Small molecule metabolites as predictors and mediators of immune response and immune therapy response

Abstract

Methods are disclosed related to identifying small molecule metabolites that serve as biomarkers of immune related adverse events (irAEs). In embodiments, treatments are disclosed that reduce the likelihood or prevent irAEs.

Description

SMALL MOLECULE METABOLITES AS PREDICTORS AND MEDIATORS OF IMMUNE RESPONSE AND IMMUNE THERAPY RESPONSECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 567,601, filed March 20, 2024, the contents of which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] Embodiments of the disclosure relate to the identification of small molecule metabolites that can serve as biomarkers of immune related adverse events (irAEs) incidence and also modulate immune response, inflammation, and autoimmunity, including immunotherapy-derived autoimmunity. In certain embodiments, a method for identifying a subject that will or is likely to develop an irAE following administration of an immunotherapy is provided, the method comprising detecting in the subject the presence of the small molecule metabolites disclosed herein. In other embodiments, a method for treating a subject for an irAE, an undesirable or aberrant immune response, immune disorder, inflammatory response, autoimmune disease or inflammation is provided, the method comprising administering or modulating one or more of the small molecule metabolites disclosed herein. In certain embodiments, the administering or modulating one or more of the small molecule metabolites disclosed herein is done via dermal administration (transdermal) of a formula including one or more of the small molecule metabolites.BACKGROUND OF THE INVENTION

[0003] Immune checkpoint blockers (ICB) are a breakthrough therapy for cancer. These transformative agents block cell-surface interactions through receptors that normally suppress antitumor T cell responses, reawakening potent endogenous immune activity against cancer cells. However, the primary cause of termination of clinical ICB therapy is treatment-associated autoimmune toxicides or immune-related adverse events (irAEs), which severely limit the efficacy of these life-saving agents. These detrimental side effects are observed in every major organ system, affecting 50-75% of patients receiving anti-CTLA4, 20-30% receiving anti-PDl and >80% receiving combination therapies. In stark contrast to treatment-associated tumor regression, which is influenced by genomic instability, immune cell infiltrate or expression of immune molecules and microbiome diversity, no modulators of treatment-associated irAEs have been identified, and the molecularmechanisms underlying irAE development and their relationship to tumor growth are poorly understood.

[0004] Mitigating irAE symptoms is an ongoing challenge in ipilimumab-treated patients, with blunting of therapeutic efficacy or discontinuation of treatment a constant concern. Treatment with ipilimumab frequently causes severe (Grade III-IV) irAE in 19-22% of monotherapy patients, with even higher incidence rates observed using combination anti-PDl or anti-PD-Ll therapy. Notably, Tpilimumab-derived irAE are likely to remain a persistent clinical challenge, owing to the substantial 10-year sustained survival rate of ipilimumab-treated melanoma patients, and the significantly improved efficacy of immune checkpoint inhibitor regimens which include ipilimumab for melanoma and other solid tumor types. Predicting the severity of irAE incidence, and mitigating adverse events without limiting anti-tumor immunity, are key challenges in tumor immunotherapy today.

[0005] Biomarker studies of irAE incidence remain incredibly limited — in fact, a preponderance of investigative attention is spent on whether irAE are biomarkers of immune checkpoint inhibitor efficacy, rather than mitigating irAE incidence. Nonetheless, a large proportion of ipilimumab-treated melanoma patients are discontinued from treatment due to the relative frequency of grade 111 / 1 V irAE, deflating the potential survival benefits of ipilimumab relative to less toxic therapeutics such as anti- PD1 / PD-L1.

[0006] Among the most common ICB -related irAEs are rheumatic complications that resemble classic autoimmunity, including inflammatory arthritis (rheumatoid arthritis, psoriatic arthritis), polymyalgia rheumatica, inflammatory myopathy, sarcoidosis, scleroderma, vasculitis, and sicca syndrome. Autoimmunity arises in both CTLA4 and PD1 knockout mice, while loss of CTLA4 is associated with autoimmune manifestations in humans, and loss of PD1 to rheumatoid arthritis. Despite similarities between ICB-related irAEs and classical autoimmunity, key distinctions have been reported, including lack of canonical autoantibodies observed in rheumatological manifestations of irAEs.

[0007] Circulating small molecule metabolites directly drive both oncogenic and immunological phenotypes. With the objective of examining metabolite modulators of clinical ICB therapy, the Applicants applied a comprehensive, high-throughput mass spectrometry technology to study late- stage melanoma and lung cancer patients undergoing anti-CTLA4 ipilimumab or anti-PDl pembrolizumab therapy. Preliminary results uncovered several new metabolite associations with treatment-associated irAEs in patients. Specifically, decreased levels of two species of lysophosphatidylcholine (LPC 16:0 and LPC 18:2) were found to be specifically linked to thedevelopment of severe irAEs in cancer patients receiving ipilimumab (anti-CTLA4) or pembrolizumab (anti-PDl) therapy. LPC comprise a diverse superfamily of bio-active lipids that regulate many aspects of inflammation and auto-immunity, however the immune-modulatory activity of these particular irAE- associated LPC isoforms has not been previously defined in vivo, and their specific relevance to human tumor immunity and clinical ICB outcomes has been unknown prior to the present disclosure. Further, Applicants have generated preliminary functional data showing that administration of purified LPC in vivo ameliorates acute symptoms of DSS-induced auto- inflammatory colitis, demonstrating supplementing LPC or modified LPC small molecules as a therapy for treating auto-immune toxicities due to cancer immunotherapy, including checkpoint therapies such as anti-CTLA4 or anti-PDl therapy.SUMMARY OF THE INVENTION

[0008] There is a need for predictive biomarkers of ICB related irAEs for immune checkpoint inhibitor outcomes that are readily accessible in a patient’s blood. The human blood circulation houses thousands of molecules, many of which remain unknown and unstudied.

[0009] The present inventors have identified predictive and dynamically regulated small molecule metabolites in bio samples from human immune checkpoint inhibitor patients and discovered that these metabolites strongly associate with the development of immune related adverse events (irAEs) in patients. The present inventors have identified small molecule metabolites associated with irAE in cancer patients undergoing cancer immunotherapy in the form of immune checkpoint inhibitors. Thus, there are presently provided small molecules that serve as biomarkers of irAE prediction and modulators of immunotherapy-derived autoimmunity without limiting therapeutic efficacy of the immunotherapy.

[0010] Thus, in one aspect, there is presently disclosed methods for identifying a subject that will or is likely to develop an immune-related adverse event following administration to the subject of a cancer therapy. In certain embodiments, the method contacting the subject or a sample isolated from the subject with an agent that detects the presence of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 in the subject or the sample, wherein the presence of the of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 at lower than baseline levels indicates that the subject has, will or is likely to have an immune-related adverse event following administration to the subject of the cancer therapy. In particular embodiments, the baseline expression is normalized mean expression of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2. In particular embodiments, the method is conducted followingadministration of the cancer therapy to the subject. In certain embodiments, the immunotherapy is a cancer immunotherapy. In particular embodiments, the method is conducted at least about three weeks following administration of the cancer therapy to the subject.

[0011] In certain alternative embodiments, the method further comprises administering one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 to the subject when a lower than baseline level of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is detected in the subject. In some embodiments, the cancer therapy is chemotherapy, immunotherapy, radiation therapy, and / or cellular therapy. In certain embodiments, the cancer therapy is immune checkpoint blocker therapy. In alternative embodiments, the immune checkpoint blocker therapy is one or more of anti-CTLA4 therapy, anti-PDl therapy, and anti-PDLl therapy. In certain embodiments, the anti-CTLA4 therapy, anti-PDl therapy, or anti-PDLl therapy comprises one or more of Ipilimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab and Durvalumab.

[0012] In another aspect, there is presently provided a method for treating an immune-related adverse event in a subject receiving cancer therapy, the method comprising administering to the subject one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2. In certain embodiments, the administering one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is performed via dermal administration (transdermal) of a formula comprising the one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2.

[0013] In an alternative aspect, there is presently provided a method of treating an immune-related adverse event in a subject receiving cancer therapy, the method comprising: a) contacting the subject or a sample isolated from the subject with an agent that detects the presence of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 in the subject or the sample; and b) administering one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 to the subject when a lower than baseline level of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is detected in the subject. In some embodiments, baseline expression is normalized mean expression of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2. In other embodiments, one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is administered to the subject following administration of the cancer therapy to the subject. In certain alternative embodiments, one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is administered to the subject at least about three weeks following administration of the cancer therapy to the subject. In certain embodiments, the administering one or both of lysophosphatidylcholine 16:0 andlysophosphatidylcholine 18:2 is performed via dermal administration (transdermal) of a formula comprising the one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2.

[0014] In certain embodiments, the cancer therapy is chemotherapy, immunotherapy, radiation therapy, and / or cellular therapy. In some embodiments, the cancer therapy is immune checkpoint blocker therapy. In some embodiments, the immune checkpoint blocker therapy is one or more of anti-CTLA4 therapy, anti-PDl therapy, and anti-PDLl therapy. In still other embodiments, the anti- CTLA4 therapy, anti-PDl therapy, or anti-PDLl therapy comprises one or more of Ipilimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab and Durvalumab.

[0015] In particular embodiments, the subject has or is being treated for one or more of an epithelial, a head, breast, endometrium, uterus, ovary, testes, neck, lung, prostate, colon, colorectal, pancreas, esophagus, liver, skin, kidney, adrenal gland and brain cancer or tumor, a metastasis or recurring tumor, cancer or neoplasia, a melanoma, a non-small cell lung cancer (NSCLC) and a head and neck squamous cell cancer (HNSCC).

[0016] In another aspect, there is presently a method of decreasing, reducing, inhibiting, suppressing, limiting, controlling or treating an undesirable or aberrant immune response, immune disorder, inflammatory response, autoimmune disease or inflammation, the method comprising administering to the subject one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2. In certain embodiments, the administering one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is performed via dermal administration (transdermal) of a formula comprising the one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2.

[0017] In alternative aspects, there is presently provided a method of decreasing, reducing, inhibiting, suppressing, limiting, controlling or treating an undesirable or aberrant immune response, immune disorder, inflammatory response, autoimmune disease or inflammation, the method comprising: a) contacting the subject or a sample isolated from the subject with an agent that detects the presence of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 in the subject or the sample; and b) administering one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 to the subject when a lower than baseline level of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is detected in the subject. In certain embodiments, the administering one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is performed via dermal administration (transdermal) of a formula comprising the one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2.

[0018] In particular embodiments, the undesirable or aberrant immune response, immune disorder, inflammatory response, autoimmune disease or inflammation comprises psoriasis, scleroderma, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, polymyalgia rheumatica, inflammatory myopathy, vasculitis, sicca syndrome, hypophysitis, diabetes mellitus, multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus (SLE), autoimmune thyroiditis, atopic dermatitis, eczematous dermatitis, psoriasis, Sjogren's Syndrome, Crohn’s disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, inflammatory bowel disease (IBD), cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener’s granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, interstitial lung fibrosis, Hashimoto's thyroiditis, autoimmune polyglandular syndrome, insulin-dependent diabetes mellitus, insulin-resistant diabetes mellitus, immune-mediated infertility, autoimmune Addison’s disease, pemphigus vulgaris, pemphigus foliaceus, dermatitis herpetiformis, autoimmune alopecia, vitiligo, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, pernicious anemia, Guillain-Barre syndrome, stiff-man syndrome, acute rheumatic fever, sympathetic ophthalmia, Goodpasture's syndrome, systemic necrotizing vasculitis, antiphospholipid syndrome or an allergy, Behcet’s disease, severe combined immunodeficiency (SCID), recombinase activating gene (RAG 1 / 2) deficiency, adenosine deaminase (ADA) deficiency, interleukin receptor common g chain ( c ) deficiency, Janus-associated kinase 3 (JAK3) deficiency and reticular dysgenesis; primary T cell immunodeficiency such as DiGeorge syndrome, Nude syndrome, T cell receptor deficiency, MHC class II deficiency, TAP-2 deficiency (MHC class I deficiency), ZAP70 tyrosine kinase deficiency and purine nucleotide phosphorylase (PNP) deficiency, antibody deficiencies, X-linked agammaglobulinemia (Bruton's tyrosine kinase deficiency), autosomal recessive agammaglobulinemia, Mu heavy chain deficiency, surrogate light chain (g5 / 14.1 ) deficiency, Hyper- IgM syndrome: X-linked (CD40 ligand deficiency) or non-X-linked, Ig heavy chain gene deletion, IgA deficiency, deficiency of IgG subclasses (with or without IgA deficiency), common variable immunodeficiency (CVID), antibody deficiency with normal immunoglobulins; transient hypogammaglobulinemia of infancy, interferon g receptor (IFNGR1, IFNGR2) deficiency, interleukin 12 or interleukin 12 receptor deficiency, immunodeficiency with thymoma, Wiskott- Aldrich syndrome (WAS protein deficiency), ataxia telangiectasia (ATM deficiency), X-linked lymphoproliferative syndrome (SH2D1 A / SAP deficiency), or hyper IgE syndrome.

[0019] In particular embodiments, the small molecule metabolite is administered or modulated in the subject in combination with administration of an immunotherapy to reduce the likelihood of an adverse immune response resulting from administration of the immunotherapy. In certain embodiments, the administering one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is performed via dermal administration (transdermal) of a formula comprising the one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2.

[0020] Tn some embodiments, the subject has cancer. In certain embodiments, a method comprises treating the subject for cancer or a side-effect or symptom or cancer or a cancer treatment

[0021] In certain embodiments, the method comprises treating an inflammatory disease or disorder or an autoimmune disease or disorder. In certain other embodiments, the method comprises treating inflammatory bowel disease or ulcerative colitis.

[0022] In some embodiments, the subject is a mammal. In alternative embodiments, the subject is a human.

[0023] In other aspects, there is presently provided a pharmaceutical composition comprising one or more of the small molecule metabolites disclosed herein or an agent that modulates one or more of the novel small molecule metabolites disclosed herein.

[0024] Certain aspects of the technology are described further in the following description, examples, claims and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings illustrate embodiments of the technology and are not limiting. For clarity and ease of illustration, the drawings are not made to scale and, in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments.

[0026] FIGs. 1A-1B: rLC-MS metabolomics. (FIG. 1A) Extracted ion chromatogram from a single rLC injection. (FIG. IB) Of the 1561 known metabolites in plasma, Applicants tested 267 for detection using rLC-MS. (FIG. 1C) 13C-glucose in plasma. (FIG. ID) Metabolite superclasses detected by rLC-MS.

[0027] FIGs. 2A-2B: Changes in LPCs with 1CB related irAEs. (FIG. 1A) Volcano plot representing odds ratio and P value for association between >8,000 metabolites and ICB related irAEs.Two LPC were found to be highly significant with notable odds ratio. (FIG. IB) Change over time in LPC 16:0 and 18:2 in patients receiving ICB with development of no, mild or severe irAEs.

[0028] FIG. 3: Circulating LPCs. Clustering of 132 LPC metabolites measured in plasma samples from the FINRISK cohort.

[0029] FIGs. 4A-4C: LPC levels in mouse models of autoimmunity. (FIG. 4A) LPC 16:0 and (FIG. 4B) LPC 18:2 levels in humanized CTLA4 mice in relation to irAE Score. (FIG. 4C) LPC levels in mice with DSS induced colitis.

[0030] FIGs. 5A-5C: LPC (18:2) protects against DSS-induced colitis. (FIG. 5A) LPC 18:2 abundance in blood 1 hour after intraperitoneal injection (25 mg / kg). (FIG. SB) Colon length and (FIG. SC) percent weight loss in control (Con), LPC 18:2 (LPC), 2.5% DSS (DSS) or DSS+LPC treated mice.

[0031] FIG. 6 : LPC supplementation does not alter ICB anti-tumor response. Bl 6 tumor volume following combination anti-CTLA4 / anti-PDl (lOOug, ICB) and / or LPC 18:2 supplementation (25mg / kg, LPC).

[0032] FIG. 7 : Power calculations. Power curves are shows for incident (blue) and prevalent (green) autoimmune disease in the FINRISK cohort.

[0033] FIG. 8: Immune cell profiling of B16 tumors by multi-color flow cytometry.

[0034] FIGs. 9A-9D: Identification of two circulating lysolipids depleted in ipilimumab toxicity and autoimmunity. (FIG. 9A) Volcano plot for irAE severity logistic regressions in ipilimumab treated melanoma patients (Cohort 1, n = 65) of 5951 ubiquitous bioactive lipid LC-MS features. Odds ratio normalized to a natural logarithm. (FIG. 9B) Tandem mass spectra of m / z 554.3019, corresponding to LPC (18:2) [M+Cl ]. (FIG. 9C) Mean excursions of LPC (18:2) in Cohort 1, error = SEM. (FIG. 9D) Natural log odds ratio of prevalent autoimmune pathologies in FINRISK-2002 (case: n = 158, control: n = 1712). Error = 95% CI.

[0035] FIGs. 9E-9G: Chemically related LPC (18:2) and LPC (16:0) in ipilimumab patient outcomes. (FIG. 9E) Tandem mass spectral chemical network of 5 irAE- associated LC-MS features corresponding to [M+Cl-] adducted LPC (18:2) and LPC (16:0). (FIG. 9F) Excursion of LPC (16:0) in Cohort 1 ipilimumab treated patient blood plasma (n = 65). Error = SEM. (FIG. 9G) Abundance of LPC (18:2) and LPC (16:0) in baseline sampling from Cohort 1 .

[0036] FIGs. 10A-10C: LPC 18:2) and LPC (16:0) loss is a conserved feature of ICB-irAE. (FIGs. 10A-10B) Maximum excursion of LPC (18:2) (FIG. 10A) and LPC (16:0) (FIG. 10B) in three cohorts of ICB-treated patients, bars = mean. (FIG. 10C) Forest plots of natural log odds ratio of irAE severity for LPC (16:0) (open) and LPC (18:2) (closed). Error = 95% CL

[0037] FIGs. 10D-10E: LPC (18:2) and LPC (16:0) excursion on different ICB treatments. (FIGs. 10D-10E) Maximum excursion of LPC (18:2) (FIG. 10D) and LPC (16:0) (FIG. 10E) in Cohort 2, broken out by anti-PDl monotherapy or anti-PDl combination ipilimumab.

[0038] FIGs. 11A-11E: LPC (18:2) supplementation is protective in two models of colitis. (FIG. 11A) LPC (18:2) (left) and LPC (16:0) (right) abundance in terminal blood of CTLA4h / hC56BL / 6 mice treated with 100 ug anti-human IgGl isotype control (hlgFc), 100 ug ipilimumab (Ipi), or 100 ug ipilimumab + 100 ug anti-mouse PD1 monoclonal antibody (Ipi + anti-PDl). Antibody treatments were administered on Day 10, 13, 16, and 19. Representative of one experiment. (FIG. 11B) LPC (18:2) abundance in terminal blood of control and 5% w / v DSS-treated C56BL / 6 mice. Representative of six independent experiments (FIG. 11C). Scatter plot of composite toxicity score in CTLA4ll / hmice with matched LPC (18:2) abundance in terminal blood. Representative of one experiment. (FIG. 11D) Toxicity score and representative H&E staining in distal colon of CTLA4h / hmice following intraperitoneal administration of 100 ug anti-human IgGl isotype control (hlgFc) or 100 ug ipilimumab + 100 ug anti-mouse PD1 monoclonal antibody (ICB), dosed as above. LPC (18:2) was administered intraperitoneally at 25 mg / kg on Days 10, 13, 16, 19, 22, and 25. Representative of one experiment (FIG. HE). Toxicity score and representative H&E staining in distal colon of DSS-treated mice. LPC (18:2) was administered at 25 mg / kg on Days 0, 2, 4, and 6 of DSS treatment. Representative of three independent experiments. Bars = mean value, significance: Student’s T-Test, * = p < 0.05, ** = p < 0.01.

[0039] FIGs. 11F-11I: Loss and supplementation of LPCs in CTLA4 humanized mice. (FIG. HF) Scatter plot of composite toxicity score in CTLA4h / hmice with matched LPC (16:0) abundance in terminal blood. Error = 95% CI. (FIG. 11G) Blood plasma LPC (18:2) following single dose intraperitoneal supplementation in C57BL / 6 mice. Bar = mean. (FIG. 11H) Toxicity scoring by four criteria in CTLA4h / hmice. Bar = mean. (FIG. HI) Blood analysis by complete blood count in Day 41 CTLA4h / hmice.

[0040] FIGs. 12A-12D: Loss and supplementation of LPCs in DSS-induced colitis. (FIGs. 12A- 12B) Percent weight loss (FIG. 12A) and colon length relative to starting body weight (FIG. 12B) in 5% DSS treated mice following LPC (18:2) supplementation. (FIG. 12C) Toxicity score in cecumand distal colon of 5% DSS treated mice following LPC (16:0) supplementation. (FIG. 12D) Colon length relative to starting body weight in 5% DSS treated mice following supplementation with saturated and unsaturated C18-LPCs.

[0041] FIGs. 13A-13B: LPC and immune feature associations in 500FG and FINRISK 2002. (FIG 13A) Heat map of LPC associations with immune cellularity by multi-color flow cytometry in 500FG (n = 488). Color = effect size with p < 0.05. (FIG. 13B) Quartiles of circulating LPC (18:2) and serum interleukin-8 (IL-8) in FINRTSK-2002.

[0042] FIGs. 14A-14E: LPC (18:2) inversely correlates with neutrophil cellularity in circulation. (FIG. 14A) Significance of association by linear regression between LPC (18:2) and various myeloid (left) and natural killer cell (right) populations in matched blood from healthy individuals (500FG, n = 491, dotted line indicates p = 0.05 following Bonferroni multiple hypothesis correction). (FIG. 14B) Quartiles of LPC (18:2) abundance and neutrophil counts as measured by multi-color flow cytometry (500FG, left) or by blood analyzer (FINRISK-2002, n = 215, right). Bars = mean. (FIG. 14C) Peripheral blood neutrophil counts in C57BL / 6 mice following treatment with 25 mg / kg of the indicated LPC. Representative of two independent experiments. Bar = mean, ** - p < 0.01. (FIG. 14D) Fold change of neutrophil counts relative to baseline sampling in Cohort 1 ipilimumab treated melanoma patients. Error = SEM. (FIG. 14E) Quartiles of LPC (18:2) abundance and neutrophil counts in ICB Cohort 1 and Cohort 2. Bars = mean. Significance is derived from linear regression including patient and timepoint as covariates to control for internal correlation.

[0043] FIGs. 15A-15B: Blood immune cellularity in ipilimumab-treated melanoma patients. (FIG. 15A) Lymphocyte, monocyte, and neutrophil-to-lymphocyte ratio fold changes by blood analyzer in Cohort 1 ipilimumab-treated patients. Error = SEM. (FIG. 15B) Immune cell counts by blood analyzed in Cohort 1 baseline sampling. Bar = mean.

[0044] FIGs. 16A-16B: LPC (18:2) supplementation and ICB response in subcutaneous mouse melanoma. (FIG. 16A) LPC (18:2) abundance in terminal blood plasma from C57BL / 6 mice bearing B16-F10 subcutaneous melanoma. (FIG. 16B) B16-F10 tumor volume following LPC (18:2) supplementation and anti-mouse CTLA4 + anti- mouse PD 1 intraperitoneal treatment.

[0045] FIGs. 17A-17D show circulating LPC 18:2 is reduced ICB-irAE Patients. Figure 17A is a volcano plot of logistic regressions for irAE in ipilimumab-treated melanoma patients (n = 65), highlighting changes in 5951 bioactive lipid metabolites. LPC 18:2 is marked in red, with odds ratio normalized to natural logarithms. FIG. 17B shows the mean excursion of LPC 18:2 in cohort 1 witherror bars representing SEM. FIG. 17C are forest plots showing the natural log odds ratio of irAE severity associated with LPC 18:2, with error bars representing 95% CI. FIG. 17D shows the maximum excursion of LPC 18:2 levels across three cohorts of ICB-treated patients, with bars indicating the mean (cl=melanoma; c2=lung cancer; c3 = mixed solid tumors).

[0046] FIGs. 18A and 18B are scatterplots showing correlation between LPC 18:2 and neutrophils in blood. FIG. 18A are scatter plots and quartile analysis reveal an inverse relationship between LPC 18:2 and neutrophils in healthy individuals, measured via multi-color flow cytometry (500FG cohort, left) and blood analyzer (FINRISK-2002 cohort, right). Linear regression indicates significant negative correlation, with bars representing mean and red lines indicating standard deviation. FIG. 18B are scatterplots showing inverse correlations are also observed in solid tumor patients (lung, breast, head and neck cancer) treated with combination ICB (cohort 2).

[0047] FIGs. 19A-19F show that LPC 18:2 suppresses inflammation in Ipilimumab-induced colitis. FIG. 19A is a schematic of CTLA-4h / h C57B1 / 6 mouse model of ICB-irAEs. FIG. 19B is a scatterplot showing LPC 18:2 concentrations in blood of CTLA4h / h mice treated with isotype control (IgG Ctrl), ipilimumab, or ipilimumab + anti-PD-1. FIG. 19C shows the relationship between LPC 18:2 concentrations and histology score in CTIA-4h / h mice. Linear regression and 95% confidence intervals depicted. FIG. 19D shows the histology scores of distal colon in CTLA4h / h mice. FIG. 19E shows the neutrophil counts and FIG. 19F shows the neutrophil scores in colon.

[0048] FIGs. 20A-20C shows the effect of LPC Supplementation on MC38 tumor growth in hCTLA4h / h / hPDlWhmice. FIG. 20A is a representative schematic of the experimental design. FIG. 20B shows the tumor growth curves showing average tumor volume over time for each treatment group. LPC supplementation in the ICB group resulted in significantly reduced tumor growth compared to ICB alone. Error bars represent SEM. FIG. 20C shows the animal weights during the study. 200,000 MC38 cells per injection.DETAILED DESCRIPTION

[0049] Among the thousands of metabolites present in human circulation, Applicants have pinpointed two specific LPC molecules, 18:2 and 16:0, as closely associated with development of severe irAEs in melanoma patients receiving ipilimumab therapy. Applicants have found that these molecules can be used, by way of example and not by way of limitation, as a biomarker for the prediction of irAE’s developed following administration of a cancer therapy, as well as in methods to treat such irAE’s as well as undesirable or aberrant immune responses, disorders, inflammatoryresponses, autoimmune diseases or inflammation generally, including inflammatory bowel disease and ulcerative colitis.

[0050] In an aspect, a method of preventing irAEs in a patient receiving a treatment for a disease is provided comprising administering a therapy to the patient capable of treating the disease and modulating a level of at least one small molecule metabolite.

[0051] In embodiments, the disease comprises a cancer. In embodiments, the cancer comprises any known cancer and any cancer yet to be discovered. In embodiments, the therapy comprises a cancer immunotherapy. In embodiments, the cancer immunotherapy is any known cancer immunotherapy or any cancer immunotherapy yet to be discovered.

[0052] In embodiments, the cancer immunotherapy comprises an immune checkpoint blocker therapy. In embodiments, the immune checkpoint blocker therapy comprises administering one or more of Ipilimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab, and Durvalumab.

[0053] In embodiments, the at least one small molecule metabolite comprises any one or more the small molecule metabolites listed in Table 5. In embodiments, the at least one small molecule metabolite comprises lysophosphatidylcholine 16:0 or lysophosphatidylcholine 18:2. In embodiments, the small molecule metabolite comprises lysophosphatidylcholine 18:2.

[0054] In embodiments, modulating the level of at least one small molecule metabolite comprises administering a pharmaceutically acceptable amount of the at least one small molecule metabolite. In embodiments, the at least one small molecule metabolite comprises lysophosphatidylcholine 18:2.

[0055] In an aspect, a method is provided comprising modulating a level of at least one small molecule metabolite in a subject to reduce likelihood of irAEs in the subject and treating the subject with a cancer immunotherapy.

[0056] In embodiments, the cancer immunotherapy comprises an immune checkpoint blocker therapy.

[0057] In embodiments, the at least one small molecule metabolite comprises any one or more of the small molecule metabolites listed in Table 5. In embodiments, the at least one small molecule metabolite comprises lysophosphatidylcholine 18:2. In embodiments, modulating a level of at least one small molecule metabolite comprises administering a pharmaceutically acceptable amount of lysophosphatidylcholine 18:2. In certain embodiments, the pharmaceutically acceptable amount oflysophosphatidylcholine 18:2 is administered via dermal administration (transdermal) of a formula comprising lysophosphatidylcholine 18:2.

[0058] In an aspect, a method of modulating an immune response in a subject is provided comprising modulating a level of at least one small molecule metabolite, wherein modulating the immune response prevents the development or reduces the likelihood of irAEs.

[0059] In embodiments, the at least one small molecule metabolite comprises lysophosphatidylcholine 18:2. In embodiments, modulating the level of the at least one small molecule metabolite comprises administering a pharmaceutically acceptable amount of lysophosphatidylcholine 18:2.

[0060] In embodiments, the method further comprises treating the subject with a cancer immunotherapy.

[0061] In embodiments, modulating the immune response comprises modulating levels of neutrophils. In embodiments, modulating levels of neutrophils results from modulating the level of the at least one small molecule metabolite.

[0062] The following examples are illustrative of procedures which can be used in various instances in carrying the disclosure into effect.

[0063] I. Definitions

[0064] As used herein, the term “small molecule” means a low molecular weight organic and / or inorganic compound. In general, a “small molecule” is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not a protein or polypeptide (e.g. , is not an oligopeptide or peptide). In some embodiments, a small molecule is not a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not a polysaccharide. In some embodiments, a small molecule does not comprise a polysaccharide (e.g., is not a glycoprotein, proteoglycan, glycolipid, etc.).

[0065] As used herein, the terms “detect” or “detecting” generally refer to obtaining information. Detecting or determining can utilize any of a variety of techniques available to those skilled in the art, including for example specific techniques explicitly referred to herein. Detecting may involve manipulation of a physical sample, consideration and / or manipulation of data or information, for example utilizing a computer or other processing unit adapted to perform a relevant analysis, and / or receiving relevant information and / or materials from a source. Detecting may also mean comparing an obtained value to a known value, such as a known test value, a known control value, or a threshold value. Detecting may also mean forming a conclusion based on the difference between the obtained value and the known value.

[0066] The terms “contact”, “contacting” or “bringing into contact” describe placement in physical association for example, in solid and / or liquid form. For lipid detection, the process may involve contacting or combining a biological sample with one or more lipid-specific reagents, such as detection probes, solvents, or derivatization agents, in a solution for analysis.

[0067] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a ligand is disclosed and discussed and a number of modifications that can be made to a number of molecules including the ligand are discussed, each and every combination and permutation of ligand and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials.

[0068] These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific form or combination of forms of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0069] All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the forms and does not pose a limitation on the scope of the forms unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0070] 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.

[0071] Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 5%; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 2%; in other forms the values can range in value either above or below the stated value in a range of approx. + / - 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied.

[0072] IL Biomarkers and Method of Use Thereof

[0073] A. Source of the Biomarkers

[0074] Biomarkers of the disclosure include LPC (18:2) and LPC (16:0). 18:2 and 16:0 are lysophospholipids. Unlike phospholipids, which have two fatty acid chains, lysophospholipids have a single fatty acid, even though their concentrations are much lower than those of conventional phospholipids in cells and tissues. Like phospholipids, lysophospholipids are classified by their polar head structure. For example, lysophosphatidylcholine (LPC) has choline in its polar head and lysophosphatidylserine (LysoPS) has L-serine. Lysophospholipids can also be classified as glycerolysophospholipids or sphingolysophospholipids depending on whether they have a glycerol or sphingosine backbone, respectively.

[0075] Any biological sample that is suitable for lipid analysis can be used for the detection of the biomarkers. Generally, biological samples that are often collected for lipid analysis are from plasma and serum, and to a lesser extent, urine. The biomarkers can be detected in a biological sample from any subject of interest. Generally, the subject of interest is a mammal such as a mouse, domestic animal, or a human, including prenatal humans. In some embodiments, the subject of interest may also include livestock (e.g., cattle, pigs, sheep, or goats), companion animals (e.g., dogs or cats), or non-human primates. The biological sample for the detection of lipid biomarkers (LPC 18:2 and / or LPC 16:0) can be a tissue sample and / or a fluid sample. In some embodiments, the biological sample for the detection of lipid biomarkers can be a tissue sample e.g., needle biopsy specimens, surgical specimens, and tissue biopsy samples. In other embodiments, the biological sample for the detection of lipid biomarkers can be a fluid sample e.g., blood, plasma, serum, cerebrospinal fluid, urine, ascitic fluid, peritoneal fluid, pleural fluid, lymph, saliva, sputum, gynecological fluids, nasal swabs, oral swabs, vaginal swabs, and other bodily fluids, secretions, and excretions. In some embodiments, the preferred sample for the detection of the lipid biomarkers include plasma and / or cerebrospinal fluid.

[0076] B. Methods of Detecting Biomarkers

[0077] 1. Extraction of Lipids

[0078] The disclosed methods of detecting the lipid biomarkers can include a step of extracting the lipid biomarkers from the biological sample. The extraction method can be chosen based on factors such as lipid structure, molecular weight, polarity, sample origin (human, animal, plant, or food), and physical state (fluid or tissue). Polarity is particularly significant in selecting extraction solvents, as it determines the efficiency of lipid recovery. Additionally, the complexity of the sample matrix poses challenges, necessitating methods to minimize matrix effects by selectively removing non-lipid components like proteins. Protein precipitation (PP) is commonly employed to remove these interferents before lipid extraction, using solvents that facilitate both processes.

[0079] Several lipid extraction techniques are available, each with its advantages and limitations. Single organic solvent extraction (SOSE), using polar solvents such as acetonitrile (ACN) or methanol (MeOH), is simple but ineffective for extracting neutral or non-polar lipids. One-phase extraction (OPE), which utilizes a combination of miscible solvents like butanol and methanol (e.g., the BUME method), has gained popularity for extracting less polar lipids due to its simplicity and effectiveness. Traditional lipid extraction methods, such as the Folch and Bligh-Dyer methods, are considered the gold standards for lipid extraction from tissues. These methods use chloroformmethanol mixtures (CHCL:MeOH) but have been modified to replace toxic solvents withalternatives like propanol, isopropanol, ethyl acetate, or ethanol. A significant modification is Matyash’ s method, which employs methyl tert-butyl ether (MTBE) to extract lipids in the upper organic phase, simplifying lipid recovery. However, MTBE's high volatility requires careful reproducibility control.

[0080] Advanced techniques such as solid-phase extraction (SPE) and solid-phase microextraction (SPME) offer targeted lipid extraction, particularly in lipid studies. SPE is frequently used as a cleanup step after liquid-liquid extraction (LLE), with silica and aminopropyl columns suitable for polar lipids and reversed-phase C8 or Cl 8 columns for non-polar lipids. A recent innovation involves superabsorbent polymer powders (SAP) in spin columns, which have demonstrated high extraction efficiency, lower detection limits, and improved reproducibility. Additionally, ultrasound-assisted extraction (UAE) has been integrated with OPE and LLE to enhance extraction efficiency, as demonstrated in methods for profiling egg yolk lipids.

[0081] Other less commonly used extraction techniques include microwave-assisted extraction (MAE), which risks degrading thermolabile analytes, and Soxhlet extraction (SE), which, despite similar recoveries to the Folch method, has drawbacks such as long extraction times, high solvent consumption, and potential degradation of sensitive lipids. A more environmentally friendly alternative is supercritical fluid extraction (SFE), which uses CO2 as a non-toxic extraction medium. SFE is highly effective for non-polar lipid extraction, and adding modifiers like ethanol, methanol, or ethyl acetate allows for polar lipid extraction. Studies have demonstrated that SFE can achieve higher extraction efficiencies than classical methods.

[0082] Beyond extraction, derivatization is often employed to chemically modify lipids, primarily in gas chromatography-mass spectrometry (GC-MS) analysis. Derivatization enhances analyte stability, selectivity, and ionization efficiency, with isotopic labeling (IL) offering additional quantification benefits. While derivatization is commonly used for fatty acid, glycerolipid, sphingolipid, phospholipid, and steroid analysis, its main drawback is the chemical alteration of lipids, which can lead to the loss of molecular information. Despite this, recent advancements in derivatization techniques, such as highly fluorescent reagents for trans-fatty acid detection, continue to expand its applications. However, the growing preference for liquid chromatography-mass spectrometry (LC-MS) over GC-MS suggests that future efforts may focus on improving derivatization methods to enhance speed and efficiency while preserving analyte integrity.

[0083] 2. Molecular Analysis

[0084] Generally, the level of the expression of the biomarker can be determined by measuring the amount of the biomarker. As discussed in more detail below, detection and measurement of LPC (18:2) and LPC (16:0) can be accomplished using instrumental techniques such as nuclear magnetic resonance (NMR), mass spectrometry, or chromatography (e.g., gas chromatography or thin-layer chromatography.

[0085] NMR spectroscopy (i.e., 1H, 13C, 31P) allows the elucidation of lipid structures as well as qualitative and quantitative analysis. For NMR analysis, extracted lipids are typically dissolved in an appropriate solvent, such as deuterated methanol (MeOH) or chloroform (CHCh), immediately before analysis for high spectral resolution and accuracy. NMR is particularly useful for analysis and characterization of membrane lipid profiles or interactions between proteins (or peptides) and lipids.

[0086] Mass spectrometry (MS) provides the same data on the analyzed samples as NMR but has a higher sensitivity than NMR. The use of MS for lipid analysis is more frequent due to the variety of techniques it offers, whether within the framework of shotgun lipid analysis or the possibility of connection with effective separation techniques such as LC or even today less used gas chromatography (GC) or thin-layer chromatography (TLC), which was used in past. In addition, a relatively large number of different ion sources or mass analyzers are commercially available for both identification and quantification or MS lipids imaging. Any suitable MS procedure can be used for the detection of the lipid biomarkers including but not limited to Reverse-phase ultra-high- performance liquid chromatography / mass spectrometry (RP-UHPLC / MS), matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), shotgun low-resolution mass spectrometry (shotgun LR-MS), shotgun high-resolution mass spectrometry (shotgun HR-MS), and ultra-high- performance supercritical fluid chromatography / mass spectrometry (UHPSFC / MS). MS-based procedures are described in further detail in for example, Salihovic, et al., Current Opinion in Chemical Biology, 76(102370), 2023; Zullig and Kofeler, Mass Spectrometry Reviews, 40: 162-176 (2021); Anh et al., Molecules, 29(24): 5934 (2024), all of which are incorporated herein by reference in their entireties.

[0087] In lipid analysis, liquid chromatography is most often used, as well as direct infusion (DI)- MS. Extracted lipids can be analyzed directly using MS without their previous separation. In the case of lipid analyses, this technique is also referred to as shotgun lipidomics. It represents a simple but powerful tool for fast, reliable, sensitive, and reproducible lipid analyses, while a triple quadrupole (QqQ) or hybrid mass analyzers like Orbitrap, quadrupole-time of flight (QTOF) or Fourier transformion cyclotron resonance (FT-ICR) can be used as mass analyzers. Nielsen and colleagues (2020) describe the use of a high-resolution mass spectrometer (HRMS) in shotgun lipidomics (Comprehensive Evaluation of a Quantitative Shotgun Lipidomics Platform for Mammalian Sample Analysis on a High-Resolution Mass Spectrometer. J. Am. Soc. Mass Spectrom. 2020, 31, 894-907). In this example, Nielsen and colleagues (2020) used a hybrid quadrupole-Orbitrap mass spectrometer with Fourier transformation (FT) equipped with nano-electrospray ionization (nano-ESI) working either in positive or negative mode to the quantitative shotgun lipidomic analysis of the mammalian sample. This approach quantification of sub-picomole levels in 35 of 38 lipid classes. Another form of mass spectrometry that can be used for lipid analysis is multi-dimensional mass spectrometrybased shotgun lipidomics (MDMS-SL) or differential mobility spectrometry (DMS).

[0088] Electrospray ionization (ESI) is widely used in flow injection-targeted metabolomics and lipid analysis due to its shorter runtime, cost-effectiveness, and improved contamination control compared to LC-MS. Samples can be introduced via an LC autosampler or continuous infusion syringes, and analyses commonly utilize tandem or high-resolution mass spectrometers, such as QTOF systems, with methods like selected ion monitoring or multiple reaction monitoring (MRM). Flow injection lipid analysis often employs class-specific internal standards (ISs) for quantification, though certain lipid subclasses, such as cholesteryl esters (CEs), require species-specific response factors. High-throughput lipid analysis has been facilitated by commercial kits, while recent advancements, such as the all-ion fragmentation approach and nano-ESI direct-infusion MS, have further improved lipid class quantification and minimized ionization suppression or enhancement effects.

[0089] In one particular form, the instrumental technique used for lipid analysis is liquid chromatography-tandem mass spectrometry (LC-TD / MS). Liquid chromatography (LC) is incorporated into tandem mass spectrometry (MS / MS) as a front-end separation technique to increase analyte resolution and reduce matrix interference before mass spectrometric detection. In LC-MS / MS, the sample is first injected into the LC system, where it is carried by a mobile phase through a chromatographic column. The column, packed with a stationary phase, separates analytes based on their physicochemical properties, such as polarity, hydrophobicity, and molecular weight. Common separation techniques include reverse-phase liquid chromatography (RPLC) for nonpolar and moderately polar compounds and hydrophilic interaction liquid chromatography (HILIC) for polar analytes. Once separated, analytes elute from the column at different retention times and are introduced into the mass spectrometer through an ionization source, such as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), which converts them into charged ions.These ions enter the first mass analyzer (MSI), where precursor ions of interest are selected based on their mass-to-charge ratio (m / z). The selected ions then undergo fragmentation in a collision cell, generating fragment ions that are analyzed in the second mass analyzer (MS2). This process provides structural information and enhances the selectivity and sensitivity of the analysis. By integrating LC with tandem MS, LC-MS / MS facilitates the precise separation, identification, and quantification of complex lipid mixtures with high specificity. Detailed description of LC-TD / MS can be found for example in Thomas, et al., Nature reviews methods primers, 2:96 (2022), the contents of which are incorporated herein by reference in its entirety.

[0090] Liquid chromatography-mass spectrometry (LC-MS) offers some advantages over flow injection MS, including the ability to separate compounds — such as isobars and isomers — based on their physicochemical properties and to mitigate ion suppression effects. In targeted metabolomics and lipid analysis, reverse-phase LC (RPLC) and hydrophilic interaction chromatography (HILIC) are commonly employed for separation. RPLC-based targeted metabolomics typically utilizes short microbore columns with fused-core particles and CIS-modified sorbents, along with acetonitrile- water or methanol-water gradients containing buffer modifiers to enhance selectivity and sensitivity. Alternative approaches, such as biphenyl column-based methods, have been developed for the absolute quantification of metabolites in complex biological samples, using multitargeted internal calibration to improve accuracy. For targeted lipid analysis, mobile phases and additives are developed for specific lipid subclasses, often incorporating strong binary solvent systems like isopropanol or tetrahydrofuran, along with formic acid, acetic acid, or ammonium-based buffers. C 18 and C8 columns are frequently used, as demonstrated in studies quantifying polyunsaturated fatty acids and oxylipins. However, methods to monitor ionization changes throughout RP gradients are sometimes employed due to challenges in RPLC lipid quantification, such as discrepancies between analytes and internal standards during ionization. Additionally, RPLC can accommodate highly polar metabolites through ion pair liquid chromatography (IP-LC), which employs ion-pairing reagents with both hydrophobic and charged functional groups to improve analyte retention. Common reagents include perfluorocarboxylic acids and alkyl sulfonic acids for cations, and ammonium or tetraalkylammonium compounds for anions. IP-LC has been used to improve the separation of phosphorylated and other highly polar metabolites.

[0091] Another MS-based technique that can be used is Mass spectrometry imaging (MSI). MSI represents a group of direct MS label-free visualization techniques that do not require sample pretreatment, as needed in other discussed methods. In MSI techniques, only a thin slice of sample is required. It is usually attached to a suitable surface and directly analyzed. In conjunction with MSI,soft ionization techniques such as desorption electrospray ionization (DESI), secondary ion MS (SIMS) or matrix assisted laser desorption / ionization (MALDI) are used.

[0092] In SIMS, the primary ion beam (Ga, Si or Cs) is accelerated to bombard the surface of the sample and to release secondary ions that can be detected by MS. SIMS has high spatial resolution and thus has the capability of analyzing surfaces of cell or tissues on the molecular level. SIMS is most commonly utilized with a TOF analyzer (TOF-SIMS). Despite the fact that applications of SIMS in lipid analysis starts much later than MALDI, developments in SIMS such as introduction of nanoSIMS or cluster ion beams (i.e., Bi3+Au3+, Au9+) that are able to reduce secondary ions fragmentation has led to improved spatial resolution and analytical sensitivity. A single-cell lipid study was performed using TOF-SIMS analysis of mammalian cells (cardiomyocytes (CMs)). TOF- SIMS surface analyses were performed using primary cluster ion beams Ar2ooo+ (for intracellular surfaces) and Bi3+(for cell surfaces and intracellular surfaces images).

[0093] In MALDI, the surface of matrix-coated sample is irradiated by a laser under vac-uum or at atmospheric pressure (AP-MALDI). For good ionization of lipids in MALDI-MSI, an appropriate matrix should be chosen, i.e., 9-aminoacridine (9-AA), 2,5-dihydroxybenzoic acid (DHB) or N-(l- naphthyl)ethylenediamine hydrochloride. In addition to MALDI, water-assisted laser desorption ionization (WALDI)-MSI represents an alternative approach where endogenous H2O is used as the MALDI matrix. Improvements in instrumentation and bioinformatics have allowed improvements in MALDI-MSI techniques to be capable of lipid classes and species identification and semiquantification with no need to use chromatographic separation; it is therefore considered as a common tool for the study of lipids.

[0094] C. Diagnosis

[0095] 1. Single Markers

[0096] The hiomarkers can be used in diagnostic tests to assess biomarker-related disease and disorder status in a subject (e.g., subjects with irAE or other conditions that would benefit from LPC (18:2) and / or LPC (16:0) treatment). For example, disease status includes, without limitation, the presence or absence of disease, the risk of developing disease, the stage of the disease, the progress of disease (e.g., progress of disease or remission of disease over time) and the effectiveness or response to treatment of disease. Based on this status, further procedures may be indicated, including additional diagnostic tests or therapeutic procedures or regimens. Representative diseases and therapies are discussed in more detail below.

[0097] The method typically involves, first, measuring the selected biomarker in a subject sample using the methods described herein, and, second, comparing the measurement with a diagnostic amount or cut-off that distinguishes a positive biomarker-related disease and disorder status from a negative biomarker-related disease and disorder status. The diagnostic amount represents a measured amount of a biomarker above which a subject is classified as having a particular status.

[0098] For example, because the LPC (18:2) and / or LPC (16:0) can be reduced compared to normal during irAE and / or other related diseases and disorders in which neutrophils are increased and / or overactive, then a measured amount below the diagnostic cutoff provides a diagnosis or status of the diseases and disorders.

[0099] As is well understood in the art, by adjusting the particular diagnostic cut-off used in an assay, one can increase sensitivity or specificity of the diagnostic assay depending on the preference of the diagnostician. The particular diagnostic cut-off can be determined, for example, by measuring the amount of the biomarker in a statistically significant number of samples from subjects with different statuses and drawing the cut-off to suit the diagnostician's desired levels of specificity and sensitivity.

[0100] 2. Combinations of Markers

[0101] While individual biomarkers are useful diagnostic biomarkers, a combination of biomarkers may provide greater predictive value of a particular status than single biomarkers alone. Specifically, the detection of a plurality of biomarkers in a sample can increase the sensitivity and / or specificity of the test. Thus, in one embodiment, two or more biomarkers can be detected and used to assess the diseases and disorders in a subject.

[0102] D. Determining Risk of Developing Disease

[0103] Methods for determining the risk of developing disease in a subject are also provided. Biomarker amounts or patterns can be characteristic of various risk states, e.g., high, medium, or low. The risk of developing a disease is determined by measuring the relevant biomarker or biomarkers and then either submitting them to a classification algorithm or comparing them with a reference amount and / or pattern of biomarkers that is associated with the particular risk level.

[0104] E. Determining Stage of Disease

[0105] Another embodiment provides methods for determining the stage of disease in a subject. Each stage of the disease can have a characteristic amount of a biomarker or relative amounts of a set of biomarkers (a pattern). The stage of a disease is determined by measuring the relevant biomarker or biomarkers and then either submitting them to a classification algorithm or comparing them with a reference amount and / or pattern of biomarkers that is associated with the particular stage.

[0106] F. Determining Course (Progression / Remission) of Disease

[0107] Still another embodiment provides methods for determining the course of disease in a subject. Disease course refers to changes in disease status over time, including disease progression (worsening) and disease regression (improvement). Over time, the amounts or relative amounts (e.g., the pattern) of the change in biomarkers. This method involves measuring one or more biomarkers in a subject at least two different time points, e.g., a first time and a second time, and comparing the change in amounts, if any. The course of disease is determined based on these comparisons. Similarly, this method is useful for determining the response to treatment. If treatment is effective, then the biomarkers will trend toward normal, while if treatment is ineffective, the biomarkers will trend toward disease indications.

[0108] G. Subject Management

[0109] In certain embodiments of the method including the detection and / or analysis of one or more biomarkers further include managing subject treatment based on the status. Such management includes the actions of the physician or clinician subsequent to disease and disorder status. For example, if a physician makes a diagnosis, then a certain regime of treatment, such as prescription or administration of chemotherapy, radiation, immunotherapy, including, but not limited to administration of the compositions discussed in more detail below, might follow. Alternatively, a diagnosis of non-disease or disorder might be followed with further testing to determine a specific disease that the patient might be suffering from. Also, if the diagnostic test gives an inconclusive result on the disease and disorder, further tests may be required.

[0110] One embodiment provides a method for selecting a subject for treatment by detecting the presence or quantity of one or more biomarkers provided herein in a sample from a subject suspected of a disease and disorder such as irAE if certain biomarkers or levels of biomarkers are detected in the sample. Such treatments can be those known to be effective and / or preferred fortreating subjects with aberrant biomarker-positive conditions (e.g., administration of LPC (18.2) and / or LPC (16:0)).

[0111] In some embodiments, the methods additionally or alternatively include identifying the subject as not having a biomarker-related disease and disorder, when the test is negative. Thus, although the subject may have cancer or another disease or disorder, the subject can be identified as negative for aberrant biomarker-related condition. Such embodiments may lead to selection of alternative treatments and may avoid treatments known to be effective or preferred for treating subjects with aberrant biomarker-positive conditions, and / or may include treatments that are known not to be effective and / or preferred for treating subjects with aberrant biomarker-positive conditions.

[0112] Additional embodiments relate to the communication of assay results or diagnoses or both to technicians, physicians or patients, for example. In certain embodiments, computers will be used to communicate assay results or diagnoses or both to interested parties, e.g.: physicians and their patients. In some embodiments, the assays will be performed or the assay results analyzed in a country or jurisdiction which differs from the country or jurisdiction to which the results or diagnoses are communicated.

[0113] In a preferred embodiment a diagnosis based on the presence or absence in a test subject of any of the disclosed biomarkers is communicated to the subject as soon as possible after the diagnosis is obtained. The diagnosis may be communicated to the subject by the subject's treating physician. Alternatively, the diagnosis may be sent to a test subject by email or communicated to the subject by phone. A computer may be used to communicate the diagnosis by email or phone. In certain embodiments, the message containing results of a diagnostic test may be generated and delivered automatically to the subject using a combination of computer hardware and software which will be familiar to artisans skilled in telecommunications. In certain embodiments all or some of the method steps, including the assaying of samples, diagnosing of diseases, and communicating of assay results or diagnoses, may be carried out in diverse (e.g., foreign) jurisdictions.

[0114] H. Biomarkers in Screening Assays

[0115] The biomarkers can be used to screen for compounds that modulate the expression of the biomarkers in vitro or in vivo, which compounds in turn may be useful in treating or preventing a biomarker-related disease and disorder in patients.

[0116] Test compounds capable of modulating the presence and / or expression of any of the biomarkers in the subject may be administered to patients who are suffering from or are at risk of developing a biomarker-related disease or disorder.

[0117] At the clinical level, screening a test compound includes obtaining samples from test subjects before and after the subjects have been exposed to a test compound. The levels in the samples of one or more of the biomarkers can be measured and analyzed to determine whether the levels of the hiomarkers change after exposure to a test compound. The samples can be analyzed by any appropriate means known to one of skill in the art including e.g., by the means described herein. In a further embodiment, the changes in the level of expression of one or more of the biomarkers can be measured using in vitro methods and materials. For example, human tissue cultured cells which express, or are capable of expressing, one or more of the biomarkers may be contacted with test compounds. Subjects who have been treated with test compounds will be routinely examined for any physiological effects which may result from the treatment. In particular, the test compounds will be evaluated for their ability to decrease disease likelihood in a subject.

[0118] I. Assessing the Effectiveness of Treatment or Risk for Developing a Biomarker-related Disease and Disorder

[0119] Methods for determining the course of a biomarker-related disease or disorder in a subject are also provided. Disease course refers to changes in disease status over time, including disease progression (worsening) and disease regression (improvement). Over time, the amounts or relative amounts (e.g., the pattern) of the biomarkers changes. Accordingly, this method involves measuring one or more biomarkers in a subject at least two different time points, e.g., a first time and a second time, and comparing the change in amounts, if any. The course of disease is determined based on these comparisons. Similarly, this method is useful for determining the response to treatment. If a treatment is effective, then the biomarkers will trend toward normal, while if treatment is ineffective, the biomarkers will trend toward disease indications.

[0120] In yet another example, the biomarkers can be used in studies to determine if the subject is at risk for developing a biomarker-related disease and disorder.

[0121] III. Methods of Treatment

[0122] Methods of treating a subject in need thereof are also provided.

[0123] The methods can stand alone or be carried out in combination with any of the other methods provided herein.

[0124] The methods typically include administering a subject in need there, e.g., a subject with irAE, and an effective amount of LPC (18.2) and / or LPC (16:0). In some forms, the subject has cancer. In some forms the subject is being treated with one or more immune checkpoint modulators (e.g., immune checkpoint blockers).

[0125] A representative but non-limiting list of cancers that the compositions can be used to treat include cancers of the blood and lymphatic system (including leukemias, Hodgkin’s lymphomas, non- Hodgkin’ s lymphomas, solitary plasmacytoma, multiple myeloma), cancers of the genitourinary system (including prostate cancer, bladder cancer, renal cancer, urethral cancer, penile cancer, testicular cancer,), cancers of the nervous system (including mengiomas, gliomas, glioblastomas, ependymomas) cancers of the head and neck (including squamous cell carcinomas of the oral cavity, nasal cavity, nasopharyngeal cavity, oropharyngeal cavity, larynx, and paranasal sinuses), lung cancers (including small cell and non-small cell lung cancer), gynecologic cancers (including cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer ovarian and fallopian tube cancer), gastrointestinal cancers (including gastric, small bowel, colorectal, liver, hepatobiliary, and pancreatic cancers), skin cancers (including melanoma, squamous cell carcinomas, and basal cell carcinomas), breast cancer (including ductal and lobular cancer and triple negative breast cancers), and pediatric cancers (including neuroblastoma, Ewing’s sarcoma, Wilms tumor, medulloblastoma). Accordingly, in some embodiments, the present disclosure relates to a method of treating breast, ovarian, colon, prostate, lung, brain, skin, liver, stomach, pancreatic or blood based cancer.

[0126] In some forms, the subject has an autoimmune or inflammatory disease or disorder, or is a subject of a transplantation.

[0127] Representative inflammatory responses or autoimmune diseases that can be treated according to the disclosed methods include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, anklosing spondylitis, antiphospholipid syndrome, autoimmune Addison’s disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (alps), autoimmune thrombocytopenic purpura (ATP), Behcet’s disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue syndrome immune deficiency, syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, Crest syndrome, Crohn’s disease, Dego’s disease, dermatomyositis, dermatomyositis - juvenile, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia - fibromyositis, grave’s disease, guillain-barre, hashimoto’s thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), Iga nephropathy, insulin dependent diabetes (Type I), juvenile arthritis, Meniere’s disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglancular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud’s phenomenon, Reiter’s syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren’s syndrome, stiff-man syndrome, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener’s granulomatosis.

[0128] The route of administration can be any suitable route including, but not limited to, oral, parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), and transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.

[0129] In some forms, the composition is administered to the subject’s skin (i.e., topically). In a more specific form, the composition is applied to the subject’s skin near and / or around and / or under the eyes. In some forms, the formulation is an eye or ocular cream, for example a skin product specifically formulated to address the delicate skin around the eyes. The formulation may also increase hydration.

[0130] In some forms, the subject has an inflammatory condition characterized by aberrant neutrophil activity. One non-limiting example is acne vulgaris, a skin disorder caused by changes in pilosebaceous units (e.g., skin structures including hair follicles and associated sebaceous glands). Other inflammatory conditions include but are not limited to cellulitis, erysipelas, impetigo, folliculitis, rosacea, drug-induced neutrophilic dermatoses, such as Sweet's syndrome.

[0131] A. Compositions

[0132] Compositions for use in the disclosed methods typically are or include LPC (18.2) and / or LPC (16:0). Also provided are delivery vehicles and pharmaceutical compositions for assisting in the delivery and persistence of an effective amount of LPC (18.2) and / or LPC (16:0) in a subject in need thereon.

[0133] B. Delivery Vehicles

[0134] The disclosed compounds can be administered and taken up into the cells of a subject with or without the aid of a delivery vehicle. Appropriate delivery vehicles for the disclosed compounds are known in the art and can be selected to suit the particular compound.

[0135] Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, Schwartzenberger et al., Blood 87:472-478 (1996)). For example in some embodiments, the compound is delivered via a liposome. Commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as well as other liposomes developed according to procedures standard in the art are well known. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.).

[0136] In some embodiments, the delivery vehicle is incorporated into or encapsulated by a nanoparticle, microparticle, micelle, synthetic lipoprotein particle, or carbon nanotube. For example, the compositions can be incorporated into a vehicle such as polymeric microparticles which provide controlled release of the compound. In some embodiments, release of the drug(s) is controlled by diffusion of the compound out of the microparticles and / or degradation of the polymeric particles by hydrolysis and / or enzymatic degradation. Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives. Polymers which are slowly soluble and form a gel in an aqueous environment, such as hydroxypropyl methylcellulose or polyethylene oxide may also be suitable as materials for drug containing microparticles. Other polymers include, but are not limited to, polyanhydrides, poly (ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybut rate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof.

[0137] C. Formulations

[0138] The disclosed compounds can be formulated in a pharmaceutical composition. Pharmaceutical compositions can be for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), enteral, transdermal (either passively or usingiontophoresis or electroporation), or transmucosal (nasal, pulmonary, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.

[0139] The compositions can be administered systemically.

[0140] Drugs can be formulated for immediate release, extended release, or modified release. A delayed release dosage form is one that releases a drug (or drugs) at a time other than promptly after administration. An extended release dosage form is one that allows at least a twofold reduction in dosing frequency as compared to that drug presented as a conventional dosage form (e.g. as a solution or prompt drug-releasing, conventional solid dosage form). A modified release dosage form is one for which the drug release characteristics of time course and / or location are chosen to accomplish therapeutic or convenience objectives not offered by conventional dosage forms such as solutions, ointments, or promptly dissolving dosage forms. Delayed release and extended release dosage forms and their combinations are types of modified release dosage forms.

[0141] Formulations are typically prepared using a pharmaceutically acceptable “carrier” composed of materials that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. The “carrier” is all components present in the pharmaceutical formulation other than the active ingredient or ingredients. The term “carrier” includes, but is not limited to, diluents, binders, lubricants, disintegrators, fillers, and coating compositions.

[0142] “Carrier” also includes all components of the coating composition which may include plasticizers, pigments, colorants, stabilizing agents, and glidants. The delayed release dosage formulations may be prepared as described in references such as “Pharmaceutical dosage form tablets”, eds. Liberman et al. (New York, Marcel Dekker, Inc., 1989), “Remington - The science and practice of pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6thEdition, Ansel et.al., (Media, PA: Williams and Wilkins, 1995) which provides information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.

[0143] The compound can be administered to a subject with or without the aid of a delivery vehicle. Appropriate delivery vehicles for the compounds are known in the art and can be selected to suit the particular active agent. For example, in some embodiments, the active agent(s) isincorporated into or encapsulated by, or bound to, a nanoparticle, microparticle, micelle, synthetic lipoprotein particle, or carbon nanotube. For example, the compositions can be incorporated into a vehicle such as polymeric particles which provide controlled release of the active agent(s). In some embodiments, release of the drug(s) is controlled by diffusion of the active agent(s) out of the particles and / or degradation of the polymeric particles by hydrolysis and / or enzymatic degradation.

[0144] Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives. Polymers which are slowly soluble and form a gel in an aqueous environment, such as hydroxypropyl methylcellulose or polyethylene oxide, may also be suitable as materials for drug containing particles or particles. Other polymers include, but are not limited to, polyanhydrides, poly (ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide- co-glycolide) (PLGA), poly-3-hydroxybut rate (PHB) and copolymers thereof, poly-4- hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof. In some embodiments, both agents are incorporated into the same particles and are formulated for release at different times and / or over different time periods. For example, in some embodiments, one of the agents is released entirely from the particles before release of the second agent begins. In other embodiments, release of the first agent begins followed by release of the second agent before the all of the first agent is released. In still other embodiments, both agents are released at the same time over the same period of time or over different periods of time.

[0145] 1. Formulations for Parenteral Administration

[0146] Compounds and pharmaceutical compositions thereof can be administered in an aqueous solution, by parenteral injection. The formulation may also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of the active agent(s) and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions include diluents sterile water, buffered saline of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and optionally, additives such as detergents and solubilizing agents (e.g., TWEEN® 20, TWEEN® 80 also referred to as POLYSORBATE® 20 or 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be lyophilized and redissolved / resuspended immediately before use. The formulation may be sterilized by, for example, filtration through a bacteria retaining filter,by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.

[0147] 2. Oral Immediate Release Formulations

[0148] Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non- gelatin capsules can prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art.

[0149] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name Eudragit® (Roth Pharma, Westerstadt, Germany), Zein, shellac, and polysaccharides.

[0150] Additionally, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.

[0151] Optional pharmaceutically acceptable excipients present in the drug-containing tablets, beads, granules or particles include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants. Diluents, also termed "fillers," are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, , dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powder sugar.

[0152] Binders are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydorxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers,aminoalkyl methacrylate copolymers, poly aery lie acid / polymethacrylic acid and polyvinylpyrrolidone.

[0153] Lubricants are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.

[0154] Disintegrants are used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as cross-linked PVP (Polyplasdone XL from GAF Chemical Corp).

[0155] Stabilizers are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions.

[0156] Surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, POLOXAMER® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.- iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

[0157] If desired, the tablets, beads granules or particles may also contain minor amount of nontoxic auxiliary substances such as wetting or emulsifying agents, dyes, pH buffering agents, and preservatives.

[0158] 3. Extended release dosage forms

[0159] The extended release formulations are generally prepared as diffusion or osmotic systems, for example, as described in “Remington - The science and practice of pharmacy” (20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000). A diffusion system typically consists of two types of de vices, reservoir and matrix, and is well known and described in the art. The matrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form. The three major types of materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include, but not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and carbopol 934, polyethylene oxides. Fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate.

[0160] Alternatively, extended release formulations can be prepared using osmotic systems or by applying a semi -permeable coating to the dosage form. In the latter case, the desired drug release profile can be achieved by combining low permeable and high permeable coating materials in suitable proportion.

[0161] The devices with different drug release mechanisms described above could be combined in a final dosage form having single or multiple units. Examples of multiple units include multilayer tablets, capsules containing tablets, beads, granules, etc.

[0162] An immediate release portion can be added to the extended release system by means of either applying an immediate release layer on top of the extended release core using coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads.

[0163] Extended release tablets containing hydrophilic polymers are prepared by techniques commonly known in the art such as direct compression, wet granulation, or dry granulation processes. Their formulations usually incorporate polymers, diluents, binders, and lubricants as well as the active pharmaceutical ingredient. The usual diluents include inert powdered substances such as any of many different kinds of starch, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphateor sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidine can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders. A lubricant is necessary in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.

[0164] Extended release tablets containing wax materials are generally prepared using methods known in the art such as a direct blend method, a congealing method, and an aqueous dispersion method. In a congealing method, the drug is mixed with a wax material and either spray- congealed or congealed and screened and processed.

[0165] 4. Delayed release dosage forms

[0166] Delayed release formulations are created by coating a solid dosage form with a film of a polymer which is insoluble in the acid environment of the stomach, and soluble in the neutral environment of small intestines.

[0167] The delayed release dosage units can be prepared, for example, by coating a drug or a drug-containing composition with a selected coating material. The drug-containing composition may be, e.g., a tablet for incorporation into a capsule, a tablet for use as an inner core in a "coated core" dosage form, or a plurality of drug-containing beads, particles or granules, for incorporation into either a tablet or capsule. Preferred coating materials include bioerodible, gradually hydrolyzable, gradually water-soluble, and / or enzymatically degradable polymers, and may be conventional "enteric" polymers. Enteric polymers, as will be appreciated by those skilled in the art, become soluble in the higher pH environment of the lower gastrointestinal tract or slowly erode as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly in the colon. Suitable coating materials for effecting delayed release include, but are not limited to, cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methylmethacrylate and / or ethyl methacrylate, and other methacrylic resins that are commercially available under the tradename EUDRAGIT®. (Rohm Pharma; Westerstadt, Germany), including EUDRAGIT®. L30D-55 and L100-55 (soluble at pH 5.5 and above), EUDRAGIT®. L-100 (soluble at pH 6.0 and above), EUDRAGIT®. S (soluble at pH 7.0 and above, as a result of a higher degree of esterification), and EUDRAGITS®. NE, RL and RS (water-insoluble polymers having different degrees of permeability and expandability); vinyl polymers and copolymers such as polyvinyl pyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate copolymer; enzymatically degradable polymers such as azo polymers, pectin, chitosan, amylose and guar gum; zein and shellac. Combinations of different coating materials may also be used. Multilayer coatings using different polymers may also be applied.

[0168] The preferred coating weights for particular coating materials may be readily determined by those skilled in the art by evaluating individual release profiles for tablets, beads and granules prepared with different quantities of various coating materials. It is the combination of materials, method and form of application that produce the desired release characteristics, which one can determine only from the clinical studies.

[0169] The coating composition may include conventional additives, such as plasticizers, pigments, colorants, stabilizing agents, glidants, etc. A plasticizer is normally present to reduce the fragility of the coating, and will generally represent about 10 wt. % to 50 wt. % relative to the dry weight of the polymer. Examples of typical plasticizers include polyethylene glycol, propylene glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate, castor oil and acetylated monoglycerides. A stabilizing agent is preferably used to stabilize particles in the dispersion. Typical stabilizing agents are nonionic emulsifiers such as sorbitan esters, polysorbates and polyvinylpyrrolidone. Glidants are recommended to reduce sticking effects during film formation and drying, and will generally represent approximately 25 wt. % to 100 wt. % of the polymer weight in the coating solution. One effective glidant is talc. Other glidants such as magnesium stearate and glycerol monostearates may also be used. Pigments such as titanium dioxide may also be used. Small quantities of an anti-foaming agent, such as a silicone (e.g., simethicone), may also be added to the coating composition.

[0170] Methods of Manufacturing delayed release dosage forms

[0171] As will be appreciated by those skilled in the art and as described in the pertinent texts and literature, a number of methods are available for preparing drug-containing tablets, beads, granules or particles that provide a variety of drug release profiles. Such methods include, but are notlimited to, the following: coating a drug or drug-containing composition with an appropriate coating material, typically although not necessarily incorporating a polymeric material, increasing drug particle size, placing the drug within a matrix, and forming complexes of the drug with a suitable complexing agent.

[0172] The delayed release dosage units may be coated with the delayed release polymer coating using conventional techniques, e.g., using a conventional coating pan, an airless spray technique, fluidized bed coating equipment (with or without a Wurster insert). For detailed information concerning materials, equipment and processes for preparing tablets and delayed release dosage forms, see Pharmaceutical Dosage Forms: Tablets, eds. Lieberman et al. (New York: Marcel Dekker, Inc., 1989), and Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 6.sup.th Ed. (Media, PA: Williams & Wilkins, 1995).

[0173] A preferred method for preparing extended release tablets is by compressing a drugcontaining blend, e.g., blend of granules, prepared using a direct blend, wet-granulation, or drygranulation process. Extended release tablets may also be molded rather than compressed, starting with a moist material containing a suitable water-soluble lubricant. However, tablets are preferably manufactured using compression rather than molding. A preferred method for forming extended release drug-containing blend is to mix drug particles directly with one or more excipients such as diluents (or fillers), binders, disintegrants, lubricants, glidants, and colorants. As an alternative to direct blending, a drug-containing blend may be prepared by using wet-granulation or dry-granulation processes. Beads containing the active agent may also be prepared by any one of a number of conventional techniques, typically starting from a fluid dispersion. For example, a typical method for preparing drug-containing beads involves dispersing or dissolving the active agent in a coating suspension or solution containing pharmaceutical excipients such as polyvinylpyrrolidone, methylcellulose, talc, metallic stearates, silicone dioxide, plasticizers or the like. The admixture is used to coat a bead core such as a sugar sphere (or so-called "non-pareil") having a size of approximately 60 to 20 mesh.

[0174] An alternative procedure for preparing drug beads is by blending drug with one or more pharmaceutically acceptable excipients, such as microcrystalline cellulose, lactose, cellulose, polyvinyl pyrrolidone, talc, magnesium stearate, a disintegrant, etc., extruding the blend, spheronizing the extrudate, drying and optionally coating to form the immediate release beads.

[0175] 5. Formulations for Mucosal and Pulmonary Administration

[0176] Active agent(s) and compositions thereof can be formulated for pulmonary or mucosal administration. The administration can include delivery of the composition to the lungs, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa. In a particular embodiment, the composition is formulated for and delivered to the subject sublingually.

[0177] In one embodiment, the compounds are formulated for pulmonary delivery, such as intranasal administration or oral inhalation. The respiratory tract is the structure involved in the exchange of gases between the atmosphere and the blood stream. The lungs are branching structures ultimately ending with the alveoli where the exchange of gases occurs. The alveolar surface area is the largest in the respiratory system and is where drug absorption occurs. The alveoli are covered by a thin epithelium without cilia or a mucus blanket and secrete surfactant phospholipids. The respiratory tract encompasses the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli. The upper and lower airways are called the conducting airways. The terminal bronchioli then divide into respiratory bronchiole, which then lead to the ultimate respiratory zone, the alveoli, or deep lung. The deep lung, or alveoli, is the primary target of inhaled therapeutic aerosols for systemic drug delivery.

[0178] Pulmonary administration of therapeutic compositions composed of low molecular weight drugs has been observed, for example, beta-androgenic antagonists to treat asthma. Other therapeutic agents that are active in the lungs have been administered systemically and targeted via pulmonary absorption. Nasal delivery is considered to be a promising technique for administration of therapeutics for the following reasons: the nose has a large surface area available for drug absorption due to the coverage of the epithelial surface by numerous microvilli, the subepithelial layer is highly vascularized, the venous blood from the nose passes directly into the systemic circulation and therefore avoids the loss of drug by first-pass metabolism in the liver, it offers lower doses, more rapid attainment of therapeutic blood levels, quicker onset of pharmacological activity, fewer side effects, high total blood flow per cm3, porous endothelial basement membrane, and it is easily accessible.

[0179] The term aerosol as used herein refers to any preparation of a fine mist of particles, which can be in solution or a suspension, whether or not it is produced using a propellant. Aerosols can be produced using standard techniques, such as ultrasonication or high-pressure treatment.

[0180] Carriers for pulmonary formulations can be divided into those for dry powder formulations and for administration as solutions. Aerosols for the delivery of therapeutic agents to the respirator}' tract are known in the art. For administration via the upper respiratory tract, the formulation can be formulated into a solution, e.g., water or isotonic saline, buffered or un-buffered, or as a suspension, for intranasal administration as drops or as a spray. Preferably, such solutions or suspensions are isotonic relative to nasal secretions and of about the same pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0. Buffers should be physiologically compatible and include, simply by way of example, phosphate buffers. For example, a representative nasal decongestant is described as being buffered to a pH of about 6.2. One skilled in the art can readily determine a suitable saline content and pH for an innocuous aqueous solution for nasal and / or upper respiratory administration.

[0181] Preferably, the aqueous solution is water, physiologically acceptable aqueous solutions containing salts and / or buffers, such as phosphate buffered saline (PBS), or any other aqueous solution acceptable for administration to an animal or human. Such solutions are well known to a person skilled in the art and include, but are not limited to, distilled water, de-ionized water, pure or ultrapure water, saline, phosphate-buffered saline (PBS). Other suitable aqueous vehicles include, but are not limited to, Ringer's solution and isotonic sodium chloride. Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

[0182] In another embodiment, solvents that are low toxicity organic (i.e. nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydrofuran, ethyl ether, and propanol may be used for the formulations. The solvent is selected based on its ability to readily aerosolize the formulation. The solvent should not detrimentally react with the compounds. An appropriate solvent should be used that dissolves the compounds or forms a suspension of the compounds. The solvent should be sufficiently volatile to enable formation of an aerosol of the solution or suspension. Additional solvents or aerosolizing agents, such as freons, can be added as desired to increase the volatility of the solution or suspension.

[0183] In one embodiment, compositions may contain minor amounts of polymers, surfactants, or other excipients well known to those of the art. In this context, “minor amounts” means no excipients are present that might affect or mediate uptake of the compounds in the lungs and that the excipients that are present are present in amount that do not adversely affect uptake of compounds in the lungs.

[0184] Dry lipid powders can be directly dispersed in ethanol because of their hydrophobic character. For lipids stored in organic solvents such as chloroform, the desired quantity of solution is placed in a vial, and the chloroform is evaporated under a stream of nitrogen to form a dry thin film on the surface of a glass vial. The film swells easily when reconstituted with ethanol. To fully disperse the lipid molecules in the organic solvent, the suspension is sonicated. Nonaqueous suspensions of lipids can also be prepared in absolute ethanol using a reusable PARI LC Jet+ nebulizer (PARI Respiratory Equipment, Monterey, CA).

[0185] Dry powder formulations (“DPFs”) with large particle size have improved flowability characteristics, such as less aggregation, easier aerosolization, and potentially less phagocytosis. Dry powder aerosols for inhalation therapy are generally produced with mean diameters primarily in the range of less than 5 microns, although a preferred range is between one and ten microns in aerodynamic diameter. Large “carrier” particles (containing no drug) have been co-delivered with therapeutic aerosols to aid in achieving efficient aerosolization among other possible benefits.

[0186] Polymeric particles may be prepared using single and double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation, simple and complex coacervation, interfacial polymerization, and other methods well known to those of ordinary skill in the art. Particles may be made using methods for making microspheres or microcapsules known in the art. The preferred methods of manufacture are by spray drying and freeze drying, which entails using a solution containing the surfactant, spraying to form droplets of the desired size, and removing the solvent.

[0187] The particles may be fabricated with the appropriate material, surface roughness, diameter and tap density for localized delivery to selected regions of the respiratory tract such as the deep lung or upper airways. For example, higher density or larger particles may be used for upper airway delivery. Similarly, a mixture of different sized particles, provided with the same or different active agents may be administered to target different regions of the lung in one administration.

[0188] 6. Topical and Transdermal Formulations

[0189] Transdermal formulations may also be prepared. These will typically be gels, ointments, lotions, sprays, or patches, all of which can be prepared using standard technology. Transdermal formulations can include penetration enhancers.

[0190] Compositions adapted for transdermal administration may be provided as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolongedperiod of time. Compositions adapted for topical administration may be provided as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For topical administration to the skin, mouth, eye or other external tissues, topical ointments or creams are preferably used. When formulated in an ointment, the LPC (i.e., LPC 18:2, LPC 16:0, or both), may be used in either a paraffinic or water-miscible ointment base. Alternatively, the LPC (i.e., LPC 18:2, LPC 16:0, or both), may be formulated in a cream with an oil-in-water base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops. In these compositions, the LPC (i.e., LPC 18:2, LPC 16:0, or both), may be dissolved or suspended in a suitable carrier, e.g., an aqueous solvent.

[0191] Formulations suitable for topical administration include liquid and I or semi-liquid preparations such as, for example, solutions, lotions, oils and / or water-in-oil emulsions such as creams, ointments and / or fats, and / or solutions and / or suspensions But is not limited thereto. The topically-administrable preparation may comprise, for example, from about 1% to about 10% (w / w) LPC (i.e., LPC 18:2, LPC 16:0, or both), the concentration of which can be as high as the solubility limit of the LPC (i.e., LPC 18:2, LPC 16:0, or both), in the solvent. Formulations for topical administration may include one or more of the additional ingredients mentioned herein.

[0192] The composition generally includes lysophosphatidylcholine 16:0 (LPC 16:0) and / or lysophosphatidylcholine 18:2 (LPC 18:2), a carrier, and one or more excipients. The lipids can be present at a concentration to facilitate effective delivery without causing irritation or disrupting the natural lipid balance of the skin. Exemplary concentrations include, but are not limited to, LPC 16:0 and / or LPC 18:2 in a range from 0.001% to 5% (w / w) of the total formulation, providing skinrepairing benefits while maintaining formulation stability.The composition can further include one or more skin-benefiting agents to improve skin texture and elasticity. Exemplary skin-benefiting agents include, but are not limited to, humectants, emollients, antioxidants, peptides, vitamins, and botanical extracts.

[0193] The composition can further include one or more humectants to improve the skin’s ability to retain moisture by attracting water molecules, and preventing dryness. Exemplary humectants include, but are not limited to, hyaluronic acid, glycerin, sorbitol, and propanediol, which complement LPC 16:0 and LPC 18:2 by further promoting hydration. In some forms, the composition includes one or more emollients to help soften and smooth the skin by reinforcing the lipid barrier and preventing transepidermal water loss. Exemplary emollients include, but are not limited to, shea butter, squalane, jojoba oil, and ceramides, which support skin repair and enhance the delivery of LPC 16:0 and LPC 18:2. In some forms, the compositions further include one or more antioxidantsto protect the skin from oxidative stress, reducing signs of aging and inflammation. Exemplary antioxidants include, but are not limited to, vitamin C, vitamin E (tocopherol), resveratrol, coenzyme Q10 (CoQlO), and green tea extract. In some forms, the compositions further include one or more peptides to support collagen synthesis and improve skin elasticity. Exemplary peptides include, but are not limited to, palmitoyl tripeptide- 1 , palmitoyl tetrapeptide-7, acetyl hexapeptide-8, and copper peptides, which enhance the bioactivity of the formulation for skin regeneration. In some forms, the compositions further include one or more vitamins to provide additional benefits, including antiinflammatory properties, skin brightening, and improved cell turnover. Exemplary vitamins include, but are not limited to, niacinamide (vitamin B3), retinol (vitamin A), and panthenol (pro-vitamin B5), which further support the lipid-restorative effects of LPC 16:0 and LPC 18:2. In some forms, the compositions further include one or more botanical extracts to provide soothing, anti-inflammatory, and skin-repairing properties. Exemplary botanical extracts include, but are not limited to, aloe vera, chamomile, licorice root extract, and centella asiatica.

[0194] A “gel” is a colloid in which the dispersed phase has combined with the continuous phase to produce a semisolid material, such as jelly.

[0195] An “oil” is a composition containing at least 95% wt of a lipophilic substance. Examples of lipophilic substances include but are not limited to naturally occurring and synthetic oils, fats, fatty acids, lecithins, triglycerides and combinations thereof.

[0196] A “continuous phase” refers to the liquid in which solids are suspended or droplets of another liquid are dispersed, and is sometimes called the external phase. This also refers to the fluid phase of a colloid within which solid or fluid particles are distributed. If the continuous phase is water (or another hydrophilic solvent), water-soluble or hydrophilic drugs will dissolve in the continuous phase (as opposed to being dispersed). In a multiphase formulation (e.g., an emulsion), the discreet phase is suspended or dispersed in the continuous phase.

[0197] “Emollients” are an externally applied agent that softens or soothes skin and are generally known in the art and listed in compendia, such as the “Handbook of Pharmaceutical Excipients”, 4thEd., Pharmaceutical Press, 2003. These include, without limitation, almond oil, castor oil, ceratonia extract, cetostearoyl alcohol, cetyl alcohol, cetyl esters wax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycol palmitostearate, glycerin, glycerin monostearate, glyceryl monooleate, isopropyl myristate, isopropyl palmitate, lanolin, lecithin, light mineral oil, medium-chain triglycerides, mineral oil and lanolin alcohols, petrolatum, petrolatum and lanolinalcohols, soybean oil, starch, stearyl alcohol, sunflower oil, xylitol and combinations thereof. In one embodiment, the emollients are ethylhexylstearate and ethylhexyl palmitate.

[0198] “Surfactants” are surface- active agents that lower surface tension and thereby increase the emulsifying, foaming, dispersing, spreading and wetting properties of a product. Suitable nonionic surfactants include emulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate, poloxamer, povidone and combinations thereof. In one embodiment, the non-ionic surfactant is stearyl alcohol.

[0199] An “emulsion” is a composition containing a mixture of non-miscible components homogenously blended together. In particular embodiments, the non-miscible components include a lipophilic component and an aqueous component. An emulsion is a preparation of one liquid distributed in small globules throughout the body of a second liquid. The dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase. When oil is the dispersed liquid and an aqueous solution is the continuous phase, it is known as an oil-in-water emulsion, whereas when water or aqueous solution is the dispersed phase and oil or oleaginous substance is the continuous phase, it is known as a water-in-oil emulsion. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, especially non-ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non-aqueous materials, particularly glycols such as propylene glycol. The oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers.

[0200] “Emulsifiers” are surface active substances which promote the suspension of one liquid in another and promote the formation of a stable mixture, or emulsion, of oil and water. Common emulsifiers are: metallic soaps, certain animal and vegetable oils, and various polar compounds. Suitable emulsifiers include acacia, anionic emulsifying wax, calcium stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolin alcohols, lecithin, medium-chain triglycerides, methylcellulose, mineral oil and lanolin alcohols, monobasic sodium phosphate, monoethanolamine, nonionic emulsifying wax, oleic acid, poloxamer, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, propylene glycol alginate, selfemulsifying glyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulfate, sorbitan esters,stearic acid, sunflower oil, tragacanth, triethanolamine, xanthan gum and combinations thereof. In one embodiment, the emulsifier is glycerol stearate.

[0201] In some embodiments, the compositions may be in the form of ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. In some embodiments, formulations of the compositions can be creams, which may further contain saturated or unsaturated fatty acids such as stearic acid, palmitic acid, oleic acid, palmito-oleic acid, cetyl or oleyl alcohols, stearic acid being particularly preferred. Creams may also contain a non-ionic surfactant, for example, polyoxy-40- stearate. In some embodiments, the LPC (i.e., LPC 18:2, LPC 16:0, or both), is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, eardrops, and eye drops are also contemplated as exemplary formulations. Additionally, the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body e.g., at or near the eye such as on the surface of the top and / or bottom eyelids. Such dosage forms can bemade by dissolving or dispensing the the LPC (i.e., LPC 18:2, LPC 16:0, or both), in the proper medium.

[0202] A “lotion” is a low- to medium-viscosity liquid formulation. A lotion can contain finely powdered substances that are in soluble in the dispersion medium through the use of suspending agents and dispersing agents. Alternatively, lotions can have as the dispersed phase liquid substances that are immiscible with the vehicle and are usually dispersed by means of emulsifying agents or other suitable stabilizers. In one embodiment, the lotion is in the form of an emulsion having a viscosity of between 100 and 1000 centistokes. The fluidity of lotions permits rapid and uniform application over a wide surface area. Lotions are typically intended to dry on the skin leaving a thin coat of their medicinal components on the skin’s surface.

[0203] A “cream” is a viscous liquid or semi-solid emulsion of either the “oil-in-water” or “water-in-oil type”. Creams may contain emulsifying agents and / or other stabilizing agents. In one embodiment, the formulation is in the form of a cream having a viscosity of greater than 1000 centistokes, typically in the range of 20,000-50,000 centistokes. Creams are often time preferred over ointments as they are generally easier to spread and easier to remove.

[0204] A sub-set of emulsions are the self-emulsifying systems. These drug delivery systems are typically capsules (hard shell or soft shell) composed of the drug dispersed or dissolved in a mixture of surfactant(s) and lipophillic liquids such as oils or other water immiscible liquids. When the capsule is exposed to an aqueous environment and the outer gelatin shell dissolves, contact between the aqueous medium and the capsule contents instantly generates very small emulsiondroplets. These typically are in the size range of micelles or nanoparticles. No mixing force is required to generate the emulsion as is typically the case in emulsion formulation processes.

[0205] The basic difference between a cream and a lotion is the viscosity, which is dependent on the amount / use of various oils and the percentage of water used to prepare the formulations. Creams are typically thicker than lotions, may have various uses and often one uses more varied oils / butters, depending upon the desired effect upon the skin. In a cream formulation, the water-base percentage is about 60-75 % and the oil-base is about 20-30 % of the total, with the other percentages being the emulsifier agent, preservatives and additives for a total of 100 %.

[0206] An “ointment” is a semisolid preparation containing an ointment base and optionally one or more active agents. Examples of suitable ointment bases include hydrocarbon bases (e.g., petrolatum, white petrolatum, yellow ointment, and mineral oil); absorption bases (hydrophilic petrolatum, anhydrous lanolin, lanolin, and cold cream); water-removable bases (e.g., hydrophilic ointment), and water-soluble bases (e.g., polyethylene glycol ointments). Pastes typically differ from ointments in that they contain a larger percentage of solids. Pastes are typically more absorptive and less greasy that ointments prepared with the same components.

[0207] A “gel” is a semisolid system containing dispersions of small or large molecules in a liquid vehicle that is rendered semisolid by the action of a thickening agent or polymeric material dissolved or suspended in the liquid vehicle. The liquid may include a lipophilic component, an aqueous component or both. Some emulsions may be gels or otherwise include a gel component. Some gels, however, are not emulsions because they do not contain a homogenized blend of immiscible components.

[0208] Suitable gelling agents include, but are not limited to, modified celluloses, such as hydroxypropyl cellulose and hydroxy ethyl cellulose; Carbopol homopolymers and copolymers; and combinations thereof. Suitable solvents in the liquid vehicle include, but are not limited to, diglycol monoethyl ether; alklene glycols, such as propylene glycol; dimethyl isosorbide; alcohols, such as isopropyl alcohol and ethanol. The solvents are typically selected for their ability to dissolve the drug. Other additives, which improve the skin feel and / or emolliency of the formulation, may also be incorporated. Examples of such additives include, but are not limited, isopropyl myristate, ethyl acetate, C12-C15 alkyl benzoates, mineral oil, squalane, cyclomethicone, capric / caprylic triglycerides, and combinations thereof.

[0209] Foams consist of an emulsion in combination with a gaseous propellant. The gaseous propellant consists primarily of hydrofluoroalkanes (HFAs). Suitable propellants include HFAs such as 1 , 1 , 1 ,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoropropane (HFA 227), but mixtures and admixtures of these and other HFAs that are currently approved or may become approved for medical use are suitable. The propellants preferably are not hydrocarbon propellant gases which can produce flammable or explosive vapors during spraying. Furthermore, the compositions preferably contain no volatile alcohols, which can produce flammable or explosive vapors during use.

[0210] Buffers are used to control pH of a composition. Preferably, the buffers buffer the composition from a pH of about 4 to a pH of about 7.5, more preferably from a pH of about 4 to a pH of about 7, and most preferably from a pH of about 5 to a pH of about 7. In a preferred embodiment, the buffer is triethanolamine.

[0211] Preservatives can be used to prevent the growth of fungi and microorganisms. Suitable antifungal and antimicrobial agents include, but are not limited to, benzoic acid, butylparaben, ethyl paraben, methyl paraben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, and thimerosal.

[0212] Additional agents that can be added to the formulation include penetration enhancers. In some embodiments, the penetration enhancer increases the solubility of the drug, improves transdermal delivery of the drug across the skin, in particular across the stratum corneum, or a combination thereof. Some penetration enhancers cause dermal irritation, dermal toxicity and dermal allergies. However, the more commonly used ones include urea, (carbonyldiamide), imidurea, N, N- diethylformamide, N-methyl-2-pyrrolidone, l-dodecal-azacyclopheptane-2-one, calcium thioglycate, 2-pyrrolidone, N,N-diethyl-m-toluamide, oleic acid and its ester derivatives, such as methyl, ethyl, propyl, isopropyl, butyl, vinyl and glycerylmonooleate, sorbitan esters, such as sorbitan monolaurate and sorbitan monooleate, other fatty acid esters such as isopropyl laurate, isopropyl myristate, isopropyl palmitate, diisopropyl adipate, propylene glycol monolaurate, propylene glycol monooleatea and non-ionic detergents such as Brij® 76 (stearyl poly(10 oxyethylene ether), Brij® 78 (stearyl poly(20)oxyethylene ether), Brij® 96 (oleyl poly(10)oxyethylene ether), and Brij® 721 (stearyl poly (21) oxyethylene ether) (ICI Americas Inc. Corp.). Chemical penetrations and methods of increasing transdermal drug delivery are described in Inayat, et al., Tropical Journal of Pharmaceutical Research, 8(2): 173-179 (2009) and Fox, et al., Molecules, 16:10507-10540 (2011).In some embodiments, the penetration enhancer is, or includes, an alcohol such ethanol, or others disclosed herein or known in the art.

[0213] Delivery of drugs by the transdermal route has been known for many years. Advantages of a transdermal drug delivery compared to other types of medication delivery such as oral, intravenous, intramuscular, etc., include avoidance of hepatic first pass metabolism, ability to discontinue administration by removal of the system, the ability to control drug delivery for a longer time than the usual gastrointestinal transit of oral dosage form, and the ability to modify the properties of the biological barrier to absorption.

[0214] Controlled release transdermal devices rely for their effect on delivery of a known flux of drug to the skin for a prolonged period of time, generally a day, several days, or a week. Two mechanisms are used to regulate the drug flux: either the drug is contained within a drug reservoir, which is separated from the skin of the wearer by a synthetic membrane, through which the drug diffuses; or the drug is held dissolved or suspended in a polymer matrix, through which the drug diffuses to the skin. Devices incorporating a reservoir will deliver a steady drug flux across the membrane as long as excess undissolved drug remains in the reservoir; matrix or monolithic devices are typically characterized by a falling drug flux with time, as the matrix layers closer to the skin are depleted of drug. Usually, reservoir patches include a porous membrane covering the reservoir of medication which can control release, while heat melting thin layers of medication embedded in the polymer matrix (e.g., the adhesive layer), can control release of drug from matrix or monolithic devices. Accordingly, the active agent can be released from a patch in a controlled fashion without necessarily being in a controlled release formulation.

[0215] Patches can include a liner which protects the patch during storage and is removed prior to use; drug or drug solution in direct contact with release liner; adhesive which serves to adhere the components of the patch together along with adhering the patch to the skin; one or more membranes, which can separate other layers, control the release of the drug from the reservoir and multi-layer patches, etc., and backing which protects the patch from the outer environment.

[0216] Common types of transdermal patches include, but are not limited to, single-layer drug- in-adhesive patches, wherein the adhesive layer contains the drug and serves to adhere the various layers of the patch together, along with the entire system to the skin, but is also responsible for the releasing of the drug; multi-layer drug-in-adhesive, wherein which is similar to a single-layer drugin-adhesive patch, but contains multiple layers, for example, a layer for immediate release of the drug and another layer for control release of drug from the reservoir; reservoir patches wherein the druglayer is a liquid compartment containing a drug solution or suspension separated by the adhesive layer; matrix patches, wherein a drug layer of a semisolid matrix containing a drug solution or suspension which is surrounded and partially overlaid by the adhesive layer; and vapor patches, wherein an adhesive layer not only serves to adhere the various layers together but also to release vapor. Methods for making transdermal patches are described in U.S. Patent Nos. 6,461,644, 6,676,961, 5,985,311, and 5,948,433.

[0217] D. Immune Checkpoint Modulators

[0218] The methods can include administering an immune checkpoint modulator to the subject, particular subjects with cancer. Immune checkpoints can be stimulatory or inhibitory, and tumors can use these checkpoints to protect themselves from immune system attacks. Currently approved checkpoint therapies block inhibitory checkpoint receptors, but investigations into therapies that activate stimulatory checkpoints are also underway. Thus, the immune checkpoint modulator can be one that blocks an inhibitory checkpoint, or activates a stimulatory checkpoint. Typically, the immune checkpoint modulator is one that induces or otherwise activates or increases an immune response against target cells for example cancer cells or infected cells. Accordingly, in some embodiments, the immune checkpoint modulator can be a chimeric antigen receptor (CAR) directed cell such as a CAR-T cell. In another embodiment, the immune checkpoint modulator can be an oncolytic virus.

[0219] In preferred embodiments, the immune checkpoint modulator blocks an inhibitory checkpoint. Blockade of negative feedback signaling to immune cells thus results in an enhanced immune response against tumors. Thus, in some embodiments the immune checkpoint modulator is administered to the subject in an effective amount to block an inhibitory checkpoint. Exemplary compounds are those that block or otherwise inhibit, for example, PD-1, PD-L1, or CTLA4.

[0220] Dosage regimens or cycles of the agents can be completely or partially overlapping, or can be sequential. Thus, the immune checkpoint modulator can be administered before, concurrently with, after or any combination thereof with a disclosed composition.

[0221] 1. PD-1 antagonists

[0222] In some embodiments, the active agents are PD-1 antagonists. Activation of T cells normally depends on an antigen- specific signal following contact of the T cell receptor (TCR) with an antigenic peptide presented via the major histocompatibility complex (MHC) while the extent of this reaction is controlled by positive and negative antigen-independent signals emanating from avariety of co-stimulatory molecules. The latter are commonly members of the CD28 / B7 family. Conversely, Programmed Death- 1 (PD- 1 ) is a member of the CD28 family of receptors that delivers a negative immune response when induced on T cells. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that decreases T cell multiplication and / or the strength and / or duration of a T cell response. Suitable PD-1 antagonists are described in U.S. Patent Nos. 8,114,845, 8,609,089, and 8,709,416, and include compounds or agents that either bind to and block a ligand of PD- 1 to interfere with or inhibit the binding of the ligand to the PD- 1 receptor, or bind directly to and block the PD- 1 receptor without inducing inhibitory signal transduction through the PD-1 receptor.

[0223] In some embodiments, the PD-1 receptor antagonist binds directly to the PD-1 receptor without triggering inhibitory signal transduction and also binds to a ligand of the PD- 1 receptor to reduce or inhibit the ligand from triggering signal transduction through the PD-1 receptor. By reducing the number and / or amount of ligands that bind to PD-1 receptor and trigger the transduction of an inhibitory signal, fewer cells are attenuated by the negative signal delivered by PD-1 signal transduction and a more robust immune response can be achieved.

[0224] It is believed that PD-1 signaling is driven by binding to a PD-1 ligand (such as B7-H1 or B7-DC) in close proximity to a peptide antigen presented by major histocompatibility complex (MHC) (see, for example, Freeman, Proc. Natl. Acad. Sci. U. S. A, 105: 10275-10276 (2008)). Therefore, proteins, antibodies or small molecules that prevent co-ligation of PD-1 and TCR on the T cell membrane are also useful PD-1 antagonists.

[0225] In preferred embodiments, the PD- 1 receptor antagonists are small molecule antagonists or antibodies that reduce or interfere with PD-1 receptor signal transduction by binding to ligands of PD-1 or to PD-1 itself, especially where co-ligation of PD-1 with TCR does not follow such binding, thereby not triggering inhibitory signal transduction through the PD-1 receptor.

[0226] Other PD-1 antagonists include antibodies that bind to PD-1 or ligands of PD-1 such as PD-L1 (also known as B7-H1) and PD-L2 (also known as B7-DC), and other antibodies.

[0227] Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following publications: PCT / IL03 / 00425 (Hardy et al., WO / 2003 / 099196); PCT / JP2006 / 309606 (Korman et al., WO / 2006 / 121168); PCT / US2008 / 008925 (Li et al., WO / 2009 / 014708); PCT / JP03 / 08420 (Honjo et al., WO / 2004 / 004771); PCT / JP04 / 00549 (Honjo et al., WO / 2004 / 072286); PCT / IB2003 / 006304 (Collins et al., WO / 2004 / 056875); PCT / US2007 / 088851(Ahmed et al., WO / 2008 / 083174); PCT / US2006 / 026046 (Korman et al., WO / 2007 / 005874); PCT / US2008 / 084923 (Terrett et al., WO / 2009 / 073533); Berger et al., Clin. Cancer Res., 14:30443051 (2008).

[0228] A specific example of an anti-PD-1 antibody is MDX-1106 (see Kosak, US 20070166281 (pub. 19 July 2007) at par. 42), a human anti-PD-1 antibody, preferably administered at a dose of 3 mg / kg.

[0229] Exemplary anti-B7-Hl antibodies include, but are not limited to, those described in the following publications: PCT / US06 / 022423 (WO / 2006 / 133396, pub. 14 December 2006); PCT / US07 / 088851 (WO / 2008 / 083174, pub. 10 July 2008); US 2006 / 0110383 (pub. 25 May 2006).

[0230] A specific example of an anti-B7-Hl antibody is MDX-1105 (WO / 2007 / 005874, published 11 January 2007)), a human anti-B7-Hl antibody.

[0231] For anti-B7-DC antibodies see 7,41 1 ,051 , 7,052,694, 7,390,888, and U.S. Published Application No. 2006 / 0099203.

[0232] The antibody can be a bi-specific antibody that includes an antibody that binds to the PD-1 receptor bridged to an antibody that binds to a ligand of PD-1, such as B7-H1. In some embodiments, the PD-1 binding portion reduces or inhibits signal transduction through the PD-1 receptor.

[0233] Other exemplary PD-1 receptor antagonists include, but are not limited to B7-DC polypeptides, including homologs and variants of these, as well as active fragments of any of the foregoing, and fusion proteins that incorporate any of these. In a preferred embodiment, the fusion protein includes the soluble portion of B7-DC coupled to the Fc portion of an antibody, such as human IgG, and does not incorporate all or part of the transmembrane portion of human B7-DC.

[0234] The PD-1 antagonist can also be a fragment of a mammalian B7-H1, preferably from mouse or primate, preferably human, wherein the fragment binds to and blocks PD- 1 but does not result in inhibitory signal transduction through PD-1. The fragments can also be part of a fusion protein, for example an Ig fusion protein.

[0235] Other useful polypeptides PD-1 antagonists include those that bind to the ligands of the PD-1 receptor. These include the PD-1 receptor protein, or soluble fragments thereof, which can bind to the PD- 1 ligands, such as B7-H 1 or B7-DC, and prevent binding to the endogenous PD- 1 receptor, thereby preventing inhibitory signal transduction. B7-H1 has also been shown to bind the proteinB7.1 (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). Such fragments also include the soluble ECD portion of the PD-1 protein that includes mutations, such as the A99L mutation, that increases binding to the natural ligands (Molnar et al., PNAS, 105: 10483-10488 (2008)). B7-1 or soluble fragments thereof, which can bind to the B7-H1 ligand and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction, are also useful.

[0236] PD-1 and B7-H1 anti-sense nucleic acids, both DNA and RNA, as well as siRNA molecules can also be PD-1 antagonists. Such anti-sense molecules prevent expression of PD- 1 on T cells as well as production of T cell ligands, such as B7-H1, PD-L1 and / or PD-L2. For example, siRNA (for example, of about 21 nucleotides in length, which is specific for the gene encoding PD- 1, or encoding a PD-1 ligand, and which oligonucleotides can be readily purchased commercially) complexed with carriers, such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119(8): 2231-2244 (2009), are readily taken up by cells that express PD-1 as well as ligands of PD-1 and reduce expression of these receptors and ligands to achieve a decrease in inhibitory signal transduction in T cells, thereby activating T cells.

[0237] Exemplary PD-1 inhibitors include, but are not limited to: Pembrolizumab (formerly MK-3475 or lambrolizumab, Keytruda, which was developed by Merck and first approved by the Food and Drug Administration in 2014 for the treatment of melanoma; Nivolumab (Opdivo) which was developed by Bristol-Myers Squibb and first approved by the FDA in 2014 for the treatment of melanoma; pidilizumab, by CureTech; AMP-224, by GlaxoSmithKline and Medlmmune; AMP-514, by GlaxoSmithKline and Medlmmune; PDR001, by Novartis; and cemiplimab, by Regeneron and Sanofi.

[0238] Exemplary PD-L1 inhibitors include, but are not limited to:

[0239] Atezolizumab (Tecentriq) is a fully humanised IgGl (immunoglobulin 1 antibody developed by Roche Genentech. In 2016, the FDA approved atezolizumab for urothelial carcinoma and non-small cell lung cancer.

[0240] Avelumab (Bavencio) is a fully human IgGl antibody developed by Merck Serono and Pfizer. Avelumab is FDA approved for the treatment of metastatic merkel-cell carcinoma. It failed phase III clinical trials for gastric cancer.

[0241] Durvalumab (Imfinzi) is a fully human IgGl antibody developed by AstraZeneca. Durvalumab is FDA approved for the treatment of urothelial carcinoma and unresectable non-small cell lung cancer after chemoradiation.

[0242] BMS-936559, by Bristol-Myers Squibb

[0243] CK-301, by Checkpoint Therapeutics See, e.g., Iwai, et al., Journal of BiomedicalScience, (2017) 24:26, DOI 10.1186 / sl2929-017-0329-9.

[0244] 2. CTLA4 antagonists

[0245] Other molecules useful in mediating the effects of T cells in an immune response are also contemplated as active agents. For example, in some embodiments, the molecule is an agent binds to an immune response mediating molecule that is not PD-1. In a preferred embodiment, the molecule is an antagonist of CTLA4, for example an antagonistic anti-CTLA4 antibody. An example of an anti-CTLA4 antibody is described in PCT / US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).

[0246] Dosages for anti-PD-1 , anti-B7-Hl , and anti-CTLA4 antibody, are known in the art and can be in the range of 0. 1 to 100 mg / kg, with shorter ranges of 1 to 50 mg / kg preferred and ranges of 10 to 20 mg / kg being more preferred. An appropriate dose for a human subject is between 5 and 15 mg / kg, with 10 mg / kg of antibody (for example, human anti-PD-1 antibody, like MDX-1106) most preferred.

[0247] Specific examples of CTLA antagonists include Ipilimumab, also known as MDX-010 or MDX-101, a human anti-CTLA4 antibody, preferably administered at a dose of about 10 mg / kg, and Tremelimumab a human anti-CTLA4 antibody, preferably administered at a dose of about 15 mg / kg. See also Sammartino, et al., Clinical Kidney Journal, 3(2): 135- 137 (2010), published online December 2009.

[0248] In other embodiments, the antagonist is a small molecule. A series of small organic compounds have been shown to bind to the B7-1 ligand to prevent binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277:7363-7368 (2002). Such small organics could be administered alone or together with an anti-CTLA4 antibody to reduce inhibitory signal transduction of T cells.

[0249] 3. Chimeric Antigen Receptor directed cells

[0250] The modulator can be a chimeric antigen receptor directed cell. The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers to a set of polypeptides, typically two in the simplest embodiments, which when in an immune effector cell, provides the cell with specificity for a cancer cell, and with intracellular signal generation. In some embodiments, a CARincludes at least an antigen binding domain such as an extracellular binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to as "an intracellular signaling domain") including afunctional signaling domain derived from a stimulatory molecule and / or costimulatory molecule as defined below. In one embodiment, the stimulatory molecule is a zeta chain (“zeta stimulatory domain”) associated with a T cell receptor complex. In one embodiment, the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one costimulatory molecule (e.g., 4-1BB (i.e., CD137), CD27 and / or CD28). In some embodiments, the CAR includes a chimeric fusion protein including an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain including a functional signaling domain derived from a stimulatory molecule. In various embodiments, CARs are fusion proteins of singlechain variable fragments (scFv) fused to a CD3-zeta transmembrane domain. However, other intracellular signaling domains such as CD28, 41 -BB and 0x40 may be used in various combinations to give the desired intracellular signal. In some embodiments, CARs disclosed herein include an extracellular binding domain.

[0251] The term “antigen binding domain” is used in the context of the present disclosure to refer to the portion of the CAR that specifically recognizes and binds to the antigen of interest. The “antigen binding domain” may be derived from a binding protein disclosed herein such as an antibody or fragment thereof. In some embodiments, the “binding domain” is a single-chain variable fragment (scFv). In certain embodiments, the “binding domain” includes the complementarity determining regions of a binding protein disclosed herein. In this embodiment, the CAR directed cell can represent the combination of a cell-penetrating antibody (assuming it penetrates a cancer cell) that induces or increase DNA damage or reduces or impairs DNA damage repair, or a combination thereof and an immune checkpoint modulator that induces, increases, or enhances an immune response. For example, the binding domain can represent the cell-penetrating antibody and the modified T-cell can represent the immune cell modulator. In another example, a CAR-directed cell disclosed herein is administered with a cell-penetrating antibody disclosed herein.

[0252] The terms “zeta” or “CD3-zeta” are used herein to define the protein provided as GenBan Acc. No. BAG36664. 1 , or the equivalent residues from a non- human species and a “zeta stimulatory domain” or alternatively a “CD3-zeta stimulatory domain” is defined as the amino acid residues from the cytoplasmic domain of the zeta chain, or functional derivatives thereof, that are sufficient to functionally transmit an initial signal necessary for T cell activation.

[0253] The term “immune effector cell,” is used herein to refer to a cell that is involved in an immune response (e.g. promotion of an immune effector response). Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloic-derived phagocytes. In some embodiments,the immune effector cell(s) is allogenic. In some embodiments, the immune effector cell(s) is autologous. In some embodiments, the immune checkpoint modulator is a CAR directed T cell (CAR-T cell). Exemplary CAR-T cells include Axicabtagene ciloleucel (KTE-C19, Axi-cel), Tisagenlecleucel, Lisocabtagene Maraleucel (liso-cel; JCAR017).

[0254] Immune effector cells such as T cells may be activated and expanded generally using methods previously described, such as for example, as described in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681 ; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041. As a general example, a population of immune effector cells e.g., T regulatory cell depleted cells, may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3 complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells.

[0255] 4. Oncolytic viruses

[0256] The modulator can be an oncolytic virus. The term “oncolytic virus” is used in the context of the present disclosure to refer to viruses that are able to infect and reduce growth of cancer cells. For example, oncolytic viruses can inhibit cell proliferation. In some embodiments, oncolytic viruses can kill cancer cells. In some embodiments, the oncolytic virus preferentially infects and inhibits growth of cancer cells compared with corresponding normal cells. In another embodiment, the oncolytic virus preferentially replicates in and inhibits growth of cancer cells compared with corresponding normal cells.

[0257] In some embodiments, the oncolytic virus is able to naturally infect and reduce growth of cancer cells. Examples of such viruses include Newcastle disease vims, vesicular stomatitis, myxoma, reovirus, sindbis, measles and coxsackievirus. Oncolytic viruses able to naturally infect and reduce growth of cancer cells generally target cancer cells by exploiting the cellular aberrations that occur in these cells. For example, oncolytic viruses may exploit surface attachment receptors, activated oncogenes such as Ras, Akt, p53 and / or interferon (IFN) pathway defects.

[0258] In another embodiment, oncolytic viruses encompassed by the present disclosure are engineered to infect and reduce growth of cancer cells. Exemplary viruses suitable for such engineering include oncolytic DNA viruses, such as adenovirus, herpes simplex vims (HSV) and Vaccinia vims; and oncolytic RNA vimses such as Lentivims, Reovims, Coxsackievirus, Seneca Valley Vims, Poliovims, Measles vims, Newcastle disease vims, Vesicular stomatitis vims (VSV)and parvovirus such as rodent protoparvoviruses H-1PV. In some embodiments, the oncolytic virus includes a backbone of an above referenced virus.

[0259] In some embodiments, tumor specificity of an oncolytic virus can be engineered to mutate or delete gene(s) required for survival of the virus in normal cells but expendable in cancer cells. For example, the oncolytic virus can be engineered by mutating or deleting a gene that encodes thymidine kinase, an enzyme needed for nucleic acid metabolism. In this example, viruses are dependent on cellular thymidine kinase expression, which is high in proliferating cancer cells hut repressed in normal cells. In another example, the oncolytic virus is engineered to include a capsid protein that binds a tumor specific cell surface molecule. In some embodiments, the capsid protein is a fibre, a penton or hexon protein. In another example, the oncolytic virus is engineered to include a tumor specific cell surface molecule for transductionally targeting a cancer cell. Exemplary tumor specific cell surface molecules can include an integrin, an EGF receptor family member, a proteoglycan, a disialoganglioside, B7-H3, CA-125, EpCAM, ICAM-1, DAF, A21, integrin- a201, vascular endothelial growth factor receptor 1 , vascular endothelial growth factor receptor 2, CEA, a tumour associated glycoprotein, CD19, CD20, CD22, CD30, CD33, CD40, CD44, CD52, CD74, CD152, CD155, MUC1, a tumour necrosis factor receptor, an insulin-like growth factor receptor, folate receptor a, transmembrane glycoprotein NMB, a C-C chemokine receptor, PSMA, RON- receptor, and cytotoxic T-lymphocyte antigen 4.

[0260] The oncolytic virus can he replication-competent. In some embodiments, the oncolytic viruses selectively replicate in cancer cells when compared with corresponding normal cells.

[0261] Conditional replication can be achieved by, for example, the insertion of a tumorspecific promoter driving the expression of a critical gene(s). Such promoters can be identified based on differences in gene expression between tumor and corresponding surrounding tissue. Exemplary native promoters include AFP, CCKAR, CEA, erbB2, Cerb2, COX2, CXCR4, E2F1, HE4, LP, MUC1, PSA, Survivin, TRP1, STAT3, hTERT and Tyr. Exemplary composite promoters include AFP / hAFP, SV40 / AFP, CEA / CEA, PSA / PSA, SV40 / Tyr and Tyr / Tyr.

[0262] Various viruses may be engineered as outlined in the above referenced examples. The oncolytic virus can be, for example, a modified HSV, Lenti virus, Baculo virus, Retrovirus, Adenovirus (AdV), Adeno-associated virus (AAV) or a recombinant form such as recombinant adeno-associated virus (rAAV) or a derivative thereof such as a self-complementary AAV (scAAV) or non-integrating AV. The oncolytic virus can be a modified HSVThe oncolytic virus can be amodified lentivirus. Other exemplary viruses include vaccina virus, vesicular stomatitis virus (VSV), measles virus and maraba virus.

[0263] In other examples, the oncolytic virus may be one of various AV or AAV serotypes. In some embodiments, the oncolytic virus is serotype 1. In another example, the oncolytic virus is serotype 2. In other examples, the oncolytic virus is serotype 3, 4, 7, 8, 9, 10, 11, 12 or 13. In another example, the oncolytic virus is serotype 5. In another example, the oncolytic virus is serotype 6.

[0264] Exemplary oncolytic viruses include T-Vec (HSV-1 ; Amgen), JX-594 (Vaccina; Sillajen), JX-594 (AdV; Cold Genesys), Reolysin (Reovirus; Oncolytics Biotech). Other examples of oncolytic viruses are disclosed in WO 2003 / 080083, WO 2005 / 086922, WO 2007 / 088229, WO 2008 / 110579, WO 2010 / 108931, WO 2010 / 128182, WO 2013 / 112942, WO 2013 / 116778, WO 2014 / 204814, WO 2015 / 077624 and WO 2015 / 166082, WO 2015 / 089280.

[0265] 5. Other Immune Checkpoint ModulatorsOther immune checkpoint targets include, but are not limited to, ICOS, 0X40, GITR, 4- 1BB, CD40, CD27-CD70, LAG3, TIM-3, TIGIT, VISTA, B7-H3, KIR, PARP, and others, and are being targeting for cancer treatment alone and in combination with anti-PD-1, anti-PD-Ll, and anti- CTLA compounds. See, for example, Iwai, et al., Journal of Biomedical Science. 24 (1): 26. doi:10.1186 / sl2929-017-0329-9; Donini, et al., JThorac Dis. 2018 May; 10(Suppl 13):S 1581-S1601. doi: 10.21037 / jtd.2018.02.79. Thus, in some embodiments, a cell-penetrating antibody is administered in combination with a compound that targets ICOS, 0X40, GITR, 4- IBB, CD40, CD27- CD70, LAG3, TIM-3, TIGIT, VISTA, B7-H3, KIR, or PARP, or a combination thereof, alone or in combination with a compound that target PD-1, PD-L1, and / or CTLA. In another embodiment, the immune checkpoint modulator is an antibody disclosed in WO 2016 / 013870.

[0266] Combination Therapies

[0267] Cancer can be treated using a variety of localized and systemic therapeutic strategies, including surgical resection, radiation therapy, chemotherapy, targeted therapies, and immunotherapies. The selection of treatment depends on tumor type, stage, genetic profile, the patient’s overall health, and available medical resources. For patients with localized cancers, surgical removal or ablation of tumors remains a preferred option, while systemic treatments are often required for advanced- stage or metastatic cancers. Chemotherapy and targeted kinase inhibitors have demonstrated effectiveness in treating advanced cancers by either directly killing tumor cells or blocking key molecular pathways essential for tumor survival and proliferation.

[0268] The disclosed methods and compositions can be used / administered alone or in combination with other standard cancer therapies. The compositions may be used as an adjunct to primary cancer treatments to increase tumor cell sensitivity to chemotherapy, radiation, or immune checkpoint inhibitors. The additional therapy may be administered simultaneously or sequentially as part of a combination treatment regimen. In some forms, therapy including the nucleic acid compositions is used between chemotherapy cycles or during drug holidays to maintain tumor suppression and prevent acquired resistance.

[0269] The disclosed methods and compositions can be used / administered in conjunction with standard cancer treatments, including but not limited to surgical intervention (tumor excision, resection, or transplant procedures), radiation therapy (external beam radiation, brachytherapy, proton therapy), chemotherapy (platinum-based agents, topoisomerase inhibitors, alkylating agents, antimetabolites, taxanes), targeted therapy (kinase inhibitors, PARP inhibitors, monoclonal antibodies, small-molecule inhibitors), and immunotherapy (immune checkpoint inhibitors, T-cell therapies, cytokine therapies). Other potential combinations include anti-angiogenesis therapy using VEGF inhibitors, COX-2 inhibitors, and tyrosine kinase inhibitors to reduce tumor vascularization and increase DNA damage accumulation in tumor cells.

[0270] As used herein, the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0271] The disclosed methods, biomarkers, and compositions, can be further understood through the following numbered paragraphs:

[0272] Paragraph 1. A method of identifying a subject that will or is likely to develop an immune- related adverse event following administration to the subject of a cancer therapy, comprising contacting the subject or a sample isolated from the subject with an agent that detects the presence of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 in the subject or the sample, wherein the presence of the of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 at lower than baseline levels indicates that the subject has, will or is likely to have an immune-related adverse event following administration to the subject of the cancer therapy.

[0273] Paragraph 2. The method of paragraph 1, wherein baseline expression is normalized mean expression of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2.

[0274] Paragraph 3. The method of paragraph 1 or paragraph 2, wherein the method is conducted following administration of the cancer therapy to the subject.

[0275] Paragraph 4. The method of any one of paragraphs 1 to 3, wherein the method is conducted at least about three weeks following administration of the cancer therapy to the subject.

[0276] Paragraph 5. The method of any one of paragraphs 1 to 4, further comprising administering one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 to the subject when a lower than baseline level of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is detected in the subject.

[0277] Paragraph 6. The method of any one of paragraphs 1 to 5, wherein the cancer therapy is chemotherapy, immunotherapy, radiation therapy, and / or cellular therapy.

[0278] Paragraph 7. The method of any one of paragraphs 1 to 6, wherein the cancer therapy is immune checkpoint blocker therapy.

[0279] Paragraph 8. The method of paragraph 7, wherein the immune checkpoint blocker therapy is one or more of anti-CTLA4 therapy, anti-PDl therapy, and anti-PDLl therapy.

[0280] Paragraph 9. The method of paragraph 8 wherein the anti-CTLA4 therapy, anti-PDl therapy, or anti-PDLl therapy comprises one or more of Ipilimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab and Durvalumab.

[0281] Paragraph 10. A method of treating an immune-related adverse event in a subject receiving cancer therapy, the method comprising administering to the subject one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2.

[0282] Paragraph 11. A method of treating an immune-related adverse event in a subject receiving cancer therapy, the method comprising: contacting the subject or a sample isolated from the subject with an agent that detects the presence of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 in the subject or the sample; and administering one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 to the subject when a lower than baseline level of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is detected in the subject.

[0283] Paragraph 12. The method of paragraph 11, wherein baseline expression is normalized mean expression of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2.

[0284] Paragraph 13. The method of any one of paragraphs 10 to 12, wherein one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is administered to the subject following administration of the cancer therapy to the subject.

[0285] Paragraph 14. The method of paragraph 13, wherein one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is administered to the subject at least about three weeks following administration of the cancer therapy to the subject.

[0286] Paragraph 15. The method of any one of paragraphs 10 to 14, wherein the cancer therapy is chemotherapy, immunotherapy, radiation therapy, and / or cellular therapy.

[0287] Paragraph 16. The method of any one of paragraphs 10 to 15, wherein the cancer therapy is immune checkpoint blocker therapy.

[0288] Paragraph 17. The method of paragraph 16, wherein the immune checkpoint blocker therapy is one or more of anti-CTLA4 therapy, anti-PDl therapy, and anti-PDLl therapy.

[0289] Paragraph 18. The method of paragraph 17 wherein the anti-CTLA4 therapy, anti-PDl therapy, or anti-PDLl therapy comprises one or more of Ipilimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab and Durvalumab.

[0290] Paragraph 19. The method of any one of paragraphs 1 to 18, wherein the subject has or is being treated for one or more of an epithelial, a head, breast, endometrium, uterus, ovary, testes,neck, lung, prostate, colon, colorectal, pancreas, esophagus, liver, skin, kidney, adrenal gland and brain cancer or tumor, a metastasis or recurring tumor, cancer or neoplasia, a melanoma, a nonsmall cell lung cancer (NSCLC) and a head and neck squamous cell cancer (HNSCC).

[0291] Paragraph 20. A method of decreasing, reducing, inhibiting, suppressing, limiting, controlling or treating an undesirable or aberrant immune response, immune disorder, inflammatory response, autoimmune disease or inflammation, the method comprising administering to the subject one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2.

[0292] Paragraph 21. A method of decreasing, reducing, inhibiting, suppressing, limiting, controlling or treating an undesirable or aberrant immune response, immune disorder, inflammatory response, autoimmune disease or inflammation, the method comprising: contacting the subject or a sample isolated from the subject with an agent that detects the presence of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 in the subject or the sample; and administering one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 to the subject when a lower than baseline level of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is detected in the subject.

[0293] Paragraph 22. The method of paragraph 20 or 21, wherein the undesirable or aberrant immune response, immune disorder, inflammatory response, autoimmune disease or inflammation comprises psoriasis, scleroderma, rheumatoid arthritis juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, polymyalgia rheumatica, inflammatory myopathy, vasculitis, sicca syndrome, hypophysitis, diabetes mellitus, multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus (SLE), autoimmune thyroiditis, atopic dermatitis, eczematous dermatitis, psoriasis, Sjogren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, inflammatory bowel disease (IBD), cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens- Johnson syndrome, idiopathic sprue, lichen planus, Graves’ disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, interstitial lung fibrosis, Hashimoto's thyroiditis, autoimmune polyglandular syndrome, insulin-dependent diabetes mellitus, insulin-resistant diabetes mellitus, immune-mediated infertility, autoimmune Addison's disease, pemphigus vulgaris, pemphigus foliaceus, dermatitis herpetiformis, autoimmune alopecia, vitiligo, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, pernicious anemia, Guillain-Barre syndrome, stiff-mansyndrome, acute rheumatic fever, sympathetic ophthalmia, Goodpasture's syndrome, systemic necrotizing vasculitis, antiphospholipid syndrome or an allergy, Behcet’s disease, severe combined immunodeficiency (SCID), recombinase activating gene (RAG 1 / 2) deficiency, adenosine deaminase (ADA) deficiency, interleukin receptor common g chain ( c ) deficiency, Janus- associated kinase 3 (JAK3) deficiency and reticular dysgenesis; primary T cell immunodeficiency such as DiGeorge syndrome, Nude syndrome, T cell receptor deficiency, MHC class II deficiency, TAP-2 deficiency (MHC class I deficiency), ZAP70 tyrosine kinase deficiency and purine nucleotide phosphorylase (PNP) deficiency, antibody deficiencies, X-linked agammaglobulinemia (Bruton's tyrosine kinase deficiency), autosomal recessive agammaglobulinemia, Mu heavy chain deficiency, surrogate light chain (g5 / 14.1 ) deficiency, Hyper- IgM syndrome: X-linked (CD40 ligand deficiency) or non-X-linked, Ig heavy chain gene deletion, IgA deficiency, deficiency of IgG subclasses (with or without IgA deficiency), common variable immunodeficiency (CVID), antibody deficiency with normal immunoglobulins; transient hypogammaglobulinemia of infancy, interferon g receptor (IFNGR1, IFNGR2) deficiency, interleukin 12 or interleukin 12 receptor deficiency, immunodeficiency with thymoma, Wiskott-Aldrich syndrome (WAS protein deficiency), ataxia telangiectasia (ATM deficiency), X-linked lymphoproliferative syndrome (SH2D1 A / SAP deficiency), or hyper IgE syndrome.

[0294] Paragraph 23. The method of paragraph 20 or paragraph 21, wherein the method comprises treating an inflammatory disease or disorder or an autoimmune disease or disorder.

[0295] Paragraph 24. The method of paragraph 23, wherein the method comprises treating inflammatory bowel disease or ulcerative colitis.

[0296] Paragraph 25. A method of preventing immune-related adverse events (irAEs) in a patient receiving a treatment for a disease, comprising: administering a therapy to the patient capable of treating the disease; and modulating a level of at least one small molecule metabolite.

[0297] Paragraph 26. The method of paragraph 25, wherein the disease comprises a cancer.

[0298] Paragraph 27. The method of paragraph 26, wherein the therapy comprises a cancer immunotherapy.

[0299] Paragraph 28. The method of paragraph 27, wherein the cancer immunotherapy comprises an immune checkpoint blocker therapy.

[0300] Paragraph 29. The method of paragraph 28, wherein the immune checkpoint blocker therapy comprises administering one or more of Ipilimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab, and Durvalumab.

[0301] Paragraph 30. The method of paragraph 25, wherein the at least one small molecule metabolite comprises any one or more the small molecule metabolites listed in Table 5.

[0302] Paragraph 31. The method of paragraph 25, wherein the at least one small molecule metabolite comprises lysophosphatidylcholine 16:0 or lysophosphatidylcholine 18:2.

[0303] Paragraph 32. The method of paragraph 31, wherein the small molecule metabolite comprises lysophosphatidylcholine 18:2.

[0304] Paragraph 33. The method of paragraph 25, wherein modulating the level of at least one small molecule metabolite comprises administering a pharmaceutically acceptable amount of the at least one small molecule metabolite.

[0305] Paragraph 34. The method of paragraph 33, wherein the at least one small molecule metabolite comprises lysophosphatidylcholine 18:2.

[0306] Paragraph 35. A method of treatment, comprising modulating a level of at least one small molecule metabolite in a subject to reduce likelihood of irAEs in the subject; and treating the subject with a cancer immunotherapy.

[0307] Paragraph 36. The method of paragraph 35, wherein the cancer immunotherapy comprises an immune checkpoint blocker therapy.

[0308] Paragraph 37. The method of paragraph 35, wherein the at least one small molecule metabolite comprises any one or more of the small molecule metabolites listed in Table 5.

[0309] Paragraph 38. The method of paragraph 35, wherein the at least one small molecule metabolite comprises lysophosphatidylcholine 18:2.

[0310] Paragraph 39. The method of paragraph 38, wherein modulating a level of at least one small molecule metabolite comprises administering a pharmaceutically acceptable amount of lysophosphatidylcholine 18:2.

[0311] Paragraph 40. A method of modulating an immune response in a subject comprising modulating a level of at least of at least one small molecule metabolite, wherein modulating the immune response prevents the development or reduces the likelihood of irAEs.

[0312] Paragraph 41. The method of paragraph 40, further comprising treating the subject with a cancer immunotherapy.

[0313] Paragraph 42. The method of paragraph 40, wherein the at least one small molecule inhibitor comprises any one or more of the small molecule inhibitors listed in Table 5.

[0314] Paragraph 43. The method of paragraph 40, wherein the at least one small molecule metabolite comprises lysophosphatidylcholine 18:2.

[0315] Paragraph 44. The method of paragraph 43, wherein modulating the level of the at least one small molecule metabolite comprises administering a pharmaceutically acceptable amount of lysophosphatidylcholine 18:2.

[0316] Paragraph 45. The method of paragraph 40, wherein modulating the immune response comprises modulating levels of neutrophils.

[0317] Paragraph 46. The method of paragraph 45, wherein modulating of neutrophils results from modulating the level of the at least one small molecule metabolite.

[0318] Paragraph 47. A method comprising: (a) detecting the level of one or more lipid biomarkers in a biological sample obtained from a subject, wherein the one or more lipid biomarkers comprise lysophosphatidylcholine 16:0 (LPC 16:0), or lysophosphatidylcholine 18:2 (LPC 18:2), or both.

[0319] Paragraph 48. The method of paragraph 47, wherein the biological sample is selected from the group consisting of blood, serum, plasma, cerebrospinal fluid, urine, saliva, or tissue extract.

[0320] Paragraph 49. The method of paragraph 47 or 48, wherein detecting the level of one or more lipid biomarkers comprises using molecular analysis, wherein the molecular analysis comprises a lipid analytical technique selected from the group consisting of mass spectrometry, liquid chromatography-mass spectrometry (LC-MS), high-performance liquid chromatography (HPLC), and nuclear magnetic resonance (NMR) spectroscopy.

[0321] Paragraph 50. The method of any one of paragraphs 47-49, further comprising comparing the detected level of lysophosphatidylcholine 16:0 (LPC 16:0) and / or lysophosphatidylcholine 18:2 (LPC 18:2) to a predetermined reference level.

[0322] Paragraph 51. The method of paragraph 50, wherein the predetermined reference level is derived from a healthy subject population or a disease-specific threshold.

[0323] Paragraph 52. The method of paragraph 47, further comprising prior to Step (a):(b) extracting the one or more lipid biomarkers from the biological sample using an extraction technique selected from the group consisting of single organic solvent extraction (SOSE), one-phase extraction (OPE), liquid-liquid extraction (LLE), solid-phase extraction (SPE), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), Soxhlet extraction (SE), supercritical fluid extraction (SFE), or a combination thereof.

[0324] Paragraph 53. The method of paragraph 52, wherein the extraction technique comprises removing non-lipid components by protein precipitation (PP) using a solvent selected from the group consisting of acetonitrile (ACN), methanol (MeOH), ethanol, isopropanol, ethyl acetate, or propanol before lipid extraction.

[0325] Paragraph 54. The method of any one of paragraphs 47-53, wherein detection is relative to a control, optionally wherein the control is a biological sample collected from a different subject or from the same subject, optionally from the same subject at a different time point.

[0326] Paragraph 55. A method of diagnosing a subject that will or is likely to develop an immune-related adverse event, optionally following administration of a cancer therapy; comprising detecting one or more lipid biomarkers according to the method of any one of paragraphs 47-54.

[0327] Paragraph 56. The method according to any one of paragraphs 1 through 9, wherein the detection is performed according to any one of paragraphs 47-54.

[0328] Paragraph 57. The method of paragraph 55 or 56, further comprising administering a treatment to the subject when the subject is determined to have an immune-related adverse event or to be at risk of developing an immune-related adverse event.

[0329] Paragraph 58. The method of paragraph 57, wherein the administration of the treatment is performed according to any one of paragraphs 10-46.

[0330] Paragraph 59. The method of paragraph 57, wherein administration to prevent or treat the immune-related adverse event comprises a composition containing lysophosphatidylcholine 16:0 (LPC 16:0), lysophosphatidylcholine 18:2 (LPC 18:2), or both.

[0331] Paragraph 60. The method of paragraph 59, wherein the composition is administered orally, intravenously, subcutaneously, intramuscularly, or topically.

[0332] Paragraph 61. The method of paragraph 59 or paragraph 60, in the form selected from the group consisting of a tablet, capsule, liquid solution, injectable formulation, gel, or cream.

[0333] Paragraph 62. The method of any one of paragraphs 59-61, wherein the composition further comprises a carrier or excipient.

[0334] Paragraph 63. The method of any one of paragraphs 59-62, wherein the comprises LPC 16:0 and / or LPC 18:2 in a concentration ranging from 0.001% to 10% (w / w) of the total composition.

[0335] Paragraph 64. The method of any one of paragraphs 59-63, wherein the composition reduces inflammation in the subject.

[0336] Paragraph 65. The method of any one of paragraphs 59-64, wherein the composition is coadministered with one or more immunosuppressive or anti-inflammatory agents to increase treatment efficacy.

[0337] Paragraph 66. The method of any one of paragraphs 59-65, wherein the composition is administered before, during, or after a cancer therapy to reduce or prevent the immune-related adverse event.

[0338] Paragraph 67. The method of any one of paragraphs 59-66, wherein the composition is personalized based on the subject’s biomarker levels detected using the method of paragraph 47.

[0339] Paragraph 68. The method of any one of paragraphs 59-67, wherein the composition is provided as part of a combination therapy with corticosteroids, monoclonal antibodies, or checkpoint inhibitor modulators.

[0340] Paragraph 69. The method of any one of paragraphs 59-68, further comprising monitoring the subject’s biomarker levels post-administration to evaluate treatment response and adjust dosage as needed.

[0341] Paragraph 70. A composition comprising: (a) an effective amount of lysophosphatidylcholine 16:0 (LPC 16:0), lysophosphatidylcholine 18:2 (LPC 18:2), or both; and (b) a carrier.

[0342] Paragraph 71. The composition of paragraph 70, further comprising one or more additional skin-benefiting agents selected from the group consisting of humectants, emollients, antioxidants, peptides, vitamins, and botanical extracts.

[0343] Paragraph 72. The composition of paragraph 70 or 71, wherein the lysophosphatidylcholine 16:0 (LPC 16:0) and / or lysophosphatidylcholine 18:2 (LPC 18:2) is present in a concentration ranging from 0.001% to 5% (w / w) of the total formulation.

[0344] Paragraph 73. The composition of any one of paragraphs 70-72, wherein the carrier comprises water, glycerin, or an oil-in-water emulsion.

[0345] Paragraph 74. The composition of any one paragraphs 70-73, further comprising one or more humectants selected from the group consisting of hyaluronic acid, glycerin, sorbitol, and propanediol.

[0346] Paragraph 75. The composition of any one of paragraphs 70-74, further comprising an emollient selected from the group consisting of shea butter, squalane, jojoba oil, and ceramides.

[0347] Paragraph 76. The composition of any one of paragraphs 70-75, further comprising an antioxidant selected from the group consisting of vitamin C, vitamin E (tocopherol), resveratrol, coenzyme Q10 (CoQlO), or green tea extract.

[0348] Paragraph 77. The composition of any one of paragraphs 70-76, further comprising a peptide selected from the group consisting of palmitoyl tripeptide- 1, palmitoyl tetrapeptide-7, acetyl hexapeptide-8, or copper peptides.

[0349] Paragraph 78. The composition of any one of paragraphs 70-77, further comprising a vitamin selected from the group consisting of niacinamide (vitamin B3), retinol (vitamin A), or panthenol (pro-vitamin B5).

[0350] Paragraph 79. The composition of any one of paragraphs 70-78, further comprising a botanical extract selected from the group consisting of om aloe vera, chamomile, licorice root extract, or centella asiatica.

[0351] Paragraph 80. The composition of any one of paragraphs 70-79, wherein the composition is suitable for administration at or near the eye, optionally to the upper and / or lower eyelids.

[0352] Paragraph 81. The composition of any one of paragraphs 70-80, wherein the composition is formulation as a gel, serum, balm, or lotion.

[0353] Paragraph 82. The composition of any one of paragraph 70-81, wherein the composition is encapsulated in liposomes or nanocarriers to increase delivery and penetration into the skin.

[0354] Paragraph 83. The composition according to any one of paragraphs 70-82, in a form suitable for topical administration.

[0355] Paragraph 84. A method of treating a subject with a disease or disorder, comprising administering to the subject, the composition of any one of paragraphs 70-83, wherein the method is performed according to any one of paragraphs 10-46, or paragraphs 47-55.

[0356] Examples

[0357] Example 1. Identification of irAE-associated metabolites.

[0358] In order to comprehensively characterize the metabolome in human plasma, an analytical approach capable of robust and rapid measurement of thousands of diverse metabolites was developed. Specifically, a next-generation, automated in-line rapid liquid chromatography (rLC) system coupled to an Agilent 6550 iFunnel quadrupole time-of-flight (QTOF) high-resolution mass spectrometer (rLC-MS) was developed. This instrument allows for analysis of an extracted plasma / serum biosample at <1 min of total analytical cycle time per sample. An extracted ion chromatogram from a human plasma sample using rLC-MS has revealed thousands of independent spectral features (FIG. 1A), with high technical reproducibility (median percent coefficient of variation of <12% across several thousand metabolite measures) and limited sample-to-sample matrix effects of <5%. To determine the breadth and sensitivity of this high-throughput rLC-MS, a secondary analysis was performed of 267 small molecules with diverse chemical characteristics and circulating concentrations, curated from a list of 1561 known circulating metabolites in the Human Metabolite and NHANES databases. These small molecules were spiked into charcoal-stripped plasma at reported physiologic conditions and the plasma assayed using the rLC-MS methods. >70% of molecules were captured with a signal-to-noise >10x, at concentrations extending from high picomolar to millimolar (FIG. IB). Dilution of an exogenous 13C-labeled standard in plasmarevealed linearity over >4 orders in magnitude concentration, consistent with traditional LC-MS, which allows for linear relative quantitation of metabolites across samples (FIG. 1C).

[0359] An additional 600 metabolites spiked into stripped plasma at a uniform concentration of 1 um showed accurate detection across diverse chemical superclasses (FIG. ID). Importantly, these approaches allow for rapid and broad characterization of multiple circulating metabolites, including many known and previously-unreported LPCs (described below) as well as thousands of other metabolites implicated in oncogenic and immune phenotypes.

[0360] Example 2: Reduced levels of distinct LPCs associate with irAEs.

[0361] To determine whether circulating metabolites may be associated with the development of irAEs in cancer patients undergoing ICB therapy, studies were peformed using a subset of plasma samples taken from a larger cohort of metastatic melanoma patients undergoing ipilimumab (anti- CTLA4) ICB monotherapy. Each patient was sampled at baseline, directly prior to receiving the first dose of ipilimumab, and then at 3-, 6-, 9- and 12- weeks post-initiation of treatment. These time-points captured acute onset of the majority of irAE toxicities commonly observed in ICB patients. Using the rLC-MS approaches -8000 spectral features were assayed representing unique small molecule metabolites. Statistical regression modeling with irAE incidence and application of Bonferroni correction for testing of multiple hypotheses was done (see Research Strategy for details of the statistical analysis), and several metabolites significantly associated with development of severe irAEs (e.g. grade III-IV) were detected (FIG. 2A).

[0362] Among the most statistically robust metabolite associations detected were two chemically- related but distinct forms of lysophosphatidylcholine (LPC) 16:0 and 18:2 (denoting fatty acid chain length and number of unsaturated bonds, respectively) whose levels in circulation were indistinguishable at baseline, but significantly decreased over time in patients who developed severe irAEs after ipilimumab therapy (FIG. 2B) including hypophysitis and colitis, thereby suggesting a potential protective role for these metabolites. These results show that particular LPC species underlie protective responses to ICB-related irAEs.

[0363] The divergent effects of LPC on human physiology and disease is likely due to the particular bio-activities of structurally distinct LPC species. Prior studies have largely focused on ~35 highly abundant circulating LPCs. Computational spectral networking of non-targeted rLC- MS data, however, revealed >100 structurally distinct LPCs species in humans, including many previously unrecognized isoforms (FIG. 3). Moreover, substantial multi-level organizational structure wasobserved, including co-clustering of circulating LPCs, reflecting common enzymatic or biochemical pathways and tissues of origin (FIG. 3).

[0364] Example 3. LPCs levels are reduced in models of ICB-related irAEs and colitis.

[0365] To determine whether changes observed in human patients with ICB-related irAEs were conserved in mice, circulating LPC levels in two relevant inflammation models were examined. In C57BL / 6 transgenic knock-in mice were engineered to express the human CTLA4 gene, which developed severe multi-organ inflammation upon treatment with ipilimumab (100 ug / mouse / injection on days 10, 13, 16, and 19 after birth), negative correlation between LPC 16:0 or 18:2 levels and irAE score was observed (FIGs. 4A-4B, n=5 mice per group), comparable to the association observed in patients with ipilimumab-related irAEs (FIGs. 2A-2B). Similarly, in a dextran sulfate sodium (DSS)- induced colitis model, corresponding decrease in circulating LPC 16:0 and 18:2 levels were also noted (FIG. 4C, n=5 mice per group), in association with development of colitis symptoms (e.g. weight loss and colonic shortening). These data demosntrate that changes in circulating LPC 16:0 and 18:2 levels are conserved in mouse models of inflammation.

[0366] Example 4. LPC supplementation ameliorates colitis.

[0367] To determine whether the decrease in circulating LPCs is causally related to the development of irAEs, or whether circulating LPCs are a passive biomarker, LPC 18:2 was supplemented in DSS colitis mice. C57BL / 6 mice were administered PBS (vehicle) or 25mg / kg 1- linoleoyl-gylcero-3- phosphocholine (LPC 18:2) every 48 hours to increase serum LPC 18:2 levels (FIG. 5A). Colitis was induced by treatment with 2.5% DSS, and after 7 days it was observed that LPC 18:2 supplementation reduced colitis-related colonic shortening (FIG. 5B) and weight loss (FIG. 5C). These data show that LPCs are protective factors that prevent the manifestation of autoimmunity and that LPC loss during ICB treatment may propagate irAEs.

[0368] Example 5. LPC supplementation does not alter ICB-related tumor regression.

[0369] It was observed that LPC 18:2 may exert protective effects in autoinflammation. Potentially, LPC 18:2 may also influence ICB-induced tumor reduction. To determine the role of LPC 18:2 in ICB-related tumor response, wild type C57BL / 6 mice were inoculated subcutaneously in the flank with LOxlO5B16 melanoma cells and treated with antibody isotype controls or combination ICB therapy (anti-CTLA4 and anti-PDl , 100 ug / mouse / injection on days 4, 7, 10, and 13). Mice were pretreated with vehicle or LPC 18:2 on days 3, 5, 7, 9, 11, and 13. Combination ICB therapy markedlyreduced tumor volume, which was unaffected by LPC supplementation (Figure 6), showing that the immune protective effect of LPC 18:2 is tumor-extrinsic.

[0370] Example 6: LPC correlates of ICB -related irAEs.

[0371] In the primary cohort of N=94 melanoma patients, regression analyses adjusted for age, sex, and baseline measures (for change models) were used to examine the association of baseline and change in each LPC species with development of: (a) any irAE (primary outcome); (b) severe irAE (Grade IILIV) versus mild (Grade I-II) or no irAE; and, (c) organ-specific irAEs (i.e. endocrine, integument, GI, or rheumatologic). LPC ‘change’ considered as area under the curve and secondarily as maximum excursion (max difference from baseline, regardless of time point) and end excursion (difference in latest timepoint value from baseline). To leverage availability of serially collected samples, established approaches to longitudinal data analyses (e.g. multivariate linear regression), incorporating an algorithm-based approach to accommodate LPC species that may demonstrate nonlinear (e.g. biphasic) increase, decrease, or both increase and decrease from baseline levels in response to immunotherapy. Such an approach can include hierarchical classifications of change patterns and be implemented in the context of functional principal components analysis (FPCA). For any LPC significantly associated with irAE, association with PCA defined clusters were examined, and Spearman rank correlation analyses was used to identify groups of inter-correlated molecules (using a threshold of r>0.50).

[0372] In the secondary cohort of N=93 patients with a diversity of solid tumors (treated with ipilimumab monotherapy or a combination therapy) and tertiary cohort of N=58 patients with melanoma or NSCLC (treated with pembrolizumab + SBRT), statistical analyses were repeated to identify irAE-associated LPCs matched in directionality and significance of association with the primary cohort. In secondary analyses, meta-analysis of pooled cohort samples was conducted, and whether any observed associations are mediated by tumor or therapy type using the Baron and Kenny’s, Sobel’s, and / or Preacher & Hayes bootstrap methods was determined. The effect modification by age and sex, and were also analyzed where appropriate.

[0373] Example 7: LPC Correlates of ICB-related Tumor Regression.

[0374] To determine the extent to which LPC associations with irAEs are common with or independent from response, baseline and change in all LPC forms analyzed herein with partial or complete response according to RECIST metrics were examined, using parallel statistical approaches as outlined above. Analyses were adjusted for age, sex, and baseline (for change models) LPC values,and are performed in each cohort separately and then pooled as a meta-analysis. Summary statistics were combined as input data for comparisons of matched metabolite-outcome associations with significance set at varying thresholds (R package Venn Diagram).

[0375] Example 8: LPC Correlates of Naturally-Occurring Autoimmunity.

[0376] Given the association between reduced LPCs and ICB-related irAEs, it was found that specific LPC isoforms were concordantly decreased in the circulation of patients who develop natural autoimmunity. This was tested in the landmark FINRISK cohort, a longitudinal community-based study initiated in 1972 in Finland to monitor trends and risk factors for cardiovascular disease in the population; this is among the longest ongoing epidemiologic studies in Europe. The FINRISK 2002 cohort was collected onset from a random sample of the population aged 25-74 years from six geographical areas of Finland, totaling 8683 participants. Since enrollment and over the last 15 years, all participants have been under surveillance for incidence of physician documented chronic diseases, including specific autoimmune diseases, enabled by collection of diagnosis codes from the Finnish National Hospital Discharge Register and Causes-of-Death Register. Autoimmune diseases include: systemic lupus erythematosus (SLE), Crohn’s disease, ulcerative colitis, rheumatoid arthritis, and mixed connective tissue disease (MCTD). Table 1 displays the characteristics for individuals with prevalent or prospective incident autoimmune disease. Plasma specimens from FINRISK participants (enrolled in 2002) have already undergone rLC-MS based study, including with measure of >100 LPC molecules, using methods described. From these studies, >100 LPCs are quantified (see LPC Identification).Table 1. FINRISK Study Samples

[0377] LPC measures were log-transformed and batch-normalized. The LPC species identified above were examined with ICB-related irAEs for relationship to prevalent autoimmune disease in regression analyses adjusting for age, sex, and batch. A nested case-control design was used torelate metabolites to risk for case status (incident autoimmune disease) versus control status (free of autoimmune disease). Cases were matched to controls 1:3 based on age (within 5 years), sex, and years from baseline to diagnosis of autoimmune disease (vs. remaining free of autoimmune disease). For any significantly associated LPC, their association with PCA defined clusters identified above was examined, and Spearman rank correlation analyses was used to identify groups of inter-correlated molecules (using a threshold of r>0.50). Effect modification by age or sex was also tested.

[0378] For the primary (N=94) or combined (N=245) cohorts with baseline and serial blood specimens available for analyses, a partial correlation of 0.157 corresponds with power of 80% with alpha=0.05 for detecting association of an LPC molecule (assuming up to 20 non-highly correlated LPC metabolites with >50% non-missing values) with a given outcome (based on events rates in Table 2); Partial correlations of similar magnitude in human studies were observed, including smaller sized cohort samples. LPC are known to be inter-correlated based upon their biosynthetic pathways (with variable missingness across a cohort experiment with repeated measures). It was found a partial correlation of 0.206 corresponding with power of 80% with alpha=0.05 for detecting the association of a metabolite representative of up to 20 informative metabolite clusters assayed using methods with prioritization of known classes of molecules; the number of clusters identifiable from >4000 metabolites (of known molecular classes) is determined using principal components analysis and alternate cluster analysis techniques. All estimates are based on a multivariable model including adjustments for age, sex, and baseline LPC measure (for change models). It was observed that an 80% power is needed to detect an association of a LPC (per 1-SD increment in log- metabolite) with prevalent autoimmune disease with a partial correlation of 0.210. Similarly, it was found that an 80% power is needed to detect an association of a given LPC (per 1- SD) with incident autoimmune disease with a partial correlation >0. 194. These also apply to the unknown metabolites, among which up to 20 clusters of inter-related analytes were identified (FIG. 7).Table 2. Study Samples

[0379] Example 9: LPC and ICB-related irAEs

[0380] To determine whether LPCs directly modulate the development of ICB- related irAEs in vivo, transgenic knock-in mice expressing two copies of the human CTLA4 gene were examined. In this model, intraperitoneal (z.p.) administration of ipilimumab mAb shortly after birth (100 ug / mouse / injection of clone MDX-010 Selleckchem on days 10, 13, 16 and 19) induced severe multiorgan inflammation in the heart, lung, kidney, liver and colon over a period of 42 days. Importantly, combination therapy with anti-PDl mAb (100 pg / mouse / injection of clone BE0033-2 Bioxcell) exacerbated irAEs, and development of toxicities correlated with the emergence of a systemic auto- reactive T cell response but not with ICB-related tumor regression. It was found that circulating LPC 16:0 and 18:2 levels were reduced upon ipilimumab treatment in CTLA4hu / hu mice and correlated with irAE score, similar to melanoma patients undergoing ipilimumab therapy. In these mice, 16:0 and 18:2 levels were increased by ip administration or dietary supplementation, or decreased by inhibiting sPLA via oral administration of LY315920 prior to treatment with antibody isotype controls, ipilimumab mAb or ipilimumab / anti-PDl mAbs, as described above. irAEs were scored by monitoring weight loss and by histopathology analysis of formalin fixed organs (e.g. heart, liver, kidney, lung and colon) to assess lymphocyte aggregation at the terminal timepoint of 42 days. Circulating CD4+ / CD8+ effector memory CD44hiCD62Llo T cells in the peripheral blood was also monitored using flow cytometry, as expansion of this subset specifically correlates with the emergence and severity of irAEs. LPC deposition in each affected organ was also assessed, as described above. Male and female mice were tested in groups of five for each experiment, with two repeats. Diminishing LPC via sPLA inhibition or abolishing LPC signaling through the GPR132 / G2A receptor will exacerbate irAE toxicities.

[0381] Example 10: LPC and Tumor Growth

[0382] Circulating LPC levels are modulated as described above prior to subcutaneous injection of 1.0 x 10^ cells B 16 cells. Tumor sizes were monitored at 4, 7, 10, 13 and 20 days after implantation using standard caliper measures and mice sacrificed to isolate tumors, ascites and peripheral blood sera at the terminal timepoint. Secreted cytokines: Anti- tumor cytokines, IFNalpha / beta, CXCL9, CXCL10, CCL5 / RANTES, TNF and IFN-gamma are measured in tumor ascites and peripheral blood sera using Nano-string (mRNA) and ELISA (protein), as described. Immune cell profiling: Should differences in tumor size be observed as a result of LPC modulation, flow cytometry analysis of single cell suspensions of B16 tumors are performed to examine immune cell composition (FIG. 8) using panels of markers for CD45+ immune cells: anti-tumor M1H0T TAM, tumor promoting M2 TAM, neutrophils, DC, CD8a DC, MDSC. T cell populations were quantified for tumor-resident CD8+ TRM cells, CD4+ Treg, NK cells, and NKT cells. ICB-related tumor regression; Upon development of palpable tumors (8mm, or ~4 days after transplantation), tumor-bearing mice were treated with isotype control antibodies or combination ICB therapy (anti-CTLA4 and anti-PDl, 100 ug / mouse / inj ection on days 4, 7, 10, and 13). Tumor size was monitored and immune phenotyping performed as above.

[0383] Example 11: LPC and Colitis

[0384] In order to examine the immune mechanisms by which LPC modulate colitis, in-depth immunological analysis of 2 mouse models was performed. In the first model, colitis was induced by providing DSS in the drinking water. This is a rapid and acute model, resulting from inflammatory injury to the intestine epithelium. Although in some experiments it was not entirely dependent on T cells, DSS colitis was worsened by treatment with anti-CTLA4 antibody or by combination therapy with anti-CTLA4 and anti-PD 1. In the second model, colitis was induced by transfer of naive CD4+ T cells, depleted of Treg, into immune deficient Ragl- / - mice. This model is progressive over 4-10 weeks and requires the activity of Thl and Th 17 cells that induce various aspects of the innate immune response.

[0385] LPC levels are modulated through sPLA enzyme inhibition using oral administration with LY315920 or injected ip with LPC 16:0 or 18:2 in the DSS model. Alternatively, because the T cell transfer model takes longer to develop and frequent ip injections may not be practical, Ragl- / - recipients were fed a high palmitic acid rich diet, a high oleic acid rich diet or combination diet beginning one week prior to initiation of colitis. For a gene -based gain of function model, LCAT over-expressing mice was studied. To induce the DSS model, mice were given 2.5% DSS in drinkingwater for seven days and followed for weight loss and clinical signs. Histology was analyzed according to a scoring system used consistently and colon length was analyzed to measure fibrosis, as well as staining for collagen deposition. To induce colitis by T cell transfer, lymph node and / or splenic CD4+ T cells were enriched using magnetic isolation followed by positive selection for naive (CD45RBhigh) CD4+ T lymphocytes by flow cytometric sorting. Approximately 5xl05cells were injected into Ragl- / - recipient mice. Induction of colitis, initially apparent by weight loss, occurred in 4-10 weeks. Primary analyses included measuring histologic signs, the weight and length of the colon, histological scoring according to standardized protocol in the lab and quantification of immune cell populations in the colon lamina propria. Male and female mice were tested in groups of five for each experiment, with two repeats. Dietary supplement or inhibitor treatment was initially provided in a prophylactic fashion, two days before DSS treatment or T cell transfer. To model checkpoint blockade, in the DSS model mice were treated with 100 pg of anti-CTLA4 or isotype controls at days -3 and day -1 before DSS. For dual antibody treatments, mice received 100 pg each of anti-CTLA4 and anti-PDl at day -1 and day 6. In the T cell transfer model, mice over expressing LCAT were crossed to Ragl- / - mice to generate double knock out recipients for either wild type or LCAT over expressing naive CD4+ T cells. Sera from mice and intestinal tissues was tested for LPC concentrations, including wild type and LCAT over expressing mice with and without colitis, with and without antibody treatment (DSS model) and under conditions in which LPC concentrations were altered as described above.

[0386] Immune cell profiling: In those cases where altering LPC affects colitis pathogenesis (including CTLA4hu / hu mice, in which colon is severely injured), mice and appropriate controls were analyzed for number and function of hematopoietic cells infiltrating the intestinal tissue by multiparameter flow cytometer and by immunohistochemistry (see FIG. 8). The focus was on the major cell types likely to be involved in colitis pathogenesis, including CD4+ T cells in the mesenteric lymph node and colonic lamina propria (the only adaptive cell in the T cell transfer model), CDllb+CDl lcintF4 / 80+ colonic macrophages and CD1 Ib-CDl lc+F4 / 80- DCs, but other cells that are relevant such as ILC, CD8 T cells, NKT which have LPC reactivity, and stromal cells were analyzed. Colon cells were activated in vitro by PMA and ionomycin and stained for intracellular cytokines. Colon fragment cultures were analyzed for cytokine production directly ex vivo.

[0387] LPC Signaling: To determine if LPC signaling directly modulates colitis, the GPR132 / G2A signaling receptor was examined, which directly binds unsaturated and saturated LPCs and is widely expressed in immune cells. For these experiments, DSS and T cell transfer colitis was examined in Gprl23 knockout mice, in which LPC levels are modulated by supplementation or pharmacology.These mice developed spontaneous, aging-associated autoimmunity, suggesting a protective role in inflammation.

[0388] Alternative pathways'. LPC may elicit indirect effects through Lysophosphatidic acid (LPA). To examine LPA, PF-8380 (30 mg / kg, Cayman Chemical), a potent inhibitor of Autotaxin (Atx)- mediated processing of LPC>LPA was orally delivered, to decrease plasma levels in vivo prior to examining immune toxicities.Materials and Methods IStudy samples (see Table 2 for patient numbers and demographics). The primary cohort includes N=94 patients enrolled at Cancer Center UK, University of Southampton with melanoma at baseline metastatic stage (MO, Mia, Mlb, Mlc, classified by tumor-node-metastasis classification), refractory to standard care and without prior treatment with IL-2 or immunotherapy. Patients received induction course of ipilimumab, at a dose of 3 mg / kg once every 3 weeks (4 treatments total). Fasting plasma was collected at baseline (0 weeks) as well as 3, 6, 9, and 12 weeks during therapy (total sample N=470), with blood sampling performed prior to ipilimumab dosing. Tumor burden was evaluated by body CT or MRI and skin lesion imaging at baseline and 12 weeks. Tumor assessments were performed at baseline and study completion, with objective response measured using RECIST 1.1 criteria. Subjects were monitored at baseline and at 3, 6, 9, 12 weeks post- therapy with strict adjudication of irAEs graded according to the NCI’s Common Terminology Criteria for Adverse Events. irAE was defined as an adverse event associated with ipilimumab exposure that was consistent with an immune phenomenon. Protocol guidelines for irAE management included corticosteroid administration, delay in a scheduled dose, or discontinuation of therapy for severe irAEs.

[0389] The secondary cohort includes N=93 patients enrolled at Moores Cancer Center, UC San Diego, an NIH designated Comprehensive Cancer Center, under Phase I clinical study designation (NCT02658890). Patients have advanced or metastatic solid tumors (melanoma=20, non-small cell lung cancer=20, head and neck squamous cell cancer=27, colorectal cancer=8, other solid tumor=19) refractory to standard care and without prior exposure to immunotherapy. Patients received mono- or combination therapy with ipilimumab plus / minus nivolumab or pembrolizumab (anti-PDl), at a dose of 2 mg / kg every 3 weeks with collection of fasting plasma samples at baseline prior to immunotherapy, as well as after 3, 6, and 12 weeks prior to dosing (N total samples=372). Tumor burden was evaluated using CT or MRI at baseline and at study completion (16 weeks post initial therapy). As with the primary cohort, tumor response was assessed using RECIST 1.1 criteria andirAEs graded according to the NCI’s Common Terminology Criteria for Adverse Events. irAEs were defined and treated according to protocol guidelines described above.

[0390] The tertiary cohort includes N=58 patients enrolled at the Smilow Cancer Center at Yale University into a Phase I clinical study (NCT02407171) for advanced melanoma (N=5) or non-small cell lung cancer (N=53) refractory to standard care and not previously exposed to immunotherapy. Patients received the pembrolizumab at a dose of 2 mg / kg once every 3 weeks with stereotactic body radiotherapy (SMRT). Fasting blood samples were collected at baseline prior to treatment (0 weeks) as well as at 3, 6, 9, 12, 15, and 24 weeks during therapy (N=total samples 406). Tumor burden evaluation was performed using CT or MRI, at baseline and at 16 weeks. Objective tumor response and irAE were assessed and treated as described for primary samples above.

[0391] rLC-MS metabolomics. The rLC-MS approach was adapted from work in traditional LC- MS metabolomics. For LPC extraction, 40 uL of plasma is transferred from 2D barcoded cryo-sample tubes using an Agilent Bravo liquid handler into a 96- well extraction plate containing 160 uL of ice- cold LC-MS -grade methanol per well. Plates were sealed, vortexed and centrifuged to precipitate proteins with supernatant containing the extracted metabolites pipetted into a clean microtiter plate for analysis. Three independent rLC separation approaches were used - a reverse phase (Cl 8 stationary phase; 98% water / 2% methanol / 0.2% acetic acid loading solvent), HILIC phase (HILIC stationary phase; 90% hexane / 10% isopropanol loading solvent), and normal phase (aminopropyl stationary phase; 90% acetonitrile / 10% water / 10 mM ammonium acetate loading solvent) - each coupled to positive and negative electrospray ionization MS. Rigorous Qc / Qa of data was performed, using ten 13C-labeled exogenous standards and synthetic standards and interval pooled plasma samples, to monitor fluctuations in extraction efficiency, instrument sensitivity, matrix artifact and mass accuracy. For analysis of distinct LPC isoforms, rLC-MS data was extracted and peaks quantified using Agilent Profinder batch analysis software using a recursive molecular feature extraction (rMFE) with secondary ‘find by ion’ algorithm, followed by statistical analysis in the Agilent Mass Profiler Pro software suite, where molecules present at <10x background or in solvent blank are removed. In addition to comprehensive measure of LPCs, using the proposed rLC-MS methods, -4000 molecules per plasma sample was additionally captured.

[0392] LPC identification. Targeted MS / MS fragmentation was performed for each mass spectral signal, and MS / MS patterns are ‘blast’ searched against an in-house MS / MS library of >3000 metabolite standards, which includes all known LPC molecules and new species identified in this study. From these studies, >100 LPCs per sample were routinely assayed and quantified (Figure 3). For any unknown molecules of interest, metabolites were searched against public spectral metabolitelibraries, including Metlin (http: / / metlin.scrips.edu), Massbank (http: / / www.massbank.jp) and HMDB (http: / / www.hmdb.ca).

[0393] Statistical analyses. Robust downstream statistical analyses for handing of large-scale metabolomics data were used to examine associations between circulating levels of LPC and ICB outcomes. Prior to all analyses, each metabolite variable was log- transformed (given the typical right- skewed distribution of most analytes detectable in human peripheral circulation), batch normalized, and then standardized to mean=0 and SD=1 to facilitate the comparison of effect sizes across LPC metabolites. Clustering methods were used, including PCA (using eigenvalue thresholds of 1) to identify groups of LPC molecules that exhibit high degrees of inter-correlation with other chemically unrelated metabolites that are co-measured in MS analysis; such small molecule ‘clusters’ may represent possible common synthetic pathways and / or tissues of origin. Changes in LPC per unit time were assessed in descriptive analyses, using mean (difference in log-analyte), fold (ratio of serial loganalyte measures), and percent change (fold change minus 1) values as described previously for the circulating human metabolome.

[0394] Example 12: LPC (18:2) and LPC (16:0) as Biomarkers of irAEs

[0395] To uncover metabolite associations with ICB-driven irAEs, circulating bio-active lipids with surrogate association to irAEs were examined. In total, 753 blood plasma samples from 150 cancer patients undergoing ICB therapy were sampled across three distinct cohorts (Table 3). The discovery cohort, corresponding to melanoma patients treated with 3mg / kg ipilimumab (Cohort 1, n = 65), displayed total irAE incidence of 56.4 percent, of which 6.4 percent constitute severe grade 111 / I V irAEs. Sampling corresponded to baseline and serial draws up to twelve weeks post-treatment, without significant sampling bias in age or sex demographics, or in tumor regression outcomes (Table 4). This sampling window directly overlapped with the reported timeline of irAE development following ipilimumab treatment. Plasma samples were analyzed using directed, non-targeted liquid chromatography mass spectrometry (LC-MS), allowing for rapid simultaneous profiling and semi- quantitative ranking of thousands of unique bio-active lipids . The maximum absolute excursion or change in abundance of each LC-MS feature was associated with the severity of ipilimumab-derived irAEs.Table 3.Table 4. Cohort 1: Ipilimumab-treated Advanced Melanoma UK Southampton00396] Approximately 6,000 LC-MS features were abundantly detected in patient plasma, of which several molecules were differentially expressed over time in association with irAE severity (FIG. 9A). Within this subset, molecules linked to severe grade III / IV irAEs were the focus. From a network analysis of targeted tandem mass spectral data, chemically-related irAE molecules (FIG. 9E) were identified against a library of chemical standards as palmitoyl-lysophosphatidylcholine (LPC (16:0)) and linoleoyl-lysophosphatidylcholine (LPC (18:2)) (FIGs. 9B and 9F), which are monoacyl metabolites of membrane phosphocholines detectable in blood at micromolar levels. LPCs comprisea super-family of bio-active second messengers implicated in immunity and inflammation. In ipilimumab-treated patients with severe irAEs, LPC (18:2) and LPC (16:0) levels significantly dropped during therapy (FIG. 9C). The association was not observed for any other LPC species, or in chemically and metabolically related lysophosphatidylethanolamines(LPE), lysophosphatidylinositols (LPI), or lysophosphatidic acids (LPA) molecules (Table 4). Importantly, LPC (18:2) and LPC (16:0) excursions were not significantly associated with RECIST 1.1 metrics of ipilimumab-driven tumor regression response (Table 4), nor did baseline abundance of LPC exhibit predictive value, although both lipids were modestly elevated at baseline in patients who developed severe irAEs (FIG. 9G). These data indicate that LPC (16:0) and LPC (18:2) are specifically depleted in patients who develop severe irAES during treatment with ipilimumab, unique among all lysolipids measured in circulation.

[0397] Depletion of LPCs from circulation occurs in inflammatory pathologies and autoimmunity. Loss of LPC (18:2) was detected in patients with systemic lupus erythematosus (SLE) and during sepsis, and is a risk factor for development of Type-II Diabetes and myocardial infarction. LPC (18:2) and LPC (16:0) abundance was measured in blood plasma from patients with prevalent autoimmune or chronic inflammatory conditions, including SLE, rheumatoid arthritis, and inflammatory bowel disease (FINRISK-2002, n = 158). Relative to healthy individuals, significantly lower LPC (18:2) and LPC (16:0) levels were observed in plasma from autoimmune patients (FIG. 9D), suggesting that LPC levels are a marker of deleterious inflammation shared in common with ipilimumab-induced irAEs.

[0398] Clinical and preclinical evidence support both overlapping and distinct mechanism of action for ipilimumab compared to other ICB antibody therapies. To explore the generalizability of the observations, two orthogonal cancer patient cohorts were examined (Table 3). First, a community- acquired cohort of mixed solid tumors (Cohort 2, n = 32), containing patients treated with ipilimumab in combination with nivolumab, or with anti-PDl therapies alone. In this cohort, reduction of LPC (18:2) and LPC (16:0) in patients who developed severe irAEs upon anti-PDl monotherapies or ipilimumab plus nivolumab combination therapy (FIGs. 10A-10B, middle panel; and FIGs. 10D- 10E). Second, in a prospective Phase I pembrolizumab trial of advanced NSCLC and melanoma patients (Cohort 3, n= 53), reduction of LPC (18:2) and LPC (16:0) was also observed in patients who developed severe treatment-associated irAEs (FIGs. 10A-10B, right panel). The effect size of LPC (16:0) and LPC (18:2) excursion on ICB is calculated to be -2.26 (CI -0.86 - -3.72) and -1.12 (- 0.25 - -2.01) log(odds ratio), respectively, for ICB-irAE associations (FIG. 10C).

[0399] Together, the data show that LPC (18:2) and LPC (16:0) are surrogate biomarkers of severe irAEs associated with ICB therapy. To further examine the relationship between LPC and ICB-driven outcomes in vivo, two relevant mouse models were studied. First, an ipilimumab-induced model of irAEs, employs humanized C57BL / 6 mice expressing the CTLA4 transgene (CTLA4h / h). CTLA4ll / hmice develop multi-organ immune toxicity from ipilimumab monotherapy or combination therapy with anti-PDl within the same therapeutic window as tumor regression response. The second model is dextran sulfate sodium (DSS)-induced colonic inflammation and colitis, a model in which CTLA4 plays a role in enhancing inflammation. Mass spectrometry analysis of terminal blood from ipilimumab and ipilimumab / anti-PD- 1 -treated CTLA4h / hmice demonstrated significant reduction of LPC (18:2) and LPC (16:0) (FIG. 11A), consistent with findings in human cancer patients. In DSS- induced colitis, reduction of LPC (18:2) and (16:0) levels was also observed in terminal blood plasma (FIG. 11B). Together, these results show that reduction in circulating LPC (18:2) and (16:0) is also observed in mouse models of ICB-driven irAEs and chemically-induced colitis.

[0400] Example 13: LPC (18:2) as a Protective Metabolite

[0401] Previous studies showed that unsaturated 18-carbon LPCs are consistently depleted in the blood of DSS-treated mice, and play a protective role in colitis. Dietary supplementation of oleic acid (Cl 8:1) or overexpression of liver stearoyl-CoA desaturase- 1 in DSS-treated mice elicited protective effects, although it is unclear whether the effect is driven by LPC (18:2), LPC (18:1), or another oleic acid metabolite. Compared to LPC 18:2, loss of LPC (16:0) was less pronounced in CTLA4h / hmice (FIG. 3A). Furthermore, reduction of LPC (18:2) was significantly correlated with a composite histological score of irAE severity (FIGs. 11C and 11F). Based upon the stronger association of LPC 18:2 and irAEs, CTLA4h / lmice were supplemented with LPC (18:2) and effects upon irAEs examined. Exogenous LPC (18:2) was administered at physiological levels via intraperitoneal injection (FIG. 11G) to CTLA4ll / hmice prior to ICB treatment. The supplemented mice experienced a reduction in colonic inflammation and leukocytic infiltration (FIG. 11D and 11H), indicating reduced ICB-associated toxicity. In DSS-induced colitis, supplementation of LPC (18:2) significantly reduced weight loss and colonic shortening (FIGs. 12A-12B), reduced intestinal inflammation, and mitigated overall intestinal damage to crypt cells (FIG. 11D). In contrast, administration of LPC (16:0) did not elicit protective effects against DSS-induced colon toxicity (FIG. 12C), nor did saturated LPC (18:0), although the monounsaturated LPC (18:1) demonstrated a modest protective effect upon colon shortening (FIG. 12D). Together, results show that circulating LPC (18:2) protects against colon inflammation.

[0402] How loss of circulating LPC (18:2) impacts immune function and irAEs is unclear. In general, mechanisms underlying ICB -driven irAEs may involve increased activity of autoreactive effector T-cells, higher auto-antibody titers, activation of innate immune cytokines, or complement- mediated damage to healthy stroma. The parameters of systemic immunity can be examined by measuring immune cells, serum cytokines, and antibody titers, with population-level variance explained at least in part by circulating metabolite variation. LPC (18:2) levels were measured in healthy human blood plasma from two large-scale, community-based cohorts (Table 5), where serum cytokines, serology, and 80 immune cell populations, measured using multi-color flow cytometry, were quantified in participants. Correlation between effector or proliferating lymphocyte populations and circulating LPC (18:2) was not observed in healthy individuals (FIG. 13A), nor were association between LPC (18:2) and immunoglobulin titer detected (Table 6). In the 500 Functional Genomes cohort (500FG, n=500), levels of LPC (18:2) were significantly inversely correlated with neutrophils and natural killer T (NKT) cells in the blood (FIG. 14A). These observations were validated using the FINRISK-2002 cohort (FINRISK-2002, n = 215), where clinical blood counts were used to measure neutrophils and other polymorphonuclear myeloid cells (FIG. 14B). Notably, circulating LPC (18:2) inversely correlated with serum IL-8, a key cytokine that drives neutrophil cellularity (Table 7 and FIG. 13B). Together, these results suggest that loss of circulating LPC (18:2) associates with aberrant innate immune activation in ICB-driven irAEs, specifically an increase in circulating neutrophils.Table 5.Table 6.Circulating LPC (18:2) associations by linear regression with serum immunoglobulin titers in 500FG (IgG [7.00-16.00 gram / Liter]; IgA [0.70-4.00 gram / Liter]; IgM [0.40-2.30 gram / Liter]; IgGl [4.90- 11.40 gram / Liter]; IgG2 [1.50-6.40 gram / Liter]; IgG3 [0.20-1.10 gram / Liter]; IgG4 [0.08-1.40 gram / Liter].Table 7. Circulating LPC (18:2) associations by linear regression with serum cytokines in FINRISK 2002 (n=2500).

[0403] Acute neutrophilia in the blood is characteristic of infections or ischemic heart failure, while chronic PR3-neutrophilia produces vasculitis and is a feature of rheumatoid arthritis. Unsaturated, 18-carbon LPCs (18:1) and (18:2) have been reported to regulate neutrophil function by inducing neutrophil superoxide release in vitro. Circulating LPC function on peripheral blood neutrophilicity has not been investigated, though albumin appears to blunt LPC (18:2) superoxide induction, supporting the study of circulating LPC function in vivo. Diminished neutrophils were observed in the blood of C57BL / 6 mice acutely treated with LPC (18:1) or LPC (18:2), but not with fully saturated LPC (18:0) (FIG. 14C) Low neutrophil-to-lymphocyte ratio (NLR) ratios are a risk factor for ICB- driven irAEs at baseline, however neutrophil measurements in longitudinal blood samples of ICB- treated patients have not been reported, and the regulatory role of circulating neutrophils has not been studied in this context. Using blood panel analyses from ipilimumab-treated melanoma patients (Cohort 1), increased neutrophil levels were observed upon therapy in patients who developed severe irAEs (FIG. 14D), in contrast to lymphocyte and monocyte excursions which did not demonstrate any relationship to immune toxicity (FIGs. 15A-15B). LPC (18:2) levels exhibited an inverse correlation with neutrophil counts in Cohorts 1 and 2 (FIG. 14E), indicating that the negative association between blood levels of LPC (18:2) and neutrophils is maintained both in healthyindividual and ICB-treated cancer patients. In CTLA4h / hmice treated with ipilimumab and anti-PDl, neutrophils accumulated in penultimate blood counts (FIG. Ill); increased neutrophil counts have also been reported in peripheral blood and colons of DSS-treated mice. These data support neutrophilia in ICB-irAE as a conserved phenomenon in mice, and neutrophil cellularity in the periphery as negatively regulated by circulating LPC (18:2).

[0404] Together, data support a functional role for LPC (18:2) as a protective metabolite for deleterious inflammation during ICB therapy. Tn patients treated with ipilimumab or pembrolizumab, LPC (18:2) is consistently reduced in those who developed severe irAEs during therapy, independently of tumor responsiveness. Indeed, LPC (18:2) was maintained at consistent levels in anti-CTLA4 / anti-PDl treated C57BL / 6 mice bearing B16 subcutaneous melanoma, and that LPC (18:2) supplementation mice had no discernible effect upon the tumor regression response to ICB treatment (FIGs. 16A-16B).

[0405] In summary, the studies demonstrate uncoupling of immune checkpoint blockade toxicity from anti-tumor efficacy via a functionally protective circulating lipid. Blood plasma LPC (18:2) levels deplete over the course of severe irAE progression, mimicking lower levels observed in other autoimmune and inflammatory pathologies. This observation builds on existing reports, where lower levels of circulating LPCs of varied acyl substitutions including LPC (18:2) are observed in prospective and prevalent blood sampling of inflammatory conditions.

[0406] The phenotypic ramifications of depleted LPCs in blood are not understood; here we describe a previously uncharacterized relationship between circulating LPC (18:2), peripheral blood neutrophils, and irAE progression. Neutrophil accumulation on ipilimumab directly correlated with irAE severity, while both healthy and ICB-treated individuals with low LPC (18:2) levels had higher circulating neutrophil counts. LPCs are cAMP-dependent modulators of superoxide release in neutrophils in an acyl saturation-dependent manner, but have not as yet been described as determinants of circulating neutrophil cellularity. It was demonstrated that LPC (18:2) supplementation in vivo rescues ICB- or chemically-induced symptoms of colitis, and negatively regulates peripheral blood neutrophil levels.This study constitutes a useful contribution to the understanding of immune checkpoint blockade toxicity and the progression of severe irAE, specifically through the lens of bioactive lipid metabolites like LPC (18:2) as significant regulators of systemic immunity.Materials and Methods II

[0407] Materials & Cell Lines. All LPCs (LPC (16:0), LPC (18:0), LPC (18: 1), and LPC (18:2)) were purchased from Avanti Polar Lipids. Dextran sulfate sodium was purchased from Affymetrix. Autotaxin inhibitor PF-8380 was purchased from Tocris. Monoclonal anti-mouse CTLA4 (CD152) and anti-PDl (CD279) antibody, as well as IgG2a isotype control antibody, were purchased from InVivoMAb.

[0408] Cohort Information. Archived and de-identified blood plasma samples were obtained in accordance with the institutional review boards of each institute (University of Southampton, Y ale University, University of California San Diego, FINRISK and 500FG IRB).

[0409] Bioactive Lipid Extraction from Blood Plasma. Na-EDTA or heparin prepared blood samples were thawed from storage at -80°C overnight in light-free conditions at 4°C. All extractions were performed in 96-well format. 20uL of each sample were mixed with 80uL of -20°C ethanol containing 20 deuterated standards to precipitate protein and extract lipid content. Samples were vortexed at 4°C and 500rpm for 15 minutes and centrifuged for 10 minutes to sediment protein content. From each supernatant, 65uL were taken and mixed with 350uL water in an Axygen 500uL retention v-bottom 96 well plate. To improve extraction, an additional 65uL of -20°C ethanol was added gently to the protein pellet, vortexed gently by hand for 15 seconds, and added to the same well.

[0410] Complete extracted sample volumes were loaded onto a Phenomenex Strata-X lOmg / mL polymeric solid phase extraction (SPE) 96-well plate pre- washed stepwise with 600 L methanol, 600uL ethanol, and equilibrated with 900pL water. Following gravity elution from each SPE well, 600,u L of 9:1 water: methanol were added as a wash, pulled through slowly at 5mmHg, and then increased to 20mmHg for 45 seconds to fully dry each SPE. Bound metabolites enriched for bioactive lipids were then eluted in 450uL ethanol into a fresh 500, u I Axygen v-bottom plate. Sample eluate was dried in a vacuum concentrator at 40°C until completely dried, before adding 50,uL per well of 75:20:5 water: methanol: acetonitrile containing lOpM CUDA as resuspension solvent. Sample plates were vortexed at 500 RPM for 10 minutes at 4°C to fully resuspend metabolites, before transferring to 300pL glass inserts in a 96-well Greiner deep well plate and immediately sealed. Samples were then immediately analyzed via LC-MS / MS.

[0411] Liquid Chromatography-Tandem Mass Spectrometry. Directed nontargeted liquid chromatography tandem mass spectrometry for the detection of bioactive lipid metabolites wasperformed as described previously24. LC-MS / MS was performed on a Thermo Vanquish UPLC system coupled to a Thermo QExactive Orbitrap mass spectrometer. Injection volume for each sample was 20pL onto a Phenomenex Kinetex C18 (1.7um particle size, 100 x 2.1 mm) column. Mobile phases were composed of; A: 70% water, 30% acetonitrile, 0.1% acetic acid, and B: 50% acetonitrile, 50% isopropanol, 0.02% acetic acid. Flow rate was a constant 0.375 mL / min, with gradient mobile phase as follows: 1% B from -1.00 minutes to 0.25 minutes, 1% to 55% B from 0.25 minutes to 5.00 minutes, 55% B to 99% B from 5.00 minutes to 5.50 minutes, and 99% B from 5.50 minutes to 7.00 minutes. Column temperature was 50°C, with a 50:25:25:0.1 water:acetonitrile:isopropanol:acetic acid needle was set to 5 seconds post-draw. Mass detection was performed with an equipped heated electrospray ionization (HESI) source with manually optimized source geometry24. Negative mode profile data was acquired for all samples, with sheath gas flow, aux gas flow, and sweep gas flow of 40, 15, and 2 units, respectively. Spray voltage was -3.5kV, and capillary and aux gas temperature were 265 and 350°C, respectively, with S-lens RF at 45. MSI scan events were in a scan range of m / z 225-650, mass resolution of 17.5k, AGC of le6 and inject time of 50ms. To assist quantification and aligning intra- and inter-cohort chromatographic drift, tandem mass spectra were acquired using collision-induced dissociation (CID). Data independent acquisition (DIA) acquired in the following four mass windows: m / z 240.7-320.7, m / z 320.7-400.7, m / z 400.7- 480.7, and m / z 480.7-560.7, with a mass resolution of 17.5, and AGC of le6, and an inject time of 40ms. Metabolite matching to LC-MS features was performed against an in-house library of bioactive lipids or by matching high quality tandem mass spectra against target features when identified.

[0412] LC-MS Data Handling. LC-MS peak identification was performed using deep neutral network-based classification as described previously. Briefly, Thermo raw to mzXML file conversion was performed using MSconvert version 3.0.9393 (ProteoWizard), from which initial bulk LC-MS feature alignment was performed using in-house, R-based landmark identification and retention time correction. Chromatographic drift-corrected mzXML were then converted into composite raster image files including m / z and retention time windows bounding putative features. High-confidence features were then identified using a trained neutral network specific for this LC-MS / MS method. Semiquantitative comparisons of LC-MS features utilized peak height intensity values.

[0413] Animal Handling. Mice were exclusively of the C57BL / 6 background, purchased originally from the lackson Laboratory. Subcutaneous tumor modeling and dextran sulfate sodium experiments were performed on mice housed in pathogen-free conditions at the La Jolla Institute for Immunology (La Jolla, CA), while CTLA411 / hICB-irAE experiments were performed at the Institutefor Human Virology at University of Maryland (Baltimore, MD). All procedures involving mice were performed according to the respective Institutional Animal Care and Use Committee.

[0414] Dextran Sulfate Sodium-Induced Colitis Model. Colitis was induced in 9-12 week C57BL / 6 using 5% dextran sulfate sodium (DSS) (Affymetrix) in drinking water. Same sex littermates were utilized; data from male mice, in which colitis was more robustly induced, are reported here, with results validated in female mice. Mice were given 5% DSS for 5 days, followed by two days on untreated water. Primary humane experimental endpoint was body weight loss in excess of 20% starting weight, measured daily. In some cases, mice were administered intraperitoneally with either control saline solution or indicated concentration of LPC. Severity of colitis was measured at experiment termination by body weight loss, colon length, and histological criteria (below).

[0415] Where indicated, blood was collected in EDTA-coated Eppendorf tubes from terminal mice by cardiac puncture, with cervical dislocation as secondary euthanasia. Blood was immediately centrifuged at 2000 RPM and 4°C for 20 minutes to collect blood plasma, which was stored at -80°C until analysis.

[0416] CTLA4 Humanized Mouse. Mice expressing human CTLA4 under the endogenous mouse Ctla4 locus, backcrossed onto the C57BL / 6 background, have been described previously40. Same sex littermate mice were used, with data from female mice where irAE presentation was most robust. Antibodies and LPC (18:2) were administered intraperitoneally at the indicated dose and time interval. Primary humane experimental endpoint was body weight loss in excess of 20%, measured every third day. Mice were humanely euthanized for toxicity scoring and blood plasma LC-MS analysis on Day 42.

[0417] Complete blood counts were collected on Day 41 (penultimate) of treatment from 50 uL of blood, collected in EDTA-coated Eppendorf tubes and analyzed by HEMAVET HV950 blood analyzer (Drew Scientific), according to manufacturer’s protocol.

[0418] Histology. Multi-organ ICB toxicity scores in CTLA4h / hmice were generated from heart, lung, salivary gland, colon, and liver.

[0419] Distal colon or cecum toxicity was determined from hematoxylin and eosin stained tissues as described by Krause et al66. Briefly, cecum and distal colon samples were collected and fixed in zinc formalin (Medical Chemical Corporation) for at least 24 hours prior to paraffin embedding. Tissue was stained with hematoxylin and eosin, and at least six representative 5um slices werecollected from each tissue for embedding on slides. Image acquisition was performed on an Axioscan Z1 platform (Zeiss), using a 20x objective lens, utilizing the Zen 2.3 software automatic scan mode. Slides were blinded and then scored on a composite of the following criteria: inflammation (0 = none, 1 = mild, 2 = moderate, 3 = severe); infiltration (0 = none, 1 = mucosal or submucosal, 2 = mucosal and submucosal, 3 = transmural); crypt damage (0 = none, 1 = basal 1 / 3 damaged, 2 = basal 2 / 3 damaged, 3 = only surface epithelium intact, 4 = entire crypt and epithelium lost); and edema (0 = none, 1 = 1.5-2x submucosal thickness, 2 = >2x submucosal thickness).

[0420] Subcutaneous Tumor Modeling. C56BL / 6 mice were used to model tumor growth and anti-tumor ICB efficacy. B16-F10 melanoma cells (1E6) were seeded in subcutaneous flanks in 9-12 week old mixed gender littermate mice. Tumor volume was measured as (A x B2) / 2, where A = the largest and B = the smallest diameter by caliper. Primary humane experimental endpoint was body weight loss in excess of 20% or tumor volume in excess of 2000mm3. Where indicated, 100 ug antimouse CTLA4 and 100 ug anti-mouse PD1 or 200 pg IgG2 isotype control antibody (InVivoMAb), or 25 mg / kg LPC (18:2), were administered intraperitoneally in 100 pL saline on Days 4, 7, 11, and 14 following tumor seeding.

[0421] Statistical Analysis. Associations between changes in bioactive lipid abundance and irAE severity were handled as ordinal logistic regressions between “none”, “Grade I / II”, and “Grade III / IV” groups; anti-tumor response associations were similarly handled as ordinal according to RECIST 1.1 metrics28of efficacy. Maximum excursion was treated as the largest absolute magnitude fold change of an LC-MS feature peak height relative to patient’s baseline sample following base-2 logarithmic transformation. Regressions included patient age and sex as covariates.

[0422] For continuous variables, including PBMC cellularity, linear regressions were performed against base-10 log transformed variables, with age, sex, and body mass index (BMI) as covariates.

[0423] Example 14: LPC 18:2 is Depleted in Solid Tumor Cancer Patients Experiencing ICB- irAEs

[0424] Analysis of thousands of distinct bio-active lipids detected in human plasma revealed LPC 18:2 as specifically reduced in the blood of patients who develop severe grade III / IV ICB-irAEs (Figures 17A-17B, data from N=65 ipilimumab-treated melanoma patients). A meta-analysis of more than 150 patients across three independent ICB cohort studies further demonstrated an association between reductions in LPC levels and severe ICB-irAEs (Figures 17C-17D).

[0425] Example 15: Lower LPC 18:2 Levels Correlate with Elevated Blood Neutrophil Counts in Healthy Individuals and ICB-irAE Patients

[0426] Circulating LPC 18:2 concentrations exhibited a significant inverse correlation with neutrophil counts in healthy individuals (Figure 18A, 500FG community cohort and FINRISK-2002 community cohort) and severe ICB-irAE patients (Figure 18B, Ipilimumab and combination ICB cohorts). In the 500FG cohort and FINRISK-2002 dataset, reduced LPC 18:2 levels correlated with elevated neutrophil counts, but not with lymphocytes, monocytes, or other immune cells (data not shown). In ICB-irAE patients, increased neutrophilia was associated with severe ICB-irAEs, consistent with the link established above between high neutrophil-to-lymphocyte ratios following ICB treatment and ICB-irAE risk see Examples 1-13). This relationship remained independent of corticosteroid treatment, highlighting a robust connection between LPC 18:2 depletion and systemic neutrophil expansion. These findings suggest LPC 18:2 as a potential indicator of severe ICB-irAEs and a contributing factor in neutrophil-driven systemic inflammation during ICB therapy.

[0427] Example 16: LPC 18:2 in Mouse Models of ICB-irAEs and Colitis

[0428] Two mouse models, ipilimumab-induced colitis in hCTLA-4h / hknock-in mice and dextran sulfate sodium (DSS) chemically-induced colitis, exhibited reduced circulating LPC 18:2 concentrations following the onset of inflammation, resembling patterns observed in human ICB- irAE patients (Figure 19A, 19B, hCTLA-4Mhshown). In ICB-treated hCTLA-4Mlmice, the extent of LPC 18:2 reduction correlated with irAE severity across untreated, ipilimumab-treated, and ipilimumab + anti-mouse PD-1 groups (Figure 19C). To assess a potential causal relationship between LPC 18:2 and severe ICB-irAEs, intraperitoneal lipid supplementation was administered in colitis models. In both models, LPC 18:2 supplementation significantly reduced ICB-induced colitis severity, as determined by histological scoring (Figure 19D, CTLA-4'l hshown). Additionally, LPC 18:2 supplementation reduced neutrophilia in both peripheral blood and the large intestine (Figure 19E-19F).

[0429] Example 17: LPC 18:2 and ICB-Driven Tumor Control

[0430] To investigate the relationship between LPC 18:2 and ICB-driven tumor control, MC38 colorectal cancer cells were subcutaneously implanted into CTLA-4h / h / PD-lh / hmice. Tumor volume was compared across control (IgG, n=3), ICB (ipilimumab + pembrolizumab, n=4), and ICB + LPC (LPC 18:2, 60 mg / kg, n=4) groups. Tumor growth was monitored daily and mice were euthanized when tumors exceeded 1500 mm3. While ICB treatment alone reduced tumor volume relative to IgGcontrols, LPC 18:2 supplementation further increased tumor shrinkage beyond the effects of ICB alone. In the ICB group, tumor regrowth occurred after day 24, indicating acquired therapy resistance, whereas no resistance was observed in the ICB + LPC group. Body weight remained stable across all groups, suggesting no systemic toxicity (Figures 20A-20C). These findings indicate that LPC 18:2 improves responses to human therapeutic ICB antibodies in ICB-irAE mice.

Claims

WHAT IS CLAIMED:

1. A method of identifying a subject that will or is likely to develop an immune-related adverse event following administration to the subject of a cancer therapy, comprising contacting the subject or a sample isolated from the subject with an agent that detects the presence of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 in the subject or the sample, wherein the presence of the of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 at lower than baseline levels indicates that the subject has, will or is likely to have an immune-related adverse event following administration to the subject of the cancer therapy.

2. The method of claim 1, wherein baseline expression is normalized mean expression of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2.

3. The method of claim 1, wherein the method is conducted following administration of the cancer therapy to the subject.

4. The method of claim 3, wherein the method is conducted at least about three weeks following administration of the cancer therapy to the subject.

5. The method of claim 4, further comprising administering one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 to the subject when a lower than baseline level of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is detected in the subject.

6. The method of claim 4, wherein the cancer therapy is chemotherapy, immunotherapy, radiation therapy, and / or cellular therapy.

7. The method of claim 5 , wherein the cancer therapy is immune checkpoint blocker therapy.

8. The method of claim 7, wherein the immune checkpoint blocker therapy is one or more of anti-CTLA4 therapy, anti-PDl therapy, and anti-PDLl therapy.

9. The method of claim 8 wherein the anti-CTLA4 therapy, anti-PDl therapy, or anti-PDLl therapy comprises one or more of Ipilimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab and Durvalumab.

10. A method of treating an immune-related adverse event in a subject receiving cancer therapy, the method comprising administering to the subject one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2.

11. A method of treating an immune-related adverse event in a subject receiving cancer therapy, the method comprising: a. contacting the subject or a sample isolated from the subject with an agent that detects the presence of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 in the subject or the sample; and b. administering one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 to the subject when a lower than baseline level of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is detected in the subject.

12. The method of claim 11, wherein baseline expression is normalized mean expression of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2.

13. The method of claim 12, wherein one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is administered to the subject following administration of the cancer therapy to the subject.

14. The method of claim 13, wherein one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is administered to the subject at least about three weeks following administration of the cancer therapy to the subject.

15. The method of claim 14, wherein the cancer therapy is chemotherapy, immunotherapy, radiation therapy, and / or cellular therapy.

16. The method of claim 15, wherein the cancer therapy is immune checkpoint blocker therapy.

17. The method of claim 16, wherein the immune checkpoint blocker therapy is one or more of anti-CTLA4 therapy, anti-PDl therapy, and anti-PDLl therapy.

18. The method of claim 17 wherein the anti-CTLA4 therapy, anti-PDl therapy, or anti-PDLl therapy comprises one or more of Ipilimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab and Durvalumab.

19. The method of claim 18, wherein the subject has or is being treated for one or more of an epithelial, a head, breast, endometrium, uterus, ovary, testes, neck, lung, prostate, colon, colorectal, pancreas, esophagus, liver, skin, kidney, adrenal gland and brain cancer or tumor, a metastasis or recurring tumor, cancer or neoplasia, a melanoma, a non- small cell lung cancer (NSCLC) and a head and neck squamous cell cancer (HNSCC).

20. A method of decreasing, reducing, inhibiting, suppressing, limiting, controlling or treating an undesirable or aberrant immune response, immune disorder, inflammatory response, autoimmune disease or inflammation, the method comprising administering to the subject one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2.

21. A method of decreasing, reducing, inhibiting, suppressing, limiting, controlling or treating an undesirable or aberrant immune response, immune disorder, inflammatory response, autoimmune disease or inflammation, the method comprising: a. contacting the subject or a sample isolated from the subject with an agent that detects the presence of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 in the subject or the sample; and b. administering one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 to the subject when a lower than baseline level of one or both of lysophosphatidylcholine 16:0 and lysophosphatidylcholine 18:2 is detected in the subject.

22. The method of claim 20 or 21, wherein the undesirable or aberrant immune response, immune disorder, inflammatory response, autoimmune disease or inflammation comprises psoriasis, scleroderma, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, polymyalgia rheumatica, inflammatory myopathy, vasculitis, sicca syndrome, hypophysitis, diabetes mellitus, multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus (SLE), autoimmune thyroiditis, atopic dermatitis, eczematous dermatitis, psoriasis, Sjogren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, inflammatory bowel disease (IBD), cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, interstitial lung fibrosis, Hashimoto's thyroiditis, autoimmune polyglandular syndrome, insulin-dependent diabetes mellitus, insulin-resistant diabetes mellitus, immune-mediated infertility, autoimmune Addison's disease, pemphigus vulgaris, pemphigus foliaceus, dermatitis herpetiformis, autoimmune alopecia, vitiligo, autoimmune hemolytic anemia, autoimmune thrombocytopenicpurpura, pernicious anemia, Guillain-Barre syndrome, stiff-man syndrome, acute rheumatic fever, sympathetic ophthalmia, Goodpasture’s syndrome, systemic necrotizing vasculitis, antiphospholipid syndrome or an allergy, Behcet’s disease, severe combined immunodeficiency (SCID), recombinase activating gene (RAG 1 / 2) deficiency, adenosine deaminase (ADA) deficiency, interleukin receptor common g chain ( c ) deficiency, Janus- associated kinase 3 (JAK3) deficiency and reticular dysgenesis; primary T cell immunodeficiency such as DiGeorge syndrome, Nude syndrome, T cell receptor deficiency, MHC class II deficiency, TAP-2 deficiency (MHC class I deficiency), ZAP70 tyrosine kinase deficiency and purine nucleotide phosphorylase (PNP) deficiency, antibody deficiencies, X-linked agammaglobulinemia (Bruton’s tyrosine kinase deficiency), autosomal recessive agammaglobulinemia, Mu heavy chain deficiency, surrogate light chain (g5 / 14. 1) deficiency, Hyper-IgM syndrome: X-linked (CD40 ligand deficiency) or non-X-linked, Ig heavy chain gene deletion, IgA deficiency, deficiency of IgG subclasses (with or without IgA deficiency), common variable immunodeficiency (CVID), antibody deficiency with normal immunoglobulins; transient hypogammaglobulinemia of infancy, interferon g receptor (IFNGR1, IFNGR2) deficiency, interleukin 12 or interleukin 12 receptor deficiency, immunodeficiency with thymoma, Wiskott-Aldrich syndrome (WAS protein deficiency), ataxia telangiectasia (ATM deficiency), X-linked lymphoproliferative syndrome (SH2D1 A / SAP deficiency), or hyper IgE syndrome.

23. The method of claim 20 or claim 21, wherein the method comprises treating an inflammatory disease or disorder or an autoimmune disease or disorder.

24. The method of claim 23, wherein the method comprises treating inflammatory bowel disease or ulcerative colitis.

25. A method of preventing immune-related adverse events (irAEs) in a patient receiving a treatment for a disease, comprising: a. administering a therapy to the patient capable of treating the disease; and b. modulating a level of at least one small molecule metabolite.

26. The method of claim 25, wherein the disease comprises a cancer.

27. The method of claim 26, wherein the therapy comprises a cancer immunotherapy.

28. The method of claim 27, wherein the cancer immunotherapy comprises an immune checkpoint blocker therapy.

29. The method of claim 28, wherein the immune checkpoint blocker therapy comprises administering one or more of Ipilimumab, Pembrolizumab, Nivolumab, Cemiplimab, Atezolizumab, Avelumab, and Durvalumab.

30. The method of claim 25, wherein the at least one small molecule metabolite comprises any one or more the small molecule metabolites listed in Table 5.

31. The method of claim 25, wherein the at least one small molecule metabolite comprises lysophosphatidylcholine 16:0 or lysophosphatidylcholine 18:2.

32. The method of claim 31, wherein the small molecule metabolite comprises lysophosphatidylcholine 18:2.

33. The method of claim 25, wherein modulating the level of at least one small molecule metabolite comprises administering a pharmaceutically acceptable amount of the at least one small molecule metabolite.

34. The method of claim 33, wherein the at least one small molecule metabolite comprises lysophosphatidylcholine 18:2.

35. A method of treatment, comprising a. modulating a level of at least one small molecule metabolite in a subject to reduce likelihood of irAEs in the subject; and b. treating the subject with a cancer immunotherapy.

36. The method of claim 35, wherein the cancer immunotherapy comprises an immune checkpoint blocker therapy.

37. The method of claim 35, wherein the at least one small molecule metabolite comprises any one or more of the small molecule metabolites listed in Table 5.

38. The method of claim 35, wherein the at least one small molecule metabolite comprises ly sophosphatidylcholine 18:2.

39. The method of claim 38, wherein modulating a level of at least one small molecule metabolite comprises administering a pharmaceutically acceptable amount of ly sophosphatidylcholine 18:2.

40. A method of modulating an immune response in a subject comprising modulating a level of at least of at least one small molecule metabolite, wherein modulating the immune response prevents the development or reduces the likelihood of irAEs.

41. The method of claim 40, further comprising treating the subject with a cancer immunotherapy.

42. The method of claim 40, wherein the at least one small molecule inhibitor comprises any one or more of the small molecule inhibitors listed in Table 5.

43. The method of claim 40, wherein the at least one small molecule metabolite comprises ly sophosphatidylcholine 18:2.

44. The method of claim 43, wherein modulating the level of the at least one small molecule metabolite comprises administering a pharmaceutically acceptable amount of lysophosphatidylcholine 18:2.

45. The method of claim 40, wherein modulating the immune response comprises modulating levels of neutrophils.

46. The method of claim 45, wherein modulating of neutrophils results from modulating the level of the at least one small molecule metabolite.

47. A method comprising:(a) detecting the level of one or more lipid biomarkers in a biological sample obtained from a subject, wherein the one or more lipid biomarkers comprise lysophosphatidylcholine 16:0 (LPC 16:0), or lysophosphatidylcholine 18:2 (LPC 18:2), or both.

48. The method of claim 47, wherein the biological sample is selected from the group consisting of blood, serum, plasma, cerebrospinal fluid, urine, saliva, or tissue extract.

49. The method of claim 47, wherein detecting the level of one or more lipid biomarkers comprises using molecular analysis, wherein the molecular analysis comprises a lipid analytical technique selected from the group consisting of mass spectrometry, liquid chromatography-mass spectrometry (LC-MS), high-performance liquid chromatography (HPLC), and nuclear magnetic resonance (NMR) spectroscopy.

50. The method of claim 47, further comprising comparing the detected level of lysophosphatidylcholine 16:0 (LPC 16:0) and / or lysophosphatidylcholine 18:2 (LPC 18:2) to a predetermined reference level.

51. The method of claim 50, wherein the predetermined reference level is derived from a healthy subject population or a disease-specific threshold.

52. The method of claim 47, further comprising prior to Step (a):(b) extracting the one or more lipid biomarkers from the biological sample using an extraction technique selected from the group consisting of single organic solvent extraction (SOSE), one-phase extraction (OPE), liquid-liquid extraction (LLE), solidphase extraction (SPE), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), Soxhlet extraction (SE), supercritical fluid extraction (SFE), or a combination thereof.

53. The method of claim 52, wherein the extraction technique comprises removing non-lipid components by protein precipitation (PP) using a solvent selected from the group consisting of acetonitrile (ACN), methanol (MeOH), ethanol, isopropanol, ethyl acetate, or propanol before lipid extraction.

54. The method of claim 47, wherein detection is relative to a control, optionally wherein the control is a biological sample collected from a different subject or from the same subject, optionally from the same subject at a different time point.

55. A method of diagnosing a subject that will or is likely to develop an immune-related adverse event, optionally following administration of a cancer therapy; comprising detecting one or more lipid biomarkers according to the method of any one of claims 47- 54.

56. The method according to any one of claims 1 through 9, wherein the detection is performed according to any one of claims 47-54.

57. The method of claim 55, further comprising administering a treatment to the subject when the subject is determined to have an immune-related adverse event or to be at risk of developing an immune-related adverse event.

58. The method of claim 57, wherein the administration of the treatment is performed according to any one of claims 10-46.

59. The method of claim 57, wherein administration to prevent or treat the immune-related adverse event comprises a composition containing lysophosphatidylcholine 16:0 (LPC 16:0), lysophosphatidylcholine 18:2 (LPC 18:2), or both.

60. The method of claim 59, wherein the composition is administered orally, intravenously, subcutaneously, intramuscularly, or topically.

61. The method of claim 59, in the form selected from the group consisting of a tablet, capsule, liquid solution, injectable formulation, gel, or cream.

62. The method of claim 59, wherein the composition further comprises a carrier or excipient.

63. The method of claim 59, wherein the comprises LPC 16:0 and / or LPC 18:2 in a concentration ranging from 0.001% to 10% (w / w) of the total composition.

64. The method of claim 59, wherein the composition reduces inflammation in the subject.

65. The method of claim 59, wherein the composition is co-administered with one or more immunosuppressive or anti-inflammatory agents to increase treatment efficacy.

66. The method of claim 59, wherein the composition is administered before, during, or after a cancer therapy to reduce or prevent the immune-related adverse event.

67. The method of claim 59, wherein the composition is personalized based on the subject’s biomarker levels detected using the method of claim 47.

68. The method of claim 59, wherein the composition is provided as part of a combination therapy with corticosteroids, monoclonal antibodies, or checkpoint inhibitor modulators.

69. The method of claim 59, further comprising monitoring the subject’s biomarker levels post-administration to evaluate treatment response and adjust dosage as needed.

70. A composition comprising:(a) an effective amount of lysophosphatidylcholine 16:0 (LPC 16:0), lysophosphatidylcholine 18:2 (LPC 18:2), or both; and(b) a carrier.

71. The composition of claim 70, further comprising one or more additional skin-benefiting agents selected from the group consisting of humectants, emollients, antioxidants, peptides, vitamins, and botanical extracts.

72. The composition of claim 70, wherein the lysophosphatidylcholine 16:0 (LPC 16:0) and / or lysophosphatidylcholine 18:2 (LPC 18:2) is present in a concentration ranging from 0.001% to 5% (w / w) of the total formulation.

73. The composition of claim 70, wherein the carrier comprises water, glycerin, or an oil-in- water emulsion.

74. The composition of claim 70, further comprising one or more humectants selected from the group consisting of hyaluronic acid, glycerin, sorbitol, and propanediol.

75. The composition of claim 70, further comprising an emollient selected from the group consisting of shea butter, squalane, jojoba oil, and ceramides.

76. The composition of claim 70, further comprising an antioxidant selected from the group consisting of vitamin C, vitamin E (tocopherol), resveratrol, coenzyme Q10 (CoQlO), or green tea extract.

77. The composition of claim 70, further comprising a peptide selected from the group consisting of palmitoyl tripeptide-1, palmitoyl tetrapep tide-7, acetyl hexapeptide- 8, or copper peptides.

78. The composition of claim 70, further comprising a vitamin selected from the group consisting of niacinamide (vitamin B3), retinol (vitamin A), or panthenol (pro-vitamin B5).

79. The composition of claim 70, further comprising a botanical extract selected from the group consisting of om aloe vera, chamomile, licorice root extract, or centella asiatica.

80. The composition of claim 70, wherein the composition is suitable for administration at or near the eye, optionally to the upper and / or lower eyelids.

81. The composition of claim 70, wherein the composition is formulation as a gel, serum, balm, or lotion.

82. The composition of claim 70, wherein the composition is encapsulated in liposomes or nanocarriers to increase delivery and penetration into the skin.

83. The composition according to claim 70, in a form suitable for topical administration.

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