Acyloxyacyl hydrolase and use thereof in the treatment of cancer

Abstract

Methods of treating cancer in a subject by administering an AOAH protein, a nucleic acid encoding an AOAH protein, or a cell (such as an immune cell) expressing an AOAH protein are provided. AOAH proteins, fusion proteins, and nucleic acids encoding the AOAH proteins or fusion proteins are also provided. Methods of treating cancer in a subject by administering a LY86 protein, a nucleic acid encoding a LY86 protein, or a cell (such as an immune cell) expressing a LY86 protein are also provided. LY86 proteins, fusion proteins, and nucleic acids encoding the LY86 proteins or fusion proteins are further provided.

Description

[0001] 4239-111527-02

[0002] ACYLOXYACYL HYDROLASE AND METHODS OF USE

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 717,036, filed November 6, 2024, which is herein incorporated by reference in its entirety.

[0005] FIELD

[0006] This disclosure relates to methods of treating cancer with an acyloxyacyl hydrolase (AOAH) protein or cells expressing an AOAH protein.

[0007] INCORPORATION OF ELECTRONIC SEQUENCE LISTING

[0008] The electronic sequence listing, submitted herewith as an XML file named 4239-111527- O2.xml (42,994 bytes), created on October 16. 2025. is herein incorporated by reference in its entirety.

[0009] BACKGROUND

[0010] Secreted proteins, such as cytokines, growth factors, and soluble enzymes, mediate broad functions, such as intercellular signaling, tissue development, and immune response. Secreted proteins can serve as therapeutics, drug targets, and biomarkers in blood tests. For example, the U.S. Food and Drug Administration approved high-dose interleukin-2 (IL-2) for treating metastatic melanoma and renal cell carcinoma in the 1990s. Meanwhile, the secreted factors vascular endothelial growth factor (VEGF) and tumor growth factor (TGF0) have been popular targets for neutralizing antibody development.

[0011] Despite a long research history, anticancer therapies targeting secreted proteins have many limitations. For example, IL -2 can activate both effector and regulatory T (Treg) cells, which activate and suppress the anti-tumor immune response, respectively. Aldiough engineering solutions may block IL-2 binding to IL-2Ra, the receptor on Tregs, such modifications may dampen die selfenhancing feedback loop of IL-2 signaling for effector T cells. Consistent with diis mechanistic limitation, few patients demonstrate an objective response to engineered IL-2. Similarly, anticytokine dierapeutics often fail to bring clinical benefits, as in the case of anti- VEGF in glioblastoma and anti-TGFp / PDLl in triggering hyper progressions. Thus, there remains a need to identify new anticancer therapies targeting secreted proteins.

[0012] SUMMARY

[0013] Methods of treating cancer in a subject using an acyloxyacyl hydrolase (AOAH) protein, a nucleic acid encoding an AOAH protein, or a cell (such as an immune cell) expressing an AOAH protein are provided. 4239-111527-02

[0014] In some aspects, the methods include administering to a subject (such as a subject with cancer) an ef'fecti ve amount of a composition including an AO AH protein or a nucleic acid encoding tire AOAH protein. In additional aspects, tire methods include administering to a subject (such as a subject with cancer) an effective amount of a composition including a fusion protein including an AOAH protein and a heterologous protein or portion thereof or a conjugate including an AOAH protein and a heterologous protein or portion thereof. In some examples, the heterologous protein is an Fc domain or an antibody or fragment thereof.

[0015] In additional aspects, the methods include administering to a subject (such as a subject with cancer) an effective amount of a composition including a T cell receptor (TCR)-T cell or a chimeric antigen receptor (CAR)-T cell expressing AOAH. wherein the TCR-T cell or CAR-T cell targets the cancer.

[0016] Modified immune cells expressing a heterologous AOAH and a TCR or CAR are also provided. In some examples, the immune cell is a T cell or a natural killer cell. In some examples, the modified immune cell is administered to a subject with cancer. In some examples, the TCR or CAR expressed by the modified immune cell specifically binds to an antigen expressed by the cancer.

[0017] Also provided are compositions including a fusion protein including an AOAH protein and a heterologous protein or portion thereof or a conjugate including an AOAH protein and a heterologous protein or portion thereof. In some examples, the heterologous protein is an Fc domain or an antibody or fragment thereof.

[0018] Also provided are lymphocyte antigen 86 (LY86) proteins and fusion proteins, nucleic acids encoding LY86 proteins or fusion proteins, and modified immune cells expressing a heterologous LY86 protein, for example, for use in methods of treating cancer.

[0019] The foregoing and other features of tire disclosure will become more apparent from the following detailed description, which proceeds with reference to tire accompanying figures.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIGS. 1A-1B illustrate Cancer Immunology Data Engine (CIDE) based on omics data from cancer immunotherapy studies. FIG. 1 A shows omics data of pre -treatment tumors with cancer immunotherapy outcomes. The middle layer presents the patient counts for each data type. The core layer shows the fraction of patients with each clinical endpoint. The outer layer presents the fraction of patients from each cancer type with cohort counts labeled outside. FIG. IB shows the workflow used to prioritize candidates from an input gene set for associations with clinical outcomes. First, users select omics data cohorts available in CIDE. The data types shown are colored as in FIG. 1 A, with sample counts labeled on each sector. Second. CIDE ranks input genes based on the median gene risk scores across selected cohorts. For top candidates selected by users, the CIDE also includes single-cell RNA-seq data for dissecting cell-lineage expression (n = 10 datasets) and cell-type specific 4239-111527-02 associations between gene expression and immunotherapy outcomes (n= 3 datasets). To probe genetic effects, the CIDE also includes CRISPR screen data in cancer cells under immunological killings, as well as links to the DepMap database for genetic screens in cancer cells and Tres database for CRISPR screens in T cells. NK: natural killer cells; Sub: sub-component of anti-tumor immunity, such as Prfl-null T cells, supernatants from Cancer - T co-cultures, or IFNy.

[0022] FIGS. 2A-2E show gene risk scores from cancer immunotherapy data predict secreted protein functions. FIG. 2A shows example survival plots. The y-axis presents the fraction of patients with survival higher than each duration (x-axis) for tumors whose target gene expression has high or low values, with cut-off determined by the best separation criterion. Z-scores and / '-values were evaluated by the two-sided Wald test in the Cox-PH regression using continuous values without any cutoffs. FIG. 2B shows gene risk scores. 37 pre -treatment tumor transcriptomics datasets (columns) with diverse types of patient immunotherapy outcomes (markers on the bottom) were collected. The risk score (Cox-PH z-score) of each gene (rows) quantifies the association between the expression level and adverse outcome. The heatmap only included the top 70 secreted proteins with significant median risk scores across cohorts. The absolute value of median risk scores was shown on the left. Pro-tumor versus anti-tumor functions (bar colors) were annotated by a double-blind literature search (Tables 4A B). Solid colors represent double -blind annotations. Transparent colors represent follow-up annotations for genes not assigned labels in the double-blind annotation. The top hierarchical tree presents the similarity among datasets measured as Pearson correlations between risk score profiles. FIG. 2C shows comparison of median risk scores between pro-tumor and anti-tumor groups. Each dot represents one gene, with colors representing the double-blind annotation. The thick line represents the median value. The bottom and top of the boxes are tire 25th and 75th percentiles, respectively (interquartile range). Whiskers encompass 1.5 times tire interquartile range. The / ?- v lue was from the two-sided Wilcoxon rank-sum test. FIG. 2D shows prediction performance of risk scores on secreted protein functions. The receiver operating characteristic (ROC) curve presents false-positive rates against true -positive rates of predicting pro-tumor versus antitumor categories from double -blind annotations based on median risk scores across all cohorts, with the diagonal line as the random expectation. FIG. 2E shows tumor-resilient T-cell (Tres) markers among prioritized secreted proteins. X-axis presents the median risk scores across immunotherapy cohorts in FIG. 2B. Y-axis presents tire median Tres scores across single -cell datasets analyzed in the Tres study. P- values were from the two-sided Wilcoxon signed-rank test comparing group values and zero.

[0023] FIGS. 3A-3K show associations between secreted protein gene expression and immunotherapy response. FIG. 3A shows risk scores for top prioritized secreted proteins. Each dot represents one clinical study, colored by cancer types. Each box plot was shown in the same format as FIG. 2C. FIG. 3B shows additional survival plot examples besides those in FIG. 2A. P-values were evaluated by the two-sided Wald test in the Cox-PH regression using continuous values without 4239-111527-02 any cutoffs. FIG. 3C shows AOAH and COLQ expression in infusion samples from anti-CD19 CAR T responders and non-responders with chronic lymphocytic leukemia. Each dot represents one patient. Values were shown in box plots as in FIG. 3 A. P- values were computed through the two- sided Wilcoxon rank-sum test, comparing values between responders and non-responders. FIG. 3D shows average AOAH expression across detection spots in spatial transcriptomics of hepatocellular carcinoma treated with anti-PDl, shown using box plots as in FIG. 3 A. FIG. 3E shows example spatial transcriptomics in hepatocellular tumors in FIG. 3D. FIGS. 3F-3G show in vivo validation of prioritized secreted proteins on anti-PDl response. Predicted anti-tumor proteins were validated using the anti-PDl resistant B16-mhgpl00 model. The predicted pro-tumor protein was validated using the anti-PDl sensitive B2905-M4 model, / '-values were computed by the two-sided Wilcoxon ranksum test at the last time point without any endpoint events (only p-values < 0.05 were shown). FIG. 3H shows endpoint-free survival plots for all mice in FIG. 3F. P-values were computed by the two-sided log-rank test comparing survival time between groups. FIGS. 3I-3J show validation of protein secretion effects by inoculating a mixture of over-expression (OE) cells and vector cells into mice. All values were shown as in FIGS. 3F-3G. P-values were computed by the two-sided Wilcoxon ranksum test at the last time point without any endpoint events, comparing the gene (or gene-vector mixture) and vector OE groups (only p-values < 0.05 were shown). FIG. 3K shows endpoint-free survival plots for all mice in FIGS. 3I-3J. All values were shown as in FIG. 3H. P-values were computed by the two-sided log-rank test, comparing the gene (or gene-vector mixture) and vector OE groups (only p-values < 0.05 were shown).

[0024] FIGS. 4A-4M show that AOAH potentiates immunotherapy efficacy in multiple tumor models. FIG. 4A shows B16F10 subcutaneous tumor growth in C57BL / 6 mice. P-values were computed by the two-sided Wilcoxon ranksum test at the last time point without any endpoint events (only p-values < 0.05 were shown). FIG. 4B shows endpoint-free survival plots for mice in FIG. 4 A. P-values were computed by the two-sided log-rank test comparing survival time between groups (only p-values < 0.05 were shown). FIG. 4C shows RIL-175 orthotopic tumor luminescence and RenCa subcutaneous tumor growth in tire C57BL / 6 mice, shown as in FIG. 4A. FIG. 4D shows endpoint- free survival plots for mice in FIG. 4C. Values were compared as in FIG. 4B. FIG. 4E shows B16- mhgplOO and B16F10 subcutaneous tumor growth in the C57BL / 6 mice who received intratumoral injection of recombinant human AOAH (rhAOAH) and vehicle, shown as in FIG. 4A. FIG. 4F shows endpoint-free survival plots for mice in FIG. 4E. FIG. 4G shows fractions of major immune cell types in the CD45+ tumor-infiltrating leukocytes isolated from B16F10 and B16mhgpl00 tumors after immune checkpoint blockade treatments. P-values were computed by two-sided unpaired student’s t test and only p-values < 0.05 were shown. FIG. 4H shows the design of plasmids used for the hydrodynamic tail vein injection (HDTVi) to induce spontaneous hepatocellular carcinoma (HCC) in mouse livers. FIG. 41 shows tumor luminescence in livers of C57BL / 6 mice. P-values were computed by the two-sided unpaired student’s t test, comparing values at the last time point. FIG. 4 J 4239-111527-02 shows the weight of livers dissected from all mice in FIG. 41. P-values were computed by the two- sided unpaired student’s t test. FIG. 4K shows representative multiplex immunofluorescence images of major leukocyte markers in the spontaneous HCC sections. FIG. 4L is a schematic of the experimental design of adoptive transfer of Aoa / z-armored pmel CD8+ T cells into the B16F10- bearing C57BL / 6 mice. FIG. 4M shows individual tumor growth and endpoint-free survival in the C57BL / 6 mice post T-cell transfer. P- values were computed by the two-sided log -rank test comparing survival time between groups.

[0025] FIGS. 5A-5H show tumor molecular landscape induced by Aoah overexpression. FIG. 5A shows examples of gene set enrichment analysis (GSEA). The X-axis presents the gene ranked by the log2 fold change (log2FC, bottom Y-axis) in each tumor model. The Y-axis on the top presents enrichment scores at each gene rank. The middle blue lines represent genes in each biological process (top title). The GSEA package reported a normalized enrichment score (NES) for each gene set. with a p-value through the two-sided permutation test (n = 1000 randomizations). FIG. 5B shows top 20 gene sets enriched in tumor models, ranked by maximum enrichment scores across all models. FIG. 5C shows Log2FC values of representative genes from enriched terms as numbered in FIG. 5B. FIG. 5D shows cell type assignments in single-cell (sc) RNA-seq data, generated from spontaneous HCC mouse tumors. Transcriptomics clusters of cells were shown using the Uniform Manifold Approximation and Projection. Cells in the T / NK cluster were further sub-clustered. PMN-MDSC: polymorphonuclear myeloid-derived suppressor cell; NK: natural killer; eDC: conventional dendritic cell; pDC: plasmacytoid dendritic cell; Treg: T regulatory cell; Tfh: T follicular helper cell. FIG. 5E shows cell fraction changes induced by Aoah. The average cell fractions in tumors (each tumor as one dot) were shown for spontaneous HCC tumors with dichotomous statuses of the OVA+SIN+SIY (OS) antigen overexpression and Aoah overexpression. FIG. 5F shows GSEA results of differential expression within each cell lineage. For each HCC tumor, the average log2(CPM / 10+l) across all cells within each lineage was computed. For each cell type, the log2FC of expression between tumors with Aoah overexpression and those with vector overexpression were computed. The GSEA results are shown as in FIG. 5B, with the antigen overexpression status marked on the bottom, for terms with at least one absolute normalized enrichment score higher than 2. FIG. 5G shows Log2FC values of representative genes from example terms in FIG. 5F. FIG. 5H shows TCR clonal expansion induced by Aoah, compared as in FIG. 5E. Within each T-cell subtype, the TCR expansion fraction was defined as the fraction of cells whose TCR clonotypes were captured in at least three distinct cells.

[0026] FIGS. 6A-6K show AOAH enhances CD8+ T-cell activation through TCR signaling. FIG. 6A shows T-cell mediated tumor killing quantified as normalized tumor-expressing mCherry fluorescence in B16 cells co-cultured with CD8+ pmel T cells. The mean and standard deviation values were computed from 4 cell culture replicates. P-values were computed through the two-way ANOVA analysis. FIG. 6B shows concentrations of cytotoxic cytokines released by mouse CD8+ T cells after activation by anti-CD3 / anti-CD28 antibodies. P-values were computed through the two- 4239-111527-02 sided paired t-test, comparing values at each concentration (n = 3 biological replicates). FIG. 6C shows the fraction of CD25+ cells among mouse CD8+ T cells after activation by anti-CD3 / anti- CD28 antibodies, / '-values were computed through the two-sided paired t-test (n - 3 biological replicates). FIG. 6D shows the concentrations of cytotoxic cytokines released by human CD8+ T cells after activation by anti-CD3 / anti-CD28 antibodies, / '-values were computed through the two- sided paired t-test, comparing values at each concentration (n = 3 biological replicates). FIG. 6E shows fractions of CD25+ cells and CD69+ cells among human CD8+ T cells after activation by anti- CD3 / anti-CD28 antibodies. P-values were computed through the two-sided paired t-test (n = 3 biological replicates). FIG. 6F shows fractions of mouse granzyme A+ and perforin-i- cytotoxic CD8+ T cells after activation by hgplOO and mgplOO-pulsed splenocytes. / '-values were computed through the two-sided paired t-test (n = 3 biological replicates). FIG. 6G shows concentrations of cytotoxic cytokines released by pmel CD8+ T cells after activation by hgplOO and mgplOO-pulsed splenocytes. P-values were computed through the two-sided paired t-test. comparing values at each concentration (n = 3 biological replicates). FIG. 6H shows significantly enriched pathways in rhAOAH-treated mouse CD8+ T cells based on RNA-seq. The normalized enrichment scores were computed by GSEA. FIG. 61 shows fractions of p-CD3c+ and p-LCK+ in mouse CD8+ T cells after activation by anti-CD3 / anti-CD28 antibodies for 10 minutes. P-values were computed through the two-sided paired t-test (n = 3 biological replicates). FIG. 6 J shows normalized luminescence intensity of human Jurkat NF AT reporter cells after activation by Immunocult at different bead to cell ratios. P-values were computed through the two-sided paired t-test (n = 3 technical replicates). FIG. 6K shows the time-dependent expression of key TCR signaling proteins in human CD8+ T cells after activation by anti-CD3 / anti-CD28 antibodies.

[0027] FIGS. 7A-7K show that AO AH depletes arachidonoyl-phosphatidylcholines to promote TCR activation. FIG. 7A shows depleted lipid species by AOAH. The y-axis represents the log2 fold change (log2FC) of MassSpec peak areas between rhAOAH and buffer treatments. Each bar represents the average log2FC across ion adducts for the same lipid with each ion as a point. Only depleted lipids with log2FC less than -1 are shown. FIG. 7B shows lipid class enrichment analysis. The log2FC of lipids within each class were compared with values of lipids outside the class by the two-sided Wilcoxon rank-sum test. Each value group was shown by a violin plot, smoothed by a kernel density estimator. Only significant lipid classes are shown (false discovery rate < 0.05). FIG. 7C shows fatty acid enrichment analysis for the PC diacyl (phosphatidylcholine) class from the T-cell culture medium. The log2FC values were compared between lipids with a fatty acid 20:4 (arachidonic acid) on the sn-2 position and other lipids by the two-sided Wilcoxon ranksum test. Values in each group were shown by violin plots as in FIG. 7B. Fatty acids on the sn-1 position were labeled for lipids with log2FC < -1. FIG. 7D shows the concentration of arachidonic acid released from mouse CD8+ T cells after rhAOAH treatment based on the competitive ELISA. P-values were computed through the two-sided paired t-test (n = 4 biological replicates). FIG. 7E shows normalized 4239-111527-02 fluorescence traces of MicroScale Thermophoresis after initiation of rhAOAH at defined time points. FIG. 7F shows the inhibition ratios of PC(16:0-20:4) on cytotoxic cytokine release from activated mouse CD8+ T cells, normalized by the control treatment. P-values were computed flirough the two- sided paired t-test, comparing values at each concentration (n = 3 biological replicates). FIG. 7G shows die inhibition ratios of PC(16:0-20:4) on cytotoxic cytokine release from activated human CD8+ T cells, shown as in FIG. 7F. FIG. 7H shows significantly down-regulated pathways in PC(16:0-20:4)-treated mouse CD8+ T cells based on RNA-seq. The normalized enrichment scores were computed by GSEA. FIG. 71 shows fractions of p-CD3c+. p-LCK+ and CD25+ in PC(16:0- 20:4) pre-treated CD8+ mouse T cells after activation by anti-CD3 / anti-CD28 antibodies. P-values were computed through the two-sided paired t-test (n = 3 biological replicates). FIG. 7J shows fractions of CD25+ and CD69+ in PC(16:0-20:4) pre-treated CD8+ human T cells after activation by anti-CD3 / anti-CD28 antibodies. P-values were computed through the two-sided paired t-test (n = 3 biological replicates). FIG. 7K shows concentrations of cytotoxic cytokines released by mouse CD8+ T cells after activation by anti-CD3 / anti-CD28 antibodies in the presence of wild type or mutant rhAOAH. P-values were computed through the two-sided paired t-test, comparing wild-type values with mutant values at each concentration (n = 3 biological replicates).

[0028] FIGS. 8A-8F show computational frameworks and analyses. FIG. 8A shows CRISPR screen phenotype scores from a co-culture between B16F10 cells and pmel T cells. Each gene’s phenotype score was defined as the log 2 fold-changes (log2FC) between treatment and control groups. The absolute log2FC values, representing the phenotype strength, were shown with violin plots that present score distributions smoothed by a kernel density estimator. The sample size in each group was labeled close to the plot. The score comparisons between secretome or membrane groups and the intracellular groups were through the two-sided Wilcoxon rank-sum tests. FIG. 8B shows histograms of Wilcoxon rank-sum z-scores comparing phenotype scores between secreted or membrane versus intracellular protein-coding genes. The Wilcoxon rank-sum tests were done as in FIG. 8A across all CRISPR screen datasets. The comparisons between z-scores and zero were through the two-sided Wilcoxon signed-rank tests, with p-values shown together with the median. The number of datasets was labeled together with the cohort title. FIG. 8C illustrates the RNA sequencing data processing pipeline. FIG. 8D illustrates the whole-exome sequencing processing pipeline. FIG. 8E shows an example plot of the tumor-resilient T-cell (Tres) model, testing whether AOAH is a Tres marker. Each dot represents a CD8 T cell from single -cell RNA-seq data generated from die tumor edge of patient #2 in a non-small cell lung cancer study. The x-axis shows immunosuppression as TGF-pi- signaling activities predicted by CytoSig. The y-axis shows cell proliferation scores computed through the KEGG cell cycle and DNA replication genes. Cells were split into high and low groups according to the AOAH expression, with a cutoff determined by the best separation criterion. The t value (Tres score) and P value were computed through the two-sided interaction test using continuous values without any cutoffs. FIG. 8F shows Tres gene scores among prioritized secreted proteins. 4239-111527-02

[0029] Each dot represents a tumor with single-cell RNA-seq data. Tres scores, indicating whether T cells with high expression of a secreted protein gene are resilient to immunosuppressive cytokine signaling, are shown through violin plots smoothed by a kernel density estimator. P- values were from the two- sided Wilcoxon signed-rank test comparing group values and zero.

[0030] FIGS. 9A-9H show associations between secreted protein coding-gene expression and cancer immunotherapy outcomes. FIG. 9A shows additional survival plot examples besides those in FIG. 2A. FIG. 9B shows correlation between AOAH expression and cytotoxic T lymphocyte (CTL) infiltration among pancreatic tumors treated with nivolumab plus chemotherapy. The CTL infiltration was estimated as tire median expression of CD8A. CD8B, GZMA, GZMB. and PRF1. FIG. 9C shows correlations with CTL infiltrations for top prioritized secreted proteins, shown as in FIG. 3A. FIG. 9D shows AOAH expression in infusion samples from adoptive T-cell transfer responders and nonresponders with melanoma. Each dot represents the infusion product for each patient. The gene expression values were shown in box plots as in FIG. 3C. P- values were computed through the two- sided Wilcoxon rank-sum test, comparing values between responders and non-responders. FIG. 9E shows gene expression in diverse cell lineages. (NK: natural killer; CAF: cancer-associated fibroblast; DC: dendritic cell; pDC: plasmacytoid DC; eDC: conventional DC). FIG. 9F shows in vitro growth of cancer cells in culture, measured by XTT assay (n = 3 cell culture replicates). The metabolic activity is measured as optical density at 492nm (read) divided by the value at 620nm (reference). FIG. 9G shows tumor growth curves for individual mice. FIG. 9H shows tumor growth curves for individual mice after inoculating a mixture of gene over-expression cells and vector cells into mice.

[0031] FIGS. 10A-10J show in vivo validation of top candidates. FIG. 10 A shows individual growth curves of subcutaneous B16-mhgpl00 tumors in NSG mice. FIG. 10B shows individual growth curves of subcutaneous B16F10 tumors in C57BL / 6 mice. FIG. 10C shows individual luminescence of orthotopic RIL-175 tumors in C57BL / 6 mice. FIG. 10D shows individual growth curves of subcutaneous RenCa tumors in C57BL / 6 mice. FIG. 10E shows individual growth curves of subcutaneous B16-mhgpl00 and B16F10 tumors treated with intratumoral injection of Vehicle / rhAOAH in C57BL / 6 mice. FIG. 10F shows body weight change of B16F10-bearing mice during rhAOAH + immune checkpoint blockade treatments. FIG. 10G shows individual growth curves of subcutaneous B16F10 tumors treated with serial concentrations of rhAOAH and survival in C57BL / 6 mice. / '-values were computed by tire two-sided log-rank test comparing survival time between groups (only -values < 0.05 were shown). FIG. 10H shows spontaneous HCC luminescence images taken by IVIS. FIG. 101 shows mouse livers dissected from mice in FIG. 10H. White arrow indicates HCC nodules. FIG. 10J shows additional representative multiplex immunofluorescence images of major tumor-infiltrating leukocytes (TIL) subtypes in the spontaneous HCC sections.

[0032] FIGS. 11A-11D show adoptive transfer of A oah -armored TCR-T cells. FIG. 11 A shows flow cytometry analysis of CD3, CD8 and TCR expression in CD45+ tumor-infiltrating leukocytes isolated from B16F10 tumors post TCR-T therapy. FIG. 1 IB shows the fractions of infiltrating CD3+, 4239-111527-02

[0033] CD8+TCR+ and CD8+TCR-T cells in B16F10 after TCR-T therapy. P- values were computed by unpaired student’s t test and only p-values < 0.05 were shown (n - 4 biological replicates). FIG. 11C shows body weight change of B16F10-bearing mice after TCR-T therapy. FIG. 11D shows representative hematoxylin and eosin images from spleens, lymph nodes and bone barrow of tumor- free C57BL / 6 mice and B16F10-bearing C57BL / 6 mice after TCR-T therapy. Scale bar = 200 pM (spleen and lymph node) and 20 pm (bone marrow).

[0034] FIGS. 12A-12G show in vivo molecular landscape induced by Aoah. FIG. 12A shows hierarchical clustering of log2FC profiles, using Pearson correlation as the distance. FIG. 12B shows IFNy activities predicted by the CytoSig package. FIG. 12C shows cell fractions for lymphocyte subtypes not included in FIG. 5E. FIG. 12D shows top 20 enriched terms from the gene ontology biological processes (GO_BP). shown as in FIG. 5F. The difference is that this analysis utilized GO_BP terms while FIG. 5F utilized KEGG terms. FIG. 12E shows cell fractions for B cells, compared as in FIG. 5E. FIG. 12F shows B-cell receptor (BCR) clonal expansion fractions, compared as in FIG. 5H. For B or plasma cells, the BCR expansion fraction was defined as the fraction of cells whose BCR clonotypes were captured in at least three distinct cells. FIG. 12G shows immunoglobulin heavy chain (IgH) class fractions.

[0035] FIGS. 13A-13M show in vitro validation on TCR activation by AOAH. FIG. 13A shows representative images of tumor cells expressing mCherry fluorescence in co-culture systems. FIG. 13B shows the T-cell cytotoxicity measured by the LDH assay in the co-culture system. P-values were computed through the two-sided paired t-test (n = 3 biological replicates). FIG. 13C shows concentrations of cytotoxic cytokines present in the co-culture system after 48 and 72hours. P-values were computed through the two-sided paired t-test (n = 3 biological replicates). FIG. 13D shows normalized mCherry fluorescence in B16-mhgpl00 and B16F10 cells co-cultured with CD8+ pmel T cells in the presence of 5 ng / mL TGF|3. P-values were computed through the two-way ANOVA analysis (n = 5 biological replicates). FIG. 13E shows rhAOAH pre-treatment prior to T-cell activation and rhAOAH treatment during T-cell activation both promoted IFNy secretion. P-values were computed through the two-sided paired t-test (n = 3 biological replicates). FIG. 13F shows flow cytometry analyses corresponding to FIGS. 6C, 6E and 6F. FIG. 13G shows the concentrations of cytotoxic cytokines released by pmel CD8+ T cells after activation by OVA-N4 and OVA-V4-pulsed splenocytes as antigen presenting cells (APCs). P-values were computed through the two-sided paired t-test, comparing values at each concentration (n = 3 biological replicates). FIG. 13H shows individual growth curves of subcutaneous Bl 6-0 VA-N4 and B16-OVA-V4 tumors and overall survival in C57BE / 6 mice. P-values were computed by the two-sided log-rank test comparing survival time between groups (only p-values < 0.05 were shown). FIG. 131 shows significantly enriched pathways in rhAOAH-treated pmel CD8+ T cells co-cultured with hgplOO-pulsed splenocytes. The normalized enrichment scores were computed by GSEA. FIG. 13J shows Log2 fold changes of key genes associated with cytotoxicity and T-cell activation based on RNA-seq of 4239-111527-02 splenocyte co-culture system. FIG. 13K shows validation of key genes associated with cytotoxicity and T-cell activation using qRT-PCR. FIG. 13L shows flow cytometry analysis of p-CD3^ and p- LCK expression in mouse CD8+ T cells after 10-min TCR activation. FIG. 13M shows uncropped raw images of western blot shown in FIG. 6K.

[0036] FIGS. 14A-14G shows lipidomics studies of AOAH-mediated TCR activation. FIG. 14A shows enriched lipid species by AOAH, shown as in FIG. 7A. Only enriched lipids with log2FC larger than 1 are shown. FIG. 14B shows normalized fluorescence traces of MicroScale Thermophoresis after initiation of rhAOAH. FIG. 14C shows the concentrations of cytotoxic cytokines released from mouse CD8+ T cells after TCR activation. FIG. 14D shows the concentrations of cytotoxic cytokines released from human CD8+ T cells after TCR activation. FIG. 14E shows the inhibition effect of PC( 18:0-20:4) on cytotoxic cytokine release from activated mouse CD8+ T cells, normalized by the control treatment. P-values were computed through the two-sided paired t-test, comparing values at each concentration (n = 3 biological replicates). FIG. 14F shows flow cytometry analysis of expressions of p-CD3L p-LCK and CD25 in mouse CD8+ T cells. FIG. 14G shows flow cytometry analysis of expressions of CD25 and CD69 in human CD8+ T cells.

[0037] FIGS. 15A-15E show that secreted protein LY86 can potentiate cancer immunotherapy efficacy. FIG. 15A shows risk scores of LY86 across 37 genome-wide immunotherapy cohorts. Each dot represents a cohort. P-values were computed by the two-sided Wilcoxon signed-rank test, comparing group values against zero. FIG. 15B shows the survival plot of LY86 activity, using data from a melanoma Ipilimumab study. The activity cutoff was selected by maximizing the difference between high and low groups. Z-scores and P-values were evaluated by the two-sided Wald test in the Cox-PH regression using continuous values without any cutoffs. FIG. 15C shows the LY86 expression across diverse immune cell lineages using average expression data from a collection of human single-cell RNA-seq studies. FIG. 15D shows the in vivo validation of Ly86 over-expression (OE) on immunodierapy response. Left panel: tumor growth curves. Error bars indicate the standard error of the mean. P-values were computed by the two-sided Wilcoxon rank sum test at die last time point widiout any endpoint events. Right panel: Kaplan-Meier curves. P-values were computed using the two-sided log-rank test, which compares survival length between groups. FIG. 15E shows the validation of die secretion effects by mixing a sub-fraction of Ly86-OE cells with vector-OE cells, shown as in FIG. 15D. P-values were computed between the Ly86 (or Ly86-vector mixture) OE and vector groups. Only p-values < 0.05 were shown.

[0038] FIG. 16 shows tumor growth and endpoint-free survival of B16-mhgpl00 tumors in C57BL / 6 mice with intraperitoneal injections of DC2.4 with Aoah or vector overexpression.

[0039] FIG. 17 shows the percentage inhibition of DC antigen-presentation and co-stimulatory markers among live CD1 lc+ cells (n = 3 biological replicates). Mouse splenocytes were pretreated with oxidized l-Palmitoyl-2-Arachidonoyl-sn-glycero-3-PhosphatidylCholine (oxPAPC) and cocultured with OT-1 CD8+T cells in the presence of 10 nM OVA antigen. 4239-111527-02

[0040] FIGS. 18A-18B show lipid peroxidation quantification based on the intracellular reactive oxygen species (ROS) level in activated mouse CD8+T cells. FIG. 18A shows flow cytometry analysis of the fluorescent shift of the lipid peroxidation sensor in mouse CD8+ T cells after TCR activation. In FIG. 18B, the y axis shows the ratio between initial lipid sensor and peroxidized sensor levels in each cell. All ratios are shown as violin plots smoothed by a kernel density estimator, p values were computed through the two-sided Wilcoxon rank-sum test, comparing median ratios between two groups (n = 3 biological replicates).

[0041] SEQUENCES

[0042] The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

[0043] SEQ ID NO: 1 is the amino acid sequence of an exemplary His-tagged AOAH protein

[0044] (italics: signal peptide; bold: His-tag; underlined: enterokinase cleavage site):

[0045] MGWSCZ / LFLVATArGVHSHHHHHHDDDDKLSNGHTCVGCVLVVSVIEQLAQVHNST VQASMERLCSYLPEKLFLKTTCYLVIDKFGSDIIKLLSADMNADVVCHTLEFCKQNT GQPLCHLYPLPKETWKFTLQKARQIVKKSP1LKYSRSGSD1CSLPVLAKICQKIKLAME QSVPFKDVDSDKYSVFPTLRGYHWRGRDCNDSDESVYPGRRPNNWDVHQDSNCNG IWGVDPKDGVPYEKKFCEGSQPRGIILLGDSAGAHFHISPEWITASQMSLNSFINLPTA LTNELDWPQLSGATGFLDSTVGIKEKSIYLRLWKRNHCNHRDYQNISRNGASSRNLK KFIESLSRNKVLDYPAIVIYAMIGNDVCSGKSDPVPAMTTPEKLYSNVMQTLKHLNS HLPNGSHVILYGLPDGTFLWDNLHNRYHPLGQLNKDMTYAQLYSFLNCLQVSPCHG WMSSNKTLRTLTSERAEQLSNTLKKIAASEKFTNFNLFYMDFAFHEIIQEWQKRGGQ PWQLIEPVDGFHPNEVALLLLADHFWKKVQLQWPQILGKENPFNPQIKQVFGDQGG H

[0046] SEQ ID NO: 2 is a nucleic acid sequence encoding an exemplary His-tagged AOAH protein:

[0047] ATGGGCTGGTCTTGCATTATCCTGTTCCTCGTTGCCACCGCCACAGGAGTCCATAG

[0048] CCATCATCACCATCACCACGACGACGATGATAAACTGAGCAACGGCCACACCTG

[0049] CGTCGGCTGCGTTTTGGTTGTATCAGTCATCGAGCAGCTTGCCCAGGTCCACAAC

[0050] TCCACCGTCCAGGCCTCTATGGAAAGGCTCTGCAGCTACCTGCCTGAGAAACTCT

[0051] TCCTGAAGACCACCTGCTACCTGGTTATTGACAAGTTCGGCTCCGATATCATCAA

[0052] GCTGCTGAGCGCTGACATGAACGCCGACGTAGTATGTCACACACTGGAGTTCTGC

[0053] AAGCAGAACACAGGCCAGCCTCTCTGTCATCTGTACCCCCTGCCTAAGGAGACCT

[0054] GGAAGTTCACCCTGCAGAAGGCCAGACAGATCGTCAAGAAGAGCCCCATCCTGA

[0055] AATACTCTAGGTCCGGCTCCGACATCTGCTCCCTGCCTGTCCTGGCTAAGATTTGC

[0056] CAGAAGATCAAGCTCGCCATGGAGCAGAGCGTCCCCTTCAAGGACGTTGACTCTG

[0057] ACAAGTACAGCGTCTTCCCTACATTGAGAGGCTACCACTGGCGGGGCAGGGACT

[0058] GCAACGACAGCGACGAGAGCGTTTACCCTGGACGGAGGCCCAACAATTGGGACG

[0059] TTCATCAGGACAGCAACTGCAACGGAATCTGGGGCGTTGATCCCAAGGACGGAG

[0060] TCCCCTACGAGAAGAAGTTTTGCGAGGGCTCCCAGCCCAGAGGCATCATCCTGCT

[0061] GGGCGACTCCGCCGGAGCCCACTTCCACATCTCCCCTGAATGGATAACAGCCAGC

[0062] CAGATGAGCCTCAACAGCTTCATCAACCTGCCCACCGCCCTGACAAACGAGCTGG

[0063] ACTGGCCCCAACTGTCCGGAGCCACCGGATTCCTGGACAGCACCGTAGGCATCAA 4239-111527-02

[0064] GGAGAAGTCTATCTACCTGAGGCTCTGGAAGAGAAACCACTGCAACCACAGGGA

[0065] CTACCAGAACATCAGCAGAAACGGCGCCAGCTCCAGAAACCTGAAAAAGTTCAT

[0066] TGAGAGCCTGAGCAGGAACAAGGTTCTCGACTACCCCGCCATCGTCATTTACGCC

[0067] ATGATCGGCAACGACGTCTGCTCCGGAAAGAGCGACCCCGTCCCCGCCATGACA

[0068] ACCCCCGAGAAGCTGTACTCAAACGTAATGCAAACACTGAAGCACCTGAACAGC

[0069] CACCTGCCTAACGGGAGCCACGTTATACTGTACGGACTGCCCGACGGAACCTTCC

[0070] TCTGGGACAACCTGCACAACAGGTACCACCCTCTGGGCCAGCTCAACAAGGACA

[0071] TGACCTACGCCCAGCTGTACTCCTTCCTGAACTGCCTGCAGGTTTCTCCTTGCCAC

[0072] GGCTGGATGAGTTCCAACAAGACCCTGAGAACCCTGACCTCCGAGAGAGCTGAG

[0073] CAGCTGTCTAACACCCTGAAGAAGATCGCTGCTTCCGAGAAGTTCACAAACTTCA

[0074] ACCTGTTCTACATGGACTTTGCTTTCCACGAGATCATCCAGGAGTGGCAGAAGCG

[0075] GGGCGGCCAGCCCTGGCAGCTGATCGAACCTGTTGACGGATTCCACCCCAACGA

[0076] GGTAGCTCTGCTGCTCCTGGCCGACCACTTCTGGAAGAAGGTACAGCTGCAATGG

[0077] CCCCAGATCCTCGGCAAGGAAAACCCTTTCAACCCTCAGATCAAGCAGGTCTTTG

[0078] GAGACCAAGGAGGTCAC

[0079] SEQ ID NO: 3 is the amino acid sequence of an exemplary His-tagged AOAH protein with

[0080] S263A mutation (italics: signal peptide; bold: His-tag; underlined: enterokinase cleavage site; bold underlined: S263A mutation):

[0081] MGWSC / fLFLVAr TGVHSHHHHHHDDDDKLSNGHTCVGCVLVVSVIEQLAQVHNST VQASMERLCSYLPEKLFLKTTCYLVIDKFGSDIIKLLSADMNADVVCHTLEFCKQNT GQPLCHLYPLPKETWKFTLQKARQIVKKSPILKYSRSGSDICSLPVLAKICQKIKLAME QSVPFKDVDSDKYSVFPTLRGYHWRGRDCNDSDESVYPGRRPNNWDVHQDSNCNG IWGVDPKDGVPYEKKFCEGSQPRGIILLGDAAGAHFHISPEWITASQMSLNSFINLPTA LTNELDWPQLSGATGFLDSTVG1KEKSIYLRLWKRNHCNHRDYQN1SRNGASSRNLK KFIESLSRNKVLDYPAIVIYAMIGNDVCSGKSDPVPAMTTPEKLYSNVMQTLKHLNS HLPNGSHVILYGLPDGTFLWDNLHNRYHPLGQLNKDMTYAQLYSFLNCLQVSPCHG WMSSNKTLRTLTSERAEQLSNTLKKIAASEKFTNFNLFYMDFAFHEIIQEWQKRGGQ PWQLIEPVDGFHPNEVALLLLADHFWKKVQLQWPQILGKENPFNPQIKQVFGDQGG H

[0082] SEQ ID NO: 4 is a nucleic acid encoding an exemplary His-tagged AOAH protein with

[0083] S263A mutation:

[0084] ATGGGCTGGTCTTGCATTATCCTGTTCCTCGTTGCCACCGCCACAGGAGTCCATAG CCATCATCACCATCACCACGACGACGATGATAAACTGAGCAACGGCCACACCTG CGTCGGCTGCGTTTTGGTTGTATCAGTCATCGAGCAGCTTGCCCAGGTCCACAAC TCCACCGTCCAGGCCTCTATGGAAAGGCTCTGCAGCTACCTGCCTGAGAAACTCT TCCTGAAGACCACCTGCTACCTGGTTATTGACAAGTTCGGCTCCGATATCATCAA GCTGCTGAGCGCTGACATGAACGCCGACGTAGTATGTCACACACTGGAGTTCTGC AAGCAGAACACAGGCCAGCCTCTCTGTCATCTGTACCCCCTGCCTAAGGAGACCT GGAAGTTCACCCTGCAGAAGGCCAGACAGATCGTCAAGAAGAGCCCCATCCTGA AATACTCTAGGTCCGGCTCCGACATCTGCTCCCTGCCTGTCCTGGCTAAGATTTGC CAGAAGATCAAGCTCGCCATGGAGCAGAGCGTCCCCTTCAAGGACGTTGACTCTG ACAAGTACAGCGTCTTCCCTACATTGAGAGGCTACCACTGGCGGGGCAGGGACT GCAACGACAGCGACGAGAGCGTTTACCCTGGACGGAGGCCCAACAATTGGGACG TTCATCAGGACAGCAACTGCAACGGAATCTGGGGCGTTGATCCCAAGGACGGAG TCCCCTACGAGAAGAAGTTTTGCGAGGGCTCCCAGCCCAGAGGCATCATCCTGCT GGGCGACGCCGCCGGAGCCCACTTCCACATCTCCCCTGAATGGATAACAGCCAGC CAGATGAGCCTCAACAGCTTCATCAACCTGCCCACCGCCCTGACAAACGAGCTGG ACTGGCCCCAACTGTCCGGAGCCACCGGATTCCTGGACAGCACCGTAGGCATCAA GGAGAAGTCTATCTACCTGAGGCTCTGGAAGAGAAACCACTGCAACCACAGGGA 4239-111527-02

[0085] CTACCAGAACATCAGCAGAAACGGCGCCAGCTCCAGAAACCTGAAAAAGTTCAT

[0086] TGAGAGCCTGAGCAGGAACAAGGTTCTCGACTACCCCGCCATCGTCATTTACGCC

[0087] ATGATCGGCAACGACGTCTGCTCCGGAAAGAGCGACCCCGTCCCCGCCATGACA

[0088] ACCCCCGAGAAGCTGTACTCAAACGTAATGCAAACACTGAAGCACCTGAACAGC

[0089] CACCTGCCTAACGGGAGCCACGTTATACTGTACGGACTGCCCGACGGAACCTTCC

[0090] TCTGGGACAACCTGCACAACAGGTACCACCCTCTGGGCCAGCTCAACAAGGACA

[0091] TGACCTACGCCCAGCTGTACTCCTTCCTGAACTGCCTGCAGGTTTCTCCTTGCCAC

[0092] GGCTGGATGAGTTCCAACAAGACCCTGAGAACCCTGACCTCCGAGAGAGCTGAG

[0093] CAGCTGTCTAACACCCTGAAGAAGATCGCTGCTTCCGAGAAGTTCACAAACTTCA

[0094] ACCTGTTCTACATGGACTTTGCTTTCCACGAGATCATCCAGGAGTGGCAGAAGCG

[0095] GGGCGGCCAGCCCTGGCAGCTGATCGAACCTGTTGACGGATTCCACCCCAACGA

[0096] GGTAGCTCTGCTGCTCCTGGCCGACCACTTCTGGAAGAAGGTACAGCTGCAATGG

[0097] CCCCAGATCCTCGGCAAGGAAAACCCTTTCAACCCTCAGATCAAGCAGGTCTTTG

[0098] GAGACCAAGGAGGTCAC

[0099] SEQ ID NO: 5 is the amino acid sequence of an IgG heavy chain fused to AOAH (fusion protein part 1) (italics: signal peptide; bold: human IgGl; underlined: (648)3 linker; bold italics:

[0100] “knob into hole” mutation):

[0101] MGWSCZ7LFLVA7ArGV / / SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLW CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTOKSLSLSPGGGGGSGGGGSGGGGSLSNGHTCVGCVLV VSVIEQLAQVHNSTVQASMERLCSYLPEKLFLKTTCYLVIDKFGSDIIKLLSADMNAD VVCHTLEFCKQNTGQPLCHLYPLPKETWKFTLQKARQ1VKKSP1LKYSRSGSDICSLP VLAKICQKIKLAMEQSVPFKDVDSDKYSVFPTLRGYHWRGRDCNDSDESVYPGRRP NNWDVHQDSNCNGIWGVDPKDGVPYEKKFCEGSQPRGIILLGDSAGAHFHISPEWIT ASQMSLNSFINLPTALTNELDWPQLSGATGFLDSTVGIKEKSIYLRLWKRNHCNHRD YQNISRNGASSRNLKKFIESLSRNKVLDYPAIVIYAMIGNDVCSGKSDPVPAMTTPEK LYSNVMQTLKHLNSHLPNGSHVILYGLPDGTFLWDNLHNRYHPLGQLNKDMTYAQ LYSFLNCLQVSPCHGWMSSNKTLRTLTSERAEQLSNTLKKIAASEKFTNFNLFYMDF AFHEIIQEWQKRGGQPWQLIEPVDGFHPNEVALLLLADHFWKKVQLQWPQILGKEN PFNPQIKQVFGDQGGH

[0102] SEQ ID NO: 6 is the nucleic acid sequence encoding fusion protein part 1 :

[0103] ATGGGATGGTCCTGCATCATTCTGTTCCTGGTGGCTACCGCAACTGGGGTACACA

[0104] GTGACAAGACTCACACCTGTCCACCCTGCCCTGCACCAGAGCTCCTGGGAGGGCC

[0105] TTCCGTATTCTTGTTTCCCCCAAAACCTAAGGACACACTGATGATTAGCCGGACTC

[0106] CTGAGGTAACATGCGTGGTGGTCGATGTCTCCCACGAGGATCCTGAAGTGAAGTT

[0107] TAACTGGTACGTCGATGGGGTCGAGGTGCACAACGCCAAGACTAAGCCTCGTGA

[0108] GGAGCAGTACAACTCTACATACAGAGTAGTGAGTGTGCTCACCGTGCTGCACCAA

[0109] GATTGGCTCAATGGCAAAGAGTACAAGTGCAAGGTCTCCAATAAAGCTCTCCCTG

[0110] CTCCCATCGAGAAAACCATAAGCAAGGCTAAAGGTCAGCCCCGCGAACCCCAGG

[0111] TGTATACCTTGCCACCATCTAGGGACGAACTGACCAAGAATCAGGTGTCCCTTtgg

[0112] TGTCTGGTGAAGGGCTTCTACCCCTCTGATATCGCCGTCGAGTGGGAAAGCAATG

[0113] GCCAGCCTGAAAACAACTATAAGACCACACCACCCGTCTTGGACAGCGACGGAT

[0114] CTTTCTTCCTGTATTCTAAGCTCACAGTGGACAAGTCCCGATGGCAGCAGGGCAA

[0115] CGTATTCAGCTGTAGTGTGATGCATGAGGCTCTGCATAACCATTACACCCAGAAA

[0116] AGCCTGAGTCTCTCTCCAGGCGGAGGCGGAGGATCCGGAGGAGGCGGTTCCGGT

[0117] GGCGGCGGATCTCTGAGCAACGGCCACACCTGCGTCGGCTGCGTTTTGGTTGTAT

[0118] CAGTCATCGAGCAGCTTGCCCAGGTCCACAACTCCACCGTCCAGGCCTCTATGGA

[0119] AAGGCTCTGCAGCTACCTGCCTGAGAAACTCTTCCTGAAGACCACCTGCTACCTG 4239-111527-02

[0120] GTTATTGACAAGTTCGGCTCCGATATCATCAAGCTGCTGAGCGCTGACATGAACG

[0121] CCGACGTAGTATGTCACACACTGGAGTTCTGCAAGCAGAACACAGGCCAGCCTCT

[0122] CTGTCATCTGTACCCCCTGCCTAAGGAGACCTGGAAGTTCACCCTGCAGAAGGCC

[0123] AGACAGATCGTCAAGAAGAGCCCCATCCTGAAATACTCTAGGTCCGGCTCCGAC

[0124] ATCTGCTCCCTGCCTGTCCTGGCTAAGATTTGCCAGAAGATCAAGCTCGCCATGG

[0125] AGCAGAGCGTCCCCTTCAAGGACGTTGACTCTGACAAGTACAGCGTCTTCCCTAC

[0126] ATTGAGAGGCTACCACTGGCGGGGCAGGGACTGCAACGACAGCGACGAGAGCGT

[0127] TTACCCTGGACGGAGGCCCAACAATTGGGACGTTCATCAGGACAGCAACTGCAA

[0128] CGGAATCTGGGGCGTTGATCCCAAGGACGGAGTCCCCTACGAGAAGAAGTTTTG

[0129] CGAGGGCTCCCAGCCCAGAGGCATCATCCTGCTGGGCGACTCCGCCGGAGCCCA

[0130] CTTCCACATCTCCCCTGAATGGATAACAGCCAGCCAGATGAGCCTCAACAGCTTC

[0131] ATCAACCTGCCCACCGCCCTGACAAACGAGCTGGACTGGCCCCAACTGTCCGGAG

[0132] CCACCGGATTCCTGGACAGCACCGTAGGCATCAAGGAGAAGTCTATCTACCTGAG

[0133] GCTCTGGAAGAGAAACCACTGCAACCACAGGGACTACCAGAACATCAGCAGAAA

[0134] CGGCGCCAGCTCCAGAAACCTGAAAAAGTTCATTGAGAGCCTGAGCAGGAACAA

[0135] GGTTCTCGACTACCCCGCCATCGTCATTTACGCCATGATCGGCAACGACGTCTGC

[0136] TCCGGAAAGAGCGACCCCGTCCCCGCCATGACAACCCCCGAGAAGCTGTACTCA

[0137] AACGTAATGCAAACACTGAAGCACCTGAACAGCCACCTGCCTAACGGGAGCCAC

[0138] GTTATACTGTACGGACTGCCCGACGGAACCTTCCTCTGGGACAACCTGCACAACA

[0139] GGTACCACCCTCTGGGCCAGCTCAACAAGGACATGACCTACGCCCAGCTGTACTC

[0140] CTTCCTGAACTGCCTGCAGGTTTCTCCTTGCCACGGCTGGATGAGTTCCAACAAG

[0141] ACCCTGAGAACCCTGACCTCCGAGAGAGCTGAGCAGCTGTCTAACACCCTGAAG

[0142] AAGATCGCTGCTTCCGAGAAGTTCACAAACTTCAACCTGTTCTACATGGACTTTG

[0143] CTTTCCACGAGATCATCCAGGAGTGGCAGAAGCGGGGCGGCCAGCCCTGGCAGC

[0144] TGATCGAACCTGTTGACGGATTCCACCCCAACGAGGTAGCTCTGCTGCTCCTGGC

[0145] CGACCACTTCTGGAAGAAGGTACAGCTGCAATGGCCCCAGATCCTCGGCAAGGA

[0146] AAACCCTTTCAACCCTCAGATCAAGCAGGTCTTTGGAGACCAAGGAGGTCAC

[0147] SEQ ID NO: 7 is the amino acid sequence of an IgGl portion to form heterodimers (fusion protein part 2) (italics: signal peptide; bold: asymmetric IgGl part; bold italics: “knob into hole” mutations; underlined: His-tag):

[0148] MGWSC / 7LFLVA7ArGV / / SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL.S CAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGHHHHHH

[0149] SEQ ID NO: 8 is a nucleic acid encoding fusion protein part 2.

[0150] ATGGGATGGTCATGTATCATCCTTTTTCTGGTAGCAACTGCAACTGGAGTACATA GCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGAC CGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGAC CCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAA GTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG GGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAC CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC CCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA CAGGTGTACACCCTGCCCCCATCCCGGGACGAGCTGACCAAGAACCAGGTCAGC CTGTCCTGCGCCGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGA GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCG ACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACG CAGAAGAGCCTCTCCCTGTCTCCGGGCCATCACCATCACCATCAT 4239-111527-02

[0151] SEQ ID NOs: 9-30 are oligonucleotide sequences (see Table 1).

[0152] SEQ ID NO: 31 is an exemplary AOAH preproprotein amino acid sequence.

[0153] MQSPWKILTVAPLFLLLSLQSSASPANDDQSRPSLSNGHTCVGCVLVVSVIEQLAQVH

[0154] NSTVQASMERLCSYLPEKLFLKTTCYLVIDKFGSDIIKLLSADMNADVVCHTLEFCKQ

[0155] NTGQPLCHLYPLPKETWKFTLQKARQIVKKSPILKYSRSGSDICSLPVLAKICQKIKLA

[0156] MEQSVPFKDVDSDKYSVFPTLRGYHWRGRDCNDSDESVYPGRRPNNWDVHQDSNC

[0157] NGIWGVDPKDGVPYEKKFCEGSQPRGIILLGDSAGAHFHISPEWITASQMSLNSFINLP

[0158] TALTNELDWPQLSGATGFLDSTVGIKEKSIYLRLWKRNHCNHRDYQNISRNGASSRN

[0159] LKKFIESLSRNKVLDYPAIVIYAMIGNDVCSGKSDPVPAMTTPEKLYSNVMQTLKHL

[0160] NSHLPNGSHVILYGLPDGTFLWDNLHNRYHPLGQLNKDMTYAQLYSFLNCLQVSPC

[0161] HGWMSSNKTLRTLTSERAEQLSNTLKKIAASEKFTNFNLFYMDFAFHEIIQEWQKRG

[0162] GQPWQLIEPVDGFHPNEVALLLLADHFWKKVQLQWPQILGKENPFNPQIKQVFGDQ

[0163] GGH

[0164] SEQ ID NO: 32 is an exemplary AOAH preproprotein encoding nucleic acid sequence.

[0165] ACTGAGCCAGGGAGCACGGAAGTTGTGCCACTGTGCAACTTGGGTTTTCTTTATC

[0166] CTGCAGTCTTTACCTCAGCAGAACCGCACACCACAGACTCCCTCCAGCTCTTTGT

[0167] GTGTGGCTCTCTCAGGGTCCAACAAGAGCAAGCTGTGGGTCTGTGAGTGTTTATG

[0168] TGTGCTTTTATTCACTTCACACTTATTGAAAAGTGTGTATGTGAGAGGGTGGGGT

[0169] GTGTGTGTCAAAGAGAGTGAGGAAGAGAAGGAGAGAGAGATCAATTGATTCTGC

[0170] AGCCTCAGCTCCAGCATCCCTCAGTTGGGAGCTTCCAAAGCCGGGTGATCACTTG

[0171] GGGTGCATAGCTCGGAGATGCAGTCCCCCTGGAAAATCCTTACGGTGGCGCCTCT

[0172] ATTCTTGCTCCTGTCTCTTCAGTCCTCGGCCTCTCCAGCCAACGATGACCAGTCCA

[0173] GGCCCAGCCTCTCGAATGGGCACACCTGTGTAGGGTGTGTGCTGGTGGTGTCTGT

[0174] AATAGAACAGCTTGCTCAAGTTCACAACTCGACGGTCCAGGCCTCGATGGAGAG

[0175] ACTGTGCAGCTACCTGCCTGAAAAACTGTTCTTGAAAACCACCTGCTATTTAGTC

[0176] ATTGACAAGTTTGGATCAGACATCATAAAACTGCTTAGCGCAGATATGAATGCTG

[0177] ATGTGGTATGTCACACTCTGGAGTTTTGTAAACAGAACACTGGCCAACCATTGTG

[0178] TCATCTCTACCCTCTTCCCAAGGAGACATGGAAATTTACACTACAGAAGGCAAGA

[0179] CAAATTGTCAAGAAGTCCCCGATTCTGAAATATTCTAGAAGTGGTTCTGACATTT

[0180] GTTCACTCCCGGTTTTGGCCAAGATCTGCCAGAAAATTAAATTAGCTATGGAACA

[0181] GTCTGTGCCATTCAAAGATGTGGATTCAGACAAATACAGCGTTTTCCCAACACTG

[0182] CGGGGCTATCACTGGCGGGGGAGAGACTGTAATGACAGCGACGAGTCAGTGTAC

[0183] CCAGGTAGAAGGCCGAACAACTGGGATGTCCATCAGGATTCAAACTGTAATGGC

[0184] ATTTGGGGTGTCGATCCAAAAGATGGAGTTCCATATGAGAAGAAATTCTGTGAAG

[0185] GTTCACAGCCCAGGGGAATCATTTTGCTGGGAGACTCAGCTGGGGCTCATTTTCA

[0186] CATCTCTCCTGAATGGATCACAGCGTCGCAGATGTCTTTGAACTCTTTCATCAATC

[0187] TACCAACAGCCCTTACCAACGAGCTTGACTGGCCCCAACTCTCTGGTGCTACAGG

[0188] ATTTCTGGACTCCACTGTTGGAATTAAAGAAAAATCTATTTACCTTCGCTTATGGA

[0189] AAAGAAACCACTGTAATCACAGGGACTACCAGAATATTTCAAGAAATGGTGCAT

[0190] CTTCCCGAAACCTGAAGAAATTTATAGAAAGCTTGTCTAGAAACAAGGTGTTGGA

[0191] CTATCCCGCCATCGTTATATATGCCATGATTGGAAATGATGTCTGCAGTGGGAAG

[0192] AGTGACCCAGTCCCAGCCATGACCACTCCTGAGAAACTCTACTCCAACGTCATGC

[0193] AGACTCTGAAGCATCTAAATTCCCACCTGCCCAATGGCAGCCATGTTATTTTGTAT

[0194] GGCTTACCAGATGGAACCTTTCTCTGGGATAATTTGCACAACAGATATCATCCTC

[0195] TCGGCCAGCTAAATAAAGACATGACCTATGCGCAGTTGTACTCCTTCCTGAACTG

[0196] CCTCCAGGTCAGCCCCTGCCACGGCTGGATGTCTTCCAACAAGACGTTGCGGACT

[0197] CTCACTTCAGAGAGAGCAGAGCAACTCTCCAACACACTGAAAAAAATTGCAGCC

[0198] AGTGAGAAATTTACAAACTTCAATCTTTTCTACATGGATTTTGCCTTCCATGAAAT

[0199] CATACAGGAGTGGCAGAAGAGAGGCGGACAGCCCTGGCAGCTCATCGAGCCCGT 4239-111527-02

[0200] GGATGGATTCCACCCCAACGAGGTGGCTTTGCTGTTGTTGGCGGATCATTTCTGG

[0201] AAAAAGGTGCAGCTCCAGTGGCCCCAAATCCTGGGAAAGGAGAATCCGTTCAAC

[0202] CCCCAGATTAAACAGGTGTTTGGAGACCAAGGCGGGCACTGAGCCTCTCAGGAG

[0203] CATGCACCCCTGGGGAGCACAGGGAGGCAGAGGCTTGGGTAAACTCATTCCACA

[0204] AACCCTATGGGGGCTGCCACGTCACAGGCCCAAAGGACTCTTCTTCAGCAGCATC

[0205] TTTGCAAAATGTCTTTCTCTCAATGAAGAGCATATCTGGACGACTGTGCAATGCT

[0206] GTGTGCTCCCGGGATCAGTAACCCTTCCGCTGTTCCTGAAATAACCTTTCATAAA

[0207] GTGCTTTGGGTGCCATTCCAAACAAGAGAGTATCTGTGCCCTTTACAGCTAATTG

[0208] TTCTAAAAGGAGTTTCTAAAAACAC

[0209] SEQ ID NO: 33 is an exemplary LY86 amino acid sequence:

[0210] MKGFTATLFLWTLIFPSCSGGGGGKAWPTHVVCSDSGLEVLYQSCDPLQDFGFSVEK CSKQLKSNINIRFGIILREDIKELFLDLALMSQGSSVLNFSYPICEAALPKFSFCGRRKG EQIYYAGPVNNPEFTIPQGEYQVLLELYTEKRSTVACANATIMCS

[0211] SEQ ID NO: 34 is an exemplary nucleic acid sequence encoding LY86:

[0212] ATGAAGGGTTTCACAGCCACTCTCTTCCTCTGGACTCTGATTTTTCCCAGCTGCAG TGGAGGCGGCGGTGGGAAAGCCTGGCCCACACACGTGGTCTGTAGCGACAGCGG CTTGGAAGTGCTCTACCAGAGTTGCGATCCATTACAAGATTTTGGCTTTTCTGTTG AAAAGTGTTCCAAGCAATTAAAATCAAATATCAACATTAGATTTGGAATTATTCT GAGAGAGGACATCAAAGAGCTTTTTCTTGACCTAGCTCTCATGTCTCAAGGCTCA TCTGTTTTGAATTTCTCCTATCCCATCTGTGAGGCGGCTCTGCCCAAGTTTTCTTTC TGTGGAAGAAGGAAAGGAGAGCAGATTTACTATGCTGGGCCTGTCAATAATCCT GAATTTACTATTCCTCAGGGAGAATACCAGGTTTTGCTGGAACTGTACACTGAAA

[0213] AACGGTCCACCGTGGCCTGTGCCAATGCTACTATCATGTGCTCCTGA

[0214] DETAILED DESCRIPTION

[0215] I. Abbreviations

[0216] AO AH acyloxyacyl hydrolase

[0217] ASGR asialoglycoprotein receptor

[0218] BCR B cell receptor

[0219] BTLA B and T lymphocyte attenuator

[0220] CAF cancer-associated fibroblast eDC conventional dendritic cell

[0221] CIDE Cancer Immunology Data Engine

[0222] CAR chimeric antigen receptor

[0223] CTL cytotoxic T lymphocyte

[0224] CTLA4 cytotoxic T lymphocyte-associated protein 4

[0225] DC dendritic cell

[0226] DNT double-negative T

[0227] EMT epithelial-mesenchymal transition

[0228] FDR false discovery rate

[0229] GPC3 glypican 3

[0230] GSEA gene set enrichment analysis 4239-111527-02

[0231] HCC hepatocellular carcinoma

[0232] ICB immune checkpoint blockade

[0233] IgH immunoglobulin heavy chain

[0234] IL interleukin

[0235] IFN interferon

[0236] LAG3 lymphocyte activation gene 3

[0237] LNP lipid nanoparticle

[0238] Log2FC log2 fold change

[0239] LY86 lymphocyte antigen 86

[0240] MST microscale thermophoresis

[0241] NES normalized enrichment score

[0242] NK natural killer

[0243] NKT natural killer T

[0244] OE over-expression

[0245] PC phosphatidylcholine

[0246] PD-1 include programmed cell death protein 1 pDC plasmacytoid dendritic cell rhAOAH recombinant human AO AH

[0247] ROC receiver operating characteristic

[0248] TCR T cell receptor

[0249] Tfh T follicular helper

[0250] TGF0 tumor growdi factor

[0251] TIL tumor-infiltrating leukocyte

[0252] TIM-3 T cell immunoglobulin and mucin domain-containing protein 3

[0253] TME tumor microenvironment

[0254] TNBC triple-negative breast cancer

[0255] TPM hanscript per million

[0256] Treg T regulatory

[0257] VEGF vascular endothelial growth factor

[0258] WES whole-exome sequencing data

[0259] II. Terms

[0260] Unless otherwise noted, technical terms are used according to conventional usage.

[0261] Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII. published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be 4239-111527-02 considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, tire materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

[0262] Acyloxyacyl hydrolase (AO AH): A secreted protein that detoxifies lipopolysaccharide by catalyzing hydrolysis of acyloxylacyl-linked fatty acyl chains from bacterial lipopolysaccharides. AOAH is produced by monocytes, macrophages, neutrophils, dendritic cells, type 1 innate lymphoid (ILC 1 ) cells and renal cortical tubule cells. AOAH has both light (small) and heavy (large) subunits that are encoded by a single locus. In some examples herein, AOAH is expressed as a preprotein (e.g., GenBank Accession No. NP 001628. 1 . SEQ ID NO: 31) that is processed to a mature protein (such as amino acids 31-571 of SEQ ID NO: 1). Nucleic acids encoding AOAH are also known and include GenBank Accession No. NM_001637.4 (SEQ ID NO: 32). Additional AOAH sequences are publicly available, such as under NCBI Gene ID 313.

[0263] Administration: To provide or give a subject an agent, such a composition disclosed herein, by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraprostatic, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0264] Antibody and Antigen Binding Fragment: An immunoglobulin, antigen binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (antigen), such as an antigen expressed by a cancer cell (for example, a tumor antigen). The term “antibody” as used herein encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), and antigen binding fragments, so long as they exhibit tire desired antigen binding activity.

[0265] Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity for the antigen. Examples of antigen binding fragments include but are not limited to Fv, Fab. Fab', Fab'-SH, F(ab')2. diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv), and multi-specific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Diibel (Eds.), Antibody Engineering, Vols. 1-2, 2nded., Springer-Verlag, 2010). Antibodies also include genetically engineered forms, such as chimeric 4239-111527-02 antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies).

[0266] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally-occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.

[0267] Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable domain genes. There are two types of light chain, lambda (X) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.

[0268] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain). In combination, the heavy and the light chain variable regions specifically bind the antigen. References to “VH” refer to the variable region of an antibody heavy chain, including that of an antigen binding fragment, such as Fv, scFv, dsFv or Fab. References to “VL” refer to the variable domain of an antibody light chain, including that of an Fv, scFv, dsFv or Fab.

[0269] The VH and VL contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs” (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5thed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991). The sequences of tire framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, e.g., the combined framework regions of tire constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

[0270] The CDRs are primarily responsible for binding to an epitope of an antigen. The amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5thed., NIH Publication No. 91-3242. Public Health Service, National Institutes of Health. U.S. Department of Health and Human Services, 1991; “Kabat” numbering scheme). Al-Lazikani et al.. (“Standard conformations for the canonical structures of immunoglobulins.” J. Mol. Bio., 273(4):927- 948, 1997; also referred to as the “Chothia” numbering scheme), and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 27(l):55-77, 2003; referred to as the “IMGT” numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to C-terminus), and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is the CDR3 from the VH of the antibody in which it is found, whereas a VL CDR1 4239-111527-02 is the CDR1 from the VL of the antibody in which it is found. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0271] A “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising tire population are identical and / or bind the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein. In some examples monoclonal antibodies are isolated from a subject. Monoclonal antibodies can have conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. See, for example, Greenfield (Ed.), Antibodies: A Laboratory Manual, 2nded. New York: Cold Spring Harbor Laboratory Press, 2014.

[0272] A “humanized” antibody or antigen binding fragment includes a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) antibody or antigen binding fragment. The non-human antibody or antigen binding fragment providing the CDRs is termed a “donor,” and the human antibody or antigen binding fragment providing tire framework is termed an “acceptor.” In one aspect, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they can be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized antibody or antigen binding fragment, except possibly the CDRs, are substantially identical to corresponding parts of natural human antibody sequences.

[0273] A “chimeric antibody” is an antibody which includes sequences derived from two different antibodies, which typically are of different species. In some examples, a chimeric antibody includes one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0274] A “fully human antibody” or “human antibody” is an antibody which includes sequences from (or derived from) the human genome, and does not include sequence from another species. In 4239-111527-02 some aspects, a human antibody includes CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using technologies for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals (see, e.g., Baibas et al. Phage display: A Laboratory Manuel. 1stEd. New York: Cold Spring Harbor Laboratory Press, 2004; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0275] Autologous: Refers to tissues, cells or nucleic acids taken from an individual’s own tissues. For example, in an autologous transfer or transplantation of immune cells (e.g., T cells or NK cells), the donor and recipient are the same person. Autologous (or “autogeneic” or “autogenous”) is related to self, or originating within an organism itself.

[0276] Cancer: A malignant tumor characterized by abnormal or uncontrolled cell growth. Other features often associated with cancer include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and suppression or aggravation of inflammatory or immunological response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc. “Metastatic disease” refers to cancer cells that have left the original tumor site and migrated to other parts of the body, for example via the bloodstream or lymph system. In some aspects herein, the cancer is the cancer is melanoma, liver cancer, renal cancer, pancreatic cancer, or lung cancer.

[0277] Chimeric antigen receptor (CAR): A chimeric molecule that includes an antigen-binding portion (such as one or more single domain antibodies or scFvs) and an intracellular domain, such as an intracellular domain from a T cell receptor (e.g. CD3Q. Typically, CARs include an antigenbinding portion(s), a transmembrane domain, and an intracellular domain. CARs also may include a hinge domain and / or a signal peptide (or leader sequence). The intracellular domain typically includes a signaling chain having an immunoreceptor tyrosine-based activation motif (IT AM), such as CD3C or FceRIy. In some instances, the intracellular' domain also includes the intracellular portion of at least one additional co-stimulatory domain, such as CD28. 4-1BB (CD137), ICOS, 0X40 (CD134), CD27 and / or DAP 10.

[0278] Conjugate: A complex of two molecules linked together, for example, linked together by a covalent bond. In one aspect, an AOAH protein is linked to a heterologous protein; for example, an antibody or antigen binding fragment that specifically binds to a target antigen (such as an antigen expressed by a tumor). The linkage can be by chemical or recombinant means. In one aspect, the linkage is chemical, wherein a reaction between the AOAH and the heterologous protein has produced a covalent bond formed between the two molecules to form one molecule. A peptide linker (e.g., a short peptide sequence) can optionally be included between the AOAH and the heterologous protein.

[0279] Conservative variants: “Conservative” amino acid substitutions are those substitutions that do not substantially affect or decrease a function of a protein, such as enzymatic activity or the ability 4239-111527-02 of the protein to interact with a target protein. For example, an AOAH protein disclosed herein can include up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conservative substitutions compared to a reference sequence and retain specific AOAH activity. The term conservative variant also includes the use of a substituted amino acid in place of an unsubstituted amino acid. Thus, a conservative substitution does not alter the basic function of a protein of interest.

[0280] Individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for instance less than 5%, in some aspects less than 1%) in an encoded sequence are also considered to be conservative variants, where the alterations result in the substitution of an amino acid with a chemically similar' amino acid.

[0281] The following are examples of amino acids that are considered to be conservative substitutions for one another:

[0282] 1) Alanine (A), Serine (S), Threonine (T);

[0283] 2) Aspartic acid (D) . Glutamic acid (E);

[0284] 3) Asparagine (N), Glutamine (Q);

[0285] 4) Arginine (R), Lysine (K);

[0286] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0287] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0288] Contacting: Placement in direct physical association; includes both in solid and in liquid form, which can take place either in vivo or in vitro. Contacting includes contact between one molecule and another molecule, for example the amino acid on tire surface of one polypeptide, such as an antigen, that contacts another polypeptide, such as an antibody or antigen binding fragment. Contacting can also include contacting a cell, for example by placing a protein (such as AOAH) in direct physical association with a cell.

[0289] Degenerate variant: In the context of the present disclosure, a “degenerate variant” refers to a polynucleotide encoding a polypeptide (such as an antibody heavy or light chain) that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a peptide are included as long as the amino acid sequence of tire peptide encoded by the nucleotide sequence is unchanged.

[0290] Effective amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be die amount of a disclosed AOAH composition necessary to inhibit or suppress growth of a tumor. In one aspect, an effective amount is the amount necessary to eliminate, reduce the size, or prevent metastasis of a tumor, such as reduce a tumor size and / or volume by at least 10%, at least 20%, at least 50%, at least 75%. at least 80%, at least 90%, at least 95%, or even 100%, and / or reduce the number and / or size / volume of metastases by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, for example as compared to a size / volume / number prior to treatment. In one aspect, an effective amount 4239-111527-02 is the amount necessary to increase the survival time of a subject with a tumor, such as increase survival time by at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months, for example as compared a survival time compared to a subject with no treatment or a different treatment. In some aspects, combinations of these effects are achieved. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in tumors) that has been shown to achieve a desired in vitro effect.

[0291] Fc region or domain: The constant region of an antibody excluding the first heavy chain constant domain. Fc region generally refers to the last two heavy chain constant domains of IgA, IgD, and IgG, and the last three heavy chain constant domains of IgE and IgM. An Fc region may also include part or all of the flexible hinge N-terminal to these domains. For IgA and IgM, an Fc region may or may not include the tailpiece, and may or may not be bound by the J chain. For IgG. the Fc region is typically understood to include immunoglobulin domains Cy2 and C 3 and optionally the lower part of the hinge between Cyl and Cy2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues following C226 or P230 to the Fc carboxyl-terminus, wherein the numbering is according to Kabat. For IgA, the Fc region includes immunoglobulin domains Ca2 and Ca3 and optionally the lower part of the hinge between Cal and Ca2. In one example, an Fc region or domain is a human IgGl Fc domain, such as amino acids 20-245 of SEQ ID NO: 3.

[0292] Fusion Protein: A single polypeptide chain including tire sequence of two or more heterologous proteins, often linked by a peptide linker. In some aspects, a fusion protein is a type of conjugate, for example, produced by recombinant methods.

[0293] Heterologous: Originating from a different genetic source. A nucleic acid molecule that is heterologous to a cell originates from a genetic source other than the cell in which it is expressed. In one specific, non-limiting example, a heterologous nucleic acid molecule encoding a protein is expressed in a cell, such as a mammalian cell. Methods for introducing a heterologous nucleic acid molecule in a cell or organism are well known in the ait, for example transformation with a nucleic acid, including electroporation, lipofection, particle gun acceleration, and homologous recombination.

[0294] Immune checkpoint inhibitor: Agents that inhibit biological pathways in specific types of immune system cells (such as T cells) and some cancer cells by blocking checkpoint proteins from binding their partner proteins. Immune checkpoint proteins inhibit T cells from killing cancer cells. When a checkpoint protein is blocked, the “inhibition” on the immune system is reduced and T cells increase their activation profile, which can lead to enhanced T cell responses against cancer cells. Examples of checkpoint proteins found in T cells or cancer cells include programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), B and T lymphocyte attenuator (BTLA), and T cell immunoglobulin and mucin domaincontaining protein 3 (TIM-3). In some aspects herein, the immune checkpoint inhibitor is a PD-1 4239-111527-02 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a BTLA antagonist, a TIM-3 antagonist, a lymphocyte activation gene 3 (LAG3) antagonist, a 4-1BB agonist, or an 0X40 agonist. In some examples, the immune checkpoint inhibitor is anti-PD-1 (such as nivolumab or pembrolizumab), anti- PD-L1 (such as atezolizumab, avelumab, or durvalumab), and / or anti-CTLA-4 (such as ipilimumab).

[0295] Isolated: A biological component (such as a nucleic acid or protein, for example an antibody or antigen binding fragment) that has been substantially separated, produced apart from, or purified away from other biological components, for example other nucleic acids and / or proteins. Thus, isolated nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids and proteins. An isolated nucleic acid or protein, for example an antibody or antigen binding fragment, can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% pure.

[0296] Linker: A bi-functional molecule that can be used to link two molecules into one contiguous molecule, for example, to link domains together or to link together heterologous proteins (for example in the form of a fusion protein). Non-limiting examples of peptide linkers include glycine-serine linkers. The terms “conjugating,” “joining,” “bonding,” or “linking” can refer to making two molecules into one contiguous molecule; for example, linking two polypeptides into one contiguous polypeptide. The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction such that there is a covalent bond formed between the two molecules to form one molecule.

[0297] Lymphocyte antigen 86 (LY86): A secreted protein involved in immune responses, inflammation and cell signaling. LY86 acts upstream of or within positive regulation of LPS- mediating signaling pathways. Exemplary LY86 protein and nucleic acid sequences are set forth herein as SEQ ID NO: 33 and SEQ ID NO: 34, respectively. Additional LY86 sequences are publicly available, such as under NCBI Gene ID 9450.

[0298] Natural killer (NK) cells: Cells of the immune system that kill target cells in the absence of a specific antigenic stimulus and without restriction according to MHC class. Target cells can be tumor cells or cells harboring viruses. NK cells are characterized by the presence of CD56 and the absence of CD3 surface markers. NK cells typically comprise approximately 10 to 15% of the mononuclear cell fraction in normal peripheral blood. Historically, NK cells were first identified by their ability to lyse certain tumor cells without prior immunization or activation. NK cells are thought to provide a “back up” protective mechanism against viruses and tumors that might escape the CTL response by down-regulating MHC class I presentation. In addition to being involved in direct cytotoxic killing, NK cells also serve a role in cytokine production, which can be important to control cancer and infection. In some examples, a “modified NK cell” is a NK cell transduced or transfected with a heterologous nucleic acid (such as one or more of the nucleic acids or vectors disclosed herein) 4239-111527-02 or expressing one or more heterologous proteins (such as an AOAH protein). The terms “modified NK cell” and “transduced NK cell” are used interchangeably in some examples herein.

[0299] Pharmaceutically acceptable carriers: Pharmaceutically acceptable carriers of use are known to those of ordinary skill in tire ail. Remington: The Science and Practice of Pharmacy, 22'1'1ecl., London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed compositions. In general, the nature of the carrier will depend on the particular mode of administration being employed. For example, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle.

[0300] In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, added preservatives (such as non-natural preservatives), pH buffering agents, and the like, for example sodium acetate or sorbitan monolaurate. In particular examples, the pharmaceutically acceptable carrier is sterile and suitable for parenteral administration to a subject for example, by injection. In some aspects, the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form such as in a selected quantity in a vial. Unit dosage forms can include one dosage or multiple dosages (for example, in a vial from which metered dosages of the agents can selectively be dispensed).

[0301] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which tire peptide or protein (such as an AOAH protein or fusion protein) is more enriched than the peptide or protein is in its original environment, such as within a cell. In one aspect, a preparation is purified such that tire protein or peptide represents at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of the total peptide or protein content of the preparation.

[0302] Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occuning or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished, for example, by chemical synthesis or by tire manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. In several aspects, a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell. The nucleic acid can be introduced, for example, on an expression vector having signals capable of expressing the protein encoded by the introduced nucleic acid or the nucleic acid can be integrated into the host cell chromosome.

[0303] Subject: Living multi-cellular vertebrate organisms, a category that includes human and nonhuman mammals, such as non-human primates, pigs, sheep, cows, dogs, cats, rodents, and the like. In one example, a subject is a human. 4239-111527-02

[0304] T cell: A white blood cell (lymphocyte) that is an important mediator of the immune response. T cells include, but are not limited to, CD4+T cells and CD8+T cells. A CD4+T lymphocyte is an immune cell that carries a marker on its surface known as “cluster of differentiation 4” (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8+T cells carry the “cluster of differentiation 8” (CD8) marker. In one aspect, a CD8+T cell is a cytotoxic T lymphocyte (CTL). In another aspect, a CD8+cell is a suppressor T cell. Activated T cells can be detected by an increase in cell proliferation and / or expression of or secretion of one or more cytokines (such as IL-2, IL-4, IL-6, IFNy. or TNFa). Activation of CD8+T cells can also be detected by an increase in cytolytic activity in response to an antigen. In some examples, a “modified T cell” is a T cell transduced with a heterologous nucleic acid (such as one or more of the nucleic acids or vectors disclosed herein) or expressing one or more heterologous proteins (such as an AOAH protein). The terms “modified T cell” and “transduced T cell” are used interchangeably in some examples herein.

[0305] T cell receptor (TCR): A heterodimeric protein on the surface of a T cell that binds an antigen (such as an antigen bound to an MHC molecule, for example, on an antigen presenting cell). TCRs include alpha and beta chains, each of which is a transmembrane glycoprotein. Each chain has variable and constant regions with homology to immunoglobulin variable and constant domains, a hinge region, a transmembrane domain, and a cytoplasmic tail. Similar to immunoglobulins, TCR gene segments rearrange during development to produce complete variable domains.

[0306] Transformed: A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformed and the like (e.g., transformation, transfection, transduction, etc.) encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transduction with viral vectors, transformation with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

[0307] Vector: A nucleic acid molecule (such as a DNA or RNA molecule) including a promoter(s) that is operably linked to the coding sequence of a protein of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and / or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication-incompetent, or a virus or bacterium or other microorganism that may be replication- competent. A vector is sometimes referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements. In some aspects, a vector includes a nucleic acid molecule encoding a disclosed AOAH protein or fusion protein. In some examples, the vector is a bacterial vector. In other examples, the vector is a viral vector, such as a nucleic acid vector having at least 4239-111527-02 some nucleic acid sequences derived from one or more viruses. In some aspects, the viral vector is a retroviral vector or an adeno-associated virus (AAV) vector.

[0308] III. AOAH Proteins, Nucleic Acids, and Modified Immune Cells

[0309] Provided herein are AOAH proteins, nucleic acids encoding AOAH proteins, and modified immune cells expressing a heterologous AOAH protein, for example, for use in methods of treating cancer.

[0310] AOAH is a secreted protein known to detoxify lipopolysaccharide by catalyzing hydrolysis of acyloxylacyl-linked fatty acyl chains from bacterial lipopolysaccharides. AOAH has both light (small) and heavy (large) subunits that are encoded by a single locus. In some examples, AOAH is expressed as a preprotein (e.g., GenBank Accession No. NP_001628.1, SEQ ID NO: 31) that is processed to a mature protein (such as amino acids 31-571 of SEQ ID NO: 1). Nucleic acids encoding AOAH are also known and include GenBank Accession No. NM_001637.4 (SEQ ID NO: 32).

[0311] In some aspects, the AOAH protein is a mature or processed form of the protein that lacks the signal peptide. In some examples, the AOAH protein does not include an N-terminal methionine; however, an N-terminal methionine can be present, for example as a result of expression in a bacterial, yeast, or mammalian system.

[0312] In some aspects, the AOAH protein has an amino acid sequence with at least 90% sequence identity (such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to amino acids 31-571 of SEQ ID NO: 1. In other examples, the AOAH protein includes or consists of the amino acid sequence of amino acids 31-571 of SEQ ID NO: 1.

[0313] In additional aspects, tire AOAH protein includes a signal peptide and / or a tag (such as for protein purification). The tag may be a histidine tag (such as a 6XHis tag), Fc-tag, glutathione S- transferase (GST)-tag, FLAG-tag, or streptavidin-binding peptide (SBP)-tag. In one example, the tag is a 6XHis tag. In some examples, the AOAH protein has an amino acid sequence with at least 90% sequence identity (such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 1. In other examples, the AOAH protein includes or consists of the amino acid sequence of SEQ ID NO: 1.

[0314] In another aspect, fusion proteins or conjugates including an AOAH protein and a heterologous protein are also provided. In some examples, the fusion protein includes a heterologous protein linked to the N-terminal of AOAH. In other examples, the fusion protein includes a heterologous protein linked to the C-terminal of AOAH. The linkage may be via a direct peptide bond between the AOAH protein and the heterologous protein, or may include a peptide linker (such as about 1-20 amino acids) between the AOAH protein and the heterologous protein. In one non- 4239-111527-02 limiting example, the linker is a (G+S) ; linker. In additional examples, the fusion protein may also include a signal peptide at the amino terminus.

[0315] In one example, the fusion protein includes an AOAH protein linked to an Fc domain. In some examples, the Fc domain has an amino acid sequence with at least 90% sequence identity (such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to amino acids 20-245 of SEQ ID NO: 5. In other examples, the Fc domain includes or consists of the amino acid sequence of amino acids 20- 245 of SEQ ID NO: 5. In some examples, the AOAH-Fc domain fusion protein includes a linker (such as a ( G S ) ; linker) between the AOAH and Fc domain portions of the fusion protein. In one example, the Fc domain is N-terminal to the AOAH protein. In some examples, the fusion protein has an amino acid sequence with at least 90% sequence identity (such as at least 90%. at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% sequence identity) to amino acids 20-801 of SEQ ID NO: 5. In other examples, the fusion protein includes or consists of the amino acid sequence of amino acids 20-801 of SEQ ID NO: 5. In further examples, the fusion protein has an amino acid sequence with at least 90% sequence identity (such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 5. In other examples, the fusion protein includes or consists of the amino acid sequence of SEQ ID NO: 5.

[0316] In other aspects, the AOAH protein may be fused or conjugated to an antibody, for example to increase targeting or specificity to the tumor microenvironment and / or tumor-draining lymph nodes. Exemplary antibodies include but are not limited to anti-PD-Ll (e.g., atezolizumab), anti- HER2 (e.g., trastuzumab), anti-PD-1 (e.g., pembrolizumab or nivolumab), anti-CTLA4 (e.g., ipilimumab), anti-mesothelin, and anti-glypican 3 (GPC3).

[0317] In some aspects, variants of the AOAH protein having one or more amino acid substitutions are provided. Amino acid substitutions, insertions, or deletions may be introduced and the products screened for a desired activity, e.g., phospholipase activity, lysophospholipase activity, diacylglycerol lipase activity, and / or acyltransferase activity (see, e.g., Munford et al., J. Biol. Client. 267:10116- 10121, 1992). Methods of detecting activity of an AOAH protein can be determined by one of skill in the art and include those described in Examples 1 and 2 and other methods known in the field (e.g., Staab et al., J. Biol. Chem. 269:23736-23742, 1994; Zou et al., eLife 10:e70938, 2021).

[0318] In some aspects, the AOAH protein includes up to 10 (such as up to 1, up to 2, up to 3, up to 4. up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence of a reference AOAH protein, such as a protein including amino acids 31-571 of SEQ ID NO: 1 or a protein including the amino acid sequence of SEQ ID NO: 31.

[0319] In additional aspects, the AOAH proteins or fusion proteins disclosed herein include one or more modifications, which in some examples may increase stability of the protein (for example, 4239-111527-02 resistance to proteolysis), solubility, and / or provide other desirable properties. While particular N- or C -terminal modifications are discussed below, each of the modifications may be at the N-terminus of the protein, the C-terminus of the protein, or both. In some examples, an AOAH protein or fusion protein is modified to include an N-terminal acetyl group and / or a C -terminal amide. In additional examples, the AOAH protein or fusion protein is modified with a polyethylene glycol (PEG) moiety (e.g., is a pegylated protein). In other examples, the modifications include one or more N-methyl amides in the linear portion of the protein. In yet further examples, a carbon chain (such as a 1-8 carbon chain) is added at the C-terminus of the protein. In still further examples, an aminoalkyl thiol (such as a C1-C8 alkyl) is added to the C-terminus of the protein. In additional examples an AOAH protein or fusion protein is cyclized. In some embodiments, the cyclization is by head to tail amino and carboxyl link (and in some examples amino acid side chain protection and deprotection before and after the cyclization, respectively). In still further examples, a modified AOAH protein or fusion protein includes N-terminal caprylic acid (sodium N-(8-[2-hydroxybenzoyl]amino) or an N-terminal fatty acid (such as C14-C18- fatty acid).

[0320] In addition to naturally occurring genetically encoded amino acids, one or more amino acid residues in the disclosed AOAH proteins or fusion proteins may be substituted with naturally occurring non-encoded amino acids and / or synthetic amino acids. In some aspects, the substitution(s) may increase resistance to proteolysis, increase oral bioavailability, or a combination thereof. In some examples, such amino acids include acylation of lysine £-amino groups; N-alkylation of arginine, histidine, or lysine; alkylation of glutamic or aspartic carboxylic acid groups; deamidation of glutamine or asparagine; -alanine and other omega-amino acids, such as 3-aminopropionic acid, 2,3- diaminopropionic acid, 4-aminobutyric acid and tire like; a-aminoisobutyric acid; e-aminohexanoic acid; 5-aminovaleric acid; N-methylglycine or sarcosine; ornithine; citrulline; penicillamine; t- butylalanine; t-butylglycine; N-methylisoleucine; phenylglycine; cyclohexylalanine; norleucine; naphthylalanine; 4-chlorophenylalanine; 2 -fluorophenylalanine; 3-fluorophenylalanine; 4- fluorophenylalanine; alpha-methyl-leucine; N-methyl-leucine; -Leucine; 1, 2,3,4- tetrahydroisoquinoline-3-carboxylic acid; [5-2 -thienylalanine; methionine sulfoxide; homoarginine; N- acetyl lysine; alpha-methyl-lysine; 2,4-diaminobutyric acid; 2, 3 -diaminobutyric acid; p- aminophenylalanine; N-methyl valine; homocysteine; homophenylalanine; homoserine; hydroxyproline; homoproline; N-methylated amino acids; peptoids (where side groups are appended to the nitrogen atom of the peptide backbone, rather than to the a-carbons); and P-peptides (where the amino group is bonded to the P carbon rather than the a-carbon).

[0321] While in certain aspects, the amino acids of the disclosed proteins are L-amino acids, in other aspects, one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, or all) L-amino acids are replaced with a D-amino acid (e.g., L-Leu^D-Leu). In some examples, including one or more D-amino acids may make the peptide more resistant to proteolysis, increase plasma half-life, and / or increase oral bioavailability. In some aspects, all amino acids in the peptide are D-amino acids. In other aspects, 4239-111527-02 one or more amino acids (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more) are D-amino acids. In some examples, at least 1, 2, or 3 C-terminal amino acids are D-amino acids. In additional examples, a modified or non-naturally occurring amino acid, non-encoded amino acid, or synthetic amino acid (such as those described above) may be a D-amino acid.

[0322] In some examples, the disclosed AOAH proteins or fusion proteins may be in the form of one or more pharmaceutically acceptable salts or esters. Pharmaceutically acceptable salts include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N' -dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N- benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. The salts may be prepared by standard procedures, for example by reacting the free acid with a suitable organic or inorganic base. Representative bases include ammonium hydroxide, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, and the like. Description of suitable pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002).

[0323] Pharmaceutically acceptable esters include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, phenyl, pyridinyl, benzyl, and the like. Pharmaceutically acceptable esters can be prepared by, for example, by treating tire compound with an appropriate amount of carboxylic acid, ester, acid chloride, acid anhydride, or mixed anhydride agent that will provide the corresponding pharmaceutically acceptable ester. Typical agents that can be used to prepare pharmaceutically acceptable esters include, for example, acetic acid, acetic anhydride, acetyl chloride, benzylhalide, benzaldehyde, benzoylchloride, methyl ethylanhydride, methyl phenylanhydride, methyl iodide, and the like.

[0324] In some examples, the AOAH fusion protein is an Fc domain fusion protein that is expressed or prepared in two parts. The two parts may be mixed together, purified, and then prepared as a composition for administering to a subject. In some examples, the first part is an AOAH protein fused to an IgG heavy chain and the second part is an IgGl portion (“asymmetric IgGl”) that can form heterodimers with the IgG heavy chain of the first part. In some examples, the first part has an amino acid sequence with at least 90% sequence identity (such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% sequence identity) to amino acids 20-801 of SEQ ID NO: 5. In other examples, the first part includes or consists of the amino acid sequence of amino acids 20-801 of SEQ ID NO: 5. In further examples, the first part has an amino acid sequence with at least 90% sequence identity (such as at least 90%, at 4239-111527-02 least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 5. In other examples, the first part includes or consists of the amino acid sequence of SEQ ID NO: 5. In some examples, the second part has an amino acid sequence with at least 90% sequence identity (such as at least 90%, at least 91%, at least 92%, at least 93%. at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to amino acids 20-245 of SEQ ID NO: 7. In other examples, the second part includes or consists of the amino acid sequence of amino acids 20-245 of SEQ ID NO: 7. In further examples, the second part has an amino acid sequence with at least 90% sequence identity (such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% sequence identity) to SEQ ID NO: 7. In other examples, the second part includes or consists of the amino acid sequence of SEQ ID NO: 7.

[0325] Nucleic acids encoding the AOAH proteins or fusion proteins are also provided. In some aspects, a nucleic acid encoding an AOAH protein has at least 85% (such as at least 85%. at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to nucleotides 91-1713 of SEQ ID NO: 2. In other examples, a nucleic acid encoding an AOAH protein includes or consists of nucleotides 91-1713 of SEQ ID NO: 2. In other aspects, a nucleic acid encoding an AOAH-Fc domain fusion protein is provided. In some examples, a nucleic acid encoding an AOAH-Fc fusion protein has at least 85% (such as at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to nucleotides 58-2403 of SEQ ID NO: 6. In other examples, a nucleic acid encoding an AOAH protein includes or consists of nucleotides 58-2403 of SEQ ID NO: 6. In further examples, a nucleic acid encoding an AOAH-Fc domain fusion protein has at least 85% (such as at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 6 or includes or consists of SEQ ID NO: 6. In additional examples, a nucleic acid encoding a second part of the AOAH Fc fusion has at least 85% (such as at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%. at least 98%, or at least 99% sequence identity) to nucleotides 58-735 of SEQ ID NO: 8. In other examples, the nucleic acid includes or consists of nucleotides 58-735 of SEQ ID NO: 8. In further examples, a nucleic acid encoding the second part of the Fc domain fusion protein has at least 85% (such as at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 8, or includes or consists of SEQ ID NO: 8.

[0326] In some aspects, a nucleic acid molecule encoding an AOAH protein is included in an expression vector (such as a viral vector) for expression in a host cell, such as a T cell or NK cell. In 4239-111527-02 some examples, the expression vector includes a promoter operably linked to the nucleic acid molecule encoding AO AH. Exemplary promoters include but are not limited to EFla promoter, CMV promoter, or SV40 promoter. In one non-limiting example, the promoter is an EFla promoter. Additional expression control sequences, such as one or more enhancers, transcription and / or translation terminators, and initiation sequences (such as a Kozak sequence) can also be included in the expression vector. In some aspects, a nucleic acid encoding AOAH is included in a viral vector. Examples of suitable virus vectors include retrovirus (e.g.. MoMLV or lentivirus), adenovirus, adeno- associated virus, vaccinia virus, and fowlpox vectors. In specific examples, the AOAH encoding nucleic acid is included in a lentiviral vector. In other examples, the vector may be a DNA vector (such as a plasmid).

[0327] Also provided are modified immune cells that express a heterologous AOAH protein and a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some aspects the modified immune cells are T cells, natural killer (NK) cells, NKT cells, double -negative T (DNT) cells, neutrophils, or macrophages. In particular examples, the modified immune cells are T cells or NK cells.

[0328] In some aspects, the immune cells are transduced or transfected with a vector (such as an expression vector) including a nucleic acid encoding an AOAH protein. In other aspects, the vector (or a DNA encoding die AOAH protein) may be introduced by contacting the cells with a nanoparticle including the vector or DNA. In some examples, the transduced or transfected cells are isolated T cells (such as primary T cells or T cells obtained from a subject), isolated NK cells (such as primary NK cells or NK cells obtained from a subject), isolated NKT cells, isolated DNT cells, isolated neutrophils, or isolated macrophages (such as primary macrophages or macrophages obtained from a subject). In some examples, the T cells, NK cells, NKT cells, DNT cells, neutrophils, or macrophages are obtained from peripheral blood. In some examples, T cells, NK cells, NKT cells, or DNT cells are also enriched, purified, and / or expanded from a sample from a subject, for example before and / or after transduction with a vector encoding AOAH and / or a TCR or CAR.

[0329] The modified immune cells may also express one or more TCRs or one or more CARs. In some aspects, the TCR or CAR specifically binds a protein expressed by a tumor (e.g., a tumor antigen). The protein specifically bound by the one or more TCRs or CARs can be selected by one of ordinary skill, based on the condition (such as type of cancer) of the subject to be treated. In some examples, the one or more TCRs or CARs specifically bind CD19, CD20, CD30, CD33, CD38, CD52, CEA, mucins, cytokeratin, VEGF, integrin aV 3, EGFR, ERBB2, SLAMF7, and / or RANKL. In other examples, the protein specifically bound by the one or more TCRs or CARs may be NY - ESO-1 (e.g., expressed in melanoma), GPC3 (e.g., expressed in hepatocellular carcinoma), mesothelin (e.g., expressed in mesothelioma, pancreatic, and triple-negative breast cancer), disialoganglioside GD2 (e.g., expressed in neuroblastoma, melanoma, osteosarcoma, breast cancer, retinoblastoma, brain 4239-111527-02 tumors, pediatric rhabdomyosarcoma, and small cell lung cancers), or CD70 (e.g., expressed in B-cell lymphoma, nasopharyngeal carcinoma, and clear cell renal cancer).

[0330] In some examples, tire immune cells may be immune cells expressing a TCR (such as an endogenous TCR) or may be modified immune cells expressing a heterologous TCR, for example, immune cells that have previously been engineered to express a heterologous TCR. In other examples, the modified immune cells expressing tire AOAH protein may be transduced or transfected with a vector encoding a TCR or may be contacted with a nanoparticle including the vector (or a DNA or mRNA encoding the TCR).

[0331] In other examples, the immune cells may be immune cells expressing a CAR, for example, immune cells that have previously been engineered to express a CAR. In other examples, the modified immune cells expressing the AOAH protein may be transduced or transfected with a vector encoding a CAR or may be contacted with a nanoparticle including the vector (or a DNA or mRNA encoding the CAR).

[0332] IV. Methods of Treating Cancer

[0333] Methods of treating cancer with a composition including an AOAH protein or fusion protein or conjugate, or nucleic acid encoding an AOAH protein or fusion protein or conjugate are provided. Methods of treating cancer with a composition including modified immune cells expressing a heterologous AOAH protein and a T cell receptor or chimeric antigen receptor are also provided.

[0334] In some aspects, the subject has a solid tumor. In some examples, the subject is administered an AOAH protein, a nucleic acid encoding an AOAH protein, or a fusion protein disclosed herein. In other aspects, the subject is administered a modified immune cell expressing AOAH and a TCR or CAR targeting an antigen expressed by the tumor. Examples of solid tumors include sarcomas (such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas), synovioma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, peritoneal cancer, esophageal cancer, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular- breast carcinoma), lung cancer, ovarian cancer, prostate cancer, liver cancer (including hepatocellular carcinoma), gastric cancer, squamous cell carcinoma (including head and neck squamous cell carcinoma), basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, fallopian tube cancer, testicular tumor, seminoma, bladder cancer, kidney cancer (such as renal cell cancer), melanoma, and CNS tumors (such as a glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyrgioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma and retinoblastoma). Solid tumors also include tumor metastases (for example, metastases to the lung, 4239-111527-02 liver, brain, or bone). In particular examples, the subject has pancreatic cancer, melanoma, kidney cancer, liver cancer (such as hepatocellular carcinoma), or lung cancer.

[0335] In other aspects, the subject has a hematological malignancy. In some examples, the subject is administered an AOAH protein or fusion protein disclosed herein. In other aspects, the subject is administered a modified immune cell expressing AOAH and a TCR or CAR targeting an antigen expressed by the hematological malignancy. Examples of hematological malignancies include leukemias, including acute leukemias (such as llq23 -positive acute leukemia, acute lymphocytic leukemia (ALL), T-cell ALL, acute myelocytic leukemia, acute myelogenous leukemia (AML), and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL)), lymphoblastic leukemia, polycythemia vera, lymphoma, diffuse large B cell lymphoma, Burkitt lymphoma, T cell lymphoma, follicular lymphoma, mantle cell lymphoma, Hodgkin disease. non-Hodgkin lymphoma, multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, myelodysplastic syndrome (MDS), hairy cell leukemia, and myelodysplasia. In some examples, the hematological malignancy is B-cell lymphoma or acute myeloid leukemia.

[0336] In some examples, a subject with cancer is administered a composition including an AOAH protein, such as an AOAH protein disclosed herein. In other examples, the subject matter is administered a composition including an AOAH fusion protein (such as an AOAH-Fc domain fusion protein) or an AOAH conjugate (such as AOAH conjugated to an antibody or portion thereof) disclosed herein. In further examples, the subject is administered a composition including a nucleic acid encoding an AOAH protein. The AOAH protein, fusion protein or conjugate, or nucleic acid can be incorporated into pharmaceutical compositions. Such compositions typically include a population of cells and a pharmaceutically acceptable carrier. A “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (see, e.g., Remington: The Science and Practice of Pharmacy, 22nded., London, UK: Pharmaceutical Press, 2013). Examples of such carriers or diluents include, but are not limited to. water, saline, Ringer’s solutions, dextrose solution, balanced salt solutions, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Supplementary active compounds can also be incorporated into the compositions. Actual methods for preparing administrable compositions include those provided in Remington: The Science and Practice of Pharmacy, 22nded., London, UK: Pharmaceutical Press, 2013.

[0337] In some aspects, a nucleic acid encoding an AOAH protein is incorporated in lipid nanoparticles. In a particular example, the AOAH encoding nucleic acid is incorporated in lipid nanoparticles (LNP) with GalNac (N-acetylgalactosamine), an asialoglycoprotein receptor (ASGR) ligand, that may achieve hepatocyte-specific LNP delivery. Hepatocytes express high levels of ASGR 4239-111527-02

[0338] (a heterodimer of ASGR1 and ASGR2 proteins). According to TCGA RNA-seq data, most hepatocellular carcinoma tumors still express high ASGR. Thus, GalNac-LNP with AOAH mRNA inside may also serve as a therapeutic approach for liver cancer.

[0339] The AOAH proteins, nucleic acids, and pharmaceutical compositions provided herein may be administered through different routes, such as oral, including buccal and sublingual, rectal, parenteral, aerosol, nasal, intravenous, intramuscular, subcutaneous, intradermal, and topical. They may be administered in different forms, including but not limited to solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles, and liposomes. In other examples, it may be desirable to administer the proteins or pharmaceutical compositions locally to the area in need of treatment, such as at or near the site of a tumor in the subject. This may be achieved by, for example, and not by way of limitation, local or regional infusion or perfusion, topical application, injection, catheter, suppository, or implant (e.g., implants formed from porous, non-porous. or gelatinous materials, including membranes, such as sialastic membranes or fibers), and the like. In some examples, the AOAH protein, fusion protein, conjugate, or nucleic acid is administered by intravenous, intratumoral, or intraperitoneal administration.

[0340] The amount of the AOAH protein, fusion protein, or conjugate that will be effective depends on the disorder (such as type of cancer) to be treated, as well as the stage of the cancer and condition of the subject. Effective amounts can be determined by standard clinical techniques. The precise amount will also depend on the route of the administration. In some aspects, the subject is administered a composition including about 0.5 mg / kg to about 50 mg / kg of the AOAH protein, fusion protein, or conjugate. In some examples, the subject is administered a composition including about 0.5 mg / kg to about 2 mg / kg, about 1 mg / kg to about 2.5 mg / kg, about 1.5 mg / kg to about 5 mg / kg, about 3 mg / kg to about 6 mg / kg, about 4 mg / kg to about 8 mg / kg, about 7 mg / kg to about 10 mg / kg, about 10 mg / kg to about 20 mg / kg, about 15 mg / kg to about 25 mg / kg, or about 25 mg / kg to about 50 mg / kg of tire AOAH protein, fusion protein, or conjugate. In some examples, the subject is administered a composition including about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg. about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg. about 7.5 mg / kg, about 8 mg / kg, about 8.5 mg / kg, about 9 mg / kg, about 9.5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, or about 50 mg / kg of the AOAH protein, fusion protein, or conjugate.

[0341] In some aspects, unit dosage forms, for example, formulations containing a dose or unit, or appropriate fraction thereof of the AOAH protein, fusion protein, or conjugate may be used. In some examples, the formulation includes about 10 mg, about 20 mg. about 25 mg, about 50 mg. about 100 mg. about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, or about 1000 mg per dose. However, 4239-111527-02 other higher or lower dosages also could be used, as can be determined by in vitro and / or in vivo testing.

[0342] Multiple doses of the composition including tire AOAH protein, fusion protein, or conjugate can be administered to a subject. For example, tire composition can be administered daily, every other day, twice per week, weekly, every other week, every three weeks, monthly, or less frequently. A skilled clinician can select an administration schedule based on the subject, the condition being treated, the previous treatment history, and other factors.

[0343] In other aspects, a subject is administered modified immune cells expressing an AOAH protein and a CAR or TCR (such as AOAH expressing CAR-T cells, CAR-NK cells, or CAR-NK-T cells). The modified immune cells can be incorporated into pharmaceutical compositions. Such compositions typically include a population of cells and a pharmaceutically acceptable carrier. A “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (see, e.g., Remington: The Science and Practice of Pharmacy, 22nded., London, UK: Pharmaceutical Press, 2013). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer’s solutions, dextrose solution, balanced salt solutions, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Supplementary active compounds can also be incorporated into the compositions. Actual methods for preparing administrable compositions include those provided in Remington: The Science and Practice of Pharmacy, 22nded., London, UK: Pharmaceutical Press, 2013.

[0344] In some examples, the composition includes about 104to 1012of the modified immune cells (for example, about 104-l 0scells, about 106-108cells, or about 106-1012cells). For example, tire composition may be prepared such that about 104to 1010modified NK cells or modified T cells cells / kg (such as about 104, 105, 106, 107, 10s, 109, or 1010cells / kg) are administered to a subject. In specific examples, the composition includes at least 104, 105, 106, or 107modified immune cells. The population of modified cells is typically administered parenterally, for example intravenously; however, other routes of administration can also be used. Appropriate routes of administration can be determined based on factors such as the subject, the condition being heated, and other factors.

[0345] Multiple doses of the population of modified immune cells can be administered to a subject. For example, the cells can be administered daily, every other day, twice per week, weekly, every other week, every three weeks, monthly, or less frequently. A skilled clinician can select an administration schedule based on the subject, the condition being treated, the previous treatment history, and other factors.

[0346] In some aspects, additional cancer therapeutics, such as chemotherapeutic agents, can be administered. The therapeutic agent can be, but is not limited to, an immune checkpoint inhibitor. Immune checkpoint inhibitors are agents that block biological pathways in specific types of immune system cells, such as, but not limited to, T cells, and some cancer cells. These immune checkpoint 4239-111527-02 proteins inhibit T cells from killing cancer cells. When a checkpoint protein is blocked, an “inhibition” on the immune system is reduced and T cells increase their activation profile, which can lead to enhanced T cell responses against cancer cells. Examples of checkpoint proteins found on T cells or cancer cells include PD-1, PD-L1, CTLA-4, BTLA, and TIM-3.

[0347] Thus, in some examples, the disclosed methods include administering a therapeutically effective amount of a PD-1 antagonist, PD-L1 antagonist, CTLA-4 antagonist, BTLA antagonist, TIM -3 antagonist, LAG3 antagonist, a 4- IBB agonist, or an 0X40 agonist to the subject. In some examples, the immune checkpoint inhibitor is and -PD-1 (such as nivolumab or pembrolizumab), anti- PD-L1 (such as atezolizumab, avelumab, or durvaluinab), and / or anti-CTLA-4 (such as ipilimumab).

[0348] V. LY86 Proteins, Nucleic Acids, and Modified Immune Cells and Methods of Use

[0349] Also provided are LY86 proteins, nucleic acids encoding LY86 proteins, and modified immune cells expressing a heterologous LY86 protein, for example, for use in methods of treating cancer.

[0350] In some aspects, the LY86 protein has an amino acid sequence with at least 90% sequence identity (such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to amino acids 20-162 of SEQ ID NO: 33. In other examples, the LY86 protein includes or consists of the amino acid sequence of amino acids 20-162 of SEQ ID NO: 33. In other examples, the LY86 protein has at least 90% sequence identity (such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to, or includes or consists of SEQ ID NO: 33.

[0351] In additional aspects, die LY86 protein includes a signal peptide and / or a tag (such as for protein purification). The tag may be a histidine tag (such as a 6XHis tag), Fc-tag, glutathione S- transferase (GST)-tag, FLAG-tag, or streptavidin-binding peptide (SBP)-tag. In one example, die tag is a 6XHis tag.

[0352] In anodier aspect, fusion proteins or conjugates including an LY86 protein and a heterologous protein are also provided. In some examples, the fusion protein includes a heterologous protein linked to the N-terminal of LY86. In other examples, the fusion protein includes a heterologous protein linked to the C-terminal of LY86. The linkage may be via a direct peptide bond between the LY86 protein and the heterologous protein, or may include a peptide linker (such as about 1-20 amino acids) between die LY86 protein and the heterologous protein. In one non-limiting example, the linker is a (GiS) . linker. In additional examples, the fusion protein may also include a signal peptide at the amino terminus.

[0353] In one example, the fusion protein includes an LY86 protein linked to an Fc domain. In some examples, the Fc domain has an amino acid sequence with at least 90% sequence identity (such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at 4239-111527-02 least 97%, at least 98%, or at least 99% sequence identity) to amino acids 20-245 of SEQ ID NO: 5. In other examples, the Fc domain includes or consists of tire amino acid sequence of amino acids 20- 245 of SEQ ID NO: 5. In some examples, the LY86-Fc domain fusion protein includes a linker (such as a (G4S)J linker) between the LY86 and Fc domain portions of the fusion protein. In one example, the Fc domain is N-terminal to the LY86 protein.

[0354] In other aspects, the LY86 protein may be fused or conjugated to an antibody, for example to increase targeting or specificity to tire tumor microenvironment and / or tumor-draining lymph nodes. Exemplary antibodies include but are not limited to anti-PD-Ll (e.g.. atezolizumab), anti-HER2 (e.g., trastuzumab), anti-PD-1 (e.g., pembrolizumab or nivolumab), anti-CTLA4 (e.g., ipilimumab), anti- mesothelin, anti-GD2, and anti-GPC3.

[0355] Nucleic acids encoding the LY86 proteins or fusion proteins are also provided. In some aspects, a nucleic acid encoding an LY86 protein has at least 85% (such as at least 85%, at least 86%, at least 87%, at least 88%, at least 89%. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 34. In other examples, a nucleic acid encoding an LY86 protein includes or consists of SEQ ID NO: 34. In other aspects, a nucleic acid encoding an LY86-Fc domain fusion protein is provided.

[0356] In some aspects, a nucleic acid molecule encoding an LY86 protein is included in an expression vector (such as a viral vector) for expression in a host cell, such as a T cell or NK cell. In some examples, the expression vector includes a promoter operably linked to the nucleic acid molecule encoding LY86. Exemplary promoters include but are not limited to EFla promoter, CMV promoter, or SV40 promoter. In one non-limiting example, the promoter is an EFla promoter. Additional expression control sequences, such as one or more enhancers, transcription and / or translation terminators, and initiation sequences (such as a Kozak sequence) can also be included in the expression vector. In some aspects, a nucleic acid encoding LY86 is included in a viral vector. Examples of suitable virus vectors include retrovirus (e.g., MoMLV or lenti virus), adenovirus, adeno- associated virus, vaccinia virus, and fowlpox vectors. In specific examples, the LY86 encoding nucleic acid is included in a lentiviral vector. In other examples, the vector may be a DNA vector (such as a plasmid).

[0357] Also provided are modified immune cells that express a heterologous LY 86 protein and a T cell receptor (TCR) or chimeric antigen receptor (CAR). In some aspects the modified immune cells are T cells, NK cells, NKT cells, DNT cells, neutrophils, or macrophages. In particular examples, the modified immune cells are T cells or NK cells.

[0358] Methods of treating cancer with a composition including an LY86 protein or fusion protein or conjugate, or nucleic acid encoding an LY86 protein or fusion protein or conjugate are provided. Methods of treating cancer with a composition including modified immune cells expressing a heterologous LY86 protein and a T cell receptor or chimeric antigen receptor are also provided. In 4239-111527-02 some examples, the subject has a solid tumor or a hematological malignancy. In some examples, the subject is administered a LY86 protein, a nucleic acid encoding an LY86 protein, or a fusion protein disclosed herein. In other aspects, the subject is administered a modified immune cell expressing LY86 and a TCR or CAR targeting an antigen expressed by tire solid tumor or hematological malignancy. The methods described in Section IV for AOAH proteins, nucleic acids, or modified immune cells can similarly be utilized with LY86 proteins, nucleic acids, or modified immune cells expressing LY86.

[0359] EXAMPLES

[0360] The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.

[0361] Example 1 Materials and Methods

[0362] This Example describes the methods utilized in Examples 2-3.

[0363] Genomics data collection and processing: Cancer immunotherapy datasets were systematically collected from published studies.

[0364] For studies with raw data available, RNA-seq and whole-exome sequencing data (WES) were uniformly processed with the in-house genomics analysis framework (FIGS. 8C-8D). The RNA-seq output contains gene and isoform levels in transcript per million (TPM) with the log2(TPM+l) transformation (FIG. 8C). The WES output contains copy number alterations (CNA) and weighted somatic mutation on gene levels (FIG. 8D).

[0365] The somatic mutation weights were computed as follows. For MuTect2, the TLOD scores were first trimmed between 0 and 200 and linearly scaled to 0 and 1. For VarScan, the SSC scores were trimmed between 0 and 255 and linearly scaled to 0 and 1. For Strelka, the QSS_NT or QSI_NT (if QSS_NT was unavailable in tire output) scores were trimmed between 0 and 3070 and then linearly scaled to 0 and 1. Finally, the mutation weight was the combined confidence among three mutation calls using the noisy-or 1 - Hi ( 1—confidence t).

[0366] For studies without raw data released, the processed data from either supplementary materials of tire paper or source code repositories were downloaded. Download sources of non-immunotherapy genomics cohorts, including TCGA, ICGC, TARGET. PRECOG, and other clinical cohorts from tire TIDE frameworks are listed in Table 1. 4239-111527-02

[0367] Table 1. Key resources table 4239-111527-02 4239-111527-02 4239-111527-02 4239-111527-02 4239-111527-02 4239-111527-02 4239-111527-02

[0368] Association computation between gene values and clinical outcomes: Among cohorts with gene values (expression, mutation, copy number alteration, promoter methylation) from pretreatment bulk tumors, the risk scores were computed according to the type of endpoints. For overall survival (OS) or progression-free survival (PFS), the Cox proportional hazard (PH) regressions were utilized, and gene risk scores were the z-scores (Coef / StdErr) from the two-sided Wald test. For RECIST outcomes, the ordinary least squares (OLS) was utilized, and gene risk scores were negative t- values (Coef / StdErr) from the two-sided student t-test. For binary outcomes, if only the binary response information was available, the two-sided Wilcoxon rank-sum test was utilized, and gene risk scores were negative z-scores. If other clinical covariates (e.g., Age, Gender, Stage) were available, the logistic regression with Firth correlation was utilized, and gene risk scores were the negative z- scores (Coef / StdErr).

[0369] For each cohort, if multiple endpoints were available, the endpoints were prioritized in this order: OS > PFS > RECIST > Binary response.

[0370] Gene prioritizations for secreted proteins: Thirty-seven non-redundant genome -wide transcriptomics datasets from pre-immunotherapy bulk tumors were utilized. The list of 1903 genes coding secreted protein was from tire Human Protein Atlas on August 10, 2022 (available at proteinatlas.org / humanproteome / tissue / secretome). Then, among 1903 genes, genes with no risk scores computed in over 5% of datasets due to the absence of gene coverage in the original study or regression failures were removed. Then, for each gene, the two-sided Wilcoxon rank-sum test comparing risk scores across datasets and zero was computed and the p-values were converted to false discovery rates (FDR) by the Benjamini-Hochberg correction.

[0371] Genes with outlier datasets whose gene risk scores were in the reverse direction compared to the overall median risk scores were also filtered. The datasets for each gene with risk scores > 2 and < -2 were counted as pos_count and neg_count, respectively. For genes with positive median risk scores, only genes where pos_count - neg_count >= 3 were kept. For genes with negative median risk scores, only genes where neg_count - pos_count >= 3 were kept. For visualization simplicity, in FIG. 4239-111527-02

[0372] 2B, only the top 70 genes ranked by absolute values of median risk scores across 37 analyses were selected.

[0373] Spatial transcriptomics data analysis: Two datasets of Visium spatial transcriptomics (ST) from hepatocellular' carcinoma patients treated with anti-PDl (Zhang el al., Genome Med. 15:72, 2023; Liu et al., J. Hepatol. 78:770-782, 2023) were downloaded. To compare the expression of

[0374] AOAH between responders and non-responders, tire counts of all spatial spots were aggregated to generate a pseudo-bulk for each ST sample, normalizing the data to log2(TPM+l). The expression level of AOAH in ST samples was visualized using the R package SpaCET.

[0375] Single-cell RNA-seq data analysis: The R package Seurat was used to process and merge eight scRNA-seq samples from hepatocellular carcinoma tumors. Quality control was conducted to filter out the cells with unique feature counts > 4000 or <200, or >15% mitochondrial counts. Then the total counts in each cell were normalized to 10,000, followed by log transformation to generate the normalized data. Uniform Manifold Approximation and Projection (UMAP) analysis was applied to the first 30 principal components (PCs) for dimensional reduction and data visualization. The cells were clustered using FindClusters from Seurat, which is a shared nearest neighbor (SNN) modularity optimization-based clustering algorithm. The marker genes of major lineage and sub-lineage can be found in Table 2. Scrublet was used to calculate doublet scores.

[0376] Table 2. Marker genes for cell type annotations in single -cell RNA-seq analysis 4239-111527-02

[0377] Lipid class enrichment analysis: For cell culture medium or pellets, the log2FC values of a lipid species were computed as the log2 fold change of MassSpec areas between rhAOAH and buffer treatments. Then, a lipid configuration was defined as tire joint of class (e.g., glycerophosphocholines, etc.) and subclass (e.g., diacyl, etc.) keys, and grouped log2FC values by the lipid configuration. For a configuration with at least three lipid species, the log2FC difference was compared between lipids within the configuration versus lipids outside the configuration by the two- sided Wilcoxon rank-sum test. The p-values were converted to false discovery rates (FDR) by the Benjamini-Hochberg correction, and FDR < 0.05 was the threshold to select significant results.

[0378] For phosphatidylcholine, the fatty acid enrichment was also analyzed at the sn-1 and sn-2 positions. For each configuration pair (fatty acid, sn position), log2FC values of lipids with this configuration were compared with values without this configuration using the two-sided Wilcoxon rank-sum test. The p-values were converted to FDR by the Benjamini-Hochberg correction, and FDR < 0.05 was the threshold to select significant results.

[0379] Animal models: NOD.Cg-Prkdcscid I12rgtmlWjl / SzJ (NSG) mice, pmel-1 transgenic mice, and OT-1 transgenic mice were bred at the National Cancer Institute, National Institutes of Health (MD, USA). C57BL / 6J and BALB / c mice were purchased from Charles River Laboratories (MA, USA), pmel-1 mice were a gift from Dr. Glenn Merlino’s lab, and OT-1 mice were a gift from Dr. Gregoire Altan-Bonnet’s lab. Female mice between 6-12-week-old were used in the study and randomized into treatment groups. The maximal tumor size permitted by the ethics committee was 2 cm in diameter, and no tumor burden exceeded that limit in the experiment.

[0380] Hydrodynamic tail vein injection (HDTVi): Two murine hepatocellular carcinoma models driven by MYC overexpression with (LucOS) or without (Luc) highly immunogenic OVA+SIN+SIY (OS) antigens and beta-catenin (encoded by CTNNB1) activation were used. Both models show resistance to anti-PDl immunotherapy. A sterile 0.9% NaCl solution / plasmid mixture was prepared as follows: 10 pg of pT3-EFla-MYC-IRES-luciferase (MYC-Luc), 10 pg of pT3-EFla-MYC-IRES- luciferase-OS (MYC-LucOS), 10 pg of pT3-N90-CTNNBl (CTNNB1), 10 pg of PKT2 / CLP-Vec, 10 pg of PKT2 / CLP-Aoa / z, and a 3:1 ratio of transposon to SB 100 transposase-encoding plasmid 4239-111527-02 dissolved in 2 mL of 0.9% NaCl solution and tail-vein injected 10% of the weight of each mouse in volume in 5-7 seconds. For the anti-PDl experiment, treatments were initiated 8-9 days after hydrodynamic delivery of plasmids, a time point that already presents malignant tumor cells. Anti- PDl (BioXCell, #BE0146, 200 pg / dose) was given twice per week for a total of 4 doses. Bioluminescence imaging was taken twice a week. Vectors for hydrodynamic delivery were produced using the EndoFree Maxi Plasmid kit (TIANGEN).

[0381] Cell lines and cell culture: Every month, all cell lines were routinely tested for mycoplasma via PCR-based method using Universal Mycoplasma Detection Kit (ATCC). B16F10, B16-mhgpl00, and RenCa cells were maintained in RPMI-1640 with GlutaMAX™ (Thermo Fisher), 10% FBS (Gibco), 20 mM HEPES (Gibco), 100 IU ml-1penicillin / streptomycin (Gibco), and 1 / 500 Plasmocin® prophylactic (Invivogen). RIL-175 cells were maintained in DMEM (Thermo Fisher) with 10% FBS (Gibco), 100 IU ml-1penicillin / streptomycin (Gibco), and 1 / 500 Plasmocin® prophylactic (Invivogen). Jurkat -Lucia™ NFAT-CD28 Cells (Invivogen) were maintained in IMDM (Thermo Fisher), 2 mM L-glutamine (Gibco), 25 mM HEPES (Gibco), 10% HI-FBS, 100 IU ml-1penicillin / streptomycin (Gibco), 100 pg / ml Normocin™, as per manufacturer instructions. All cell lines were cultured in a humidified, 5% CO2, 37°C incubator.

[0382] Mouse CD8+ T cell isolation: Spleens from 8-10-week-old C57BL / 6 mice were minced on a 70-pm cell strainer (Falcon) to dissociate splenocytes. After centrifugation, the splenocytes were incubated in ACK lysing buffer (Gibco) for 2 minutes on ice to lysate the red blood cells (RBCs), neutralized by RPML1640 with 10% FBS, and centrifuged at 300xg for 5 minutes. The splenocytes were then resuspended in EasySep buffer (STEMCELL Technologies) at 1E7 cells / mL concentration. Mouse CD8+ T cells were isolated using the EasySep™ Mouse CD8+ T Cell Isolation Kit (STEMCELL Technologies), as per manufacturer instructions.

[0383] Human CD8+ T cell isolation: The collection of peripheral blood from healthy donors was approved and performed at the NIH Blood Center. All donors consented to their blood being used in the study. Blood samples were mixed with FicolLPaque PLUS (Cytiva), and this mixture was centrifuged at 400xg with the lowest acceleration and deceleration speed for 25 minutes at room temperature. The peripheral blood mononuclear cells (PBMC) were carefully collected using a Pasteur pipette, centrifuged at 300xg for 10 minutes, and washed with PBS containing 1% FBS (Gibco) and 0.5% EDTA. After centrifugation, the PBMCs were incubated in ACK lysing buffer (Gibco) for 5 minutes at room temperature to lyse RBCs and then neutralized by RPMI-1640 with 10% FBS and centrifuged at 300xg for 5 minutes. The PBMCs were then resuspended in EasySep™ buffer (STEMCELL Technologies) at 1E7 cells / mL concentration. Human CD8+ T cells were isolated using the EasySep™ Human CD8+ T Cell Isolation Kit (STEMCELL Technologies), as per manufacturer instructions. 4239-111527-02

[0384] Mouse and human CD8+ T cell culture and activation: Mouse and human CD8+ T cells were cultured in TexMACS™ medium (Miltenyi) supplemented with 10% HI-FBS and 50 pM - mercaptoethanol (Gibco) at 1-1.5E6 cells / mL, and incubated at 37°C, 5% CO2. Prior to TCR activation, 1.5E6 cells / mL CD8+ T cells were pre-treated with 10 pg / mL rhAOAH or Vehicle in the complete T-cell medium without rhIL-2 for 20-22 hours.

[0385] For antibody-based activation assays, 1 pg / mL, 0.5 pg / mL, and 0.1 pg / mL anti-mouse / human CD3 antibody (Tonbo) in PBS was coated in a 96-well plate at 50 pL per well and incubated at 37°C for 2 hours. After incubation, the coated wells were washed with sterile PBS twice and 3E5 CD8+ T cells in 200 pL complete T-cell medium supplemented with 100 lU / mL recombinant human IL-2 (R&D systems), and 0.5 pg / mL anti-mouse / human CD28 antibody were seeded into each well.

[0386] The CD8+ T cells were activated for a short period of time (5, 10, and 20 minutes) for evaluation of TCR signaling cascades, and activated for a longer period time (24 hours) for quantification of the fraction of activated T cells and release of cytotoxic cytokines.

[0387] For the antigen-specific activation assays, splenocytes isolated from 8-10-week-old C57BL / 6 mice were pulsed with hgplOO (Genescript), mgplOO (Genescript), OVA-N4, OVA-V4 (gifts from Gregoire Atlan-Bonnet lab) antigenic peptides at varied concentrations (1 pM, 100 nM, 10 nM, 1 nM) in complete RPML1640 at 37°C for 2 hours. After incubating, the pulsed splenocytes were washed with complete RPML1640 twice and centrifuged at 300xg for 5 minutes. The pulsed splenocytes were seeded into a round-bottom 96-well plates at 3E5 / 100 pL per well complete T-cell medium with 100 lU / mL recombinant human IL-2. The pmel and OT-1 mouse CD8+ T cells were mixed with the pulsed splenocytes at 1E5 / 100 pL per well. The antigen-specific CD8+ T cells were activated for 24 and 48 hours for quantification of the fraction of cytotoxic T cells and release of cytotoxic cytokines.

[0388] Lentivirus transfection and transduction: Full-length cDNA of luciferase and murine Aoah were constructed into pLenti6.3 / V5-TOPO (Invitrogen, K5310-00) and pReceiver-Lvl56-EFl- Vector (GeneCopoeia, EX-Lvl56) by homologous recombination, respectively. The plasmid DNA was transformed into One Shot™ Stbl3™ Chemically Competent E. coli (Invitrogen) via heat shock, and was then purified with QIAprep Spin Miniprep Kit (QIAGEN), as per manufacturer instructions. 1E6 per well Lenti-X™ 293T cells (Takara) were seeded onto a 6-well plate. At 24 hours after cell seeding, two packaging plasmids psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259), together with the pReceiver-Lvl56-EFl-Vector or pReceiver-Lvl56-EFl-Acw Cr7 / / Co / / Adamts7 plasmid, were transfected into the 293T cells using the Lipofectamine 3000 transfection reagent (Invitrogen), according to the manufacturer’s protocol.

[0389] Culture medium was replaced with Opti-MEM™ with GlutaMAX™ (Gibco) supplemented with 1:500 ViralBoost Reagent (Alstem) 6 hours after transfection. The lentivirus was harvested by spinning down the viral supernatant at l,000xg, 4°C for 15 minutes to remove the cell debris at 48 and 72 hours post transfection. For lentivirus transduction, B16-mhgpl00, B16F10, B2095-M4, RIL- 175, and RenCa cells were mixed with lentivirus at the 1:1 dilution with culture medium, 10 pg / mL 4239-111527-02 polybrene (Sigma-Aldrich) was added to the mixture for 24-48 hours before refreshing the medium. Two days after infection, puromycin (Sigma-Aldrich) was added for the selection and maintenance of cells with gene over-expression. RIL-175-Aoa / z cells co-infected with lentivirus harboring luciferase were further selected using blasticidin (Sigma-Aldrich).

[0390] AO AH recombinant human protein production: Hie recombinant human AOAH proteins (rhAOAH) with His-tag (SEQ ID NO: 1) or fusion protein (SEQ ID NOs: 5 and 7) were purchased from a Contract Research Development company (WuXi Biologies). Briefly, human recombinant proteins were purified from the 1 L cell-culture supernatant of HEK293 cells with AOAH construct overexpression. The final buffer was PBS pH7.4 with 10% glycerol. To produce AOAH Fc-fusion protein, part 1 (SEQ ID NO: 5) and part 2 (SEQ ID NO: 7) were produced and mixed in equal HEK293 supernatant volume. The expected product (dimer of part 1 and part 2) was further purified by protein A column followed by size exclusion chromatography.

[0391] RT-qPCR: Total RNA was extracted by using the PureLink™ RNA Mini Kit (Invitrogen). and cDNA was synthesized through reverse transcription using the SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). RT-qPCR was performed using the SYBR™ Green PCR Master Mix (Applied Biosystems) and corresponding primers, and Ct values were detected by StepOne Plus Real-time PCR system. Raw data were processed using SDS 19.1 software, and the relative mRNA expression level was normalized to the internal ribosomal reference gene Rpll3a or Actb. The primer sequences (5’-3’) are listed in Table 1.

[0392] Tumor inoculation and ICB treatment: For subcutaneous tumor models, 1E5 B16F10 / B16mhgpl00, or 5E5 Renca cells were injected subcutaneously into 7-8-week-old female C57BL / 6J and BALB / c mice, respectively. For orthotopic tumor models, a skin incision of -2.0 cm was made on the abdomen in the liver region of anesthetized mice. 1.2E5 luciferase-labeled RIL-175 cells were injected into the sub-capsular space of the liver of 7-8-week-old female C57BL / 6J mice.

[0393] Anti-PDl antibody (10 mg / kg) was given intraperitoneally to tumor -bearing mice twice a week for a total of 4 doses, starting on day 5 (RIL-175), day 6 (B16F10), day 7 (RenCa), or day 10 (B16-mligpl00) post tumor inoculation. Anti-CTLA4 antibody (10 mg / kg) was given to B16F10- bearing mice twice a week for a total of 4 doses, starting on the next day following the 1st anti-PDl treatment. Alternatively, control groups of mice received IgG treatment. For AOAH treatment models, human recombinant AOAH (WuXi Biologies) stored in 10% glycerol PBS (5 mg / kg) was given intratumorally to Bl 6F 10 and B16-mhgpl00-bearing mice once every two days for a total of 10 doses, starting on day 5 post tumor inoculation. Alternatively, the control group of mice received an intratumoral injection of vehicle (10% glycerol in PBS).

[0394] Tumor volume was measured based on the formula: (LengthxWidth2) / 2. Progression of orthotopic tumor burden and treatment effects in mice were monitored in vivo by bioluminescence imaging twice per week. Mice were anesthetized with vaporized isoflurane followed by intraperitoneal injection of D-luciferin (150 mg / kg, GoldBio), and luciferase activities were measured 4239-111527-02 with the IVIS system (PerkinElmer). Mice were euthanized according to a pre -determined survival endpoint defined as tumor volume > 2000 mm3or the tumor length (longer diameter) > 20 mm. This endpoint is compiled with ethical regulations of animal studies at NIH, according to the animal protocol CDSL-001, or conducted under protocol 23-480, approved by the Committee of the Use of Live Animals in Teaching and Research at the University of Hong Kong.

[0395] Flow cytometry: B 16-mhgpl00 and B16F10 tumors were first dissociated by 250 pg / mL Liberase™ TM Research Grade (Sigma-Aldrich) and 100 pg / mL DNAse I (Gibco) at 37°C for 30 minutes with vortexing every 10 minutes. The dissociated cells were filtered through a 70-pm cell strainer, centrifuged at 300xg for 10 minutes, and resuspended in EasySep Buffer. The CD45+ tumor-infiltrating leukocytes (TILs) were isolated from the cell mixture by using EasySep™ Mouse TIL (CD45) Positive Selection Kit (STEMCELL Technologies), as per manufacturer instructions. The TILs were washed with Cell Staining Buffer (BioLegend) prior to antibody staining. Mouse and human CD8+ T cells were collected from culture plates after TCR activation, centrifuged at 300xg for 5 minutes, and washed with Cell Staining Buffer. TILs or CD8+ T cells were incubated with a fluorochrome-conjugated antibody cocktail at <1E6 cells / 100 pL on ice for 20 minutes. The cells were further stained with DAPI at 0.2 pg / mL in 1 mL Cell Staining Buffer at room temperature for 5 minutes. The stained cells were washed with Cell Staining Buffer twice prior to flow cytometry analysis. All the flow cytometry experiments were performed on FACSymphony™ A5 (BD Biosciences), and results were analyzed by FlowJo™ Software vl 1.

[0396] Multiplex immunohistochemistry staining: Spontaneous hepatocellular (HCC) nodules and B16F10 tumors were carefully dissected from mouse livers and skin, respectively. They were then fixed in 10% neutral buffered formalin at room temperature overnight, dehydrated, and embedded into paraffin blocks.

[0397] For HCC tumors: Dewaxing, antigen retrieval, blocking, serial antibody incubation, and DAPI staining were performed on the HCC paraffin sections by using the 7-color mIHC staining kit (Servicebio Technology), as per manufacturer instructions. Panoramic scanning of mIHC sections was done using a Nikon Eclipse Cl fluorescence microscope. The fluorochromes used included SpAqua, SpGreen. SpGold, SpOrange, Spied, Cy5, Cy5.5, and DAPI. The antibodies used for mIHC staining were listed in Table 1.

[0398] For B16F10 tumors: 4-plex fluorescent staining was accomplished using a sequence of triple staining on an autostainer followed by a combination of manual and automated staining for the 4th target. Triple fluorescent staining was performed on the Leica Biosystems Bond RX autostainer using the Bond Polymer Refine Kit (Leica Biosystems DS9800), with omission of the PostPrimary reagent, DAB and hematoxylin. After antigen retrieval with citrate (Bond Epitope Retrieval 1), sections were incubated for 30 minutes with CD8a (eBioscience #14-0195-82, 1:25), followed by ImmPRESS® HRP-conjugated goat anti-rat (Vector Labs) and OPAL Fluorophore 570 (AKOYA). The CD8a antibody complex was stripped by heating with Bond Epitope Retrieval 1. Sections were then 4239-111527-02 incubated for 30 minutes with PEP8h (non-commercial, 1 : 500) , followed by the Bond Polymer reagent and OPAL Fluorophore 480. The PEP8h antibody complex was stripped by heating with Bond Epitope Retrieval 1. Sections were then incubated for 60 minutes with CD3 (Bio-Rad #MCA1477, 1:50), followed by ImmPRESS® HRP-conjugated goat anti-rat (Vector Labs) and OPAL Fluorophore 620. The CD3 antibody complex was stripped by heating with Bond Epitope Retrieval 1. Sections were then incubated overnight at 4°C with CDllc (Cell Signaling Technology #97585, 1:350), followed by the Bond Polymer reagent and OPAL Fluorophore 520. Sections were stained with DAPI and coverslipped with Prolong Gold AntiFade Reagent (Invitrogen). Images were captured using the AKOYA PhenoImager whole slide scanner.

[0399] Retrovirus transduction on pmel CD8+ T cells: Full-length cDNA of murine Aoah was constructed into MSCV-IRES-Thyl.l (a gift from Chuan Wu lab at NIH). The plasmid DNA transformation, purification, and Lenti-X™ 293T cell seeding procedures were the same as those in lentivirus preparation. At 24 hours after cell seeding, pLC and Gag, together with the MSCV-IRES- Thyl.l empty vector or MSCV-IRES-Thy I . I - / V / plasmid were transfected into the 293T cells using the Lipofectamine 3000 transfection reagent (Invitrogen), as per manufacturer instructions. Culture medium was replaced with Opti-MEM™ with GlutaMAX™ (Gibco) supplemented with 1:500 ViralBoost Reagent (Alstem) 6 hours after transfection. The retrovirus was harvested by spinning down the viral supernatant at 3000 rpm, 4°C for 30 minutes to remove the cell debris at 48 and 72 hours post-transfection. The retrovirus was concentrated lOOx by using the Retrovirus Precipitation Reagent (Alstem).

[0400] Fresh AoaA-low (verified by qRT-PCR, Ct > 30) CD8+ T cells from 8-10-week-old pmel mice were activated by Dynabeads™ Mouse T-Activator CD3 / CD28 (Gibco) at 1:1 beads-to-cells ratio in presence of 100 lU / mL rhIL-2 for 24 hours. The activated CD8+ T cells were transduced by the lOx fresh retrovirus in warm T-cell medium supplemented with 100 lU / mL rhIL-2 and 10 pg / mL polybrene, by spinf ection at 2000xg, 32 °C for 2 hours. The transduced T cells were incubated at 37°C overnight and subsequently the Thy 1.1+ population was isolated by using mouse CD90.1 MicroBeads (Miltenyi), as per manufacturer instructions. The expression and secretion of Aoah in mouse pmel CD8+ T cells were validated by qRT-PCR and ELISA.

[0401] Adoptive T-cell transfer: Pmel CD8+ T cells were expanded for 7-10 days in a complete T- cell medium with 100 lU / mL rhIL-2 after retrovirus transduction. 1E5 B16F10 cells were subcutaneously inoculated into 7-8- week-old C57BL / 6 mice. One day before T-cell transfer (Day 8 or 9 post tumor inoculation), B16F10-bearing C57BL / 6 mice were irradiated with a dose of 600 cGy and randomized into two groups. The next day, 2E6 vector and Aoa / i-armored pmel CD8+ T cells were transferred intravenously into the mice, and 10,000 IU / 0.5 mL rhIL-2 was administered intraperitoneally into the mice twice a day for 3 consecutive days.

[0402] Incucyte tumor-killing assay: Pmel CD8+ T cells were briefly activated by 1 pg / mL anti- CD3+ 0.5 pg / mL anti-CD28 antibodies in the presence of 10 pg / mL rhAOAH or buffer vehicle for 24 4239-111527-02 hours. The activated CD8+ T cells were expanded for 3-4 days prior to co-culture. mCherry-labeled B16-inhgpl00 and B16F10 cells were seeded into a 96-well plate at a density of 2.5E3 cells / well. Following 24-hour culture, the expanded CD8+ T cells were seeded into each well at different effector-to-target ratios in a complete T-cell medium with 100 lU / mL rhIL-2 and 10 pg / mL rhAOAH or vehicle. The plates were imaged, and the red fluorescence of tumor cells was quantified every 6 hours for 72 hours using the Incucyte® S3 Live -Cell Analysis System (Essen Bioscience).

[0403] Western blot: Human CD8+ T cells were pre-heated with 10 pg / mL rhAOAH and an equal volume of buffer vehicle in a complete T-cell medium at a density of 2E6 cells / mL. Following a 20- hour pre-treatment, the CD8+ T cells were resuspended in the complete T-cell medium supplemented with 50 lU / mL rhIL-2. The cells were treated again with 10 pg / mL rhAOAH and Vehicle as previously described and further activated with a 1:500 dilution of human T Cell TransAct™ (Miltenyi) for 5, 10, and 20 minutes at 37°C. The T cells were collected and centrifuged at 300 rpm for 5 minutes at 4°C, followed by one wash with lx ice-cold PBS. Subsequently, the T cells were lysed in Pierce RIPA buffer (Thermo Fisher), supplemented with a protease inhibitor cocktail (MCE) and a phosphatase inhibitor cocktail (MCE). The protein lysates were quantified using the Bradford protein assay, separated by SDS-PAGE, and transferred to an activated PVDF membrane (Millipore). The membrane was incubated with the primary antibody for 16-18 hours at 4°C, followed by an HRP-conjugated secondary antibody for 1 hour at room temperature. Each antibody incubation was followed by 3 times of 10-minute washing with lx TBST. Blots were visualized using enhanced chemiluminescence (Bio-Rad) and X-ray film. The antibodies used are listed in Table 1.

[0404] NFAT luciferase assay: Jurkat-Lucia™ NFAT-CD28 cells were purchased from Invivogen (jktl-nfat-cd28) and cultured in the recommended growth medium with selective antibiotics. The Jurkat cells were pre-treated with 10 pg / mL rhAOAH or buffer vehicle in the complete IMDM without antibiotics for 20-22 hours. The NFAT induction was followed by the manufacturer’s instructions: In a 96-well plate, 3E5 Jurkat cells were activated with Immunocult (Stem Cell Technologies) at 12.5 pl / mL (1:2), 6.25 pl / mL (1:4), 3.125 pl / mL (1:8) and 1.5625 pl / mL (1:16) or mock in 200 pL IMDM with 10 pg / mL rhAOAH or vehicle per well. After 24-hour activation, the supernatants were collected and centrifuged to remove cell debris. The luminescence of the supernatants was quantified by SpectraMax iD5 (Molecular- Devices) and normalized by the baseline luminescence of the supernatants from unactivated Jurkat cells.

[0405] Untargeted lipidomics using mass spectrometry: Mouse CD8+ T cells were pre-treated with 10 pg / mL rhAOAH or buffer vehicle at 2E6 cells / mL in a complete T-cell medium without rhlL- 2 for 22-24 hours. After pre-treatment, the cell culture supernatants were collected, centrifuged and snap-frozen in liquid nitrogen. T cells were collected, centrifuged, washed with ice-cold saline and snap-frozen in liquid nitrogen prior to sample submission.

[0406] For cell culture supernatants, the sample was extracted with chloroform: methanol (2:1, v / v). The sample was then centrifuged at 3000xg for 5 minutes. The non-polar phase was aliquoted out 4239-111527-02 and dried under nitrogen. For cell pellets, 5 mL chloroform: methanol (2:1, v / v) was added to 5 million cell pellets. The sample was then sonicated under ice chilled probe sonicator for 20 seconds, cooled down for 10 seconds and another sonication for 20 seconds. Hie sample was then centrifuged at 3000xg for 5 minutes. Then, 1.5 mL supernatant was aliquot out and dried under nitrogen. The sample was then reconstituted with 50 pL IPA: methanol: chloroform (1: 1:0.2, v / v). Then, 3 pL was injected into the LC-MS / MS system.

[0407] The chromatographic separation was earned out on a Vanquish UPLC (Thermo Fisher, Waltham, MA, USA). Mobile phases used were 10 mM ammonium formate with 0.1% formic acid in acetonitrile and water, v / v 6:4 (A), and 10 mM ammonium formate with 0.1% formic acid in acetonitrile and IPA 1:9 (B). The column was a Thermo Fisher Accucore C30 (2.1x150 mm, 2.6 pm). An injection volume of 3 pL and a flow rate of 0.26 mL per minute were used. The column oven temperature was set at 45 °C. The gradient started at 30% B and was increased to 43% B in 2 minutes, then increased to 55% B in 2.1 minutes, 65% B in 12 minutes, 85% B in 18 minutes, and 100% B in 20 minutes, then held for 5 minutes and decreased linearly to 30% B for re-equilibration time at starting conditions.

[0408] The mass spectrometry analysis was processed using an Orbitrap Exploris 120 mass spectrometer Thermo Fisher (Waltham, MA, USA) equipped with a HESI II probe in polar switching mode with source parameters set as follows: sheath gas flow rate, 60; auxiliary gas flow rate, 17; sweep gas flow rate, 1; spray voltage, +3.5 / -3.0kV; capillary temperature, 275 °C; S-lens RF level, 70; and heater temperature, 325°C. Data were collected at dd-MS2 mode. Data analysis was performed using Lipidsearch (Thermo Fisher Scientific / Mitsui Knowledge Industries) with the default parameters for Orbitrap MS Product Search and Alignment. After alignment, raw peak areas for all identified lipids were exported to Excel files.

[0409] Microscale thermophoresis (MST) assay: The 2 mM rhAOAH solution in saline and 100 nM MST dye solution in saline were prepared prior to the MST test. rhAOAH and dye solutions were mixed in a 1:1 volume ratio (400 pL final volume) and incubated for 30 minutes at room temperature, then centrifuged at 15,000 g for 10 minutes at4°C. phosphatidylcholine (PC)(16:0-20:4) and PC(18:0-20:4) stocks in chloroform were dried under a fume hood before being resuspended in 5 pL ethanol. Samples were diluted to 2 mM, 800 pM, 400 pM, 40 pM. and 4 pM with 0.9% saline containing fatty acid-free BSA (0.2 mg / ml final concentration) and Triton X-100 (0.09%). 15 pL of 1 mM rhAOAH was added to an equal volume of each dilution and mixed to form final dilutions of 1 mM, 400 pM. 200 pM, 20 pM. and 2 pM (8.33%, 3.33%, 1.67%, 0.17%, and 0.017% ethanol in saline as mock, respectively). 15 pL of 1 mM rhAOAH was simultaneously added to an equal volume of saline buffer to create corresponding ethanol dilutions as a control. Samples were incubated for 15 minutes at room temperature and then loaded into standard MST capillaries, then run using the red channel excitation and detection at an LED intensity of 50%. 4239-111527-02

[0410] Phosphatidylcholine treatment assay: 16:0-20:4 phosphatidylcholine (PC) and 18:0-20:4 PC were purchased from Avanti Polar Lipids. The PCs in chloroform were dried under a tissue culture hood for 1-2 hours. The dried PCs were first readily dissolved by 5 pL 70% ethanol and diluted to 500 pM stock using a complete T-cell medium. Mouse and human CD8+ T cells were pretreated with different concentrations of PCs (30 pM. 60 pM, 120 pM, and 200 pM) in the presence of 10 pg / mL rhAOAH or vehicle matched with ethanol concentrations for 20-22 hours. After pretreatment, the CD8+ T cells were activated by anti-CD3 / CD28 antibodies following the same procedure as described in “Mouse and human CD8+ T cell culture and activation” above.

[0411] Example 2 Cancer Immunology Data Engine

[0412] The Cancer Immunology Data Engine (CIDE) incorporates 80 omics datasets spanning 5897 bulk-tumor pre-treatment samples with immunotherapy outcomes from 17 solid tumor types (FIG. 1 A). CIDE includes gene expression, copy number alteration, somatic mutation, and DNA methylation data (FIG. 1 A), which was processed uniformly by the developed in-house framework (FIGS. 8C-8D). CIDE integrates the most comprehensive data among existing immuno-oncology databases (Table 3). The CRI iAtlas was not included in the comparison, as only web frameworks that allow input of any gene names, instead of pre-computed signatures, were included.

[0413] Table 3. Comparison of cancer immunotherapy web frameworks on sample numbers on genomewide experiments

[0414] Only numbers from pre-treatment tumor were considered in the CIDE framework, and on-treatment and post-treatment tumors were excluded. 4239-111527-02

[0415] * The TIDE study utilized a T-cell dysfunction model to compute marker scores using bulk tumor cohorts without immunotherapy treatments. However, die sample numbers were not included for die T-cell dysfunction model and only tumors treated with cancer immuno therapies were considered.

[0416] # Only genome -wide sequencing data were included and targeted panel sequencing datasets were excluded in the numbers.

[0417] Users can input any gene, and CIDE outputs risk scores, calculated as the association between gene values (expression, mutation, copy number, or promoter methylation) and clinical outcomes across omics cohorts. Users have die option of inputting multiple genes and choosing to query any subset of data cohorts (Input 1 and 2 in FIG. IB) from cancer immunodierapy studies (FIG. 1A) and other omics projects (e.g., TCGA, ICGC, TARGET, PRECOG, TIDE). CIDE then ranks input genes by median risk score across the selected cohorts and outputs gene candidates associated with low and high risks (Output 1 in FIG. IB). For user-selected genes, CIDE then queries single-cell datasets (13 currently) to identify cell lineages underlying the reported bulk-level associations (Output 2 in FIG. IB). To further probe the function of the input genes, CIDE also returns phenotype scores derived from 74 CRISPR screens in cancer cells under diverse immunological killing pressures (Output 3 in FIG. IB), hi addition, CIDE provides hyperlinks to external databases hosting genetic screens of T- cell fitness and cancer cell line growth. This analysis allows users to evaluate phenotypes (typically cell growth) caused by perturbation of a candidate gene in cancer or T cells during the anti-tumor immune response.

[0418] For all human genes covered in CIDE, risk associations between the expression levels and cancer immunotherapy outcomes were computed (Methods, Examples in FIG. 2A). The focus was on 1903 genes encoding secreted proteins for further analysis. Ninety genes were prioritized based on tiieir CIDE median risk score across 37 genome-wide bulk transcriptomics datasets from pretreatment tumors (FDR < 0.05, FIG. 2B). Sixty-eight genes were significantly associated with favorable immunotherapy outcomes, while 22 were associated with adverse outcomes (FIG. 2B, Table 4A and 4B). Among the results were well-known regulators of cancer immunodierapy response, such as IFNG, FLT3LG, and CXCL13 (FIG. 2B).

[0419] Table 4 A. Gene function annotation (negative risk scores)

[0420] The table includes double-blind annotations for statistical evaluations and follow-up annotations (bold) to identify missing literature in the first-round double-blind annotation.

[0421] TME: tumor microenvironment; DC: dendritic cell; ICB: immune checkpoint blockade; Treg: T- regulatory cells; NK: natural killer; EMT: epithelial-mesenchymal transition; HCC: hepatocellular carcinoma; TNBC: triple-negative breast cancer. 4239-111527-02 4239-111527-02 4239-111527-02 4239-111527-02 4239-111527-02 4239-111527-02 4239-111527-02 4239-111527-02 4239-111527-02 4239-111527-02

[0422] Table 4B. Gene function annotation (positive risk scores)

[0423] The table includes double-blind annotations for statistical evaluations and follow-up annotations (bold) to identify missing literature in the first-round double-blind annotation. TME: tumor microenvironment; DC: dendritic cell; ICB: immune checkpoint blockade; Treg: T- regulatory cells; NK: natural killer; EMT: epithelial-mesenchymal transition; HCC: hepatocellular carcinoma; TNBC: triple-negative breast cancer. 4239-111527-02

[0424] Gene risk scores represent associations, not causality. To explore causality, the 90 identified genes were shuffled, the “favorable” or “adverse” labels were removed, and a blinded colleague was recruited to annotate each gene as “pro-” or “anti-” tumor based on publications that perturbed each 4239-111527-02 gene and measured effects on tumor progression (FIG. 2B left bars and Tables 4A-4B). Median gene risk scores output by CIDE across immunotherapy cohorts reliably predicted pro-tumor versus antitumor effects of identified genes (FIGS. 2C-2D).

[0425] Encouraged by tire high prediction performance of gene risk scores on anti-tumor versus protumor functions of secreted proteins (FIGS. 2C-2D). focus was placed on genes without any known function in cancer to identify new secreted regulators of tumor immunity (Stars in FIG. 2B).

[0426] Foui' genes were selected for further study: AO AH, ADAMTS7, COLQ, and CR1L. The selection criteria included having no previously characterized function in cancer, prioritization by CIDE, and the previous tumor-resilient T-cell (Tres) model (Zhang et al., Nat. Med. 28:1421-1431, 2022), and interest in exploring emerging areas of cancer immunology. Specifically, AO AH ranked first among candidates with negative risk associations with cancer immunotherapy outcomes, while ADAMTS7 ranked first for positive risk (FIG. 2B). The Tres model identifies marker genes of CD8+ T cells resilient in immunosuppressive solid tumors. Among secreted proteins with no known cancer roles, only AOAH and COLQ had significantly positive Tres scores (FIG. 2E and FIGS. 8E-8F), indicating that T cells with high AOAH or COLQ expression tend to be more resilient in solid tumors than those with low levels. Finally, the complement activation regulator CR1L was added to explore the emerging role of the complement system in cancer progression.

[0427] The expression levels of these candidates were significantly associated with favorable (AOAH, CR1L, COLQ) or adverse (ADAMTS7) cancer immunotherapy outcomes in diverse solid tumor types (FIGS. 3A-3B). AOAH had the most significant associations with immunotherapy outcomes in multiple solid tumor types (FIG, 3A), such as pancreatic, melanoma, renal, and hepatocellular carcinomas (FIGS. 2A, 3B, and 9 A). Tumors with high AOAH expression had high cytotoxic T-cell infiltration levels (FIGS. 9B-9C). Consistent with AOAH and COLQ having positive Tres scores (FIG. 2E), therapeutic T cells infused into cellular immunotherapy responders have higher AOAH and COLQ expression levels than expression levels from non-responders (FIGS. 3C and 9D).

[0428] Single-cell RNA-seq data showed that these four candidates are expressed in diverse cell lineages (FIG. 9E). Top candidate AOAH is highly expressed in natural killer cells, monocytes, dendritic cells, and CD8+ T cells. To examine tire spatial distribution of AOAH in tumors, spatial transcriptomics data from tumors treated with immune checkpoint blockades was searched for and two anti-PDl studies covering 15 hepatocellular carcinoma tumors (Zhang et al., Genome Med. 15:72, 2023; Achar et al., Science 376:880-884, 2022) were identified. AOAH expression levels were consistently higher among responders than non-responder tumors (FIG. 3D), and spatial transcriptomics data showed that the AOAH expression spans the entire tumor region (FIG. 3E). 4239-111527-02

[0429] Example 3

[0430] Validation of AOAII in Mouse Tumor Models

[0431] Motivated by the associations between clinical outcomes and expression of AOAH, COLQ, CR1L, and ADAMTS7, experimental validation of their functions in anti-tumor immune response using mouse tumor models was undertaken. First. Aoah. Colq. and Crll (all three predicted as antitumor) were overexpressed in the anti-PDl resistant B16-mhgpl00 cell line and Adamts7 (predicted as pro-tumor) was overexpressed in the anti-PDl sensitive B2905-M4 cell line via stable lentiviral transductions. There was no significant change in cell proliferation upon overexpression of any genes (FIG. 9F). indicating that these secreted proteins might not alter cell intrinsic phenotypes. Next, subcutaneous tumor injection into C57BL / 6 mice was performed and they were treated with anti-PDl and IgG control. Aoah strongly suppressed, and Adamts7 strongly stimulated tumor progression in both IgG and anti-PDl treatment groups, while Colq and Crll suppressed tumor growth in both groups but achieved statistical significance only in the anti-PDl groups (FIGS. 3F-3H, and 9G).

[0432] To further establish that secreted proteins mediated these effects, a mixture of 10% Aoah- overexpression (OE), Colq-OE, Crll -OE, or 20% Adamts7-OE cells to 90% or 80% control cells was inoculated into mice. For each mixture, an effect similar to 100% OE cells on the anti-PDl response was observed (FIGS. 3I-3K and 9H). These results suggest that Aoah, Colq, Crll, and Adamts7 expression in a minority of cells can modulate anti-PDl efficacy by affecting immune response in the entire tumor microenvironment.

[0433] Among the four secreted proteins, Aoah was the top computational candidate and had the strongest in vivo effects. Accordingly, four in vivo experiments characterizing the anti-tumor effects of AOAH were designed.

[0434] The first experiment evaluated the synergy between Aoah-OE and immune checkpoint blockades (ICB) in multiple syngeneic tumor models. Aoah was overexpressed in the B16F10 melanoma cell line, which is even more resistant to anti-PDl therapy than the B16-mhgpl00 cell line utilized in the previous section (FIGS. 3F-3K). Aoa / z-overexpression (OE) in B16F10 tumors resulted in a remarkable increase in the efficacy of anti-PDl monotherapy and combination therapy using anti- PDl and anti-CTLA4 (FIGS. 4A-4B) but did not result in tumor shrinkage or survival advantages in combination with IgG treatment or in immunodeficient mice (FIGS. 10A-10B). Inoculation of 10% Aoah-OE cells with 90% Vector-OE cells was sufficient to promote ICB (FIGS. 4A-4B). Using the gene OE approach, Aoah also potentiated anti-PDl responses in the RIL-175 hepatocellular carcinoma and the Renca renal cancer models (FIGS. 4C-4D and 10C-10D). This result is consistent with the high correlation between AOAH expression and favorable outcomes in liver and renal cancer patients upon ICB (FIG. 9A).

[0435] The second experiment administered recombinant human AOAH protein (rhAOAH) to B16F10 and B16mhgpl00 mice. His-tag or Fc-fusion recombinant human AOAH was produced in HEK293 cells and each protein was isolated to >99.7% purity with endotoxin levels <0.1EU / mg. 4239-111527-02

[0436] Intratumoral administration of 2-5 mg / kg rhAOAH protein in mice significantly suppressed tumor progression and synergized with anti-PDl and anti-PDl&CTLA4 combination treatment without causing any noticeable weight loss (FIGS. 4E-4F and 10E-10G). After ICB treatments, tire composition of CD45+ tumor-infiltrating leukocytes isolated from B16-mhgpl00 and B16F10 tumors was analyzed. There was higher infiltrations of CD8+ T cells and CDllc+ dendritic cells (DCs) in Aoah-OE and rhAOAH-treated tumors (FIGS. 4G-4H), suggesting that AOAH could promote CD8+ T-cell expansion and antigen presentation.

[0437] The third experiment examined the effects of AOAH in spontaneous hepatocellular carcinoma (HCC) models, which induce tumors from mouse hepatocytes driven by MYC and P-catenin (FIG. 41). Compared to syngeneic models used previously, this model develops tumors spontaneously, which is more similar to how human cancers arise and therefore may be more physiologically relevant. High expression of Aoah inhibited tumor progression in both OVA+SIN+SIY (OS) antigen- high and OS antigen-null HCC (FIGS. 4J-4K and 10H-10I), demonstrating inhibition of spontaneous tumors regardless of antigen strength. Multiplex immunofluorescence imaging showed that Aoah- high HCC nodules had higher infiltrations of CD8+ T cells and that CD1 lc+ DCs were preferentially enriched when Aoah was overexpressed in OS-antigen induced nodules (FIG. 4K and 10J) .

[0438] The fourth experiment tested whether AOAH augments adoptive T-cell transfer in solid tumors. Aoah was overexpressed in pmel CD8+ TCR-T cells that target B16F10 cells (FIG. 4L). The B16F10-bearing mice received non-lethal radiation for lympho-depletion before T-cell transfer, which mimicked the standard procedure for human patients (FIG. 4L). Compared to vector-armored T cells, Aoa / i-armored TCR-T cells exhibited more potent B16F10 tumor suppression and prolonged mouse survival (FIG. 4M). Soon after T-cell transfer, Aoa / i-armored T cells induced more CD8+ TCR+ T cell infiltrations into tumors but not more CD8+ TCR-T cells (FIGS. 11A-11B). Aoa / / -armored TCR T cells did not induce noticeable weight loss or lymphoid tissue damage (FIGS. 11C-11D). Thus, AOAH bolsters adoptive T-cell transfer and induces antigen recognition in solid tumors.

[0439] RNA-seq was performed on tumors from the hepatocellular carcinoma (MYC-Luc with no antigen and MYC-LucOS with OS antigens) and melanoma (B16-mhgpl00 and B16F10) mouse models. Changes in gene expression among tire four models, computed as log2 fold change (log2FC) of gene expression between Aoah-OE and vector-OE tumors, were superficially divergent (FIG. 12A). Thus, gene set enrichment analysis (GSEA) was carried out, screening for common pathways whose gene members are induced by Aoah (FIG. 5A). Top pathways enriched across all models include arachidonic acid metabolism, antigen processing and presentation, T-cell receptor (TCR) signaling, IFNy response, and other pathways related to these terms (FIGS. 5B-5C).

[0440] The enrichment of antigen processing and presentation and TCR signaling (FIGS. 5A-5B) corroborate positive correlations between AOAH expression and cytotoxic T-cell infiltration across human tumors (FIGS. 9B-9C) and the AoaA-enhanced infiltrations of CD8 T cells and dendritic cells in mouse tumors (FIG. 4G). The enrichment of IFNy response (FIGS. 5B and 12B) suggests IFNy 4239-111527-02 release as a consequence of TCR activation. Perhaps most significant is the enrichment of arachidonic acid (AA) metabolism upon Aoah overexpression (FIGS. 5A-5B). Arachidonic acid is often linked to phospholipids, and the release of arachidonic acid triggers downstream responses in livers. The GSEA result, along with tire known lipase activity of AOAH, suggests that AOAH may function in tumors as a phospholipase.

[0441] To specifically study the effects of Aoah overexpression (OE) on immune cells, single-cell RNA-seq was performed on CD45+ cells isolated from the MYC-Luc spontaneous hepatocellula' (HCC) tumors (FIG. 5D and Table 2) induced with and without OS antigens. For both conditions, tumors overexpressing Aoah contained higher fractions of T and NK cells and lower fractions of polymorphonuclear myeloid-derived suppressor cells (FIG. 5E). Among the T / NK cells, cytotoxic CDS T and type-1 T helper cells were consistently higher among tumors with Aoah OE. However, the abundance of other cell types, such as T regulatory cells, does not correlate with Aoah overexpression (FIGS. 5E and 12E).

[0442] For each immune cell subtype, the log2FC of gene expression upon Aoah OE was computed. GSEA revealed pathways related to adaptive immune activation (FIG. 5F). For example, “antigen processing and presentation,” enriched in the bulk transcriptomics analysis, was also broadly enriched in log2FC profiles of monocytes, macrophages, and exhausted CD8 T cells (Star 1 in FIG. 5F). The pathway “cell adhesion molecules” was enriched in many T-cell subtypes (Star 2 in FIG. 5F). Representative genes supporting this enrichment are Itga4 and It gal (FIG. 5G), which previous studies have shown are upregulated in activated T cells and bind to the intercellular and vascular cell adhesion molecules on endothelial cells to promote T-cell migration to inflamed tissues.

[0443] A third notable enrichment is the oxidative phosphorylation pathway in T / NK cells, proliferating T cells, monocytes, and macrophages (Star 3 in FIGS. 5F and 12D). Previous studies showed that impaired oxidative phosphorylation limits tire self-renewal of T cells exposed to persistent antigens by limiting nucleotide triphosphate synthesis. The data here suggest that Aoah induction can protect oxidative phosphorylation (FIG. 5F) and nucleotide triphosphate biosynthesis (FIG. 12D), thus enhancing the anti-tumor efficacy of chronically stimulated T cells.

[0444] T-cell receptors (TCR) and B-cell receptors (BCR) were also analyzed. TCR clonal expansion was consistently elevated among cytotoxic CD8 T, type-1 T helper, and T proliferating cells in tumors with Aoah OE compared to tumors with vector OE (FIG. 5H). Other cells, such as Treg, did not present consistent TCR clonal expansion (FIG. 5H). For B and plasma cells, the singlecell profiling showed no consistent trends of B-cell fractions, BCR clonal expansion, or immunoglobulin class switch (FIGS. 12E-12G). Thus, the single-cell analysis suggests that Aoah promotes T-cell infiltration and activation in spontaneous HCC tumor models.

[0445] The bulk and single-cell transcriptomics analyses above suggested that AOAH potentiates CD8+ T cells in tumors. This suggests that AOAH might promote TCR activation and antigen 4239-111527-02 recognition, and this hypothesis was tested using three in vitro models for human and mouse CD8 T cells and one in vivo model for mouse tumor cells with different antigen immunogenicities.

[0446] The first model is an antigen-specific tumor -killing model, pitting pmel CD8+ T cells against B16-mhgpl00 and B16F10 cells. The antigen-specific T cells treated with rhAOAH killed antigenpositive tumor cells more effectively (FIGS. 6A and 13A) and exhibited higher cytotoxicity and proinflammatory cytokine secretion (FIGS. 13B-13C). The Tres model predicted that ACZ4 / / -positive T cells were resilient to immunosuppressive TGFp (FIGS. 8E-8F). Indeed, rhAOAH treatment promoted T-cell mediated tumor killing in the presence of TGFp (5 ng / mL, FIGS. 13A and 13D).

[0447] The second model utilizes anti-CD3 / CD28 antibodies to trigger TCR signal transduction in mouse and human CD8+ T cells. The rhAOAH treatment significantly enhanced IFNy secretion, and rhAOAH pretreatment before TCR activation further promoted cytokine secretion (FIG. 13E). The rhAOAH treatment remarkably stimulated mouse and human donor T-cell activation, as shown by increased cytotoxic cytokine secretions and activation markers (FIGS. 6B-6E and 13F). To further verify AOAH effects on human TCR stimulation, a T leukemia Jurkat cell line that stably expresses an NF AT -inducible luciferase reporter was utilized to quantify TCR-induced NF AT transcription by luminescence. The rhAOAH treatment stimulated NF AT activity across various anti-CD3 / CD28 stimulation levels (FIG. 6J).

[0448] The TCR stimulation effect of AOAH was more evident at low anti-CD3 concentrations (FIGS. 6B, 6D), indicating that AOAH may be particularly effective in potentiating T cells experiencing weak TCR signals. Consistent with this, treatment of mouse and human CD8+ T cells with rhAOAH resulted in phosphorylation of TCR-induced downstream factors, including CD3L LCK, ZAP-70, and LAT early in the activation timeline (FIGS. 61, 6K, 13M). RNA-seq of rhAOAH- treated CD8+ T cells activated by anti-CD3 / CD28 antibodies showed up-regulation of cell cycle and TCR activation-related pathways such as glycolysis and mTORCl signaling (FIG. 6H).

[0449] The third model was an MHCI-dependent co-culture system in which antigen-specific CD8 T cells interact with splenocytes pulsed with antigens of various immunogenicity levels (FIG. 6F). The rhAOAH treatment significantly enhanced the T-cell cytotoxicity and pro-inflammatory cytokine secretion for intermediate (hgplOO), strong (OVA-N4), and weak (mgplOO and OVA-V4) antigens (FIGS. 6F-6G and 13G). Particularly, rhAOAH consistently promoted IFNy secretion for antigens with low immunogenicity and density (FIGS. 6G and 13G). RNA-seq of splenocyte co-cultures treated with rhAOAH showed enrichment of antigen presentation via MHCI, interferon signaling, inflammatory responses, cell cycle, glycolysis, and mTORCl signaling (FIGS. 13I-13K), which was consistent with most pathways in CD8+ T cells activated by anti-CD3 / CD28.

[0450] The fourth model tested the AOAH effect on the in vivo growth of B16 tumors expressing the weak (OVA-V4) and strong (OVA-N4) antigens evaluated by model three (FIG. 13G). Aoah overexpression in tumors suppressed tumor progression in both B16-OVA-V4 (weak antigen) and B16-OVA-N4 (strong antigen) models (FIGS. 13H). This result is consistent with previous in vivo 4239-111527-02 results in B16 cells expressing the weak mgplOO antigen (FIGS. 4A-4B) and the intermediate hgplOO antigen (FIGS. 3F-3K), confirming that AOAH promotes in vivo TCR antigen recognition over a wide range of immunogenicity levels.

[0451] The RNA-seq analysis showed that Aoah overexpression in liver tumors activated arachidonic acid metabolism (FIGS. 5A-5B). Also, previous studies showed that AOAH can function as a phospholipase, cutting fatty acids on both sn-1 and sn-2 positions of glycerophospholipids (Munford et al., J. Biol. Chern, 267:10116-21. 1992). Therefore, the mechanism of AOAH-enhanced activation of T cells may be related to its phospholipase activity. To test this hypothesis, untargeted lipidomics was performed on the cell culture medium and pellets for T cells treated with rhAOAH and vehicle control (FIGS. 7 A and 14A).

[0452] To evaluate whether AOAH preferentially depletes or enriches any lipid categories, a lipid class enrichment analysis that compares the log2FC values of lipids with a similar configuration against values of lipids without the configuration was performed. Only four lipid classes in the T-cell culture medium passed the false discovery rate (FDR) threshold of less than 0.05 (FIG. 7B). The depletion of diacyl glycerophosphocholine (PC), also named phosphatidylcholine, by AOAH achieved the highest statistical significance (FIG. 7B).

[0453] Phosphatidylcholine has two fatty acids connected on the sn-1 and sn-2 positions by ester links. The fatty acids enriched at each location were further analyzed and only one enrichment was found: the 20:4 (arachidonic acid) on the sn-2 position (FIG. 7C). Higher arachidonic acid release from rhAOAH-treated mouse CD8+ T cells was independently verified by competitive ELISA (FIG. 7D). These results were consistent with the enrichment of Arachidonic acid metabolism in the transcriptomics analysis (FIGS. 5A-5B).

[0454] Among phosphatidylcholines (PC) from human plasma, 16:0 and 18:0 are the two most abundant fatty acid species in the sn-1 position. The binding between AOAH and PC(16:0-20:4) or PC(18:0-20:4) was confirmed by microscale thermophoresis assays (FIGS. 7E and 14B). The AOAH binding strength on PC(16:0-20:4) was higher than those on PC(18:0-20:4) (FIG. 7E), which is consistent with PC(16:0-20:4) as one of the top lipids depleted upon AOAH treatment (FIG. 7C).

[0455] Consistent with these results, previous in vitro studies demonstrated AOAH as a phospholipase. The phosphatidylcholine hydrolysis on tire sn-2 position will produce lyso- phosphatidylcholine (LPC). The untargeted lipidomics analysis showed that AOAH treatment depleted both PC(16:0-20:4) and its hydrolysis product LPC mono-acyl 16:0 (FIGS. 7A and 7C). This result is consistent with AOAH’s capability as both phospholipase and lyso-phospholipase. A recent study demonstrated LPC(16:0) as an immunosuppressive lipid by suppressing T-cell motility (Zhang et al., Sci. Immunol. 9:eadh2334, 2024). Collectively, these data suggest that AOAH can completely hydrolyze the immunosuppressive PC(16:0-20:4) to promote T-cell functions. In contrast to the depletion of LPC mono-acyl (16:0), LPC mono-alkyl species are enriched upon AOAH 4239-111527-02 treatment (FIG. 7B), suggesting that AO AH cannot hydrolyze alkyl lipids in which fatty acids are connected to the glycerol moiety through an ether link.

[0456] To evaluate the effects of PC(16:0-20:4) and PC(18:0-20:4) on TCR activation, mouse CD8+ T cells were preheated with both lipids in a serum-free medium to avoid interference from preexisting lipids in the fetal bovine serum (FBS). The result demonstrated that both PC(16:0-C20:4) and PC(18:0-C20:4) can diminish T-cell activation, as shown by decreased secretion of IFNy TNFa, and IL-2 (FIG. 14C). The rhAOAH treatment rescued T-cell suppression from PC(16:0-20:4) from low to high lipid concentrations and suppression from PC(18:0-20:4) at low (60 pM) but not at a high (200 pM) concentrations (FIGS. 7F and 14C). Consistent with the mouse results, in human CD8+ T cells, PC(16:0-20:4) significantly inhibited the secretion of IFNY and TNFa after TCR stimulation, which was rescued by rhAOAH treatment (FIGS. 7G and 14D).

[0457] RNA-seq on mouse CD8+ T cells heated with PC( 16:0-20:4) showed down-regulation of the cell cycle, glycolysis, and mTORCl signaling (FIG. 7H). In contrast, the same pathways were up- regulated by rhAOAH heatment (FIG. 6H). Further, the inhibitory effect of PC(16:0-20:4) on TCR and T-cell activation was confirmed by flow analysis in mouse and human T cells (FIGS. 71-7 J and 14F-14G).

[0458] A previous crystal structure study of AOAH binding on the bacteria lipopolysaccharide delineated the catalytic site as Serine 263, initiating a nucleophilic attack on the substrate’s carbonyl carbon (Gorelik et al., Proc. Natl. Acad. Sci. USA 115:E896-E905, 2018). AOAH may also depend on the same site to promote T-cell functions. Ums, a point mutation of Serine 263 to Alanine was inhoduced. The Ser263-Ala mutant rhAOAH had significantly less enhancing effect on the T-cell activation than the wildtype protein (FIG. 7K).

[0459] Example 4 Lymphocyte antigen 86

[0460] Lymphocyte antigen 86 (LY86) is associated with favorable clinical immunotherapy outcomes across multiple cancer types (FIG. 15A and B) and does not have known roles in cancer. LY86 is expressed in B cells and monocytes (FIG. 15C).

[0461] Ly86 was overexpressed in B16F10 mouse melanoma cells (RT-qPCR fold change = 229.12 compared to vector control), a tumor model resistant to immune checkpoint blockade (ICB). Then, subcutaneous inoculation of B16F10 tumors in C57BL / 6 mice was performed and the mice were intraperitoneally treated with anti-PD-1 and anti-CTLA4. Ly86 overexpression significantly sensitized B16F10 tumors to ICB relative to vector control (FIG. 15D).

[0462] To test whether the observed effects of Ly86 overexpression (OE) are non-cell-autonomous, mixtures of Ly<%>-OE:vector-only cells were created at 10:90 and 20:80 ratios. Injection of these mixes showed that 10% Ly86-OE cells promoted ICB efficacy, with 20% achieving an ICB- enhancing effect comparable to Ly86 overexpression in 100% of cancer cells (FIG. 15E). These 4239-111527-02 results indicate that Ly86 can promote ICB efficacy in a non-cell-autonomous manner when overexpressed.

[0463] Example 5

[0464] AO AH potentiates intraperitoneal transfer of dendritic cell line

[0465] Aoah was overexpressed (OE) in the mouse dendritic (DC) line DC2.4. Aoah-OE and vector- OE DC2.4 cells were injected into B16-mhgpl00-bearing mice intraperitoneally. The median tumor volume with injections of Aoah-OE DC2.4 was 12.7% of that with vector-OE DC2.4 (FIG. 16, onesided Wilcoxon rank-sum p value = 0.055).

[0466] Example 6

[0467] AOAH prevents oxidized phosphatidylcholine from inhibiting dendritic cell activation

[0468] Mouse splenocytes (as antigen-presenting cells) were pretreated with oxidized 1 -palmitoyl -2- arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC) and co-cultured with OT-1 CD8+T cells in the presence of OVA antigens. oxPAPC significantly diminished the expression of MHC-I, MHC-II, CD40, and CD80 on CDllc+DCs among splenocytes, deteriorating their antigen presentation and costimulatory functions. As predicted, rhAOAH treatment reverted the inhibitory effect of oxPAPC on CD11+DCs (FIG. 17).

[0469] Example 7

[0470] AOAH recombinant protein reduces lipid peroxidation of activated T cells

[0471] It is demonstrated in the Examples above that AOAH can rescue T cells from suppressions induced by arachidonyl phosphatidylcholines (PC). The arachidonyl moiety on the PC sn-2 position can trigger lipid peroxidation. In addition, reactive oxygen species (ROS) are produced at high levels during T cell activation and can enhance lipid peroxidation via oxidation of PCs, thereby inhibiting T cell functions. As shown in FIGS. 18A-18B, rhAOAH treatment reduced lipid peroxidation in CD8+ T cells, explaining AOAH rescue of T cells from arachidonyl PCs.

[0472] It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from tire spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

4239-111527-02We claim:

1. A method of treating cancer in a subject, comprising administering to the subject an effective amount of a composition comprising: an acyloxyacyl hydrolase (AO AH) protein or a nucleic acid encoding the AO AH protein; a fusion protein comprising an AO AH protein and a heterologous protein or portion thereof; a conjugate comprising an AO AH protein and a heterologous protein or portion thereof; or a T cell receptor (TCR)-T cell or a chimeric antigen receptor (CAR)-T cell expressing AOAH, wherein the TCR-T cell or CAR-T cell targets an antigen expressed by the cancer.

2. The method of claim 1 , wherein the AOAH protein comprises an amino acid sequence with at least 90% sequence identity to amino acids 31-571 of SEQ ID NO: 1.

3. The method of claim 2, wherein the AOAH protein comprises the amino acid sequence of amino acids 31-571 of SEQ ID NO: 1.

4. The method of any one of claims 1 to 3, wherein the heterologous protein comprises an Fc domain or an antibody or fragment thereof.

5. The method of any one of claims 1 to 4, wherein the composition is administered intratumorally or parenterally.

6. A modified immune cell expressing a heterologous acyloxyacyl hydrolase (AOAH) and a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

7. The modified immune cell of claim 6. wherein the immune cell is a T cell or a natural killer cell.

8. The modified immune cell of claim 6 or claim 7, wherein tire AOAH protein comprises an amino acid sequence with at least 90% sequence identity to amino acids 31-571 of SEQ ID NO: 1.

9. The modified immune cell of claim 8. wherein tire AOAH protein comprises the amino acid sequence of amino acids 31-571 of SEQ ID NO: 1.

10. A method of treating cancer in a subject, comprising administering to the subject an effective amount of the modified immune cell of any one of claims 6 to 9.4239-111527-0211. The method of claim 10, wherein the TCR or CAR specifically binds an antigen expressed by the cancer.

12. The method of claim 10 or claim 11, wherein the immune cell is administered intravenously.

13. The method of any one of claims 1 to 5 and 10 to 12, further comprising administering to the subject an immune checkpoint inhibitor.

14. The method of claim 13, wherein the immune checkpoint inhibitor is anti-CTLA-4, anti-PD- 1. anti-PD-Ll. or any combination thereof.

15. The method of any one of claims 1 to 5 and 10 to 14, wherein the cancer is melanoma, liver cancer, renal cancer, pancreatic cancer, or lung cancer.

16. A composition comprising: a fusion protein comprising an acyloxyacyl hydrolase (AOAH) protein and a heterologous protein or portion thereof; or a conjugate comprising an AOAH protein and a heterologous protein or portion thereof.

17. The composition of claim 16, wherein the heterologous protein comprises an Fc domain or portion thereof, or an antibody or fragment thereof.

18. The composition of claim 16 or claim 17, wherein the AOAH protein comprises an amino acid sequence with at least 90% sequence identity to amino acids 31-571 of SEQ ID NO: 1.

19. The composition of claim 18, wherein the AOAH protein comprises the amino acid sequence of amino acids 31-571 of SEQ ID NO: 1.

20. The composition of claim 17. wherein the Fc domain or portion thereof comprises an amino acid sequence with at least 90% sequence identity to amino acids 20-245 of SEQ ID NO: 5.

21. The composition of claim 20. wherein the Fc domain or portion thereof comprises the amino acid sequence of amino acids 20-245 of SEQ ID NO: 5.

22. A method of treating cancer in a subject, comprising administering to the subject an effective amount of a composition comprising: a lymphocyte antigen 86 (LY86) protein or a nucleic acid encoding a LY86 protein;4239-111527-02 a fusion protein comprising a LY86 protein and a heterologous protein or portion thereof; a conjugate comprising a LY86 protein and a heterologous protein or portion thereof; a T cell receptor (TCR)-T cell or a chimeric antigen receptor (CAR)-T cell expressing LY86, wherein the TCR-T cell or CAR-T cell targets an antigen expressed by the cancer.

23. The method of claim 22, wherein the LY86 protein comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 33.

24. The method of claim 22 or claim 23, wherein the heterologous protein comprises an Fc domain or an antibody or fragment thereof.

25. A modified immune cell expressing a heterologous lymphocyte antigen 86 (LY86) protein and a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

26. The modified immune cell of claim 25, wherein the immune cell is a T cell or a natural killer cell.

27. The modified immune cell of claim 25 or claim 26, wherein the LY86 protein comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 33.

28. A method of treating cancer in a subject, comprising administering to the subject an effective amount of die modified immune cell of any one of claims 25 to 27.

29. The method of claim 28, wherein die TCR or CAR specifically binds to an antigen expressed by the cancer.

30. The method of claim 28 or claim 29, wherein the composition is administered intravenously.

31. The method of any one of claims 22 to 24 and 28 to 30, further comprising administering to the subject an immune checkpoint inhibitor.

32. A composition comprising: a fusion protein comprising a lymphocyte antigen 86 (LY86) protein and a heterologous protein or portion thereof; or a conjugate comprising a LY86 protein and a heterologous protein or portion thereof.4239-111527-0233. The composition of claim 32, wherein the heterologous protein comprises an Fc domain or portion thereof, or an antibody or fragment thereof.

Images