Compositions and methods for diagnosing and treating immune-related adverse events associated with immune checkpoint inhibitor treatment

Abstract

The present disclosure generally relates to compositions and methods for predicting or diagnosing immune-related adverse events (irAE) before, during, or after immune checkpoint inhibitor (ICI) treatment in a subject with cancer. The method includes assessment of immune cell types, cytokines, autoantibodies or any combination thereof.

Description

Attorney Docket No. UTSD.P4287WO / 1001338856 PATENT COOPERATION TREAT (PCT) APPLICATION COMPOSITIONS AND METHODS FOR DIAGNOSING AND TREATING IMMUNE- RELATED ADVERSE EVENTS ASSOCIATED WITH IMMUNE CHECKPOINT INHIBITOR TREATMENT Inventors: David E. Gerber Shaheen Khan Assignee: THE BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM 210 West 7th Street, Austin, TX 78701 Entity: SmallAttorney Docket No. UTSD.P4287WO / 1001338856 TITLE COMPOSITIONS AND METHODS FOR DIAGNOSING AND TREATING IMMUNE- RELATED ADVERSE EVENTS ASSOCIATED WITH IMMUNE CHECKPOINT INHIBITOR TREATMENT CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 638,757 filed April 25, 2024, the contents of which is hereby incorporated by reference in its entirety. ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant number AI156189 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND 1. Field

[0003] The present disclosure relates to identification of biomarkers for predicting, diagnosing, or monitoring immune-related adverse events associated with immune checkpoint inhibitor therapy. 2. Discussion of Related Art

[0004] Immune checkpoint inhibitors (ICI) are highly effective therapies that have revolutionized the treatment of numerous cancers. However, in some cases these agents cause autoimmune toxicities termed immune-related adverse events (irAEs). In contrast to the predictability and limited scope of chemotherapy and molecularly targeted therapy toxicities, irAEs may occur at any point during—and occasionally even well after—ICI therapy. They may affect almost any organ system, including potentially fatal cardiovascular and neurologic events in rare cases. In recent years, ICI indications have expanded from refractory, advanced malignancies to earlier-stage, curable tumors. At the same time, combination ICI regimens, which are associated with far higher irAE rates, have received approval for melanoma, kidney cancer, lung cancer, liver cancer, and mesothelioma. Taken together, these trends have only heightened the clinical importance of irAEs.

[0005] Further complicating ICI administration are profound challenges in monitoring and diagnosing irAEs. Although irAE may affect a plethora of organs, the rarity of certain types such as myocarditis renders longitudinal, extensive multi-organ monitoring costly andAttorney Docket No. UTSD.P4287WO / 1001338856 impractical. Indeed, expert guidelines generally limit routine surveillance to standard chemistries and thyroid function tests, which may detect kidney, liver, and certain endocrine toxicities but little else. Once irAEs do occur, accurate and timely recognition, evaluation, and treatment remain elusive. Similar to non-ICI-related autoimmune diseases—which may require incorporation of clinical history, physical examination findings, serologies, radiographic studies, and occasionally histologic sampling to render a diagnosis—it appears that irAEs are far more difficult to diagnose than chemotherapy- or targeted therapy-related toxicities. Failure to detect and treat irAE quickly could result in high-grade events. Although lower grade irAEs have been associated with ICI efficacy, the acute clinical deterioration caused by severe irAEs may overshadow any potential benefit. Given the expanding use of ICI therapy across cancer types and stages, the ability to predict and accurately diagnose irAEs represents a key clinical priority in the field of immuno-oncology. SUMMARY

[0006] In some aspects, provided herein is a method of predicting the risk of developing and / or diagnosing immune-related adverse events (irAE) associated with immune checkpoint inhibitor (ICI) treatment in a subject comprising: providing a sample from the subject; assessing the frequencies of one or more immune cell types in the sample, wherein the one or more immune cell types comprise natural killer (NK) cells, CD4 proliferating cells,proliferating T and NK cells, gamma / delta T cells ( T cells), plasmablasts, double-negativeT (dNT) cells, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cells, NKT cells, HLA-DR+ CD4 memory effector T cells, CD4 central memory cells, or any combination thereof; and predicting risk for developing and / or diagnosing irAE in the subject, wherein the subject is predicted as having a high risk of developing or diagnosed with having irAE if the frequencies of the one or more immune cell types are different in the subject when compared to the frequencies in a control sample.

[0007] In further aspects, the disclosure encompasses a method of monitoring the risk of developing irAE associated with ICI treatment in a subject comprising: providing a sample from the subject, assessing the frequencies of one or more immune cell types in the sample, wherein the one or more immune cell types comprise natural killer (NK) cell, CD4 proliferatingcell, proliferating T and NK cell, gamma / delta T cell ( T cell), plasmablast, double-negativeT (dNT) cell, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof, and monitoring risk for developing irAE in the subject, wherein the subject is predicted as having a high risk of developing irAE if the frequencies of the one or more immune cell types are different in the subject when compared to the frequencies in a control sample.Attorney Docket No. UTSD.P4287WO / 1001338856

[0008] In some aspects, the irAE comprises ocular toxicity, rash, dermatitis, pruritus, colitis, hepatitis, nephritis, arthritis, myositis, myocarditis, pneumonitis, thyroiditis, hypophysitis, adrenalitis, gastritis, pancreatitis, vasculitis, diabetes, myasthenia gravis, encephalitis, peripheral neuropathy, meningitis, hemolytic anemia, thrombocytopenia, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), aplastic anemia, pure red cell aplasia, and / or neutropenia.

[0009] In some aspects, the assessment of frequencies of one or more immune cell types is performed before ICI treatment to predict the subject’s risk of developing irAE. In some aspects, the assessment of frequencies of one or more immune cell types is performed during ICI treatment to diagnose the subject’s risk of developing irAE. In some aspects, the assessment of one or more frequencies of immune cell types is performed after ICI treatment to monitor the subject’s risk for developing irAE or the subject’s recovery from irAE.

[0010] In some aspects, the subject is predicted as high risk of developing irAE if the frequency of NK cell is decreased in the sample prior to ICI treatment (baseline) when compared to the frequencies in a control sample. In some aspects, the subject is predicted as high risk of developing irAE if the frequencies of CD4 proliferating cell, proliferating T and NKcell, gamma / delta T cell ( T cell), plasmablast, dNT cell, CXCR3 T cells, CD57+ T cells,CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof are elevated in the sample prior to ICI treatment (baseline) when compared to the frequencies in a control sample. In some aspects of the method, the subject is diagnosed with irAE if the frequencies of dNT cells, CD4proliferating cells, proliferating T and NK cells, gamma / delta T cells ( T cells), plasmablasts,or any combination thereof are elevated after initiation of ICI treatment, when compared to the frequencies in a control sample.

[0011] In some aspects, the sample is whole blood, serum, plasma, cerebrospinal fluid, pleural fluid, pericardial fluid, peritoneal fluid, bone marrow, or tissue, urine, cerebrospinal fluid (CSF), or other body fluid.

[0012] In some aspects, the ICI treatment is administered as part of a cancer treatment. In some aspects, the ICI treatment comprises administration of an inhibitor of PD-1, PD-L1, TIM- 3, LAG- 3, CTLA-4, CSF- 1R, or any combination thereof.

[0013] In some aspects of the method, assessing the frequencies of one or more immune cells (step b) comprises mass cytometry (cytometry by time-of-flight CyTOF), scRNA- sequencing, immune profiling assay, flow cytometry, fluorescence-activated cell sorting (FACS), and / or immunomagnetic separation.Attorney Docket No. UTSD.P4287WO / 1001338856

[0014] In certain aspects, the method further comprises assessing the expression of cytokines in the sample from the subject, wherein the cytokines are one or more of C-X-C motif chemokine ligand 13 (CXCL13), C-X-C motif chemokine ligand 9 (CXCL9), C-X-C motifchemokine ligand 10 (CXCL10), Interleukin-1 beta (IL1 ), Tumor necrosis factor (TNF ),Chemokine ligand 3 (CCL3), Chemokine ligand 8 (CXCL8), Fos Proto-Oncogene (FOS), Chemokine ligands 4 (CCL4), TNF alpha induced protein 3 (TNFAIP3), TNF alpha induced protein 6 (TNFAIP6), Testis Expressed 14 (TEX14), C-X-C motif chemokine ligand 2 (CXCL2), Cluster of Differentiation 83 (CD83) or any combination thereof. In some aspects, theassessment comprises identifying if the expression of IL1 , TNF , CCL3, CXCL8, or anycombination thereof are elevated in the sample from the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, theassessment comprises identifying if the expression of IL1 , CXCL8, CXCL9, CXCL10, FOS,CCL4, CCL3, TNFAIP3, TNFAIP6, TEX14, CXCL2, CD83, or any combination thereof are elevated in the sample from the subject after initiation of ICI treatment when compared to the expression in a control sample.

[0015] In certain aspects, the method further comprises assessing the level of antinuclear antibodies (ANA) in the sample from the subject. In some aspects, the assessment comprises assessing if the level of ANA is elevated in the sample from the subject when compared to the level in the control sample.

[0016] In some aspects, the method further comprises repeating one or more steps disclosed herein at a second time point, thereby permitting determination of a change in the subject’s risk of developing irAE and / or diagnosis of irAE in the subject.

[0017] In some aspects, the method further comprises predicting the subject as having low risk: if the frequency of NK cell is increased in the sample from the subject prior to ICI treatment (baseline) when compared to the frequency in the control sample; if the frequencies of CD4proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell), plasmablast, dNTcell, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof are decreased in the sample from the subject prior to ICI treatment (baseline) when compared to the frequencies in the control sample; and / or if the frequencies of dNT cell, CD4 proliferatingcell, proliferating T and NK cell, gamma / delta T cell ( T cell), plasmablast, or anycombination thereof is decreased in the sample from the subject after initiation of ICI treatment, when compared to frequencies in the control sample.

[0018] In further aspects, the method comprises treating the subject with an ICI therapy if the subject is predicted to have a low risk for developing irAE. In some aspects, the methodAttorney Docket No. UTSD.P4287WO / 1001338856 encompasses treating the subject predicted as having a high risk of developing irAE with a non-ICI therapy or treating said subject with a ICI therapy and an irAE mitigating therapy, wherein the irAE mitigating therapy is selected from corticosteroids (e.g., prednisone, methylprednisolone, dexamethasone, budesonide), TNF inhibitors (e.g., infliximab), or hormone replacement (e.g., hydrocortisone, levothyroxine) BRAF inhibitor (e.g., vemurafenib, dabrafenib, and encorafenib), MEK inhibitor (e.g., binimetinib, cobimetinib, selumetinib, trametinib), CXCL8 inhibitors (e.g., repertaxin), or any combination thereof.

[0019] The disclosure further encompasses a method of treating a subject with cancer comprising: providing a sample from the subject; assessing the frequencies of one or more immune cell types in the sample, wherein the one or more immune cell types comprise naturalkiller (NK) cells, CD4 proliferating cells, proliferating T and NK cells, gamma / delta T cells (T cells), plasmablasts, double-negative T (dNT) cells, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cells, NKT cells, HLA-DR+ CD4 memory effector T cells, CD4 central memory cells, or any combination thereof. In such aspects, the method comprises predicting the subject’s risk of developing irAE, wherein the subject is diagnosed as: low risk if: the frequency of NK cell is increased in the sample prior to ICI treatment (baseline) when compared to the frequency in a control sample; and / or (II) the frequencies of CD4 proliferatingcell, proliferating T and NK cell, gamma / delta T cell ( T cell), plasmablast, dNT cell, CXCR3T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof are decreased in the sample prior to ICI treatment (baseline) when compared to the frequencies in a control sample. In some aspects, the method comprises predicting the subject’s risk of developing irAE, wherein the subject is diagnosed as high risk if the frequency of NK cell is decreased in the sample prior to ICI treatment (baseline) when compared to the frequency in a control sample; and / or the frequencies of CD4 proliferating cell, proliferating T and NK cell,gamma / delta T cell ( T cell), plasmablast, dNT cell, CXCR3 T cells, CD57+ T cells, CD57+NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, CD4 central memory or any combination thereof are elevated in the sample prior to ICI treatment (baseline) when compared to the frequencies in a control sample. The method further comprises treating the subject with: an ICI therapy if subject is diagnosed as low risk of developing irAE, a non-ICI therapy if the subject is diagnosed as high risk of developing irAE; or an ICI therapy and an irAE mitigating therapy if the subject is diagnosed as high risk of developing irAE.

[0020] In some aspects, the assessment further comprises assessing the expression of a cytokine in the sample provided by the subject, wherein the cytokine is CXCL13, CXCL9,CXCL10, IL1 , TNF , CCL3, CXCL8, FOS, CCL4, TNFAIP3, TNFAIP6, TEX14, CXCL2,Attorney Docket No. UTSD.P4287WO / 1001338856 CD83, or any combination thereof. In some aspects, the assessment comprises identifying ifthe expression of IL1 , TNF , CCL3, CXCL8, or any combination thereof are elevated in thesample from the subject prior to ICI treatment (baseline) when compared to the expression in the control sample. In some aspects, the assessment comprises identifying if the expressionof IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4, CCL3, TNFAIP3, TNFAIP6, TEX14, CXCL2,CD83, or any combination thereof is elevated in the sample from the subject after initiation of ICI treatment when compared to the expression in the control sample. In some aspects, the assessment further comprises assessing the level of antinuclear antibodies (ANA) in the sample from the subject. In some aspects, the assessment comprises assessing if the level of ANA is elevated in the sample from the subject when compared to the level in the control sample. In some aspects, the disclosed ICI therapy comprises administration of an inhibitor of PD-1, PD-L1, TIM-3, LAG- 3, CTLA-4, CSF- 1R, or any combinations thereof.

[0021] In some aspects, the disclosure further encompasses a method of identifying the presence of at least one differentially frequent immune cell type with associated with irAE in a biological sample of a subject with cancer, comprising: providing a sample from the subject; and assessing the frequencies of one or more immune cell types in the sample, wherein the assessment comprises assessing the frequencies of one or more immune cell types comprises assessing one or more of natural killer (NK) cell, CD4 proliferating cell, proliferatingT and NK cell, gamma / delta T cell ( T cell), plasmablast, double-negative T (dNT) cell,CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA- DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof. In some aspects, the subject is planning, undergoing, or has completed immune checkpoint inhibitor (ICI) treatment. In some aspects of the method, irAE comprises ocular toxicity, rash, dermatitis, pruritus, colitis, hepatitis, nephritis, arthritis, myositis, myocarditis, pneumonitis, thyroiditis, hypophysitis, adrenalitis, gastritis, pancreatitis, vasculitis, diabetes, myasthenia gravis, encephalitis, peripheral neuropathy, meningitis, hemolytic anemia, thrombocytopenia, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), aplastic anemia, pure red cell aplasia, and / or neutropenia.

[0022] In some aspects, the method further comprises assessing the expression of a cytokine in the sample provided by the subject, wherein the cytokine is CXCL13, CXCL9, CXCL10,IL1 , TNF , CCL3, CXCL8, FOS, CCL4, TNFAIP3, TNFAIP6, TEX14, CXCL2, CD83, or anycombination thereof. In some aspects, the method further comprises assessing the level of antinuclear antibodies (ANA) in the sample from the subject. In some aspects, assessing the frequencies of immune cells comprises mass cytometry (cytometry by time-of-flight CyTOF), scRNA-sequencing, immune profiling assay, flow cytometry, fluorescence-activated cell sorting (FACS), or immunomagnetic separation.Attorney Docket No. UTSD.P4287WO / 1001338856 BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG.1 depicts the study design. The study included 164 cancer patients receiving ICI therapy (anti-PD1 or anti-PDL1 or combination (anti-PD1 + anti-CTLA4)). Blood was collected at baseline before the initiation of ICI therapy and post-immunotherapy (2-3 weeks (“wks”) and 6-8 wks). Peripheral blood mononuclear cells (PBMCs) were used for single-cell RNA sequencing and cytometry by time of flight (CyTOF) assays. Serum was used to assess levels of antinuclear antibodies (ANA) and 40 cytokines.

[0024] FIGs.2A-2I show single-cell RNA sequencing (ScRNA-seq) analysis of pre- and post- ICI therapy blood samples. FIG. 2A shows scRNA-seq experimental design of 72 blood samples (36 pairs) cancer patients at baseline before the initiation of ICI therapy and 6-8 wks post immunotherapy initiation. FIG.2B shows uniform manifold approximation and projection (UMAP) of PBMCs annotated by Azimuth Reference shown by major immune cell types. FIG. 2C shows UMAP of PBMCs annotated by Azimuth Reference shown by 30 immune cell subsets. FIG. 2D shows the abundance of natural killer (NK; CD56 dim) cells shown as a percentage of total PBMCs at baseline (BL) before the initiation of immunotherapy in patients that did not develop irAEs (No irAE) versus patients that developed irAE (irAE) guided by Azimuth Reference. FIG.2E shows the abundance of CD4 proliferating T cells shown as a percentage of total PBMCs at baseline (BL) before the initiation of immunotherapy in patients that did not develop irAEs (No irAE) versus patients that developed irAE (irAE) guided by Azimuth Reference. FIG.2F shows the abundance of plasmablasts shown as a percentage of total PBMCs at baseline (BL) before the initiation of immunotherapy in patients that did not develop irAEs (No irAE) versus patients that developed irAE (irAE) guided by Azimuth Reference. FIG.2G shows the abundance of double negative T cells shown as a percentage of total PBMCs at baseline (BL) before the initiation of immunotherapy in patients that did not develop irAEs (No irAE) versus patients that developed irAE (irAE) guided by Azimuth Reference. FIG.2H shows the spearman correlation of 27 cell clusters identified by Azimuth Reference with irAE showing Spearman r value. FIG.2I shows the spearman correlation of 27 cell clusters identified by Azimuth Reference with irAE showing p-value. Significant clusters are marked in blue, red, purple, and aqua circles, while non-significant clusters are shown in grey circles.

[0025] FIGs.3A-3H show the association of unsupervised cell clusters with irAEs. FIG.3A shows UMAP of 25 cell states identified by unsupervised clustering shown according to cell type. FIG.3B shows a frequency plot showing the proportion of 20 clusters at BL and 6-8 wk post-ICI therapy identified by unsupervised clustering in irAE versus No irAE group. FIG.3C shows the abundance of cluster 18 is T / NK Mki67 positive cells, shown as a percentage of total PBMCs in two different patient groups at baseline (BL) before the initiation ofAttorney Docket No. UTSD.P4287WO / 1001338856 immunotherapy identified by unsupervised clustering. FIG.3D shows the abundance of cluster 6 that corresponds to NK CD56 dim cells, shown as a percentage of total PBMCs in two different patient groups at baseline (BL) before the initiation of immunotherapy identified by unsupervised clustering. FIG. 3E shows spearman correlation of cell clusters identified by unsupervised clustering with irAE showing two-tailed p-value. Significant clusters (Cluster 18 and 6) are marked in blue and purple circles, respectively, while non-significant clusters are in grey circles. FIG.3F shows a summary of r and p values for four significant clusters. FIGs. 3G-3H depict receiver operating characteristic (ROC) curve plot of cluster 18 (Mki67+ve T / NK) (FIG.3G) and cluster 6 (NK) (FIG.3H) cells at baseline for predicting irAEs.

[0026] FIGs.4A-4L show CyTOF analysis of PBMC samples at baseline before ICI therapy. FIG. 4A shows the CyTOF experimental design of 128 blood samples (64 pairs) from 64 cancer patients at baseline before the initiation of ICI therapy and 6-8 wks post immunotherapy. FIG. 4B shows Opt-SNE of 30 cell clusters identified by unsupervised consensus meta-clustering using Euclidean as a distance metric shown according to major immune cell types. FIG. 4C shows Opt-SNE of 30 cell clusters identified by unsupervised consensus meta-clustering using Euclidean as a distance metric shown according to 30 immune cell subsets. FIG.4D shows a volcano plot showing significant clusters (blue circles) differentially abundant in irAE versus no irAE at baseline with false discovery rate (FDR) <0.1. FIG. 4E shows the abundance of significant clusters K10-plasmablast identified by unsupervised clustering using Edge R (FDR<0.1) shown as a percentage of total PBMCs baseline (BL) in patients with no irAE versus irAE at baseline. FIG.4F shows the abundance of keratin 17 – double negative T – gamma delta T cells (K17-dNT- T) identified by unsupervised clustering using Edge R (FDR< 0.1) shown as a percentage of total PBMCs at baseline (BL) in patients with no irAE versus irAE at baseline. FIG.4G shows the abundance of cluster K2-NK identified by unsupervised clustering using Edge R (FDR<0.1) shown as a percentage of total PBMCs at baseline (BL) in patients with no irAE versus irAE at baseline. FIG.4H shows the abundance of significant cluster plasmablasts identified by manual gating strategy in patients with irAEs versus no irAEs. FIG.4I shows the abundance of significant cluster CD8 terminal effector cells identified by manual gating strategy in irAEs versus no irAEs. FIG. 4J shows the abundance of significant cluster NKT cells identified by manual gating strategy in irAEs versus no irAEs. FIG.4K shows the abundance of significant cluster activated CD4 effector memory T cells (TEM) identified by manual gating strategy in irAEs versus no irAEs. FIG.4L shows the median HLA-DR expression on CD4 effector memory T cells in patients with no irAE versus irAE (p < 0.05).

[0027] FIGs.5A-5E show the association of B cell subsets with irAEs. FIG.5A shows the abundance of Naïve B cells as a percentage of total PBMCs at baseline (BL) and post-Attorney Docket No. UTSD.P4287WO / 1001338856 immunotherapy in patients that did not develop irAEs (No irAE) versus patients that developed irAE (irAE) guided by Azimuth Reference. FIG.5B shows CyTOF analysis of 128 PBMCs (64 pairs) from 64 cancer patients at BL and 6-8 wks post immunotherapy showing the abundance of Naïve B cells as a percentage of total PBMCs in patients with no irAE versus irAE identified by manual gating strategy. FIG.5C shows CyTOF analysis of 128 PBMCs (64 pairs) from 64 cancer patients at BL and 6-8 wks post-immunotherapy showing the abundance of plasmablasts as a percentage of total PBMCs in patients with no irAE versus irAE identified by manual gating strategy. FIG. 5D shows the comparison of serum conc. of chemokine CXCL13in 164 patients at baseline (BL) and post-immunotherapy (6-8 wks). (P<0.05). FIG. 5E shows the comparison of serum conc. of antinuclear antibodies (ANA) in 107 patients at baseline (BL) and post-immunotherapy (at 2-4 wks and 6-8 wks).

[0028] FIG.6A shows the abundance of CD16-positive monocytes cells as a percentage of total PBMCs at baseline (BL) and post-immunotherapy in patients that did not develop irAEs (No irAE) versus patients that developed irAE (irAE) guided by Azimuth Reference.

[0029] FIG.6B shows pseudobulk expression profiles for differential expression analysis at baseline in irAE compared to the no irAE group as a volcano plot for CD16+ monocytes.

[0030] FIG.6C shows pseudobulk expression profiles for differential expression analysis at baseline in irAE compared to the no irAE group as a volcano plot for CD14+ monocytes.

[0031] FIG.6D shows pseudobulk expression profiles for differential expression analysis in irAE compared to the No irAE group at baseline is shown as a volcano plot for CD16+ monocytes at 6-8 wks.

[0032] FIG.6E shows pseudobulk expression profiles for differential expression analysis in irAE compared to the no irAE group at baseline is shown as a volcano plot for CD14+ monocytes at 6-8 wks. Significantly upregulated genes are shown in blue circles, significantly downregulated genes in red dots, and genes with no significant change are shown in grey.

[0033] FIG.6F shows the comparison of serum conc. of chemokine CXCL9 in 164 patients at baseline (BL) and post-immunotherapy (6-8 weeks). (P<0.05).

[0034] FIG.6G shows the comparison of serum conc. of chemokine CXCL10 in 164 patients at baseline (BL) and post-immunotherapy (6-8 weeks). (P<0.05).

[0035] FIGs. 7A-7H show single-cell RNA sequencing (scRNA-seq) analysis of PBMCs at baseline and post-ICI therapy. FIG. 7A shows UMAP of 23 cell states identified by unsupervised clustering shown according to cell type. FIG. 7B provides a frequency plot showing the proportion of 23 clusters identified by unsupervised clustering in irAE versus no- irAE group at BL and 6-8 weeks post-ICI therapy. FIG. 7C provides a dot plot showingAttorney Docket No. UTSD.P4287WO / 1001338856 canonical marker gene expression for 23 unsupervised clusters. Arrows point to cluster 2 (NK) and Cluster 18 (Mki67+). FIG.7D show the abundance of cluster 18 (Mki67+), shown as a percentage of total PBMCs in two groups at baseline (BL) identified by unsupervised clustering. FIG.7E shows the abundance of NK (CD56dim) cells; FIG.7F shows the abundance of CD4 proliferating T cells; FIG. 7G shows the abundance of plasmablasts; and FIG. 7H shows the abundance combined proliferating lymphocyte cluster (CD4, CD8 and NK).

[0036] FIGs.8A-8O show mass cytometry (CyTOF) analysis of PBMC samples at baseline and post-ICI therapy. FIG.8A shows CyTOF experimental design of 110 blood samples (55 pairs) from 55 cancer patients (N=12 no-irAE and N=43 irAE) at baseline (BL) before the initiation of ICI therapy and 6-8 weeks post immunotherapy. FIG.8B shows Opt-SNE of 30 cell clusters identified by unsupervised consensus meta-clustering of CYTOF data using Euclidean as a distance metric shown according to major immune cell types. FIG.8C shows Opt-SNE of 30 cell clusters identified by unsupervised consensus meta-clustering of CYTOF data using Euclidean as a distance metric shown according to 30 immune cell subsets. FIG. 8D provides a heatmap showing expression of 35 cell surface immune markers used in the CYTOF panel in 30 metaclusters. FIG.8E provides a volcano plot of CyTOF data showing significant metaclusters clusters (aqua circles) differentially abundant in irAE versus no-irAE at baseline with (P < 0.05). FIG. 8F provides the abundance of significant cluster for K10 plasmablast; FIG.8G provides the abundance of significant cluster of NK K2; FIG.8H provides the abundance of significant cluster for CD8 terminal Effector K4, FIG. 8I provides theabundance of significant cluster for K8, TCR T; FIG.8J provides the abundance of significantcluster for K13, CD8 terminal effector, identified by unsupervised clustering of CyTOF data using Edge R (P < 0.05) shown as a percentage of total PBMCs in no-irAE versus irAE cases at BL. FIG. 8K provides the abundance of significant cluster NKT. FIG. 8L provides the abundance of significant CD57 positive clusters. FIG. 8M provides the abundance of significant cluster identified by manual gating strategy in irAE versus no-irAE cases of CD8+KLRG1+CCR7- CD8 effector memory cluster identified by unsupervised T / NK subclustering analysis of scRNA-seq data. FIG.8N shows significant increase in abundance of cluster K21 (CD4 memory) in irAE and no-irAE cases post-immunotherapy in all cases. FIG. 8O shows significant increase in abundance of cluster K21 (CD4 memory) in irAE and no-irAE cases post-immunotherapy inmelanoma cases by Wilcoxon matched-pairs signed rank test of CyTOF data.

[0037] FIGs. 9A-9N show baseline autoimmune-like, proliferative and inflammatory state associated with irAE patients. FIG.9A shows the abundance of plasmablasts as a percentage of total PBMCs at baseline (BL) based on the grade of irAE. FIG.9B shows a comparison of plasma concentration of antinuclear antibodies (ANA) (N=65 total; N=22 no-irAE and N=43Attorney Docket No. UTSD.P4287WO / 1001338856 irAE) at BL and post-immunotherapy (at 2-4 weeks and 6-8 weeks). FIG. 9C shows a comparison of plasma concentration of ANA in 65 patients based on irAE grade at BL and post-immunotherapy. G0-1, Grade 0-1; G2, Grade 2; G3, Grade 3. FIG.9D shows abundance of CXCR3+Mki67+cell cluster as a percentage of total PBMCs at BL based on the grade of irAE. FIG.9E provides pseudobulk scRNA-seq differential gene expression analysis of T and NK cells in irAE compared to the no-irAE group at baseline is shown as a volcano plot. Significantly upregulated genes are shown in blue dots, significantly downregulated genes in red dots, and genes with no change are shown in grey. FIG.9F shows a heat map depicting logFC and p values for the 3-gene signature in T and NK cells comparing irAE versus No irAE group at baseline (adjusted P < 0.05 and 2 fold change). FIG.9G provides MsigDB pathwayenrichment analysis for T / NK cells at baseline. Arrow shows the TNF Signaling as the highlyenriched pathway in irAEs at baseline. FIG.9H shows GSEA enrichment analysis for T / NK cells showing TNF signaling as the most enriched pathway at baseline. FIG. 9I shows abundance of CXCR3+Mki67+cluster identified by high-resolution unsupervised clustering of scRNA-seq data. FIG.9J shows abundance of CXCR3+CD4+memory cluster identified by high-resolution unsupervised clustering of scRNA-seq data. FIG. 9K shows abundance of CXCR3+CD8+memory clusters in the scRNA-seq data at baseline. FIG.9L shows CXCR3 gene expression in T and NK cells at baseline. FIG.9M shows the abundance of CXCR3+CD8+clusters at baseline identified in the CyTOF data. FIG. 9N shows the abundance of CXCR3+CD4+memory clusters at baseline identified in the CyTOF data.

[0038] FIGs.10A-10P show single cell RNA sequencing analysis of myeloid cells in patients with and without irAE. FIG. 10A provides pseudobulk differential expression analysis of unsupervised myeloid cell clusters of scRNA-seq data in irAE versus no-irAE group at baseline is shown as a volcano plot (adjusted P < 0.05 and fold change 1.5). Significantly upregulated genes are shown in blue dots, significantly downregulated genes in red dots, and genes with no change are shown in grey. FIG.10B provides Gene Set Enrichment Analysis (GSEA) of myeloid cells at baseline in irAE patients. FIG.10C provides MsigDB pathway enrichment in of myeloid cells at baseline in irAE patients. Arrows in panel C show the TNF Signaling and inflammatory response pathways as highly enriched in myeloid cells in irAE group at baseline. FIG.10D provides heatmap of selected upregulated genes in myeloid cells in irAE versus no- irAE at baseline (adjusted P < 0.05 and fold change 1.5). FIG.10E provides gene expression changes of CXCL10, CCL3, CCL4, SPARC and SIGLEC1 in myeloid cells in irAE versus no- irAE cases post-therapy (adjusted P < 0.05 and fold change 1.5). FIG.10F provides gene expression changes of TNF and IRF4 in monocytes in No-irAE cases post-therapy compared to baseline (adjusted P < 0.05 and fold change 1.5). FIG.10G provides heatmap of selected upregulated and downregulated genes in Azimuth guided CD14+classical monocytes in irAEAttorney Docket No. UTSD.P4287WO / 1001338856 versus no-irAE at baseline (adjusted P < 0.05 and fold change 1.5). FIG.10H provides Gene Ontology analysis (GO) analysis in CD14+classical monocytes in irAE versus no-irAE at baseline. FIG. 10I provides a heatmap of selected upregulated genes in Azimuth guided CD14+and CD16+classical monocytes in irAE versus No irAE at post-therapy (adjusted P < 0.05 and fold change 1.5). FIG.10J provides a heatmap of selected upregulated genes in Azimuth guided CD14+and CD16+classical monocytes in No irAE at post-therapy compared to baseline (adjusted P < 0.05 and fold change 1.5). FIG.10K provides unsupervised sub- clustering analysis of all myeloid cells (without pDCs). FIG.10L provides a dot plot showing gene expression markers for 16 unsupervised Louvain clusters for myeloid cells. FIG.10M shows significantly upregulated cytokines / chemokine ligands and their receptors at baseline in irAE patients compared to No irAE patients at baseline (adjusted P < 0.05 and fold change 1.5). FIG.10N provides a trajectory analysis of myeloid cells using Monocle 3. FIG.10O shows continuous embedding depicting the fraction of myeloid cells expressing CXCL8 and IL1B genes and their corresponding gene expression changes between irAE and No irAE patients at baseline in myeloid cells. FIG.10P shows abundance of TGFBIhighcluster 13 in irAE versus No irAE patients at baseline shown as a percentage of total myeloid cells.

[0039] FIGs. 11A-11J show a comparison of plasma concentrations of chemokines at baseline (BL) and post-immunotherapy (6-8 weeks). FIG. 11A provides a comparison of plasma concentrations of CXCL10 at baseline (BL) and post-immunotherapy (6-8 weeks). FIG. 11B provides a comparison of plasma concentrations of CXCL9 at baseline (BL) and post-immunotherapy (6-8 weeks) in all patients. FIG.11C provides a comparison of plasma concentrations of CXCL10 at baseline (BL) and post-immunotherapy (6-8 weeks) in PD / PDL1 treated patients. FIG. 11D provides a comparison of plasma concentrations of CXCL9 at baseline (BL) and post-immunotherapy (6-8 weeks) in PD1 / PDL1-treated patients. FIG.11E provides a comparison of plasma concentrations of CXCL10 at baseline (BL) and post- immunotherapy (6-8 weeks) in combination (CTLA4+PD1)-treated patients. FIG.11F provides a comparison of plasma concentrations of CXCL9 at baseline (BL) and post-immunotherapy (6-8 weeks) in combination (CTLA4+PD1)-treated patients. FIG.11G provides a comparison of plasma concentrations of CXCL8 at baseline (BL) and post immunotherapy (6-8 weeks) in all patients. FIG.11H provides a comparison of plasma concentrations of CXCL8 at baseline (BL) and post-immunotherapy (6-8 weeks) in PD1 / PDL1-treated patients. FIG.11I provides a comparison of plasma concentrations of CXCL8 at baseline (BL) and post-immunotherapy in combination (CTLA4+PD1)-treated patients. FIG.11J provides a dot plot showing comparison of relative gene expression values of chemokines CXCL10, CXCL9 and CXCL8 at baseline (BL) and post-immunotherapy (6-8 weeks) in myeloid cells in scRNA-seq data. Number of patients for comparison of plasma conc. of chemokines: All patients (N=146 total; N=79 irAEAttorney Docket No. UTSD.P4287WO / 1001338856 and N=67 no-irAE); PD1 / PDL1-treated patients (N=122 total; N=59 No irAE and N=63 irAE); patients treated with combination CTLA4+PD1 therapy (N=18 total; N=6 No irAE and N=12 irAE).

[0040] FIG.12 provides a dot plot showing the top five marker genes for 23 Louvain clusters identified by unsupervised clustering of PBMC scRNA-seq data.

[0041] FIG.13 provides a heatmap showing the top five marker genes for 23 Louvain clusters identified by unsupervised clustering of PBMC scRNA-seq data.

[0042] FIGs.14A-14C shows mass cytometry analysis of PBMCs in cancer patients receiving immune checkpoint inhibitory (ICI) therapy. FIG. 14A shows mass cytometry analysis of PBMCs in cancer patients receiving PD1 / PD-L1-treated patients (N=46 total; N=11 No irAE and N=35 irAE). FIG.14B shows mass cytometry analysis of PBMCs in melanoma patient cohort (N=19 total; N=5 No irAE and N=14 irAE). FIG.14C shows mass cytometry analysis in non-small cell lung cancer (NSCLC) patient cohort (N=21 total; N=3 No irAE and N=18 irAE).The abundance of metaclusters K4-CD8 terminal Effector, K8-TCR T and K13-CD8 terminalEffector, identified by unsupervised clustering shown as a percentage of total PBMCs in patients with no irAE versus irAE at baseline (BL) before the initiation of therapy.

[0043] FIG. 15A shows manual gating strategies for CyTOF data analysis (PBMC) are illustrated for plasmablast.

[0044] FIG. 15B shows manual gating strategies for CyTOF data analysis (PBMC) are illustrated for NKT.

[0045] FIG. 15C shows manual gating strategies for CyTOF data analysis (PBMC) are illustrated for CD8 terminal effector memory T cells.

[0046] FIG. 16A shows abundance of clusters in irAE patients versus No irAE patients by Wilcoxon matched-pairs signed rank test identified by unsupervised clustering shown as a percentage of total PBMCs in K6, B-cells.

[0047] FIG. 16B shows abundance of clusters in irAE patients versus No irAE patients by Wilcoxon matched-pairs signed rank test identified by unsupervised clustering shown as a percentage of total PBMCs in K5, CD8+.

[0048] FIG. 16C shows abundance of clusters in irAE patients versus No irAE patients by Wilcoxon matched-pairs signed rank test identified by unsupervised clustering shown as a percentage of total PBMCs in K18, CD8+.Attorney Docket No. UTSD.P4287WO / 1001338856

[0049] FIG. 16D shows abundance of clusters in irAE patients versus No irAE patients by Wilcoxon matched-pairs signed rank test identified by unsupervised clustering shown as a percentage of total PBMCs in K22, CD4+CD57.

[0050] FIG. 16E shows abundance of clusters in irAE patients versus No irAE patients by Wilcoxon matched-pairs signed rank test identified by unsupervised clustering shown as a percentage of total PBMCs in K28-CD8+.

[0051] The drawing figures do not limit the present disclosure to the specific embodiments disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain embodiments of the present disclosure. DETAILED DESCRIPTION

[0052] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized, and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.

[0053] Provided herein are methods of predicting, diagnosing, and / or monitoring immune- related adverse events (irAE) in a subject undergoing or planning to undergo immune checkpoint inhibitor (ICI) treatment. The present disclosure is based in part on the surprising determination that subjects develop a unique immune cell, a cytokine, and / or antinuclear antibodies (ANA) profile at baseline (pre-treatment), and / or after initiation of ICI treatment, that correlates with irAE during ICI treatment, in subjects with cancer. These immune cells, cytokine, and / or antinuclear antibody (ANA) profiles can be used as biomarkers for predicting, diagnosing, and / or monitoring irAE during ICI treatment and help guide more effective cancer treatment strategies with reduced toxic side effects, especially associated with ICI treatment. I. Terminology

[0054] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred aspects and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.Attorney Docket No. UTSD.P4287WO / 1001338856

[0055] As used in the specification, articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0056] “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result. The term “about” in association with a numerical value means that the numerical value can vary plus or minus by 5% or less of the numerical value.

[0057] Throughout this specification, unless the context requires otherwise, the word “comprise” and “include” and variations (e.g., “comprises,” “comprising,” “includes,” “including”) will be understood to imply the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other integer or step or group of integers or steps.

[0058] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0059] Moreover, the present disclosure also contemplates that in some aspects, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B, and C, it is specifically intended that any of A, B, or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0060] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise-Indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0061] As used herein, “treatment,” “therapy,” and / or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and / or the remission of the disease, disorder or condition.Attorney Docket No. UTSD.P4287WO / 1001338856

[0062] As used herein, “prevent” or “prevention” refers to eliminating or delaying the onset of a particular disease, disorder or physiological condition, or to the reduction of the degree of severity of a particular disease, disorder or physiological condition, relative to the time and / or degree of onset or severity in the absence of intervention.

[0063] As used herein, “individual,” “subject,” “host,” and “patient” can be used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, prophylaxis or therapy is desired, for example, humans, pets, livestock, horses or other animals. As used herein, the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals. The term “nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. In some aspects, the subject can be a human. In other aspects, the subject can be a human in need of treating a cancer.

[0064] As used herein “immune-related adverse event” or “irAE” is diverse toxicities, side- effects, or problems associated with cancer immunotherapy. Immunotherapy include therapy using immunotoxins, T-cell transfer, chimeric antigen receptors, antibodies, immune system modulators, immune checkpoint inhibitors, and / or other immunotherapies known to those of skill in the art.

[0065] As used herein “immune checkpoint inhibitor” is a drug that blocks immune checkpoints. These checkpoints are a normal part of the immune system and keep immune responses from being too strong. By blocking them, these drugs allow immune cells to respond more strongly, for e.g. to cancer. Immune checkpoint inhibitors work by preventing cancer cells from turning T-cells (white blood cells that detect infections and abnormalities) off. Non- limiting examples of immune checkpoint inhibitors include inhibitors of PD-1, PD-L1, TIM-3, LAG- 3, CTLA-4, CSF- 1R, and any combination thereof. The immune checkpoint receptors may be, without limitation, on tumor cells or immune cells such as T cells, monocytes, microglia, and macrophages. The agents which assert immune checkpoint blockade may be small chemical entities or polymers, or antibodies, antibody fragments, single chain antibodies or other antibody constructs, including, but not limited to, bispecific antibodies and diabodies. Immune checkpoint inhibitors which may be used according to the disclosure include any that disrupt the inhibitory interaction of cytotoxic T cells and tumor cells. These include but are not limited to anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA4 antibodies, anti-LAG-3 antibodies, and anti-TIM-3 antibodies. The inhibitor need not be an antibody but can be a small molecule or other polymer. If the inhibitor is an antibody it can be a polyclonal, monoclonal, fragment, single chain, or other antibody variant construct. Inhibitors may target any immune checkpoint known in the art, including but not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3,Attorney Docket No. UTSD.P4287WO / 1001338856 B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, CSF-1R, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, CD28, CD86, CD69, CD48, CD113, CEACAM-1, Galectin-1, TIGIT, GPR56, CD48, GARP, PD1H, LAIR1, TIM1, TIM4, and the B-7 family of ligands. Combinations of inhibitors for a single target immune checkpoint or different inhibitors for different immune checkpoints may be used. Illustrative examples of immune checkpoint inhibitors include CTLA-4 blocking antibodies (ipilimumab (Yervoy®), tremelimumab (Imjuno®)), PD-1 inhibitors (pembrolizumab (Keytruda®)), nivolumab (Opdivo®), cemiplimab (Libtayo®), CT- 011 (pidilizumab), AMP224, PD-L1 inhibitors (atezolizumab (Tecentriq®)), avelumab (Bavencio®), durvalumab (Imfinzi®), BMS- 936559, Lag3 inhibitors (Rrlatlimab), combination of Lag3 and PD1 inhibitor, PD-1 inhibitor nivolumab (Opdualag®), OX40 inhibitor (MEDI6469), and CD160 inhibitor (BY55). Non-limiting examples of inhibitors of CSF-1R include PLX3397, PLX486, RG7155, AMG820, ARRY-382, FPA008, IMC-CS4, JNJ-40346527, and MCS 110. The terms “ICI treatment,” “ICI therapy,” “ICI compounds,” and the like, refer to one or more ICI (or the use thereof) disclosed herein or known to those of skill in the art.

[0066] As used herein “transcript” or “RNA transcript” or “RNA” is messenger RNA (mRNA) molecule.

[0067] As used herein “antinuclear antibodies” or “ANA” are autoantibodies that bind to contents of the cell nucleus.

[0068] As used herein “cytokine” is a broad category of small proteins that are important in cell signaling. Release of cytokine has an effect on the behavior of cells around them. Cytokines are involved in autocrine signaling, paracrine signaling, and endocrine signaling as immunomodulating molecules. Non-limiting examples of cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines are produced by a variety of cell types including immune cells like macrophages, monocytes, dendritic cells, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells; and a given cytokine may be produced by more than one type of cell.

[0069] As used herein “immune cell” is a cell which develops from stem cells in the bone marrow and become different types of white blood cells. Immune cells include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, and lymphocytes (B cells and T cells).

[0070] As used herein “frequency” refers to the amount of a particular analyte (e.g., immune cell subset) present in the sample. The amount may be a number, ratio, proportion, or a percentage of the analyte when compared to the control sample or determined using a standard curve. In some aspects, frequency of immune cell is provided as a percentage ofAttorney Docket No. UTSD.P4287WO / 1001338856 total PBMCs for each cell subset. The amount may be an absolute amount or a relative amount.

[0071] As used herein “expression” or “expression level” or “level of expression” refers to amount of a particular analyte (e.g., antibody or cytokine) present in the sample. The amount may be a concentration, number, ratio, proportion, or a percentage of the analyte when compared to the control sample or determined using a standard curve. The amount may be an absolute amount or a relative amount.

[0072] As used herein “cancer” may be one or more neoplasm or cancer. The neoplasm may be malignant or benign, the cancer may be primary or metastatic; the neoplasm or cancer may be early stage or late stage. Non-limiting examples of neoplasms or cancers include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma (childhood cerebellar or cerebral), basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brainstem glioma, brain tumors (cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic gliomas), breast cancer, bronchial adenomas / carcinoids, Burkitt lymphoma, carcinoid tumors (childhood, gastrointestinal), carcinoma of unknown primary, central nervous system lymphoma (primary), cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing’s sarcoma in the Ewing family of tumors, extracranial germ cell tumor (childhood), extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancers (intraocular melanoma, retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumors (childhood extracranial, extragonadal, ovarian), gestational trophoblastic tumor, gliomas (adult, childhood brain stem, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic), gastric carcinoid, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma (childhood), intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer (renal cell cancer), laryngeal cancer, leukemias (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myelogenous, hairy cell), lip and oral cavity cancer, liver cancer (primary), lung cancers (non-small cell, small cell), lymphomas (AIDS- related, non-Hodgkin, primary central nervous system), macroglobulinemia (Waldenström), malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma (childhood), melanoma, intraocular melanoma, merkel cell carcinoma, mesotheliomas (adult malignant,Attorney Docket No. UTSD.P4287WO / 1001338856 childhood), metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome (childhood), multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, myelogenous leukemia (chronic), myeloid leukemias (adult acute, childhood acute), multiple myeloma, myeloproliferative disorders (chronic), nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic cancer (islet cell), paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors (childhood), pituitary adenoma, plasma cell neoplasia, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma (childhood), salivary gland cancer, sarcoma (Ewing family of tumors, Kaposi, soft tissue, uterine), Sézary syndrome, skin cancers (nonmelanoma, melanoma), skin carcinoma (Merkel cell), small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer with occult primary (metastatic), stomach cancer, supratentorial primitive neuroectodermal tumor (childhood), T-Cell lymphoma (cutaneous), testicular cancer, throat cancer, thymoma (childhood), thymoma and thymic carcinoma, thyroid cancer, thyroid cancer (childhood), transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor (gestational), unknown primary site (adult, childhood), ureter and renal pelvis transitional cell cancer, urethral cancer, uterine cancer (endometrial), uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma (childhood), vulvar cancer, and Wilms tumor (childhood).

[0073] As used herein, treatment of cancer can comprise increased inhibition of cancer progression and / or metastases, inhibition of an increase in tumor volume, a reduction in tumor volume and / or growth, a reduction in tumor growth rate, an eradication of a tumor and / or cancer cell, or any combination thereof. In some aspects, the treatment can also prolong the survival of a subject, improve the prognosis and / or improve the quality of life of the subject.

[0074] As used herein, a biological sample may be from any biological tissue, fluid, or cell from the subject. The sample can be solid or fluid. The sample can be a heterogeneous cell population. Non-limiting examples of suitable biological samples include sputum, serum, blood, blood cells (e.g., white cells), a biopsy, urine, peritoneal fluid, pleural fluid, or cells derived therefrom. The biopsy can be a fine needle aspirate biopsy, a core needle biopsy, a vacuum assisted biopsy, an open surgical biopsy, a shave biopsy, a punch biopsy, anAttorney Docket No. UTSD.P4287WO / 1001338856 incisional biopsy, a curettage biopsy, or a deep shave biopsy. Biological samples may also include sections of tissues, such as frozen sections or formalin fixed sections taken for histological purposes. A sample can be a tumor tissue, tissue surrounding a tumor, or non- tumor tissue. Methods of collecting a biological sample from a subject are well known in the art. In some aspects, the biological sample can be a peripheral blood sample. In some aspects, the biological sample can be peripheral blood mononuclear cell (PBMC).

[0075] Sample from the subject can be procured one or more times, before, during, and / or after diagnosis. In some aspects, samples can be procured from the subject before, during, and / or after treatment of cancer, wherein the cancer treatment comprises ICI treatment. In some aspects, sample can be procured from the subject prior to the start of ICI treatment. In some aspects, sample can be procured from the subject undergoing ICI treatment, before onset of irAE. In other aspects, sample can be procured after onset of irAE in a subject. In some aspects, sample can be procured before, during and / or after administration of a non-ICI cancer treatment, or ICI treatment combined with steroid treatment, for monitoring the treatment for irAE. Additionally, samples can be procured repeatedly at multiple stages after initial sample procurement, to determine and / or monitor irAE in a subject.

[0076] In some aspects, the sample can be obtained from the subject prior to the initiation of ICI treatment. In some aspects, the sample can be obtained from the subject about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, or more after the initiation of the ICI treatment. In some aspects, the sample can be obtained at about 2-4 weeks after the initiation of the ICI treatment. In some aspects, the sample can be obtained at about 6-8 weeks after the initiation of the ICI treatment.

[0077] In some aspects, control sample can be procured from a healthy subject and / or a subject undergoing ICI treatment but has a low risk of developing irAE or ICI toxicity. In some aspects, the control sample can comprise non-cancer cells. In some aspects, the non-cancer cells can be from the same tissue type as the cancer cells. For example, if the cancer cells are from breast cancer, then the non-cancer cells can be from healthy breast tissue. In some aspects, the control can comprise an average levels of the biomarker profile in a sample from a subject before onset of cancer. In some aspects, control sample can be a sample from the subject prior to diagnosis or treatment. In certain aspects, the biomarker profile can be measured in a person or persons other than the subject with cancer. In some aspects, the control a person or persons with similar characteristics to the subject with cancer. In some aspects, the control can be an average of the combination of disclosed biomarker levels from different healthy sources (e.g., more than one healthy control subject and / or more than oneAttorney Docket No. UTSD.P4287WO / 1001338856 subject has a low risk of developing irAE). In some aspects, the control sample can be pooled sample. In some aspects, the control sample is procured from a subject with low risk of developing irAE.

[0078] As used herein, a subject that has a low risk of developing irAE can be a subject or population that does not develop irAE with ICI treatment. In an aspect, a subject that has a low risk of developing irAE can be a subject or population that does not develop irAE with ICI treatment as determined through retrospective analysis to not develop irAE with ICI treatment. II. Biomarkers

[0079] The present disclosure provides immunological characteristics of ICI-related irAE and insights into the biological profiles of ICI-related irAE. These profiles can be used as biomarkers to provide rationales for potential cancer treatment options. Immune cell profile

[0080] In some aspects, the present disclosure provides a method of predicting the risk of developing and / or diagnosing immune-related adverse events (irAE) associated with immune checkpoint inhibitor (ICI) treatment in a subject. The method comprises providing a sample from the subject, assessing the frequencies of one or more immune cell types in the sample; and predicting the risk for developing / diagnosing irAE in the subject. In some aspects, assessment of immune cell types comprises comparing the frequencies of one or more immune cell types in the sample from the subject to the frequencies the same immune cell type in a control sample. In some aspects, immune cell type profile related to ICI-associated irAE comprises an immune cell type profile wherein the frequencies of one or more immune cell types are elevated in the subject when compared to the frequencies in a control sample. In some aspects, immune cell type profile related to ICI-associated irAE comprises an immune cell type profile wherein the frequencies of one or more immune cell types are decreased in the subject when compared to the frequency in a control sample.

[0081] In some aspects, the immune cell type profile comprises one or more of an immune cell types disclosed in FIG.2C, or combinations thereof. In some aspects, the immune cell type profile comprises of baseline or a pre-treatment immune cell type profile that correlates with future irAE. In some aspects, the immune cell type profile comprises immune cell type profile that correlates with onset of irAE during ICI treatment. In some aspects, the immune cell type profile is assessed after ICI treatment to monitor the subject’s risk for developing irAE or the subject’s recovery from irAE. The disclosed immune cell types can be used as biomarker to assess the risk of a subject developing irAE before, during or after ICI treatment. In some aspects, the subject is planning to undergo or is undergoing ICI treatment as part of a cancer therapy.Attorney Docket No. UTSD.P4287WO / 1001338856

[0082] In some aspects, an immune cell subset or an immune cell cluster comprises an immune cell type. In some aspects, the frequency of the one or more immune cell types are determined as the frequency of an immune cell subset. In such aspects, the frequency of an immune cell subset is determined as percentage of total PBMC cells. In some aspects, the frequency of the one or more immune cell types are determined as the frequency of immune cell cluster. In such aspects, the frequency is determined as the abundance of cell clusters as a percentage of total CD45+ cells.

[0083] In some aspects, one or more immune cell types in the immune cell profile have an elevated frequency at baseline or pre-treatment. In some aspects, the immune cell type having an elevated frequency at baseline or pre-treatment has frequency level of at least about .01%, at least about 0.05%, at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% greater as compared to the frequency in a control sample. In some aspects, the immune cell type having an elevated frequency at baseline or pre-treatment has frequency level of at least about 5% greater as compared to the frequency in a control sample.

[0084] In some aspects, one or more immune cell types in the immune cell profile have a decreased frequency at baseline or pre-treatment. In some aspects, the immune cell type having a decreased frequency at baseline or pre-treatment has frequency level of at least about .01%, at least about 0.05%, at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% lower as compared to the frequency in a control sample. In some aspects, the immune cell type having a decreased frequency at baseline or pre-treatment has frequency level of at least about 5% lower as compared to the frequency in a control sample.

[0085] In some aspects, during prediction and / or diagnosis, the abundance of immune cells in an immune cell subset can be determined from a sample by determining the percentage of immune cells in the sample that fall into the subset. (For example, X% of the immune cells in the sample are subset 1.) The percentage of immune cells in a given subset can then be compared to the percentage of immune cells in that subset in a control sample. In someAttorney Docket No. UTSD.P4287WO / 1001338856 aspects, the abundance (e.g., percentage) of immune cells in the one or more immune cell subset can be present at least about 0.1 percentage points (for example, 1.0% as compared to 1.1%), at least about 0.2 percentage points, at least about 0.3 percentage points, at least about 0.4 percentage points, at least about 0.5 percentage points, at least about 0.6 percentage points, at least about 0.7 percentage points, at least about 0.8 percentage points, at least about 0.9 percentage points, at least about 1 percentage point, at least about 2 percentage points, at least about 3 percentage points, at least about 4 percentage points, at least about 5 percentage points, at least about 6 percentage points, at least about 7 percentage points, at least about 8 percentage points, at least about 9 percentage points, at least about 10 percentage points, at least about 15 percentage points, at least about 20 percentage points, at least about 25 percentage points, at least about 30 percentage points, at least about 35 percentage points, at least about 40 percentage points, at least about 45 percentage points, at least about 50 percentage points, at least about 55 percentage points, at least about 60 percentage points, at least about 65 percentage points, at least about 70 percentage points, at least about 75 percentage points, at least about 80 percentage points, at least about 85 percentage points, at least about 90 percentage points, at least about 95 percentage points, at least about 99 percentage points, or at least about 100 percentage points lower as compared to the abundance of the immune cells in the one or more immune cell subset in control sample.

[0086] In some aspects, during prediction and / or diagnosis, the abundance of immune cells in an immune cell subset can be determined from a sample by determining the percentage of immune cells in the sample that fall into the subset. (For example, X% of the immune cells in the sample are subset 1.) The percentage of immune cells in a given subset can then be compared to the percentage of immune cells in that subset in control sample. In some aspects, the abundance (e.g., percentage) of immune cells in the one or more immune cell subset can be present at least about 0.1 percentage points (for example, 1.0% as compared to 1.1%), at least about 0.2 percentage points, at least about 0.3 percentage points, at least about 0.4 percentage points, at least about 0.5 percentage points, at least about 0.6 percentage points, at least about 0.7 percentage points, at least about 0.8 percentage points, at least about 0.9 percentage points, at least about 1 percentage point, at least about 2 percentage points, at least about 3 percentage points, at least about 4 percentage points, at least about 5 percentage points, at least about 6 percentage points, at least about 7 percentage points, at least about 8 percentage points, at least about 9 percentage points, at least about 10 percentage points, at least about 15 percentage points, at least about 20 percentage points, at least about 25 percentage points, at least about 30 percentage points, at least about 35 percentage points, at least about 40 percentage points, at least about 45Attorney Docket No. UTSD.P4287WO / 1001338856 percentage points, at least about 50 percentage points, at least about 55 percentage points, at least about 60 percentage points, at least about 65 percentage points, at least about 70 percentage points, at least about 75 percentage points, at least about 80 percentage points, at least about 85 percentage points, at least about 90 percentage points, at least about 95 percentage points, at least about 99 percentage points, or at least about 100 percentage points greater as compared to the abundance of the immune cells in the one or more immune cell subset in control sample.

[0087] In some aspects, the immune cell type comprises one or more of natural killer (NK)cell, CD4 proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell),plasmablasts, double-negative T (dNT) cell, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof.

[0088] In some aspects, a subject can be predicted as high risk of developing irAE if the frequency of NK cell is decreased in the sample prior to ICI treatment (baseline) when compared to the frequency in a control sample.

[0089] In some aspects, the subject can be predicted as high risk of developing irAE if the frequencies of one or more of CD4 proliferating cell, proliferating T and NK cell, gamma / deltaT cell ( T cell or gdT cell), plasmablast, dNT cell, CXCR3 T cells, CD57+ T cells, CD57+NKT cells, CD8 terminal effector cell, NKT cells, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof are elevated in the sample from the subject prior to ICI treatment (baseline) when compared to the frequencies in a control sample. In some aspects, the subject can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of CD4 proliferating cells is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of proliferating T and NK cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of immune cell type gdT cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of plasmablasts is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of dNT cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can beAttorney Docket No. UTSD.P4287WO / 1001338856 predicted as having a high risk of developing or diagnosed with having irAE if the frequency of CXCR3 T cells is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of CD57+ T cells is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of CD57+ NKT cells is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of CD8 terminal effector cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of proliferating NKT cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of HLA-DR+ CD4 memory effector T cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of CD4 central memory cells is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as high risk of developing irAE if the frequencies of CD4 proliferating cell, proliferating T and NK cell,gamma / delta T cell ( T cell or gdT cell), plasmablast, dNT cell, CXCR3 T cells, CD57+ Tcells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, and CD4 central memory cell are elevated in the sample from the subject prior to ICI treatment (baseline) when compared to the frequencies in a control sample.

[0090] In some aspects, the subject can be predicted or diagnosed with high risk of developing irAE if the frequencies of one or more of CD4 proliferating cell, proliferating T and NK cell,gamma / delta T cell ( T cell or gdT cell), plasmablast, dNT cell, CXCR3 T cells, CD57+ Tcells, CD57+ NKT cells, CD8 terminal effector cell, NKT cells, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof, are elevated and / or if the frequency of NK cell is decreased when compared to frequencies, in a control sample, at baseline, or before initiation of ICI treatment.

[0091] In some aspects, the subject can be predicted as having a high risk of developing or diagnosed with having irAE if the frequencies of one or more of dNT cell, proliferating T andNK cell, gamma / delta T cell ( T cell), plasmablast, or any combination thereof are elevatedAttorney Docket No. UTSD.P4287WO / 1001338856 in the sample from the subject after initiation of ICI treatment, when compared to the frequencies in a control sample. In some aspects, the subject can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of dNT cell is elevated in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a high risk of developing or diagnosed with irAE if the frequency of proliferating T and NK cell is elevated in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a high risk ofdeveloping or diagnosed with having irAE if the frequency of gamma / delta T cell ( T cell) iselevated in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of plasmablast is elevated in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a high risk of developing or diagnosed with having irAE if the frequencies of dNT cell, proliferating T and NKcell, gamma / delta T cell ( T cell), and plasmablast are elevated in the sample from thesubject after initiation of ICI treatment, when compared to the frequencies in a control sample.

[0092] In some aspects, a subject can be predicted as low risk of developing irAE if the frequency of NK cell is increased in the sample from the subject prior to ICI treatment (baseline) when compared to the frequency in a control sample.

[0093] In some aspects, the subject can be predicted as low risk of developing irAE if the frequencies of one or more of CD4 proliferating cell, proliferating T and NK cell, gamma / deltaT cell ( T cell or gdT cell), plasmablast, dNT cell, CXCR3 T cells, CD57+ T cells, CD57+NKT cells, CD8 terminal effector cell, NKT cells, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof are decreased in the sample from the subject prior to ICI treatment (baseline) when compared to the frequencies in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of CD4 proliferating cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of immune cell type T and NK cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of gdT cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE ifAttorney Docket No. UTSD.P4287WO / 1001338856 the frequency of plasmablast is decreased prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of dNT cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of CXCR3 T cells is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of CD57+ T cells is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of CD57+ NKT cells is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of CD8 terminal effector cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of proliferating NKT cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of HLA-DR+ CD4 memory effector T cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of CD4 central memory cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as low risk of developing irAE if the frequencies of CD4proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell or gdT cell),plasmablast, dNT cell, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, and CD4 central memory cell are decreased in the sample from the subject prior to ICI treatment (baseline) when compared to the frequencies in a control sample.

[0094] In some aspects, the subject can be predicted or diagnosed with low risk of developing irAE if the frequencies of one or more of CD4 proliferating cell, proliferating T and NK cell,gamma / delta T cell ( T cell or gdT cell), plasmablast, dNT cell, CXCR3 T cells, CD57+ Tcells, CD57+ NKT cells, CD8 terminal effector cell, NKT cells, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof, are decreased and / or if theAttorney Docket No. UTSD.P4287WO / 1001338856 frequency of NK cell is increased when compared to frequencies, in a control sample, at baseline, or before initiation of ICI treatment.

[0095] In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of one or more of dNT cell, proliferating T and NKcell, gamma / delta T cell ( T cell), plasmablast, or any combination thereof is decreased inthe sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of dNT cell is decreased in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with irAE if the frequency of proliferating T and NK cells is decreased in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk ofdeveloping or diagnosed with having irAE if the frequency of gamma / delta T cell ( T cell) isdecreased in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of plasmablast is decreased in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of dNT cell, proliferatingT and NK cell, gamma / delta T cell ( T cell), and plasmablast are decreased in the samplefrom the subject after initiation of ICI treatment, when compared to the frequency in a control sample.

[0096] In some aspects, the present disclosure provides a method of monitoring the risk of developing immune-related adverse events (irAE) associated with immune checkpoint inhibitor (ICI) treatment in a subject. The method comprises providing a sample from the subject, assessing the frequencies of one or more immune cell types in the sample from the subject; and predicting the risk for developing / diagnosing irAE in the subject being monitored. In some aspects, assessment of immune cell type comprises comparing the frequencies of one or more immune cell types in the sample from the subject to the frequencies the same immune cell type in a control sample. In some aspects, immune cell type profile related to ICI- associated irAE comprises an immune cell type profile wherein the frequencies of one or more immune cell types are elevated in the subject when compared to the frequencies in a control sample. In some aspects, immune cell type profile related to ICI-associated irAE comprises an immune cell type profile wherein the frequencies of one or more immune cell types areAttorney Docket No. UTSD.P4287WO / 1001338856 decreased in the sample from the subject when compared to the frequencies in a control sample.

[0097] In some aspects, the immune cell type profile used for the method of monitoring comprises one or more of an immune cell types disclosed in FIG.2C, or combinations thereof. In some aspects, the immune cell type profile used for the method of monitoring comprises of baseline or a pre-treatment immune cell type profile that correlates with future irAE. In some aspects, the immune cell type profile used for the method of monitoring comprises immune cell type profile that correlates with onset of irAE during ICI treatment. In some aspects, the immune cell type used for the method of monitoring comprises wherein the immune cell type profile is assessed after ICI treatment to monitor the subject’s risk for developing irAE or the subject’s recovery from irAE. The disclosed immune cell types can be used as biomarker to monitor the risk of a subject developing irAE during or after ICI treatment. In some aspects, the subject is planning to undergo or is undergoing ICI treatment as part of a cancer therapy.

[0098] In some aspects, during monitoring, the immune cell subset having an elevated level which appear at baseline or pre-treatment has an elevated expression level of at least about .01%, at least about 0.05%, at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% greater as compared to sample. In some aspects, the immune cell type having an elevated frequency at baseline or pre-treatment has frequency level of at least about 5% greater as compared to the frequency in a control sample.

[0099] In some aspects, during monitoring the immune cell subset having decreased level which appear at baseline or pre-treatment has a decreased expression level of at least about .01%, at least about 0.05%, at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least 100% lower as compared to as compared to sample. In some aspects, the immune cell type having a decreased frequency at baseline or pre-treatment has frequency level of at least about 5% lower as compared to the frequency in a control sample.Attorney Docket No. UTSD.P4287WO / 1001338856

[0100] In some aspects, during monitoring, the abundance of immune cells in an immune cell subset can be determined from a sample by determining the percentage of immune cells in the sample that fall into the subset. (For example, X% of the immune cells in the sample are subset 1.) The percentage of immune cells in a given subset can then be compared to the percentage of immune cells in that subset in a control sample. In some aspects, the abundance (e.g., percentage) of immune cells in the one or more immune cell subset can be present at least about 0.1 percentage points (for example, 1.0% as compared to 1.1%), at least about 0.2 percentage points, at least about 0.3 percentage points, at least about 0.4 percentage points, at least about 0.5 percentage points, at least about 0.6 percentage points, at least about 0.7 percentage points, at least about 0.8 percentage points, at least about 0.9 percentage points, at least about 1 percentage point, at least about 2 percentage points, at least about 3 percentage points, at least about 4 percentage points, at least about 5 percentage points, at least about 6 percentage points, at least about 7 percentage points, at least about 8 percentage points, at least about 9 percentage points, at least about 10 percentage points, at least about 15 percentage points, at least about 20 percentage points, at least about 25 percentage points, at least about 30 percentage points, at least about 35 percentage points, at least about 40 percentage points, at least about 45 percentage points, at least about 50 percentage points, at least about 55 percentage points, at least about 60 percentage points, at least about 65 percentage points, at least about 70 percentage points, at least about 75 percentage points, at least about 80 percentage points, at least about 85 percentage points, at least about 90 percentage points, at least about 95 percentage points, at least about 99 percentage points, or at least about 100 percentage points lower as compared to the abundance of the immune cells in the one or more immune cell subset in control sample.

[0101] In some aspects, during monitoring, the abundance of immune cells in an immune cell subset can be determined from a sample by determining the percentage of immune cells in the sample that fall into the subset. (For example, X% of the immune cells in the sample are subset 1.) The percentage of immune cells in a given subset can then be compared to the percentage of immune cells in that subset in control sample. In some aspects, the abundance (e.g., percentage) of immune cells in the one or more immune cell subset can be present at least about 0.1 percentage points (for example, 1.0% as compared to 1.1%), at least about 0.2 percentage points, at least about 0.3 percentage points, at least about 0.4 percentage points, at least about 0.5 percentage points, at least about 0.6 percentage points, at least about 0.7 percentage points, at least about 0.8 percentage points, at least about 0.9 percentage points, at least about 1 percentage point, at least about 2 percentage points, at least about 3 percentage points, at least about 4 percentage points, at least about 5Attorney Docket No. UTSD.P4287WO / 1001338856 percentage points, at least about 6 percentage points, at least about 7 percentage points, at least about 8 percentage points, at least about 9 percentage points, at least about 10 percentage points, at least about 15 percentage points, at least about 20 percentage points, at least about 25 percentage points, at least about 30 percentage points, at least about 35 percentage points, at least about 40 percentage points, at least about 45 percentage points, at least about 50 percentage points, at least about 55 percentage points, at least about 60 percentage points, at least about 65 percentage points, at least about 70 percentage points, at least about 75 percentage points, at least about 80 percentage points, at least about 85 percentage points, at least about 90 percentage points, at least about 95 percentage points, at least about 99 percentage points, or at least about 100 percentage points greater as compared to the abundance of the immune cells in the one or more immune cell subset in control sample.

[0102] In some aspects, the immune cell type used for the method of monitoring comprises one or more of natural killer (NK) cell, CD4 proliferating cell, proliferating T and NK cell,gamma / delta T cell ( T cell), plasmablasts, double-negative T (dNT) cell, CD8 terminaleffector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof.

[0103] In some aspects, a subject being monitored can be predicted as high risk of developing irAE if the frequency of NK cell is decreased in the sample from the subject prior to ICI treatment (baseline) when compared to the frequency in a control sample.

[0104] In some aspects, the subject being monitored can be predicted as having a high risk of developing irAE if the frequencies of one or more of CD4 proliferating cell, proliferating Tand NK cell, gamma / delta T cell ( T cell or gdT cell), plasmablast, dNT cell, CXCR3 T cells,CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cells, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof are elevated in the sample from the subject prior to ICI treatment (baseline) when compared to the frequencies in a control sample. In some aspects, the subject being monitored can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of CD4 proliferating cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of proliferating T and NK cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of gdT cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subjectAttorney Docket No. UTSD.P4287WO / 1001338856 being monitored can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of plasmablast is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of dNT cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of CXCR3 T cells is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of CD57+ T cells is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of CD57+ NKT cells is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of CD8 terminal effector cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of proliferating NKT cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of HLA-DR+ CD4 memory effector T cells is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of CD4 central memory cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as high risk of developing irAE if the frequencies of CD4proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell or gdT cell),plasmablast, dNT cell, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, and CD4 central memory cell are elevated in the sample from the subject prior to ICI treatment (baseline) when compared to the frequencies in a control sample.

[0105] In some aspects, the subject being monitored can have a high risk of developing irAE if the frequencies of one or more of CD4 proliferating cell, proliferating T and NK cell,Attorney Docket No. UTSD.P4287WO / 1001338856gamma / delta T cell ( T cell or gdT cell), plasmablast, dNT cell, CXCR3 T cells, CD57+ Tcells, CD57+ NKT cells, CD8 terminal effector cell, NKT cells, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof, are elevated and / or if the frequency of NK cell is decreased when compared to frequencies, in a control sample, at baseline, or before initiation of ICI treatment.

[0106] In some aspects, the subject being monitored can be predicted as having a high risk of developing or diagnosed with having irAE if the frequencies of one or more of immune celltype dNT cell, proliferating T and NK cell, gamma / delta T cell ( T cell), plasmablast, or anycombination thereof are elevated in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of dNT cell is elevated in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a high risk of developing or diagnosed with irAE if the frequency of proliferating T and NK cells is elevated in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a high risk of developing ordiagnosed with having irAE if the frequency of gamma / delta T cell ( T cell) is elevated in thesample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a high risk of developing or diagnosed with having irAE if the frequency of plasmablast is elevated in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a high risk of developing or diagnosed with having irAE if the frequencies of dNTcell, proliferating T and NK cell, gamma / delta T cell ( T cell), and plasmablast are elevatedin the sample from the subject after initiation of ICI treatment, when compared to the frequencies in a control sample.

[0107] In some aspects, the subject being monitored can be predicted as low risk of developing irAE if the frequency of NK cell is increased in the sample from the subject prior to ICI treatment (baseline) when compared to the frequency in a control sample.

[0108] In some aspects, the subject being monitored can be predicted as low risk of developing irAE if the frequencies of one or more of CD4 proliferating cell, proliferating T andNK cell, gamma / delta T cell ( T cell or gdT cell), plasmablast, dNT cell, CXCR3 T cells,CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof is decreased in the sample from the subject prior to ICI treatment (baseline) when compared to the frequency inAttorney Docket No. UTSD.P4287WO / 1001338856 a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of CD4 proliferating cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of proliferating T and NK cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of gdT cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of plasmablast is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of dNT cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of CXCR3 T cells is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of CD57+ T cells is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of CD57+ NKT cells is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of CD8 terminal effector cells is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of proliferating NKT cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of HLA-DR+ CD4 memory effector T cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of CD4 central memory cell is decreased in the sample from the subject prior to ICI treatment,Attorney Docket No. UTSD.P4287WO / 1001338856 when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as low risk of developing irAE if the frequencies of CD4proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell or gdT cell),plasmablast, dNT cell, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, and CD4 central memory cell are decreased in the sample from the subject prior to ICI treatment (baseline) when compared to the frequency in a control sample.

[0109] In some aspects, a subject being monitored can be predicted or diagnosed with low risk of developing irAE if the frequencies of one or more of CD4 proliferating cell, proliferatingT and NK cell, gamma / delta T cell ( T cell or gdT cell), plasmablast, dNT cell, CXCR3 Tcells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cells, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof, are decreased and / or if the frequency of NK cell is increased when compared to frequencies, in a control sample, at baseline, or before initiation of ICI treatment.

[0110] In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequencies of one or more of dNT cell,proliferating T and NK cell, gamma / delta T cell ( T cell), plasmablast, or any combinationthereof is decreased after initiation of ICI treatment, when compared to the frequencies in a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of dNT cell is decreased in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with irAE if the frequency of proliferating T and NK cells is decreased in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency ofgamma / delta T cell ( T cell) is decreased in the sample from the subject after initiation ofICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of plasmablast is decreased in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject being monitored can be predicted as having a low risk of developing or diagnosed with having irAE if the frequencies of dNT cell, proliferating T and NK cell,gamma / delta T cell ( T cell), and plasmablast are decreased in the sample from the subjectafter initiation of ICI treatment, when compared to the frequencies in a control sample.Attorney Docket No. UTSD.P4287WO / 1001338856

[0111] Any known method in the art can be used to determine the frequency of an immune cell subset. By way of non-limiting examples, frequency of an immune cell subset can be measured mass cytometry (cytometry by time-of-flight CyTOF), scRNA-sequencing, immune profiling assay, flow cytometry, fluorescence-activated cell sorting (FACS), and / or immunomagnetic separation. Samples tested can comprise whole blood, serum, plasma, cerebrospinal fluid, pleural fluid, pericardial fluid, peritoneal fluid, bone marrow, or tissue, urine, cerebrospinal fluid (CSF), or other body fluid. Samples can be obtained from the subject before, during, and / or after ICI treatment, and determination of the frequency of an immune cell subset can be performed, to assess the risk of irAE. In some aspects, samples can be obtained before the initiation of ICI treatment, and / or about 6-8 weeks after the initiation of ICI treatment. In some aspects, the immune cell subset is determined in peripheral blood mononuclear cells (PBMCs). The risk assessment comprises predicting, diagnosing, or monitoring ICI associated irAE.

[0112] In further aspects, the disclosed method comprises calculating the sensitivity and specificity for one or more of the disclosed immune cells to diagnose, predict or monitor ICI treatment associated irAE. Sensitivity and specificity can be determined using any known methods in the art, non-limiting examples include Fisher Discriminant Analysis (FDA) and support vector machines (SVM). In some aspects, the method can further comprise calculating the area under the curve (AUC) of the receiver operating characteristic (ROC) curve for the one or more immune cells using a calculated sensitivity and specificity.

[0113] In some aspects, the AUC determined for one or more of the disclosed immune cells can be in about 0.6- to about 0.9. In some aspects, AUC for one or more of the disclosed immune cells can be about 0.60, about 0.65, about 0.70, about 0.75, about 0.80, about 0.85, or about 0.90. In some aspects, AUC for the proliferative T cells can be about 0.81. In some aspects, AUC for the NK cell cluster is about 0.77. Cytokine profile

[0114] In some aspects, the present disclosure further provides a profile comprising one or more cytokines associated with ICI-related irAE. In some aspects, the profile comprises one or more cytokines with an elevated level of expression in a subject with irAE prior to the initiation of ICI treatment (baseline) when compared to a level of expression in a control sample. In some aspects, the profile comprises one or more cytokines with an elevated level of expression in a subject with irAE after initiation of ICI treatment when compared to a level of expression in a control sample.

[0115] In some aspects, the profile comprises one or more cytokines with an elevated level of expression in a subject with irAE at pre-treatment or baseline when compared to level ofAttorney Docket No. UTSD.P4287WO / 1001338856 expression in a control sample. In some aspects, the profile comprises one or more cytokines with an elevated level of expression in a subject with irAE after initiation of ICI treatment when compared to level of expression in a control sample. The one or more cytokines disclosed herein can be used as biomarker to assess the risk of a subject developing irAE before, during or after ICI treatment. In some aspects, the subject is planning to undergo or is undergoing ICI treatment as part of a cancer therapy.

[0116] In some aspects, the present disclosure provides a method comprising predicting or diagnosing the subject as having a high risk of developing irAE if the level of expression of one or more cytokines, are elevated when compared to the level of expression in a control sample. The method comprises providing a sample from the subject pretreatment or at baseline, and / or after initiation of ICI treatment, determining the level of expression of one or more cytokines in the sample, and predicting risk for developing or diagnosing as having irAE in the subject.

[0117] In some aspects, the expression of one or more cytokines can be determined as expression level of the gene encoding the cytokine. In some aspects, the expression of one or more cytokines can be determined as the concentration of the corresponding protein of the cytokine.

[0118] In some aspects, during predicting or diagnosing the expression of the cytokines can be determined in monocytes isolated from a sample obtained from the subject. In some aspects, the monocytes can be peripheral blood monocytes. In some aspects, the monocytes can be one or more of CD14+ classical monocytes, CD16+ non-classical monocytes, or any combination thereof. In some aspects, the monocytes can be CD14+ classical monocytes. In some aspects, the monocytes can be CD16+ non-classical monocytes. In some aspects, the monocytes can be CD14+ classical monocytes and CD16+ non-classical monocytes. In some aspects, the expression of cytokines can be determined from a serum sample obtained from the subject.

[0119] In some aspects, during predicting or diagnosing the risk of irAE, one or more of the disclosed cytokines have an elevated expression of at least about .01%, at least about 0.05%, at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% greater as compared to the expression in a control sample. In some aspects, the disclosed cytokines canAttorney Docket No. UTSD.P4287WO / 1001338856 have an elevated expression of at least about 5%, at least 25% lower, at least 50% lower, or 100% lower as compared to the expression in a control sample.

[0120] In some aspects, during predicting or diagnosing the risk of irAE predicting or diagnosing the risk of irAE one or more of the disclosed cytokines have an elevated expression having a log fold change value from about 0.1 to about 5. For example, a log fold change value can be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.

[0121] In some aspects, during predicting or diagnosing the risk of irAE, one or more of the disclosed cytokines can be expressed at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% lower as compared to the expression in a control sample. In some aspects, one or more of the disclosed cytokines can be at least about 5%, at least 25% lower, at least 50% lower, or 100% lower, as compared to the expression in a control sample.

[0122] In some aspects, during predicting or diagnosing the risk of irAE predicting or diagnosing the risk of irAE one or more of the disclosed cytokines have a decreased expression level having a log fold change value from about -0.1 to about -5. For example, a log fold change value can be about -0.1, -0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -0.9, -1, -1.1, - 1.2, -1.3, -1.4, -1.5, -1.6, -1.7, -1.8, -1.9, -2, -2.1, -2.2, -2.3, -2.4, -2.5, -2.6, -2.7, -2.8, -2.9, -3, -3.1, -3.2, -3.3, -3.4, -3.5, -3.6, -3.7, -3.8, -3.9, -4, -4.1, -4.2, -4.3, -4.4, -4.5, -4.6, -4.7, -4.8, - 4.9, or -5.

[0123] In some aspects, the expression of one or more of the disclosed cytokines can be measured by determining the concentration of the cytokine. In such aspects, the concentration of the cytokines can be determined using a standard curve. In some aspects, the concentration of the cytokines can be determined using a fit of a standard curve such as for e.g., mean fluorescence intensity versus pg / ml. In some aspects, during prediction or diagnosis, one or more of the disclosed cytokines have a concentration of about 10 pg / ml, about 20 pg / ml, about 30 pg / ml, about 40 pg / ml, about 50 pg / ml, about 60 pg / ml, about 70 pg / ml, about 80 pg / ml,Attorney Docket No. UTSD.P4287WO / 1001338856 about 90 pg / ml, about 100 pg / ml, about 120 pg / ml, about 140 pg / ml, about 150 pg / ml, about 160 pg / ml, about 180 pg / ml, about 200 pg / ml, about 300 pg / ml, about 400 pg / ml, about 500 pg / ml, about 600 pg / ml, about 700 pg / ml, or about 800 pg / ml.

[0124] In some aspects, the disclosed cytokines are one or more of C-X-C motif chemokine ligand 13 (CXCL13), C-X-C motif chemokine ligand 9 (CXCL9), C-X-C motif chemokine ligand10 (CXCL10), Interleukin-1 beta (IL1 ), Tumor necrosis factor (TNF ), Chemokine ligand 3(CCL3), Chemokine ligand 8 (CXCL8), Fos Proto-Oncogene (FOS), Chemokine ligands 4 (CCL4), TNF alpha induced protein 3 (TNFAIP3), TNF alpha induced protein 6 (TNFAIP6), Testis Expressed 14 (TEX14), C-X-C motif chemokine ligand 2 (CXCL2), Cluster of Differentiation 83 (CD83), or any combination thereof.

[0125] In some aspects, the method of predicting or diagnosing a subject as having a high risk of developing irAE associated with ICI treatment comprises assessing the expression ofCXCL13, CXCL9, CXCL10, IL1 , TNF , CCL3, CXCL8, FOS, CCL4, TNFAIP3, TNFAIP6,TEX14, CXCL2, CD83, or any combination thereof.

[0126] In some aspects, the subject can be predicted or diagnosed as having a high risk ofdeveloping irAE if the expression of one or more of IL1 , TNF , CCL3, CXCL8, or anycombination thereof, are elevated in the sample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subjectis predicted or diagnosed as having a high risk of developing irAE if the expression of IL1 iselevated in the sample provided by the subject prior to ICI treatment (baseline) when compared to expression in a control sample. In some aspects, the subject is predicted ordiagnosed as having a high risk of developing irAE if the expression of TNF is elevated inthe sample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject is predicted or diagnosed as having a high risk of developing irAE if the expression of CCL3 is elevated in the sample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject is predicted or diagnosed as having a high risk of developing irAE if the expression of CXCL8 is elevated in the sample provided by the subject prior to ICI treatment (baseline) when compared to expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a high risk of developing irAE ifthe expression of IL1 , TNF , CCL3, and CXCL8 are elevated in the sample provided by thesubject prior to ICI treatment (baseline) when compared to the expression in a control sample.

[0127] In some aspects, the subject can be predicted or diagnosed as having a high risk ofdeveloping irAE if the expression of one or more of IL1 , CXCL8, CXCL9, CXCL10, FOS,CCL4, CCL3, TNFAIP3, TNFAIP6, TEX14, CXCL2, CD83, or any combination thereof areAttorney Docket No. UTSD.P4287WO / 1001338856 elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted ordiagnosed as having a high risk of developing irAE if the expression of IL1 is elevated in thesample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a high risk of developing irAE if the expression of CXCL8 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a high risk of developing irAE if the expression of CXCL9 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a high risk of developing irAE if the expression of CXCL10 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a high risk of developing irAE if the expression of FOS is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a high risk of developing irAE if the expression of CCL4 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a high risk of developing irAE if the expression of CCL3 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a high risk of developing irAE if the expression of TNFAIP3 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a high risk of developing irAE if the expression of TNFAIP6 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the level of gene expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a high risk of developing irAE if the expression of TEX14 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a high risk of developing irAE if the expression of CXCL2 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a high risk of developing irAE if the expression of CD83 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a high risk of developing irAE if the expression ofAttorney Docket No. UTSD.P4287WO / 1001338856one or more of IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4, CCL3, TNFAIP3, TNFAIP6,TEX14, CXCL2, and CD83 are elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample.

[0128] In some aspects, the subject can be predicted or diagnosed as having a low risk ofdeveloping irAE if the expression of one or more of IL1 , TNF , CCL3, CXCL8, or anycombination thereof, is decreased in the sample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subjectis predicted or diagnosed as having a low risk of developing irAE if the expression of IL1 isdecreased in the sample provided by the subject prior to ICI treatment (baseline) when compared to expression in a control sample. In some aspects, the subject is predicted ordiagnosed as having a low risk of developing irAE if the expression of TNF is decreased inthe sample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject is predicted or diagnosed as having a low risk of developing irAE if the expression of CCL3 is decreased in the sample provided by the subject prior to ICI treatment (baseline) when compared to expression in a control sample. In some aspects, the subject is predicted or diagnosed as having a low risk of developing irAE if the expression of CXCL8 is decreased in the sample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a low risk of developing irAE ifthe expression of IL1 , TNF , CCL3, and CXCL8 are decreased in the sample provided bythe subject prior to ICI treatment (baseline) when compared to the expression in a control sample.

[0129] In some aspects, the subject can be predicted or diagnosed as having a low risk ofdeveloping irAE if the expression of one or more of IL1 , CXCL8, CXCL9, CXCL10, FOS,CCL4, CCL3, TNFAIP3, TNFAIP6, TEX14, CXCL2, CD83, or any combination thereof are decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predictedor diagnosed as having a low risk of developing irAE if the expression of IL1 is decreased inthe sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a low risk of developing irAE if the expression of CXCL8 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a low risk of developing irAE if the expression of CXCL9 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a low risk of developingAttorney Docket No. UTSD.P4287WO / 1001338856 irAE if the expression of CXCL10 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a low risk of developing irAE if the expression of FOS is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a low risk of developing irAE if the expression of CCL4 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a low risk of developing irAE if the expression of CCL3 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a low risk of developing irAE if the expression of TNFAIP3 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a low risk of developing irAE if the expression of TNFAIP6 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a low risk of developing irAE if the expression of TEX14 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a low risk of developing irAE if the expression of CXCL2 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a low risk of developing irAE if the expression of CD83 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be predicted or diagnosed as having a low risk of developingirAE if the expression of one or more of IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4, CCL3,TNFAIP3, TNFAIP6, TEX14, CXCL2, and CD83 are decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample.

[0130] In some aspects, provided herein is a method of monitoring the risk of a subject for developing irAE. In some aspects, a subject being monitored has a high risk of developing irAE if the level of expression of one or more cytokines are elevated when compared to the level of expression in a control sample. In some aspects, the method comprises providing a sample from the subject after initiation of ICI treatment, determining the level of expression of one or more cytokines in the sample, and predicting risk for developing or diagnosing as having irAE in the subject, or the subject’s recovery from irAE.Attorney Docket No. UTSD.P4287WO / 1001338856

[0131] In some aspects, the expression of one or more cytokines can be determined as expression level of the gene encoding the cytokines. In some aspects, the expression of one or more cytokines can be determined as the concentration of the corresponding protein of the cytokine.

[0132] In some aspects, during monitoring the expression of the cytokines can be determined in monocytes isolated from a sample from the subject. In some aspects, the monocytes can be peripheral blood monocytes. In some aspects, the monocytes can be one or more of CD14+ classical monocytes, CD16+ non-classical monocytes, or any combination thereof. In some aspects, the monocytes can be CD14+ classical monocytes. In some aspects, the monocytes can be CD16+ non-classical monocytes. In some aspects, the monocytes can be CD14+ classical monocytes and CD16+ non-classical monocytes. In some aspects, the expression of cytokines can be determined from a serum sample obtained from the subject.

[0133] In some aspects, during monitoring, one or more of the disclosed cytokines have an elevated expression of at least about .01%, at least about 0.05%, at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% greater as compared to control sample. In some aspects, the disclosed cytokines can have an elevated expression of at least about 5%, at least 25% lower, at least 50% lower, or 100% lower as compared to the expression in a control sample.

[0134] In some aspects, during monitoring, one or more of the disclosed cytokines have an elevated expression having a log fold change value from about 0.1 to about 5. For example, a log fold change value can be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.

[0135] In some aspects, during monitoring, one or more of the disclosed cytokines can be expressed at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at leastAttorney Docket No. UTSD.P4287WO / 1001338856 about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% lower as compared to control sample. In some aspects, one or more of the disclosed cytokines can be at least about 5%, at least 25% lower, at least 50% lower, or 100% lower, as compared to the expression in a control sample.

[0136] In some aspects, during monitoring, one or more of the disclosed cytokine has a decreased expression level having a log fold change value from about -0.1 to about -5. For example, a log fold change value can be about -0.1, -0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -0.9, -1, -1.1, -1.2, -1.3, -1.4, -1.5, -1.6, -1.7, -1.8, -1.9, -2, -2.1, -2.2, -2.3, -2.4, -2.5, -2.6, -2.7, -2.8, -2.9, -3, -3.1, -3.2, -3.3, -3.4, -3.5, -3.6, -3.7, -3.8, -3.9, -4, -4.1, -4.2, -4.3, -4.4, -4.5, -4.6, -4.7, -4.8, -4.9, or -5

[0137] In some aspects, the expression of one or more of the disclosed cytokines can be measured by determining the concentration of the cytokines. In such aspects, the concentration of the cytokine can be determined using a standard curve. In some aspects, the concentration of the cytokines can be determined using a fit of a standard curve for mean fluorescence intensity versus pg / ml. In some aspects, during monitoring, one or more of the disclosed cytokines have a concentration of about 10 pg / ml, about 20 pg / ml, about 30 pg / ml, about 40 pg / ml, about 50 pg / ml, about 60 pg / ml, about 70 pg / ml, about 80 pg / ml, about 90 pg / ml, about 100 pg / ml, about 120 pg / ml, about 140 pg / ml, about 150 pg / ml, about 160 pg / ml, about 180 pg / ml, about 200 pg / ml, about 300 pg / ml, about 400 pg / ml, about 500 pg / ml, about 600 pg / ml, about 700 pg / ml, or about 800 pg / ml.

[0138] In some aspects, the method of monitoring the risk of developing irAE associated with ICI treatment comprises assessing one or more cytokines, in a sample provided by the subject.In some aspects, the disclosed cytokines is one or more of CXCL13, CXCL9, CXCL10, IL1 ,TNF , CCL3, CXCL8, FOS, CCL4, TNFAIP3, TNFAIP6, TEX14, CXCL2, CD83, or anycombination thereof.

[0139] In some aspects, the method of monitoring the risk of developing irAE associated withICI treatment comprises assessing the expression of CXCL13, CXCL9, CXCL10, IL1 , TNF ,CCL3, CXCL8, FOS, CCL4, TNFAIP3, TNFAIP6, TEX14, CXCL2, CD83, or any combination thereof.

[0140] In some aspects, the subject being monitored can have a high risk of developing irAEif the expression of one or more of IL1 , TNF , CCL3, CXCL8, or any combination thereof,are elevated in the sample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject being monitoredcan have a high risk of developing irAE if the expression of IL1 is elevated in the sampleAttorney Docket No. UTSD.P4287WO / 1001338856 provided by the subject prior to ICI treatment (baseline) when compared to expression in a control sample. In some aspects, the subject being monitored can have a high risk ofdeveloping irAE if the expression of TNF is elevated in the sample provided by the subjectprior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject being monitored can have a high risk of developing irAE if the expression of CCL3 is elevated in the sample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject being monitored can have a high risk of developing irAE if the expression of CXCL8 is elevated in the sample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject being monitored can have a highrisk of developing irAE if the expression of IL1 , TNF , CCL3, and CXCL8 are elevated in thesample provided by the subject prior to ICI treatment (baseline) compared to the expression in a control sample.

[0141] In some aspects, the subject being monitored can have a high risk of developing irAEif the expression of one or more of IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4, CCL3,TNFAIP3, TNFAIP6, TEX14, CXCL2, CD83, or any combination thereof are elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a highrisk of developing irAE if the expression of IL1 is elevated in the sample provided by thesubject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a high risk of developing irAE if the expression of CXCL8 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a high risk of developing irAE if the expression of CXCL9 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a high risk of developing irAE if the expression of CXCL10 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a high risk of developing irAE if the expression of FOS is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a high risk of developing irAE if the expression of CCL4 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a high risk of developing irAE if the expression of CCL3 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample.Attorney Docket No. UTSD.P4287WO / 1001338856 In some aspects, the subject being monitored can have a high risk of developing irAE if the expression of TNFAIP3 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a high risk of developing irAE if the expression of TNFAIP6 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the level of expression in a control sample. In some aspects, the subject being monitored can have a high risk of developing irAE if the expression of TEX14 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a high risk of developing irAE if the expression of CXCL2 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a high risk of developing irAE if the expression of CD83 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitoredcan have a high risk of developing irAE if the expression of one or more of IL1 , CXCL8,CXCL9, CXCL10, FOS, CCL4, CCL3, TNFAIP3, TNFAIP6, TEX14, CXCL2, and CD83 are elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample.

[0142] In some aspects, the subject being monitored can have a low risk of developing irAEif the expression of one or more of IL1 , TNF , CCL3, CXCL8, or any combination thereof, isdecreased in the sample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject being monitoredcan have a low risk of developing irAE if the expression of IL1 is decreased in the sampleprovided by the subject prior to ICI treatment (baseline) when compared to expression in a control sample. In some aspects, the subject being monitored can have a low risk ofdeveloping irAE if the expression of TNF is decreased in the sample provided by the subjectprior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject being monitored can have a low risk of developing irAE if the expression of CCL3 is decreased in the sample provided by the subject prior to ICI treatment (baseline) when compared to expression in a control sample. In some aspects, the subject being monitored can have a low risk of developing irAE if the expression of CXCL8 is decreased in the sample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject being monitored can have a lowrisk of developing irAE if the expression of IL1 , TNF , CCL3, and CXCL8 are decreased inthe sample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample.Attorney Docket No. UTSD.P4287WO / 1001338856

[0143] In some aspects, the subject being monitored can have a low risk of developing irAEif the expression of one or more of IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4, CCL3,TNFAIP3, TNFAIP6, TEX14, CXCL2, CD83, or any combination thereof are decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a lowrisk of developing irAE if the expression of IL1 is decreased in the sample provided by thesubject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a low risk of developing irAE if the expression of CXCL8 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a low risk of developing irAE if the expression of CXCL9 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a low risk of developing irAE if the expression of CXCL10 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a low risk of developing irAE if the expression of FOS is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a low risk of developing irAE if the expression of CCL4 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a low risk of developing irAE if the expression of CCL3 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a low risk of developing irAE if the expression of TNFAIP3 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a low risk of developing irAE if the expression of TNFAIP6 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a low risk of developing irAE if the expression of TEX14 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a low risk of developing irAE if the expression of CXCL2 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject being monitored can have a low risk of developing irAE if the expression of CD83 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In someAttorney Docket No. UTSD.P4287WO / 1001338856 aspects, the subject being monitored can have a low risk of developing irAE if the expressionof one or more of IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4, CCL3, TNFAIP3, TNFAIP6,TEX14, CXCL2, and CD83 are decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample.

[0144] Detection, quantification and / or monitoring of a cytokine can be conducted using well known methods in the art including RNA sequencing (for e.g., single cell RNA sequencing)enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot. In some aspects, assessment of a cytokine level can be performed using readily available commercial panels (for e.g., Bio-Plex Pro Human Chemokine 40-plex Panel, Bio-Rad Laboratories, Hercules, California). Concentrations of a cytokine can be determined on the basis of the fit of a provided standard curve for mean fluorescence intensity. Samples to be tested may comprise whole blood, serum, plasma, urine, CSF or other suitable body fluid. Samples can be obtained from the subject before, during and / or after ICI treatment, and quantification of a cytokine concentration can be performed, to assess the risk of irAE. In some aspects, the samples are obtained from the subject before the initiation of ICI treatment. The risk assessment comprises predicting, diagnosing, or monitoring ICI associated irAE. Antinuclear antibody profile

[0145] In some aspects, the present disclosure provides an antinuclear antibody profile associated with ICI-related irAE. In some aspects, the antinuclear antibody profile comprises an elevated level of antinuclear antibody in sample from a subject with irAE as compared to a control sample.

[0146] In some aspects, the antinuclear antibody profile comprises a baseline or a pre- treatment antinuclear antibody that correlates with future toxicity. In some aspects, the profile comprises an antinuclear antibody with an elevated level in a subject with irAE after initiation of ICI treatment when compared to level in a control sample. In some aspects, the antinuclear antibody can be used as biomarkers to assess the risk of a subject developing irAE before, during or after ICI treatment.

[0147] In some aspects, the method of predicting or diagnosing a subject as having a high risk of developing irAE associated with ICI treatment comprises assessing the level of antinuclear antibody. In some aspects, the subject is predicted or diagnosed as having a high risk of developing irAE if the level of antinuclear antibody, is elevated in the sample from a subject when compared to the level in a control sample. In some aspects, the subject is predicted or diagnosed as having a high risk of developing irAE if the level of antinuclear antibody, is elevated in the sample from a subject prior to ICI treatment (baseline) whenAttorney Docket No. UTSD.P4287WO / 1001338856 compared to the level in a control sample. In some aspects, the subject is predicted or diagnosed as having a high risk of developing irAE if the level of antinuclear antibody, is elevated in the sample from a subject after initiation of ICI treatment, when compared to the level in a control sample.

[0148] In some aspects, during prediction or diagnosis, an antinuclear antibody can have an elevated level of at least about .01%, at least about 0.05%, at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% greater when compared to the level in a control sample.

[0149] In some aspects, during prediction or diagnosis, an antinuclear antibody can an average concentration level of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100, when compared to average concentration in a control sample.

[0150] In some aspects, the disclosure provides an antinuclear antibody profile for monitoring the risk of developing irAE associated with ICI treatment in a subject. In some aspects, the antinuclear antibody profile comprises an antinuclear antibody with an elevated level in a sample from the subject with irAE as compared to the level in a control sample.

[0151] In some aspects, the method of monitoring a subject for a risk of developing irAE associated with ICI treatment comprises assessing the level of antinuclear antibody. In some aspects, during monitoring a subject can have a high risk of developing irAE if the level of antinuclear antibody, is elevated in the sample from a subject when compared to the level in a control sample. In some aspects, during monitoring a subject can have a high risk of developing irAE if the level of antinuclear antibody, is elevated in the sample from a subject prior to ICI treatment (baseline) when compared to the level in a control sample. In some aspects, during monitoring a subject can have a high risk of developing irAE if the level ofAttorney Docket No. UTSD.P4287WO / 1001338856 antinuclear antibody, is elevated in the sample from a subject after initiation of ICI treatment, when compared to the level in a control sample.

[0152] In some aspects, during monitoring, an antinuclear antibody can have an elevated level of at least about .01%, at least about 0.05%, at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% greater when compared to the level in a control sample.

[0153] In some aspects, during monitoring, an antinuclear antibody can an average concentration level of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100, when compared to average concentration in a control sample.

[0154] Detection, quantification and / or monitoring of antinuclear antibody can be conducted using well known methods in the art including assays using immunofluorescence, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot. In some aspects, assessing of antinuclear antibody levels is performed using readily available assays (for e.g., QUANTA lite ANA assay). Concentrations of antinuclear antibodies can be determined on the basis of the fit in a provided standard curve. Samples to be tested may comprise whole blood, serum, plasma, urine, CSF or other suitable body fluid. In some aspects, the sample is a serum sample. Samples can be obtained from the subject before, during and / or after ICI treatment, and quantification of antinuclear antibody level can be performed, to assess the risk of irAE. In some aspects, the samples are obtained from the subject before the initiation of an ICI treatment. In some aspects, the samples are obtained after the initiation of an ICI treatment. The risk assessment comprises predicting, diagnosing, or monitoring ICI associated irAE. Combination profilesAttorney Docket No. UTSD.P4287WO / 1001338856

[0155] In some aspects, the disclosure further provides a profile for diagnosing and / or predicting the risk of ICI associated irAE, which comprises one or more of an immune cell profile, a cytokine profile, an antinuclear antibody profile, or any combination thereof. The profile comprises one or more of immune cell types, cytokines, and / or autoantibodies disclosed herein, or combinations thereof. In some aspects, the profile comprises a baseline or a pre-treatment one or more of immune cell types, cytokines, autoantibodies, any combinations thereof, that correlates with future toxicity. In some aspects, one or more of immune cell types, cytokines, and / or autoantibodies, or any combination thereof are used as biomarkers to assess the risk of a subject developing irAE before, during, or after ICI treatment.

[0156] In some aspects, the method for diagnosis and / or prediction comprises providing a sample from the subject, assessing one or more of immune cell types, cytokines, and / or autoantibodies, or any combination thereof, in the sample, and predicting risk for developing / diagnosing irAE in the subject. In some aspects, a subject can be predicted as having a high risk for developing or diagnosed as having irAE associated with ICI treatment, if the profile comprises one or more of immune cell types, cytokines and / or autoantibodies, or any combination thereof, at an elevated frequencies, expression, or levels in the sample from the subject than frequencies, expression or levels in a control sample. In some aspects, a subject can be predicted as having a low risk for developing or diagnosed as having irAE associated with ICI treatment, if the profile comprises one or more of immune cell types, cytokines and / or autoantibodies or any combination thereof, at a lower frequencies, expression, or levels in the sample from the subject, when compared to the frequencies, expression, or levels in a control sample.

[0157] In some aspects, the subject predicted or diagnosed with high risk of developing irAE is associated with frequencies, expression, or levels of one or more of NK cell, CD4proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell), plasmablast,double-negative T (dNT) cell, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memoryeffector T cell, CD4 central memory cell, CXCL13, CXCL9, CXCL10, IL1 , TNF , CCL3,CXCL8, FOS, CCL4, TNFAIP3, TNFAIP6, TEX14, CXCL2, CD83, antinuclear antibody, or any combination thereof.

[0158] In some aspects, the subject can be predicted or diagnosed with high risk of developing irAE if the frequencies, expression, or levels of one or more of CD4 proliferating cell,proliferating T and NK cell, gamma / delta T cell ( T cell or gdT cell), plasmablast, dNT cell,CD8 terminal effector cell, NKT cells, HLA-DR+ CD4 memory effector T cell, CD4 centralmemory cell, IL1 , TNF , CCL3, CXCL8, antinuclear antibody, or any combination thereof,Attorney Docket No. UTSD.P4287WO / 1001338856 are elevated and / or if the frequency of NK cell is decreased when compared to frequencies, expression, or levels in a control sample, at baseline, or before initiation of ICI treatment.

[0159] In some aspects, the subject can be predicted or diagnosed with high risk of developing irAE if the frequencies, expression, or levels of one or more of dNT cell, proliferating T and NKcell, gamma / delta T cell ( T cell), plasmablast, IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4,CCL3, TNFAIP3, TNFAIP6, TEX14, CXCL2, CD83, antinuclear antibody, or any combination thereof, are elevated when compared to frequencies, expression, or levels in a control sample, after initiation of ICI treatment.

[0160] In some aspects, the subject predicted or diagnosed with low risk of developing irAE is associated with frequencies, expression, or levels of one or more of NK cell, CD4proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell), plasmablast,double-negative T (dNT) cell, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memoryeffector T cell, CD4 central memory cell, CXCL13, CXCL9, CXCL10, IL1 , TNF , CCL3,CXCL8, FOS, CCL4, TNFAIP3, TNFAIP6, TEX14, CXCL2, CD83, antinuclear antibody, or any combination thereof.

[0161] In some aspects, the subject can be predicted or diagnosed with low risk of developing irAE if the frequencies, expression, or levels of one or more of CD4 proliferating cell,proliferating T and NK cell, gamma / delta T cell ( T cell or gdT cell), plasmablast, dNT cell,CD8 terminal effector cell, NKT cells, HLA-DR+ CD4 memory effector T cell, CD4 centralmemory cell, IL1 , TNF , CCL3, CXCL8, antinuclear antibody, or any combination thereof,are lower and / or if the frequency of NK cell is elevated when compared to frequencies, expression, or levels in a control sample, at baseline, or before initiation of ICI treatment.

[0162] In some aspects, the subject can be predicted or diagnosed with low risk of developing irAE if the frequencies, expression, or levels of one or more of dNT cell, proliferating T and NKcell, gamma / delta T cell ( T cell), plasmablast, IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4,CCL3, TNFAIP3, TNFAIP6, TEX14, CXCL2, CD83, antinuclear antibody, or any combination thereof, are decreased when compared to frequencies, expression, or levels in a control sample, after initiation of ICI treatment.

[0163] In some aspects, the disclosure further provides a profile for monitoring the risk of ICI associated irAE, which comprises one or more of an immune cell profile, a cytokine, profile, an antinuclear antibody profile, or any combination thereof. The profile comprises one or more of immune cell types, cytokines, and / or autoantibodies disclosed herein, or combinations thereof. In some aspects, the profile comprises a baseline or a pre-treatment one or more of immune cell types, cytokines, autoantibodies, any combinations thereof, that correlates with future toxicity. In some aspects, one or more of immune cell types, cytokines, and / orAttorney Docket No. UTSD.P4287WO / 1001338856 autoantibodies, or any combination thereof are used as biomarkers to monitor the risk of a subject developing irAE before, during, or after ICI treatment.

[0164] In some aspects, the method for monitoring comprises providing a sample from the subject, assessing one or more of immune cell types, cytokines, and / or autoantibodies, or any combination thereof, in the sample, and predicting risk for developing / diagnosing irAE in the subject. In some aspects, a subject can be predicted as having a high risk for developing or diagnosed as having irAE associated with ICI treatment, if the profile comprises one or more of immune cell types, cytokines, and / or autoantibodies, or any combination thereof, at an elevated frequencies, expression, or levels in the sample from the subject than frequencies, expression or levels in a control sample. In some aspects, a subject can be predicted as having a low risk for developing or diagnosed as having irAE associated with ICI treatment, if the profile comprises one or more of immune cell types, cytokines, autoantibodies or any combination thereof, at a lower frequencies, expression, or levels in the sample from the subject, when compared to the frequencies, expression, or levels in a control sample.

[0165] In some aspects, the subject being monitored can have a high risk of developing irAE by determining the frequencies, expression, or levels of one or more of NK cell, CD4proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell), plasmablast,double-negative T (dNT) cell, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell,CXCL13, CXCL9, CXCL10, IL1 , TNF , CCL3, CXCL8, FOS, CCL4, TNFAIP3, TNFAIP6,TEX14, CXCL2, CD83, antinuclear antibody, or any combination thereof.

[0166] In some aspects, the subject being monitored can have a high risk of developing irAE if the frequencies, expression, or levels of one or more of CD4 proliferating cell, proliferatingT and NK cell, gamma / delta T cell ( T cell or gdT cell), plasmablast, dNT cell, CXCR3 Tcells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cells, HLA-DR+ CD4memory effector T cell, CD4 central memory cell, IL1 , TNF , CCL3, CXCL8, antinuclearantibody, or any combination thereof, are elevated and / or if the frequency of NK cell is decreased when compared to frequencies, expression, or levels in a control sample, at baseline, or before initiation of ICI treatment.

[0167] In some aspects, the subject being monitored can have a high risk of developing irAE if the frequencies, expression, or levels of one or more of dNT cell, proliferating T and NK cell,gamma / delta T cell ( T cell), plasmablast, IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4,CCL3, TNFAIP3, TNFAIP6, TEX14, CXCL2, CD83, antinuclear antibody, or any combination thereof, are elevated when compared to frequencies, expression, or levels in a control sample, after initiation of ICI treatment.Attorney Docket No. UTSD.P4287WO / 1001338856

[0168] In some aspects, the subject being monitored can have a low risk of developing irAE is associated with frequencies, expression, or levels of one or more of NK cell, CD4proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell), plasmablast,double-negative T (dNT) cell, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell,CXCL13, CXCL9, CXCL10, IL1 , TNF , CCL3, CXCL8, FOS, CCL4, TNFAIP3, TNFAIP6,TEX14, CXCL2, CD83, antinuclear antibody, or any combination thereof.

[0169] In some aspects, the subject being monitored can have a low risk of developing irAE if the frequencies, expression, or levels of one or more of CD4 proliferating cell, proliferatingT and NK cell, gamma / delta T cell ( T cell or gdT cell), plasmablast, dNT cell, CXCR3 Tcells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cells, HLA-DR+ CD4memory effector T cell, CD4 central memory cell, IL1 , TNF , CCL3, CXCL8, antinuclearantibody, or any combination thereof, are lower and / or if the frequency of NK cell is elevated when compared to frequencies, expression, or levels in a control sample, at baseline, or before initiation of ICI treatment.

[0170] In some aspects, the subject being monitored can have a low risk of developing irAE if the frequencies, expression, or levels of one or more of dNT cell, proliferating T and NK cell,gamma / delta T cell ( T cell), plasmablast, IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4,CCL3, TNFAIP3, TNFAIP6, TEX14, CXCL2, CD83, antinuclear antibody, or any combination thereof, are decreased when compared to frequencies, expression, or levels in a control sample, after initiation of ICI treatment. III. Methods of Treatment

[0171] The present disclosure further provides a method of treating a subject with cancer. The method comprises assessing one or more of immune cell types, cytokines, autoantibodies or any combination thereof or any combinations thereof and assigning a risk level for developing irAE. In some aspects, risk assessment is done before, or during ICI treatment. In some aspects, the risk assessment comprises predicting, diagnosing, or monitoring ICI associated irAE. If the profile correlates with irAE or future development of irAE, the subjects are assigned high risk for developing irAE. If the profile correlates with low or no risk of irAE the subjects are assigned a low risk for developing irAE.

[0172] In some aspects, the ICI treatment comprises one or more ICI treatment disclosed herein. The progression of treatment of cancer using ICI is guided by the profile assessments and assigned risk levels. Based on the profile assessments and assigned risk levels, the ICI treatment can be continued, withdrawn, or modified accordingly.Attorney Docket No. UTSD.P4287WO / 1001338856

[0173] In some aspects, the method for treating cancer comprises providing a sample from a subject, assessing one or more of the immune cell types, cytokines, autoantibodies or any combination thereof, treating the subject with an ICI treatment if subject is predicted, diagnosed, or monitored as low risk for irAE. In some aspects, the subject receives a non-ICI treatment if the subject is predicted, diagnosed or monitored as high risk. In some aspects, the subject receives an ICI treatment and an irAE mitigating treatment if the subject is diagnosed as high risk.

[0174] In some aspects, ICI treatment comprises administration of an inhibitor of PD-1, PD- L1, TIM-3, LAG- 3, CTLA-4, CSF- 1R, or any combinations thereof. In some aspects, exemplary irAE mitigating therapy can include corticosteroids (e.g., prednisone, methylprednisolone, dexamethasone, budesonide), TNF inhibitors (e.g., infliximab), or hormone replacement (e.g., hydrocortisone, levothyroxine), and CXCL8 inhibitors (e.g., repertaxin).

[0175] In some aspects, the methods disclosed herein to assess the risk of a subject developing irAE can comprise assessing the risk of one or more other ICI associated irAE including ocular toxicity, rash, dermatitis, pruritus, colitis, hepatitis, nephritis, arthritis, myositis, myocarditis, pneumonitis, thyroiditis, hypophysitis, adrenalitis, gastritis, pancreatitis, vasculitis, diabetes, myasthenia gravis, encephalitis, peripheral neuropathy, meningitis, hemolytic anemia, thrombocytopenia, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), aplastic anemia, pure red cell aplasia, neutropenia, diarrhea or another gastrointestinal disorder, pure red cell aplasia, microcytic anemia, lupus, autoimmune nephritism, autoimmune hepatitis, pneumonitis, pericarditis, endocrinopathy, Addison’s disease, hypogonadism, Sjogren’s syndrome, and / or type I diabetes.

[0176] In some aspects, based on the severity of irAE, ICI treatment may be suspended, with consideration of resuming when symptoms of irAE revert. In some aspects, ICI treatment administered to a subject may be modified. In such aspects, the dosage of ICI treatment may be reduced or skipped. Additionally, irAE mitigating therapy for e.g., corticosteroids may be administered. Subjects may be administered a high-dose corticosteroids (for e.g., prednisone 1 to 2 mg / kg / d or methylprednisolone 1 to 2 mg / kg / d), which may be tapered over the course of at least 4 to 6 weeks. In some aspects, infliximab or other immunosuppressive therapy may be administered either individually, or in combination with other irAE mitigating therapies. In some aspects, irAE mitigating therapy may be administered sequentially or simultaneously with the ICI treatment. In some aspects, permanent discontinuation of ICI may be recommended. In some aspects, a non-ICI treatment may be recommended.Attorney Docket No. UTSD.P4287WO / 1001338856

[0177] In some aspects, corticosteroids include, for example, betamethasone sodium phosphate, desonide sodium phosphate, dexamethasone sodium phosphate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, methylprednisolone disodium phosphate, methylprednisolone sodium succinate, prednisolone sodium phosphate, prednisolone sodium succinate, prednisolamate hydrochloride, prednisone disodium phosphate, prednisone sodium succinate, triamcinolone acetonide disodium phosphate and triamcinolone acetonide disodium phosphate, alclomethasone dipropionate, amcinonide, beclomethasone monopropionate, betamethasone 17-valerate, ciclomethasone, clobetasol propionate, clobetasone butyrate, deprodone propionate, desonide, desoxymethasone, dexamethasone acetate, diflucortolone valerate, diflurasone diacetate, diflucortolone, difluprednate, flumetasone pivalate, flunisolide, fluocinolone acetonide acetate, fluocinonide, fluocortolone pivalate, fluormetholone acetate, fluprednidene acetate, halcinonide, halometasone, hydrocortisone acetate, medrysone, methylprednisolone acetate, mometasone furoate, parametasone acetate, prednicarbate, prednisolone acetate, prednylidene, rimexolone, tixocortol pivalate and triamcinolone hexacetonide.

[0178] In some aspects, a non-ICI therapy non-limiting examples of which can include chemotherapy, hormonal therapy, small molecule therapy, toxin therapy, prodrug-activating enzyme therapy, biologic therapy, surgical therapy, anti-angiogenic therapy, targeted therapy, epigenetic therapy, demethylation therapy, histone deacetylase inhibitor therapy, differentiation therapy, radiation therapy, stem cell transplantation and / or any combination thereof.

[0179] Cancer therapeutic agents or chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-Attorney Docket No. UTSD.P4287WO / 1001338856 fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5- FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK.RTM.; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2’,2“-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (”Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTEPvE™, Pvhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; trastuzumab, docetaxel, platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin); ONTAKT™ (denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Further cancer therapeutic agents include sorafenib and other protein kinase inhibitors such as afatinib, axitinib, bevacizumab, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, trastuzumab, vandetanib, vemurafenib, and sunitinib; sirolimus (rapamycin), everolimus and other mTOR inhibitors. Examples of additional chemotherapeutic agents include topoisomerase I inhibitors (e.g., irinotecan, topotecan, camptothecin and analogs or metabolites thereof, and doxorubicin); topoisomerase II inhibitors (e.g., etoposide, teniposide, and daunorubicin); alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, decarbazine, methotrexate, mitomycin C, and cyclophosphamide);Attorney Docket No. UTSD.P4287WO / 1001338856 DNA intercalators (e.g., cisplatin, oxaliplatin, and carboplatin); DNA intercalators and free radical generators such as bleomycin; and nucleoside mimetics (e.g., 5- fluorouracil, capecitibine, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea). Exemplary chemotherapeutic agents that disrupt cell replication include: paclitaxel, docetaxel, and related analogs; vincristine, vinblastin, and related analogs; thalidomide, lenalidomide, and related analogs (e.g., CC-5013 and CC- 4047); protein tyrosine kinase inhibitors (e.g., imatinib mesylate and gefitinib); proteasome inhibitors (e.g.,bortezomib); NF- inhibitors, including inhibitors of kinase.

[0180] In some aspects, modifying a ICI treatment may include recommending a decreased dosing level of an ICI, to be about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% less than the typical dose for a particular indication or individual (e.g., about 3 mg / kg, about 2.5 mg / kg, about 2 mg / kg, about 1.5 mg / kg, about 1 mg / kg, about 0.9 mg / kg, about 0.8 mg / kg, about 0.7 mg / kg, about 0.6 mg / kg, about 0.5 mg / kg, about 0.4 mg / kg, about 0.3 mg / kg, about 0.2 mg / kg, about 0.1 mg / kg, about 0.05 mg / kg, about 0.01 mg / kg, about 0.005 mg / kg), or about 1.5×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, or 10× less ICI than the ICI dose for a particular indication or for individual. Such modification to the ICI treatment may be undertaken either alone or in conjunction with corticosteroids or other cancer treatments.

[0181] In another aspect, a reduced frequency of administration of an ICI treatment can be recommended. For example, such a reduced frequency of administration of an ICI treatment may include an administration as follows: about once every 3 weeks, about once every 3.5 weeks, about once every 4 weeks, about once every 4.5 weeks, about once every 5 weeks, about once every 5.5 weeks, about once every 6 weeks, about once every 6.5 weeks, about once every 7 weeks, about once every 7.5 weeks, about once every 8 weeks, about once every 8.5 weeks, about once every 9 weeks, about once every 2.5 months, about once every 3 months, about once every 3.5 months, about once every 4 months, about once every 4.5 months, about once every 5 months, about once every 5.5 months, and about once every 6 months. In some aspects, the reduced frequency of administration may be recommended either alone or in conjunction with corticosteroids or other cancer treatment.

[0182] In some aspects, the disclosed methods prevent, reduce, and / or treat one or more ICI associated irAE and / or one or more symptoms associated with irAE. In some aspects, the methods disclosed herein prevent, reduce, and / or treat one or more ICI associated irAE and / or one or more symptoms associated with irAE by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or more.Attorney Docket No. UTSD.P4287WO / 1001338856

[0183] In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequency of NK cell is decreased prior to initiation of ICI treatment (baseline), when compared to the frequency in a control sample.

[0184] In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequencies of one ormore of CD4 proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell or gdTcell), plasmablast, dNT cell, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cells, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof are elevated in the sample from the subject prior to ICI treatment (baseline) when compared to the frequencies in a control sample.

[0185] In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment, if the frequency of CD4 proliferating cells is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequency of proliferating T and NK cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequency of immune cell type gdT cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequency of plasmablasts is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequency of dNT cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequency of CXCR3 T cells is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequency of CD57+ T cells is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequency of CD57+ NKTAttorney Docket No. UTSD.P4287WO / 1001338856 cells is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequency of CD8 terminal effector cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequency of proliferating NKT cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequency of HLA-DR+ CD4 memory effector T cell is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with a non- ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequency of CD4 central memory cells is elevated in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequencies of CD4 proliferating cell, proliferating T andNK cell, gamma / delta T cell ( T cell or gdT cell), plasmablast, dNT cell, CXCR3 T cells,CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, and CD4 central memory cell are elevated in the sample from the subject prior to ICI treatment (baseline) when compared to the frequencies in a control sample.

[0186] In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequencies of one ormore of CD4 proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell or gdTcell), plasmablast, dNT cell, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cells, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof, are elevated and / or if the frequency of NK cell is decreased when compared to frequencies, in a control sample, at baseline, or before initiation of ICI treatment.

[0187] In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequencies of one ormore of dNT cell, proliferating T and NK cell, gamma / delta T cell ( T cell), plasmablast, orany combination thereof are elevated in the sample from the subject after initiation of ICI treatment, when compared to the frequencies in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequency of dNT cell is elevated in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a controlAttorney Docket No. UTSD.P4287WO / 1001338856 sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequency of proliferating T and NK cell is elevated in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigatingtreatment if the frequency of gamma / delta T cell ( T cell) is elevated in the sample from thesubject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequency of plasmablast is elevated in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequenciesof dNT cell, proliferating T and NK cell, gamma / delta T cell ( T cell), and plasmablast areelevated in the sample from the subject after initiation of ICI treatment, when compared to the frequencies in a control sample.

[0188] In some aspects, a subject can be treated with an ICI treatment if the frequency of NK cell is increased in the sample from the subject prior to ICI treatment (baseline) when compared to the frequency in a control sample.

[0189] In some aspects, the subject can be treated with an ICI treatment if the frequencies ofone or more of CD4 proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cellor gdT cell), plasmablast, dNT cell, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cells, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof are in the sample from the subject prior to ICI treatment (baseline) when compared to the frequencies in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequency of CD4 proliferating cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequency of immune cell type T and NK cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequency of gdT cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequency of plasmablast is decreased prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of dNT cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In someAttorney Docket No. UTSD.P4287WO / 1001338856 aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of CXCR3 T cells is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of CD57+ T cells is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be predicted as having a low risk of developing or diagnosed with having irAE if the frequency of CD57+ NKT cells is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequency of CD8 terminal effector cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequency of proliferating NKT cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequency of HLA-DR+ CD4 memory effector T cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequency of CD4 central memory cell is decreased in the sample from the subject prior to ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequencies of CD4 proliferatingcell, proliferating T and NK cell, gamma / delta T cell ( T cell or gdT cell), plasmablast, dNTcell, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector are decreased when compared to frequency, in a control sample, at baseline, or before initiation of ICI treatment.

[0190] In some aspects, the subject can be treated with an ICI treatment if the frequencies ofone or more of CD4 proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cellor gdT cell), plasmablast, dNT cell, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cells, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof, are decreased and / or if the frequency of NK cell is increased when compared to frequencies, in a control sample, at baseline, or before initiation of ICI treatment.

[0191] In some aspects, the subject can be treated with an ICI treatment if the frequencies ofone or more of dNT cell, proliferating T and NK cell, gamma / delta T cell ( T cell),plasmablast, or any combination thereof are decreased in the sample from the subject after initiation of ICI treatment, when compared to the frequencies in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequency of dNT cell isAttorney Docket No. UTSD.P4287WO / 1001338856 decreased in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequency of proliferating T and NK cells is decreased in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequency ofgamma / delta T cell ( T cell) is decreased in the sample from the subject after initiation ofICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequency of plasmablast is decreased in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if thefrequency of dNT cell, proliferating T and NK cell, gamma / delta T cell ( T cell), andplasmablast are decreased in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample.

[0192] In some aspects, the subject can be treated with an ICI treatment if the frequencies ofone or more of CD4 proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cellor gdT cell), plasmablast, dNT cell, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cells, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof, are decreased and / or if the frequency of NK cell is increased when compared to frequencies, in a control sample, at baseline, or before initiation of ICI treatment.

[0193] In some aspects, the subject can be treated with an ICI treatment if the frequency ofone or more of dNT cell, proliferating T and NK cell, gamma / delta T cell ( T cell),plasmablast, or any combination thereof is decreased in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequency of dNT cell is decreased in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequency of proliferating T and NK cells is decreased in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequency ofgamma / delta T cell ( T cell) is decreased in the sample from the subject after initiation ofICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if the frequency of plasmablast is decreased in the sample from the subject after initiation of ICI treatment, when compared to the frequency in a control sample. In some aspects, the subject can be treated with an ICI treatment if thefrequencies of dNT cell, proliferating T and NK cell, gamma / delta T cell ( T cell), andAttorney Docket No. UTSD.P4287WO / 1001338856 plasmablast are decreased in the sample from the subject after initiation of ICI treatment, when compared to the frequencies in a control sample.

[0194] In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the expression of one ormore of IL1 , TNF , CCL3, CXCL8, or any combination thereof, are elevated in the sampleprovided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the expressionof IL1 is elevated in the sample provided by the subject prior to ICI treatment (baseline) whencompared to expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigatingtreatment if the expression of TNF is elevated in the sample provided by the subject prior toICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the expression of CCL3 is elevated in the sample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the expression of CXCL8 is elevated in the sample provided by the subject prior to ICI treatment (baseline) when compared to expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and anirAE mitigating treatment if the expression of IL1 , TNF , CCL3, and CXCL8 are elevated inthe sample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample.

[0195] In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the expression of one ormore of IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4, CCL3, TNFAIP3, TNFAIP6, TEX14,CXCL2, CD83, or any combination thereof are elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or anICI treatment and an irAE mitigating treatment if the expression of IL1 is elevated in thesample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the expression of CXCL8 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subjectAttorney Docket No. UTSD.P4287WO / 1001338856 can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the expression of CXCL9 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the expression of CXCL10 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the expression of FOS is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the expression of CCL4 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the expression of CCL3 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the expression of TNFAIP3 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the expression of TNFAIP6 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the level of gene expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the expression of TEX14 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the expression of CXCL2 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the expression of CD83 is elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if theexpression of one or more of IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4, CCL3, TNFAIP3,Attorney Docket No. UTSD.P4287WO / 1001338856 TNFAIP6, TEX14, CXCL2, and CD83 are elevated in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample.

[0196] In some aspects, the subject can be treated with an ICI treatment, if the expression ofone or more of IL1 , TNF , CCL3, CXCL8, or any combination thereof, are decreased in thesample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject can be treated with an ICItreatment if the expression of IL1 is decreased in the sample provided by the subject prior toICI treatment (baseline) when compared to expression in a control sample. In some aspects,the subject can be treated with an ICI treatment if the expression of TNF is decreased in thesample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject can be treated with an ICI treatment if the expression of CCL3 is decreased in the sample provided by the subject prior to ICI treatment (baseline) when compared to expression in a control sample. In some aspects, the subject can be treated with an ICI treatment if the expression of CXCL8 is decreased in the sample provided by the subject prior to ICI treatment (baseline) when compared to the expression in a control sample. In some aspects, the subject can be treated with an ICItreatment if the expression of IL1 , TNF , CCL3, and CXCL8 are decreased in the sampleprovided by the subject prior to ICI treatment (baseline) compared to the expression in a control sample.

[0197] In some aspects, the subject can be treated with an ICI treatment if the expression ofone or more of IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4, CCL3, TNFAIP3, TNFAIP6,TEX14, CXCL2, CD83, or any combination thereof are decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with an ICI treatment if the expression ofIL1 is decreased in the sample provided by the subject after initiation of ICI treatment whencompared to the expression in a control sample. In some aspects, the subject can be treated with an ICI treatment if the expression of CXCL8 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with an ICI treatment if the expression of CXCL9 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with an ICI treatment if the expression of CXCL10 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with an ICI treatment if the expression of FOS is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treatedAttorney Docket No. UTSD.P4287WO / 1001338856 with an ICI treatment if the expression of CCL4 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with an ICI treatment if the expression of CCL3 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with an ICI treatment if the expression of TNFAIP3 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with an ICI treatment if the expression of TNFAIP6 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with an ICI treatment if the expression of TEX14 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with an ICI treatment if the expression of CXCL2 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the can be treated with an ICI treatment if the expression of CD83 is decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample. In some aspects, the subject can be treated with an ICI treatment if the expression of one or more ofIL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4, CCL3, TNFAIP3, TNFAIP6, TEX14, CXCL2, andCD83 are decreased in the sample provided by the subject after initiation of ICI treatment when compared to the expression in a control sample.

[0198] In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the level of antinuclear antibody, is elevated in the sample from a subject when compared to the level in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment, if the level of antinuclear antibody, is elevated in the sample from a subject prior to ICI treatment (baseline) when compared to the level in a control sample. In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment, if the level of antinuclear antibody, is elevated in the sample from a subject after initiation of ICI treatment, when compared to the level in a control sample.

[0199] In some aspects, the subject can be treated with an ICI treatment, if the level of antinuclear antibody, is decreased in the sample from a subject when compared to the level in a control sample. In some aspects, the subject can be treated with an ICI treatment, if the level of antinuclear antibody, is decreased in the sample from a subject prior to ICI treatment (baseline) when compared to the level in a control sample. In some aspects, the subject canAttorney Docket No. UTSD.P4287WO / 1001338856 be treated with an ICI treatment, if the level of antinuclear antibody, is decreased in the sample from a subject after initiation of ICI treatment, when compared to the level in a control sample.

[0200] In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment based on frequencies, expression, or levels of one or more of NK cell, CD4 proliferating cell, proliferating T and NKcell, gamma / delta T cell ( T cell), plasmablast, double-negative T (dNT) cell, CXCR3 T cells,CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memoryeffector T cell, CD4 central memory cell, CXCL13, CXCL9, CXCL10, IL1 , TNF , CCL3,CXCL8, FOS, CCL4, TNFAIP3, TNFAIP6, TEX14, CXCL2, CD83, antinuclear antibody, or any combination thereof.

[0201] In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequencies, expression, or levels of one or more of CD4 proliferating cell, proliferating T and NK cell, gamma / delta Tcell ( T cell or gdT cell), plasmablast, dNT cell, CXCR3 T cells, CD57+ T cells, CD57+ NKTcells, CD8 terminal effector cell, NKT cells, HLA-DR+ CD4 memory effector T cell, CD4 centralmemory cell, IL1 , TNF , CCL3, CXCL8, antinuclear antibody, or any combination thereof,are elevated and / or if the frequency of NK cell is decreased when compared to frequencies, expression, or levels in a control sample, at baseline, or before initiation of ICI treatment.

[0202] In some aspects, the subject can be treated with a non-ICI treatment, a modified ICI treatment, or an ICI treatment and an irAE mitigating treatment if the frequencies, expression,or levels of one or more of dNT cell, proliferating T and NK cell, gamma / delta T cell ( T cell),plasmablast, IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4, CCL3, TNFAIP3, TNFAIP6,TEX14, CXCL2, CD83, antinuclear antibody, or any combination thereof, are elevated when compared to frequencies, expression, or levels in a control sample, after initiation of ICI treatment.

[0203] In some aspects, the subject treated with an ICI treatment based on the frequencies, expression, or levels of one or more of NK cell, CD4 proliferating cell, proliferating T and NKcell, gamma / delta T cell ( T cell), plasmablast, double-negative T (dNT) cell, CXCR3 T cells,CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memoryeffector T cell, CD4 central memory cell, CXCL13, CXCL9, CXCL10, IL1 , TNF , CCL3,CXCL8, FOS, CCL4, TNFAIP3, TNFAIP6, TEX14, CXCL2, CD83, antinuclear antibody, or any combination thereof.

[0204] In some aspects, the subject can be treated with an ICI treatment if the frequencies, expression, or levels of one or more of CD4 proliferating cell, proliferating T and NK cell,gamma / delta T cell ( T cell or gdT cell), plasmablast, dNT cell, CXCR3 T cells, CD57+ TAttorney Docket No. UTSD.P4287WO / 1001338856 cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cells, HLA-DR+ CD4 memory effectorT cell, CD4 central memory cell, IL1 , TNF , CCL3, CXCL8, antinuclear antibody, or anycombination thereof, are lower and / or if the frequency of NK cell is elevated when compared to frequencies, expression, or levels in a control sample, at baseline, or before initiation of ICI treatment.

[0205] In some aspects, the subject can be treated with an ICI treatment if the frequencies, expression, or levels of one or more of dNT cell, proliferating T and NK cell, gamma / delta Tcell ( T cell), plasmablast, IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4, CCL3, TNFAIP3,TNFAIP6, TEX14, CXCL2, CD83, antinuclear antibody, or any combination thereof, are decreased when compared to frequencies, expression, or levels in a control sample, after initiation of ICI treatment.

[0206] In some aspects, the disclosed method for treatment using one or more of an immune cell profile, a cytokine profile, an antinuclear antibody profile, or any combination thereof, can further comprise repeating one or more steps of the disclosed assessment and / or modifying one or more steps of the disclosed treatment. In an aspect, the disclosed method can further comprise modifying one or more of the administrations of the ICI compounds. In some aspects the method can comprise modifying one or more of steps of administration. For example, in an aspect, the method can be altered by changing the amount of one or more of the ICI compounds, thereof administered to a subject, or by changing the frequency of administration of one or more of the ICI compounds thereof to a subject, or by changing the duration of time one or more of the ICI compounds administered to a subject. In some aspects, the ICI compounds comprise one or more ICI compounds disclosed herein. In some aspects, the assessment using one or more of an immune cell profile, a cytokine profile, an antinuclear antibody profile, or any combination thereof, can be repeated one or more times at the start of the treatment. In some aspects, the assessment using one or more of an immune cell profile, a cytokine profile, an antinuclear antibody profile or any combination thereof, can be repeated one or more times during the treatment. In some aspects, the assessment using one or more of an immune cell profile, a cytokine profile, an antinuclear antibody profile, or any combination thereof, can be repeated one or more times after the treatment is completed.

[0207] In some aspects, the assessments can be automated using computer software analytical programs. The present disclosure provides computer implemented methods of detaining, comparing, and analyzing patterns of frequencies, expression or levels of immune cells, cytokines, antinuclear antibodies, or any combination thereof, in order to assess the risk of irAE in a subject. The analytical programs can be interfaced with, for example, programs that are part of an automated immune cells, cytokines, and / or antinuclear antibodies, detection or quantification system so that data from the automated detection or quantification systemAttorney Docket No. UTSD.P4287WO / 1001338856 can fed directly to the analytical programs. Computer implemented programs can be implemented to output, for example, the identity of immune cells, cytokines, and / or antinuclear antibodies in the sample and the degree of upregulation or downregulation. The interface between the analytical programs may be direct or indirect. In some aspects, the programs of this disclosure can be designed to accept information on the detection or quantification of immune cells, cytokines, and / or antinuclear antibodies, are able to implement data analysis, and output risk assessments. In some aspects the programs of disclosure can further output treatment strategies. IV. Kits

[0208] The present disclosure provides kits for use in assessing risk of irAE and / or treating cancer, as described herein. Such kits can include one or more containers comprising ready- to-use microarray chips, or other detection devices, computer software data analysis for assessing risk and determining treatment strategies. In some aspects, kits can further include one or more containers comprising active agents for treatment of cancer or mitigating the toxicity.

[0209] Having described several aspects, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the present disclosure. Additionally, a number of well-known processes and elements have not been described in order to avoid unnecessarily obscuring the present disclosure. Accordingly, this description should not be taken as limiting the scope of the present disclosure.

[0210] Those skilled in the art will appreciate that the presently disclosed aspects teach by way of example and not by limitation. Therefore, the matter contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the method and assemblies, which, as a matter of language, might be said to fall there between. EXAMPLES

[0211] The following examples are included to demonstrate preferred aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which areAttorney Docket No. UTSD.P4287WO / 1001338856 disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure. Example 1: Patient, Materials, and Methods Clinical data sources and collection

[0212] Clinical data and longitudinal specimens were collected from patients enrolled in an institutional prospective immunotherapy cohort. This study was approved by the UT Southwestern Institutional Review Board (IRB #STU 082015-053). All patients provided informed consent, and the study was performed in accordance with the Declaration of Helsinki. This study enrolled patients aged 18 years with any type of cancer planned for but not yet started on ICI (including anti-programmed death 1 [PD1], PD1 ligand [PDL1], and cytotoxic T lymphocyte antigen 4 [CTLA4] monoclonal antibodies, administered as single-agent therapy or in combinations). At the time of case selection, anti-lymphocyte antigen 3 (LAG3) antibodies had not been FDA approved and are therefore not included in the patient sample. Data collected included age; sex; race / ethnicity; cancer type and stage, tumor characteristics, treatment information, and irAE. Due to challenges in determining the occurrence, type, timing, and severity of irAE, two oncologists (MSVI, DH) experienced in ICI administration and monitoring reviewed each case for toxicities, with discrepancies reviewed and adjudicated by a third experienced clinician (DEG). Enrolled patients underwent initial blood collection at pre- prior to ICI therapy, after 1 treatment cycle (either 2 weeks orirAE were categorized as clinically significant (common terminology criteria for adverse events [CTCAE] grade 2) or not clinically significant (CTCAE grade 1). This threshold was chosen because, in general, grade 2 or greater toxicity implies need for systemic medical intervention, whereas grade 1 toxicity is generally asymptomatic or minimally symptomatic and requires neither ICI modification nor specific systemic treatment. Blood samples were centrifuged at 3000 rpm at 4 °C for 15 minutes to obtain plasma and isolate PBMCs. Single-cell RNA sequencing and data analysis

[0213] Single-cell RNA sequencing (scRNA-seq) data was produced on a total of ~500,000 cells across 80 samples. All 80 PBMC patient samples in this study were processed for ScRNA-seq on 10X Chromium instrument. Up to ~8,000 cells per sample were loaded on a 10X chromium controller, and single-cell libraries were generated and sequenced on Illumina Novaseq 6000 sequencer using paired-end (PE) sequencing to generate approx. 50,000 paired-end reads per cell. All 80 single-cell libraries were generated using 5- prime gene expression 10X assay in Dr. Khan’s laboratory. All primary data alignments were performed using standard 10X Genomics pipelines, and tertiary data analysis was performed usingAttorney Docket No. UTSD.P4287WO / 1001338856 several ScRNA-seq programs, including Seurat and Monocle 2. High dimensional analysis was performed, including quality control, integration, and batch normalization for all 80 samples to generate an integrated normalized scRNA-seq dataset. First, a filter was used to detect empty droplets and identify real cells based on the number of unique molecular identifiers (#UMIs) per cell. The cutoff was automatically calculated as the inflection point of the knee plot. The minimum threshold was set to 500. A filter to eliminate cells was then applied with high mitochondrial read. The cutoff range was et to 3 median absolute deviations above the median. All outliers were excluded where the number of expressed genes per cell and the number of UMIs per cell did not have a linear relationship. Cells with a high probability of being a doublet were then eliminated from the datasets using the ScDblfinder program. Integrating 80 samples for batch correction to identify shared cell states across samples was performed using Harmony and Seurat. These were similar both integration methods. Normalization and dimensionalityusing SCTransform o ion and identify different cell stat ell clusters were identified in the further used as a reference to id top 30 principal components an um fold change and FDR>0.05. RNA-Sequenci

[0214] RNA wa (PBMCs) using Trizol and RNAe encia, CA). RNA was treated with as verified using Agilent Bioanaly used to perform RNA-SEQ using e manufacturer’s instructions. The000 or Novaseq 6000 using a standard single-end protocol. Reads were aligned with TopHat2 to the UCSC Homo sapiens reference genome hg19. Transcripts were assessed, and quantities were determined using Cufflinks v2.0.2. Comparisons of expression levels were made using fragments per kilobase of transcript per million mapped reads (FPKM) values with Cuffdiff from the Cufflinks pack. Differential gene expression analysis was performed using Deseq2. RNA- seq data for pathway and functional analysis was analyzed utilizing the David platform. CIBERSORTx was then applied to bulk RNA-seq data to determine the leukocyte composition. Cytokine / chemokine and ANA analysisAttorney Docket No. UTSD.P4287WO / 1001338856

[0215] Monitoring of cytokine and chemokine levels was performed using Bio-Plex Pro Human Chemokine 40-plex Panel (Bio-Rad Laboratories, Hercules, California) according to the manufacturer’s instructions using a Luminex 200 System. The list of cytokines and chemokines are provided in Table 1. Bio-Plex Manager™ 6.1 software was used for data analysis. Concentrations of cytokines and chemokines (pg / mL) were determined on the basis of the fit of a standard curve for mean fluorescence intensity versus pg / ml. The serum levels of Antinuclear IgG antibodies were assessed using QUANTA lite ANA assay (INOVA diagnostics) in cancer patients at baseline, 2-3 weeks, and 6 weeks post ICI therapy. All serum samples were run in duplicates, and analysis was performed using GraphPad PRISM. Results were expressed as the mean ± standard error (SE). Table 1: Cytokines and chemokines included in the analysis Cytokines and Chemokines 6Ckine / CCL21 ENA-78 / CXCL5 IFN-y CTACK / CCL27 BCA-1 / CXCL13 IL-1b Eotaxin / CCL11 Fractalkine / CX3CL1 IL-2 Eotaxin-2 / CCL24 GCP-2 / CXCL6 IL-4 Eotaxin-3 / CCL26 Gro-a / CXCL1 IL-6 I-309 / CCL1 Gro-b / CXCL2 IL-10 MCP-1 / CCL2 SCYB16 / CXCL16 IL-16 MCP-2 / CCL8 SDF-1a+b / CXCL12 GM-CSFMCP-3 / CCL7 IP-10 / CXCL10 MIF MCP-4 / CCL13 I-TAC / CXCL11MDC / CCL22 MIG / CXCL9MIP-1a / CCL3 IL-8 / CXCL8MIP-1d / CCL15MIP-3a / CCL20MIP-3b / CCL19MPIF-1 / CCL23Mass cytometry

[0216] A panel of 36 antibodies for metal isotope-labeled conjugates, Maxpar Direct Immune Profiling Assay Panel by Fluidigm were used for staining Cryopreserved PBMCs derived from patients receiving ICI therapy. Cells were analyzed on a Helios mass cytometer (Fluidigm). Data were normalized and analyzed with gating on live, singlets CD45+ cells using the OptSNE, FlowSOM and CITRUS algorithms using OMIQ.ai (Omiq, Santa Clara, California). Equal sampling of 30,0 ere analyzed. Statistical analysis of cell abunda aphpad Prism software.Attorney Docket No. UTSD.P4287WO / 1001338856 Statistical analysis

[0217] Statistical signifi s software (Version 29) and GraphPad Prism S USA). Student’s t test, the Mann-Whitney U t . For toxicity analyses, because irAEs may o th immune checkpoint inhibitors, patients were they had been followed without evidence of to ressed as the mean ± standard error (SE). Fo was set at P < 0.05.Example 2: Clinical characterization of patients and study design.

[0218] In this study, 248 cancer patients receiving ICI therapy who had been enrolled on an institutional immunotherapy registry were studied. The study was approved by the UT Southwestern Institutional Review Board (STU 082015-053) and all patients provided written, informed consent prior to undergoing any study-related treatments. Blood was collected from patients at pre-treatment baseline (BL), 2-3 weeks after ICI initiation, and 6-8 weeks after ICI initiation. We isolated peripheral blood mononuclear cells (PBMCs) to perform 5’ scRNA-seq using 10X chromium technology and mass cytometry by time of flight (CyTOF) using Helios. Serum was isolated to perform multiplex cytokine and ANA assays.

[0219] The current analysis irAE was categorized as clinically significant (common terminology criteria for adverse events [CTCAE] grade 2) or not clinically significant (CTCAE grade 1). This threshold was chosen because grade 2 or greater toxicity generally implies the need for systemic medical intervention, whereas grade 1 toxicity usually refers to asymptomatic events that require neither ICI modification nor specific intervention.

[0220] FIG.1 shows th horts included in each analysis, including cyt ass cytometry (N=64), and single-cell RNA (S demographic, cancer, treatment, and toxicity atients received either anti-PD1 / PDL1 therapy eceiving a combination with anti-CTLA4. Table 2: Characteristi treatment, and irAEs.Lung 140 (56) Melanoma 57 (23)Attorney Docket No. UTSD.P4287WO / 1001338856 Kidney 18 (7) Head / Neck 12 (5) Other 21 (9) Treatment No of Patients PD1 / PDL-1 214 (86) PD1 + CTLA-4 34 (14) Immune toxicity No of Patients irAE 129 (52) No irAE 119 (48) Example 3: Baseline and dynamic abundance of immune cell subsets associated with irAEs

[0221] 5’ single-cell RNA sequencing (scRNA-seq) was performed using the 10X chromium 5’ immune profiling assay on paired baseline and 6-8 week samples. For this experiment, 40 patients were identified, of whom 28 had experienced various types of grade 2 irAE (Table 3). The 6-8 week post-ICI initiation timepoint was selected because (1) earlier studies suggested that it provided more useful comparison with pre-treatment baseline, and (2) most. Table 3: Characteristics of patients for single cell RNA sequencing: Cancer, treatment, and irAEs Type of Cancer No of Patients Lung 15 Melanoma 16 Kidney 2 Head / Neck 2 Other 3 Treatment No of Patients PD1 / PDL-1 33 PD1 + CTLA-4 5 Immune toxicity No of Patients irAEs 28 No irAE 8Attorney Docket No. UTSD.P4287WO / 1001338856, immune cell types (FIG.2C). All platelets and erythrocytes were eliminated for downstream analysis. The frequencies were calculated as a percentage of total PBMCs for each cell subset and compared it in patients with and without irAEs at baseline and 6-8 wks post immunotherapy.

[0223] Four cell clusters were identified that significantly correlated with the development of irAEs. The natural killer (NK) cell cluster was significantly less abundant in patients with irAEs at baseline, while CD4 proliferating, plasmablasts, and double-negative T (dNT) cells were significantly greater at baseline in patients with irAEs (FIG.2D-2G). The association of all 30 immune cell states were assessed by performing a Spearman correlation analysis, with CD4 proliferating, NK, plasmablasts, and dNT demonstrating a p-value less than 0.05 (FIG.2H-2I and Table 4). Table 4: Spearman correlation analysis CD4 dnT NK Plasmablast proliferating r 0.329 0.411 -0.392 0.336 P(two-tailed) 0.04 0.013 0.018 0.04

[0224] The dataset was also analyzed using an unsupervised clustering strategy incorporating 30 principal components. After data integration, normalization, and dimensionality reduction, 21 distinct cell clusters were identified across 72 patient samples representing all major immune cell lineages (FIG. 3A). The proportion of 21 clusters is depicted in FIG.3B. Frequency calculations as a percentage of total PBMCs revealed a clear increase in the abundance of cell cluster 18 (Ki67 positive T / NK cell cluster) at baseline in patients with irAEs (FIG.3C). In contrast, cluster 6, resembling the transcriptional state of NK cells, was significantly lower in patients with irAEs (FIG. 3D). These results confirmed the findings from the Azimuth reference-guided cell annotation. Spearman correlation analysis of all baseline clusters identified cluster 6 and cluster 18 as the most significantly associated with irAEs (FIG. 3E-3F and Table 5). Unsupervised sub-clustering of T and B cells were also performed, which clearly showed a greater abundance of plasmablasts and dNT cell subclusters in patients who developed irAE, as observed by reference-guided clustering. Table 5: Spearman correlation analysis for cluster 18 and 6Attorney Docket No. UTSD.P4287WO / 1001338856 Proliferating T / NK Cluster NK Cluster 6 18 r 0.329 0.411 P(two-tailed) 0.04 0.013

[0225] In an analysis of T and NK cell clusters at the post-ICI initiation time point, an enhanced shift in effector and proliferative cell populations in patients who developed irAE was observed. In both the irAE and non-irAE groups, data analysis showed a significant increase in the frequency of circulating dNT cells and proliferative CD4 clusters following ICI initiation. However, the abundance of both clusters was significantly higher in the irAE group. Notably, the ratio of CD4 effector memory to central memory T cells in the blood was more than two- fold higher in irAE patients. Additionally, scRNA-seq gene expression analysis revealedsignificantly higher T- and NK cell production of IFN and TNF in patients with irAEs. Therewere no significant changes in the abundance of NK cell clusters after ICI initiation.

[0226] To assess the potential of the pre-treatment baseline abundance of NK and proliferating T cell clusters as candidate biomarkers, the area under curve (AUC) by receiver operating characteristic (ROC) analysis was determined (FIG. 3G-3H). The AUC for the proliferative T cell cluster was 0.817 (P<0.001). The AUC for the NK cell cluster was 0.772 (P=0.003), suggesting potential utility for the prediction of future irAE occurrence. Example 4: Mass cytometry analysis of immune subsets associated with irAEs

[0227] To corroborate the scRNA-seq findings, CyTOF analysis was performed in a larger group of 64 patient samples (N=12 No irAE and N=52 irAE) that had paired BL and 6-8 weeks post-ICI initiation timepoints available to assess the cell surface expression of 36 immune markers (FIG.4A). After normalization, data was analyzed with gating on live singlets CD45+ cells using OptSNE and Flowsom. Unsupervised consensus meta-clustering using Euclidean as a distance metric was performed. This analysis yielded 30 distinct cell clusters, including major immune cell subsets (B cells, CD4 T cells, CD8 T cells, NK cells, and myeloid cells). (FIG.4B-4C). The abundance of cell clusters was then calculated as a percentage of total CD45+ cells. Edge R analysis comparing the abundance of cell clusters between irAE and no irAE groups revealed nine significant clusters (FIG.4D). Mirroring the findings from scRNA- seq data, the abundance of cluster K10, which resembled plasmablasts, and cluster K17, which resembled double negative gamma delta cells (dNT- T), were significantly greater in the irAE group (false discovery rate [FDR]<0.05) (FIG. 4E-4F). In contrast, decreased abundance of cluster K2 was observed, which resembled NK cells, in alignment with the scRNA-seq data (FIG.4G).Attorney Docket No. UTSD.P4287WO / 1001338856

[0228] Unsupervised clustering of cytometry by time of flight (CyTOF) data revealed greater abundance of NKT-like and CD8 terminal effector cells at baseline in patients who developed irAEs. Additional analysis was performed using an extensive manual gating strategy to confirm the CyTOF results, particularly for plasmablasts, which are very low in abundance from unsupervised clustering. Among all cell clusters, manual gating revealed a significantly increased abundance of plasmablasts, CD8 terminal Effector, and NKT cells, and a reduced abundance of NK cells at baseline, further confirming the results of scRNA-seq and unsupervised CyTOF data analysis (FIG.4H-4J). In addition, manual gating revealed a greater baseline abundance of a small cluster of HLA-DR+ CD4 memory effector T cell population (P<0.05) (FIG. 4K). Significantly higher HLA-DR expression was also observed on CD4 effector memory T cells in patients with irAEs (FIG.4L). Example 5: Baseline subclinical / clinical autoimmunity linked to irAEs

[0229] Because the scRNA-seq data revealed a significantly greater baseline abundance of plasmablasts in patients with irAEs, the role of the B cell compartment in irAEs was further studies by performing an in-depth scRNA-seq analysis to sub-cluster B cells across 72 samples. Overall, any significant differences were not observed in baseline or post-ICI initiation abundance of B cell clusters between patients with and without irAEs. Although, an increased abundance was observed of naïve B cells at baseline and post-ICI initiation in patients with irAEs, neither timepoint reached statistical significance (FIG. 5A-5B). In both irAE and no-irAE cases, naïve, intermediate, and memory B cells showed reduced circulating frequencies after ICI initiation—the CYTOF data analysis of B cells aligned with the single-cell RNA sequencing results. Next performed scRNA-seq differential gene expression and pathway analysis was performed between irAE and no-irAE groups for naïve, intermediate, and memory B cells. No significant changes were observed in any major pathways before or after therapy. In contrast to most B cell clusters, both scRNA-seq and CyTOF data revealed an increase in the frequency of plasmablasts after ICI initiation in all patients, with levels significantly higher in patients with irAEs (FIG.5C).

[0230] Next, serum levels of 40 cytokines and chemokines were evaluated using a multiplex cytokine panel in 164 patients (N=492 samples across three timepoints: BL, 2-3 weeks, 6-8 weeks). The multiplex data analysis revealed significantly enhanced induction of serum CXCL13 (P=0.004) cytokine at 6-8 wks in the irAE group versus the no-irAE group (P=0.8) (FIG.5D). Plasma levels of CXCL13 chemokine has been shown to be a marker for germinal cell activity. The data thus suggest that increased germinal cell activity is potentially associated with irAEs. In addition to CXCL13, the cytokine data analysis also revealed an enhanced induction of serum CXCL9 and CXCL10 cytokines at 6-8 wks in the irAE group as shown in FIG.6E-6F.Attorney Docket No. UTSD.P4287WO / 1001338856

[0231] Given these results, it was determined whether antinuclear antibodies (ANA)—a hallmark of systemic lupus erythematosus and a feature of several other autoimmune diseases—are associated with irAEs in the disclosed cohort. To evaluate this, the serum ANA levels in 107 patients with matched BL, was assessed in 2-3 weeks, and 6-8 weeks samples (FIG. 5E). It was found that baseline ANA levels were significantly higher in patients who developed irAEs compared to patients without irAEs. ANA levels remained unchanged at both post-ICI initiation time points.10, 15, 16, and 17). Azimuth reference-guided scRNA-seq data analysis was then performed, which identified two clusters of monocytes: CD14+ classical monocytes and CD16+ non- classical monocytes. Results from both supervised and unsupervised clustering analyses showed no significant changes in the abundance of myeloid cell clusters after ICI initiation in either patient group. A numerically lower baseline abundance of circulating CD16+ monocytes was observed in patients with irAEs, although the difference did not reach statistical significance (FIG. 6A). Notably, after ICI initiation, the frequency of this cluster slightly decreased in the no-irAE group but increased in the irAE group.

[0233] Next, differential single-cell gene expression was analyzed to compare myeloid cells from irAE versus no irAE group at baseline and post-immunotherapy. Pseudobulk differential analysis revealed significant changes (p<0.05) in 102 genes in non-classical monocytes andtime point. It was observed a significantly higher number of genes differentially expressed in irAE compared to No-irAE cases versus what was observed at baseline between the two groups. As determined by Dseq and depicted in volcano plots (FIG.6D-6E), more than 450 genes in classical monocytes (Cluster 15) and more than 450 genes in non-classical monocytes (Cluster 10) had significantly different expressions (FDR<0.05) in irAE compared to no-irAE cases. Among these, an upregulation of an inflammatory gene signature wasAttorney Docket No. UTSD.P4287WO / 1001338856 observed again in classical (Cluster 15) and non-classical monocytes (Cluster 10) after ICI initiation in patients with irAEs which was much more enhanced. The top significantlyupregulated proinflammatory genes were IL1 , CXCL8, and several cytokines andchemokines involved in the recruitment of T cells to sites of organ inflammation, includingIFN -inducible CXCL9 and CXCL10 (FIG.6F and Table 6).Table 6: List of genes significantly upregulated in CD16+ monocytes Gene Name Log FC P.Value CXCL8 4.32 3.71E-05 IL1B 3.44 4.76E-10 FOS 2.74 4.97E-07 CCL4 2.17 0.000572277 CXCL10 2.11 9.98E-06 CCL3 1.79 0.004662315 TNFAIP3 1.21 3.88E-07

[0234] GO enrichment and multiple pathway analysis revealed that inflammatory response, leukocyte migration, and TNF signaling were the top pathways significantly upregulated in irAE patients compared to patients without irAEs at 6-8 wks post-initiation. In addition, as previously mentioned, the data analysis of 40 cytokines / chemokines in 164 patients with available matched baseline, 2-3 week, and 6-8 week post-ICI initiation time points had revealed strikingly higher induction of CXCL9 and CXCL10 levels in the serum of irAE patients (p<0.004 for CXCL9 and p< 0.002 for CXCL10) compared to the no-irAE group (p< 0.08 (ns) for CXCL9 and p< 0.14 (ns) for CXCL10) at the 6-8 week timepoint, supporting the single-cell gene expression data in monocytes (FIG.6F-6G and Table 7). Table 7: List of genes significantly upregulated in CD14+ monocytes Gene Name Log FC P.Value CXCL8 4.40 0.001584 CCL3 4.17 5.07E-06 IL1B 3.33 2.19E-0 TNFAIP3 2.58 1.88E-05 TEX14 2.54 0.000663 CXCL2 2.16 0.00246 CD83 2.10 0.000267 CCL4 2.04 0.007355 CXCL9 1.45 0.056

[0235] The scRNA-seq gene expression results implied that the proinflammatory monocyte population in the blood of irAE patients was the source of these increased serum levels ofIFN inducible chemokines CXCL9 and CXCL10 cytokines upon ICI initiation. These findingsAttorney Docket No. UTSD.P4287WO / 1001338856 also strongly suggested that monocyte gene signature and levels of CXCL9 and CXCL10 in peripheral blood can be used as potential biomarkers of irAE risk. Example 7: Summary of examples 1-6

[0236] With ICI use extending from refractory, metastatic cancers to curative settings and more toxic ICI combination regimens being approved for multiple tumour types, irAE represents a major clinical concern. These autoimmune toxicities remain largely unpredictable, potentially affecting almost any organ system and occurring at any point during treatment. Yet, after the enrolment of thousands of patients in hundreds of ICI clinical trials over the past decade, the only consistent clinical observation in this area is that patients with pre-existing autoimmune disease appear to be at risk for autoimmune disease flare and have greater risk of irAEs when treated with ICI. Furthermore, there is little agreement as to the optimal means to monitor patients for irAE once they have started ICI therapy.

[0237] To address these issues, a comprehensive multidimensional cellular and molecular analysis was performed of the host immune system in patients treated with ICI by performing diverse assays on baseline and post-ICI initiation peripheral blood samples. These studies revealed a number of key observations: (1) an increased pre-treatment baseline abundance of plasmablasts, distinct proliferative T / NK cell clusters, NKT, CD8 terminal effectors and double negative T cells, as well as a lower frequency of natural killer cells, among patients who developed clinically significant irAE; (2) patients with irAEs showed a significantly enhanced shift of the peripheral immune system to an activated and inflammatory phenotype with an increased abundance of proliferative and activated CD4 effector populations, monocytes with inflammatory gene signatures, and significantly enhanced levels of the proinflammatory chemokines CXCL9, CXL10, and CXCL13; (3) myeloid cells from patients developing irAE show a significantly upregulated proinflammatory gene expression and cytokine profile following therapy that results in enhanced recruitment of immune cells to sites of inflammation; and (4) patients who developed irAEs had significantly higher levels of serum ANA levels both before and after ICI initiation. Considered together, these results suggested that underlying subclinical autoimmunity, compromised self-tolerance, and a proinflammatory immune state were linked to future irAEs.

[0238] The comprehensive analysis disclosed herein revealed associations of plasmablast abundance, ANA, and serum levels of CXCL13 cytokine with irAEs which suggested an autoimmune-like state in patients with irAEs. The disclosed study shed light on the potential roles of B cell-mediated immune responses in irAEs.

[0239] The ScRNA-seq and cytokine results disclosed herein demonstrated a direct involvement of myeloid cells in the peripheral blood in mediating irAEs. It was shown that theAttorney Docket No. UTSD.P4287WO / 1001338856 monocytes from irAE patients are more proinflammatory at baseline and post-ICI initiation withincreased gene expression of TNF and IL1 . A significant upregulation of CXCL9 andCXCL10 gene expression was observed in the monocytes from irAE patients suggesting that these monocytes were the source of increased serum levels of chemokines CXCL9 and CXCL10. These upregulated chemokines, in turn, result in enhanced recruitment of activated CD4 T cells to the sites of organ destruction. One possibility for irAE monocytes to be proinflammatory could be the activations signals received from the activated CD4 effector memory cluster that was significantly more abundant in irAE patients. It is also possible that monocytes could be providing activation signals to T Cells. Another finding in the study disclosed herein was the lower abundance of NK cells correlating with irAE patients. NK cells are innate lymphoid cells that bridge innate and adaptive immunity by producing cytokines and chemokines and play a critical role in antitumor immunity. Notably, the abundance of NK proliferating cells was much higher in irAE patients, although it did not reach significance. Whether these NK cells have a direct role in irAE remains to be studied. Another observation was a strikingly higher abundance of double negative T (dNT) cells at baseline in irAE patients that were TCF7+ve and LEF1 +ve. Although several studies have reported the expansion of DNT cells in various autoimmune diseases and observed their presence at sites of inflammation, it is essential to note that the biology of DNT cells is highly complex. In addition, studies have reported that DNT cells can exert protumor or antitumor effects depending on the tumor type. Given these findings, it is improbable that one particular cell type is driving irAEs, and could be that there is a complex interplay between multiple innate and adaptive immune cell subsets that results in an autoimmune-like state in irAE patients at baseline and which shifts to an enhanced inflammatory state following ICI therapy.

[0240] The results disclosed herein can be incorporated into the selection of patients, treatment regimens subsets from these assays can be test

[0241] In summary yses demonstrated irAE-associated ch uggested the utility of monocyte gene ating CD4 T cells, plasmablasts, dNT s. In this way, the present study rev l state that led to hyperinflammation he comprehensive approach led to t ing immunological determinants that revent toxicities in patients receiving IAttorney Docket No. UTSD.P4287WO / 1001338856 Example 8: Clinic Examples 10-14

[0242] 162 cancer d in a prospective institutional immun proved by the UT Southwestern Instit ts provided written, informed consent ood samples were collected at pre-tre n. Peripheral blood mononuclear cells ing 10X chromium technology and ma lasma was isolated to perform multipl orized as clinically significant (commo 2) or not clinically significant (CTCAE 2 or greater toxicity generally implies th, de 1 toxicity usually refers to asymptomatic events that require neither ICI modification nor specific intervention. FIG.1 outlines the overall experimental strategy and patient cohorts included in each analysis, including cytokines / chemokines (N=146), ANA (N=65), CyTOF (N=55), and scRNA-seq (N=36). The overall numbers and selection of cases for each assay reflect cost / resources, specimen availability, and an effort to maximize the overlap of cases across assays. Table 8 provides demographic, cancer, treatment, and toxicity characteristics for the study cohort and Table 9 provides demographics, cancer type, ICI treatment, irAE type, irAE grade, and assays performed in all 162 cancer patients in the study cohort. All patients received anti-PD1 / PDL1 therapy; 87% received monotherapy, and the remainder combination therapy, most commonly with anti-CTLA4. Table 8: Characteristics of Patients in Study Cohort Characteristic Number (%) Sex Female 58 (36) Male 104 (64) Race-ethnicity Asian 4 (2) Black or African American 12 (7) Hispanic White 12 (7) Non-Hispanic White 126 (78) Unknown 8 (5) Cancer typeAttorney Docket No. UTSD.P4287WO / 1001338856 Non-small cell lung cancer 81 (50)Attorney Docket No. UTSD.P4287WO / 1001338856 tri.A ner giro onNo eNn pPy nHp,pyt tEn bAe abababarimm m m m ,ttnIa ululululeeCIr ovTi ovni ovni ovninmta lll learg lag llag lagtrIemsnuremsnuremsnuremsnureCcIn e-nLlc -,a p ln nLlcn-nLlcn-nLlcnCyt oNec aCo lNec aCo lea o leaeNcC NcC pytr eec eti e e ehtih tih tihcns aR W W aci W W ct,sisr ytc o c o c o c oceiticiina niitna niitnniitnnithpy caaprnh TpsaLpsaLTapsaLTapsaLraa tE O NiHro iHroO NiHroO NiHrogre hohmtcre e e ee)I o xe lalalalaDC-Im (ut / SM M M M 9relottinei9 9 9 9bbita eg7-6-6-7-ah P0 0 0 0TnIA7 6 6 7Attorney Docket No. UTSD.P4287WO / 1001338856Ar gp pp e r ppeiro y eHltbrlayhocnpedeh yHoNoCnpt+ nebab b b bmtma a a am aumulm oulm oulmulI eilo oCIriTpviivnivnivninlllll lr agn ag lag lagemurmn rmn rmn rcs e s u e s u e s u enae- L c - L c - L c - L cpnCyt ollnNec anllnCoNec anllnCoNec anllnCoNec aC ec ete eaih tih ti ehtihR W W W W yticciin oni cio cio ciontnni nni nnihtTap aa tLTp aa tLp aa tLTp aLE OsNiOsHroNisHro ioOsHrNiHrola ellelaxema am SeF eM MeF ee9g59 9 9-6 6 70-0- -A5 60607Attorney Docket No. UTSD.P4287WO / 1001338856nemubam m bamtzi ul mzui ulzilm ao l o ouler zeovzezeovTta binta bta binlalill l llr r ag lnr ag lag lage / e ems u emsnuremsnuremnuree httddcn- Llc - Llc - Llcs- Llcpy otr c aUartlBanCo lnNec anCo lnNec anCo lnNec anlnCoNec aC eti ehti ehti ehti ehtihW W W W W cin oni cin oncioncioncionTa tia Ta tn ia Ta tn iaa tn iaa taOpsiLOpsiLOpsiLTOpsiLTps LN HroN HroN HroN HroO NiHrolalalalamem m melaF eeF eeF eeF eM 959 9 9 9-6-6 7 60 0-0- -5 6 60706Attorney Docket No. UTSD.P4287WO / 1001338856uzil m b borubzi a al m mboz ulmulea eovovpmta bininil lllle lleh agtmnurCmagl onmn ros-eci s u esa Lla c - L ceMm noo ln n r nl Nec aCeRaCa o lnNec aC rtoeteteope dihih tihN RetW W W cin oani cioptnni cioncionsaiLTa tn iaa tn iaa taOpsiLTOps LTps LHroN HroNiHroO NiHroella ela elaam M M MeF e969 9 9-6-6 50 0-0-6 6 605Attorney Docket No. UTSD.P4287WO / 1001338856mbumu lm amil oulliovziet ovmpi n a binulllo emllag lagmal re Cl onmnuremnureulqe c aics- LCnSs anCerRanCa olcs-lnLNec anColclnNec aC rtoetope di eti etieh h hN RtW W W oni cio cio ciotnnitnnitnniaa a a tLTroOpsaLTpsaLTpsaLNiHroO NiHroO NiHroellaa elam e M MeF e96939 9- -6-60 0 0-6 3 606Attorney Docket No. UTSD.P4287WO / 1001338856ens p p ppp p l pParyhehyh oNyHydhioeh oCeDocyhb u azil bu azibamuollrmom obvimb ulrneaobvb ulpmim neaovpmina lll l lmo ag lag lag lagnm asnuremsnuremnuremnurel-nLlcn-nLlcsn-nLlcsn- LlceMo lNec a lCoNec aCo lNec anCo lnNec aC eti e e e ehtih tih tih tihW W W W W cin oTa ni cioptnni cin oni cin oni cin onia Tapta Tapta Tapta TaptaOsiLOs L s L s L s LN HroNiHroO NiHroO NiHroO NiHroellela ellaa ameam M MF eMeF e979 9 9 9-7-5 6 50 0-0- -7 7 50605Attorney Docket No. UTSD.P4287WO / 1001338856ne amum mza u umtmuillomzuill zioloIa o z o z zCeIrvTientva bientea bta blllllllllr agn r agn r agn r ag lnr agemu emu emum mn rcs- cs s e s u e s u en e nLln-nLlcn-nLlcn- Llc - LlcapCyt o lNec aCo lNec aCo lnNec aCo ln nNec aCo lnNec aC ec etete e eaihih tih tih tihR W W W W W yticc o ciinniin oni cin oni cin oni cionnhtTaOptsaiLTa taOpsiLTa taOpsiLTa tn iaOpsiLTa taOpsiLE N HroN HroN HroN HroN Hrolala ellelaxemema am SF eeF eM MeF ee9 9g4-690 -69 90 -80-80- A4 6 6 808Attorney Docket No. UTSD.P4287WO / 1001338856u a ziu lz b moilarorumzibb bua mbambllaoovzemepmepminta blrla llllragn rmagn age emu eomurmn rd cs- cn s-e s u ed nLlalLlc - LlcalBanlnenln nlnCoNec aC MoNec aCoNec aC eti ehtika irka irka irhcacir e ncacir e nc cir e nW WlBrofAnmAaclBrofAnmAaaclBrofAnmAaccin o cio n d cio cioaniptna nitwo etrna nitna nitsaiLTpsaLnk to opTpsaLTp aLHroO NiHro nUN / eO R NioOsHrNiHroel lla elaam e Meam FeMeF e979 9 9 9-7-6 6 60 0-0- -7 7 60606Attorney Docket No. UTSD.P4287WO / 1001338856a babamubzi amulm oulli oom zulv v eovninta binuo dullagam ml rnea oml remnureoaulqe c d ka lCnSs aaCecHe u e cs- LCnNqSs anColcnlnaNec aleC M eti ehti ehti ehtihW W W W cin oani cioptnni cioncionsaa tn iaa tn iaa taiLTOpsiLpsiLTps LHroN HroHroO NiHroellaa elameelaM MF eM 97999 9- -5-60 0 0-7 9 506Attorney Docket No. UTSD.P4287WO / 1001338856oo retr pe l n opepypy , yN Cd aHocapN H H, Hm m a + mb b bu u uzi a a azil zlo ilo m zu mlumu roet ovmilliobrbvimb beameaa binpi n pmpm ga a lla an rm m g mu eonomsnureoLlclnaec al neal- L cnenllnaleC M MoNec aC M eti ehti ehtika ir ehctacl irofeA nmnihW W WBrAacW cin oani cioptnni cin oni cin oni cin onisaa taa t a t a tiLTOps LTpsaLTpsaLTpsaLHroNiHroO NiHroO NiHroO NiHroela ella elaameelaM M MF eM 979 9 9 9-3-6 7 60 0-0- -7 3 60706Attorney Docket No. UTSD.P4287WO / 1001338856aba oiy tntamyr acif,htaEae eAr gn nmr ua nemier fu sitilop epehniro oNoNeDnPrPdsAnIocyh,* oN t + a + nebabmtmamu uzibabuzimzillo alr mmoraumul ozb umul bICeilioembailombaIrvTpiinta beipmpviinepm lllla llragn r agn r aoig magem cs u emu e mn omnurenae-pnLlcsn-nLlcnoc alnoisc-nLlcnCyt o lNec aCo lNec a rCaSh rCac o lNec aC ec etai ehti ehti ehti ehtihR W W W W W yticciin oni cin oni cin oni cin oni cionnhtTaptaa t a t a tnaitLTp aLp a Tp a Tp aEOsNiOsHroNisHro iLHroOsNiLHroOsNiLHrox ela ela eelame lame laSMF eMF eM e9g79 9 9-6 4 6950-0-0- -A7 6 40605Attorney Docket No. UTSD.P4287WO / 1001338856Ar gpiro e y pH myh o y e yN MnP mo pCaPehtnebauziubl zaboilomamtmurbrb ulamulIaemilmbmb vroCIriTpieapmeapmuvdina lla l armo ag lnrmagn rmecnae nm pas u eoms u eonl- L cnena - L c aollncalenC MollnNc aleCytM Ne eC M ec etai ehti ehtika i hcac rl irfe netihR W W WBro A nmAacW yticciin oni cioncioncioncionn a tn ih atp Ta tn iaa tn iaa tn iaa taEsiLOpsHroNiLTOpsHroNiLTOpsHroNiLTps LHroO NiHrolala ellelaxem ma am SeF eeF eM MeF ee9g49-69 9 90 -70-7 50-0- A4 6 7 705Attorney Docket No. UTSD.P4287WO / 1001338856r i oHh hOp PHP am t + u u + nebabazil z b bmtmumuo ilaro abr maumu mIa milobbbb mloulCe li vmamaili eIrTpiinepmepmpviinvalllllr agn r ag illneage tmu emur htmn rc ss- cs-ec os u en e aLlLlsa - Llcap enlnanlnenlnCytrBoNecCoNec aC Mmo oNec aC ec etai ehtinehai ti ehtihR W WsA W W yticciin onniwdet cioncioncionn a thaont ro naitn iaa tn iaa tatTEOpsNiL kHro n opUN / e TOpsR NiLpsHro iLTps LHroO NiHrolala ellelaxem ma am SeF eeF eM MeF ee9g59-59 9 90 -70-6 60-0- A5 5 7 606Attorney Docket No. UTSD.P4287WO / 1001338856t + neb7a + ab+ ab 31b m babmtmumaulmu- Da u4C / mzui allomumuIaCemIr iliovl -iTpiienvtanBaB ovzemilliov1inta bpiinlllllll la el lrgn r agn rmagn r agemu emu e Cl onm mn rcs- cs- c ai s u e s u en eLlLlc - Llc - LlcapnCyt o ln nNec aCo ln n r nNec aCeRaCa o ln nNec aCo lnNec aC eckca i ci r etetetets a a r e nihihihihci RlBrofAnmAacW W W W tsir ye t ti cioncioncio cio ciocci naitn i nni nni nniaa taa taa t a ta nTpLTpLTp p a Tp ar hOsiOsiOs L s L s La thEN HroN HroNiHro iHroO NiHrcoro e emxlu eala ela ela elat / SM M M M M tneit9 0aeg7-69 9 90 -60-7 80-0- P A7 5 6 708Attorney Docket No. UTSD.P4287WO / 1001338856m bulari umu rbaovi bamb illiovmbamnCapiinepm g i nta llaurLeareem gcomnurelc rln cnn saal-nLlcnec a nCac eP cMo lNec aC etikhca i ac rl irfe neti ehtihWBro A nmAacW W cin oani cioptna ni cio ciotna nitna nitsaiLTpsaLpsaLTp aLHroO NiHro iOsHroNiHroela ela elaeM M M 97969 9- -6-70 0 0-7 6 607Attorney Docket No. UTSD.P4287WO / 1001338856t u + neziu lzibababababtolomamr rm m m m abbbulul umululI emambaovovilo oCIrTepmepminiinpviivninlla l l ar agdn rn uom lelememu eamreoCre Cr ocs- c d llcnllll e nnaeL kpnollnaaec aeuqeCnaale agmn cuna agmn cunaaleCytNecC H NS sC MS LCS LC M ec etai ehtin hk / rntop etdi ehti ehtihR W WnUwo oNeRetW W W yticc o n diinni cin oniwet cin oni cin oni cionnhtTa ta Ta taont ro Ta ta Ta tn ia Ta taEOpsNiLOpsHroNiL kHro n opUN / eOpsR NiLOpsHroNiLps LHroO NiHrolax eela ela ela elamelaSM M M MeF eM e9 9 9g6-70-790 -69 90 -50-30- A6 7 7 6 503Attorney Docket No. UTSD.P4287WO / 1001338856r mrmmbb uambeamuil bul uliovmbao leiinevmpmppminvaa a a lllllonomo a elm mmgn rCmoamgn ral neal neasl-uLecla nics-uLecenM M Molln n r nlnNec aCeRaCa o lNec aC eti ehti ehti ehti ehtinhaiW W W W WsAcin oani cin oncioncioncioncionptisaa tn i n i n i n iiLTOpsaa t a t a t a tiLTOpsaLTp aLTp aLTp aLHoNiOsHoNiOs sHroNr rHroNiHroO NiHroella elaame e eelalalaM MF eM M M 999 9 9 9 9-70-4-7-6-6-90704070606Attorney Docket No. UTSD.P4287WO / 1001338856+bab b buz buzma a a ilu m mumumuora iloil bmu rbliolpvii olvni ovnimblneaovmbapminepm llremdn uamamamg e Cl oon amr o o on c aid kall ecn n nu nLancrCeRaaCa ecHe u e aCn le ale aleNqSs aC M M M naieti etin / roe esh hkn nwttope ditieh hAW W UoN RtW W cin o cio cio n d cio c oaniptnnisaiLTarOptnnisaiLTarOptw sao etnrnniitnniiLr k tno ope TaOpsaiLTarOptsaiLHoN HoN HoUN / R N HoN Hrolame ela eelalamla elaF eM MeF eM M 989597969 9- - - -6-80 0 0 0 0-8 5 7 6 608Attorney Docket No. UTSD.P4287WO / 1001338856oavvairvurmiliovn dudpiina lll llmo an mg lnr amgn r eCm asl-uLecs-uLecl oanicenMollnNec anCollnNec anCerRaCaeti ea ir ehtikhcacl ir e n tihW WBrofAnmAacW cin o c o caniinniin oni cin onptsaiLTarOptsaiLTa taOpsiLTaitaOpsiLHoN HroN HroN Hroellalaamee mlaMF eeF eM 93969 9- -7-70 0 0-3 6 707Attorney Docket No. UTSD.P4287WO / 1001338856emtmmu o oaulmullarbrber ovovvirmbambaTin nudepmepm alll l lmo aenmg lsn r amg lnr ag lnr agn r-uLecs-u em cs-u em cs-u ecp al nlln nLlln nLlln nLllnyetMoNec aCoNec aCoNec aCoNec aC etikhca i acl r i rrfe nmnaieti etish hWB o A n Aac AW W cin oni cin oni cioncioncionTa ta Ta tn ia Ta tn iaa tn iaa taOpsiLOpsiLOpsiLTps LTps LN HroN HroN HroO NiHroO NiHroela ela ellaameelaM M MF eM 97959 9 9- -6-6 60 0 0-0-7 5 6 606Attorney Docket No. UTSD.P4287WO / 1001338856gr poy ,H mn miArPdspAnI y a eHr,ntpra oNoNoN tnu eziu lz buzuzuzoilo a ilo ilo ilmtrabrbmulrbrobrbermbeambeaovmbambambaT pmpminepmepmepm lll lamg la l an r ag lnag lmag me s-uLem cs-uremn r omn r ocs-u ecn s u e npnl ylnLt oNec anlln nLllnal- LenlclnaleCoNec aCoNec aC MoNec aC M kca iracl irfeeti eti etetetmn h hihihihBro A n AacW W W W W cin o cio cio cio cio cioTa niptnni nni nni nn naTapta Tapta Tapta Taitna TaitaOs L s L s L s Lps Lpos LNiHroO NiHroO NiHroO NiHroO NiO HrNiHroellelele alaa a alamem M M M MF eeF e979 9 9 9 9-7-4 6 6 40 0-0-0- -7 7 4 60604Attorney Docket No. UTSD.P4287WO / 1001338856ubzilauzomil iu lzilr pororbb i b bambammbambamepmeCepmepm gllln agulo uag ileurLemnuree oml remnurehtlcs-lnLec anColcnlnaa lNec atCu uCsqe cs- LCnSs anColc olnNec ase aC Mmoeti e ehtih ti ehti ehtihW W W W W oni citn oni cin oncioncionaLTa tia Ta tn ia Ta tn iaa taroOpsNiLOpsHroNiLOpsHroNiLTOps LHroNiHroellaameela ela elaMF eM M M 9796979 9- - -8-80 0 0 0-7 6 7 808Attorney Docket No. UTSD.P4287WO / 1001338856n u eziu ubu lbazil zi abazimt ormolrolr mmorIabb ul bbbbumul bbCemaovmamailomaIrTepminepmeipmpviinepm lla l l l ar ag lnrmag lnag lememu eomuremn rCr ocs- cn s- cs-u ell e nnaeLpnllnalLenlln nLlclnagn cnaleCyt oNec aC MoNec aCoNec aCmSuL aC M ec eti ehti ehti ehti ehti ehtisa hci R W W W W W W tsir ye t ti cioncioncio cio cio ciocci narn aitnaitna nitna nitna nitnnitahtTpsaLTpsaLTp aLTp aLTp aLTap aLhEO NiO HroNioOsHrNiHroOsNiHroOsNiHroOsNiHrcoro x ellalalamelelmu eat / SMemamaF eeF eMeF eM tneit9 9 9 9 9 9ae 7-4-5-5-7-6- PgA070405050706Attorney Docket No. UTSD.P4287WO / 1001338856nezilabazi ababmt olo armmr mm m ab umul b uululICeIrmbTeailiovpmpiimb mneailiovpmpii ovnina armdulle lmlageomcnononamlnlre Cl onimsnureaep ale aldeakc au e cnancr-nLlclnCytM MeHeNqC Ss aCeRaCa oNec aC ec etetetetesaihihihih tihci R W W W W W tsir ye tctiio cio c o c o c occi nnitnniitnniitnniitnnita nTap aaLTp aaLTp aaLTp a Tap ar hOsiOsis s L s La thEN HroN HroO NiHroO NiHroO NiHrcoro x elelele eua a al lmea at / SM M M M M tneit9 9 9 9 9ae 8-6-6-7-5- PgA0806060705Attorney Docket No. UTSD.P4287WO / 1001338856mulmumormulolul bvi ovb ul avni ovnimaov rnepminudaoi a lllallaotlmognnmomgnaurgemnursaamgn rlalnis s e l s u ec- L c - L c b - L ceoMh rnCac ollnNec anCollnNec aoil anllnC G moNec aC eti e e e e ehtih tih tih tih tihW W W W W W cin oani cioncioncioncioncionptn i n i n i n i n isaa tiLTOpsaa t a t a t a tiLTOpsaiLTOpsaLTp aLTp aLHoNiOsHoNiOsHroN HroNr rHroNiHroellalalalalaamemem m m MF e F eeF eeF eeF e96069895929- - - - -60 0 0 0 0-6 5 8 5 206Attorney Docket No. UTSD.P4287WO / 1001338856tn+ eb maubzaa mbuzim ilmil utmzalu aorpi ilmu rbIal i oeo b m zlombCIrviabe etvi eaT nCaCa b n pm i rtleardleen uaca o emdn uomrCr o amrc r n d ll ell e nll enaepcnacakc au e cnagcnaldakc au e cnCytaPc eHeC NqSs aCmnSuL a eC MeHeNqC Ss aC ec eti eti eti eti etisa h h h h hci R W W W W W tsir ye tc o c o c o c oticciina niitnniinniinni cin oniaa taa taa t a ta nTp Tp Tp Tp a Tp ar htOsiLrOsiLrOsiLrOsiLrOsiLahcEN HoN HoN HoN HoN Hroro x ele e e eualal l lmea a at / SM M M M M tneit9 9 9 9 9ae 6-7-7-5-5- PgA0607070505Attorney Docket No. UTSD.P4287WO / 1001338856tbnau + em z b bababmil iltpai orbma aummuullamuler membeamiliovvirnu ovTCpmpi dindulll la al lna omrgemnurmageomnur agemnurd llcs- cn s- cs-eekae LlalLlLlcpa cun nln nln nlnyt eHeNqC Ss aCoNec a eC MoNec aCoNec aC nk / rnn oeti etin / rUwtoopNe d h hkRetW Wn n o kca i creUwtoopNe d aRetlBr irofAnmnAacnwd c o c n doetnr inniiotnnitwo et crin onitk tno opTap aaLTp aLnk to opTap aLUN / eOsR NiOsHroNiHro nUN / eOsR NiHroela ela ellalaam m M M MeF eeF e97969 9 9-5 7 50-0-0- -7 6 50705Attorney Docket No. UTSD.P4287WO / 1001338856r i o P h hH Cp h h nDP AnI dt+ neb+ ababab baba abamtmum mm mu mulm amulo ulmul av ulI eili voviloroCIrTpiiniinpviinuvdina a llarrafcomomagn rmecnaeopnam emo utnoanms u eonAn ecct ele al- L cenaR M MollnNc aleCytd i h eC M ec etai ehtin / roehktn nt pdi ehtihR W W Uwo oNeRetW W yticciin onniwden dtwet cioncionn a thaont roont ro naitn iaa tatTEOpsNiL kHro n opUN / ekRn opUN / e TOps LTps LR NiHroO NiHrolalxemeelaameela elaS F eMF eM M e9g59-79 9 90 -60-5 60-0- A5 7 6 506Attorney Docket No. UTSD.P4287WO / 1001338856mtrm mul lIabCeIrmb ulTeao ul avvozp mi ovnir enudta blla lllr agdualee mncurm neo a om nae s- L l rgemnure Crepnlcnald ka le cs- L c llagcCyt o lnNec a eaC MecHe uCnNqSs anllnCoNec a nnCmSuL aC e i cetai ehti ehtikhca aci re netiW Wlr hR WBrofAnmAacW yticc oiinni cin oni cin oni cioncionnhtTa ta Ta ta Ta tn iaa tn iaa taEOpsNiLOpsHroNiLOpsHroNiLTOpsHroNiLps LHro iHrolax ee me la ela ela elaS F eM M M M e9 9 9g4-60-790 -790 -60- A4 6 7 706Attorney Docket No. UTSD.P4287WO / 1001338856n u eziu u u lbazil zi auz zimt ol mil lrbmuorobrula oral b av no bI embaombambrbmbCIrTevpi e eau maeamn pmpmd +mpm lll l lr agalag lalgemlem e m csn-urmLeocnmsn-urLemncs-urcLeoct oani Cl oanin e nlnal nln nlnr c cap l a e lC M Nc a lC Nc ap alr n rCyt oNec oeoeCeHul aCa eRaCaec etetetetn / roetaihihihihknt p ihW W W WnUoNe dRwoRetW yticciin oni cioncioncioncioncionn a tn ih aa tn iaa tn iaa tn i n iaa taa tatTEOpsNiLTOpsHroNiLTOpsHroNiLTOps Lps LTps LHroNiHro iHroO NiHrolalxemeela elaamela elaS F eM MeF eM M e9 9g8-790 -79 9 90 -60-6 60-0- A8 7 7 6 606Attorney Docket No. UTSD.P4287WO / 1001338856yr -0 2- -o2 0 0Ege eAt dera aderaderaderadrari cG G G G G tsfeoh egidaEHrgAri 0 2 3 0 0noderdty,echtr sit ssefaf eiplo,siitinbaynhctEae / , ito sititnoms am imuapeneAr giro opNy dHiroeednp eHonNoN tnuzuzu u u eilo il zoil zoil zoilmtrbr r rora bbbbbbbbICeIrm Team p meam p meam p meambapmepm lla alragem csnurm mdn ueo o enonamlnlrcm eot oniae- Lpnlclnaale ald kea c au e c aCn praclrCyt oNecC M MeHeNqSs aCeHul aCaec eteteteteaihihihih tihR W W W W W yticciin oni cio cio cio cionhtTaptnni nni nni nnia Ta taa taa taa taEOsNiLHroOpsNiLTHroOpsNiLTHroOpsNiLTHroOpsNiLHroelalalaxe lam m melaSMeF eeF eeF eM e9g89-69697950-0- - -A8 6060705Attorney Docket No. UTSD.P4287WO / 1001338856 A N A enikotyCX XdemrFoOfrTeypCX XsyaAsNsRqAcses Xyr + + +o2 2 2Ege e e etd d dArararaiacG GrG tsfeoh egidaEHrgAri 3 2 3nodem dtecsisefdiafbaorayigl sinsEa ihtoar titaAr gtil hiro opCytrpHa, eH tnu ezil babamt ormum abblulI emaovoCIrTepmivninlla a arg m memnur o ocnae s- Lepnlcnlnanle aleCyt oNec aC M M ec eti ehti etis a h hci R W W Wtsir yetctiio cio ciocci nni nni nniarnh Ta taOps LTaptsaa tLTpsaLa th ENiHroO NiHrOircoN Hor lo e a emxu e lamlat / SMeF eMtneitae9g69-6940- -PA60604Attorney Docket No. UTSD.P4287WO / 1001338856 Example 9: Baseline abundance of immune cell subsets associated with irAEs

[0243] A 5’ scRNA-seq was performed using the 10X chromium immune profiling assay on paired baseline (BL) and 6-8 week samples. For this experiment, 40 patients were identified, of whom 30 had experienced various types of grade 2 irAE. A 6-8 weeks post-ICI initiation timepoint was chosen because (1) previous studies suggested that it provided more useful comparison with pre-treatment baseline than do earlier post-ICI initiation timepoints and (2) most irAEs occur after this time, thereby rendering this timepoint potentially useful for prediction of future events. After applying quality control filters, 36 matched sample pairs (BL and 6-8 weeks) yielded high-quality transcriptomes, of which four were technical replicates (1 no-irAE and 3 irAE) (see FIG.2A, Example 3). Following data integration, normalization, and dimensionality reduction, an initial unsupervised clustering analysis at a lower resolution identified 23 distinct cell clusters across samples. These clusters represented all major immune cell lineages, including B cells, CD4 T cells, CD8 T cells, NK cells, monocytes, and dendritic cells (DCs) (FIG.7A). The proportion of these 23 unsupervised Louvain clusters are shown in FIG.7B, and the expression of canonical marker genes is presented as a dot plot in FIG.7C. Cell annotations based on the expression of top five marker genes as well as a heat map of top marker genes for each unsupervised cluster is provided in FIG.12 and FIG.13. Frequency calculations as a percentage of total PBMCs revealed a significant increase in the abundance of proliferating lymphocyte cluster (cluster 18, characterized by Mki67+ cells) (FIG. 7D). Conversely, NK cell cluster (cluster 6) was reduced in irAE patients. In addition, cluster 22, which resembled plasma cell / plasmablast-like clusters showed increased frequency in irAE patients at baseline.

[0244] To confirm these findings, a reference-guided cell annotation analysis was performed with Azimuth on the integrated datasets of 72 patient samples (as previously presented in FIG. 2B and 2C). Consistent with the unsupervised clustering results, three cell clusters were identified to be significantly correlated with the development of irAEs. The NK cell cluster— specifically CD56dimCD16+ NK cells—was significantly less abundant in patients irAE patients at baseline, whereas CD4 proliferating T cells and plasmablasts were significantly more abundant (FIGs.7E-7G). Moreover, when combining Azimuth-guided CD4, CD8, and NK proliferating clusters, the aggregated proliferating lymphocyte cluster was most strongly correlated with irAEs at baseline (FIG.7H) mirroring the unsupervised clustering data. These results demonstrate an activated immune system is present in patients predisposed to irAE. Next, the baseline abundance of this proliferating lymphocyte cell cluster was evaluated to see whether it might serve as a candidate biomarker. Receiver operating characteristic (ROC) analysis yielded an area under the curve (AUC) of 0.78 (P < 0.0001) for the CD4 proliferating cluster, indicating its potential clinical utility in predicting future irAE occurrence.Attorney Docket No. UTSD.P4287WO / 1001338856 Example 10: Mass cytometry (CyTOF) analysis reveals distinct immune subsets associated with irAEs

[0245] To further understand the systemic immune landscape in patients who develop irAEs, the cell surface expression of immune markers was assessed in blood using mass cytometry. Specifically, CyTOF analysis was performed on PBMC samples from an expanded cohort of 55 patients (43 irAE, 12 no-irAE) with paired baseline and 6–8 weeks post-ICI time points using a panel of 36 antibodies (FIG.8A). Metaclustering analysis yielded 30 distinct immunecell clusters—including subsets of B cells, CD4 T cells, CD8 T cells, NK cells, and myeloidcells (FIGs.8B–8C). A corresponding heat map of cell surface marker expression is shown in FIG.8D.

[0246] The abundance of each cell cluster as a percentage of total CD45 cells was nextcalculated. EdgeR analysis comparing irAE and no-irAE groups at baseline identified elevensignificant clusters in OMIQ with P < 0.05 (FIG.8E). Specifically, seven clusters (K4-CD8, K8-TCR , K9-B cell, K10-plasmablast, K13-CD8, K17-TCR , K25-CD8) were increased in irAEpatients, whereas four clusters (K2-NK, K16-CD123 , K28-CD8, K29-CD4) were decreased.Consistent with the scRNA-seq findings, plasmablasts (cluster K10) were elevated (P = 0.01)while NK cells (cluster K2, CD56 CD57 ) were reduced (P = 0.007) in the irAE group (FIGs.8F–8G). Further stratification by cancer and therapy type revealed that clusters K4, K8, and K13 exhibited the most significant differences in irAE patients (FIGs. 8H–8J). Clusters K4(CD8 CCR7 CD45RA CD27 CD28 CD57 ) and K13(CD8 CCR7 CD45RA CD27 CD28 CD57 ) resembled terminal effector memory CD8 Tcells, while cluster K8 comprised double-negative TCR CD57 cells. Extended CyTOFanalyses by therapy and cancer type are presented in FIGs.14A-14C (NSCLC, melanoma, and PD1 / PDL1 therapy). Although the limited number of patients receiving combination immunotherapy precluded detailed analysis of that subgroup, the directionality and magnitude of subgroup findings largely mirrored those of the overall cohort. For additional rigor, manually gating analysis of the CyTOF data was performed. A greater baseline abundance of the NKTcell population (FIG. 8K) and a striking increase in CD57 T cells, including CD57 CD8 andCD8 CD57 NKT populations in irAE patients (FIG. 8L) were identified. The manual gatingstrategy is shown in FIGs.15A-15C. Of note, the two highly correlated clusters identified bymetaclustering CD8 terminal effector cell cluster K4 and TCR cluster K13 were CD57 . Anincreased abundance of HLA-DR CD38 CD4 memory T cells in irAE patients at baseline wasalso observed. To validate the increase in CD8 effector cells in scRNA-seq data, high-resolution subclustering of CD8 and NK cells was performed, which revealed a significantlyhigher abundance of CD8 KLRG1 CCR7 effector memory clusters in irAE patients (FIG.8M).Collectively, our CyTOF analysis at baseline revealed a T cell profile skewed toward anAttorney Docket No. UTSD.P4287WO / 1001338856activated effector / terminal effector phenotype (CD57 ), increased plasmablasts, and reducedCD56dimCD16 NK cells in patients with irAEs.

[0247] Next, treatment-induced changes were investigated by comparing the abundance of metaclusters between irAE and no-irAE groups at 6-8 weeks post-ICI initiation. Following ICIinitiation, the abundance of CD8 effector clusters (K4 and K13) and TCR cluster K8remained significantly higher in irAE patients, whereas the abundance of NK cell cluster K2 remained significantly lower. CD57+ T and NKT cell populations remained significantly higher in the irAE group post-therapy. Using the Wilcoxon matched-pairs signed rank test, longitudinal changes in the abundance of CyTOF metaclusters relative to baseline within eachgroup were further assessed. This analysis revealed a significant increase in the CD4memory cluster (K21, CD4 CD45RO CD45RA CD27 CD28 CD57 ) exclusively in irAE cases(P = 0.003 vs. P = 0.38 in no-irAE), particularly in melanoma patients (FIGs.8N–8O). After ICIinitiation, several populations decreased including memory B cells (cluster K6), exhausted Tcells (CD57 PD1 CD4 cluster K22 and CD57 PD1 CD8 cluster K5), NKT cells(CD8 CD161 CD57 cluster K28), and an intermediate naïve / memory CD8 cluster (K18)(FIGs. 16A-16E). Interestingly, these decreases were significant only in irAE cases, suggesting an enhanced activated state post-therapy. In summary, the CyTOF analysis revealed an activated and skewed T cell compartment in irAE patients at baseline characterized by an effector / terminal effector phenotype with increased plasmablasts and reduced NK cells. Example 11: Baseline proinflammatory autoimmune-like state associated with irAEs

[0248] Because the scRNA-seq and CyTOF analyses revealed a high baseline abundance of plasmablasts in patients with irAEs, the B cell compartment was further examined by performing differential gene expression and pathway analysis between irAE and no-irAE groups for naïve and memory B cells, but no significant changes in major pathways before or after therapy were observed. A significant increase in plasmablasts was observed in patients with irAEs treated with combination (CTLA4+PD1) therapy. It was next investigated whether a higher baseline abundance of plasmablasts correlated with irAE severity. An increased baseline abundance of plasmablast was most strongly associated with Grade 3 irAEs, which typically require hospitalization (FIG.9A). Given these findings, plasma antinuclear antibody (ANA) levels was next assessed — a non-specific clinical biomarker for systemic lupus erythematosus and other autoimmune diseases in 65 patients at BL, 2–3 weeks, and 6–8 weeks samples (FIG. 9B). Baseline ANA levels were significantly higher in patients who subsequently developed irAEs compared to those without irAEs, and these ANA levels remained unchanged across both post-ICI initiation time points. Furthermore, when examiningAttorney Docket No. UTSD.P4287WO / 1001338856 ANA levels according to irAE grade, levels were highest for Grade 3 cases at all time points (FIG. 9C). These ANA and plasmablast findings suggest that potentially a pre-existing autoimmune-like state exists in irAE patients. To determine whether this autoimmune-like state correlates with a more proliferative and proinflammatory phenotype, a grade-based analysis of the proliferating lymphocyte cluster (Mki67+) identified in the scRNA seq data was performed. This highly proliferative lymphocyte cluster, which was CXCR3+HLADR+ was most significantly associated with higher-grade irAEs (FIG.9D).

[0249] Next, gene expression changes were evaluated in T and NK cells at baseline. Pseudobulk differential analysis between irAE and no-irAE group at baseline revealed significant changes (adjusted P < 0.05 and fold change 1.5) in over 300 genes, as depicted in volcano plot (FIG.9E). Notably, IFNG, TNF, and CXCR3 were among the top upregulated genes in patients who later developed irAEs (FIG.9F). Gene Set Enrichment Analysis (GSEA) and MsigDB Pathway enrichment identified TNF signaling as the most significantly upregulated pathway in T cells of irAE patients at baseline (FIGs.9G-9H). In contrast, genes associated with T cell inhibition and immunosuppression (CXCR4 and SIGLEC7) were downregulated in irAE patients. Interestingly, the autoimmune regulator (AIRE) gene wassignificantly upregulated (fold change > 2; adjusted P = 0.04) upon treatment only in the no-irAE group, suggesting a potential role in enhanced self-tolerance and prevention of irAEs. Given that CXCR3 is a homing receptor for CXCL9, CXCL10, and CXCL11 and is expressed on activated / effector T cells with a propensity to migrate to inflammatory sites, scRNA-seq data at a higher resolution was examined and additional manual gating performed of the CYTOF data. This higher-resolution unsupervised analysis of PBMC scRNA-seq replicated our earlier finding of an increased Mki67+CXCR3+HLADR+cells in irAE patients and identified a higher abundance of CXCR3+CD4+IL32+ memory cluster associated with irAEs, thereby supporting the increased CXCR3 gene expression data (FIGs.9I-9J). Sub-clustering analysis of CD8 / NK / Mki67 clusters also showed an increased abundance of activated CXCR3- expressing CD8 memory clusters expressing CXCR3 in the irAE patients at baseline (FIG. 9K). An overall increase in CXCR3 gene expression in scRNA-seq data at baseline is depicted in FIG. 9L. Manual gating of T cell subsets in the CyTOF data likewise revealed a higherabundance of CXCR3 CD4 and CXCR3 CD8 effector memory cells in irAE patients atbaseline (FIGs.9M-9N). Taken together, these findings suggest a preexisting autoimmune- like proinflammatory state in patients who develop irAEs, characterized by elevated baseline ANA levels, increased plasmablasts, and a higher abundance of activated / proliferativeCXCR3+ lymphocytes and CXCR3 T memory and effector memory cells. Additionally, anupregulated 3-gene signature was identified —IFNG, TNF, and CXCR3—in T / NK cells atAttorney Docket No. UTSD.P4287WO / 1001338856 baseline that strongly associates with the subsequent development of irAEs and may serve as a biomarker to predict irAEs. Example 12: Distinct features of myeloid cells and cytokine / chemokines associated with irAEs

[0250] With most irAE studies focusing on the T cell compartment, the role of myeloid cell function in irAE development remains understudied. To investigate this area, unsupervised clustering analysis of myeloid cells in PBMC scRNA-seq data was first performed (FIG.2B), which revealed eight distinct myeloid subsets (Clusters 6, 9, 10, 11, 13, 17, 19, and 21) defined by the expression of canonical myeloid markers. These included six monocyte clusters—twoCD16 clusters (10 and 19), three CD14 clusters (6, 9, and 13), and one intermediatemonocyte cluster (11) as well as a plasmacytoid dendritic cell (pDC) cluster (21) and a conventional dendritic cell (cDC) cluster (17). Differential single-cell gene expression analyses were then performed in these myeloid populations, comparing irAE versus no-irAE cases both at baseline and after ICI initiation. Pseudobulk differential expression analysis revealed significant changes in over 250 genes (adjusted P < 0.05 and fold change 1.5), including upregulation of IL1B and CXCL8 and downregulation of the immunosuppressive gene SIGLEC-10 and complement genes C1QA, C1QB, and C1QC in irAE patients at baseline (FIG. 10A). Gene Set Enrichment Analysis (GSEA) and MsigDB pathway enrichment identified TNF signaling and inflammatory response as the two significantly upregulated pathways in irAE patients at baseline (FIGs.10B–10C). A Heatmap of selected upregulated genes (adjusted P < 0.05 and fold change 1.5) involved in TNF signaling and inflammatory responses is shown in FIG.10D. At post-therapy, significant upregulation of chemokine genes(CXCL10, CCL3, and CCL4) in CD16 monocytes from irAE patients was observed, whereasimmunosuppressive gene SPARC was downregulated (FIG. 10E). Intriguingly, TNF was significantly upregulated at 6–8 weeks post-therapy compared to baseline exclusively in No irAE patients (FIG.10F). To further validate these findings and elucidate the role of monocytes, an Azimuth reference-guided analysis of peripheral blood monocytes was performed, whichidentified two clusters: CD14 classical monocytes and CD16 non-classical monocytes.Differential single-cell gene expression analysis between irAE and no-irAE cases in these monocyte clusters revealed significant changes (adjusted P < 0.05 and fold change 1.5) in over 250 genes in classical monocytes at baseline, including upregulation of proinflammatory genes IL1B, TNF, and CXCL8. TNF signaling emerged as the top enriched pathway in irAE patients, mirroring the unsupervised clustering results. Gene Ontology analysis further showed significant upregulation of genes involved in inflammatory responses,leukocyte / monocyte chemotaxis, and migration in CD14 monocytes, while type I interferonsignaling and complement genes were downregulated in irAE patients at baseline (FIGs.10G–Attorney Docket No. UTSD.P4287WO / 1001338856 10H). Post-ICI initiation, over 450 differentially expressed genes were detected in classical monocytes and more than 150 genes in non-classical monocytes (adjusted P < 0.05 and fold change 1.5). Consistent with these earlier findings, upregulation of several chemokines was observed, for example involved in recruiting T cells and monocytes to sites of organinflammation—including CCL3, CCL4, and IFN -inducible CXCL10 in irAE patients followingICI treatment. Heatmap depicting significant gene expression (adjusted P < 0.05 and fold change 1.5) between irAE and no-irAE patients at baseline is shown in FIG.10I. Notably, IL18 was significantly upregulated in irAE patients’ post-therapy. Given that IL18 activates Tand NK cells and strongly induces IFN production, its increased expression may contributeto hyperinflammation in irAE patients. Importantly, no-irAE patients displayed elevated expression of a distinct set of genes involved in macrophage migration / chemotaxis post-ICItreatment including SLAMF8, CCR2, MST1, PTK2, C5, and CCL5 in CD14 monocytes, andTNF, HES1, CCL5, KLF13, and NOTCH1 in CD16 monocytes indicative of a pro-tumorigenicmyeloid reprogramming. Heat map of these significant genes (adjusted P < 0.05 and fold change 1.5) is depicted in FIG.10J.

[0251] To gain a deeper understanding of myeloid cell involvement in irAEs, all myeloid cells (excluding pDCs) were further subsetted and higher-resolution unsupervised subclusteringanalysis performed, which revealed 16 distinct clusters (FIG. 10K). These included CD14monocyte clusters (C4, C10, C11, C12), an IFN-activated CD14 cluster (C5), S100A12highCD14 clusters (C1 and C3), an IL1Bhigh CD14 cluster (C2), a TGFBIhigh CD14 cluster(C13), CD14lowHLAhigh clusters (C8 and C9), CD16 monocyte clusters (C0, C7, and C14),an IFN-activated CD16 cluster (C15), and a cDC cluster (C6) as defined by the markersshown in FIG.10L. As before, the irAE patients showed significant baseline upregulation of IL1B and CXCL8, with increased CCL3, CCL4, and CXCL10 post-therapy (adjusted P < 0.05 and fold change 1.5). Examining all cytokines / chemokine ligands and their receptors at baseline revealed ILB, CCL3L1, CCR2, CXCL2, CXCL3, CXCL8, IL6R, IL6ST, TNFSF14 and TNFRSF10D significantly upregulated (adjusted P < 0.05 and fold change 1.5) in irAE patients (FIG. 10M). Trajectory analysis indicated that CD14+ monocytes differentiate into three distinct branches: DC-like (C6), monocyte / macrophage-like (C5, C13) and the most differentiated CD16+CD14- state (C0, C14, C15) (FIG.10N). Although no significant changes were observed in the overall abundance of myeloid clusters as a percentage of total PBMCs at baseline or after ICI initiation, a greater fraction of myeloid cells expressed IL1B in irAE patients compared to No irAE patients at baseline (20.23% vs. 11.69%, respectively), as shown in FIG.10O. Similarly, the proportion of myeloid cells expressing CXCL8 was higher in irAE patients than in no-irAE patients at baseline (13.73% vs. 7.98%, respectively), as depicted in FIG. 10O. In addition, the TGFBIhigh cluster (C13), expressingAttorney Docket No. UTSD.P4287WO / 1001338856 immunosuppressive markers TGFB1, CCR2, and STAB1, was more abundant in no-irAE patients at baseline as a percentage of total myeloid cells (FIG.10P). A trend toward increased frequency of cDC cluster (C5) was also noted in irAE patients at baseline, although this association did not reach significance.

[0252] Given this enhanced cytokine and chemokine gene expression profile in irAE patients, plasma levels of 40 cytokines was next evaluated and chemokines using a multiplex cytokine panel in 146 patients at three-time points: pre-treatment baseline, 2-3 weeks, and 6-8 weeks post-ICI initiation. This analysis revealed a complex and dynamic serum cytokine profile. Aspreviously reported by our group and others, 35-36 IFN -inducible chemokines CXCL9 andCXCL10 were elevated in both irAE and no-irAE groups following ICI therapy relative to baseline. However, irAE patients showed a significantly greater increase in plasma CXCL10 at 6–8 weeks (fold change 1.99, P=0.008) compared to the no-irAE group (fold change 1.48, P=0.09; FIG. 11A). Similarly, CXCL9 levels displayed a more pronounced fold induction in irAE patients at 6–8 weeks (FIG.11B). This heightened induction of CXCL9 and CXCL10 was observed in patients receiving both single-agent and combination therapy (FIGs.11C-11F).

[0253] In contrast, serum CXCL8 levels at 6–8 weeks post-ICI were significantly lower in irAE patients than in those without irAEs (FIGs.11G-11I). Notably, changes in CXCL8, CXCL9, and CXCL10 in serum reflected their respective gene expression levels in myeloid cells (FIG. 11J), implying that circulating monocytes may partially account for the source of these serumcytokines. Additionally, IFN levels tended to rise while IL-6 levels tended to decrease in irAEpatients following ICI therapy, although these trends did not reach statistical significance. Overall, results from serum cytokine data suggest that heightened induction of CXCL9 and CXCL10, coupled with low CXCL8 at 6 weeks, could potentially serve as a predictive biomarker signature for irAE risk. In summary, these findings indicate that a higher IL1B and CXCL8 gene signature in myeloid cells at baseline strongly correlates with irAE development. After ICI is administered, monocytes in irAE patients undergo a distinct reprogramming—partlyvia enhanced IFN signaling—leading to increased production of IFN -inducible cytokinesCXCL9 and CXCL10 and downregulation of CXCL8. This shift potentially drives the recruitment of CXCR3+ proliferating and activated effector T cells (highly enriched in irAE patients), which may be self-reactive, through the CXCL10–CXCR3 axis, ultimately precipitating irAEs. In contrast, immunosuppressive immune profile was observed in no-irAE patients at baseline and distinct set of genes upregulated upon ICI therapy. We also observed therapy-induced increases in TNF and CXCL8 gene expression in myeloid cells in No irAE patients.Attorney Docket No. UTSD.P4287WO / 1001338856 Example 13: Discussion of Examples

[0254] With ICI use extending from refractory, metastatic cancers to curative settings and more toxic ICI combination regimens being approved for multiple tumor types, irAEs continue to represent a major clinical concern. These autoimmune toxicities remain largely unpredictable, potentially affecting almost any organ system and occurring at any point during treatment. Yet, after the enrollment of thousands of patients in hundreds of ICI clinical trials over the past decade, the only consistent clinical observation in this area is that patients with pre-existing autoimmune disease appear to be at risk for autoimmune disease flare and have greater risk of irAEs when treated with ICI.However, the molecular mechanisms underlying irAEs remain poorly understood. To address these issues and knowledge gaps, a comprehensive multidimensional cellular and molecular analysis of the host peripheral immune system in a large and diverse cohort of ICI-treated patients was conducted. Using various assays on longitudinal peripheral blood samples, our study revealed several key findings. Patients who eventually developed irAEs had (1) an increased baseline abundance of plasmablast and significantly elevated plasma ANA levels; (2) a skewed baseline T cell profile characterized by increased proliferative and activated CXCR3+ T cells, CD57+ T cells and NKT cells along with a lower baseline abundance of CD56dimCD16+ NK cells; (3) distinct upregulated gene signatures at baseline in T / NK cells (CXCR3, TNF and IL1B) and in myeloid cells (IL1B and CXCL8) coupled with enhanced TNF signaling and higher fraction of IL1B+ve myeloid cells; (4) a significant shift post-therapy towards an activated and inflammatory immune state, marked by increased CD4 memory populations, upregulated IL18 andsignificantly elevated IFN- inducible proinflammatory chemokines CXCL9 and CXCL10coupled with reduced CXCL8 levels in myeloid cells. In contrast, no-irAE patients exhibited an immunosuppressive profile characterized by increased baseline abundance of TGFBIhigh myeloid cluster enriched for immunosuppressive markers such as STAB1 as well as posttreatment upregulation of TNF and AIRE, accompanied by distinct myeloid pro- tumorigenic reprogramming including SPARC upregulation. Together, these findings suggest that underlying subclinical autoimmunity and a proliferative activated proinflammatory immune state predispose patients to irAEs.

[0255] This study is notable for its large size of the study cohort (which permitted subset analyses for some assays) and the comprehensive nature of the correlative studies performed (allowing comparison of findings across methodologies). Additional strengths include the high- quality of clinical data (particularly the occurrence, grade, and type of irAE), the use of real- world cases, and the focus on pre-treatment baseline blood samples, the results of which could be incorporated into the up-front selection of patients, treatment regimens, and monitoring strategies. The numerous novel observations in the present study could reflect theAttorney Docket No. UTSD.P4287WO / 1001338856 relatively large sample size, the diversity of cancer types, and the inclusion of grade 2 irAE (in contrast to certain prior reports incorporating only grade 3 toxicities). We chose to incorporate grade 2 events because (1) they are medically significant, requiring adjustment of ICI and / or administration of immunosuppression or other therapies; (2) in some cases, they may become permanent; and (3) if not recognized and acted upon, they could progress to higher-grade toxicities. Stated otherwise, the distinction between grade 2 and grade 3 irAE could represent chance differences in timing, monitoring, or clinician behaviour rather than true biological or clinical differences.

[0256] Recently efforts have aimed to elucidate the biological underpinnings of irAE has emerged. In a scRNA-seq analysis of ICI-treated melanoma patients, Lozano et al. identified an activated CD4 effector memory subset who went on to develop high-grade irAE. Using mass cytometry, Nunez et al. reported early on-treatment expansion of Ki67+CD8+ T cells and T regulatory cells (Tregs) in lung cancer and melanoma. Our comprehensive analysis extends these findings, revealing associations between an increased baseline abundance of plasmablasts, proliferating activated Mki67+CXCR3+HLADR+ T cells, and high ANA levels, suggesting a pre-existing autoimmune-like state predisposing patients to irAEs. Furthermore, our AUC analysis indicates that baseline proliferating lymphocyte abundance may serve as a predictive biomarker for irAEs. Prior studies have linked plasmablast expansion to autoimmune diseases such as systemic lupus erythematosus (SLE). Given our plasmabalst findings, it will be important to consider future mechanistic studies to explore the possible role of B cell-mediated immune responses in irAE development. A particular notable finding in our study is the reduced baseline abundance of CD57-CD56dimCD16+NK cells in irAE cases, warranting future investigations into its role in irAE pathogenesis. Several previous studies have identified CD8 effector and terminal effector T cells in irAE-affected tissues in colitis, arthritis, myocarditis, and thyroiditis. This study builds upon this by demonstrating strikingly higher abundance of circulating CD8 effector and terminal effector T cells in irAE patients at baseline characterized by CCR7-CD45RA+ expression and enrichment for CCL5, KLRG1, GZMK, and CXCR3 gene markers. This suggest that these blood circulating CD8 T cells may serve as precursors to tissue-infiltrating CD8 clusters in irAEs. Importantly, no previous studies have compared pre-treatment peripheral blood from patients with and without irAEs, making these findings the first to identify a novel association between preexisting circulating CD8 terminal effector / effector memory cells and irAEs. Another key finding in this dataset is themarkedly high levels of CD57+ T cells, NKT cells, and TCR cells in irAE patients prior to ICItherapy initiation. Specifically, the increased abundance of CD57+CD45RA+CXCR3+CCR7- CD8 T cells correlated most strongly with irAE development. CD57 is a marker of terminally differentiated cells with high cytotoxic potential but reduced proliferative capacity and its roleAttorney Docket No. UTSD.P4287WO / 1001338856 in cancer is emerging. Several autoimmune diseases, including Graves’ disease, ankylosing spondylitis, and rheumatoid arthritis, are characterized by an elevated frequency of CD57+ T cells in circulation. The results in the Examples above suggest that CD57+CXCR3+CD8 T cells, with the propensity to be recruited to tissues via CXCL9 and CXCL10, may play a crucial role in irAEs. In addition to increased CXCR3+Mki67+ proliferating T cells and CXCR3+CD57+CD8 T cells, an increased baseline frequency of CXCR3+IL32+ CD4 memory cells in irAE patients was also identified. Furthermore, at baseline, T / NK cells from irAE patients exhibited enrichment for the TNF signaling pathway and displayed a distinct three- gene signature (TNF-CXCR3-IFNG) that could serve as a predictive biomarker for irAEs. Collectively, our findings highlight the role of peripheral CXCR3 as a key mediator and potential biomarker of irAEs. Conversely, no-irAE patients exhibited a more immunosuppressive and immune-evasive T / NK profile at baseline, characterized by upregulation of CXCR4 and SIGLEC7, which may contribute to impaired T cell activity and reduced responsiveness to ICI therapy.55-59 Additionally, no irAE patients demonstrated AIRE upregulation following treatment, suggesting enhanced self-tolerance and protection against irAEs.

[0257] The scRNA-seq and cytokine analyses also highlight the critical role of peripheral blood myeloid cells in mediating irAEs, an area that has not been extensively explored. Specifically, a proinflammatory monocyte phenotype was identified in irAE patients at baseline, characterized by elevated IL1B and CXCL8 gene expression and increased IL1B+ monocyte fractions. This suggests that baseline IL1B-high monocytes, along with the CXCL8-IL1B signature, could serve as predictive biomarkers for irAEs. Furthermore, a significant upregulation of CXCL9 and CXCL10 gene expression in monocytes was observed, coupled with a marked increase in their serum levels following ICI initiation in irAE patients. This finding suggests a key role for these chemokines in the recruitment of activated CXCR3+ T cells, which were highly enriched in irAE patients and may contribute to self-reactivity at sites of organ damage, ultimately leading to irAE development. One potential explanation for the enhanced proinflammatory monocyte response in irAE patients is the activation signals received from proliferative T cells and the activated T effector memory clusters, which were significantly more abundant in irAE patients. Additionally, enhanced TNF signaling and increased IFNG gene expression was observed in T / NK cells. Conversely, monocytes in irAE patients exhibited upregulated IL18 gene expression upon ICI initiation. Given that IL18activates T and NK cells and is a potent inducer of IFN production, its increased expressionmay contribute to hyperinflammation in irAE patients. Thus, the disclosed findings suggest bidirectional interactions between monocytes and T cells that may drive irAE pathogenesis through the IFNG / CXCL10 / CXCR3 axis. In contrast, a more immunosuppressive and immune

Claims

Attorney Docket No. UTSD.P4287WO / 1001338856 CLAIMS What is claimed is:

1. A method of predicting the risk of developing and / or diagnosing immune-related adverse events (irAE) associated with immune checkpoint inhibitor (ICI) treatment in a subject comprising: a. providing a sample from the subject; b. assessing the frequencies of one or more immune cell types in the sample, wherein the one or more immune cell types comprise natural killer (NK) cells, CD4 proliferating cells, proliferating T and NK cells, gamma / delta T cells ( Tcells), plasmablasts, double-negative T (dNT) cells, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cells, NKT cells, HLA-DR+ CD4 memory effector T cells, CD4 central memory cells, or any combination thereof; and c. predicting risk for developing and / or diagnosing irAE in the subject, wherein the subject is predicted as having a high risk of developing or diagnosed with having irAE if the frequencies of the one or more immune cell types are different in the subject when compared to the frequencies in a control sample.

2. A method of monitoring the risk of developing irAE associated with ICI treatment in a subject comprising: a. providing a sample from the subject; b. assessing the frequencies of one or more immune cell types in the sample, wherein the one or more immune cell types comprise natural killer (NK) cells, CD4 proliferating cells, proliferating T and NK cells, gamma / delta T cells ( Tcells), plasmablasts, double-negative T (dNT) cells, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cells, NKT cells, HLA-DR+ CD4 memory effector T cells, CD4 central memory cells, or any combination thereof; and c. monitoring risk for developing irAE in the subject, wherein the subject is predicted as having a high risk of developing irAE if the frequencies of the one or more immune cell types are different in the subject when compared to the frequencies in a control sample.

3. The method of claim 1 or claim 2, wherein the irAE comprises ocular toxicity, rash, dermatitis, pruritus, colitis, hepatitis, nephritis, arthritis, myositis, myocarditis, 129Attorney Docket No. UTSD.P4287WO / 1001338856 pneumonitis, thyroiditis, hypophysitis, adrenalitis, gastritis, pancreatitis, vasculitis, diabetes, myasthenia gravis, encephalitis, peripheral neuropathy, meningitis, hemolytic anemia, thrombocytopenia, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), aplastic anemia, pure red cell aplasia, and / or neutropenia.

4. The method of claim 1, wherein the assessment of frequencies of one or more immune cell types is performed before ICI treatment to predict the subject’s risk of developing irAE.

5. The method of claim 1, wherein the assessment of frequencies of one or more immune cell types is performed during ICI treatment to diagnose the subject’s risk of developing irAE.

6. The method of claim 2, wherein the assessment of one or more frequencies of immune cell types is performed after ICI treatment to monitor the subject’s risk for developing irAE or the subject’s recovery from irAE.

7. The method of claim 1 or claim 2, wherein the subject is predicted as high risk of developing irAE if the frequency of NK cell is decreased in the sample prior to ICI treatment (baseline) when compared to the frequencies in a control sample.

8. The method of claim 1 or claim 2, wherein the subject is predicted as high risk of developing irAE if the frequencies of CD4 proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell), plasmablast, dNT cell, CXCR3 T cells, CD57+ T cells,CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof are elevated in the sample prior to ICI treatment (baseline) when compared to the frequencies in a control sample.

9. The method of claim 1 or claim 2, wherein the subject is diagnosed with irAE if the frequencies of dNT cells, CD4 proliferating cells, proliferating T and NK cells, gamma / delta T cells ( T cells), plasmablasts, or any combination thereof areelevated after initiation of ICI treatment, when compared to the frequencies in a control sample.

10. The method of claim 1 or claim 2, wherein the sample is whole blood, serum, plasma, cerebrospinal fluid, pleural fluid, pericardial fluid, peritoneal fluid, bone marrow, or tissue, urine, cerebrospinal fluid (CSF), or other body fluid.

11. The method of claim 1 or claim 2, wherein said ICI treatment is administered as part of a cancer treatment. 130Attorney Docket No. UTSD.P4287WO / 1001338856 12. The method of claim 11, wherein said ICI treatment comprises administration of an inhibitor of PD-1, PD-L1, TIM-3, LAG- 3, CTLA-4, CSF- 1R, or any combination thereof.

13. The method of claim 1 or claim 2, wherein assessing the frequencies of one or more immune cells (step b) comprises mass cytometry (cytometry by time-of-flight CyTOF), scRNA-sequencing, immune profiling assay, flow cytometry, fluorescence-activated cell sorting (FACS), and / or immunomagnetic separation.

14. The method of claim 1 or claim 2, wherein the method further comprises assessing the expression of cytokines in the sample from the subject, wherein the cytokines are one or more of C-X-C motif chemokine ligand 13 (CXCL13), C-X-C motif chemokine ligand 9(CXCL9), C-X-C motif chemokine ligand 10 (CXCL10), Interleukin-1 beta (IL1 ),Tumor necrosis factor (TNF ), Chemokine ligand 3 (CCL3), Chemokine ligand 8(CXCL8), Fos Proto-Oncogene (FOS), Chemokine ligands 4 (CCL4), TNF alpha induced protein 3 (TNFAIP3), TNF alpha induced protein 6 (TNFAIP6), Testis Expressed 14 (TEX14), C-X-C motif chemokine ligand 2 (CXCL2), Cluster of Differentiation 83 (CD83) or any combination thereof.

15. The method of claim 14, wherein the assessment comprises identifying if the expression of IL1 , TNF , CCL3, CXCL8, or any combination thereof are elevated inthe sample from the subject prior to ICI treatment (baseline) when compared to the expression in a control sample.

16. The method of claim 14, wherein the assessment comprises identifying if the expression of IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4, CCL3, TNFAIP3,TNFAIP6, TEX14, CXCL2, CD83, or any combination thereof are elevated in the sample from the subject after initiation of ICI treatment when compared to the expression in a control sample.

17. The method of any one of claims 1, 2 and 14, wherein the method further comprises assessing the level of antinuclear antibodies (ANA) in the sample from the subject.

18. The method of claim 17, wherein the assessment comprises assessing if the level of ANA is elevated in the sample from the subject when compared to the level in the control sample.

19. The method of claim 1 or claim 2, further comprising repeating steps (a)-(c) at a second time point, thereby permitting determination of a change in the subject’s risk of developing irAE and / or diagnosis of irAE in the subject.

20. The method of claim 1 or claim 2, further comprising predicting the subject as having low risk: 131Attorney Docket No. UTSD.P4287WO / 1001338856 (a) if the frequency of NK cell is increased in the sample from the subject prior to ICI treatment (baseline) when compared to the frequency in the control sample; (b) if the frequencies of CD4 proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell), plasmablast, dNT cell, CXCR3 Tcells, CD57+ T cells,CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof are decreased in the sample from the subject prior to ICI treatment (baseline) when compared to the frequencies in the control sample; and / or (c) if the frequencies of dNT cell, CD4 proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell), plasmablast, or any combination thereof isdecreased in the sample from the subject after initiation of ICI treatment, when compared to frequencies in the control sample.

21. The method of claim 20, further comprising treating the subject with an ICI therapy if the subject is predicted to have a low risk for developing irAE.

22. The method of claim 1 or claim 2, further comprising treating the subject predicted as having a high risk of developing irAE with a non-ICI therapy or treating said subject with a ICI therapy and an irAE mitigating therapy, wherein the irAE mitigating therapy is selected from corticosteroids (e.g., prednisone, methylprednisolone, dexamethasone, budesonide), TNF inhibitors (e.g., infliximab), or hormone replacement (e.g., hydrocortisone, levothyroxine) BRAF inhibitor (e.g. vemurafenib, dabrafenib, and encorafenib), MEK inhibitor (e.g. binimetinib, cobimetinib, selumetinib, trametinib), CXCL8 inhibitors (e.g. repertaxin), or any combination thereof.

23. A method of treating a subject with cancer comprising: (a) providing a sample from the subject; (b) assessing the frequencies of one or more immune cell types in the sample, wherein the one or more immune cell types comprise natural killer (NK) cells, CD4 proliferating cells, proliferating T and NK cells, gamma / delta T cells ( T cells), plasmablasts,double-negative T (dNT) cells, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cells, NKT cells, HLA-DR+ CD4 memory effector T cells, CD4 central memory cells, or any combination thereof; (c) predicting the subject’s risk of developing irAE, wherein the subject is diagnosed as: (i) low risk if: 132Attorney Docket No. UTSD.P4287WO / 1001338856 (I) the frequency of NK cell is increased in the sample prior to ICI treatment (baseline) when compared to the frequency in a control sample; and / or (II) the frequencies of CD4 proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell), plasmablast, dNT cell, CXCR3 T cellsCD57+ Tcells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof are decreased in the sample prior to ICI treatment (baseline) when compared to the frequencies in a control sample; and / or (ii) high risk if: (I) the frequency of NK cell is decreased in the sample prior to ICI treatment (baseline) when compared to the frequency in a control sample; and / or (II) the frequencies of CD4 proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell), plasmablast, dNT cell, CXCR3 T cells,CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA- DR+ CD4 memory effector T cell, CD4 central memory or any combination thereof are elevated in the sample prior to ICI treatment (baseline) when compared to the frequencies in a control sample; and (d) treating the subject with: (i) an ICI therapy if subject is diagnosed as low risk of developing irAE; (ii) a non-ICI therapy if the subject is diagnosed as high risk of developing irAE; or (iii) an ICI therapy and an irAE mitigating therapy if the subject is diagnosed as high risk of developing irAE.

24. The method of claim 23, wherein the assessment further comprises assessing the expression of a cytokine in the sample provided by the subject, wherein the cytokine is CXCL13, CXCL9, CXCL10, IL1 , TNF , CCL3, CXCL8, FOS, CCL4, TNFAIP3,TNFAIP6, TEX14, CXCL2, CD83, or any combination thereof.

25. The method of claim 24, wherein the assessment comprises identifying if the expression of IL1 , TNF , CCL3, CXCL8, or any combination thereof are elevated inthe sample from the subject prior to ICI treatment (baseline) when compared to the expression in the control sample. 133Attorney Docket No. UTSD.P4287WO / 1001338856 26. The method of claim 24 or claim 25, wherein the assessment comprises identifying if the expression of IL1 , CXCL8, CXCL9, CXCL10, FOS, CCL4, CCL3, TNFAIP3,TNFAIP6, TEX14, CXCL2, CD83, or any combination thereof is elevated in the sample from the subject after initiation of ICI treatment when compared to the expression in the control sample 27. The method of claim 19 or claim 21, wherein the assessment further comprises assessing the level of antinuclear antibodies (ANA) in the sample from the subject.

28. The method of claim 25, wherein the assessment comprises assessing if the level of ANA is elevated in the sample from the subject when compared to the level in the control sample.

29. The method of claim 21, wherein said ICI therapy comprises administration of an inhibitor of PD-1, PD-L1, TIM-3, LAG- 3, CTLA-4, CSF- 1R, or any combinations thereof.

30. A method of identifying the presence of at least one differentially frequent immune cell type with associated with irAE in a biological sample of a subject with cancer, the method comprising: a. providing a sample from the subject; and b. assessing the frequencies of one or more immune cell types in the sample, wherein the assessment comprises assessing the frequencies of one or more immune cell types comprises assessing one or more of natural killer (NK) cell, CD4 proliferating cell, proliferating T and NK cell, gamma / delta T cell ( T cell),plasmablast, double-negative T (dNT) cell, CXCR3 T cells, CD57+ T cells, CD57+ NKT cells, CD8 terminal effector cell, NKT cell, HLA-DR+ CD4 memory effector T cell, CD4 central memory cell, or any combination thereof.

31. The method of claim 30, wherein the subject is planning, undergoing, or has completed immune checkpoint inhibitor (ICI) treatment.

32. The method of claim 30, wherein irAE comprises ocular toxicity, rash, dermatitis, pruritus, colitis, hepatitis, nephritis, arthritis, myositis, myocarditis, pneumonitis, thyroiditis, hypophysitis, adrenalitis, gastritis, pancreatitis, vasculitis, diabetes, myasthenia gravis, encephalitis, peripheral neuropathy, meningitis, hemolytic anemia, thrombocytopenia, hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), aplastic anemia, pure red cell aplasia, and / or neutropenia.

33. The method of claim 30, wherein the method further comprises assessing the expression of a cytokine in the sample provided by the subject, wherein the cytokine 134Attorney Docket No. UTSD.P4287WO / 1001338856 is CXCL13, CXCL9, CXCL10, IL1 , TNF , CCL3, CXCL8, FOS, CCL4, TNFAIP3,TNFAIP6, TEX14, CXCL2, CD83, or any combination thereof.

34. The method of claim 30, wherein the method further comprises assessing the level of antinuclear antibodies (ANA) in the sample from the subject.

35. The method of claim 30, wherein assessing the frequencies of immune cells comprises mass cytometry (cytometry by time-of-flight CyTOF), scRNA-sequencing, immune profiling assay, flow cytometry, fluorescence-activated cell sorting (FACS), or immunomagnetic separation. 135

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