolytic virus is serotype 1. In another example, the oncolytic virus is serotype 2. In other examples, the oncolytic virus is serotype 3, 4, 7, 8, 9, 10, 11, 12 or 13. In another example, the oncolytic virus is serotype 5. In another example, the oncolytic virus is serotype 6.
[0745] Exemplary oncolytic viruses include T-Vcc (HSV-1; Amgen), JX-594 (Vaccina; Sillajen), JX-594 (AdV; Cold Genesys), Reolysin (Reovirus; Oncolytics Biotech). Other examples of oncolytic viruses are disclosed in WO 2003 / 080083, WO 2005 / 086922, WO 2007 / 088229, WO 2008 / 110579, WO 2010 / 108931, WO 2010 / 128182, WO 2013 / 112942, WO 2013 / 116778, WO 2014 / 204814, WO 2015 / 077624 and WO 2015 / 166082, WO 2015 / 089280.
[0746] 5. Other Immune Checkpoint Modulators
[0747] Other immune checkpoint targets include, but are not limited to, ICOS, 0X40, GITR, 4-1BB, CD40, CD27-CD70, LAG3, TIM-3, TIGIT, VISTA, B7-H3, KIR, PARP, and others, and are being targeting for cancer treatment alone and in combination with anti-PD-1, anti-PD-L1, and anti-CTLA compounds. See, for example, Iwai, et al., Journal of Biomedical Science. 24 (1): 26. doi:10.1186 / sl2929-017-0329-9; Donini, et al., J Thorac Dis. 2018 May;10(Suppl 13): S 1581-S 1601. doi: 10.21037 / jtd.2018.02.79. Thus, in some forms, a cellpenetrating antibody is administered in combination with a compound that targets ICOS, 0X40, GITR, 4- IBB, CD40, CD27-CD70, LAG3, TIM-3, TIGIT, VISTA, B7-H3, KIR, or PARP, or a combination thereof, alone or in combination with a compound that target PD-1,
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[0750] PD-L1, and / or CTLA. In another form, the immune checkpoint modulator is an antibody disclosed in WO 2016 / 013870.
[0751] The disclosed invention can be further understood by the following numbered paragraphs:
[0752] 1. A method of enhancing, increasing, accelerating or / and otherwise improving an immune response in a subject in need thereof comprising administering the subject an effective amount of a compound that increases expression and / or levels of one or more transporters associated with antigen processing (TAP) proteins, optionally selected from TAP2, TAPI, and combinations thereof, and / or reduces expression and / or levels of Suppressor of Cytokine Signaling 1 (SOSC1) and / or interleukin 4 (IL-4), and optionally further comprising administering the subject an immune checkpoint modulator.
[0753] 2. The method of paragraph 1, wherein the subject has cancer, optionally wherein the cancer is Non-Small Cell Lung Cancer (NSCLC), or an infection.
[0754] 3. The method of paragraphs 1 and 2, wherein the compound increases the level of TAP2 and / or TAPI protein in cells of the subject, optionally wherein the cells are cancer cells.
[0755] 4. The method of any one of paragraphs 1-3, wherein the compound is TAP2 or TAPI, or functional fragment or variant thereof, or a nucleic acid encoding the TAP2 or TAPI or functional fragment or variant, or any combination thereof.
[0756] 5. The method of any one of paragraphs 1-4, wherein the compound is Dasatinib, Vinorelbine, Docetaxel, Vinblastine, Vindesine, Mitomycin, Colchicine, Digoxin, Ouabain, Pyrithione Zinc, Oxibendazole, Albendazole, Fenbendazole, Podofilox, Gentian, Emetine, Oxamflatin, BML-281, SAHA / Vorinostat, or another compound of Figures 7B, 7C, 7D, or 7E.
[0757] 6. A method of reducing or otherwise preventing an immune response in a subject in need thereof comprising administering the subject an effective amount of a compound that decreases expression and / or levels of one or more transporters associated with antigen processing (TAP) proteins, optionally selected from TAP2, TAPI, and combinations thereof, and / or reduces expression and / or levels of Suppressor of Cytokine Signaling 1 (SOSC1) and / or interleukin 4 (IL-4), optionally wherein the compound is selected from aminopterin, pemetrexed, dinitolmide, amprolium, and the compounds of Figures 7N-7O.
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[0760] 7. The method of paragraph 6, wherein the subject has an autoimmune or inflammatory disease or disorder, transplant rejection optionally wherein the transplant comprises cells, tissue, and / or organs, and / or graft verse host disease (GVHD).
[0761] 8. The method of paragraphs 6 or 7, wherein the compound reduces the level of TAP2 and / or TAPI protein in cells of the subject, optionally wherein the cells at the site of autoimmunity or inflammation, transplant, or GVHD.
[0762] 9. The method of any one of paragraphs 5-7, wherein the compound is a functional nucleic acid that targets the TAP2 and / or TAPI gene or a gene product thereof.
[0763] 10. A method of selecting a subject for treatment comprising detecting the level of TAP2, TAPI, SOSC1, and / or IL-4 in a sample from the subject, and selecting the subject for treatment when any one or more of TAP2, TAPI, SOSC1, and IL-4 is higher or lower than a control.
[0764] 11. The method of paragraph 10, wherein a level of TAP2 lower than the control indicates the subject is in need of an increased immune response.
[0765] 12. The method of paragraphs 10 or 11, wherein a level of TAPI lower than the control indicates the subject is in need of an increased immune response.
[0766] 13. The method of any one of paragraphs 10-12, wherein a level of SOCS1 higher than the control indicates the subject is in need of an increased immune response.
[0767] 14. The method of any one of paragraphs 10-13, wherein a level of IL-4 higher than the control indicates the subject is in need of an increased immune response.
[0768] 15. The method of any one of paragraphs 10-14, wherein a level of TAP2 higher than the control indicates the subject is in need of an reduced immune response.
[0769] 16. The method of any one of paragraphs 10-15, wherein a level of TAPI higher than the control indicates the subject is in need of an increased reduced response.
[0770] 17. The method of any one of paragraphs 10-16, wherein a level of SOCS1 lower than the control indicates the subject is in need of an reduced immune response.
[0771] 18. The method of any one of paragraphs 10-17, wherein a level of IL-4 lower than the control indicates the subject is in need of an reduced immune response.
[0772] 19. The method of any one of paragraphs 10-18, wherein the TAP2, TAPI, SOSC1, and / or IL-4 level is detected by measuring one or more of TAP2, TAPI, SOSC1, and / or IL-4 mRNA and / or protein.
[0773] 20. The method of any one of paragraphs 10-19, wherein the sample is a tissue sample, optionally a tumor biopsy.
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[0776] 21. The method of any one of paragraphs 10-20 further comprising treating the subject to increase or decrease an immune response.
[0777] 22. The method of paragraph 21 according to the method of any one of paragraphs 1-9.
[0778] 23. A method of enhancing, increasing, accelerating or / and otherwise improving an immune response in a subject in need thereof comprising administering the subject an effective amount of a compound that and / or decreases the intranuclear level or bioavailability of basic peptides and / or increases the level or bioavailability of intranuclear acidic peptides in cancer or infected cells or other target cells in which an increased sensitivity to an immune and / or inflammatory response thereto is desired, and optionally further comprising administering the subject an immune checkpoint modulator, optionally wherein the subject has cancer, optionally wherein the cancer is Non-Small Cell Lung Cancer (NSCLC), or an infection.
[0779] 24. A method of reducing or otherwise preventing an immune response in a subject in need thereof comprising administering the subject an effective amount of a compound that increases the level or bioavailability of intranuclear basic peptides and / or decreases the level or bioavailability of intranuclear acidic peptides in autoimmune and / or transplanted cells / tissue or other target cells in which an reduced sensitivity to an immune and / or inflammatory response thereto is desired, optionally wherein the subject has an autoimmune or inflammatory disease or disorder, or a transplant an optionally is in danger of rejection thereof optionally wherein the transplant comprises cells, tissue, and / or organs, and / or graft verse host disease (GVHD).
[0780] 25. A method of modulating gene expression in cells comprising modulating the level or bioavailability of basic and / or acid peptides in the nucleus of the cells.
[0781] 26. The method of any one of paragraphs 23-25 wherein the compound that increases level or bioavailability of basic peptides and / or that decreases levels or bioavailability of acidic peptides is (i) a plurality of the same or different basic peptides or a nucleic acid encoding the same.
[0782] 27. The method of any one of paragraphs 23-25 wherein the compound that increases level or bioavailability of acidic peptides and / or that decreases levels or bioavailability of basic peptides is a plurality of the same or different acidic peptides or a nucleic acid encoding the same.
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[0785] 28. The method of any one of paragraphs 23-25, wherein the compound is one that targets microproteins and / or induced formation of defective ribosomal products, optionally wherein the compound is one that modulates protein synthesis optionally causing premature chain termination during translation.
[0786] 29. The method of any one of paragraphs 23-25, wherein the compound is an ERAP1 and / or ERAP2 inhibitor or activator.
[0787] 30. The method of any one of paragraphs 23-25, wherein the compound increases protein degradation via the Ubiquitin-Proteasome System (UPS).
[0788] 33. A composition or method according to any of this disclosure herein including but not limited to a text, figures, and combinations thereof.
[0789] 34. A composition or method according to the any of experiments provided herein.
[0790] 35. A pharmaceutical composition comprising an effective amount of the compound of any one of the foregoing paragraphs for use the method of any one of the foregoing paragraphs.
[0791] Examples
[0792] Material and Methods
[0793] Patients, cohorts, and tissue microarrays
[0794] Formalin-fixed paraffin-embedded (FFPE) samples from seven previously reported retrospective collections of primary NSCLCs represented in tissue microarrays (TMAs) were analyzed(Datar et al., 2021). The first collection includes samples from 342 patients with NSCLC collected at Sotiria General Hospital and Patras University General Hospital (Greece) between 1991 and 2001 (cohort #1). Another TMA-based cohort collected included pretreatment biopsy or resection samples from 375 patients with NSCLC at Yale between 1999 and 2007 (cohort #2). NSCLC samples from patients treated with PD- 1 axis blockers were retrospectively collected from Yale University in 2 independent cohorts (cohort #3, n=107 and cohort #4, n=75). Additional NSCLC collection includes samples from 259 patients with NSCLC seen at Yale Pathology between 1988 and 2012 (cohort #5). The 6thcollection included pretreatment biopsy or resection samples from 189 patients with NSCLC, seen at Yale Pathology (New Haven, CT) between 1988 and 2003 (cohort #6). The last collection includes primary resected lung adenocarcinomas from 130 patients collected between 2020-2022 at Yale University that were clinically tested for EGFR and KRAS mutations (cohort #7). All cases in the cohorts were reviewed by a local pathologist using
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[0797] hematoxylin and eosin-stained preparations and tumor histology variant was confirmed by morphology analysis. Tumor cores for TMA construction were obtained from case areas selected by a pathologist to represent the disease. Tumor core selection was not based on specific tumor segments or location. Clinicopathologic information from patients in both cohorts was collected from clinical records and pathology reports. All the studies were conducted in accordance with recognized ethical guidelines (e.g., Declaration of Helsinki, CIOMS, Belmont Report, U. S. Common Rule) and tissue and clinical information were used after approval from the Yale Human Investigation Committee protocols 9505008219, 1412015109, 1608018220, and 1603017333, which approved the patient consent forms or waiver of consent.
[0798] Analysis of public datasets
[0799] To determine the frequency and type of mutations in TAP2 occurring in NSCLC, analysis of data from publicly available TCGA lung cancer cohorts was performed. The analysis of variants was performed using paired germline DNA from each case as a reference using the cBioPortal bioinformatic pipeline (Cerami et al., 2012; Gao et al., 2013). Genomic alterations considered to be deleterious included missense mutations, truncating mutations, and deep (e.g., homozygous) deletions. Analysis of the cohorts included both primary lung adenocarcinomas and squamous cell carcinomas. Comparative analysis of mRNA expression of the TAPI, TAP2, IFNy, IL-4, and IL-8 from TCGA NSCLC cohort was conducted using batch-normalized RNA sequencing at transcript levels expressed as RNA-Seq by expectation-maximization units.
[0800] Multiplexed quantitative immunofluorescence (mQIF)
[0801] Multiplexed QIF protocols for FFPE tissue specimens were developed for simultaneous detection of the cell permeant nuclear dye 4',6-diamidino-2-phenylindole (DAPI), pancytokeratin (CK), TAPI, and TAP2 using isotype-specific antibodies and different fluorescence conjugates as described previously by our group(Datar et al., 2021; Henick et al., 2022; Schalper et al., 2017). For the multiplexed staining, sections were deparaffinized and subjected to antigen retrieval using EDTA Buffer (Sigma-Aldrich; pH = 8) and boiled for 20 minutes at 97°C in a pressure-boiling container (PT Module, Lab Vision). Slides were then sequentially incubated at room temperature with dual Endogenous Peroxidase Block (Dako, No. S2003) for 10 minutes and with a blocking solution containing 0.3% BSA in 0.05% Tween solution for 30 minutes. Secondary antibodies and fluorescence reagents used were anti-rabbit Envision (K4003, Dako) biotinylated
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[0804] Tyramide / Streptavidin-Alexa750 conjugate (PerkinElmer), anti-mouse IgG2a antibody (Abeam) with Cy3 plus (PerkinElmer), goat anti-rabbit (Abeam), and anti-mouse IgGl antibody (Abeam) with Cy5-tyramide (PerkinElmer). Residual horseradish peroxidase activity between incubations with secondary antibodies was eliminated by exposing the slides twice for 10 minutes at room temperature to a solution containing benzoic hydrazide (0.136 mg) and hydrogen peroxide (50 pL). To determine the reproducibility of the QIF assay, serial sections from the index TMA containing positive and negative controls were measured at different timepoints.
[0805] Tissue fluorescence measurement and scoring
[0806] Quantitative measurement of the fluorescence signal was performed using the AQUA method that enables objective and sensitive measurement of targets within user-defined tissue compartments (Datar et al., 2021; Henick et al., 2022; Schalper et al., 2017). The TMA slides were scanned using an automated HistoRx PM2000 multispectral slide scanner with 20x magnification and autoexposure configuration. Briefly, the QIF score of each target in CK- positive (CK+) cancer cell compartment was calculated by dividing the target pixel intensities by the area of CK positivity. Scores were normalized to the exposure time and bit depth at which the images were captured, allowing scores collected at different exposure times to be comparable. Markers were also measured in the CK-negative stromal cell tissue compartment by collecting the signal score in the area defined by DAPI staining and lacking CK+pixels. A cancer- cell / stromal cell ratio of the markers <1 or <1.4 was considered as cancer cell-selective marker downregulation for specific analyses. The specific marker staining patterns and cancer cell- specific loss was confirmed by visual inspection by trained personnel.
[0807] RNAscope™ coupled immunohistochemistry (IHC)
[0808] Multiplexed QIF protocols for FFPE tissue specimens were developed for simultaneous detection of the IL-4 mRNA (red), CDllb mRNA (white) TAP2 protein (yellow), cytokeratin protein (CK, green) using RNAscope™ assay followed by immunohistochemistry. In brief, the FFPE tissue sections were deparaffinized, endogenous peroxidase activity was quenched with hydrogen peroxide followed by target retrieval and protease plus treatment. The RNAscope™ assay was then performed using the RNAscope™ Multiplex Fluorescent V2 kit containing IL-4 and GDI lb in Cl and C2 respectively. Post RNA probing, the slides were sequentially stained for TAP2, and CK proteins followed by DAPI nuclear staining. The TMA slides were scanned using an
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[0811] automated Akoya Vectra Polaris slide scanner with 20x magnification and autoexposure configuration. Quantitative measurement of the fluorescence signal was performed using the AQUA method that enables objective and sensitive measurement of targets within user-defined tissue compartments(Datar et al., 2021; Henick et al., 2022; Schalper et al., 2017).
[0812] Cell culture and transfection
[0813] The lung adenocarcinoma cells lines A549 (HLA-A2+, KRAS mutant), PC9 (HLA-A2+, EGFR mutant), H1975 (HLA-A2", EGFR mutant) and H520 (HLA-A2-, KRAS / EGFR wild type) were originally purchased from the American Type Culture Collection (ATCC). PC9, H1975 and H520 cells were grown in RPMI-1640 culture media and A549 cells were grown in DMEM culture media. All the culture media were supplemented with 10% fetal bovine serum and Penicillin-streptomycin antibiotic cocktails. Cells were incubated in a humidified incubator supplied with 5% CO2. Exponentially grown cells were used in this study. Cell lines were authenticated every 3-6 months using the GenePrint® 10 System in the Yale University DNA Analysis Facility. Cells were also periodically tested for mycoplasma contamination.
[0814] To mimic cancer-cell downregulation of TAPI & TAP2, TAPI and TAP2 expression was transiently reduced in A549 and PC9 cells. Briefly, cells were transfected with lOOnM scrambled ON- TARGETplus SMARTpool small interfering RNA (siRNA) against TAPI and TAP2 (Dharmacon) (4 pooled siRNAs for each gene) using lipofectamine 3000 reagent (Invitrogen). Post forty-eight hours of transfection the knockdown efficacy was confirmed at RNA and protein levels.
[0815] For overexpression studies, 250ng of N-terminal FLAG tag TAP2 constructs (Genecopoeia, Cat# EX-Z2364-M11) were transfected to A549 cells using lipofectamine 3000 reagent. Cells were incubated for 48 hours. Transfection efficiency was confirmed by intracellular staining of FLAG tag (CST, cat# 8146S) and subsequent analysis was performed using FCS Express software version 7 (Devovo software). Data shown include average results from at least 4 replicates from more than two independent experiments.
[0816] Generation of TAP2 knockout cells
[0817] A549 cells were transfected with pSpCas9 BB-2A-GFP (GenScript, Cat# SC1678) containing TAP2 targeting gRNA sequence 5’-GCGCCTTGTACCTGCTCGTA-3’ (SEQ ID NO: 16). Cells were incubated for 48hours followed by screening of GFP positive cells using cell sorter (BD Biosciences). GFP positive cells were serially diluted to obtain monoclonal cell population. Monoclonal TAP2 knockout clones were confirmed by TAP2 mRNA
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[0820] expression, immunofluorescence, and DNA sequencing analysis. Data shown include average results from at least 4 replicates from more than two independent experiments.
[0821] Quantitative Real time PCR (qRT-PCR)
[0822] The total mRNA was isolated using Trizol reagent (Thermo Fisher Scientific Cat# 15596026). cDNA was prepared from Ipg of total mRNA using iScript cDNA Synthesis Kit (Bio-Rad, Cat#1708891) and qRT-PCR was performed using iQ SYBR Green Super Mix (Bio-Rad, Cat #1708880) with normalization to GAPDH. Primer sequences as per Table 5. Data shown include average results from at least 4 replicates from more than two independent experiments.
[0823] Table 5: Real-time PCR primer details.
[0824] Genes Primers SEQ ID NO:
[0825] Forward: 5’- TACAAGATGGCTCAGCCGATA -3’ SEQ ID NO: 17 Human TAPI
[0826] Reverse: 5’- ACCTGTCTGGTTCTGTTGGAA -3’ SEQ ID NO: 18 Forward: 5 ’ -T A A ATGCCAATGTGCTCTTG-3 ’ SEQ ID NO: 19 Human TAP2
[0827] Reverse: 5’- AAGCACTTCCTGATGGCGG-3’ SEQ ID NO:20 Forward: 5’- TGCACCACCAACTGCTTAGC -3’ SEQ ID NO:21 Human GAPDH
[0828] Reverse: 5’- GGCATGGACTGTGGTCATGAG -3’ SEQ ID NO: 22
[0829]
[0830] NanoString analysis
[0831] The differential gene expression analysis with approximately 770 genes was performed using the nCounter PanCancer Pathways Panel (NanoString Technologies). Post pretreatment the concentration and purity of each RNA sample from A549 was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific). The 200ng total RNA transcripts were hybridized with PanCancer reporter probes included in the 770-plex PanCancer 10360 panel. Post
[0832] hybridization, samples were placed into the nCounter MAX / FLEX System Prep Station to measure RNA counts. Finally, the RNA counts were normalized for technical efficiency by the geometric mean of internal control probes using nSolver 4.0 Advanced Analysis software. The differential expression data represents normalized counts transformed to log2 scale.
[0833] Human Phospho-Kinase Array
[0834] A549 cells were transfected with lOOnM scrambled ON-TARGETplus SMARTpool small interfering RNA (siRNA) against TAPI and TAP2 (Dharmacon) (4 pooled siRNAs
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[0837] for each gene) using lipofectamine 3000 reagent (Invitrogen). Post forty-eight hours of transfection the cells were treated with IFNy+TNFa (20ng / ml each) for 24hours. Post incubation, the expression levels of phospho kinases were examined using the Proteome Profiler Human Phospho-Kinase Array Kit (ARY003C, R& D Systems, Inc. USA) according to the manufacturer’s instructions. Data shown include average results from at least 4 replicates from more than two independent experiments.
[0838] Acid stripping and peptide reloading assay
[0839] Cells (2 x 106 / ml) were incubated with citric acid-Na2HPO4 buffer (0.263 M citric acid and 0.123 M Na2HPO4 containing 1% BSA (pH 3)) for 2 minutes, washed three times with 5 ml of culture medium. Further, cells were incubated with HER2369-377 and MAGE3271-279 peptides (50µM and 100µM) in a CO2 incubator for 2 hours. Finally, cells were washed and analyzed using antibodies mAb 4F10 specific to HLA-A2 bound to the HER2 nonamer peptide KIFGSLAFL (corresponding to amino acids 369-377 of the full-length HER2 protein (HER2369-377)) (Krokhotin et al., 2019) and mAb 12B6 specific to HLA-A2 bound to the MAGE3 nonamer peptide FLWGPRALV (corresponding to amino acids 271-279 of the full-length MAGE3 protein (MAGE3271-279))(Wu et al., 2018) and examined on a LSR II flow cytometer (BD Biosciences). Subsequent analysis was performed using FCS Express software version 7 (Devovo software). Data shown include average results from at least 4 replicates from more than two independent experiments.
[0840] Flow cytometry analysis
[0841] For intracellular staining: post treatment cells were fixed with 4%PFA for 15 minutes and permeabilized with 100% methanol for 30 minutes on ice. Cells were washed three times with FACS buffer (2% fetal bovine serum + 0.5% bovine serum albumin) followed by incubation with antibodies targeting TAPI (this study), TAP2 (abeam, Cat# abl80611) and SOCS 1 (abeam, Cat# ab9870) for Ihour in ice. Cells were washed three times with FACS buffer followed by staining with APC conjugated secondary antibodies and evaluated on a LSR II flow cytometer (BD Biosciences). Subsequent analysis was performed using FCS Express software version 7 (Devovo software Data shown include average results from at least 4 replicates from more than two independent experiments.
[0842] Single cell isolation and TECS preparation from primary NSCLC tumor tissues The fresh human lung tumor samples were washed with RPMI-1640 culture medium, cut into small pieces of approximately 2-3 mm3and incubated with tissue digestion enzyme mix (Miltenyi Biotec, Cat#130-108-339) over gentleMACS
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[0845] Dissociator (Miltenyi Biotec.) for 20 minutes. The digested tissue suspension was passed through cell strainer and centrifuged at 300g for 7minutes. The cell pellet was dissolved in RPMI-1640 medium and cell viability was measured using 0.4% trypan blue solution. The autologous CD3 T-cells (TESC; CD3 T-cell eliminated cell suspension) were eliminated from the tumor cell mixture using CD3 enrichment microbeads (Miltenyi Biotec, Cat# 130-050-101) as per manufacturer instructions. For flow analysis, the tumor single cell suspension was incubated with Fc blocker (Biolegend, Cat# 422302) for 15min and further incubated with EpCAM (Biolegend, Cat# 324252), CD3 (BD Biosciences, Cat# 555333), Annexin-V (Biolegend, Cat# 640922), CD8 (BD Biosciences, Cat# 566852) CDllb (BD Biosciences, Cat# 563839) and CD25 (BD Biosciences, Cat# 560987) for 25 minutes. Cells were acquired using LSR II flow cytometer (BD Biosciences). Subsequent analysis was performed using FCS Express software version 7 (Devovo software).
[0846] ATACseq and bulkRNA sequencing
[0847] Bulk ATAC-seq reads were trimmed using TrimGalore (https: / / github.com / FelixKrueger / TrimGalore) and aligned to the human genome (Hg38 assembly) using Bowtie2(v2.3.4.3) (Langmead and Salzberg, 2012). Sambamba (v0.7.1) (Tarasov ct al., 2015) used to sort the bam file and unpaired reads were removed. PCR duplicates were removed using Picard (vl.31) mark duplicates function (http: / / broadinstitute.github.io / picard / ) and further mitochondrial reads were removed using Samtools(v.1.12) view function (Li et al., 2009). All downstream analyses were performed on these filtered reads. Peak calling and differential accessible peaks were identified using MACS2 (V2.2.7.1) (Zhang et al., 2008) with default parameters except -nomodel, extsize 200 -SPMR. Further filtering of enriched peaks performed based on the peaks with adjusted p- value <0.05 and fold enrichment score. Further differentially enriched peaks were annotated using ChlPseeker R package (Yu et al., 2015). Genes with peaks located around 3kb proximity were considered as promoter associated peaks and enriched differential peaks regions were used to identify motif identification using motif discovery function in MEME Suite (Bailey et al., 2015). Motifs identified in our gene of interest, TAP2 were used for further transcription factors and its binding site using AnimalTFDB(v3.0) (Hu et al., 2019). Annotated transcription factors were further filtered based on qvalue <0.05 and high score. For the purpose of visualization, genome coverage files from filtered final bam files were generated using Bedtools(v2.30.0) (Quinlan and Hall, 2010) and each position was normalized by dividing the total library size and multiplying by 106. Further bedgraph files
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[0850] were converted into bigWig format using the bedGraphToBigWig command from the UCSC genome browser tool. Integrated genome viewer (IGV) (v2.13.2) used for peaks visualization and comparison.
[0851] Ex vivo tumor cell killing assay using autologous tumor / immune cell co-cultures Single-cell suspension from the NSCLC tumor tissues samples were incubated for 16hours in a CO2 incubator followed by stimulation with IFNy and TNFa (20ng / ml each) or recombinant IL-4 (20ng / ml) for 24 hours. Post incubation, cells were treated with anti-IL-4Ra (5pg / ml) and / or pembrolizumab (5pg / ml) for an additional 72hours. To analyze tumor cell death, cells were stained with surface markers (EpCAM, Annexin-V, CD8, CD25; as mentioned above) and acquired using LSR II flow cytometer (BD Biosciences). Subsequent analysis was performed using FCS Express software version 7 (Devovo software).
[0852] In vitro tumor killing assay, cytotoxicity assay and cell proliferation assay Cells were either transfected with scrambled siRNA or siRNA against TAPI (siTAPl), TAP2 (siTAP2) and TAP with TAP2 (siTAPl+TAP2) using lipofectamine 3000 reagent (Invitrogen). After 48 hours of transfection, cells were stimulated with alone IFNy or IFNy with TNF for an additional 24 hours. Post incubations, cells were incubated with anti-HER2 / neu T cells (Cellero, specifically recognize 369-377 amino acid region of the HER2 protein) at a 2:1 and 5:1 effector- to-target ratio for 72hours. Annexin V staining assay was carried out using FITC Annexin V Apoptosis Detection Kit (BioVision, Cat# K101) as per manufacturer instruction and analyzed by flow cytometer (BD Biosciences). Subsequent analysis was performed using FCS Express software version 7 (Devovo software). Cytotoxicity assay was carried out using CyQUANT™ LDH Cytotoxicity Assay Kit (ThermoFisher Scientific, Cat#C20302) as per manufacturer instruction. Plates were acquired using Tecan microplate reader. Data represents % LDH release.
[0853] Cell viability assay was performed in a 96 well plate using CyQUANT™ MTT Cell Viability Assay (ThermoFisher Scientific, Cat# V13L54) as per manufacturer instruction. Plates wereacquired using Tecan microplate reader. Data represents % cell viability Data shown include average results from at least 4 replicates from more than two independent experiments.
[0854] High throughput drug screening
[0855] A549 cells were screened with the Enzo 640 FDA-approved drugs, Pharmakon-1600 (Microsource Discovery Systems) and Enzo Epigenetics library available at the Yale Center
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[0858] for Molecular Discovery. Briefly, cells were plated at 2000 cells / well in 20µl in glass bottom 384-well imaging plates (Cellvis, P384-1.5H-N) and incubated at 37°C for 24 hours.
[0859] 20nL of 10 mM DMSO stock of each screening compound (or DMSO alone vehicle control used as Negative Control) was transferred using Echo Acoustic Dispenser (Labcyte, now Beckman) to assay plates containing 20µl A549 cells for a final screening compound concentration of lOpM (0.1% DMSO final). After 24 hours of compound treatment, cells were gently washed with 30µl of PBS and fixed with 4% (v / v) paraformaldehyde (Electron Microscopy Sciences 15710-S) diluted in PBS at room temperature for 20 min. Cells were washed twice with 30pl PBS, then permeabilized with 30µl of 0.5% (v / v) Triton X-100 in PBS for 5 min. Following fixation and permeabilization, cells were washed twice with 30µl PBS and incubated with 30µl of blocking buffer consisting of 10% (v / v) FBS (Gibco, 16140-071) diluted in PBS, for 1 hour at room temperature. Primary antibody solution was prepared by diluting TAP2 (TAP2 Rb polyAb, Abeam Cat# abl80611) or PD-L1 (PD-L1 Rb mAb, CST, Cat# 13684S) antibody at 1:250 (v / v) in blocking buffer. After blocking, cells were incubated with 20µl of antibody solution (either TAP2 or PD-L1) at 4°C overnight. Next day, cells were washed twice with 30pl PBS and incubated with 20µl secondary antibody solution, consisting of 1:1000 (v / v) goat anti-rabbit AlexaFluor 488 (Invitrogen Al 1034), 1:1000 Hoechst 33342 dye (Invitrogen, H1399) and 1:5000 HCS CellMask Deep Red Stain (Invitrogen, H32721) in blocking buffer, for 1 h in the dark at room temperature. Cells were washed twice with 30µl PBS and 50µl of PBS was added to each well and scanned with a GE Healthcare IN Cell Analyzer 2200. Image analysis was conducted using a custom pipeline in CellProfiler 3.1.9 (cellprofiler.org). Activity cut-off for hit selection was determined by using three standard deviations from the mean of the whole sample population.
[0860] Statistical analysis
[0861] QIF signals between compartments were analyzed using linear regression, correlation functions, and expressed as regression / correlation coefficients. For experiments including numerous fields of view (FOV) per case slide, the top 10% marker scores in each preparation were analyzed. Patient characteristics were compared using the student t test for continuous variables and χ2 test for categorical variables. Survival functions were compared using Kaplan-Meier estimates and statistical significance was determined using the log-rank test. Stratification of cases for clinicopathologic associations and survival analysis was conducted using a cancer-cell / stromal TAPI or TAP2 ratio of 1.41 as cut-
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[0864] point that was the highest ratio seen in a cohort of non-tumor lung specimens (not shown). Correlation studies were performed calculating linear regression coefficients and / or Spearman rho-rank functions. Associations between the markers and statistical significance were determined using JMP Pro. vl 1 and GraphPad Prism v7.0a software. Significance was assessed using two-tailed Student’s t-test. Differences were considered to be statistically significant when P < 0.05.
[0865] Results
[0866] Cancer-cell selective downregulation of TAP2 is common in human NSCLC A multiplexed quantitative immunofluorescence (mQIF) panel was established for simultaneous detection of the markers DAPI for all nucleated cells, cytokeratin (CK) for tumor epithelial cells, CD8 for effector tumor infiltrating lymphocytes (TILs), TAPI and TAP2 proteins to analyze formalin-fixed paraffin embedded (FFPE) tumor specimens (see methods. Using this assay, 1,290 pre-treatment NSCLCs were studied from 7 independent cohorts of patients treated with and without immune checkpoint blockers and represented in tissue microarrays (cohort #1, n=287; cohort #2, n=288; cohort #3 n=81; cohort 4, n=56; cohort 5, n=259; cohort 6, n=189; and cohort 7, n=130, see Tables 1-4).
[0867] TfiKk I: of TAJ5expression (txtfe-niiawcfeapy treated cnaoits) mfh s::nic:oparfi«k-gia SratkJisfd anatexis by Chi-Snpiare test.
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[0879] Table 3: Association of TAP expression (non-immunotherapy treated cohorts) with clinicopathologic variables. Statistical analysis by Chi-Square test.
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[0887] Table 4: Association of TAP expression (non-immunotherapy treated cohorts) with clinicopathologic variables. Statistical analysis by Chi-Square test.
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[0897] As shown in Figure 1A, most NSCLCs showed comparable cytoplasmic expression of TAPI and TAP2 proteins in both CK-expressing cancer-cells and in CK-negative stromal cells. However, a subset of cases showed marked reduction of TAPI and / or TAP2 protein expression in malignant cancer-cells, but with preservation of the markers in the neighboring non-tumor stromal cells and intratumoral CD8+ TILs, supporting a cancer-cell selective protein downregulation (Figure IB and 9A-9B).
[0898] To systematically identify cases with cancer-cell TAPI and / or TAP2 protein downregulation across the cohorts using an objective strategy, and to account for the possible impact of local proinflammatory signals and differences in the pre-analytic variables across specimens, the levels of TAPI and TAP2 were measured in CK+ cancer-cells and CK- nontumor stromal cells from each case using an automated compartment-based fluorescence colocalization algorithm (AQUA®). Tumor samples with lower TAPI or TAP2 protein signal in CK+ cancer-cells than in the neighboring CK- non-malignant stromal cells from the same specimen were considered as having cancer-cell selective protein downregulation. Using this strategy and as shown in Figures 1C-1E illustrating Cohorts #1-4 and Figure 9C showing Cohorts #5-7, 6.1% of cases across the cohorts showed TAPI downregulation (range 0.2-12.1% in Cohorts #1-7), 24.2% showed TAP2 downregulation (range 18.4-29.6% in Cohorts #1-7) and 18.4% displayed concurrent TAPI and TAP2 downregulation (range 13-33.9% in Cohorts #1-7).
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[0901] Cancer-cell TAP2 downregulation is associated with low TILs and worse outcomes after treatment with immune checkpoint blockers
[0902] Next, and to assess the possible impact of TAPI and TAP2 protein expression on local adaptive anti-tumor responses, the levels of CD8+ TILs were compared across cases with and without cancer-cell TAPI and / or TAP2 downregulation. As shown in Figures 1F-1G showing Cohorts #1-4 and Figure 9D showing Cohorts #5-7, tumors with downregulation of TAPI, TAP2 or TAPI & TAP2 showed lower CD8+ TIL levels than specimens with unaltered TAP1 / 2 expression. Although these differences were inconsistent across the cohorts, tumors with lower TAP2 protein alone showed a more pronounced difference in CD8+ TILs relative to cases without TAP 1 / 2 downregulation that reached statistical significance in four of the seven studied cohorts (range 6.6-33.3% lower CD8+ TIL signal in Cohorts #1-7). Statistical power limitations associated with small sample size of each of the TAP 1 / 2 downregulation tumor sub-groups, (particularly cases with TAPI downregulation alone) could contribute to the differences seen across the cohorts.
[0903] To assess the clinical significance of cancer-cell TAPI or TAP2 protein downregulation in NSCLC, the association of these phenotypes with major clinicopathologic variables including the patient age, gender, clinical stage, NSCLC histology and smoking status; as well as with the presence of clinically actionable oncogenic driver mutations in lung adenocarcinomas were studied. As shown in Tables 1-4, there were no consistent associations across the cohorts. In addition, the frequency of cancer-cell TAPI and / or TAP2 downregulation was comparable across lung adenocarcinomas harboring activating mutations in EGFR or KRAS. These results support that TAPI and TAP2 protein downregulation does not strongly segregate with specific patient / tumor characteristics and can occur in any NSCLC subtype.
[0904] To determine the impact of cancer-cell TAPI and TAP2 protein downregulation in treatment- specific outcomes, the overall survival (OS) of patients in NSCLC cohorts was studied with or without baseline / pre-treatment cancer-cell TAP 1 / 2 protein downregulation treated with standard chemotherapy (Cohorts #1, 2, 5 and, 6) or after treatment with PD-1 axis blockers (Cohorts #3 & 4). The survival analysis stratifying cases based on the presence of lower TAPI or TAP2 levels in malignant than in stromal cells from the same sample (e.g. a tumor / stromal signal ratio <1) showed no consistent associations across the cohorts. It was therefore considered that the existence of a biological threshold of TAP 1 / 2 proteins levels is needed to mediate a clinical impact and a more stringent cut-point based
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[0907] on the maximum epithelial / stromal ratio observed in a set of morphologically normal lung tissue specimens (e.g. epithelial / stromal ratio <1.4) was explored. As shown in Figures 1H-II (Cohorts #1-4) and 10A-10B (Cohorts #5 & 6), the level of cancer-cell TAPI downregulation had no consistent association with OS regardless of the treatment type. However, low levels of cancer-cell TAP2 were consistently associated with shorter OS only in the cohorts of patients treated with PD-1 axis blockers (Figures 1J-1K) (Cohort #3; HR 2.4 [CI: 1.02-5.66] and Cohort #4; HR 4.6 [CI: 2.17-9.86], Together, these results indicate that TAP2 is more commonly downregulated than TAPI in human NSCLCs and indicates a negative predictive role of cancer-cell TAP2 downregulation for immunotherapy.
[0908] TAP2 expression modulates the surface HLA class-I antigen levels in lung cancer cells
[0909] In non-malignant cells, TAPI and TAP2 form a heteromeric transporter in the ER membrane mediating the active transport of short immunoproteasome-derived peptides into the ER lumen for subsequent loading onto HLA molecules (Figure 2A) (Dhatchinamoorthy et al., 2021; Sadagopan et al., 2022). Therefore, disruption in TAP1 / 2 proteins in malignant cells could affect the levels of surface peptide HLA-complcxcs and reduce the tumor recognition by cognate effector tumor-antigen specific T-cells. To determine the impact of TAP1 / 2 proteins on the cancer-cell surface antigen levels an assay was established to measure specific class-I peptide- HLA complexes by flow cytometry briefly described in Figure 2B. The surface peptide-HLA complexes were measured in lung cancer-cells with or without TAPI and / or TAP2 downregulation using siRNA, and before or after stimulation with proinflammatory cytokines IFNy and TNFa that are able to increase TAP1 / 2 expression and are thought to be present in the tumor microenvironment (Maggs et al., 2021) (Figures 2B, 114A-11D and 12A). The HLA-A2+ human lung adenocarcinoma A549 (KRAS mutant) or PC9 cells (EGFR mutant) with endogenous TAP1 / 2, HER2 and MAGE3 protein expression were analyzed by flow cytometry with the mAb 4F10 specific to HLA-A2 bound to the HER2 peptide KIFGSLAFL corresponding to amino acids 369-377 of the full-length HER2 protein (HER2369-377) (Krokhotin et al., 2019) or the mAb 12B6 specific to HLA-A2 bound to the MAGE3 nonamer FLWGPRALV corresponding to amino acids 271-279 of the full-length MAGE3 protein (MAGE3271-279) (Wu et al., 2018). As shown in the Figures 12B-12C, the siRNA prominently reduced TAPI and TAP2 mRNA and protein expression in lung cancer cells and also limited the increase induced by
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[0912] IFNy alone or IFNy + TNFa. For malignant cells, the baseline surface HLA-A2-HER2369-377 and HLA-A2-MAGE3271-279 peptide complex levels in A549 cells were relatively low and prominently increased following treatment with proinflammatory cytokines (Figures 2C-2E and 2F-2H). In control / untreated cells, TAPI and / or TAP2 downregulation induced a ~5-25% reduction in the levels of surface peptide- HLA-A2 complexes that was more pronounced for TAP2 and reached statistical significance only in cells with concurrent TAP1 / TAP2 downregulation for HLA-A2-HER2369-377 (Figure 2C), and in those with TAP2 reduction alone or TAP1 / TAP2 reduction for HLA-A2-MAGE3271-279 levels (Figure 2F). In addition, TAP2 downregulation consistently decreased the levels of surface peptide-HLA complexes after treatment with proinflammatory cytokines (Figures 2D-E and 2G-2H). TAPI downregulation alone had lower impact on the surface antigen levels after cytokine stimulation that was inconsistent across the different peptide-HLA complexes analyzed, and the concurrent downregulation of both TAP1 and TAP2 produced an effect comparable to TAP2 reduction alone supporting a dominant effect this protein. A similar impact of TAPI and / or TAP2 downregulation on the levels of surface peptide-HLA complexes with or without cytokine treatments was seen in the human lung adenocarcinoma cells PC9 expressing higher endogenous TAP2, as well as ~2 fold higher baseline HLA-A2-HER2369-377 and HLA-A2- MAGE3271-279 levels supporting the consistency of the findings (Figures 2I-2K and 2L-2N).
[0913] Due to the more prominent effect of TAP2 reduction, and to assess the possible contribution of residual TAP2 expression after siRNA-based silencing, the surface peptide-HLA complex levels in A549 cells were measured with targeted TAP2 elimination using CRISPR / Cas9-based gene editing. As shown in the Figure 12D, TAP2 elimination was associated with undetectable TAP2 protein levels with or without cytokine stimulation. Similar to the knockdown experiments using siRNA, TAP2 abrogation only slightly reduced the levels of HLA-A2-HER2369-377 and HLA-A2-MAGE3271-279 peptide complexes in unstimulated A549 cells, but prominently prevented the increase induced by treatment with IFNy or IFNy + TNFa (Figures 2O-2P). No detectable changes in the surface peptide complexes were seen in human H1975 and H520 lung cancer cells lacking HLA-A2 and / or HER2 protein expression after different experimental perturbations (Figures 11C-11D). Together, these results demonstrate that TAP2 reduction can limit the
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[0916] levels of surface HLA class-I antigens in lung cancer cells, particularly under proinflammatory conditions.
[0917] TAP2 downregulation protects lung cancer cells from tumor antigen-specific T-cell killing
[0918] To determine the role of cancer-cell TAP1 / 2 expression in adaptive immune evasion, A549 cells were co-cultured with or without TAPI and / or TAP2 downregulation with human HLA- A2+ / CD8+ T-cells expressing the αβTCRs recognizing the HLA-A2-HER2369-377 peptide complexes (Figure 3A). Then, and as outlined in Figure 3B, cancer-cell apoptosis was measured via surface Annexin V staining of EpCAM+ malignant cells as well as cancer-cell viability by LDH release and MTT assay in preparations before or after stimulation with proinflammatory cytokines, and using different effector to target cell ratios. Of note, treatment with cytokines in these experiments were applied only to cancer-cells before co-culturing and they were removed / washed before adding T-cells to avoid direct lymphocyte stimulation. As shown in Figures 3C-3F, virtually no Annexin V positivity was detected in preparations containing only cancer-cells with or without TAP1 / 2 silencing or after pre-incubation with IFNy or IFNy +TNFa. In samples containing A549 and CD8+ T-cells in different ratios, the downregulation of TAPI in cancer-cells induced no detectable changes in the tumor-antigen specific T-cell killing in control / unstimulated preparations (Figure 3D), and only partially suppressed the cancer-cell apoptosis in samples pre-incubated with proinflammatory cytokines (Figures 3E-3F). In contrast, TAP2 downregulation induced a more prominent reduction in the apoptosis of unstimulated A549 cells and almost totally prevented the tumor-antigen specific T-cell mediated killing induced by proinflammatory cytokines (Figures 3E-3F). The concurrent downregulation of both TAPI and TAP2 produced an effect comparable to TAP2 downregulation alone.
[0919] To assess the possible contribution of alternative cancer-cell death mechanisms beyond apoptosis, experiments were conducted using two independent cell viability measurements: LDH release and MTT staining. As shown in Figures 3G-3I and 3J-3L, the results obtained were similar to those measuring Annexin V with limited impact of TAPI downregulation alone on T- cell mediated cancer-cell killing, a more prominent effect of TAP2 downregulation, and a comparable effect of TAP2 vs concurrent TAP1 / TAP2 silencing. Similar results were also obtained using PC9 lung cancer cells as T-cell targets using all the cell apoptosis / cell viability assays (Figures 3M-3V), as well as in A549 cells
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[0922] with TAP2 gene elimination using CRISPR / Cas9-based editing (Figures 3W-3Y).
[0923] Together, these results demonstrate that TAP2 reduction mediates, and is sufficient to induce tumor antigen-specific T-cell immune evasion in lung cancer cells.
[0924] TAP2 downregulation alters proinflammatory responses of lung cancer cells via SOCS1
[0925] Because of the prominent impact of TAP2 silencing in reducing the surface peptide-HLA complexes and the T-cell killing of lung cancer cells after treatment with proinflammatory cytokines, experiments were designed to test a possible role of TAP2 in modulating intracellular inflammatory responses. To assess this, the immune transcriptomic profiles of control and TAP2-downregulated A549 lung cancer cells were compared. As shown in Figure 4A, downregulation of TAP2 prominently reduced the baseline expression of multiple proinflammatory TNF superfamily genes (e.g., TNFSF4), HLA class-I APM transcripts (e.g. HLA- A, B, C, TAPI, PSMB8, PSMB9 and PSMB10) and intracellular signaling regulators (e.g. STAT1, STAT2, IRF2, JAK3, STAT6 and CD40). Moreover, TAP2 downregulation was also associated with reduced expression of chemokines involved in T-and NK-cell recruitment and activation including CXCL9 and IL-15 (Tokunaga et al., 2018; Waldmann et al., 2020), which could at least partially explain the lower CD8+ TIL levels seen in TAP2-defi cient tumors (Figures 1F-1G). Notably, TAP2 silencing did not reduce the expression of IFNy receptor IFNGR1 or the TNFa receptors TNFRSF1A and TNFRSF1B. The analysis of transcripts significantly upregulated after TAP2 reduction identified SOCS1 (suppressor of cytokine signaling 7) as the top upregulated gene and this molecule has been previously shown in multiple studies to effectively suppress the IFNy / STAT pathway and TNFa signaling (Alspach et al., 2019; He et al., 2006; Liau et al., 2018). Consistent with increased SOCS1 mRNA expression, SOCS1 protein was also significantly higher in A549 cells after TAP2 downregulation by flow cytometry analysis (Figure 4B). To further study the association between TAP2 and SOCS1, A549 cells were transfected with full-length human TAP2 containing a short amino-acid tag located in the N-terminal domain allowing its selective identification (FLAG-TAP2). As shown in Figures 4C and 4D, TAP2 overexpression significantly reduced SOCS1 protein levels supporting the bi-directional modulation of both proteins in lung cancer cells.
[0926] Together, these results show...
Claims
ATTORNEY DOCKET NO. YU 8874 PCTCLAIMSWe claim:
1. A method of enhancing, increasing, accelerating or / and otherwise improving an immune response in a subject in need thereof comprising administering the subject an effective amount of a compound that increases expression and / or levels of one or more transporters associated with antigen processing (TAP) proteins, optionally selected from TAP2, TAPI, and combinations thereof, and / or reduces expression and / or levels of Suppressor of Cytokine Signaling 1 (SOSC1) and / or interleukin 4 (IL-4), and optionally further comprising administering the subject an immune checkpoint modulator.
2. The method of claim 1, wherein the subject has cancer, optionally wherein the cancer is Non-Small Cell Lung Cancer (NSCLC), or an infection.
3. The method of claims 1 and 2, wherein the compound increases the level of TAP2 and / or TAPI protein in cells of the subject, optionally wherein the cells are cancer cells.
4. The method of any one of claims 1-3, wherein the compound is TAP2 or TAPI, or functional fragment or variant thereof, or a nucleic acid encoding the TAP2 or TAPI or functional fragment or variant, or any combination thereof.
5. The method of any one of claims 1-4, wherein the compound is Dasatinib, Vinorelbine, Docetaxel, Vinblastine, Vindesine, Mitomycin, Colchicine, Digoxin, Ouabain, Pyrithione Zinc, Oxibendazole, Albendazole, Fenbendazole, Podofilox, Gentian, Emetine, Oxamflatin, BML-281, SAHA / Vorinostat, or another compound of Figures 7B, 7C, 7D, or 7E.
6. A method of reducing or otherwise preventing an immune response in a subject in need thereof comprising administering the subject an effective amount of a compound that decreases expression and / or levels of one or more transporters associated with antigen processing (TAP) proteins, optionally selected from TAP2, TAPI, and combinations thereof, and / or reduces expression and / or levels of Suppressor of Cytokine Signaling 1 (SOSC1) and / or interleukin 4 (IL-4), optionally wherein the compound is selected from aminopterin, pemetrexed, dinitolmide, amprolium, and the compounds of Figures 7N-7O.
7. The method of claim 6, wherein the subject has an autoimmune or inflammatory disease or disorder, transplant rejection optionally wherein the transplant comprises cells, tissue, and / or organs, and / or graft verse host disease (GVHD).13845836485.1ATTORNEY DOCKET NO. YU 8874 PCT8. The method of claims 6 or 7, wherein the compound reduces the level of TAP2 and / or TAPI protein in cells of the subject, optionally wherein the cells at the site of autoimmunity or inflammation, transplant, or GVHD.
9. The method of any one of claims 5-7, wherein the compound is a functional nucleic acid that targets the TAP2 and / or TAPI gene or a gene product thereof.
10. A method of selecting a subject for treatment comprising detecting the level of TAP2, TAPI, SOSC1, and / or IL-4 in a sample from the subject, and selecting the subject for treatment when any one or more of TAP2, TAPI, SOSC1, and IL-4 is higher or lower than a control.
11. The method of claim 10, wherein a level of TAP2 lower than the control indicates the subject is in need of an increased immune response.
12. The method of claims 10 or 11, wherein a level of TAPI lower than the control indicates the subject is in need of an increased immune response.
13. The method of any one of claims 10-12, wherein a level of SOCS1 higher than the control indicates the subject is in need of an increased immune response.
14. The method of any one of claims 10-13, wherein a level of IL-4 higher than the control indicates the subject is in need of an increased immune response.
15. The method of any one of claims 10-14, wherein a level of TAP2 higher than the control indicates the subject is in need of an reduced immune response.
16. The method of any one of claims 10-15, wherein a level of TAPI higher than the control indicates the subject is in need of an increased reduced response.
17. The method of any one of claims 10-16, wherein a level of SOCS1 lower than the control indicates the subject is in need of an reduced immune response.
18. The method of any one of claims 10-17, wherein a level of IL-4 lower than the control indicates the subject is in need of an reduced immune response.
19. The method of any one of claims 10-18, wherein the TAP2, TAPI, SOSC1, and / or IL-4 level is detected by measuring one or more of TAP2, TAPI, SOSC1, and / or IL-4 mRNA and / or protein.
20. The method of any one of claims 10-19, wherein the sample is a tissue sample, optionally a tumor biopsy.
21. The method of any one of claims 10-20 further comprising treating the subject to increase or decrease an immune response.
22. The method of claim 21 according to the method of any one of claims 1 -9.13945836485.1ATTORNEY DOCKET NO. YU 8874 PCT23. A method of enhancing, increasing, accelerating or / and otherwise improving an immune response in a subject in need thereof comprising administering the subject an effective amount of a compound that and / or decreases the intranuclear level or bioavailability of basic peptides and / or increases the level or bio availability of intranuclear acidic peptides in cancer or infected cells or other target cells in which an increased sensitivity to an immune and / or inflammatory response thereto is desired, and optionally further comprising administering the subject an immune checkpoint modulator, optionally wherein the subject has cancer, optionally wherein the cancer is Non-Small Cell Lung Cancer (NSCLC), or an infection.
24. A method of reducing or otherwise preventing an immune response in a subject in need thereof comprising administering the subject an effective amount of a compound that increases the level or bioavailability of intranuclear basic peptides and / or decreases the level or bioavailability of intranuclear acidic peptides in autoimmune and / or transplanted cells / tissue or other target cells in which an reduced sensitivity to an immune and / or inflammatory response thereto is desired, optionally wherein the subject has an autoimmune or inflammatory disease or disorder, or a transplant an optionally is in danger of rejection thereof optionally wherein the transplant comprises cells, tissue, and / or organs, and / or graft verse host disease (GVHD).
25. A method of modulating gene expression in cells comprising modulating the level or bioavailability of basic and / or acid peptides in the nucleus of the cells.
26. The method of any one of claims 23-25 wherein the compound that increases level or bioavailability of basic peptides and / or that decreases levels or bioavailability of acidic peptides is (i) a plurality of the same or different basic peptides or a nucleic acid encoding the same.
27. The method of any one of claims 23-25 wherein the compound that increases level or bioavailability of acidic peptides and / or that decreases levels or bioavailability of basic peptides is a plurality of the same or different acidic peptides or a nucleic acid encoding the same.
28. The method of any one of claims 23-25, wherein the compound is one that targets microproteins and / or induced formation of defective ribosomal products, optionally wherein the compound is one that modulates protein synthesis optionally causing premature chain termination during translation.14045836485.1ATTORNEY DOCKET NO. YU 8874 PCT29. The method of any one of claims 23-25, wherein the compound is an ERAP1 and / or ERAP2 inhibitor or activator.
30. The method of any one of claims 23-25, wherein the compound increases protein degradation via the Ubiquitin-Protcasomc System (UPS).14145836485.1