Methods of Treating Disease Using MALT1 Inhibitors

Abstract

Provided herein is MALT1 inhibitor and administering MALT1 inhibitor to subject, so that the effectiveness of MALT1 inhibitor is decoupled from the reduction of regulatory T cell.Therefore, MALT1 inhibitor and administering method of MALT1 inhibitor allow disease (for example, autoimmune disease) treatment while avoiding the reduction of Treg.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 288,081, filed December 10, 2021, U.S. Provisional Patent Application No. 63 / 288,083, filed December 10, 2021, U.S. Provisional Patent Application No. 63 / 288,085, filed December 10, 2021, U.S. Provisional Patent Application No. 63 / 306,655, filed February 4, 2022, U.S. Provisional Patent Application No. 63 / 306,657, filed February 4, 2022, and U.S. Provisional Patent Application No. 63 / 306,660, filed February 4, 2022, the entire disclosures of each of which are incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] 2. Background of the Invention Mucosa associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is an intracellular signaling protein known from innate immune cells (e.g., natural killer cells NK, dendritic cells DC, and mast cells) and adaptive immune cells (e.g., T cells and B cells). MALT1 plays an essential role in influencing immune responses. For example, in T cell receptor signaling, MALT1 mediates nuclear factor κΒ (NFKB) signaling, leading to T cell activation and proliferation. Thus, MALT1 is of interest in the mechanisms of autoimmune and inflammatory pathologies. Additionally, constitutive (dysregulated) MALT1 activity is associated with cancers, such as MALT lymphoma and activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL). Modulators of MALT1 activity may be useful as potential therapeutic agents. Summary of the Invention [Means for solving the problem]

[0003] Summary of the Invention Provided herein are compounds designed to act as MALT1 inhibitors.Further disclosed herein is the administration of MALT1 inhibitors in subjects, such that the effectiveness of MALT1 inhibitors is decoupled from the reduction of regulatory T cells (Treg).Tregs generally control immune responses to self and foreign antigens, and aid in the prevention of autoimmune diseases.Therefore, MALT1 inhibitors and methods of administering MALT1 inhibitors allow for the treatment of diseases (e.g., autoimmune diseases) while avoiding the reduction of Treg.In a specific embodiment, MALT1 inhibitors are effective for the treatment of chronic diseases or disorders, including any of chronic graft-versus-host disease (cGHVD), delayed hypersensitivity, psoriatic arthritis, primary sclerosing cholangitis, multiple sclerosis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis, lupus, Sjogren's syndrome, scleritis, or rheumatoid arthritis.

[0004] Disclosed herein are methods of treating a chronic disorder, the methods comprising administering a MALT1 inhibitor to a subject, wherein after administration to the subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 4 hours to about 20 hours per 24 hours. In various embodiments, after administration to the subject, the MALT1 inhibitor achieves a time above the blood concentration target of about 6 hours to about 18 hours per 24 hours. In various embodiments, after administration to the subject, the MALT1 inhibitor achieves a time above the blood concentration target of about 8 hours to about 16 hours per 24 hours. In various embodiments, after administration to the subject, the MALT1 inhibitor achieves a time above the blood concentration target of about 10 hours to about 14 hours per 24 hours.

[0005] In various embodiments, the MALT1 inhibitor is administered at a dose of about 1 mg / kg to about 6 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 2 mg / kg to about 5 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 3 mg / kg. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 12 hours to about 24 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 16 hours to about 24 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 22 hours to about 24 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 1 hour to about 15 hours per 24 hours. In various embodiments, following administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 3 hours to about 12 hours per 24 hours. In various embodiments, following administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 6 hours to about 10 hours per 24 hours.

[0006] In various embodiments, the MALT1 inhibitor is administered at a dose of about 8 mg / kg to about 20 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 9 mg / kg to about 15 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 10 mg / kg. In various embodiments, following administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 6 hours to about 24 hours per 24 hours. In various embodiments, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 12 hours to about 24 hours per 24 hours. In various embodiments, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 18 hours to about 24 hours per 24 hours. In various embodiments, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 21 hours to about 24 hours per 24 hours.

[0007] In various embodiments, the MALT1 inhibitor is administered at a dose of about 8 mg / kg to about 20 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 9 mg / kg to about 15 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 10 mg / kg.

[0008] 1. A method of treating a chronic disorder, the method comprising administering to a subject a MALT1 inhibitor, wherein after administration to the subject, the MALT1 inhibitor has a log 10 Additionally disclosed herein are methods for achieving a log AUC (AUC) of about 1.0 μg*hr / mL to about 1.75 μg*hr / mL after administration to a subject. 10 In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a log AUC of about 1.25 μg*hr / mL to about 1.50 μg*hr / mL. 10 In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 60% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 70% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 80% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 90% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 95% of the level prior to administration.

[0009] Additionally disclosed herein is a method of treating a chronic disorder, the method comprising administering to a subject a MALT1 inhibitor, wherein the MALT1 inhibitor is administered at a dose of about 1 mg / kg to about 100 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 20 mg / kg to about 40 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 25 mg / kg to about 35 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 30 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 5 mg / kg to about 15 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 8 mg / kg to about 12 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 10 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 2 mg / kg to about 10 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 2 mg / kg to about 5 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 3 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 1 mg / kg to about 5 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 1 mg / kg to about 3 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 1 mg / kg. In various embodiments, the MALT1 inhibitor is administered intravenously. In various embodiments, the MALT1 inhibitor is administered locally.

[0010] In various embodiments, the MALT1 inhibitor is administered daily for 5-20 days. In various embodiments, the MALT1 inhibitor is administered daily for 5-8 days. In various embodiments, the MALT1 inhibitor is administered daily for 7 days. In various embodiments, the MALT1 inhibitor is administered daily for 10-15 days. In various embodiments, the MALT1 inhibitor is administered daily for 14 days. In various embodiments, the MALT1 inhibitor is administered over one or more cycles, a cycle comprising administering the MALT1 inhibitor once daily for 1-2 weeks followed by 1-2 weeks of no treatment. In various embodiments, a cycle comprises administering the MALT1 inhibitor once daily for 2 weeks followed by 1 week of no treatment. In various embodiments, the reduction in the level of Tregs in subjects with a chronic disorder following administration of the MALT1 inhibitor is less compared to the reduction in the level of Tregs in healthy subjects given the MALT1 inhibitor.

[0011] Additionally disclosed herein is a method of treating a chronic disorder, the method comprising administering a MALT1 inhibitor to a subject, the subject having previously been identified as having elevated IL-2 compared to a reference.Additionally disclosed herein is a method of treating a chronic disorder, the method comprising administering a MALT1 inhibitor to a subject, the subject having previously been identified as having elevated IL-15 compared to a reference.Additionally disclosed herein is a method of treating a chronic disorder, the method comprising administering a MALT1 inhibitor to a subject, the subject having previously been identified as having elevated IL-7 compared to a reference.

[0012] Additionally disclosed herein is a method of treating a chronic disorder, the method comprising administering to a subject a MALT1 inhibitor in combination with a second agent comprising any one of IL-2, IL-15, and IL-7. In various embodiments, the second agent comprises IL-2, and the IL-2 is administered at a dose of about 10,000 International Units (IU) to about 50,000 International Units (IU). In various embodiments, the second agent comprises IL-2, and the second agent is administered at a dose of about 20,000 International Units (IU) to about 40,000 International Units (IU). In various embodiments, the second agent comprises IL-2, and the second agent is administered at a dose of about 30,000 International Units (IU). In various embodiments, the second agent comprises IL-2, and the IL-2 is administered at a dose of about 100,000 International Units (IU) to about 5 million International Units (IU). In various embodiments, the second agent comprises IL-2, and the IL-2 is administered at a dose of about 500,000 International Units (IU) to about 4.5 million International Units (IU). In various embodiments, the second agent comprises IL-2, and the IL-2 is administered at a dose of about 1 million International Units (IU) to about 4 million International Units (IU). In various embodiments, the second agent comprises IL-2, and the IL-2 is administered at a dose of about 2 million International Units (IU) to about 3 million International Units (IU). In various embodiments, the second agent comprises IL-2, and the IL-2 is administered at a dose of about 3 million International Units (IU).

[0013] Additionally disclosed herein is a method for treating chronic disorders, the method comprising administering a MALT1 inhibitor to a subject, and the effectiveness of the MALT1 inhibitor is decoupled from its depleting effect on Treg. In various embodiments, the effectiveness of the MALT1 inhibitor is represented by at least about 50% reduction in clinical score, and the depleting effect of the MALT1 inhibitor on Treg is represented by at least about 40% reduction in Treg.

[0014] Additionally disclosed herein is a method of treating a chronic disorder, the method comprising administering a MALT1 inhibitor to a subject, wherein after administration of the MALT1 inhibitor to the subject, the subject's Treg level remains at least 60% of the level prior to administration. In various embodiments, after administration of the MALT1 inhibitor to the subject, the subject's Treg level remains at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the level prior to administration. In various embodiments, the chronic disorder is graft-versus-host disease (GHVD). In various embodiments, the graft-versus-host disease (GHVD) is scleroderma GVHD (scGVHD). In various embodiments, the chronic disorder is psoriatic arthritis. In various embodiments, the chronic disorder is primary sclerosing cholangitis. In various embodiments, the chronic disorder is multiple sclerosis. In various embodiments, the chronic disorder is inflammatory bowel disease. In various embodiments, the inflammatory bowel disease is Crohn's disease. In various embodiments, the chronic disorder is ulcerative colitis. In various embodiments, the chronic disorder is psoriasis. In various embodiments, the chronic disorder is lupus. In various embodiments, the chronic disorder is Sjogren's syndrome. In various embodiments, the chronic disorder is scleritis. In various embodiments, the chronic disorder is rheumatoid arthritis. In various embodiments, the chronic disorder is delayed hypersensitivity.

[0015] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and accompanying drawings. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 depicts an exemplary method for identifying candidate subjects for receiving a MALT1 inhibitor, according to one embodiment. [Diagram 2] FIG. 2 depicts restoration of IL-2 signaling (pStat5) in MALT1 conditional knockout mice with the addition of exogenous IL-2. [Figure 3A]FIG. 3A depicts the structure of an exemplary MALT1 inhibitor. [Figure 3B] Figure 3B shows suppression of proinflammatory cytokines-IFNγ, IL-2, and TNFα from CD45RO+ memory T cells activated via crosslinking of the T cell receptor (α-CD3 / -CD28 / -CD2) for 24 h in the presence of increasing concentrations of MALT1 inhibitors. [Figure 3C] FIG. 3C shows suppression of IL-17a from CD45RO+ memory T cells activated via T cell receptor crosslinking + costimulation (α-CD3 / -CD28 / -CD2) for 48 h in the presence of increasing concentrations of MALT1 inhibitors. [Figure 3D] FIG. 3D shows that human B cell proliferation induced via IgM / CD40L stimulation was attenuated by inhibition of MALT1. [Figure 3E] FIG. 3E shows that IL-6 and TNFα production from immune complex-stimulated human monocyte-derived macrophages was inhibited using MALT1 inhibition. [Figure 3F] Figure 3F shows a dose-dependent impact on protease activity by MALT1i-dependent cleavage of HOIL-1 substrates as determined by immunoblotting. The shaded area corresponds to the concentration of MALT1i required to achieve 50-90% target coverage as determined from a human whole blood assay. [Figure 4A] Figure 4A shows that clinical scores were reduced in rats (n=8 / group) treated with MALT1i prior to immunization with collagen (days 0, 7) at the indicated doses once daily for 4 weeks (prevention, left). Clinical scores measured three times per week starting on day 14 are plotted for each group (mean±SEM). Total disease burden over time is represented by plotting the calculated area under the curve (AUC) across treatment groups (right). [Figure 4B]Figure 4B shows the clinical scores of animals immunized with collagen, randomized on day 12, and treated with MALT1i for 2 weeks starting on day 14 (treatment regimen). Clinical scores measured 3 times per week are plotted for each group (mean ± SEM) (left). Total disease burden over time is represented by plotting the calculated area under the curve (AUC) across treatment groups (right). Significant differences from vehicle-treated groups were calculated via one-way ANOVA using Graphpad Prism; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4C] Figure 4C shows plasma concentrations of MALT1 inhibitors measured between 0 and 24 hours after the last dose. Rat whole blood potency (IC50 and IC90, see Table 2) values ​​are indicated as dotted lines. [Figure 4D] Figure 4D shows that therapeutic treatment with a MALT1 inhibitor suppresses proinflammatory cytokine and autoantibody production in a rat model of collagen-induced arthritis (CIA). Proinflammatory cytokines were measured at the end of the study in whole blood and plasma (top row) and synovium (bottom row) taken from knee joints, respectively. Cytokines that showed significant changes in the MALT1i-treated group compared to vehicle are plotted. Significant differences from the vehicle-treated group were calculated via one-way ANOVA using Graphpad Prism; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4E] Figure 4E shows that MALT1 inhibition resulted in a dose-dependent reduction in antigen-specific autoantibody (α-collagen IgG) levels while sparing total IgG antibody titers. Statistical analysis was performed using one-way ANOVA (*p<0.05, **p<0.01). [Figure 5A]Figure 5A and Figure 5B show that MALT1i-induced reduction of splenic Tregs is significantly more sensitive in healthy animals compared to diseased animals. In particular, Figure 5A shows that naive healthy animals were treated with MALT1i for 2 weeks in parallel with animals in rat CIA studies. The frequency of splenic Tregs was compared between healthy (H) and diseased (D) animals by flow cytometry (n=8 / group). The significance between similar dosing groups in healthy and diseased animals was calculated using the Mann-Whitney test using Graphpad Prism software; **p<0.01, ***p<0.001. [Figure 5B] Figure 5A and Figure 5B show that MALT1i-induced reduction of splenic Tregs is significantly more sensitive in healthy animals compared to diseased animals. In particular, Figure 5A shows that naive healthy animals were treated with MALT1i for 2 weeks in parallel with animals in rat CIA study. The frequency of splenic Tregs was compared between healthy (H) and diseased (D) animals by flow cytometry (n=8 / group). The significance between similar dosing groups in healthy and diseased animals was calculated using the Mann-Whitney test using Graphpad Prism software; **p<0.01, ***p<0.001. [Figure 6A] FIG. 6A depicts the pharmacokinetics of the REO-981 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model over a 24-hour period following dosing. [Figure 6B] FIG. 6B depicts the endpoint clinical scores at various doses of REO-981 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. [Figure 6C] Figure 6C depicts the levels of Tregs following administration of REO-981 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. The frequency of splenic Tregs was significantly reduced at the highest dose of MALT1 inhibition. [Figure 6D]Figure 6D depicts the clinical score and percent reduction in Treg after administration of REO-981 MALT1 inhibitor with respect to compound exposure (PK) expressed as area under the curve (AUC) for a given dose. Plasma MALT1i concentration (x-axis) expressed as AUC0-24h is plotted against the corresponding clinical score (percent reduction compared to vehicle) and splenic Treg frequency (percent reduction compared to naive) (y-axis). Curve fitting for AUC / clinical score and AUC / Treg relationships was performed using Graphpad Prism to show the dose-related uncoupling of efficacy and Treg reduction. [Figure 6E] Figure 6E shows that pooled exposure-response data from four separate MALT1 inhibitors show a consistent dose-related uncoupling of efficacy and Treg reduction. Statistical analysis was performed using one-way ANOVA (***p<0.001). [Figure 6F] FIG. 6F depicts the percent reduction in clinical scores and Tregs following administration of the REO-981 MALT1 inhibitor according to Cmax. [Figure 6G] FIG. 6G depicts the percent reduction in clinical scores and Tregs following administration of the REO-981 MALT1 inhibitor according to Ctrough (trough concentration). [Figure 7A] FIG. 7A depicts the pharmacokinetics of the REO-528 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model over a 24-hour period following dosing. [Figure 7B] FIG. 7B depicts the endpoint clinical scores at various doses of REO-528 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. [Figure 7C] FIG. 7C depicts the levels of Tregs following administration of REO-528 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. [Figure 7D]FIG. 7D depicts the percent reduction in clinical scores and Tregs following administration of the REO-528 MALT1 inhibitor with respect to compound exposure (PK) expressed as area under the curve (AUC) for a given dose. [Figure 7E] FIG. 7E depicts the percent reduction in clinical scores and Tregs following administration of the REO-528 MALT1 inhibitor according to Cmax. [Figure 7F] FIG. 7F depicts the percent reduction in clinical scores and Tregs following administration of the REO-528 MALT1 inhibitor according to Ctrough (trough concentration). [Figure 8A] FIG. 8A depicts the pharmacokinetics of the REO-538 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model over a 24-hour period following dosing. [Figure 8B] FIG. 8B depicts the endpoint clinical scores at various doses of REO-538 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. [Figure 8C] FIG. 8C depicts the levels of Tregs following administration of REO-538 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. [Figure 8D] FIG. 8D depicts the percent reduction in clinical scores and Tregs following administration of the REO-538 MALT1 inhibitor as a function of compound exposure expressed as area under the curve (AUC). [Figure 9A] FIG. 9A depicts the pharmacokinetics of the REO-703 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model over a 24-hour period following dosing. [Figure 9B] FIG. 9B depicts the endpoint clinical scores at various doses of REO-703 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. [Figure 9C] FIG. 9C depicts the levels of Tregs following administration of the REO-703 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. [Figure 9D]FIG. 9D depicts the percent reduction in clinical scores and Tregs following administration of the REO-703 MALT1 inhibitor as a function of compound exposure expressed as area under the curve (AUC). [Figure 10A] FIG. 10A depicts the pharmacokinetics of the REO-076 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model over a 24-hour period following dosing. [Figure 10B] FIG. 10B depicts the endpoint clinical scores at various doses of REO-076 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. [Figure 10C] FIG. 10C depicts the levels of Tregs following administration of the REO-076 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. [Figure 10D] FIG. 10D depicts the percent reduction in clinical scores and Tregs following administration of the REO-076 MALT1 inhibitor as a function of compound exposure expressed as area under the curve (AUC). [Figure 11] FIG. 11 shows the qualitative characterization of various MALT1 inhibitors relative to their respective efficacy and Treg influence observed in a rat collagen-induced arthritis (CIA) model. [Figure 12A] FIG. 12A shows dose-dependent efficacy of REO-528 and REO-703 MALT1 inhibitors in a rat CIA model. [Figure 12B] FIG. 12B shows the levels of Tregs following administration of REO-528 and REO-703 MALT1 inhibitors in a rat CIA model. [Figure 12C] FIG. 12C shows the plasma pharmacokinetics of REO-528. [Figure 12D] FIG. 12D shows the plasma pharmacokinetics of REO-703. [Figure 13A] FIG. 13A shows the endpoint clinical scores across different dosing regimens with the REO-538 MALT inhibitor. [Figure 13B]FIG. 13B shows the pharmacokinetics of REO-538 over a 24 hour period post-dosing across different dosing regimens. [Figure 13C] FIG. 13C shows the levels of IL-1β, IL-6, KC / GRO, and TNFα in synovial fluid following administration of REO-538. [Figure 14] FIG. 14 shows the percentage reduction in clinical scores and Tregs after administration of the REO-528 MALT1 inhibitor according to the area under the curve (AUC). [Figure 15] FIG. 15 shows dosing strategies for REO-528 MALT1 inhibitor, including combination REO-528+IL-2 therapy. [Figure 16A] FIG. 16A shows the percentage suppression of naive CD4+ T cells by human Tregs co-cultured with varying ratios of Tregs to naive CD4+ T cells in the presence of the indicated concentrations of MALT1i. [Figure 16B] FIG. 16B is a histogram representation of FoxP3 MFI from nTregs stimulated with Dynabeads. [Figure 16C] FIG. 16C shows CTV tracing of naive CD4+ T cells activated with dynabeads for 3 days in culture in the presence of 1, 0.3, and 0.01 μM MALT1i. [Figure 16D] Figure 16D is a quantification of the percent proliferation of naive CD4 T cells from Figure 16C. Data are representative of two independent experiments with two donors and two technical replicates per donor. [Figure 16E] Figures 16E and 16F show the levels of pSTAT5(Y694) measured from Tregs pretreated with the indicated concentrations of MALT1i or 1 μM tofacitinib followed by 25 IU IL-2. Data are representative of two independent experiments with two donors and two technical replicates per donor. [Figure 16F]Figures 16E and 16F show the levels of pSTAT5(Y694) measured from Tregs pretreated with the indicated concentrations of MALT1i or 1 μM tofacitinib followed by 25 IU IL-2. Data are representative of two independent experiments with two donors and two technical replicates per donor. [Figure 17A] FIG. 17A depicts Treg levels following administration of a MALT1 inhibitor in naive mice. [Figure 17B] FIG. 17B shows the pharmacokinetic profile of the MALT1 inhibitor on day 28. [Figure 17C] FIG. 17C shows the IC50 and Kd values ​​of MALT1 inhibitors. [Figure 18A] FIG. 18A depicts Treg levels following administration of the REO-981 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model or in healthy animals. [Figure 18B] FIG. 18B depicts the endpoint clinical scores following administration of the REO-981 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. [Figure 18C] FIG. 18C depicts the pharmacokinetic (PK) profile following administration of the REO-981 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. [Figure 18D] Figure 18D depicts the clinical score and percent reduction in Tregs after administration of REO-981 MALT1 inhibitor as a function of compound exposure, expressed as area under the curve (AUC). Plasma MALT1i concentration (x-axis), expressed as AUC0-24h, is plotted against the corresponding clinical score (percent reduction compared to vehicle) and splenic Treg frequency (percent reduction compared to naive) (y-axis). Curve fitting for AUC / clinical score and AUC / Treg relationships was performed using Graphpad Prism to show the dose-related uncoupling of efficacy and Treg reduction. [Figure 19]Figure 19 shows that pooled exposure-response data from four separate MALT1 inhibitors show consistent dose-related uncoupling of efficacy and Treg reduction. Statistical analysis was performed using one-way ANOVA (***p<0.001). [Figure 20A] FIG. 20A depicts the mean clinical scores following administration of a MALT1 inhibitor in a prophylactic experimental autoimmune encephalomyelitis (EAE) model. [Figure 20B] FIG. 20B depicts the pharmacokinetic (PK) profile following administration of a MALT1 inhibitor in a prophylactic experimental autoimmune encephalomyelitis (EAE) model. [Figure 20C] FIG. 20C depicts the mean clinical scores following administration of a MALT1 inhibitor in a therapeutic experimental autoimmune encephalomyelitis (EAE) model. [Figure 20D] FIG. 20D depicts the pharmacokinetic (PK) profile following administration of a MALT1 inhibitor in a therapeutic experimental autoimmune encephalomyelitis (EAE) model. [Figure 21A] FIG. 21A depicts the mean GVHD scores following administration of a MALT1 inhibitor in a mouse GVHD model. [Figure 21B] FIG. 21B depicts the pharmacokinetic (PK) profile following administration of a MALT1 inhibitor in a mouse GVHD model. [Figure 22A] Figures 22A and 22B show Kaplan-Meier curves of overall survival (OS) and progression-free survival (PFS), respectively, in a mouse model of scleroderma GVHD (scGVHD) following administration of a MALT1 inhibitor. [Figure 22B] Figures 22A and 22B show Kaplan-Meier curves of overall survival (OS) and progression-free survival (PFS), respectively, in a mouse model of scleroderma GVHD (scGVHD) following administration of a MALT1 inhibitor. [Figure 22C] 22C-22E show the levels of T follicular helper cells (TFH) cells, germinal center (GC) B cells, and Treg cells in each of the different treatment groups. [Figure 22D]22C-22E show the levels of T follicular helper cells (TFH) cells, germinal center (GC) B cells, and Treg cells in each of the different treatment groups. [Figure 22E] 22C-22E show the levels of T follicular helper cells (TFH) cells, germinal center (GC) B cells, and Treg cells in each of the different treatment groups. [Figure 22F] FIG. 22F shows the percentage change in ear thickness as an indicator of delayed-type hypersensitivity following administration of a MALT1 inhibitor. [Figure 23] FIG. 23 depicts Treg levels following administration of a MALT1 inhibitor in a mouse accelerated lupus model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description definition As used herein, the term "about" refers to a value within 10% above or below the stated value. For example, the term "about 5 nM" refers to a range of 4.5 nM to 5.5 nM.

[0018] As used herein, the term "sample" refers to a specimen taken from a subject (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or skin), pancreatic juice, chorionic villus sample, and cells). For example, a sample may be blood taken from a subject to determine the level of one or more biomarkers, for example, to determine whether the subject is a candidate subject.

[0019] As used herein, "pharmaceutical acceptable carrier" refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound that is formulated with it.The pharmaceutical acceptable carrier, adjuvant, or vehicle that may be used in the compositions described herein includes, but is not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0020] As used herein, "pharmaceutical acceptable salt" refers to a salt that is suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, within the scope of sound medical judgment, and is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutical acceptable salts in detail in J. Pharmaceutical Sciences (1977) vol. 66:1-19. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutical acceptable, non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. Other pharma- ceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogensulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N-alkylated salts such as lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate salts. + (C 1-4Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharma- ceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed, where appropriate, using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkylsulfonate, and arylsulfonate counterions.

[0021] As used herein, a "subject" to which administration is contemplated includes, but is not limited to, humans (i.e., male or female of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults)) and / or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

[0022] Disease, disorder, and condition are used interchangeably herein.

[0023] As used herein, and unless otherwise specified, the terms "treat", "treating" and "treatment" contemplate actions taken while a subject is suffering from a specified disease, disorder or condition that reduce the severity of the disease, disorder or condition or delay or slow the progression of the disease, disorder or condition ("therapeutic treatment"), and also contemplate actions taken before a subject begins to suffer from a specified disease, disorder or condition ("prophylactic treatment").

[0024] As used herein, the "effective amount" of a compound refers to an amount sufficient to induce desired biological response.As will be understood by those skilled in the art, the effective amount of the compound of the present invention can vary depending on factors such as the desired biological end point, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, health and condition of the subject.The effective amount includes therapeutic and prophylactic treatment.

[0025] As used herein, and unless otherwise specified, a "therapeutically effective amount" of a compound is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder or condition, or to delay or minimize one or more symptoms associated with a disease, disorder or condition.A therapeutically effective amount of a compound refers to the amount of a therapeutic agent that, alone or in combination with other therapies, provides a therapeutic benefit in the treatment of a disease, disorder or condition.The term "therapeutically effective amount" can include an amount that improves overall therapy, reduces or avoids the symptoms or causes of a disease or condition, or enhances the therapeutic effectiveness of another therapeutic agent.

[0026] Methods of Treating a Disease or Disorder The present invention is based, in part, on the discovery that a MALT1 inhibitor can be administered to a subject suffering from a disease (e.g., an autoimmune disease), and that upon administration of the MALT1 inhibitor, the effectiveness of the MALT1 inhibitor against the disease is decoupled from its depleting effect on Treg cells.

[0027] In a specific embodiment, the effectiveness of MALT1 inhibitors against disease (e.g., autoimmune disease) is measured according to endpoint clinical scores. An example of an endpoint clinical score is the Clinical Disease Activity Index (CDAI), which is a useful clinical composite score for patients with rheumatoid arthritis. CDAI is the sum of four outcome parameters: tender and swollen joint counts (28 joints are evaluated) and global assessment of disease activity by the patient and physician (visual analog scale of 0-10 cm). Possible scores range from 0 to 17. (Source: American College of Rheumatology). An additional example of an endpoint clinical score is the CIA clinical score, which measures erythema and swollen joints, approximating the human composite score, the swollen joint count of CDAI.

[0028] In a specific embodiment, the efficacy of the MALT1 inhibitor against a disease (e.g., an autoimmune disease) is measured according to the change in the endpoint clinical score. In a specific embodiment, the reduction of Treg cells is measured according to the level of Treg cells after administration of the MALT1 inhibitor compared to the level of Treg cells before administration of the MALT1 inhibitor.

[0029] In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 30% change (e.g., increase or decrease) in clinical score and a less than 50% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 30% change (e.g., increase or decrease) in clinical score and a less than 40% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 30% change (e.g., increase or decrease) in clinical score and a less than 30% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 30% change (e.g., increase or decrease) in clinical score and a less than 30% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 30% change (e.g., increase or decrease) in clinical score and a less than 25% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 30% change (e.g., increase or decrease) in clinical score and a less than 20% decrease in Treg levels. In various embodiments, the efficacy of a MALT1 inhibitor against a disease is decoupled from its depleting effect on Treg cells if administration of the MALT1 inhibitor results in a greater than 30% change (e.g., increase or decrease) in clinical score and less than a 10% decrease in Treg levels.In various embodiments, the efficacy of a MALT1 inhibitor against a disease is decoupled from its depleting effect on Treg cells if administration of the MALT1 inhibitor results in a greater than 30% change (e.g., increase or decrease) in clinical score and less than a 5% decrease in Treg levels.

[0030] In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 40% change (e.g., increase or decrease) in clinical score and a less than 50% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 40% change (e.g., increase or decrease) in clinical score and a less than 40% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 40% change (e.g., increase or decrease) in clinical score and a less than 30% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a change (e.g., increase or decrease) in clinical score of more than 40% and a decrease in Treg level of less than 30%. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a change (e.g., increase or decrease) in clinical score of more than 40% and a decrease in Treg level of less than 25%. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a change (e.g., increase or decrease) in clinical score of more than 40% and a decrease in Treg level of less than 20%. In various embodiments, the efficacy of a MALT1 inhibitor against a disease is decoupled from its depleting effect on Treg cells if administration of the MALT1 inhibitor results in a greater than 40% change (e.g., increase or decrease) in clinical score and less than a 10% decrease in Treg levels.In various embodiments, the efficacy of a MALT1 inhibitor against a disease is decoupled from its depleting effect on Treg cells if administration of the MALT1 inhibitor results in a greater than 40% change (e.g., increase or decrease) in clinical score and less than a 5% decrease in Treg levels.

[0031] In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 50% change (e.g., increase or decrease) in clinical score and a less than 50% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 50% change (e.g., increase or decrease) in clinical score and a less than 40% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 50% change (e.g., increase or decrease) in clinical score and a less than 30% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 50% change (e.g., increase or decrease) in clinical score and a less than 30% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 50% change (e.g., increase or decrease) in clinical score and a less than 25% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 50% change (e.g., increase or decrease) in clinical score and a less than 20% decrease in Treg levels. In various embodiments, the efficacy of a MALT1 inhibitor against a disease is decoupled from its depleting effect on Treg cells if administration of the MALT1 inhibitor results in a greater than 50% change (e.g., increase or decrease) in clinical score and less than a 10% decrease in Treg levels.In various embodiments, the efficacy of a MALT1 inhibitor against a disease is decoupled from its depleting effect on Treg cells if administration of the MALT1 inhibitor results in a greater than 50% change (e.g., increase or decrease) in clinical score and less than a 5% decrease in Treg levels.

[0032] In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 60% change (e.g., increase or decrease) in clinical score and a less than 50% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 60% change (e.g., increase or decrease) in clinical score and a less than 40% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 60% change (e.g., increase or decrease) in clinical score and a less than 30% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 60% change (e.g., increase or decrease) in clinical score and a less than 30% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 60% change (e.g., increase or decrease) in clinical score and a less than 25% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 60% change (e.g., increase or decrease) in clinical score and a less than 20% decrease in Treg levels. In various embodiments, the efficacy of a MALT1 inhibitor against a disease is decoupled from its depleting effect on Treg cells if administration of the MALT1 inhibitor results in a greater than 60% change (e.g., increase or decrease) in clinical score and less than a 10% decrease in Treg levels.In various embodiments, the efficacy of a MALT1 inhibitor against a disease is decoupled from its depleting effect on Treg cells if administration of the MALT1 inhibitor results in a greater than 60% change (e.g., increase or decrease) in clinical score and less than a 5% decrease in Treg levels.

[0033] In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 70% change (e.g., increase or decrease) in clinical score and a less than 50% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 70% change (e.g., increase or decrease) in clinical score and a less than 40% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 70% change (e.g., increase or decrease) in clinical score and a less than 30% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 70% change (e.g., increase or decrease) in clinical score and a less than 30% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 70% change (e.g., increase or decrease) in clinical score and a less than 25% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 70% change (e.g., increase or decrease) in clinical score and a less than 20% decrease in Treg levels. In various embodiments, the efficacy of a MALT1 inhibitor against a disease is decoupled from its depleting effect on Treg cells if administration of the MALT1 inhibitor results in a greater than 70% change (e.g., increase or decrease) in clinical score and less than a 10% decrease in Treg levels.In various embodiments, the efficacy of a MALT1 inhibitor against a disease is decoupled from its depleting effect on Treg cells if administration of the MALT1 inhibitor results in a greater than 70% change (e.g., increase or decrease) in clinical score and less than a 5% decrease in Treg levels.

[0034] In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 80% change (e.g., increase or decrease) in clinical score and a less than 50% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 80% change (e.g., increase or decrease) in clinical score and a less than 40% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 80% change (e.g., increase or decrease) in clinical score and a less than 30% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 80% change (e.g., increase or decrease) in clinical score and a less than 30% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 80% change (e.g., increase or decrease) in clinical score and a less than 25% decrease in Treg levels. In various embodiments, the effectiveness of the MALT1 inhibitor against a disease is decoupled from the depleting effect on Treg cells when administration of the MALT1 inhibitor results in a greater than 80% change (e.g., increase or decrease) in clinical score and a less than 20% decrease in Treg levels. In various embodiments, the efficacy of a MALT1 inhibitor against a disease is decoupled from its depleting effect on Treg cells if administration of the MALT1 inhibitor results in a greater than 80% change (e.g., increase or decrease) in clinical score and less than a 10% decrease in Treg levels.In various embodiments, the efficacy of a MALT1 inhibitor against a disease is decoupled from its depleting effect on Treg cells if administration of the MALT1 inhibitor results in a greater than 80% change (e.g., increase or decrease) in clinical score and less than a 5% decrease in Treg levels.

[0035] In various embodiments, the effectiveness of the MALT1 inhibitor against the disease is decoupled from the depleting effect on Treg cells based on the administration or dosing of the MALT1 inhibitor. In various embodiments, the MALT1 inhibitor is administered to the subject at either 1 mg / kg, 3 mg / kg, 10 mg / kg, 30 mg / kg, or 100 mg / kg, so that the effectiveness of the MALT1 inhibitor against the disease is decoupled from the depleting effect on Treg cells. In various embodiments, the MALT1 inhibitor is administered to the subject every day for about 14 days, and then the MALT1 inhibitor is not administered to the subject for about 7 days. In such an embodiment, the on / off administration of the MALT1 inhibitor decouples the effectiveness of the MALT1 inhibitor against the disease from the depleting effect on Treg cells.

[0036] In various embodiments, after administration of the MALT1 inhibitor to a subject, the MALT1 inhibitor achieves a unique pharmacokinetic (PK) profile that decouples the efficacy of the MALT1 inhibitor against a disease from its depleting effect on Treg cells. In various embodiments, after administration of the MALT1 inhibitor to a subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 12 hours to about 24 hours per 24 hours. In various embodiments, after administration of the MALT1 inhibitor to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 3 hours to about 12 hours per 24 hours. Time above IC50 and time above IC90 blood concentration target levels are described in further detail herein. In various embodiments, the time above IC50 blood concentration target and / or time above IC90 blood concentration target decouples the efficacy of the MALT1 inhibitor against a disease from its depleting effect on Treg cells.

[0037] In various embodiments, following administration of the MALT1 inhibitor to a subject, the MALT1 inhibitor exhibits a log 10 In various embodiments, after administration of the MALT1 inhibitor to a subject, the MALT1 inhibitor achieves a log AUC value of 2.0 to 4.0 ng / mL. 10 In such embodiments, the log (C) value achieved by the MALT1 inhibitor after administration is 10 (AUC) or log 10 (Cmax) decouples the efficacy of MALT1 inhibitors against disease from their depleting effect on Treg cells.

[0038] MALT1 inhibitor administration and dosing A contemplated MALT1 inhibitor may be administered to a subject, such as a mammalian subject (e.g., a human subject), by one or more routes of administration. In various embodiments, a contemplated MALT1 inhibitor may be administered to a subject by intravenous, intraperitoneal, intramuscular, intraarterial, or subcutaneous injection, among others. In various embodiments, a contemplated MALT1 inhibitor may be administered to a subject via local administration. For example, for brain-related diseases (e.g., multiple sclerosis), a contemplated MALT1 inhibitor may be administered intracranially, intracerebrally, or intracerebroventricularly. For example, for brain-related diseases (e.g., multiple sclerosis), a contemplated MALT1 inhibitor may be administered intracranially, intracerebrally, or intracerebroventricularly. For example, for skin or joint-related diseases (e.g., psoriatic arthritis, psoriasis, lupus, or rheumatoid arthritis), a contemplated MALT1 inhibitor may be administered locally (e.g., administered topically to the skin or injected locally into a joint).

[0039] In some embodiments, the MALT1 inhibitor (e.g., a MALT1 inhibitor disclosed herein, such as in Table 1A or Table 1B) is administered at a dose of about 0.1 mg / kg to about 100 mg / kg, e.g., about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, 50 mg / kg, 51 mg / kg, 52 mg / kg, 53 mg / kg, 54 mg / kg, 55 mg g / kg, 12mg / kg, 13mg / kg, 14mg / kg, 15mg / kg, 16mg / kg, 17mg / kg, 18mg / kg, 19mg / kg, 20mg / kg, 21mg / kg, 22mg / kg, 23mg / kg, 24mg / kg, 25mg / kg, 26mg / kg, 27mg / kg, 28mg / kg, 29mg / kg, 30mg / kg, 31mg / kg, 32mg / kg, 33mg / kg, 34mg / kg, 35mg / kg, 36mg / kg, 37mg / kg, 38mg / kg, 39mg / kg, 40mg / kg, 41mg / k g, 42mg / kg, 43mg / kg, 44mg / kg, 45mg / kg, 46mg / kg, 47mg / kg, 48mg / kg, 49mg / kg, 50mg / kg, 51mg / kg, 52mg / kg, 53mg / kg, 54mg / kg, 55mg / kg, 56mg / kg , 57mg / kg, 58mg / kg, 59mg / kg, 60mg / kg, 61mg / kg, 62mg / kg, 63mg / kg, 64mg / kg, 65mg / kg, 66mg / kg, 67mg / kg, 68mg / kg, 69mg / kg, 70mg / kg, 71mg / kg, 7 Administered at doses of 2mg / kg, 73mg / kg, 74mg / kg, 75mg / kg, 76mg / kg, 77mg / kg, 78mg / kg, 79mg / kg, 80mg / kg, 81mg / kg, 82mg / kg, 83mg / kg, 84mg / kg, 85mg / kg, 86mg / kg, 87mg / kg, 88mg / kg, 89mg / kg, 90mg / kg, 91mg / kg, 92mg / kg, 93mg / kg, 94mg / kg, 95mg / kg, 96mg / kg, 97mg / kg, 98mg / kg, 99mg / kg, or 100mg / kg.

[0040] In various embodiments, the MALT1 inhibitor is administered at a dose of about 1 mg / kg to about 6 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 2 mg / kg to about 5 mg / kg. In various embodiments, the dose is about 3 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 8 mg / kg to about 20 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 9 mg / kg to about 15 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 10 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 8 mg / kg to about 20 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 9 mg / kg to about 15 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 10 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 8 mg / kg to about 20 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 9 mg / kg to about 15 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 10 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 20 mg / kg to about 40 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 25 mg / kg to about 35 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 30 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 50 mg / kg to about 100 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 60 mg / kg to about 100 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 70 mg / kg to about 100 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 80 mg / kg to about 100 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 90 mg / kg to about 100 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 100 mg / kg.

[0041] In various embodiments, a MALT1 inhibitor (e.g., a MALT1 inhibitor disclosed herein, such as in Table 1A or Table 1B) may be administered one or more times daily, weekly, monthly, or yearly to a subject depending on factors such as, for example, the subject's age, weight, sex, the subject's diet, and the subject's excretion rate. In specific embodiments, the MALT1 inhibitor is administered daily. In various embodiments, the MALT1 inhibitor is administered daily for 5-20 days. In various embodiments, the MALT1 inhibitor is administered daily for 5-8 days. In various embodiments, the MALT1 inhibitor is administered daily for 7 days. In various embodiments, the MALT1 inhibitor is administered daily for 10-15 days. In various embodiments, the MALT1 inhibitor is administered daily for 14 days.

[0042] In various embodiments, a MALT1 inhibitor (e.g., a MALT1 inhibitor disclosed herein, such as in Table 1A or Table 1B) may be administered to a subject over one or more cycles. In various embodiments, a cycle can include a first time period during which the MALT1 inhibitor is administered to the subject, followed by a second time period during which the MALT1 inhibitor is withheld from the subject. In various embodiments, the MALT1 inhibitor is administered over one or more cycles, a cycle comprising administering the MALT1 inhibitor once daily for 1-2 weeks, followed by 1-2 weeks of treatment without the MALT1 inhibitor. In various embodiments, the MALT1 inhibitor is administered over one or more cycles, a cycle comprising administering the MALT1 inhibitor once daily for 10-14 days, followed by 7-10 days of treatment without the MALT1 inhibitor. In various embodiments, the MALT1 inhibitor is administered over one or more cycles, a cycle comprising administering the MALT1 inhibitor once daily for 12-14 days, followed by 7-9 days of treatment without the MALT1 inhibitor. In various embodiments, the MALT1 inhibitor is administered for one or more cycles, where a cycle comprises administering the MALT1 inhibitor once daily for about two weeks, followed by about one week of treatment without the MALT1 inhibitor. In various embodiments, the treatment without the MALT1 inhibitor is no treatment.

[0043] Combination therapy The MALT1 inhibitors described herein may be administered in combination with another agent or therapy.The subject to whom the MALT1 inhibitors disclosed herein are administered may have a disease, disorder, or condition, or symptoms thereof, that would benefit from treatment with another agent or therapy.

[0044] In some embodiments, the MALT1 inhibitors described herein may be administered as the sole active ingredient or in combination with other drugs, e.g., immunosuppressive or immunomodulatory agents or other anti-inflammatory agents, or chemotherapeutic agents, e.g., malignant cell anti-proliferative agents, e.g., as adjuvants, for the treatment or prevention of allo- or xenograft acute or chronic rejection or inflammatory or autoimmune disorders. For example, the MALT1 inhibitors disclosed herein may be used in combination with calcineurin inhibitors, such as cyclosporine A or FK 506; mTOR inhibitors, such as rapamycin, 40-0-(2-hydroxyethyl)-rapamycin, biolimus-7 or biolimus-9; ascomycins with immunosuppressive properties, such as ABT-281, ASM981; corticosteroids; cyclophosphamide; azathioprene; methotrexate; leflunomide; mizoribine; mycophenolic acid or salts; mycophenolate mofetil; or IL-1 beta inhibitors.

[0045] In some embodiments, a MALT1 inhibitor described herein is combined with a co-agent that is a PI3K inhibitor.

[0046] In some embodiments, a MALT1 inhibitor described herein is combined with a co-agent that affects BTK (Bruton's tyrosine kinase).

[0047] For the treatment of oncology diseases, the MALT1 inhibitors described herein may be used in combination with B cell modulating agents, such as rituximab, ofatumumab, BTK or SYK inhibitors, inhibitors of PKC, PI3K, PDK, PIM, JAK and rmTOR, and BH3 mimetics.

[0048] In various embodiments, the MALT1 inhibitors described herein may be administered in combination with one or more cytokines. In various embodiments, the MALT1 inhibitors described herein may be administered in combination with IL-2. In various embodiments, the MALT1 inhibitors described herein may be administered in combination with IL-15. In various embodiments, the MALT1 inhibitors described herein may be administered in combination with IL7.

[0049] In specific embodiments, the MALT1 inhibitors described herein may be administered in combination with IL-2, where IL-2 is administered at a low dose to avoid toxicity. In various embodiments, the low dose of IL-2 is about 10,000 International Units (IU) to about 50,000 International Units (IU). In specific embodiments, the MALT1 inhibitors described herein may be administered in combination with IL-2, where IL-2 is administered at a dose of about 20,000 International Units (IU) to about 40,000 International Units (IU). In specific embodiments, the MALT1 inhibitors described herein may be administered in combination with IL-2, where IL-2 is administered at a dose of about 30,000 International Units (IU). In various embodiments, the low dose of IL-2 is about 100,000 International Units (IU) to about 5 million International Units (IU). In various embodiments, the low dose of IL-2 is about 500,000 International Units (IU) to about 4.5 million International Units (IU). In various embodiments, the low dose of IL-2 is about 1 million International Units (IU) to about 4 million International Units (IU). In various embodiments, the low dose of IL-2 is about 2 million International Units (IU) to about 3 million International Units (IU). In various embodiments, the low dose of IL-2 is about 3 million International Units (IU).

[0050] In some embodiments, the MALT1 inhibitors described herein may be administered simultaneously with, before or after one or more other therapeutic agents. In some embodiments, the MALT1 inhibitors described herein may be administered separately from other agents, by the same or different route of administration, or together in the same pharmaceutical composition as other agents. In various embodiments, the MALT1 inhibitor is administered to a subject simultaneously with a second agent. In such embodiments, the MALT1 inhibitor may be co-formulated with the second agent. For example, the MALT1 inhibitor is combined with the second agent as a single pharmaceutical composition prior to administration to a subject.

[0051] In various embodiments, the MALT1 inhibitor is administered to the subject prior to administration of the second agent. In various embodiments, the MALT1 inhibitor is administered to the subject at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours prior to administration of the second agent. In various embodiments, the MALT1 inhibitor is administered to the subject at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days prior to administration of the second agent.

[0052] In various embodiments, the MALT1 inhibitor is administered to the subject after administration of the second agent.In various embodiments, the MALT1 inhibitor is administered to the subject at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours after administration of the second agent.In various embodiments, the MALT1 inhibitor is administered to the subject at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days prior to administration of the second agent.

[0053] Characteristics after administration of MALT1 inhibitors Disclosed herein is a method for administering a MALT1 inhibitor to a subject, wherein the MALT1 inhibitor achieves specific characteristics in the subject after administration to the subject. Exemplary characteristics include pharmacokinetic (PK) profile, pharmacodynamic (PD) profile, Treg level, or Treg reduction percentage.

[0054] In various embodiments, the MALT1 inhibitor achieves a unique pharmacokinetic (PK) or pharmacodynamic (PD) profile in the subject. In various embodiments, the PK profile or PD profile in the subject allows the effectiveness of the MALT1 inhibitor to be decoupled from its depleting effect on regulatory T cells (Treg). In a specific embodiment, the plasma PK profile of the MALT1 inhibitor allows the effectiveness of the MALT1 inhibitor to be decoupled from its depleting effect on regulatory T cells (Treg).

[0055] In various embodiments, the plasma PK profile of the MALT1 inhibitor refers to the time above the IC50 blood concentration target after administration to a subject. As used herein, "time above the IC50 blood concentration target" refers to the time period after administration during which the concentration of the MALT1 inhibitor in the blood of a subject exceeds the IC50 value. As used herein, IC50 value refers to the concentration of a compound (e.g., a MALT1 inhibitor) that results in 50% inhibition of a target-related process. In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 100 ng / mL to 1000 ng / mL. In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 100 ng / mL to 500 ng / mL. In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 100 ng / mL to 400 ng / mL. In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 100 ng / mL to 300 ng / mL. In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 100 ng / mL to 250 ng / mL. In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 100 ng / mL to 200 ng / mL. In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 200 ng / mL to 1000 ng / mL. In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 200 ng / mL to 500 ng / mL. In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 200 ng / mL to 400 ng / mL. In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 200 ng / mL to 300 ng / mL. In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 200 ng / mL to 250 ng / mL. In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 300 ng / mL to 1000 ng / mL. In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 300 ng / mL to 500 ng / mL. In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 300 ng / mL to 400 ng / mL.In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 400 ng / mL to 1000 ng / mL. In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 400 ng / mL to 500 ng / mL. In various embodiments, the IC50 blood concentration target of the MALT1 inhibitor is 500 ng / mL to 1000 ng / mL.

[0056] In various embodiments, the plasma PK profile of the MALT1 inhibitor refers to the time above the IC90 blood concentration target after administration to a subject. As used herein, "time above IC90 blood concentration target" refers to the time period after administration during which the concentration of the MALT1 inhibitor in the blood of a subject exceeds the IC90 value. As used herein, the IC90 value refers to the concentration of a compound (e.g., a MALT1 inhibitor) that results in 90% inhibition of a target-related process. In general, the IC90 value is considered to be the concentration of a compound (e.g., a MALT1 inhibitor) that results in complete target coverage. In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 1000ng / mL to 10000ng / mL. In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 1000ng / mL to 5000ng / mL. In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 1000ng / mL to 4000ng / mL. In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 1000 ng / mL to 3000 ng / mL. In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 1000 ng / mL to 2500 ng / mL. In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 1000 ng / mL to 2000 ng / mL. In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 2000 ng / mL to 10000 ng / mL. In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 2000 ng / mL to 5000 ng / mL. In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 2000 ng / mL to 4000 ng / mL. In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 2000 ng / mL to 3000 ng / mL. In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 2000 ng / mL to 2500 ng / mL. In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 3000 ng / mL to 10000 ng / mL. In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 3000 ng / mL to 5000 ng / mL.In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 3000 ng / mL to 4000 ng / mL. In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 4000 ng / mL to 10000 ng / mL. In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 4000 ng / mL to 5000 ng / mL. In various embodiments, the IC90 blood concentration target of the MALT1 inhibitor is 5000 ng / mL to 10000 ng / mL.

[0057] In various embodiments, following administration of the MALT1 inhibitor, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 4 hours to about 20 hours per 24 hours. In various embodiments, following administration of the MALT1 inhibitor, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 6 hours to about 18 hours per 24 hours. In various embodiments, following administration of the MALT1 inhibitor, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 8 hours to about 16 hours per 24 hours. In various embodiments, following administration of the MALT1 inhibitor, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 10 hours to about 14 hours per 24 hours. In various embodiments, following administration of the MALT1 inhibitor, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 12 hours to about 24 hours per 24 hours. In various embodiments, following administration to a subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 16 hours to about 24 hours per 24 hours. In various embodiments, following administration to a subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 22 hours to about 24 hours per 24 hours.

[0058] In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 1 hour to about 15 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 3 hours to about 12 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 6 hours to about 10 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 6 hours to about 24 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 12 hours to about 24 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 18 hours to about 24 hours per 24 hours. In various embodiments, following administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 21 hours to about 24 hours per 24 hours.

[0059] Exemplary IC50 and IC90 values ​​for MALT1 inhibitors are shown below.

[0060] [Table 1]

[0061] In various embodiments, the plasma PK profile of the MALT1 inhibitor refers to the area under the curve (AUC) value after administration of the MALT1 inhibitor. In general, the AUC value represents the exposure to the MALT1 inhibitor experienced by the subject. In various embodiments, after administration to a subject, the MALT1 inhibitor has a log 10 In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a log AUC of about 1.0 μg*hr / mL to about 1.75 μg*hr / mL. 10In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a log AUC of about 1.25 μg*hr / mL to about 1.50 μg*hr / mL. 10 Achieve (AUC).

[0062] In various embodiments, the plasma PK profile of the MALT1 inhibitor refers to the Cmax value after administration of the MALT1 inhibitor. Generally, the Cmax value represents the maximum observed concentration of MALT1. In various embodiments, after administration to a subject, the MALT1 inhibitor has a log 10 In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a log C value of 2.0 to 2.5 ng / mL. 10 In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a log C value of 2.5 to 3.0 ng / mL. 10 In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a log Cmax value of 3.0 to 3.5 ng / mL. 10 In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a log C value of 3.0 to 4.0 ng / mL. 10 In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a log C value of 3.5 to 4.0 ng / mL. 10 (Cmax) value is achieved.

[0063] In various embodiments, after administration of a MALT1 inhibitor, the level of Tregs in the subject remains close to the level of Tregs prior to administration of the MALT1 inhibitor. Thus, administration of a MALT1 inhibitor does not deplete the level of Tregs in the subject. In various embodiments, following administration of a MALT1 inhibitor to a subject, the level of Tregs in the subject remains at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 60% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 70% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 80% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 90% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 95% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 99% of the level prior to administration.

[0064] Methods for identifying candidates for treatment with MALT1 inhibitors Disclosed herein is a method for identifying candidate subjects to be selected for administration of MALT1 inhibitor. Generally, candidate subjects represent subjects that may respond favorably to MALT inhibitor. For example, after administration of MALT1 inhibitor in candidate subjects, the effectiveness of MALT1 inhibitor may be decoupled from its depleting effect on Treg cells. In various embodiments, candidate subjects given MALT1 inhibitor may respond more favorably than non-candidate subjects. In various embodiments, candidate subjects given MALT1 inhibitor experience improved effectiveness due to MALT1 inhibitor compared to non-candidate subjects. In various embodiments, candidate subjects given MALT1 inhibitor experience reduced Treg reduction compared to non-candidate subjects.

[0065] In various embodiments, when administered in a candidate subject, the MALT1 inhibitor reduces the log 10 Compared with the corresponding range of AUC values, the efficacy of MALT1 inhibitors and the Treg reduction effect are decoupled log 10 In various embodiments, when administered in candidate subjects, the MALT1 inhibitor exhibits a greater range of AUC values ​​than when the MALT1 inhibitor is administered in non-candidate subjects. 10 Compared to the corresponding range of Cmax values, the efficacy of MALT1 inhibitors and the Treg-reducing effect are decoupled log 10 In various embodiments, when administered in candidate subjects, the MALT1 inhibitor exhibits a greater range of Cmax values ​​than when the MALT1 inhibitor is administered in non-candidate subjects. 10 Compared to the corresponding range of Ctrough values, the efficacy of MALT1 inhibitors and the Treg reduction effect are decoupled log 10 A larger range of Ctrough values ​​is present.

[0066] In various embodiments, the MALT1 inhibitor administered in a candidate subject is log 10 Compared with the corresponding range of AUC values, the efficacy of MALT1 inhibitors and the Treg reduction effect are decoupled log 10 In various embodiments, the MALT1 inhibitor administered in the candidate subject exhibits at least a 10% increase in the range of AUC values ​​compared to when the MALT1 inhibitor is administered in the non-candidate subject. 10 Compared with the corresponding range of AUC values, the efficacy of MALT1 inhibitors and the Treg reduction effect are decoupled log 10 In a specific embodiment, the MALT1 inhibitor administered in the candidate subject exhibits at least a 20% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, at least a 80% increase, at least a 90% increase, at least a 100% increase, at least a 110% increase, at least a 120% increase, at least a 130% increase, at least a 140% increase, at least a 150% increase, at least a 160% increase, at least a 170% increase, at least a 180% increase, at least a 190% increase, at least a 200% increase, at least a 300% increase, at least a 400% increase, or at least a 500% increase in the range of AUC values. 10 Compared with the corresponding range of AUC values, the efficacy of MALT1 inhibitors and the Treg reduction effect are decoupled log 10 In various embodiments, the MALT1 inhibitor administered in the candidate subject exhibits at least a 50% increase in the range of AUC values ​​compared to when the MALT1 inhibitor is administered in the non-candidate subject. 10 Compared with the corresponding range of AUC values, the efficacy of MALT1 inhibitors and the Treg reduction effect are decoupled log 10 There is at least a 100% increase in the range of AUC values.

[0067] Referring now to FIG. 1, FIG. 1 depicts an exemplary method 100 for identifying a candidate subject for administering a MALT1 inhibitor according to one embodiment. FIG. 1 introduces a subject 110, who is categorized as either a candidate subject 140 or a non-candidate subject 145 by undergoing the method depicted in FIG. 1. In various embodiments, the assay 120 is performed on a sample obtained from the subject. As used herein, a "sample" or a "test sample" can include a single cell or a plurality of cells or a fragment of a cell or an aliquot of a bodily fluid, e.g., a blood sample, taken from a subject by means including venipuncture, excretion, ejaculation, massage, biopsy, needle aspiration, lavage sample, scraping, surgical incision, or intervention or other means known in the art. The sample can be obtained by the individual or by a third party, e.g., a medical professional. Examples of medical professionals include doctors, paramedics, nurses, first responders, psychologists, phlebotomists, medical physics personnel, nurse practitioners, surgeons, dentists, and any other obvious medical professionals as would be known to one of ordinary skill in the art.

[0068] In various embodiments, the sample is tested to determine the value of one or more biomarkers by performing an assay 120, where the assay 120 may be a marker quantification assay that determines a quantitative expression value of one or more biomarkers from a test sample. The assay 120 may be an immunoassay, and more particularly a multiplex immunoassay, examples of which are described in more detail below. Expression levels of various biomarkers may be obtained in a single run using a single test sample obtained from the subject 110. The quantified expression values ​​of the biomarkers may then be evaluated (e.g., evaluating associated characteristics 130) to categorize the subject 110 as a candidate subject 140 or a non-candidate subject 145.

[0069] In various embodiments, the assay 120 for one or more biomarkers includes a DNA assay, a microarray, a polymerase chain reaction (PCR), RT-PCR, a Southern blot, a Northern blot, an antibody binding assay, an enzyme-linked immunosorbent assay (ELISA), flow cytometry, a protein assay, a Western blot, nephelometry, turbidimetry, chromatography, mass spectrometry, an immunoassay including, but not limited to, an RIA, immunofluorescence, immunochemiluminescence, immunoelectrochemiluminescence, or competitive immunoassay. In various embodiments, the information from the assay 120 can be quantitative. In various embodiments, the information from the assay 120 can be qualitative, such as an observation of a pattern or fluorescence, which can be translated into a quantitative indication by a user or automatically by a reader or computer system.

[0070] Various immunoassays designed to quantify markers can be used in screening, including multiplex assays. Measurement of the concentration of target markers in a sample or fraction thereof can be achieved by various specific assays. For example, traditional sandwich-type assays can be used in formats such as arrays, ELISA, RIA, etc. Other immunoassays include Ouchterlony plates, which provide simple determination of antibody binding. Additionally, Western blots can be performed on protein gels or protein spots on filters using detection systems specific to the markers as desired, conveniently using labeling methods.

[0071] Protein-based analysis using an antibody that specifically binds to a polypeptide (e.g., a marker) can be used to quantify marker levels in a test sample obtained from a subject. In various embodiments, the antibody that binds to the marker can be a monoclonal antibody. In various embodiments, the antibody that binds to the marker can be a polyclonal antibody. For multiplex analysis of markers, an array containing one or more marker affinity reagents, e.g., antibodies, can be generated. Such arrays can be constructed to include antibodies against the markers. Detection can utilize one or a panel of marker affinity reagents, e.g., a panel or cocktail of affinity reagents specific for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more markers.

[0072] In various embodiments, a sample obtained from subject 110 may be processed prior to implementation of assay 120 (e.g., an immunoassay). In various embodiments, processing the sample allows implementation of assay 120 to more accurately assess the expression level of one or more biomarkers in the sample.

[0073] In various embodiments, a sample from a subject may be processed to extract biomarkers from the sample. In one embodiment, the sample may undergo phase separation to separate the biomarkers from other portions of the sample. For example, the sample may undergo centrifugation (e.g., pelleting or density gradient centrifugation) to separate larger and / or denser entities (e.g., cells and other macromolecules) in the sample from the biomarkers. Other examples include filtration (e.g., ultrafiltration) to phase separate the biomarkers from other portions of the sample.

[0074] In various embodiments, a sample from a subject may be processed to produce a sub-sample that contains a fraction of the biomarkers that were in the sample. In various embodiments, producing a fraction of the biomarkers may involve performing a protein fractionation procedure. One example of a protein fractionation procedure includes chromatography (e.g., gel filtration, ion exchange, hydrophobic chromatography, or affinity chromatography). In a specific embodiment, the protein fractionation procedure involves affinity purification or immunoprecipitation in which an antibody specific for the biomarker binds. Such an antibody may be immobilized on a support, such as a magnetic or nanoparticle, or a plate.

[0075] In various embodiments, a sample from a subject 110 is processed to extract biomarkers from the sample and further processed to produce a sub-sample containing a fraction of the extracted biomarkers. Overall, this allows for a purified sub-sample of a biomarker of particular interest. Therefore, implementation of an assay (e.g., an immunoassay) to assess the expression level of a biomarker of particular interest can be more accurate and of higher quality.

[0076] In various embodiments, the assay 120 determines quantitative expression values ​​of biomarkers including any of IL-2, IL-7, and IL-15.

[0077] In various embodiments, the assay 120 determines the level of one or more cells in a sample from the subject 110. In various embodiments, the assay 120 may determine the level of one or more immune cells in a sample from the subject 110. In specific embodiments, the assay 120 may determine the level of any one of lymphocytes, NK cells, Th17 cells, Treg cells, white blood cells, platelet count, hemoglobin, red blood cell count, erythrocyte sedimentation rate. In various embodiments, information from the assay 120 may be used to calculate a score for the subject 110, such as a disease activity score (DAS). Exemplary methods for determining the levels of any one of lymphocytes, NK cells, Th17 cells, Treg cells, white blood cells, platelet count, hemoglobin, red blood cell count, erythrocyte sedimentation rate, and / or for determining a Disease Activity Score (DAS) are described in further detail in Li et al., “Increased Serum Interleukin-2 Levels Are Associated with Abnormal Peripheral Blood Natural Killer Cell Levels in Patients with Active Rheumatoid Arthritis.” Mediators of Inflammation, Vol. 2020, Article ID 6108342, p. 15, 2020, which is incorporated by reference in its entirety.

[0078] 1, in various embodiments, step 130 involves evaluating relevant features, including the expression value of any one of the biomarkers (e.g., IL-2, IL-15, and IL-7). Depending on the evaluation at step 130, the subject is categorized as a candidate subject 140 or a non-candidate subject 145.

[0079] In specific embodiments, the subject 110 is determined to be a candidate subject 140 if the evaluation identifies the subject as having elevated IL-2. In specific embodiments, the evaluation identifies the subject as having elevated IL-2 if the subject has a level of IL-2 that is higher than a reference value. In various embodiments, the reference value represents IL-2 values ​​corresponding to multiple reference individuals, e.g., healthy individuals.

[0080] Now referring to FIG. 2, FIG. 2 depicts the restoration of IL-2 signaling (pStat5) in MALT1 conditional knockout mice with the addition of exogenous IL-2. Further details of the restoration of IL-2 signaling in MALT1 conditional knockout mice with the addition of exogenous IL-2 are described in Cheng et al., “MALT1 Protease is Critical in Maintaining Function of Regulatory T Cells and May be a Therapeutic Target for Antitumor Immunity,” J Immunol 2019; 202:3008-3019, which is incorporated herein by reference in its entirety. Notably, IL-2 signaling (pStat5) is impaired in MALT1 conditional knockout in mice. MALT1 KO in mice can simulate the administration of MALT1 inhibitors at high concentrations that deplete Treg levels. Moreover, the addition of exogenous IL-2 restores pStat5 signaling. This therefore suggests that cytokines in the inflammatory milieu (e.g., IL-2) can rescue the effects of complete MALT1 blockade (e.g., caused by administration of high concentrations of MALT1 inhibitors). Given that the reduction in Treg levels is a consequence of MALT1 blockade, the presence or addition of IL-2 can reverse and rescue the Treg reduction.

[0081] In specific embodiments, the subject 110 is determined to be a candidate subject 140 if the evaluation identifies the subject as having elevated IL-7. In specific embodiments, the evaluation identifies the subject as having elevated IL-7 if the subject has a level of IL-7 that is higher than a reference value. In various embodiments, the reference value represents IL-7 values ​​corresponding to multiple reference individuals, e.g., healthy individuals.

[0082] In specific embodiments, the subject 110 is determined to be a candidate subject 140 if the evaluation identifies the subject as having elevated IL-15. In specific embodiments, the evaluation identifies the subject as having elevated IL-15 if the subject has an IL-15 value that is higher than a reference value. In various embodiments, the reference value represents an IL-15 value corresponding to a number of reference individuals, e.g., healthy individuals.

[0083] MALT1 inhibitors Disclosed herein is a MALT1 inhibitor useful as a therapeutic agent for treating diseases, such as autoimmune diseases. Disclosed herein is a method of administering a MALT1 inhibitor to a subject, in which the effectiveness of the MALT1 inhibitor against a disease is decoupled from Treg reduction. Exemplary MALT1 inhibitors are detailed in FIG. 3A, Table 1A, and / or Table 1B. In various embodiments, any of the MALT1 inhibitors shown in Table 1A or Table 1B can be administered to a subject. Additional exemplary MALT1 inhibitors are described in WO2021138298 and WO2021207343, each of which is incorporated herein by reference in its entirety. Further examples of MALT1 inhibitors include JNJ-67856633, CTX-177, and MLT-943. Additional exemplary MALT1 inhibitors are described in WO 2018119036, U.S. Patent Publication No. 20210332045, WO 202134004, WO 2020111087, WO 2018020474, WO 2017040304, WO 2015181747, Fontan, L. et al., Chemically Induced Degradation of MALT1 to Treat B-Cell Lymphomas. Blood 2019; 134:2073, and Hamp, I. et al., A patent review of MALT1 inhibitors (2013-present), Expert Opinion on Therapeutic Patents, Vol. 31:12, (2021), pp. 1079-1096.

[0084] [Table 2-1]

[0085] [Table 2-2]

[0086]

Table 3-1

[0087]

Table 3-2

[0088]

Table 3-3

[0089]

Table 3-4

[0090]

Table 3-5

[0091]

Table 3-6

[0092]

Table 3-7

[0093]

Table 3-8

[0094]

Table 3-9

[0095]

Table 3-10

[0096]

Table 3-11

[0097]

Table 3-12

[0098]

Table 3-13

[0099]

Table 3-14

[0100]

Table 3-15

[0101]

Table 3-16

[0102]

Table 3-17

[0103]

Table 3-18

[0104]

Table 3-19

[0105]

Table 3-20

[0106] Pharmaceutical Compositions The compound provided according to the present invention is usually administered in the form of pharmaceutical composition.The present invention thus provides pharmaceutical composition that contains one or more of the described compounds, or its pharma-ceutically acceptable salt or ester as active ingredient, and one or more pharma-ceutically acceptable excipients, carriers including inert solid diluents and fillers, diluents including sterile aqueous solutions and various organic solvents, permeation enhancers, solubilizers and adjuvants.The pharmaceutical composition can be administered alone or in combination with other therapeutic agents.Such compositions are prepared in a manner well known in the pharmaceutical art (see, for example, Remington's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, Pa. 17th Ed. (1985); and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (Edited by GS Banker & CT Rhodes)).

[0107] The pharmaceutical compositions may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar uses, including rectal, buccal, intranasal and transdermal routes, intraarterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, oral, topical, as an inhalant, or via impregnated or coated devices such as stents or cylindrical polymers inserted into an artery, for example as described in those patents and patent applications incorporated by reference.

[0108] The forms in which the novel composition of the present invention can be incorporated for administration by injection include aqueous or oily suspensions or emulsions using elixirs, mannitol, dextrose, or sterile aqueous solutions, and similar pharmaceutical vehicles, as well as sesame oil, corn oil, cottonseed oil, or peanut oil. Aqueous solutions in saline are also conventionally used for injection, but are less preferred in the context of the present invention. Ethanol, glycerol, propylene glycol, and liquid polyethylene glycol, etc. (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils can also be used. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.

[0109] Sterile injectable solution is prepared by incorporating the compound according to the present invention in the required amount in a suitable solvent with various other components as listed above as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating various sterilized active components into a sterile vehicle that contains basic dispersion medium and other components as listed above.In the case of sterile powder for preparing sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying technology, which produces powder of active component and any additional desired component from its solution that has been previously sterile filtered.

[0110] Oral administration is another route for administration of the compounds according to the present invention. Administration may be via capsules or enteric coated tablets, etc. In the preparation of pharmaceutical compositions containing at least one compound described herein, the active ingredient is usually diluted with an excipient and / or enclosed in a carrier, which may be in the form of a capsule, sachet, paper or other container. When an excipient serves as a diluent, it can be in the form of a solid, semi-solid, or liquid material (as described above) that acts as a vehicle, carrier or medium for the active ingredient. Thus, the composition can be in the form of a tablet, pill, powder, lozenge, sachet, cachet, elixir, suspension, emulsion, solution, syrup, aerosol (as a solid or in a liquid medium), e.g., ointment, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders, containing up to 10% by weight of the active compound.

[0111] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose.The formulations may additionally include lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives, such as methyl and propylhydroxy-benzoates; sweetening agents; and flavoring agents.

[0112] The compositions of the present invention can be formulated to provide rapid, sustained or delayed release of active ingredient after administration to a patient by using procedures known in the art. Controlled release drug delivery systems for oral administration include osmotic pump systems and dissolution systems containing polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled release systems are given in U.S. Pat. Nos. 3,845,770; 4,326,525; 4,902,514; and 5,616,345. Another formulation for use in the methods of the present invention employs transdermal delivery devices ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for delivery of pharmaceutical agents is well known in the art. See, for example, U.S. Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

[0113] The composition is preferably formulated in unit dosage form.The term "unit dosage form" refers to a physically separate unit suitable as a unit dosage for human subjects and other mammals, each unit containing a predetermined amount of active material calculated to produce desired therapeutic effect in association with suitable pharmaceutical excipients (e.g., tablet, capsule, ampoule).Compound is generally administered in a medicamentously effective amount.

[0114] To prepare solid compositions, such as tablets, the main active ingredient is mixed with pharmaceutical excipients to form a preformulated solid composition containing a homogeneous mixture of the compound of the present invention. When referring to these preformulated compositions as homogeneous, it is meant that the active ingredient is evenly distributed throughout the composition, so that the composition can be easily subdivided into equally effective unit dose forms, such as tablets, pills and capsules.

[0115] The tablet or pill of the present invention may be coated or otherwise compounded to provide a dosage form that provides the advantage of prolonged action or to protect against the acidic conditions of the stomach.For example, the tablet or pill may comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.The two components may be separated by an enteric layer that serves to resist disintegration in the stomach and to allow the inner component to pass intact into the duodenum or to be delayed in release.Various materials can be used for such enteric layers or coatings, including a number of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0116] Compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable, aqueous or organic solvents, or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as listed above. Preferably, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions, preferably in pharma- ceutically acceptable solvents, may be nebulized by use of an inert gas. Nebulized solutions may be inhaled directly from the nebulizing device, or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from a device that delivers the formulation in an appropriate manner.

[0117] In some embodiments, the pharmaceutical composition comprises a disclosed compound, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0118] Exemplary Diseases or Disorders The compounds and compositions described herein are generally useful for modulating MALT1 and for treating diseases or disorders, particularly diseases or disorders that are sensitive to modulation of MALT1 proteolytic and / or autoproteolytic activity. In some embodiments, the compounds and compositions described herein are useful for inhibiting MALT1. In some embodiments, it is contemplated that the compounds and compositions of the present invention may be useful in treating diseases, disorders, or conditions characterized by deregulated NF-kB activation, such as autoimmune disorders, immunological disorders, inflammatory disorders, allergic disorders, respiratory disorders, and oncological disorders. In some embodiments, it is contemplated that the compounds and compositions of the present invention may be useful in treating chronic diseases or disorders, such as chronic autoimmune disorders, chronic immunological disorders, or chronic inflammatory disorders.

[0119] In typical embodiments, the present invention is intended to encompass the compounds disclosed herein, as well as pharma- ceutically acceptable salts, pharma- ceutically acceptable esters, tautomeric forms, polymorphs, and prodrugs of such compounds. In some embodiments, the present invention includes pharma- ceutically acceptable addition salts, pharma- ceutically acceptable esters, solvates (e.g., hydrates) of addition salts, tautomeric forms, polymorphs, enantiomers, mixtures of enantiomers, stereoisomers, or mixtures of stereoisomers (either pure or as racemic or non-racemic mixtures) of the compounds described herein.

[0120] In some embodiments, the autoimmune and inflammatory disorder is selected from the group consisting of arthritis, ankylosing spondylitis, inflammatory bowel disease, ulcerative colitis, gastritis, pancreatitis, Crohn's disease, celiac disease, primary sclerosing cholangitis, multiple sclerosis, Sjogren's syndrome, systemic sclerosis, scleritis, systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, rheumatic fever, gout, organ or graft rejection, acute or chronic graft versus host disease, chronic allograft rejection, Behcet's disease, budo Selected from uveitis, psoriasis, psoriatic arthritis, BENTA disease, polymyositis, dermatitis, atopic dermatitis, dermatomyositis, acne vulgaris, myasthenia gravis, hidradenitis suppurativa, Graves' disease, Hashimoto's thyroiditis, Sjogren's syndrome, and antibody-mediated vasculitis syndromes, including bullous disorders (e.g., pemphigus vulgaris), ANCA-associated vasculitis, Henoch-Schönlein purpura, and immune complex vasculitis (primary or secondary to infection or cancer).

[0121] In some embodiments, the oncological disorder is selected from the group consisting of carcinoma, sarcoma, lymphoma, leukemia and germ cell tumors, adenocarcinoma, bladder cancer, clear cell carcinoma, skin cancer, brain cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, brain tumor, breast cancer, gastric cancer, germ cell tumor, glioblastoma, hepatic adenoma, Hodgkin's lymphoma, liver cancer, kidney cancer, lung cancer, pancreatic cancer, head / neck / pharyngeal cancer, ovarian cancer, skin tumor, prostate cancer, renal cell carcinoma, stomach cancer, and ovarian cancer. cancer), blood cancer, medulloblastoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), mantle cell lymphoma, marginal zone lymphoma, T-cell lymphoma, especially Sézary syndrome, mycosis fungoides, cutaneous T-cell lymphoma, T-cell acute lymphoblastic leukemia, melanoma, mucosa-associated lymphoid tissue (MALT) lymphoma, multiple myeloma, plasma cell neoplasm, lentigo maligna melanoma, acral lentigo melanoma, squamous cell carcinoma, chronic myeloid leukemia, myeloid leukemia, superficial melanoma, acral lentigo melanoma Selected from melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft tissue melanoma, melanoma with small nevus-like cells, melanoma with Spitz features, uveal melanoma, precursor T-cell, leukemia / lymphoma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, follicular lymphoma, chronic lymphocytic leukemia / lymphoma, Burkitt lymphoma, mycosis fungoides, peripheral T-cell lymphoma, nodular sclerosing type of Hodgkin lymphoma, mixed cellular subtype of Hodgkin lymphoma, non-small cell lung cancer, large cell carcinoma, and small cell lung cancer.

[0122] In some embodiments, the oncological disorder is cancer in the form of tumor or blood-borne cancer. In some embodiments, the tumor is a solid tumor. In some embodiments, the tumor is malignant and / or metastatic. In some embodiments, the tumor is selected from adenoma, adenocarcinoma, blastoma (e.g., hepatoblastoma, glioblastoma, neuroblastoma and retinoblastoma), carcinoma (e.g., colorectal or hepatocellular carcinoma, pancreatic, prostate, gastric, esophageal, cervical, and head and neck cancer, and adenocarcinoma), desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, germ cell tumor, lymphoma, leukemia, sarcoma (e.g., Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, or any other soft tissue sarcoma), Wilms' tumor, lung tumor, colon tumor, lymphatic tumor, breast tumor, or melanoma.

[0123] In some embodiments, the allergic disorder is selected from contact dermatitis, celiac disease, asthma, hypersensitivity to house dust mites, pollen and related allergens, and beryllium disease.

[0124] In some embodiments, the respiratory disorder is selected from asthma, bronchitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, pulmonary edema, pulmonary embolism, pneumonia, pulmonary sarcoidosis, silicosis, pulmonary fibrosis, respiratory failure, acute respiratory distress syndrome, primary pulmonary hypertension, and emphysema.

[0125] In some embodiments, the compounds and compositions of the invention may be useful in the treatment of rheumatoid arthritis, systemic lupus erythematosus, vasculitic conditions, allergic diseases, asthma, chronic obstructive pulmonary disease (COPD), acute or chronic transplant rejection, graft-versus-host disease, cancers or solid tumors of hematopoietic origin, chronic myeloid leukemia, myeloid leukemia, non-Hodgkin's lymphoma or other B-cell lymphomas.

[0126] In a specific embodiment, the compounds and compositions of the present invention may be useful in treating chronic diseases or disorders, such as chronic autoimmune disorders, chronic immunological disorders, or chronic inflammatory disorders. Examples of chronic diseases or disorders include chronic graft-versus-host disease (cGHVD), psoriatic arthritis, primary sclerosing cholangitis, multiple sclerosis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis, lupus, Sjogren's syndrome, scleritis, or rheumatoid arthritis.

[0127] In particular embodiments, the compounds and compositions of the present invention may be useful in the treatment of chronic graft-versus-host disease (cGHVD). In particular embodiments, the compounds and compositions of the present invention may be useful in the treatment of psoriatic arthritis. In particular embodiments, the compounds and compositions of the present invention may be useful in the treatment of primary sclerosing cholangitis. In particular embodiments, the compounds and compositions of the present invention may be useful in the treatment of multiple sclerosis. In particular embodiments, the compounds and compositions of the present invention may be useful in the treatment of inflammatory bowel disease. In particular embodiments, the compounds and compositions of the present invention may be useful in the treatment of Crohn's disease. In particular embodiments, the compounds and compositions of the present invention may be useful in the treatment of ulcerative colitis. In particular embodiments, the compounds and compositions of the present invention may be useful in the treatment of psoriasis. In particular embodiments, the compounds and compositions of the present invention may be useful in the treatment of lupus. In particular embodiments, the compounds and compositions of the present invention may be useful in the treatment of Sjogren's syndrome. In particular embodiments, the compounds and compositions of the present invention may be useful in the treatment of scleritis. In particular embodiments, the compounds and compositions of the present invention may be useful in the treatment of rheumatoid arthritis.

[0128] Additional Embodiments Disclosed herein are methods of treating a chronic disorder, the methods comprising administering a MALT1 inhibitor to a subject, wherein after administration to the subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 4 hours to about 20 hours per 24 hours. In various embodiments, after administration to the subject, the MALT1 inhibitor achieves a time above the blood concentration target of about 6 hours to about 18 hours per 24 hours. In various embodiments, after administration to the subject, the MALT1 inhibitor achieves a time above the blood concentration target of about 8 hours to about 16 hours per 24 hours. In various embodiments, after administration to the subject, the MALT1 inhibitor achieves a time above the blood concentration target of about 10 hours to about 14 hours per 24 hours. In various embodiments, the MALT1 inhibitor is administered at a dose of about 1 mg / kg to about 6 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 2 mg / kg to about 5 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 3 mg / kg.

[0129] In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 12 hours to about 24 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 16 hours to about 24 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 22 hours to about 24 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 1 hour to about 15 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 3 hours to about 12 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 6 hours to about 10 hours per 24 hours.

[0130] In various embodiments, the MALT1 inhibitor is administered at a dose of about 8 mg / kg to about 20 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 9 mg / kg to about 15 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 10 mg / kg. In various embodiments, a method comprising administering a MALT1 inhibitor to a subject, wherein after administration to the subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 6 hours to about 24 hours per 24 hours. In various embodiments, after administration to the subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 12 hours to about 24 hours per 24 hours. In various embodiments, after administration to the subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 18 hours to about 24 hours per 24 hours. In various embodiments, following administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 21 hours to about 24 hours per 24 hours.

[0131] In various embodiments, the MALT1 inhibitor is administered at a dose of about 8 mg / kg to about 20 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 9 mg / kg to about 15 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 10 mg / kg.

[0132] 1. A method of treating a chronic disorder, the method comprising administering to a subject a MALT1 inhibitor, wherein after administration to the subject, the MALT1 inhibitor has a log 10 Additionally disclosed herein are methods for achieving a log AUC (AUC) of about 1.0 μg*hr / mL to about 1.75 μg*hr / mL after administration to a subject. 10 In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a log AUC of about 1.25 μg*hr / mL to about 1.50 μg*hr / mL. 10In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 60% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 70% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 80% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 90% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 95% of the level prior to administration.

[0133] Additionally disclosed herein is a method of treating a chronic disorder, the method comprising administering to a subject a MALT1 inhibitor, wherein the MALT1 inhibitor is administered at a dose of about 1 mg / kg to about 100 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 20 mg / kg to about 40 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 25 mg / kg to about 35 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 30 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 5 mg / kg to about 15 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 8 mg / kg to about 12 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 10 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 2 mg / kg to about 10 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 2 mg / kg to about 5 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 3 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 1 mg / kg to about 5 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 1 mg / kg to about 3 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 1 mg / kg. In various embodiments, the MALT1 inhibitor is administered intravenously. In various embodiments, the MALT1 inhibitor is administered locally.

[0134] In various embodiments, the MALT1 inhibitor is administered daily for 5-20 days. In various embodiments, the MALT1 inhibitor is administered daily for 5-8 days. In various embodiments, the MALT1 inhibitor is administered daily for 7 days. In various embodiments, the MALT1 inhibitor is administered daily for 10-15 days. In various embodiments, the MALT1 inhibitor is administered daily for 14 days. In various embodiments, the MALT1 inhibitor is administered over one or more cycles, a cycle comprising administering the MALT1 inhibitor once daily for 1-2 weeks, followed by 1-2 weeks of no treatment. In various embodiments, a cycle comprises administering the MALT1 inhibitor once daily for 2 weeks, followed by 1 week of no treatment.

[0135] In various embodiments, the reduction in the level of Tregs in subjects with a chronic disorder following administration of a MALT1 inhibitor is lower compared to the reduction in the level of Tregs in healthy subjects given a MALT1 inhibitor.

[0136] Disclosed herein are methods of treating a chronic disorder, the methods comprising administering a MALT1 inhibitor to a subject, wherein after administration to the subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 4 hours to about 20 hours per 24 hours. In various embodiments, after administration to the subject, the MALT1 inhibitor achieves a time above the blood concentration target of about 6 hours to about 18 hours per 24 hours. In various embodiments, after administration to the subject, the MALT1 inhibitor achieves a time above the blood concentration target of about 8 hours to about 16 hours per 24 hours. In various embodiments, after administration to the subject, the MALT1 inhibitor achieves a time above the blood concentration target of about 10 hours to about 14 hours per 24 hours. In various embodiments, the MALT1 inhibitor is administered at a dose of about 1 mg / kg to about 6 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 2 mg / kg to about 5 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 3 mg / kg.

[0137] In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 12 hours to about 24 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 16 hours to about 24 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of about 22 hours to about 24 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 1 hour to about 15 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 3 hours to about 12 hours per 24 hours. In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 6 hours to about 10 hours per 24 hours.

[0138] In various embodiments, the MALT1 inhibitor is administered at a dose of about 8 mg / kg to about 20 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 9 mg / kg to about 15 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 10 mg / kg. In various embodiments, a method comprising administering a MALT1 inhibitor to a subject, wherein after administration to the subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 6 hours to about 24 hours per 24 hours. In various embodiments, after administration to the subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 12 hours to about 24 hours per 24 hours. In various embodiments, after administration to the subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 18 hours to about 24 hours per 24 hours. In various embodiments, following administration to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of about 21 hours to about 24 hours per 24 hours.

[0139] In various embodiments, the MALT1 inhibitor is administered at a dose of about 8 mg / kg to about 20 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 9 mg / kg to about 15 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 10 mg / kg.

[0140] 1. A method of treating a chronic disorder, the method comprising administering to a subject a MALT1 inhibitor, wherein after administration to the subject, the MALT1 inhibitor has a log 10 Additionally disclosed herein are methods for achieving a log AUC (AUC) of about 1.0 μg*hr / mL to about 1.75 μg*hr / mL after administration to a subject. 10 In various embodiments, after administration to a subject, the MALT1 inhibitor achieves a log AUC of about 1.25 μg*hr / mL to about 1.50 μg*hr / mL. 10 In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 60% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 70% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 80% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 90% of the level prior to administration. In various embodiments, after administration of a MALT1 inhibitor to a subject, the subject's Treg level remains at least 95% of the level prior to administration.

[0141] Additionally disclosed herein is a method of treating a chronic disorder, the method comprising administering to a subject a MALT1 inhibitor, wherein the MALT1 inhibitor is administered at a dose of about 1 mg / kg to about 100 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 20 mg / kg to about 40 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 25 mg / kg to about 35 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 30 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 5 mg / kg to about 15 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 8 mg / kg to about 12 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 10 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 2 mg / kg to about 10 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 2 mg / kg to about 5 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 3 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 1 mg / kg to about 5 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 1 mg / kg to about 3 mg / kg. In various embodiments, the MALT1 inhibitor is administered at a dose of about 1 mg / kg. In various embodiments, the MALT1 inhibitor is administered intravenously. In various embodiments, the MALT1 inhibitor is administered locally.

[0142] In various embodiments, the MALT1 inhibitor is administered daily for 5-20 days. In various embodiments, the MALT1 inhibitor is administered daily for 5-8 days. In various embodiments, the MALT1 inhibitor is administered daily for 7 days. In various embodiments, the MALT1 inhibitor is administered daily for 10-15 days. In various embodiments, the MALT1 inhibitor is administered daily for 14 days. In various embodiments, the MALT1 inhibitor is administered over one or more cycles, a cycle comprising administering the MALT1 inhibitor once daily for 1-2 weeks, followed by 1-2 weeks of no treatment. In various embodiments, a cycle comprises administering the MALT1 inhibitor once daily for 2 weeks, followed by 1 week of no treatment.

[0143] In various embodiments, the reduction in the level of Tregs in subjects with a chronic disorder following administration of a MALT1 inhibitor is lower compared to the reduction in the level of Tregs in healthy subjects given a MALT1 inhibitor. EXAMPLES

[0144] The following examples are set forth to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used, made, and evaluated, are intended to be purely illustrative, and are not intended to limit the scope of the invention.

[0145] Example 1: Materials and Methods Involving the Use of MALT1 Inhibitors Subsequent examples (eg, Examples 2-6) involve the use of MALT1 inhibitors. Example 1 herein describes materials and methods for carrying out the experiments of Examples 2-6.

[0146] Compound synthesis and characterization The lead compound used in this study (referred to herein as "REO-981" and referred to in the Examples as "MALT1i") was synthesized as per the reported protocol described in Martin, K. et al., Pharmacological Inhibition of MALT1 Protease Leads to a Progressive IPEX-Like Pathology. Front Immunol (2020) 11:745, which is incorporated herein by reference in its entirety. Biochemical characterization of the compounds was performed via measurement of enzymatic activity against the target. The relative and longitudinal effects of the compounds across MALT1 scaffolding and paracaspase / protease function were measured using MALT1i-treated activated primary human CD4 + This was determined by immunoblotting of cell lysates prepared from T cells.

[0147] Whole Blood Potency Assay Heparinized human whole blood was obtained from healthy donors (Research Blood Components, Watertown, MA). Blood samples were diluted 1:1 with RPMI 1640 (ThermoFisher Scientific, Waltham, MA) and aliquoted into 96-well plates in a final volume of 100 μl. After treating samples with different concentrations of MALT1i from 0 to 30 μM in a volume of 10 μl and incubating for 1 h at 37°C, stimulation of the treated blood was performed with α-CD3 (clone UCHT1, Thermofisher) and α-CD28 (clone CD28.2, Thermofisher) at 1 μg / ml each in a volume of 10 μl and incubated for 48 h at 37°C + 5% CO2. Plates were spun at 470×g for 10 min and approximately 40 μl of supernatant (plasma) was separated from each well and stored at −80°C prior to cytokine analysis.

[0148] Heparinized whole blood was obtained from naïve 8-10 week old Sprague Dawley rats (Charles River Laboratories, Wilmington, MA). Processing of blood samples diluted 1:1 was performed similarly to human whole blood. After treatment with MALT1i samples were incubated with different concentrations of MALT1i from 0 to 30 μM. After the initial incubation, blood was stimulated with 10 μl of phorbol 12-myristate 13-acetate (PMA) / ionomycin (Sigma, St. Louis, MO) at concentrations of 25 ng / ml and 1 μg / ml, respectively, as previously described. Plates were then incubated for 6 h at 37°C + 5% CO2, spun down, and supernatants separated as described for the human whole blood assay. Samples were stored at -80°C prior to cytokine analysis.

[0149] Jurkat IL-2 assay Jurkat human T cell line (ATCC, clone E6.1) was exposed to a range of concentrations of MALT1i and assessed for survival and inhibition of cytokine expression following cell activation. Cells were cultured in RPMI / 10% FBS (Thermofisher) and were diluted at 3 × 10 per ml. 6 MALT1i cells were maintained at a concentration of 100 / mL. Different concentrations of MALT1i were stamped onto 384-well plates (PerkinElmer, Waltham, MA) by ECHO, after which the cells were plated in fresh medium and incubated for 30 min before stimulation with soluble α-CD3 / CD28 / CD2 (ImmunoCult, Stemcell Technologies, Vancouver, Canada) for 24 h. Supernatants were collected as described above and either processed immediately for cytokine analysis or stored at -80°C. To assess the viability of compound-treated cells, cells were lysed with CTG reagent (Promega, Madison, WI) and measured by luminometer.

[0150] Primary human cell assays All CD4 +T cells were isolated from human donor PBMCs using the EasySep kit (Stemcell) and preincubated with REO-981 (0–5 μM) for 30 min at 37°C and 5% CO2 before stimulation with ImmunoCult Human CD3 / CD28 T-cell activator (Stemcell) as per the manufacturer's protocol.

[0151] Memory CD4 + T cells were isolated from human donor PBMCs using the EasySep kit (Stemcell) and incubated overnight in complete RPMI medium (Thermofisher) supplemented with 10% FBS (Atlanta Biologics), 10 mM HEPES, 2 mM GlutaMAX, 1 mM Na-pyruvate, and 1x MEM non-essential amino acids (Thermofisher). Cells were treated with varying ratios of MALT1i (0–30 μM) for 30 min at 37°C, 5% CO2, followed by stimulation with ImmunoCult Human CD3 / CD28 / CD2 T-cell activator (Stemcell).

[0152] Human monocytes were isolated from human donors using an EasySep kit (Stemcell) and differentiated into macrophages in RPMI-1640 medium supplemented with 10% FBS, 2 mM GlutaMAX, 100 IU / ml penicillin and 100 IU / ml streptomycin (Thermofisher) for 6 days in Petri dishes in the presence of 50 g / ml M-CSF (Peprotech, Cranbury, NJ). After differentiation, cells were harvested using Accutase solution (Stemcell Technologies) and incubated in 96-well plates for 2 h at 37 °C and 5% CO2. Cells were treated with varying ratios of MALT1i (0–30 μM) for 30 min at 37 °C before stimulation with 50 μg / ml depleted-zymosan (Invivogen, San Diego, CA) or IgG immune complexes. For preparation of IgG immune complexes, 92 μg / ml anti-human IgG (Jackson Immunoresearch, West Grove, PA) was gently placed on top of 10 μg / ml human IgG (Bio-Rad, Hercules, CA) and incubated for 1 h at 37°C and 5% CO2 before stimulating the cells.

[0153] Freshly isolated total CD19 from human PBMCs + B cells (Stemcell) were labeled with 2 μM Cell Trace Violet (ThermoFisher) and incubated on a Nutator for 5 min. Cells were then washed, plated, and treated with MALT1i (0–1 μM) for 30 min at 37°C, 5% CO2, followed by stimulation with 50 μg / ml soluble F(ab')2 anti-human IgA+IgG+IgM(H+L) (Jackson Immunoresearch, West Grove, PA) and 100 ng / ml soluble recombinant CD40L (TNFSF5) (ThermoFisher) for 4 days. Proliferation was assessed by flow cytometry.

[0154] Rat collagen-induced arthritis model Adult female Lewis rats (Charles River) weighing 180-200 g were immunized subcutaneously with bovine type II collagen (Chondrex, Woodinville, WA) / incomplete Freund's adjuvant (Sigma) emulsion prepared as per manufacturer's protocol (Chondrex) on days 0 and 7. MALT1i doses for oral administration were prepared by suspending the compound in 0.5% Na-carboxymethylcellulose / 0.5% Tween-80 in water (vehicle). For prophylactic treatment, animals were dosed via oral gavage on day 0 prior to immunization with collagen and continued once daily (qd) for 4 weeks. For therapeutic treatment, animals were randomized as per clinical disease scoring on day 14 and qd dosing of compound via oral gavage was performed for 2 weeks. Vehicle-treated and naïve animals were used as controls. Clinical scores and joint swelling (hind paw volume) were measured before the first dose on day 0 and then three times weekly until the end of the study. Body weight measurements were taken three times weekly to assess compound tolerability. Criteria (on a scale of 0-4 per paw) were as follows: 0, no evidence of erythema and swelling; 1, erythema and mild swelling limited to the mid-foot (tarsus) or ankle joint; 2, erythema and mild swelling extending from the ankle to the midfoot; 3, erythema and moderate swelling extending from the ankle to the mid-foot joint; 4, erythema and severe swelling involving the ankle, paws, and toes. After 2-3 weeks of dosing, representative animals were bled at different time intervals over a 0-24 hour time period to assess compound exposure levels in plasma via LC / MS.

[0155] For isolation of synovial fluid prior to study termination, animals were anesthetized and the skin of the hind limb was separated to ensure exposure of the knee joint. A small incision was made near the top of the knee joint, the joint cavity was rinsed twice with 60 μl of phosphate-buffered saline (PBS), and approximately 50-60 μl of synovial fluid was collected. Synovial fluid samples were centrifuged and the supernatants were stored at -80°C prior to cytokine analysis. At study termination, plasma was prepared from whole blood and stored at -80°C prior to cytokine analysis and measurement of total and anti-collagen IgG. Single cell suspensions were prepared from harvested spleens according to standard methods, and Treg frequencies were measured by flow cytometry-based immunophenotypic analysis of freshly isolated cells.

[0156] Immunophenotyping Harvested spleens were homogenized in cold PBS and cell debris was removed by passing through a 70 μM cell strainer. The cell suspension was centrifuged at 300 × g for 5 min at 4 °C and the pellet was treated with 1X RBC lysis buffer as per the manufacturer's protocol (Thermofisher). The washed cell pellet was diluted with 5–10 × 10 6Cells were resuspended at a final concentration of cells / ml and 100 μl of cell suspension was processed for immunophenotypic analysis. Briefly, cells were first stained with Fixable Live-Dead dye (BD Biosciences, Franklin Park, NJ) followed by FcR blockade with rat anti-CD32 antibody (BD) as per manufacturer's protocol. Cells were then stained for 30 min on ice with a panel of fluorescently labeled antibodies (BD unless otherwise stated) targeting rat immune cell surface markers including CD45 (clone OX-1, pan-lymphocyte marker), CD3 (clone 1F4, T cells), CD45RA (clone OX-33, B cells), CD11b / c (clone OX-42, monocytes / macrophages), CD4 (clone OX-35, helper T cells), CD8 (clone OX-8, cytotoxic T cells), and CD25 (clone OX-39, high affinity IL-2 receptor α). Cells were washed with cold FACS wash buffer (BD) and fixed overnight at 4°C with 1x FoxP3 / Transcription Factor Fixation Buffer (Thermofisher). Cells were subsequently permeabilized by washing with 1x FoxP3 / Transcription Factor Permeabilization and Wash Buffer (Thermofisher) and then stained for intracellular FoxP3 (clone 150D, Biolegend, San Diego, CA), a canonical Treg marker, for 30 min. Cells were washed 2-3 times with permeabilization and wash buffer as previously described and finally resuspended in FACS wash buffer. Samples were acquired on a BD LSRFortessa flow cytometer and data were analyzed using FlowJo software (BD).

[0157] Cytokine analysis All cytokine measurements were performed as per manufacturer's protocols using commercially available kits. Supernatants from human whole blood and cell assays were quantified using the human Proinflammatory Panel 1 (human) kit on a Sector Imager 6000 reader (Meso Scale Discovery, Rockville, MD). IL-17a was quantified using the V-Plex Human IL-17a kit on a Sector Imager 6000 reader (Meso Scale Discovery, Rockville, MD). Supernatants from rat whole blood assays were analyzed for IL-2 levels using the rat IL-2 Duoset ELISA kit (R&D). Plasma and synovial fluid samples from the rat CIA study were quantified for proinflammatory cytokines using the rat V-PLEX Proinflammatory Panel 2 kit (MSD).

[0158] Anti-collagen antibody and total IgG Rat CIA plasma was analyzed for anti-collagen antibodies using a Rat anti-bovine Type II Collagen IgG Antibody ELISA Kit (Chondrex). The assay was performed according to the manufacturer's protocol and samples were diluted 1:100,000. Rat plasma was analyzed for total IgG antibodies using a Rat total IgG Uncoated ELISA Kit (Invitrogen, Waltham, MA). The assay was performed according to the manufacturer's protocol and samples were diluted 1:400,000.

[0159] nTreg isolation, expansion, and in vitro suppression assay Human CD4 + CD127 low CD25 +Natural nTregs (nTregs) were isolated from human donor PBMCs using the Regulatory T-Cell Isolation Kit (Stemcell) and cultured for expansion. Briefly, nTregs were activated with Dynabeads Human T-Activator CD3 / CD28 (Thermofisher) at a 1:1 bead-to-cell ratio in complete RPMI medium supplemented with 10% FBS, 10 mM HEPES, 2 mM GlutaMAX, 1 mM Na-pyruvate, and 1x MEM non-essential amino acids (Thermofisher). After 2 days of culture, the culture volume was doubled and IL-2 was added at a final concentration of 300 IU (Peprotech). On days 5 and 7, cells were expanded in the presence of 300 IU IL-2. On day 9, cells were restimulated with Dynabeads at a 1:1 bead-to-cell ratio. On day 13, nTregs were harvested and beads were magnetically removed for downstream Treg suppression assays.

[0160] For in vitro Treg suppression assays, naive CD4 T cells were assayed by measuring Cell Trace Violet (CTV) (Thermofisher) dilutions in the presence of varying ratios of autologous nTregs. + T cell proliferation was assessed. Briefly, Human Naive CD4 + Naive CD4 T cells were isolated from human donor PBMCs using the T-cell Isolation Kit (Stemcell). + T cells were isolated and labeled with CTV according to the manufacturer's protocol. Naïve CD4 T cells were cultured using Dynabeads Human T-Activator CD3 / CD28 beads at a bead-to-cell ratio of 1:8 in complete RPMI in the presence of varying ratios of MALT1i-treated nTregs for 3 days. +Cells were activated. Cells were then processed for flow analysis of proliferation. Briefly, cells were first stained with Fixable Live-Dead dye (BD) and then stained with the following panel of fluorescently labeled antibodies (Biolegend unless otherwise stated): CD4 (clone RPA-T4) and CD25 (clone M-A251) for 20 min at 4°C. Cells were subsequently stained for intracellular FoxP3 (clone 236A / E7, Invitrogen) using Foxp3 Transcription Factor Staining Buffer Set (eBiosciences) according to the manufacturer's instructions. Cells were analyzed on a BD LSRFortessa flow cytometer. Percent inhibition was calculated using the following formula: % inhibition = ((% naive T cell proliferation) - (% naive T cell + Treg proliferation)) / (% naive T cell proliferation) x 100.

[0161] pSTAT5 measurement in nTregs Purified Tregs were expanded as indicated above. On day 12, cells were harvested and incubated with various concentrations of MALT1i for 18 h in serum-free RPMI at 37°C and 5% CO2. Tregs were then stimulated with 25 IU IL-2 (Peprotech) for 15 min, followed by fixation with 2% paraformaldehyde. Cells were then permeabilized with 90% methanol and stained with anti-pSTAT5 antibody (Y694, clone 47, BD) at a dilution of 1:100. pSTAT5 levels were analyzed on a BD LSRFortessa flow cytometer.

[0162] MALT1 biochemical assay Inhibitor potency was assessed by measuring the enzymatic activity of full-length MALT1 at various concentrations of compound. The enzymatic assay consisted of a single substrate reaction monitoring the release of a fluorescent dye upon cleavage of the peptide substrate. The peptide substrate has the following sequence: Ac-Leu-Arg-Ser-Arg-Rh110-dPro (custom synthesis from WuXi AppTec, Shanghai, China). The assay buffer consisted of 50 mM Hepes, pH 7.5, 0.8 M sodium citrate, 1 mM DTT, 0.004% tween-20, and 0.005% bovine serum albumin (BSA). Steady-state kinetic analysis of peptide substrate binding yielded a Michaelis-Menten constant (K) of 150 μM. M ) resulting in a 384-well F-bottom polypropylene, black microplate (Greiner Bio_One, Cat. No. 781209) containing 15 nM enzyme and 30 μM peptide substrate. The reaction was quenched after 60 min with the addition of iodoacetic acid to a final concentration of 10 mM. Total fluorescence was measured using an Envision (PerkinElmer) with fluorescence excitation at 485 nm and emission at 520 nm.

[0163] For potency determination, 1 μL of serially diluted compound (in 100% DMSO) was pre-incubated with 40 μL of enzyme for 30 min. The reaction was initiated by the addition of 10 μL of peptide substrate. Relative fluorescence units were converted to percent inhibition by using 0% and 100% inhibition controls as references. The 100% inhibition control was (S)-1-(5-chloro-6-(2H-1,2,3-triazol-2-yl)pyridin-3-yl)-3-(2-chloro-7-(1-methoxyethyl)pyrazolo[1,5-a]pyrimidin-6-yl)urea (IC) at a final concentration of 1 μM. 50 = 15 nM) and the 0% inhibition control consisted of 2% DMSO. IC was calculated by fitting the concentration-response curves to a four-parameter logistic equation in GraphPad Prism. 50 The value was calculated.

[0164] Immunoblotting analysis of MALT1 function Primary human CD4 + T cells were negatively selected from human donor PBMCs (EasySep Human CD4 +T cells were isolated by T cell isolation kit (Stemcell Technologies). T cells were preincubated with 0–30 μM MALT1i or DMSO as a control for 30 min before stimulation with 50 ng / mL phorbol 12-myristate 13-acetate (Sigma) and 1.34 mM ionomycin (Sigma). Whole cell lysates were prepared in lysis buffer (CellLyticMT Cell Lysis reagent (Sigma)) containing HALT protease and phosphatase inhibitors (ThermoFisher Scientific) and 10 nM MG-132 (Sigma). Proteins were separated on NuPAGE 4–12% Bis-Tris denaturing gels (Invitrogen, Waltham, MA) and transferred onto nitrocellulose membranes (iBLOT2, Invitrogen). After blocking for 1 h at room temperature, blots were incubated in primary antibodies overnight, washed with PBS + 0.05% Tween-20, incubated in secondary antibodies for 2 h, washed, and imaged using a LI-COR Odyssey infrared imager (LI-COR Biosciences, Lincoln, NE). Antibodies were diluted in Intercept TBS-T blocking buffer (LI-COR). Bands were quantified using ImageStudio software (LI-COR Biosciences). Primary antibodies used were anti-HOIL-1 (Millipore Sigma, MABC576), anti-BCL10 (Abcam, 33905), anti-pIKKab (Cell Signaling Technologies, 2078), anti-pJNK (Cell Signaling Technologies, 4668), and anti-COX-IV (Cell Signaling Technologies, 4668).The secondary antibodies used were anti-mouse IgG H+L DyLight 680 (Cell Signaling Technologies, 5470) and anti-rabbit IgG H+L DyLight 800 (Cell Signaling Technologies, 5151).

[0165] statistics All statistical calculations were performed using Graphpad Prism 9.2.0. Data points from most readouts, e.g., cytokine expression, cell proliferation, disease scores, antibody levels, etc., were expressed as mean ± standard error of the mean (SEM). Significant differences (p<0.05) between unpaired observations were calculated using one-way ANOVA and corrected for multiple comparisons using Dunnett's test. Comparisons between two groups (e.g., healthy vs. arthritic animals) were performed using the Mann-Whitney test. Cytokine expression data in treatment groups were expressed as a percentage of DMSO control normalized to 100% activity. IC 50 and I.C. 90 Potential estimates of values ​​were performed in GraphPad Prism using a four-parameter fit. Plasma concentrations of MALT1i (AUC 0-24h ) to the corresponding effects observed in terms of clinical scores and / or reduction in splenic Tregs to generate pharmacokinetic-pharmacodynamic (PK-PD) correlations.

[0166] Example 2: Pharmacological modulation of MALT1 attenuates multiple immune effector functions in primary human cells We assessed the impact of MALT1i (REO-MALT1 inhibitor) on inflammatory processes associated with adaptive and innate immune cells activated via stimulation of distinct ITAM-containing immunoreceptors (Figures 3B-3E and Table 2). MALT1i inhibited the upregulation of CD45RO activated in vitro by α-CD3 / -CD28 / -CD2 in a dose-dependent manner. +MALT1i inhibited the release of proinflammatory cytokines (IFNγ, IL-2, and TNFα) and IL-17A from memory T cells (Figures 3B and 3C, respectively). MALT1i also attenuated B cell proliferation stimulated via co-crosslinking of BCR / CD40L with anti-human IgA / IgG / IgM and recombinant CD40L (Figure 3D). In myeloid cells, MALT1i effectively suppressed proinflammatory cytokine expression from human monocyte-derived macrophages differentiated in vitro and stimulated via FcγR with anti-human IgG / human IgG-derived immune complexes (Figure 3E and Table 2). Macrophages stimulated with bacterial lipopolysaccharide (LPS), which does not signal through an ITAM-dependent mechanism, were insensitive to MALT1i. In contrast, macrophages stimulated with depleted zymosan, which signals through the ITAM-dependent Dectin-1 receptor, were sensitive to MALT1 inhibition. Taken together, these data indicate that inhibition of MALT1 with an allosteric inhibitor can selectively suppress ITAM-driven autoimmune and inflammatory processes by multiple immune cell types in a stimulus-limited manner.

[0167] [Table 4]

[0168] To understand the dose-dependent relationship between MALT1i efficacy and Treg effects, a surrogate indicator of in vivo target engagement was developed using a whole blood assay. Spike-in of MALT1i was followed by TCR stimulation (MALT1-dependent pathway) and measurement of cytokine secretion in rat and human whole blood. This assay takes into account plasma protein binding and allows understanding of inhibitor potency in complex mixtures of cells. As shown in Table 3, pretreatment of human whole blood with MALT1i and subsequent co-stimulation of TCR with antibodies directed against CD3 and CD28, or PMA / ionomycin (surrogate ITAM signaling; data not shown), resulted in suppression of pro-inflammatory cytokine production. Several cytokines were found to be affected, including IL-2, IFNγ, and TNFα, but IL-2 was shown to be the most sensitive to MALT1 inhibition. IL-2 responses with PMA / ionomycin showed similar ICs compared to anti-CD3 / 28 stimulation. 50 showed.

[0169] [Table 5]

[0170] We next assessed the relationship between cytokine PD effects and MALTi target engagement by measuring the dose-dependent effect of MALT1i on the proteolytic cleavage of known MALT1 substrates. Immunoblotting was used to monitor cleavage of the MALT1 substrate HOIL-1 across a wide range of MALT1i concentrations encompassing 50-90% target coverage as determined using a human whole blood assay. Human whole blood IL-2 IC 50 At compound concentrations comparable to those of the control, protease function was affected by >80% ( Fig. 3F ).

[0171] Example 3: Treatment with MALT1i ameliorates disease pathogenesis in rat collagen-induced arthritis The effect of MALTi administration in a rat CIA model was evaluated. Oral administration of MALT1i demonstrated a dose-dependent reduction in disease activity scores in both preventative (Figure 4A) and therapeutic (Figure 4B) dosing regimens, as indicated by reductions in clinical scores over time and lower overall disease burden. Therapeutic MALT1i administration dose-dependently and significantly reduced disease scores at all doses tested, with greater than 50% reductions observed at the 3 and 10 mg / kg doses. Analysis of MALT1i levels in the plasma of animals treated with the compound demonstrated that MALT1i maintained 90% target coverage (rat whole blood IC 100 / mL) for approximately 10 hours over a 24-hour dosing period. 90 , Table 3) was shown to be effective at concentrations resulting in a 100% inhibition of 5'-hydroxytryptamine (Figure 4C).

[0172] The effector phase in the rat CIA model is characterized by inflammation in the joints caused by a systemic inflammatory response and by neutrophil accumulation and deposition of antigen-antibody immune complexes with eventual activation of the complement cascade. Proinflammatory cytokines (e.g., TNFα, IL-1β) secreted by activated macrophages have also been implicated in maintaining inflammation.

[0173] At the end of the study, the highest dose of MALT1i significantly reduced IL-6 levels in plasma. Significant inhibition of KC-GRO, a neutrophil chemoattractant produced by activated macrophages, was also observed in both plasma and synovial fluid at all doses tested. A significant reduction in TNFα in synovial fluid was also observed at all doses (Figure 4D). Furthermore, analysis of plasma for anti-collagen antibodies, which have been linked to immune complex formation, showed a significant reduction with MALT1i treatment (Figure 4E). There was no effect on total IgG levels among all treatment groups compared to naïve animals, suggesting that this level of target coverage does not lead to complete immunosuppression, although more systemic effects remain to be defined in subsequent studies. These data support a significant reduction in IC over the entire 24-h period. 90Maintenance of systemic MALT1i exposure equivalent to coverage indicated that it was not required to suppress known disease drivers of chronic inflammation, suggesting a lack of broad immunosuppression.

[0174] We next compared the effect of MALT1i on the peripheral Treg compartment in naive and CIA rats with active disease. To match the dosing paradigm of the CIA model, healthy rats were treated with MALT1i for 14 days, and Tregs were measured in healthy rats and CIA rats treated with the same three doses of MALT1i for the same period. Treg numbers are slightly elevated in the context of disease, and the reduction in Treg numbers in the spleens of rats treated with MALT1i was significantly more reduced at any given dose than Treg numbers from the spleens of rats with active CIA (Figure 5A). This observation was further confirmed with exposure-response analysis, which showed that at similar plasma drug concentrations, the effect of Treg reduction was more pronounced in naive animals compared to diseased animals (Figure 5B).

[0175] Example 4: Pharmacokinetics and Pharmacodynamics of MALT1 Inhibitors The effects of MALT1 inhibitors were analyzed to determine whether efficacy could be achieved at effective concentrations of MALT1i without affecting the Treg compartment. In this example, MALT1 inhibitors, including REO-981 MALT1 inhibitor, were administered to rats in the collagen-induced arthritis (CIA) rat model and analyzed according to the methods described in Example 1.

[0176] FIG. 6A depicts the single-dose pharmacokinetics of REO-981 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. REO-981 MALT1 inhibitor was administered at three doses (1 mg / kg (mpk), 3 mg / kg (mpk), and 10 mg / kg (mpk)). FIG. 6A shows the blood PK of REO-981 MALT1 inhibitor up to 24 hours after administration. Additionally, FIG. 6A shows the IC90 value (see horizontal dotted line). Here, rats administered 3 mg / kg or 10 mg / kg of REO-981 MALT1 inhibitor exhibited blood concentration levels of MALT1 inhibitor that exceeded the IC90 value for the entire 24 hours. Additionally, rats administered a low dose of 1 mg / kg of REO-981 MALT1 inhibitor also exhibited blood concentration levels of MALT1 inhibitor that exceeded the IC90 value for more than 20 of the 24 hours.

[0177] Figure 6B depicts the endpoint clinical scores at various doses of the REO-981 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model, where a 1 mg / kg dose of REO-981 resulted in a 38% reduction in clinical score, a 3 mg / kg dose of REO-981 resulted in a 57% reduction in clinical score, and a 10 mg / kg dose of REO-981 resulted in an 80% reduction in clinical score (reduction compared to vehicle treatment).

[0178] Figure 6C depicts Treg levels following administration of the REO-981 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model, where a 1 mg / kg dose of REO-981 resulted in a 22% reduction in Treg levels, a 3 mg / kg dose of REO-981 resulted in a 30% reduction in Treg levels, and a 10 mg / kg dose of REO-981 resulted in a 50% reduction in Treg levels (reduction compared to vehicle).

[0179] FIG. 6D depicts the percent reduction in clinical scores and Tregs after administration of the REO-981 MALT1 inhibitor according to the area under the curve (AUC). Here, FIG. 6D shows the decoupling of efficacy of the REO-981 MALT1 inhibitor and reduction in Treg levels. Exposure-response analysis revealed the uncoupling of efficacy in CIA from Treg reduction (FIG. 4D). Notably, a drug concentration AUC of 31,500 ng*hr / ml achieved a 50% effect on disease scores, and a drug concentration AUC of 155,000 ng*hr / ml reduced Treg numbers by 50% compared to naive animals (FIG. 6D). In other words, as shown in FIG. 6D, a log 10 In terms of AUC values, the REO-981 MALT1 inhibitor exhibits efficacy in the form of approximately a 50% reduction in clinical scores. Moreover, a log 10 In terms of AUC values, the REO-981 MALT1 inhibitor exhibits a reduction in Tregs in the form of an approximately 40% reduction in Treg levels. Therefore, as an example, a log 10 The AUC range represents the range in which the REO-981 MALT1 inhibitor achieves decoupling of MALT1 inhibitor efficacy and reduction of Treg levels.

[0180] To confirm that this uncoupling of efficacy from Treg reduction was not dependent on the specific compound tested, exposure-response from four distinct MALT1 inhibitors was further analyzed. Figure 6E shows combination data using four structurally distinct MALT1 inhibitors depicting the uncoupling of efficacy and Treg reduction. In general, the drug concentrations required to achieve significant effects on efficacy were 3-5 times lower than those required for reduction in Treg numbers (Figure 6E), indicating that the uncoupling of efficacy in CIA from reduction in Treg is a generalizable feature of allosteric inhibition of MALT1.

[0181] FIG. 6F depicts the percent reduction in clinical scores and Tregs following administration of the REO-981 MALT1 inhibitor according to Cmax. As shown in FIG. 6F, a log 10 At Cmax values, the REO-981 MALT1 inhibitor exhibits efficacy in the form of approximately 50% reduction in clinical scores. Moreover, a log 10 At Cmax values, the REO-981 MALT1 inhibitor exhibits a reduction in Tregs in the form of about a 40% reduction in Treg levels. Thus, for example, a log 10 The Cmax range represents the range in which the REO-981 MALT1 inhibitor achieves decoupling of MALT1 inhibitor efficacy and reduction of Treg levels.

[0182] FIG. 6G depicts the percent reduction in clinical scores and Tregs following administration of REO-981 MALT1 inhibitor according to Ctrough (trough concentration). As shown in FIG. 6G, a log 10 In terms of Ctrough values, the REO-981 MALT1 inhibitor exhibits efficacy in the form of approximately a 50% reduction in clinical scores. Moreover, a log 10 In terms of Ctrough values, the REO-981 MALT1 inhibitor exhibits a reduction in Tregs in the form of an approximately 40% reduction in Treg levels. Thus, for example, a log 10 The Ctrough range represents the range in which the REO-981 MALT1 inhibitor achieves decoupling of MALT1 inhibitor efficacy and reduction of Treg levels.

[0183] Overall, in this example, we analyzed the impact of REO-981 MALT1i on splenic Treg numbers in a CIA model to test the impact of MALT1i on Tregs in the disease. + FoxP3 +There is a statistically significant reduction in Tregs. This is in contrast to disease scores, as well as synovial fluid and plasma cytokine and chemokine concentrations, where significant reductions in disease scores were observed at all doses tested. There was further evidence of uncoupling of efficacy from Treg reduction when plasma drug concentrations (AUC) over time were plotted against effects on clinical scores and Treg numbers (Figure 6D). There was a clear separation in the two measured effects: an AUC of 31,500 ng*h / ml resulted in a 50% reduction in disease scores, whereas an AUC of 155,000 ng*h / ml was required for a 50% reduction in Treg numbers. These data indicate that efficacy was achieved at a concentration of MALT1i that was approximately 5-fold lower than the concentration that reduced Tregs. Additional evidence of uncoupling was observed in the sensitivity of Treg reduction to MALT1 inhibitor treatment in diseased rats versus naïve rats. Naïve animals were observed to have a greater magnitude of Treg reduction at any given dose compared to diseased animals. This was true when drug exposure was measured: for any given unit of exposure, there was a greater impact on Treg numbers in healthy compared to diseased animals, thus ruling out the possibility that dose differences were a consequence of changes in drug exposure.

[0184] There may be different mechanistic explanations for the uncoupling of efficacy and Treg reduction. Analysis of drug target coverage can provide insight into the mechanism of uncoupling. Inhibition of IL-2 is tightly linked to MALT1 signaling and may qualify as a proximal pharmacodynamic event following target modulation and therefore may be used as a marker for target engagement. The amount of target coverage required for efficacy with the tested MALT1i was determined by the IL-2 IC over the 24-h dosing period. 90 (Figure 6C). Analysis of target coverage for several other compounds demonstrated that IC 90The time to achieve the above-mentioned efficacy ranged from 4 to 12 h (data not shown). The ability of MALT1i to affect multiple pathogenic factors (e.g., TNFα, IL-6, KC-GRO) may underlie the observed efficacy in the absence of total target coverage over a 24-h period. Therefore, the resulting additive effect may require lower target coverage (e.g., IC of multiple cellular effects) to achieve a significant effect on disease scores. 50 In contrast, the impact on Tregs may be due to inhibition of only a single factor, e.g., IL-2, which may not be fully affected at effective doses. Consistent with this hypothesis, the reduction in Treg numbers was consistent with the IL-2 IC over the entire dosing period, regardless of the compound used. 90 Thus, dosing paradigms in which MALT1 is not sufficiently inhibited for the duration of the dosing interval may spare enough MALT1-driven IL-2 to maintain Tregs, as predicted by increased sensitivity of Tregs to lower concentrations of IL-2, but not enough to expand pathogenic effector T cell populations. 90 Above 100% (Figure 6C), IL-2 may become limiting and affect Treg maintenance, again explaining the difference in susceptibility to Treg reduction in naive compared to inflamed animals.

[0185] Example 5: Additional Pharmacokinetics and Pharmacodynamics of REO-528 and REO-703 MALT1 Inhibitors In this example, MALT1 inhibitors, including the MALT1 inhibitors REO-528, REO-538, REO-076, and REO-703, were administered to rats in a collagen-induced arthritis (CIA) rat model and analyzed according to the methods described in Example 1.

[0186] FIG. 7A depicts the single-dose pharmacokinetics of REO-528 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. REO-528 MALT1 inhibitor was administered at three doses (3 mg / kg (mpk), 10 mg / kg (mpk), and 30 mg / kg (mpk)). FIG. 7A shows the blood PK of REO-528 MALT1 inhibitor up to 24 hours after administration. Additionally, FIG. 7A shows the estimated IC90 value (see horizontal dotted line). Here, rats administered 30 mg / kg of REO-528 MALT1 inhibitor exhibited blood concentration levels of MALT1 inhibitor that exceeded the IC90 value for the entire 24 hours. Additionally, rats administered 10 mg / kg dose of REO-528 MALT1 inhibitor exhibited blood concentration levels of MALT1 inhibitor that exceeded the IC90 value for more than 12 hours out of the 24 hours.

[0187] Figure 7B depicts the endpoint clinical scores at various doses of REO-528 MALT1 inhibitor in rat collagen-induced arthritis (CIA) model. The dotted line in Figure 7B indicates the clinical score corresponding to 50% symptom reduction. Here, REO-528 at a dose of 3 mg / kg resulted in a 24% reduction in clinical score, REO-528 at a dose of 10 mg / kg resulted in a 59% reduction in clinical score, and REO-528 at a dose of 30 mg / kg resulted in a 74% reduction in clinical score (reduction compared to vehicle treatment).

[0188] Figure 7C depicts Treg levels following administration of the REO-528 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model, where a 3 mg / kg dose of REO-528 resulted in a 3% reduction in Treg levels, a 10 mg / kg dose of REO-528 resulted in a 36% reduction in Treg levels, and a 30 mg / kg dose of REO-528 resulted in a 37% reduction in Treg levels (reduction compared to vehicle).

[0189] FIG. 7D depicts the percent reduction in clinical scores and Tregs after administration of REO-528 MALT1 inhibitor according to area under the curve (AUC). Here, FIG. 7D shows the decoupling of efficacy of REO-528 MALT1 inhibitor and reduction in Treg levels. As shown in FIG. 7D, a log 10 In terms of AUC values, the REO-528 MALT1 inhibitor exhibits efficacy in the form of approximately a 50% reduction in clinical scores. Moreover, a log 10 In terms of AUC values, the REO-528 MALT1 inhibitor exhibits a reduction in Tregs in the form of an approximately 40% reduction in Treg levels. Therefore, as an example, a log 10 The AUC range represents the range in which the REO-528 MALT1 inhibitor achieves decoupling of MALT1 inhibitor efficacy and reduction of Treg levels.

[0190] FIG. 7E depicts the percent reduction in clinical scores and Tregs after administration of REO-528 MALT1 inhibitor according to Cmax. As shown in FIG. 7E, a log 10 At Cmax values, the REO-528 MALT1 inhibitor exhibits efficacy in the form of approximately 50% reduction in clinical scores. Moreover, a log 10 At Cmax values, the REO-528 MALT1 inhibitor exhibits a reduction in Tregs in the form of about a 40% reduction in Treg levels. Thus, for example, a log 10 The Cmax range represents the range in which the REO-528 MALT1 inhibitor achieves decoupling of MALT1 inhibitor efficacy and reduction of Treg levels.

[0191] FIG. 7F depicts the percent reduction in clinical scores and Tregs following administration of REO-528 MALT1 inhibitor according to Ctrough (trough concentration). As shown in FIG. 7F, a log 10In terms of Ctrough values, the REO-528 MALT1 inhibitor exhibits efficacy in the form of a reduction in clinical scores of about 50%. Moreover, a log 10 In terms of Ctrough values, the REO-528 MALT1 inhibitor exhibits a reduction in Tregs in the form of an approximately 50% reduction in Treg levels. Thus, for example, a log 10 The Ctrough range represents the range in which the REO-528 MALT1 inhibitor achieves decoupling of MALT1 inhibitor efficacy and reduction of Treg levels.

[0192] FIG. 8A depicts the single-dose pharmacokinetics of REO-538 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. REO-538 MALT1 inhibitor was administered at four doses (0.3 mg / kg (mpk), 1 mg / kg (mpk), 3 mg / kg (mpk), and 10 mg / kg (mpk)). FIG. 8A shows the blood PK of REO-538 MALT1 inhibitor up to 24 hours after administration. Additionally, FIG. 8A shows the estimated IC90 value (see horizontal dotted line). Here, rats administered 10 mg / kg REO-538 MALT1 inhibitor exhibited blood concentration levels of MALT1 inhibitor above the IC90 value for almost the entire 24 hours. Rats administered the REO-538 MALT1 inhibitor at a dose of 3 mg / kg exhibited blood levels of the MALT1 inhibitor that exceeded the IC90 value for more than 6 hours out of a 24-hour period.

[0193] Figure 8B depicts the endpoint clinical scores at various doses of REO-538 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model, where a 0.3 mg / kg dose of REO-538 resulted in a 26% reduction in clinical score, a 1 mg / kg dose of REO-538 resulted in a 59% reduction in clinical score, a 3 mg / kg dose of REO-538 resulted in a 65% reduction in clinical score, and a 10 mg / kg dose of REO-538 resulted in an 80% reduction in clinical score (reduction compared to vehicle treatment).

[0194] 8C depicts Treg levels following administration of the REO-538 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model, where a 1 mg / kg dose of REO-538 resulted in a 1% reduction in Treg levels, while a 10 mg / kg dose of REO-538 resulted in a 49% reduction in Treg levels (reduction compared to vehicle).

[0195] FIG. 8D depicts the percent reduction in clinical scores and Tregs after administration of REO-538 MALT1 inhibitor according to area under the curve (AUC). Here, FIG. 8D shows the decoupling of efficacy of REO-538 MALT1 inhibitor and reduction in Treg levels. As shown in FIG. 8D, a log 10 In terms of AUC values, the REO-538 MALT1 inhibitor exhibits efficacy in the form of approximately a 50% reduction in clinical scores. Moreover, a log 10 In terms of AUC values, the REO-538 MALT1 inhibitor exhibits a reduction in Tregs in the form of an approximately 40% reduction in Treg levels. Therefore, as an example, the log 10 The AUC range represents the range in which the REO-538 MALT1 inhibitor achieves decoupling of MALT1 inhibitor efficacy and reduction of Treg levels.

[0196] FIG. 9A depicts the single-dose pharmacokinetics of REO-703 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. REO-703 MALT1 inhibitor was administered at three doses (3 mg / kg (mpk), 10 mg / kg (mpk), and 30 mg / kg (mpk)). FIG. 9A shows the blood PK of REO-703 MALT1 inhibitor up to 24 hours after administration. Additionally, FIG. 9A shows the estimated IC90 value (see horizontal dotted line). Here, rats administered 30 mg / kg of REO-703 MALT1 inhibitor exhibited blood concentration levels of MALT1 inhibitor that exceeded the IC90 value for about 12 hours out of the entire 24 hours. Rats administered 10 mg / kg dose of REO-703 MALT1 inhibitor exhibited blood concentration levels of MALT1 inhibitor that exceeded the IC90 value for about 6 hours out of the 24 hours.

[0197] 9B depicts the endpoint clinical scores at various doses of the REO-703 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model, where a 3 mg / kg dose of REO-703 resulted in a 35% reduction in clinical score, a 10 mg / kg dose of REO-703 resulted in a 57% reduction in clinical score, and a 30 mg / kg dose of REO-703 resulted in a 60% reduction in clinical score (reduction compared to vehicle treatment).

[0198] Figure 9C depicts the levels of Tregs following administration of the REO-703 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model, where none of the doses of REO-703 resulted in a statistically significant reduction in Treg levels (compared to vehicle).

[0199] FIG. 9D depicts the percent reduction in clinical scores and Tregs after administration of REO-703 MALT1 inhibitor according to area under the curve (AUC). Here, FIG. 9D shows the decoupling of efficacy of REO-703 MALT1 inhibitor and reduction in Treg levels. As shown in FIG. 9D, a log 10 In AUC values, the REO-703 MALT1 inhibitor exhibits efficacy in the form of about a 50% reduction in clinical scores. Additionally, the REO-703 MALT1 inhibitor did not exhibit a reduction in Tregs. Thus, by way of example, a log 10 The AUC range represents the range in which the REO-703 MALT1 inhibitor achieves decoupling of MALT1 inhibitor efficacy and reduction of Treg levels.

[0200] Figure 10A depicts the single-dose pharmacokinetics of REO-076 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model. REO-076 MALT1 inhibitor was administered at three doses (3 mg / kg (mpk), 10 mg / kg (mpk), and 30 mg / kg (mpk)). Figure 10A shows the blood PK of REO-076 MALT1 inhibitor up to 24 hours after administration.

[0201] Figure 10B depicts the endpoint clinical scores at various doses of REO-076 MALT1 inhibitor in rat collagen-induced arthritis (CIA) model. The dotted line in Figure 10B indicates the clinical score corresponding to 50% symptom reduction. Here, REO-076 at a dose of 3 mg / kg results in a 23% reduction in clinical score, REO-076 at a dose of 10 mg / kg results in a 42% reduction in clinical score, and REO-076 at a dose of 30 mg / kg results in a 74% reduction in clinical score (reduction compared to vehicle treatment).

[0202] 10C depicts the levels of Tregs following administration of the REO-076 MALT1 inhibitor in a rat collagen-induced arthritis (CIA) model, where a 3 mg / kg dose of REO-076 resulted in a 24% reduction in Treg levels, a 10 mg / kg dose of REO-076 resulted in a 30% reduction in Treg levels, and a 30 mg / kg dose of REO-076 resulted in a 40% reduction in Treg levels (reduction compared to vehicle).

[0203] FIG. 10D depicts the percent reduction in clinical scores and Tregs after administration of REO-076 MALT1 inhibitor according to area under the curve (AUC). As shown in FIG. 10D, a log 10 In terms of AUC values, the REO-076 MALT1 inhibitor exhibits efficacy in the form of a reduction of approximately 50% in clinical scores. Moreover, a log 10 In terms of AUC values, the REO-076 MALT1 inhibitor exhibits a reduction in Tregs in the form of an approximately 40% reduction in Treg levels. Therefore, as an example, a log 10 The AUC range represents the range in which the REO-076 MALT1 inhibitor achieves decoupling of MALT1 inhibitor efficacy and reduction of Treg levels.

[0204] Figure 11 shows the qualitative efficacy and Treg influence results of each of the various MALT1 inhibitors. It is noted that both REO-538 and REO-528 exhibit high efficacy and low Treg influence, i.e., they exhibit a log 10 It may have a larger range of AUC values.

[0205] Figure 12A shows the dose-dependent efficacy of REO-528 and REO-703 MALT1 inhibitors in a rat CIA model, where both REO-528 and -703 demonstrated efficacy in a rat CIA model in a dose-dependent manner.

[0206] Figure 12B shows the levels of Tregs after administration of REO-528 and REO-703 MALT1 inhibitors in a rat CIA model. Notably, a dose-dependent reduction in Tregs was observed with REO-528, but not with REO-703.

[0207] Figure 12C shows the plasma pharmacokinetics of REO-528. Figure 12D shows the plasma pharmacokinetics of REO-703. Plasma PK suggests a shorter time-on-target (e.g., blood concentration of MALT1 inhibitor above IC90 value) for REO-703 compared to REO-538. Notably, at a dose of 30 mg / kg, REO-528 achieved a concentration above IC90 value for almost all of 24 hours after administration. In comparison, at a dose of 30 mg / kg, REO-703 achieved a concentration above IC90 value for about 4 hours within 24 hours after administration.

[0208] Example 6: PK and PD of REO-538 MALT1 inhibitor administered according to various dosing regimens In this example, REO-538 MALT1 inhibitor was administered to rats in a collagen-induced arthritis (CIA) rat model and analyzed according to the methods described in Example 1.

[0209] At time=0, rats were dosed via oral gavage with 1) naive, 2) vehicle, or 3) MALT1 inhibitor. Rats dosed with MALT1 inhibitor were dosed according to one of three dosing regimens: 1) 3 mg / kg per day for 14 days (qd×14) (referred to as the 3 mg / kg group in this example), 2) 10 mg / kg per day for 14 days (qd×14) (referred to as the 10 mg / kg group in this example), 3) 10 mg / kg per day for 2 days (qd×2) followed by 3 mg / kg per day for 12 days (qd×12) (referred to as the 10+3 mg / kg group in this example). To analyze cytokine levels, a-CD3 / CD28 stimulation of 200 ml of heparinized whole blood (diluted 1:1) was performed for 24 hours, followed by separation of plasma. Cytokines in plasma and synovial fluid were measured by an electrochemiluminescence-based detection method (Meso scale detection, MSD) and via ELISA.

[0210] Figure 13A shows the endpoint clinical scores across different dosing regimens with the REO-538 MALT inhibitor. Here, REO-538 at a dose of 3 mg / kg per day for 14 days resulted in a 25% reduction in clinical score. REO-538 at a dose of 10 mg / kg per day for 14 days resulted in a 70% reduction in clinical score. REO-538 at 10 mg / kg per day for 2 days followed by 3 mg / kg per day for 12 days resulted in a 43% reduction in clinical score (reduction compared to vehicle treatment).

[0211] FIG. 13B shows the single-dose pharmacokinetics of REO-538 across different dosing regimens. FIG. 13B shows the blood PK of the REO-538 MALT1 inhibitor up to 24 hours after dosing (day 28). Additionally, FIG. 13B shows the estimated IC50 values ​​(log 1 of approximately 200 ng / mL). 10 value) and IC90 value (log 10 The dotted line above the value indicates the

[0212] FIG. 13C shows the levels of IL-1β, IL-6, KC / GRO, and TNFα in synovial fluid after administration of REO-538. In general, the cytokine levels of IL-1β and IL-6 in synovial fluid were lower for the 10 mg / kg and 10+3 mg / kg groups compared to the 3 mg / kg group, while the 3 mg / kg group showed similar levels of IL-1β and IL-6 compared to the vehicle group. Additionally, each of the 3 mg / kg, 10 mg / kg, and 10+3 mg / kg groups exhibited lower levels of KC / GRO and TNFα compared to the vehicle group.

[0213] Example 7: Further examples of decoupling of MALT1 inhibitor efficacy and Treg reducing effect In this example, REO-528 MALT1 inhibitor was administered to rats in a collagen-induced arthritis (CIA) rat model.

[0214] Adult female Lewis rats (180-200 g body weight) were immunized subcutaneously with type II bovine collagen / incomplete Freund's adjuvant emulsion on days 0 and 7. On day 14, rats were randomized based on clinical disease scores in the different treatment groups. Rats were given a once-daily (qd) dose of either compound via oral gavage or vehicle (0.5% carboxymethylcellulose-Na+0.5% Tween 80 in water, suspension) for 2 weeks. Naive rats were used as controls. Body weight measurements were taken 3 times per week to assess compound tolerability. Clinical scores and joint swelling (hind paw volume) were measured before the first dose on day 0 and then 3 times per week until the end of the study. Clinical scores measured on a scale of 0-4 per paw are listed in the table below.

[0215] [Table 6]

[0216] After 14 days of dosing, a subset of rats was bled at different time intervals (15 min, 1 h, 2 h, 6 h, 12 h, and 24 h after the last dose) to assess compound exposure levels in plasma. At the end of the study on day 28, whole blood CBC was measured. Plasma cytokine and anti-collagen antibody levels were measured by ELISA. Spleen cells were collected and immunophenotypic analysis was assessed by flow cytometry.

[0217] FIG. 14 depicts the percent reduction in clinical scores and Tregs after administration of REO-528 MALT1 inhibitor according to area under the curve (AUC). Here, FIG. 14 shows the decoupling of efficacy of REO-528 MALT1 inhibitor and reduction in Treg levels. As shown in FIG. 14, a log 10 In terms of AUC values, the REO-528 MALT1 inhibitor exhibits efficacy in the form of approximately a 50% reduction in clinical scores. Moreover, a log 10 In terms of AUC values, the REO-528 MALT1 inhibitor exhibits a reduction in Tregs in the form of an approximately 40% reduction in Treg levels. Therefore, as an example, the log 10 The AUC range represents the range in which the REO-528 MALT1 inhibitor achieves decoupling of MALT1 inhibitor efficacy and reduction of Treg levels.

[0218] Example 8: Identification of Candidate Subjects for Administration of MALT1 Inhibitors A test sample is obtained from a subject (e.g., a human subject) on day 0 to determine whether the subject is a candidate or non-candidate (as described in FIG. 1). A blood sample is obtained and an immunoassay is performed to determine the level of IL-2 in the blood sample. The quantitative expression value of IL-2 from the test sample is compared with a reference value, where the reference value is the average expression of IL-2 in blood samples obtained from healthy patients.

[0219] A subject whose test sample has a quantitative expression value of IL-2 above the reference value is categorized as a candidate subject. A subject whose test sample has a quantitative expression value of IL-2 below the reference value is categorized as a non-candidate subject. A MALT1 inhibitor (e.g., REO-528 MALT1 inhibitor) is administered to the candidate subject. After administration of the MALT1 inhibitor to the candidate subject, a decoupling of the efficacy of the MALT1 inhibitor and the reduction of Treg is observed. In particular, a wide range is observed (e.g., log 2 , which represents the range in which the REO-528 MALT1 inhibitor achieves decoupling of the efficacy of the MALT1 inhibitor and the reduction of Treg levels). 10 AUC range), where candidate subjects (e.g., subjects having a quantitative expression value of IL-2 above the reference value) exhibit greater resistance to Treg reduction as a result of the presence of higher IL-2 levels.

[0220] Example 9: Additional dosing regimen of MALT1 inhibitor (REO-528) Figure 15 shows a dosing strategy for REO-528 MALT1 inhibitor, including combination REO-528 + IL-2 therapy. The purpose of this dosing strategy is to evaluate the reversibility of Treg reduction following MALT1 inhibitor administration and to determine whether IL-2 or intermittent MALT1 inhibitor dosing mediates Treg rescue.

[0221] Dosing with vehicle (n=10) or REO-528 compound (n=25) via oral gavage at 30 mg / kg (mpk) begins on day 1. After 7 days of dosing, 5 mice per group (vehicle and compound) are withdrawn within 24 hours of the last dose (day 8) and analyzed for: Splenic immunophenotype analysis including Teff and Treg 24-hour a-CD3 / CD28 stimulation of 200 ml heparinized whole blood (diluted 1:1), plasma separated, and cytokines analyzed; unstimulated controls included Plasma PK analysis for compounds only

[0222] Dosing continues as illustrated for 5 days (days 8-12) with 5 mice per new group. In particular, 1) 5 mice are treated daily with REO-528, 2) 5 mice are treated with a combination of REO-528 and recombinant mouse IL-2 (Peprotech, cat# 212-12) administered daily via the intraperitoneal route at 30,000 IU / animal (approximately 2 mg), 3) 5 mice are treated with recombinant mouse IL-2 alone, 4) 5 mice are treated every other day (QOD) with REO-528, and 5) 5 mice are treated with vehicle.

[0223] After 5 days of dosing (day 13), the animals are sacrificed and a readout similar to that of day 8 is performed. Additionally, the weight and body condition of the mice are monitored.

[0224] Example 10: Addition of IL-2 increases IL-2R signaling despite MALT1 inhibition To assess whether MALT1 inhibition (e.g., using the REO-981 MALT1 inhibitor) directly affects Treg suppressive function, an in vitro Treg suppression assay was used. + CD127 low CD25 + Tregs were isolated from human donor PBMCs using the Regulatory T-Cell Isolation Kit (Stemcell) and expanded in culture as previously described (16). Briefly, Tregs were activated with Dynabeads Human T-Activator CD3 / CD28 (Thermofisher) at a 1:1 bead-to-cell ratio in complete RPMI medium supplemented with 10% FBS, 10 mM HEPES, 2 mM GlutaMAX, 1 mM Na-pyruvate, and 1x MEM non-essential amino acids (Thermofisher). After 2 days of culture, the culture volume was doubled and IL-2 was added to a final concentration of 300 IU (Peprotech). On days 5 and 7, cells were expanded in the presence of 300 IU IL-2. On day 9, cells were restimulated with Dynabeads at a 1:1 bead-to-cell ratio. On day 13, Tregs were harvested and beads were magnetically removed for downstream Treg suppression assays.

[0225] For in vitro Treg suppression assays, naive CD4 T cells were assayed by measuring Cell Trace Violet (CTV) (Thermofisher) dilutions in the presence of varying ratios of autologous nTregs. + T cell proliferation was assessed. Briefly, Human Naive CD4 + Naive CD4 T cells were isolated from human donor PBMCs using the T-cell Isolation Kit (Stemcell). + T cells were isolated and labeled with CTV according to the manufacturer's protocol. Naïve CD4 T cells were transfected with Dynabeads Human T-Activator CD3 / CD28 beads at a bead-to-cell ratio of 1:8 in the presence of varying ratios of MALT1i-treated nTregs in complete RPMI for 3 days. + Cells were activated. Cells were then processed for flow analysis of proliferation. Briefly, cells were first stained with Fixable Live-Dead dye (BD) and then stained with the following panel of fluorescently labeled antibodies (Biolegend unless otherwise stated): CD4 (clone RPA-T4) and CD25 (clone M-A251) for 20 min at 4°C. Cells were subsequently stained for intracellular FoxP3 (clone 236A / E7, Invitrogen) using Foxp3 Transcription Factor Staining Buffer Set (eBiosciences) according to the manufacturer's instructions. Cells were analyzed on a BD LSRFortessa flow cytometer. Percent inhibition was calculated using the following formula: % inhibition = ((% naive T cell proliferation) - (% naive T cell + Treg proliferation)) / (% naive T cell proliferation) x 100.

[0226] MALT1 inhibition was induced by surface CD3 / CD28 stimulation using Dynabeads to inhibit naive CD4 + MALT1i did not reduce the ability of human Tregs to suppress T cell proliferation (Figure 16A). MALT1i also had no effect on FoxP3 expression in Tregs (Figure 16B). Responder CD4 +At lower ratios of Tregs to T cells, higher compound concentrations increased the apparent suppressive capacity of Tregs. This observation was consistent with anti-CD3 / 28 stimulated CD4 + This may reflect the effect of the compound on the proliferation of T cell responders. Indeed, labeled responder CD4+ cells cultured with higher concentrations of MALT1i in the absence of co-cultured Tregs showed a significantly higher proliferation rate. + T cells showed a reduced proliferative capacity (Figures 16C and 16D). Overall, these data suggest that MALT1 inhibition reduces the proliferation and proliferation capacity of naive CD4 T cells in vitro. + This suggests that it does not directly affect the ability of Tregs to suppress T cell proliferation, nor does it affect Treg stability via downregulation of FOXP3.

[0227] We next investigated the effect of MALT1i on IL-2R signaling, which results in phosphorylation of STAT5 (pSTAT5) and is dependent on JAK1 / 3. Therefore, we measured IL-2R activity using a flow cytometric readout of pSTAT5, and the JAK1 / 3 inhibitor tofacitinib served as a positive control for inhibition of pSTAT5.

[0228] Purified Tregs were expanded as indicated above. On day 12, cells were harvested and incubated with various concentrations of MALT1i for 18 h in serum-free RPMI at 37°C and 5% CO2. Tregs were then stimulated with 25 IU IL-2 (Peprotech) for 15 min, followed by fixation with 2% paraformaldehyde. Cells were then permeabilized with 90% methanol and stained with anti-pSTAT5 antibody (Y694, clone 47, BD) at a dilution of 1:100. pSTAT5 levels were analyzed on a BD LSRFortessa flow cytometer.

[0229] Interestingly, Tregs appeared to have elevated basal levels of pSTAT5, which was reduced with tofacitinib treatment. IL-2 treatment increased pSTAT5 levels in all conditions except the tofacitinib-treated group. Importantly, no effect of MALT1i was observed in either basal or IL-2-treated cells, suggesting that MALT1 does not play a key role in this pathway (Figures 16E and 16F) and that chronic inhibition of MALT1 abrogates IL-2R-dependent signaling in Tregs. Furthermore, these results indicate that IL-2 treatment can increase or restore IL-2R-dependent signaling in Tregs despite MALT1 inhibition.

[0230] Example 11: Administration of MALT1 inhibitors reduces the levels of Tregs In this example, MALT1 inhibitors, including the MALT1 inhibitors REO-751 and REO-095, were administered to naive mice to examine the impact of MALT1 inhibitors on Treg levels. Mice were dosed with REO-751 or REO-095 for 4 weeks, and spleen cells were analyzed for Treg levels. In particular, REO-751 or REO-095 MALT1 inhibitors were dosed at one of 1 mg / kg (mpk), 10 mg / kg (mpk), or 100 mg / kg (mpk).

[0231] FIG. 17A depicts Treg levels after administration of MALT1 inhibitors in naive mice. The % reduction in Treg levels is shown compared to vehicle control. In general, REO-751 exhibited a higher reduction in Treg levels compared to REO-095. Notably, REO-751 mediated a significantly less than 2-fold reduction in Tregs than REO-095. At a dose of 100 mg / kg, REO-751 reduced Treg levels by 76%, while at the same dose of 100 mg / kg, REO-095 reduced Treg levels by 32%.

[0232] Furthermore, the pharmacokinetic (PK) profiles of REO-751 and REO-095 were examined to determine whether the PK profiles could explain the differences in Treg reduction. Figure 17B shows the pharmacokinetic profile of MALT1 inhibitors at day 28, where similar exposure levels were observed between both REO-751 and REO-095.

[0233] Figure 17C shows the IC50 and Kd values ​​of MALT1 inhibitors. Notably, REO-751 exhibits increased potency (e.g., lower IC50 and lower Kd values) compared to REO-095. Here, increased potency and affinity can determine the effect on Treg levels. These results suggest that acceptable efficacy can be achieved according to unique potency / affinity characteristics while ensuring that Treg levels are not overly depleted.

[0234] Example 12: Administration of REO-981 MALT1 inhibitor for the treatment of arthritis The effects of MALT1 inhibitors were analyzed to determine whether efficacy could be achieved without affecting the Treg compartment at effective concentrations of MALT1i. In this example, MALT1 inhibitors, including the REO-981 MALT1 inhibitor, were administered to rats in a collagen-induced arthritis (CIA) rat model and analyzed.

[0235] Adult female Lewis rats (Charles River) weighing 180-200 g were immunized subcutaneously with bovine type II collagen (Chondrex, Woodinville, WA) / incomplete Freund's adjuvant (Sigma) emulsion prepared as per manufacturer's protocol (Chondrex) on days 0 and 7. MALT1i doses for oral administration were prepared by suspending the compound in 0.5% Na-carboxymethylcellulose / 0.5% Tween-80 in water (vehicle). For prophylactic treatment, animals were dosed via oral gavage on day 0 prior to immunization with collagen and continued once daily (qd) for 4 weeks. For therapeutic treatment, animals were randomized as per clinical disease scoring on day 14 and qd dosing of compound via oral gavage was performed for 2 weeks. Vehicle-treated and naïve animals were used as controls. Clinical scores and joint swelling (hind paw volume) were measured before the first dose on day 0 and then three times weekly until the end of the study. Body weight measurements were taken three times weekly to assess compound tolerability. Criteria (on a scale of 0-4 per paw) were as follows: 0, no evidence of erythema and swelling; 1, erythema and mild swelling limited to the midfoot (tarsus) or ankle joint; 2, erythema and mild swelling extending from the ankle to the midfoot; 3, erythema and moderate swelling extending from the ankle to the midfoot joint; 4, erythema and severe swelling involving the ankle, paws, and toes. After 2-3 weeks of dosing, representative animals were bled at different time intervals over a 0-24 hour time period to assess compound exposure levels in plasma via LC / MS.

[0236] In particular, the REO-981 MALT1 inhibitor was administered to healthy or diseased rats to examine the impact of MALT1 inhibitors on Treg levels. Here, diseased rats refer to collagen-induced arthritis (CIA) model rats. At time=0, rats were dosed with 1) naive, 2) vehicle, 3) MALT1 inhibitor (1 mg / kg, 3 mg / kg, or 10 mg / kg), or 4) tofacitinib. Blood was obtained from rats at various time intervals after administration (e.g., 15 min, 1 h, 2 h, 6 h, 12 h, and 24 h). Blood pharmacokinetics (PK) and pharmacodynamics (PD) of MALT1 inhibitors were calculated from blood samples. Endpoint clinical scores were determined by monitoring the behavior of rats after administration. Furthermore, rats were sacrificed on day 28 after administration for splenic immunophenotyping analysis, including determination of Treg levels.

[0237] FIG. 18A depicts Treg levels after administration of MALT1 inhibitors in a rat collagen-induced arthritis (CIA) model or healthy animals. Here, a dose-dependent reduction in regulatory T cells was observed in both diseased and healthy animals treated with REO-981. It is noted that diseased rats given 10 mg / kg REO-981 experienced a 50% reduction in Treg cells, while healthy rats given the lower 1 mg / kg REO-981 dose also experienced a 50% reduction in Treg cells. This suggests that healthy animals were able to upregulate T REG These results suggest that the IL-1 signaling pathway is more sensitive to MALT1 inhibition in terms of modulation.

[0238] Figure 18B depicts the endpoint clinical score after administration of MALT1 inhibitor in rat collagen-induced arthritis (CIA) model. Additionally, Figure 18C depicts the pharmacokinetic (PK) profile after administration of MALT1 inhibitor in rat collagen-induced arthritis (CIA) model. Referring to Figure 18B, REO-981 achieved a dose-dependent reduction in clinical score. Administration of 10 mg / kg REO-981 MALT1 inhibitor achieved an 80% reduction in endpoint clinical score, similar to the 75% reduction achieved by 5 mg / kg tofacitinib dose.

[0239] Referring to Figure 18C, both the 3 mg / kg and 10 mg / kg doses of REO-981 achieved plasma concentrations above the IC90 value for 24 hours after dosing. Additionally, the 1 mg / kg dose of REO-981 achieved plasma concentrations above the IC90 value for approximately 12 of the 24 hours after dosing.

[0240] FIG. 18D depicts the percent reduction in clinical scores and Tregs after administration of REO-981 MALT1 inhibitor according to area under the curve (AUC). Here, FIG. 18D shows the decoupling of efficacy of REO-981 MALT1 inhibitor and depletion of Treg levels. Exposure-response analysis revealed the uncoupling of efficacy in CIA from Treg reduction (FIG. 4D). In particular, we observed that a drug concentration AUC of 31,500 ng*h / ml achieved a 50% effect on disease scores and a drug concentration AUC of 155,000 ng*h / ml reduced Treg numbers by 50% compared to naive animals (FIG. 18D). In other words, as shown in FIG. 18D, a log 10 In terms of AUC values, the REO-981 MALT1 inhibitor exhibits efficacy in the form of approximately a 50% reduction in clinical scores. Moreover, a log 10In terms of AUC values, the REO-981 MALT1 inhibitor exhibits Treg depletion in the form of approximately a 40% reduction in Treg levels. Thus, for example, a log 10 The AUC range represents the range in which the REO-981 MALT1 inhibitor achieves decoupling of MALT1 inhibitor efficacy and depletion of Treg levels.

[0241] To confirm that this uncoupling of efficacy from Treg reduction was not dependent on the specific compound tested, exposure-response from four distinct MALT1 inhibitors was further analyzed. Figure 19 shows combination data using four structurally distinct MALT1 inhibitors depicting the uncoupling of efficacy and Treg reduction. In general, the drug concentrations required to achieve significant effects on efficacy were 3-5 times lower than those required for reduction in Treg numbers (Figure 19), indicating that the uncoupling of efficacy in CIA from reduction in Treg is a generalizable feature of allosteric inhibition of MALT1.

[0242] Overall, in this example, we analyzed the impact of REO-981 MALT1i on splenic Treg numbers in a CIA model to test the impact of MALT1i on Tregs in the disease. + FoxP3 +There is a statistically significant reduction in Tregs. This is in contrast to disease scores, as well as synovial fluid and plasma cytokine and chemokine concentrations, where a significant reduction in disease scores was observed at all doses tested. There was further evidence of uncoupling of efficacy from Treg reduction when plasma drug concentrations over time (AUC) were plotted against the effect on clinical scores and Treg numbers (Figure 18D). There was a clear separation in the two measured effects: an AUC of 31,500 ng*h / ml resulted in a 50% reduction in disease scores, whereas an AUC of 155,000 ng*h / ml was required for a 50% reduction in Treg numbers. These data indicate that efficacy was achieved at approximately 5-fold lower concentrations of MALT1i than those that reduced Tregs.

[0243] Example 13: Administration of MALT1 inhibitors for the treatment of multiple sclerosis In this example, REO-751 MALT1 inhibitor was administered to mice with different disease models (e.g., preventive experimental autoimmune encephalomyelitis (EAE) model, therapeutic experimental autoimmune encephalomyelitis (EAE)) to investigate the impact of administration of MALT1 inhibitor. Induction of EAE in mice is performed via injection of neuropeptide (MOG35-55) on day 0, and disease in the form of paralysis is detected 7-10 days later. The preventive dosing regimen refers to the initiation of treatment with MALT1 inhibitor on day 0, prior to the induction of disease. The therapeutic dosing regimen refers to the initiation of treatment with MALT inhibitor on day 12, after disease is detected in mice. Mice were sampled at the endpoint 24 hours after the last dose. Clinical scores were determined by monitoring the behavior of mice after dosing according to the following grading system:

[0244] [Table 7]

[0245] Figure 20A depicts the mean clinical score after administration of a MALT1 inhibitor in a preventive experimental autoimmune encephalomyelitis (EAE) model. Figure 20B depicts the pharmacokinetic (PK) profile after administration of a MALT1 inhibitor in a preventive experimental autoimmune encephalomyelitis (EAE) model. Figure 20C depicts the mean clinical score after administration of a MALT1 inhibitor in a therapeutic experimental autoimmune encephalomyelitis (EAE) model. Figure 20D depicts the pharmacokinetic (PK) profile after administration of a MALT1 inhibitor in a therapeutic experimental autoimmune encephalomyelitis (EAE) model.

[0246] In general, efficacy in the rat EAE model was observed in both preventive and therapeutic treatment conditions. No effect on Tregs was observed after treatment with MALT1 inhibitors (data not shown). For a REO-951 dose of 10 mg / kg, the concentration of REO-951 MALT1 inhibitor was approximately 50% lower than that observed in naive mice.

[0247] Example 14: Administration of MALT1 inhibitors for the treatment of graft-versus-host disease (GVHD) In this example, a dose of 100 mg / kg of the REO-751 MALT1 inhibitor was administered to mice in a graft-versus-host disease (GVHD) model to investigate the impact of administration of the MALT1 inhibitor. The effect of the MALT1 inhibitor was evaluated in scGVHD and delayed-type hypersensitivity (DTH) models. For the scGVHD model, bone marrow (1E7 cells) and spleen cells (4E6) from LP / J donors were transferred into C57Bl / 6 recipients that had been irradiated a total of 8.5 Gy the previous day. For the DTH model, female Balb / c mice were immunized subcutaneously with keyhole limpet hemocyanin (KLH) on day 0 and challenged intradermally with KLH 7 days later.

[0248] At time=0, mice were dosed with 1) naive, 2) vehicle, 3) REO-751 MALT1 inhibitor (100 mg / kg), or 4) ruxolitinib (60 mg / kg). Blood was obtained from the mice at various time intervals after dosing (e.g., 15 min, 1 h, 2 h, 6 h, 12 h, and 24 h). Blood pharmacokinetics (PK) and pharmacodynamics (PD) of the MALT1 inhibitor were calculated from the blood samples. GVHD scores were determined by monitoring the behavior of the mice after dosing according to the following grading system:

[0249] [Table 8]

[0250] Figure 21A depicts the mean GVHD score after administration of MALT1 inhibitor in a mouse GVHD model. Figure 21B depicts the pharmacokinetic (PK) profile after administration of MALT1 inhibitor in a mouse GVHD model. In general, as shown in Figure 21A, the efficacy of REO-751 MALT1 inhibitor was observed in the GVHD model. A REO-751 dose of 100 mg / kg achieved a GVHD score similar to that of ruxolitinib (60 mg / kg). No difference in Tregs was observed with treatment. As shown in Figure 21B, the concentration of REO-751 at 24 hours (day 55) after administration was 385 ng / mL. This PK concentration at this endpoint is lower than that of naive mice.

[0251] Figures 22A-22F show the results after administration of MALT1 inhibitors. Here, "Cpd1" and "Cpd2" shown in Figures 22A-22F refer to the REO-751 MALT1 inhibitor. Figures 22A and 22B show the overall survival (OS) and progression-free survival (PFS) Kaplan-Meier curves, respectively, in a mouse model of scleroderma GVHD (scGVHD) after administration of a MALT1 inhibitor. In particular, Figure 22A shows the survival for all animals during a scGVHD study in which mice were given 1) vehicle, 2) 100 mg / kg REO-751 orally once daily, or 3) 60 mg / kg ruxolitinib twice daily from day 21 to day 56 (after cell transfer). The naive control group received neither irradiation nor cell transfer. Referring to Figure 22B, Figure 22B shows PFS, defined as an increase in GVHD score of greater than 2 compared to the GVHD score on day 21, where 40% of mice achieved progression-free survival at the end (e.g., day 56), representing an improvement over the naïve and ruxolitinib groups.

[0252] 22C-22E show the T follicular helper cells (T FH 22C-22E show the levels of Treg cells, germinal center (GC) B cells, and Treg cells in the spleen, as measured by flow cytometry. Notably, administration of a MALT1 inhibitor significantly increased the levels of Treg cells compared to vehicle and ruxolitinib. FH Ruxolitinib reduced the percentage of GC B cells and GC B cells, but did not reduce the percentage of Tregs compared to vehicle and ruxolitinib.

[0253] Now referring to Figure 22F, Figure 22F shows the percentage change in ear thickness as an index of delayed hypersensitivity after administration of MALT1 inhibitor. In particular, Figure 22F shows the ear thickness before challenge (per mouse) and 48 hours after KLH challenge normalized to naive / vehicle values. Figure 22F shows that administration of MALT1 inhibitor prevented DTH response, indicating that T cell-driven responses in the skin, an important target tissue in scGVHD, can be attenuated with MALT1 blockade.

[0254] Example 15: Administration of MALT1 Inhibitors for the Treatment of Lupus In this example, the REO-528 MALT1 inhibitor was administered to mice in an accelerated lupus nephritis model (IFN-alpha accelerated) to investigate the impact of administration of a MALT1 inhibitor. Mice were divided into six groups: 1) naïve, 2) vehicle (weeks 13-18), 3) REO538 (3 mg / kg) administered daily from weeks 13-18, 4) REO-538 (30 mg / kg) administered daily from weeks 13-18, 5) REO-538 (30 mg / kg) administered daily from weeks 15-18, and 6) 1 mg / kg BID dexamethasone (Dex) from weeks 15-18. Groups 2-6 were also administered an AAV encoding mIFNα to accelerate lupus nephritis disease. Mice were sacrificed after administration for splenic immunophenotypic analysis, including determination of levels of Tregs.

[0255] Figure 23 depicts Treg levels after administration of REO-538 in a mouse accelerated lupus model. REO-538 reduces Treg levels in a dose-dependent manner (e.g., 30mg / kg REO-538 reduces Treg levels more than 3mg / kg REO-538). Additionally, the % Treg cells (in splenocytes) tend to be higher than in vehicle-treated accelerated lupus animals. MALT1 treatment reduces Tregs compared to vehicle, but back toward naive levels.

Claims

1. 1. Use of a MALT1 inhibitor for the manufacture of a pharmaceutical composition for treating a chronic disorder, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor achieves a time above an IC50 blood concentration target of 4 to 20 hours per 24 hours.

2. The use described in claim 1, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor achieves a time above the blood concentration target of 6 to 18 hours per 24 hours.

3. The use described in claim 1, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor achieves a time above the blood concentration target of 8 to 16 hours per 24 hours.

4. The use described in claim 1, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor achieves a time above the blood concentration target of 10 to 14 hours per 24 hours.

5. The use according to any one of claims 1 to 4, wherein the dose of the MALT1 inhibitor is 1 mg / kg to 6 mg / kg.

6. The use according to any one of claims 1 to 4, wherein the dose of the MALT1 inhibitor is 2 mg / kg to 5 mg / kg.

7. The use according to any one of claims 1 to 4, wherein the dose of the MALT1 inhibitor is 3 mg / kg.

8. 1. Use of a MALT1 inhibitor for the manufacture of a pharmaceutical composition for treating a chronic disorder, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor achieves a time per 24-hour period above an IC50 blood concentration target of 12 to 24 hours.

9. The use of claim 8, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of 16 to 24 hours per 24 hours.

10. The use of claim 8, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor achieves a time above the IC50 blood concentration target of 22 to 24 hours per 24 hours.

11. The use of claim 8, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of 1 to 15 hours per 24 hours.

12. The use of claim 8, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of 3 to 12 hours per 24 hours.

13. The use of claim 8, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of 6 to 10 hours per 24 hours.

14. The use according to any one of claims 8 to 13, wherein the dose of the MALT1 inhibitor is 8 mg / kg to 20 mg / kg.

15. The use according to any one of claims 8 to 13, wherein the dose of the MALT1 inhibitor is 9 mg / kg to 15 mg / kg.

16. The use according to any one of claims 8 to 13, wherein the dose of the MALT1 inhibitor is 10 mg / kg.

17. 1. Use of a MALT1 inhibitor for the manufacture of a pharmaceutical composition for treating a chronic disorder, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor achieves a time per 24-hour period above an IC90 blood concentration target of 6 hours to 24 hours.

18. The use of claim 17, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of 12 to 24 hours per 24 hours.

19. The use of claim 17, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of 18 to 24 hours per 24 hours.

20. The use of claim 17, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor achieves a time above the IC90 blood concentration target of 21 to 24 hours per 24 hours.

21. The use according to any one of claims 17 to 20, wherein the dose of the MALT1 inhibitor is 8 mg / kg to 20 mg / kg.

22. The use according to any one of claims 17 to 20, wherein the dose of the MALT1 inhibitor is 9 mg / kg to 15 mg / kg.

23. The use according to any one of claims 17 to 20, wherein the dose of the MALT1 inhibitor is 10 mg / kg.

24. 1. Use of a MALT1 inhibitor for the manufacture of a pharmaceutical composition for treating a chronic disorder, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor has a log IL-1 activity of 0.5 μg*hr / mL to 2.0 μg*hr / mL. １０ (AUC) is achieved using.

25. The method of claim 25, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor exhibits a log IL-1 activity of 1.0 μg*hr / mL to 1.75 μg*hr / mL. １０ 25. The use of claim 24, wherein the AUC is achieved.

26. The method of claim 26, wherein after administration of the pharmaceutical composition to a subject, the MALT1 inhibitor exhibits a log IL-1 activity of 1.25 μg*hr / mL to 1.50 μg*hr / mL. １０ 26. The use according to claim 24 or 25, which achieves (AUC).

27. The use of claim 24 or 25, wherein after administration of the pharmaceutical composition to a subject, the subject's Treg levels remain at least 60% of the levels prior to administration.

28. The use of claim 24 or 25, wherein after administration of the pharmaceutical composition to a subject, the subject's Treg levels remain at least 70% of the levels prior to administration.

29. The use of claim 24 or 25, wherein after administration of the pharmaceutical composition to a subject, the subject's Treg levels remain at least 80% of the levels prior to administration.

30. The use of claim 24 or 25, wherein after administration of the pharmaceutical composition to a subject, the subject's Treg levels remain at least 90% of the levels prior to administration.

31. The use of claim 24 or 25, wherein after administration of the pharmaceutical composition to a subject, the subject's Treg levels remain at least 95% of the levels prior to administration.

32. 1. Use of a MALT1 inhibitor for the manufacture of a pharmaceutical composition for treating a chronic disorder, wherein the dose of the MALT1 inhibitor is 1 mg / kg to 100 mg / kg.

33. 33. The use according to claim 32, wherein the dose of the MALT1 inhibitor is 20 mg / kg to 40 mg / kg.

34. The use according to claim 32 or 33, wherein the dose of the MALT1 inhibitor is 25 mg / kg to 35 mg / kg.

35. 34. The use according to claim 32 or 33, wherein the dose of the MALT1 inhibitor is 30 mg / kg.

36. 33. The use according to claim 32, wherein the dose of the MALT1 inhibitor is 5 mg / kg to 15 mg / kg.

37. The use according to claim 32 or 36, wherein the dose of the MALT1 inhibitor is 8 mg / kg to 12 mg / kg.

38. 37. The use of claim 32 or 36, wherein the dose of the MALT1 inhibitor is 10 mg / kg.

39. 33. The use according to claim 32, wherein the dose of the MALT1 inhibitor is 2 mg / kg to 10 mg / kg.

40. 40. The use according to claim 32 or 39, wherein the dose of the MALT1 inhibitor is 2 mg / kg to 5 mg / kg.

41. 40. The use of claim 32 or 39, wherein the dose of the MALT1 inhibitor is 3 mg / kg.

42. 33. The use according to claim 32, wherein the dose of the MALT1 inhibitor is 1 mg / kg to 5 mg / kg.

43. The use according to claim 32 or 42, wherein the dose of the MALT1 inhibitor is 1 mg / kg to 3 mg / kg.

44. 43. The use of claim 32 or 42, wherein the dose of the MALT1 inhibitor is 1 mg / kg.

45. The use of claim 32 or 42, wherein the pharmaceutical composition is administered intravenously.

46. ​​The use of claim 32 or 42, wherein the pharmaceutical composition is administered topically.

47. The use of claim 32, wherein the pharmaceutical composition is administered daily for 5 to 20 days.

48. The use of claim 32, wherein the pharmaceutical composition is administered daily for 5 to 8 days.

49. The use of claim 32, wherein the pharmaceutical composition is administered daily for 7 days.

50. The use of claim 32, wherein the pharmaceutical composition is administered daily for 10 to 15 days.

51. The use of claim 32, wherein the pharmaceutical composition is administered daily for 14 days.

52. The use of claim 32, wherein the pharmaceutical composition is administered over one or more cycles, a cycle comprising administering the pharmaceutical composition once daily for 1 to 2 weeks, followed by 1 to 2 weeks without treatment.

53. 53. The use of claim 52, wherein the cycle comprises administering the pharmaceutical composition once daily for two weeks, followed by one week of no treatment.

54. The use of claim 1, wherein the reduction in Treg levels in the subject with the chronic disorder after administration of the pharmaceutical composition is less than the reduction in Treg levels in healthy subjects given the MALT1 inhibitor.

55. 1. Use of a MALT1 inhibitor for the manufacture of a pharmaceutical composition for treating a chronic disorder, wherein the subject to whom the pharmaceutical composition is administered has previously been identified as having elevated IL-2 compared to a reference.

56. 1. Use of a MALT1 inhibitor for the manufacture of a pharmaceutical composition for treating a chronic disorder, wherein the subject to whom the pharmaceutical composition is administered has previously been identified as having IL-15 compared to a reference.

57. 1. Use of a MALT1 inhibitor for the manufacture of a pharmaceutical composition for treating a chronic disorder, wherein the subject to whom the pharmaceutical composition is administered has previously been identified as having elevated IL-7 compared to a reference.

58. Use of a MALT1 inhibitor for the manufacture of a pharmaceutical composition for treating a chronic disorder, the pharmaceutical composition comprising a MALT1 inhibitor in combination with a second agent comprising any one of IL-2, IL-15, and IL-7.

59. 59. The use of claim 58, wherein the second agent comprises IL-2, and the dose of IL-2 is between 10,000 international units (IU) and 50,000 international units (IU).

60. 59. The use of claim 58, wherein the second agent comprises IL-2, and the dose of IL-2 is between 20,000 international units (IU) and 40,000 international units (IU).

61. 59. The use of claim 58, wherein the second agent comprises IL-2 and the dose of the IL-2 is 30,000 international units (IU).

62. 59. The use of claim 58, wherein the second agent comprises IL-2, and the dose of the IL-2 is from 100,000 international units (IU) to 5 million international units (IU).

63. 59. The use of claim 58, wherein the second agent comprises IL-2, and the dose of the IL-2 is between 500,000 international units (IU) and 4.5 million international units (IU).

64. 59. The use of claim 58, wherein the second agent comprises IL-2, and the dose of the IL-2 is between 1 million international units (IU) and 4 million international units (IU).

65. 59. The use of claim 58, wherein the second agent comprises IL-2, and the dose of IL-2 is between 2 million international units (IU) and 3 million international units (IU).

66. 59. The use of claim 58, wherein the second agent comprises IL-2 and the dose of IL-2 is 3 million international units (IU).

67. 10. Use of a MALT1 inhibitor for the manufacture of a pharmaceutical composition for treating a chronic disorder, wherein the effectiveness of the MALT1 inhibitor is decoupled from its depleting effect on Tregs.

68. The use of claim 67, wherein the efficacy of the MALT1 inhibitor is represented by at least a 50% reduction in clinical score, and the depleting effect of the MALT1 inhibitor on Tregs is represented by at least a 40% reduction in Tregs.

69. 1. Use of a MALT1 inhibitor for the manufacture of a pharmaceutical composition for treating a chronic disorder, wherein after administration of the pharmaceutical composition to a subject, the subject's Treg level remains at least 60% of the level prior to administration.

70. 70. The use of claim 69, wherein after administration of the pharmaceutical composition to the subject, the level of Tregs in the subject remains at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the level prior to administration.

71. 2. The use according to claim 1, wherein the chronic disorder is graft-versus-host disease (GHVD).

72. 72. The use of claim 71, wherein the graft-versus-host disease (GHVD) is scleroderma GVHD (scGVHD).

73. 2. The use according to claim 1, wherein the chronic disorder is psoriatic arthritis.

74. 2. The use according to claim 1, wherein the chronic disorder is primary sclerosing cholangitis.

75. 2. The use according to claim 1, wherein the chronic disorder is multiple sclerosis.

76. 2. The use according to claim 1, wherein the chronic disorder is inflammatory bowel disease.

77. 77. The use of claim 76, wherein the inflammatory bowel disease is Crohn's disease.

78. 77. The use of claim 76, wherein the inflammatory bowel disease is ulcerative colitis.

79. 2. The use according to claim 1, wherein the chronic disorder is psoriasis.

80. 2. The use of claim 1, wherein the chronic disorder is lupus.

81. 2. The use according to claim 1, wherein the chronic disorder is Sjogren's syndrome.

82. The use according to claim 1, wherein the chronic disorder is scleritis.

83. 2. The use according to claim 1, wherein the chronic disorder is rheumatoid arthritis.

84. 2. The use according to claim 1, wherein the chronic disorder is delayed-type hypersensitivity.

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