Treatment of major depressive disorder with immunotherapeutic antibodies

Abstract

Disclosed is a method for the treatment of major depressive disorder (MDD), which includes administering to a human subject suffering from MDD, a therapeutically effective amount of an antibody selected from fezakinumab, ustekinumab, gusacitinib and dupilumab, and combinations thereof.

Description

Attorney Docket: 27527-0234WO1TREATMENT OF MAJOR DEPRESSIVE DISORDER WITH IMMUNOTHERAPEUTIC ANTIBODIESFIELD OF THE INVENTION

[0001] The present disclosure relates to the treatment of neuropsychiatric disorders, including major depressive disorder, by the administration of immunotherapeutic drugs. More particularly, this disclosure also pertains to the treatment of major depressive disorder by the administration of immunotherapeutic antibodies.BACKGROUND OF THE INVENTION

[0002] Major depressive disorder (MDD) is a prevalent neuropsychiatric disorder with significant morbidity and mortality, due to a risk for suicide and various medical conditions like cardiovascular disorders and cancer [1, 2], Despite the availability' of effective pharmacological therapies for MDD. more than a third of patients do not achieve full remission following treatment with the standard antidepressant medications [3], One of the many reasons for this insufficient treatment outcome is our limited understanding of the underlying etiopathological mechanisms of MDD [4, 5],

[0003] There is increasing evidence of a close interaction between the brain and immune system contributing to the pathophysiology of stress-related disorders, including MDD [6], Pre-clinical animal models have shown that various forms of stress lead to changes in circulating immune cells and secreted proteins and that blocking specific targets can prevent stress-induced behavioral changes [7-9], In humans, it is now well-established that around 20-30% of patients with MDD show a blood signature with features of low-grade inflammation: In particular, interleukin (IL-) 6, tumor necrosis factor (TNF-) alpha, and C-reactive protein (CRP) have been reported to be elevated among MDD patients compared to healthy individuals

[0010] , On a cellular level, increases in total leukocytes and in various cells of both myeloid (e.g., neutrophils and monocytes) and lymphoid lineages (e.g., T-helper cells) have been reported

[0011] .

[0004] Despite the large number of studies describing immune changes in MDD, few clinical studies have tested the efficacy of anti-inflammatory drugs in reducing symptoms of depression. In fact, infliximab, a TNF-a inhibitor, is the only biologic that has been tested directly in patients with primary mood disorders, with evidence of greater benefit forAttorney Docket: 27527-0234WO1depression in those with higher circulating levels of CRP

[0012] and lipid markers

[0013] , Thus, despite these growing insights into immunological alterations in MDD. the therapeutic role of immunomodulation has been largely unexplored.

[0005] This stands in stark contrast to other immune-inflammatory disorders such as atopic dermatitis (AD) or psoriasis. AD is the most common inflammatory skin disorder affecting up to 10% of adults

[0014] , characterized by pruritic and erythematous lesions most commonly affecting the flexor surfaces, face, and scalp

[0015] , Psoriasis, which affects about 3% of the population

[0016] , characteristically presents with well-demarcated plaques with silvery white scales, accompanied with pruritus and nail changes. While both AD and psoriasis are heterogeneous with involvement of multiple immune axes, dysregulation of T-helper cells is a key feature [17-26], In recent years, several immunomodulatory’ therapies have been approved for these disorders. Most prominently, dupilumab, which targets the IL-4 receptor a subunit (IL-4Ra) and thus inhibits the Th2 pathway, was the first monoclonal antibody approved for the treatment of moderate-to-severe AD [27-30], Dupilumab has demonstrated clinical efficacy, a favorable safety profile, and reversal of the inflamed molecular signature of AD lesional skin [31-33], Other biologics, such as the IL-22 inhibitor fezakinumab and small molecule JAK / STAT inhibitors, are in the pipeline or approved for AD [34. 35], The approved treatments for psoriasis largely target the Thl7 / IL-23 axis, including inhibitors of IL-17A (e.g., secukinumab), IL-23pl9 (e.g., risankizumab), and IL-12 / IL-23 p40 (e.g., ustekinumab). These targeted therapies have been shown to down regulate key Thl7 and Thl -related markers in the skin and serum [36-38],SUMMARY OF THE INVENTION

[0006] In an embodiment, a method for the treatment of maj or depressive disorder (MDD), includes administering to a human subject suffering from MDD, a therapeutically effective amount of an antibody selected from fezakinumab, ustekinumab, gusacitinib and dupilumab, and combinations thereof.In an embodiment, a method for the treatment of major depressive disorder, includes administering to a human subject suffering from MDD. a therapeutically effective amount of an anti-IL-4R antibody. In another embodiment, the anti-IL-4R antibody is dupilumab.Attorney Docket: 27527-0234WO1In an embodiment, a method for the treatment of major depressive disorder, includes administering to a human subject suffering from MDD, a therapeutically effective amount of an anti-TGM2 antibody. In another embodiment, the anti-TGM2 antibody is zampilimab.

[0007] In an embodiment, a method for the treatment of major depressive disorder includes the steps of (a) determining that ahuman subject is suffering from MDD; and (b) administering to the human subject a therapeutically effective amount of an antibody selected from fezakinumab, ustekinumab, gusacitinib and dupilumab, and combinations thereof. In another embodiment, the antibody is dupilumab.

[0008] In an embodiment, a method for the treatment of major depressive disorder in a human subject, includes the steps of (a) diagnosing that a human subject is suffering from major depressive disorder; and (b) administering to the human subject a therapeutically effective amount of an antibody selected from fezakinumab, ustekinumab, gusacitinib and dupilumab, and combinations thereof. In another embodiment, the antibody is dupilumab. In another embodiment, the human subject is diagnosed as suffering from major depressive disorder based on assessment by the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders - 5thedition.

[0009] In an embodiment according to any one of the previous embodiments, the major depressive disorder is treatment resistant depression.

[0010] In an embodiment according to any one of the previous embodiments, the antibody is administered at a dosage of from 50 to 500 mg. In another embodiment, the dosage is administered daily, weekly or monthly. In another embodiment, the antibody is administered by injection. In another embodiment, the antibody is administered at a daily dose of between 40 and 80 mg.BRIEF DESCRIPTION OF THE FIGURES

[0011] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0012] FIG. 1. All differentially expressed proteins. Heatmap of serum proteins detected on Olink that are differentially expressed in either major depressive disorder (MDD), atopic dermatitis (AD), or psoriasis (PSO) versus healthy controls (HC), with criteria of FC>1.5 andAttorney Docket: 27527-0234WO1P<0.05. Table shows FCs in MDD versus HC, AD versus HC, PSO versus HC, MDD versus AD, and MDD versus PSO. ***P<0.001, **P<0.01. *P<0.05.

[0013] FIG. 2. Pathway analyses. Mean z-scores of immune-related pathways in healthy controls (HC). study participants with major depressive disorder (MDD), atopic dermatitis (AD), and psoriasis (PSO). Pathways include A. markers of CD4+ T-cells, and proteins up-regulated by B. IFN-a, C. IFN-y, D. Th2, and E. co-regulated by IL-17 and TNF-a. Black symbols indicate significance of comparison with HC; red symbols indicate significance of comparison with MDD. ***P<0.001, **P<0.01, *P<0.05.

[0014] FIG. 3. Drug improvement percentages of the major depressive disorder (MDD) signature. Percentage improvement of MDD signature with various drugs at weeks 2, 4, 12, and 16 (W2, W4, W12, W16) from three different datasets: A. the proteomic MDD signature measured by Olink, and the MDD signature obtained from whole-blood sequencing from previously published datasets from B. Cathomas et al.

[0051] and C. Mostafavi et al.

[0052] ,

[0015] FIG. 4. Heatmap of major depressive disorder (MDD) signature modulation by dupilumab therapy versus placebo. The proteins listed reflect the proteomic MDD signature, as defined by FC>1.2 and P<0.05 for the MDD versus controls comparison. The color of the heatmap represents the normalized mean expression level of these proteins in a previously published dataset of atopic dermatitis patients treated with dupilumab and placebo

[0029] , Heatmap shows lesional skin at week 0 (W0), week 4 (W4), and week 16 (W16) and non-lesional skin at weeks 0 and 16 in the placebo and dupilumab groups. The table shows FCs at week 4 and week 16 versus week 0. ***P<0.001, **P<0.01, *P<0.05.

[0016] FIG.5. Pharmacological blocking of IL-4R in mice during chronic social defeat stress (CSDS) promotes stress-resilience. Mice that received an anti-IL-4R antibody every second day during a 10-day CSDS paradigm showed less avoidance behavior compared to mice that received control IgG as indicated by A. social interaction ratio and B. absolute time spent interacting with a target aggressor (AGG) mouse. C. The proportion of mice that were resilient after CSDS was higher in the anti-IL-4R antibody group compared to the IgG group. D. There was no difference in locomotion between the groups. Statistics: Two-tailed t-test (Figs. A and D), 2-way ANOVA(Fig. B), Chi-squared (Fig. C). **P<0.01, *P<0.05, ns=not significant.

[0017] FIG. 6. Number of shared and uniquely differentially expressed proteins between major depressive disorder, atopic dermatitis, and psoriasis, compared to healthy controls. Red indicates upregulated and blue indicates downregulated proteins.Attorney Docket: 27527-0234WO1

[0018] FIG.7. Provides P-values adjusted for multiple comparisons using the Benjamini-Hochberg procedure to control for false discovery rate (FDR).DETAILED DESCRIPTION

[0019] Given the successful translational approach in dermatology, where disease immunophenotyping led to advances in understanding pathogenesis and expansion of the therapeutic pipeline, the systemic immune activation in MDD could potentially be similarly leveraged to facilitate the development of more specific and effective treatments. We therefore performed in-depth proteomic profiling to compare the serum inflammatory signature in patients with MDD, AD, psoriasis, and healthy controls (HCs). Using an in-silico approach, we also implemented a drug improvement analysis, testing whether common biologies used in dermatology may also alleviate the observed dysregulated MDD proteomic signature. Because the MDD signature showed Th2 dysregulation and significant improvement with dupilumab, using a mouse model of chronic social defeat stress (CSDS), we hypothesized that pharmacologically blocking the activated Th2 pathway by neutralizing circulating IL-4Ra with a monoclonal antibody may prevent social stress-induced behavioral alterations. Overall, our data support the potential pathogenicity of the Th2 axis in MDD, supporting further exploration of specific immune targeting of Th2 as a novel, disease-modulating treatment strategy. Furthermore, the here applied back-translational drug repurposing strategy' could be applied to other disease conditions and medications.METHODSPatient characteristics

[0020] All subjects provided consent under protocols approved by the Icahn School of Medicine at Mount Sinai Institutional Review Board. MDD was assessed by the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders-Fifth Edition (SCID-5)

[0039] , Participants with MDD completed the Quick Inventory of Depressive Symptomatology-self report (QIDS-SR) to measure depressive symptom severity

[0040] , AD and psoriasis patients were recruited from outpatient dermatology clinics in Mount Sinai and all had a dermatologist-confirmed diagnosis with symptoms lasting >1 year. To meet criteria for moderate-to-severe disease, all AD and psoriasis patients had body surface area of >10%.Attorney Docket: 27527-0234WO1Disease severity was further characterized using SCORing of Atopic Dermatitis (SCORAD)

[0041] and Psoriasis Activity Scoring Index (PASI)

[0042] for AD and psoriasis, respectively.

[0021] MDD and HC subjects had no history of atopy or co-occurring primary inflammatory disorders. AD, psoriasis, and HC subjects had no prior clinical diagnosis of MDD. Other exclusion criteria applying to all enrolled subjects include active infection, current substances of abuse, other unstable medical illnesses, history of immunosuppression, and concomitant use of anti-inflammatory medications. AD and psoriasis subjects were also specifically screened to exclude the use of systemic steroids, immunosuppressants, or phototherapy within 4 weeks of sample collection and the use of topical steroids or calcineurin inhibitors within 2 weeks of sample collection.Blood Samples

[0022] On the day of the blood draw, patients were fasted for at least 6 hours. Blood was drawn into Vacutainer Gold Top 5 mL Silica Gel tubes (BD, #365968). Blood was allowed to clot for >30 min, then centrifuged at 1300 g for 15 min at 4°C, then aliquoted and stored at -80 °C. Serum aliquots from subjects were analyzed using the high-throughput proteomic Olink Proseek multiplex assay (Olink Proteomics, Uppsala, Sweden), as previously described [43-48], Serum levels of 353 proteins derived from four panels (General Inflammation, Neurological Inflammation, Cardiovascular Disease II, Cardiovascular Disease III) were quantified.Mouse chronic social defeat stress (CSDS) and social interaction test

[0023] All animal experiments were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee. Seven-week-old C57BL / 6J (Stock#: 000664) mice were purchased from The Jackson Laboratory'. 4-6-month-old male retired CD-I breeders (Charles River Laboratories, Crl: CDl[ICR]) were used as aggressors for male CSDS. Mice were habituated to the animal facility for at least 1 week. Animals were maintained on a 12-hour light / dark cycle (lights on at 7am, lights off at 7pm) with ad libitum access to food and water. For the social interaction test, mice were allowed to acclimate to the testing room for at least 1 hour. Before each defeat, CD-I mice were screened for aggressive behavior for three consecutive days based on previously described criteria

[0049] , Two days before the start of the defeat, CD-I mice were housed on one side of a perforated Plexiglas partition. During 10 consecutive days of CSDS, experimental mice (7-8-week-old) wereAttorney Docket: 27527-0234WO1subjected to direct physical interaction with a CD-I mouse for 10 minutes per day, and the rest of the day placed on the other side of the Plexiglas divider, allowing for sensory but not direct physical contact. Experimental mice received 8 pg / kg rat anti-mouse IL4-R antibody (CD124, clone: mIL4R-Ml [RUO], BD Biosciences, #552288) or isotype control rat anti-mouse IgG2a K (clone: R35-95, Fisher Scientific, # 554687) every second day throughout CSDS. Antibodies were administered intraperitoneally 20 minutes before the defeat bout. After the last day of defeat, stressed and unstressed control mice were singly housed. All stressed mice were carefully examined for wounding during the CSDS experiments and mice with excessive wounding were excluded. SI test was performed 24 hours after the last defeat session. After a 1-hour habituation period to the behavioral suite (red light conditions), mice were placed into a Plexiglas arena (42cm3, Nationwide Plastics) with a small, meshed enclosure on one end. For the first 2.5 minutes, the experimental mouse freely explored the arena. The mouse was then removed from the arena, which was subsequently cleaned with 70% ethanol. Then, a novel social target (CD-I mouse) was placed into the enclosure and the experimental mouse was placed back into the arena for another 2.5 minutes. Locomotor activity was tracked and recorded using aNoldus Etho vision System (Noldus Information Technology Inc, Version 11.0, Leesburg, VA). SI ratio was calculated as the ratio between the time the experimental mouse spent in the vicinity of the enclosure (SI zone) when a target mouse was present versus absent. Mice with an SI ratio of >1 show a behavioral profile similar to unstressed control mice and were termed “resilient / ’ while mice with an SI ratio <1 were termed “susceptible.”Statistical Analysis

[0024] Analyses were performed with R language (www. R-project.org) using publicly available Bioconductor packages (www.bioconductor.org). Fold changes (FCs) were estimated, and hypothesis testing was conducted using contrasts under the general framework for linear models using the limma package, adjusting for age, gender, and body mass index (BMI) as covariates. P-values adjusted for multiple comparisons using the Benjamini-Hochberg procedure to control for false discovery rate (FDR) are available in Figure 7. Proteins with |FC|>1.2 and P<0.05 or FDR<0.05 were considered differentially expressed. Pathway analyses were performed using the GSVA package. Correlations with clinical parameters were evaluated using Spearman correlations.

[0025] Publicly available microarray datasets of AD clinical trials were analyzed as previously described

[0050] , Expression values were modeled with mixed-effect models, withAttorney Docket: 27527-0234WO1treatment and time as fixed factors and a random effect for each patient. Drug improvement percentages were calculated at available timepoints by the following formula: -100 * (Expj.swF. F. Kx - Expi.s. WEEK. o) / (Expi.s. WEEK.0 - EXPNT. WEEK. O). This analysis was performed with the differentially expressed proteins (DEPs) as above and with differentially expressed genes derived from published whole-blood RNA-sequencing datasets [51, 52], Statistical analyses of the mouse studies were performed with GraphPad Prism software (Version 9, GraphPad Software Inc.). For mouse experiments, outliers were identified using the Grubbs' test and excluded from analyses.RESULTS

[0026] We enrolled 25 patients with MDD, 30 with AD, 21 with psoriasis, and 32 HCs. No significant differences were noted in age, gender, race, or BMI among any comparison groups (Table 1)Table 1: Demographic TableHealthy (n=32) MDD AD (n=30) Psoriasis P-Value (n=25) (n=21)Attorney Docket: 27527-0234WO1AD, Atopic dermatitis; BMI, Body mass index; MDD, Major depressive disorder; PASI, Psoriasis Area and Severity Index; QIDS-SR, Quick Inventory of Depressive Symptomatology-self report; SCORAD, Scoring Atopic Dermatitis; SD, Standard deviation

[0027] To characterize the systemic immune changes of MDD in comparison to inflammatory’ skin conditions, we used the Olink high-throughput proteomic assay to measure 353 serum proteins in patients with MDD, AD, psoriasis, and HCs. We identified 77 (64 up, 13 down) DEPs in MDD, 88 (87 up, 1 down) in AD, and 98 (84 up, 4 down) in psoriasis (Figure 6, Figure 7). The numbers of unique DEPs in MDD, AD, and psoriasis, respectively, were 45 (32 up, 13 down), 31 (30 up, 1 down), and 43 (39 up. 4 down). MDD shared 23 up-regulated proteins with AD and 21 with psoriasis.MDD proteomic signature shows both unique and common features with AD and psoriasis

[0028] The proteins with the most pronounced dysregulation in either MDD, AD. or psoriasis compared to controls are depicted in an unsupervised hierarchical clustering heatmap (Figure 1, Figure 7). In general, the clustering algorithm divided the markers by disease category’ into three broad groups, suggesting that MDD, AD, and psoriasis each harbor distinct proteomic features.

[0029] Proteins with unique or preferential dysregulation in MDD included those related to activation of T-cells and other immune cells (CD244, CD40, LAT), Thl7 differentiation (CXCL1), negative immune regulation (CD274, GAL8), apoptosis (CASP3, STK4), inflammasome activity modulation (STAMBP), and lipid metabolism (MANF, DECR1, NAAA). The unique MDD signature also encompassed markers with previously implicated roles in neuronal proliferation and differentiation (TGM2, AXIN1) [53-55], neural inflammation and neurodegeneration (SIRT2, IKBKG) [56-59]. and metabolism of potentially neurotoxic products (GLO1)

[0060] , Additionally, several proteins that promote vascular inflammation and angiogenesis (PDGFB, CXCL5, PECAM1, VWF, F2R, FUR, LRPAP1, ITGB1BP2), with possible roles in atherosclerotic plaque development and / or cardiovascular disease [61-64], were also increased in MDD, but not AD or psoriasis.

[0030] Although MDD serum demonstrated a distinct inflammatory signature with abnormalities that were not detected in AD or psoriasis, several dysregulated proteins were also shared between MDD and the inflammatory skin diseases. The Th2 chemokines CCL13 andAttorney Docket: 27527-0234WO1CCL17, which predictably were most dysregulated in AD, were also significantly increased in MDD (Figure 1, Figure 7). Thl markers (CXCL9, CXCL10. CXCL11) were increased in all three diseases, with CXCL11 showing the highest FCHs in MDD, while CXCL9 and CXCL10 were higher in AD and psoriasis. Several markers of T-cell activation that have also been implicated in atherosclerosis were elevated either in all three conditions (CD40LG, TNFSF14 / LIGHT) or in MDD and AD (IL-16) [44, 45], Other protein increases that MDD shared with AD and / or psoriasis included KYNU, which promotes Th 17 differentiation, CASP8, which is pro-apoptotic, and several markers implicated in angiogenesis and / or neuronal development / function (SELP, CLEC1B, SRC, PLAT, PLXNB3, NMNAT1) [65-69], An additional subset of markers were dysregulated in the inflammatory skin conditions but not in MDD (Figure 1. Figure 7).

[0031] The shared immune signature between MDD and inflammatory skin diseases was further reinforced by pathway enrichment analyses using previously published immune-related datasets (Figure 2) [70-72], MDD, AD, and psoriasis all demonstrated significant enrichment in proteins related to CD4+T-cells and Th2 activation (Figure 2A, C). All three conditions also showed up-regulation in proteins that are inducible by IFN-a, IFN-y, and IL-17 / TNF-a (Figure 2B. D, E).

[0032] Spearman correlation coefficient analyses were performed to assess the association between serum markers and QIDS-SR (Table 2). QIDS-SR demonstrated strong positive correlations with ACP5, whose up-regulation in neurons has been linked to depression-like behavior in mice

[0073] , Thl markers (IFNG, CXCL10), innate immunity cytokine IL-2, and growth factor FGF21 were also positively correlated with QIDS-SR.Table 2QIDS-SRProtein R P-valueACP5 0.48464551 0.01407859CXCL10 0.42187164 0.03567956FGF21 0.41914234 0.03701865SCGB3A2 0.41680294 0.03819792IFNG 0.39262915 0.05220782TNFRSF11 0.38678065 0.05613447B IL2 0.38171195 0.05971974CX3CL1 0.37040486 0.06835459ARTN 0.36611596 0.07186913IL18R1 0.35948766 0.07757011Attorney Docket: 27527-0234WO1CCL7 0.35792806 0.07896009IL33 0.35363916 0.08288002CCL19 0.34623106 0.0899948CD163 0.34428157 0.09194111IL12B 0.34194217 0.09431812IL10RA 0.33687347 0.09962553The inflammatory signature of MDD serum is reversible by dupilumab and other systemic therapeutics

[0033] For AD, several systemic therapies have been evaluated in clinical trials in patients with moderate-to-severe disease, with accompanying mechanistic studies that measured molecular changes in the skin before and after treatment [29, 74-77], These include the broad immunosuppressant cyclosporine and targeted immunotherapeutic agents (e.g., dupilumab, fezakinumab, ustekinumab, gusacitinib) [29, 74-77], Due to some shared features in their blood proteomic phenotypes, we were interested in determining whether therapeutics that have been previously utilized in AD may have any effect on the MDD inflammatory signature. Thus, using a similar approach to a previous publication in which we aggregated the molecular data from different clinical trials of AD [29, 50, 74-77], we compared the relative effects of multiple therapeutic agents in AD on the MDD serum inflammatory signature (Figure 3A). Overall, cyclosporine showed the highest consistent median percentage improvement in the MDD signature compared to baseline (week 2: 99.8%, week 12: 101.2%). Dupilumab showed a median improvement in the MDD signature of 58.1% at week 4, which increased to 112.9% by week 16, making it the highest of any of the therapeutics evaluated. Percentage improvements ranged from 40-80% for fezakinumab, ustekinumab, and gusacitinib at both the 40mg and 80mg daily doses. The analysis was repeated for gene products that were differentially expressed in MDD versus controls obtained from publicly available whole-blood RNA-sequencing datasets [51, 52], with similar results (Figure 3B-C).

[0034] For the drug improvement analysis, the data for dupilumab were derived from a placebo-controlled, double-blind trial that evaluated the molecular lesional and non-lesional skin profiles of AD patients at baseline and after weeks 4 and 16 of treatment

[0029] , We next took the set of markers that were dysregulated in MDD and assessed their expression levels in the skin of AD patients from this trial at different treatment time points. The effects of both dupilumab and placebo on the MDD serum proteomic signature at weeks 0, 4, and 16 areAttorney Docket: 27527-0234WO1summarized in Figure 4. Overall, there was a cluster of markers from the MDD signature that showed elevations in lesional AD compared to non-lesional AD at baseline, and then showed incremental decreases with dupilumab therapy, with diminished or no changes in the placebo group. Predictably, the Th2 chemokines CCL 13 and CCL 17, which were elevated in both MDD and AD, showed significant decreases at weeks 4 and 16 in the dupilumab group, but not in placebo. Other markers that were part of the MDD proteomic signature that showed a trend effect with dupilumab (but not placebo) at week 4 and / or 16 compared to baseline included markers of T-cell / NK-cell activation or cytotoxicity (CD6, CD40, CD244, GZMA), Thl7 (CXCL1, KYNU), negative regulation (CD274), inflammatory proteases (MMP9), and vascular or neurologic development, function and inflammation (SELP, FUR, TGM2, CTSB, HBEGF).Anti-IL4R antibody blockade in CSDS mouse models decreased social avoidance and improved resilience

[0035] Finally, we tested whether, similar to the mechanism of action of dupilumab, pharmacologically blocking IL-4R in mice could prevent stress-induced behavioral alterations (Figure 5). Applying a back-translational approach, we subjected mice to CSDS. The CSDS paradigm is one of the best-validated mouse models of psychosocial stress. It consists of the experimental mouse being subordinated by an aggressive CD-I mouse through a combination of physical contact and sensory exposure over 10 days. A majority of stressed mice develop a behavioral phenotype characterized by social avoidance

[0078] , impaired social reward

[0079] , and reduced preference for natural reward (anhedonia)

[0078] , They also develop several physiological disturbances such as metabolic syndrome, systemic inflammation, and gastrointestinal disturbances [7, 80, 81], These mice are termed stress-susceptible (SUS mice). However, a subset of animals shows behavioral and physiological profiles similar to unstressed control (CON) mice, which are termed resilient (RES). To pharmacologically block the IL-4R, we injected an anti-IL-4R antibody, or an isotype control every second day 20 minutes before the defeat bout during 10 days of CSDS. This pharmacological strategy has been successfully applied previously in murine cancer research

[0082] , Mice that received an IL-4R antibody showed less social avoidance behavior compared to mice that received control IgG (Figures 5 A, B). The proportion of mice that were resilient after CSDS was higher in the anti-IL-4R antibody group compared to the IgG group (Figure 5C). There was no difference in locomotion between the groups (Figure 5D). Taken together, these data suggest that the IL-4R pathway isAttorney Docket: 27527-0234WO1causally involved in the pathophysiology of stress-induced behavioral changes, and that blocking it could constitute a druggable target to treat stress-disorders.DISCUSSION

[0036] Here, we present the first study that compares the serum proteomic phenotype of MDD to AD and psoriasis. In contrast to MDD where treatment options remain limited and focus primarily on pharmacological strategies targeting central nervous system neurotransmitters, recent advances in understanding the mechanisms of AD and psoriasis have led to the development of novel disease-modulating targeted therapeutics, such as dupilumab for AD [29, 44], Here we demonstrate that the circulatory inflammatory MDD signature shares Th2 skewing and other immune and neurovascular abnormalities with AD, which may be suppressible by IL-4R antagonism as evidenced by both prior mechanistic studies of dupilumab in AD patients and our proof of principle pre-clinical data that pharmacologically blocking IL-4R promotes stress-resilience.

[0037] We compared the blood immune profile of MDD to two inflammatory skin diseases that are mediated by distinct T-cell pathways. AD is primarily mediated by Th2 / Th22, with variable involvement of Thl / Thl7, while psoriasis is primarily Thl7 mediated [44, 83-85], A recent meta-analysis has indeed shown an increase in CD4+T-helper cells

[0011] and there is substantial evidence of multi-cytokine polarization in depression [51, 52, 86-94], suggesting that like AD and psoriasis, MDD pathogenesis may also involve preferential activation of T-helper cells. Our proteomic study illustrates significant up-regulation of proteins related to T-cell / NK cell activation and cytotoxicity (CD244. CD40, LAT), Th2 (CCL13, CCL17), Thl (CXCL9. CXCL10, CXCL11), and Th 17 (CXCL1, KYNU). Although Thl / Th2 imbalances have been reported in MDD, with Th2 generally linked to acute, mild stress while Thl has been more associated with chronic, repetitive stress, the exact role of Thl / Th2 to MDD pathogenesis has been inconsistently described

[0088] , On one hand, anti-inflammatory properties of Th2 were proposed, with enhanced depressive-like behaviors reported in IL-4-deficient mice

[0095] , while mice with knockout in T-bet, aThl-specific transcription factor, showed decreased depressive-like behaviors

[0096] , though these findings were limited to mouse models. Contrasting studies showed that MDD patients were shown to have higher levels of Th2 cytokines IL-4 and IL- 13 in the blood with decreased IFN-y

[0097] , and to induce Th2 cytokine production in a severity-dependent manner in patients who also have atopy

[0098] , While Th2 was predictably highest in AD, the elevations in both Th2 (CCL13, CCL17) and Thl (CXCL9, CXCL10, CXCL11) inAttorney Docket: 27527-0234WO1our MDD cohort suggest that the activation of these chemokines may drive the recruitment of pro-inflammatory immune cells including Thl / Th2 cells to the CNS, contributing to disease pathogenesis. CCL17 was also shown to be produced by hippocampal neurons in murine models and may be a neuromodulatory chemokine that regulates hippocampal synaptic transmission

[0099] , Multiple reports have linked Thl7 activity to depression [90, 93, 94, 100, 101], While several Thl7 markers (CCL20, PI3) that were highest in psoriasis were unchanged in MDD. MDD also harbored a specific pattern of Th 17 polarization with elevations only with KYNU and CXCL1. KYNU regulates tryptophan metabolism via the kynurenine pathway, where overactivation leading to the accumulation of neurotoxic metabolites has been associated with depressogenic effects on the brain and elevated risk of cardiovascular disease [102-106], CXCL1 was shown to promote depressive-like behaviors and hippocampal apoptosis when overexpressed in mouse models

[0092] , A previous Olink study also identified CXCL1 and several other DEPs in our MDD cohort (CASP8, STAMBP, CD244, CXCL5) to be significantly associated with anxiety and depression in patients with chronic pain

[0107] , Overall, the immune profile of MDD with features of Th2, Thl, and Th 17 polarization bears similarities particularly to the AD phenotype, suggesting the possible utility of repurposing systemic therapies from AD for managing depression.

[0038] In a direct comparison among various broad and targeted systemic therapies that were studied in clinical trials of AD, we found that the inflammation in MDD shows potential for modulation by medications used in AD to varying degrees. Predictably, cyclosporine showed the highest overall improvement in the MDD disease signature across the two time points tested at weeks 2 and 12, as given its broad immunosuppressive properties, it has been described as a criterion standard for reversing the inflammatory profile of AD

[0074] , However, cyclosporine is also associated with side effects like hypertension and nephrotoxicity, limiting its use for long-term disease modification. The greatest percentage improvement in the MDD signature at a single timepoint, surpassing even cyclosporine, was seen for dupilumab at week 12. Beyond the cardinal Th2 chemokines CCL13 and CCL17, several other inflammatory proteins that were elevated in MDD were observed to also be increased in the AD cutaneous transcriptome and suppressed after dupilumab therapy

[0029] , These include markers of T-cell / Thl7 activation (CD6, CD244, CXCL1. KYNU) and vascular and neurologic inflammation (SELP, FUR, TGM2). While dupilumab directly modulates the Th2 axis, there may be additional downstream effects on other inflammatory features that could either be pathogenic or prognostically important in MDD. Notably, in pooled data from two phase 3Attorney Docket: 27527-0234WO1clinical trials of moderate-to-severe AD, dupilumab led to rapid improvement in anxiety and depression as measured by Hospital Anxiety and Depression Scale (HADS) at week 2 with persistence through week 16

[0108] , Similar improvements in depressive symptoms were also observed in other conditions, including asthma and prurigo nodularis [109, 110], This improvement could be due to alleviation in disease symptoms, but also may be due to the direct effect of dupilumab on the pro-inflammatory products that are dysregulated in these disease states. Thus, dupilumab. a specific IL-4R antagonist with a favorable safety profile that has been paradigm-shifting in the treatment of AD, could potentially be repurposed for the treatment of depression. These findings were further validated in a mouse model of CSDS, where pharmacologically blocking IL-4R during CSDS alleviated stress-induced social avoidance.

[0039] Proteomic profiling of blood from patients with MDD revealed a highly inflammatory phenotype, with profound immune activation and dysregulation. This inflammatory state is supported by elevations in products of apoptosis (CASP3, CASP8, STK4) that were observed in MDD, but lacking or diminished in AD and psoriasis. The important role of apoptosis in MDD is consistent with previous reports that chronic stress may exacerbate neuronal cell death and precipitate depression [111, 112], Several markers in the unique MDD proteomic signature are important to neuronal development, function, and inflammation, some of which were previously linked to depression and other psychiatric and neurologic conditions. For example, TGM2 was found to be increased in the prefrontal cortex of mice exposed to chronic stress and postmortem in depressed suicide subjects and has been identified as a potential therapeutic target in MDD

[0053] , AXIN1 is a scaffold protein that regulates neuronal proliferation, differentiation, and migration during cerebral cortical development, and has been implicated in autism [54, 55], Increases in GLO1, which clears toxic byproducts from the CNS, has been associated with anxiety and depression-like behavior in mice with evidence of amelioration by GLO1 inhibition [60, 113, 114],

[0040] Previously, using the Olink analysis platform with a similar panel of proteins to assess the blood of AD and psoriasis patients, we identified many proteins involved in angiogenesis and vascular remodeling that have been implicated in atherosclerosis and cardiovascular morbidity and / or mortality [44, 45], MDD shares some of these abnormalities with the skin diseases (IL-16, TNFSF14 / LIGHT, SELP), but also harbors unique features of vascular inflammation (PDGFB. PECAM1, VWF). Additional examples of markers that were exclusively dysregulated in MDD include F2R, a thrombin receptor found on platelets andAttorney Docket: 27527-0234WO1endothelial cells that promotes angiogenesis in response to stimulation by VEGF

[0061] , LRPAP1. which has a role in LDL / VLDL absorption by endothelial cells and thus promotes atherosclerotic plaque formation

[0063] , and ITGB1BP2, which is implicated in hypertension and compensatory muscle responses in hypertrophic cardiomyopathy

[0063] , These findings suggest that like in AD and psoriasis, MDD may contribute to a pro-atherogenic state that confers an increased risk of cardiovascular disease, which may predictably be further accentuated in patients with greater levels of perceived stress.

[0041] Although Olink is a multiplex platform allowing simultaneous quantification of hundreds of proteins, it has decreased sensitivity to detect changes in certain cytokines like IL-4 and IL-13, which were below the limit of detection. Additionally, data about depressive symptoms, which could further elucidate which blood markers may be associated with depression, were not collected from AD and psoriasis patients, who notably did not have any diagnoses of clinical depression. Finally, the potential effects of dupilumab on the MDD serum signature were only evaluated in a retroactive AD cohort with supportive data from a mouse model, while the actual clinical and in situ transcriptomic / proteomic effects of dupilumab on patients with MDD are still unknown.

[0042] In summary, the serum of patients with MDD contains unique up-regulation of neuroinflammatory and vascular inflammation or cardiovascular risk proteins, but also shares immune abnormalities with inflammatory skin diseases, most notably the common Th2 skewing, with AD. These immune abnormalities appear to be modifiable by IL-4R antagonism, as supported by a retroactive analysis in AD skin treated with dupilumab and in a mouse model of CSDS, although human studies and clinical trials are needed to further elucidate the pathogenicity of the Th2 axis in MDD. Our findings, together with the rapid expansion of targeted therapeutics that has revolutionized the treatment paradigm in inflammatory skin diseases, provide a rationale for employing a similar strategy in psychiatry, where specific immunotherapeutic targeting may lead to new personalized treatment regimens for MDD and beyond.Attorney Docket: 27527-0234WO1REFERENCES1. 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Claims

Attorney Docket: 27527-0234WO1CLAIMS1. A method for the treatment of major depressive disorder (MDD), comprising administering to a human subject suffering from MDD, a therapeutically effective amount of an antibody selected from fezakinumab, ustekinumab, gusacitinib and dupilumab, and combinations thereof.

2. A method for the treatment of major depressive disorder (MDD), comprising administering to a human subject suffering from MDD. a therapeutically effective amount of an anti-IL-4R antibody.

3. The method of claim 2, wherein the anti-IL-4R antibody is dupilumab.

4. A method for the treatment of major depressive disorder (MDD), comprising administering to a human subject suffering from MDD. a therapeutically effective amount of an anti-TGM2 antibody.

5. The method of claim 4, wherein the anti-TGM2 antibody is zampilimab.

6. A method for the treatment of major depressive disorder (MDD), comprising:a. determining that a human subject is suffering from MDD; andb. administering to said human subject a therapeutically effective amount of an antibody selected from fezakinumab, ustekinumab, gusacitinib and dupilumab, and combinations thereof.

7. A method for the treatment of major depressive disorder (MDD), comprising:a. determining that a human subject is suffering from MDD; andb. administering to said human subject a therapeutically effective amount of dupilumab.Attorney Docket: 27527-0234WO18. A method for the treatment of major depressive disorder (MDD) in a human subject, comprising:a. diagnosing that a human subject is suffering from MDD; andb. administering to said human subject a therapeutically effective amount of an antibody selected from fezakinumab, ustekinumab, gusacitinib and dupilumab, and combinations thereof.

9. A method for the treatment of major depressive disorder (MDD) in a human subject, comprising:a. diagnosing that a human subject is suffering from MDD; andb. administering to said human subject a therapeutically effective amount of dupilumab.

10. The method of claim 8 or 9, wherein the human subject is diagnosed as suffering from MDD based on assessment by the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders - 5thedition.

11. The method according to any of the previous claims, wherein the major depressive disorder is treatment resistant depression.

12. The method according to any of the previous claims, wherein the antibody is administered at a dosage of from 50 to 500 mg.

13. The method of claim 12, wherein the antibody is administered daily, weekly or monthly.

14. The method of claim 12, wherein the antibody is administered at a daily dose of between 40 and 80 mg.Attorney Docket: 27527-0234WO115, The method of claim 12, wherein the antibody is administered by injection.

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