Immunoadsorption therapy

Abstract

To provide further methods for treating autoimmune diseases and other lymphocyte mediated diseases.SOLUTION: A pharmaceutical composition comprising a glucocorticoid is used in treating a lymphocyte mediated disease in a subject. The treatment comprises administering a dose of the pharmaceutical composition to the subject to deliver the glucocorticoid at a dose equivalent to 6-26 mg / kg human equivalent dose (HED) of dexamethasone base. The pharmaceutical composition also comprises one or more pharmaceutically acceptable carriers, preservatives, and / or chelating agents. The lymphocyte mediated disease is selected from among an autoimmune disease, cancer, residual HIV disease, a graft versus host disease, and an allergic disorder.SELECTED DRAWING: Figure 11

Description

[Technical field]

[0001] The present invention relates to compositions for use in the treatment of diseases by immunoablation. In particular, the compositions of the present invention may be for use in the treatment of diseases mediated by immune cells such as lymphocytes. [Background technology]

[0002] The inventors have previously discovered that high concentrations of glucocorticoids can be used to condition patients to enhance the efficacy of cellular immunotherapy, such as adoptive T cell therapy. This is described in International Patent Application PCT / US2018 / 025517 (published as WO2018 / 183927). In that application, the inventors described the toxicity associated with chemotherapy and radiation-mediated preconditioning, which is believed to non-selectively destroy splenic cellularity. The inventors have provided glucocorticoids (a subclass of steroids) and other non-toxic lymphodepleting agents in acute doses to help cancer patients undergoing cellular immunotherapy.

[0003] WO2018 / 183927 describes that high doses of glucocorticoids can cause lymphoid tissue ablation and reduce binding of cellular immunotherapy to lymphoid tissue (particularly germinal centers and marginal zones in lymph nodes and germinal centers and marginal zones in the spleen). WO2018 / 183927 further describes that high doses of glucocorticoids also lymphodeplete peripheral blood lymphocytes via a biological mechanism (as opposed to the cytotoxic mechanism of preconditioning with chemotherapy or radiation therapy).

[0004] Previous studies that have examined the use of steroids to precondition patients prior to ACT have shown that this approach is ineffective. Hinrichs (J Immunother. 2005 Nov-Dec;28(6):517-24.) evaluated dexamethasone as a preconditioning treatment prior to ACT. Hinrichs demonstrated that compared with total body irradiation (TBI), HED of 0.8 mg / kg administered on days -6, -4, and -2 produced lymphodepletion equivalent to 5 Gy TBI. Hinrichs demonstrated that pretreatment with intraperitoneal systemic dexamethasone at 10 mg / kg (HED 0.81 mg / kg) on ​​days -6, -4, and -2 prior to ACT induced equivalent lymphodepletion compared to radiation, but this pretreatment did not enhance tumor killing by ACT. Hinrichs, in contrast, discloses that radiation pretreatment enhanced tumor killing by ACT. In the Hinrichs paper, dexamethasone was said to cause lymphodepletion of the spleen as shown by a 99% reduction in splenic cellularity. However, while Hinrichs reported 99% lymphodepletion, no enhancement of tumor killing by ACT was observed. Hinrichs, in contrast, observes that radiation enhances tumor killing by ACT. Experiments to replicate the lymphodepletion reported by Hinrichs, however, demonstrate that the Hinrichs dose of 10 mg / kg (HED 0.81 mg / kg) intraperitoneal dexamethasone on days -6, -4, and -2 does not effectively lymphodeplete peripheral blood lymphocytes. At the Hinrichs dose, only peripheral blood B lymphocytes were significantly lymphodepleted, a 65% reduction in live events measured by flow cytometry in CD3-CD19+ cells, from 10680 (vehicle control) to 3733. In contrast, CD3+ T lymphocytes were reduced from 3370 to 2441 live events, a non-significant 33% reduction.CD3+CD4+ T lymphocytes decreased from 1779 to 902 surviving events, a non-significant 50% decrease; CD3+CD8_ T lymphocytes decreased from 1318 to 1277 surviving events, a non-significant 3% decrease; CD3+CD4+CD25+FoxP3+ Tregs decreased from 198 to 70 surviving events, a non-significant 65% decrease; and natural killer (NK) cells decreased from 1153 to 958 surviving events, a non-significant 17% decrease. ***

[0005] Autoimmunity is a phenomenon in which the immune system launches an abnormal attack against the subject's own components. (In healthy individuals, the immune system avoids harmful autoimmune reactions by establishing tolerance to the subject's own components.) Diseases resulting from harmful autoimmune reactions are called autoimmune diseases. Different autoimmune diseases affect different parts of the body. They can be debilitating (e.g., in the case of rheumatoid arthritis, which affects the joints), neurodegenerative / neurodestructive (e.g., in the case of multiple sclerosis), and in some cases, such as diabetes, are associated with significant mortality (Thomas et al., 2010).

[0006] The pathogenesis of autoimmune disorders is broadly explained by the crucial role of T and B lymphocytes in inappropriately recognizing self-antigens and initiating cell-mediated and / or humoral responses, which result in inflammatory tissue and vascular damage (Sullivan et al., 2010; Shlomchik et al., 2001).

[0007] Autoimmune diseases are very often treated with long-term administration of immunosuppressants such as steroids. For example, pemphigus patients have been treated with 100 mg dexamethasone by 2-hour IV infusion daily for 3 days (Pasricha et al., 2008). This dose is not lymphodepleting. Pemphigus patients were treated in this manner every 28 days until cured, which took 3-12 months. The relapse rate was 15% and all patients went into remission with further dexamethasone (Dexa) treatment. This dose of Dexa is approximately 1-2 mg / kg. Although it helps manage autoimmune diseases and reduce symptoms, such treatment regimens are not curative and are associated with several long-term side effects and increased risk of infection (Patt et al., 2013).

[0008] To control immune damage, lymphodepleting therapies are increasingly being tested. One attractive premise for such therapies is that they can “reboot” the immune system and restore immune tolerance (Lu et al., 2011). However, the tolerance-acquiring ability of lymphodepleting therapies is still debated. The debate is exemplified by the conflicting evidence from multiple studies on anti-thymocyte globulin (ATG), a prototype immune depleting drug, especially on whether it induces CD4+ CD25+ Foxp3+ regulatory T (Treg) cells (Lu et al., 2011). To understand the impact of ATG on T cells at the clonal level in vivo, Lu et al. investigated the effect of anti-mouse thymocyte globulin (mATG) in a reductionist model in which the T lymphocyte repertoire consists of a single clone of pathological T effector (Teff) cells specific for a physiological self-antigen. mATG treatment led to the peripheral induction of antigen-specific Treg cells from the primarily monoclonal Teff repertoire, independent of thymic involvement. This de novo Treg cell induction consistently occurred in local draining lymph nodes, and persistence of induced Treg cells in the blood correlated with long-term protection from autoimmune destruction (Lu et al., 2011), thus providing in vivo evidence of clonal conversion of pathological autoantigen-specific Teff cells to Treg cells in the setting of immune depletion therapy. ***

[0009] Type 1 diabetes (T1D) is an autoimmune disease that progressively leads to the exhaustion of insulin-secreting β-cells and ultimately to clinically significant hyperglycemia and metabolic instability (Atkinson et al., 2014). Overall, T1D accounts for approximately 5% of diabetes cases and affects approximately 20 million people worldwide (Menke et al., 2013). Approximately 1.25 million Americans have T1D, and an estimated 40,000 new cases are diagnosed in the United States each year (American Diabetes Association, Diabetes Care 37, 2014). T1D is associated with an annual economic burden of $14.4 billion in the United States, taking into account indirect costs such as medical expenses and lost income.

[0010] Therapeutic insulin and other treatments based on exogenous hypoglycemic agents do not cure T1D but merely provide a solution to manage blood glucose levels, leaving patients with variable blood glucose levels and susceptible to the development of micro- and macrovascular diabetic complications (Peng et al., 2018).

[0011] Safe therapeutic interventions to remove autoimmune substrates from diabetic patients are lacking. Autoimmunity in T1D involves a multi-pronged immune response (Snarski et al., 2016; Cantu-Rodriguez et al., 2016). Thus, antigen-specific immunotherapy based on the use of antibodies, fusion proteins, cytokines, regulatory T cells, and small molecule inhibitors only results in some degree of β-cell preservation and blood glucose level reduction in patients with T1D (Kim et al., 2013). Even in combination, immunotherapy targeting specific components of the autoimmune repertoire could not guarantee the restoration of insulin independence (Bone et al., 2017).

[0012] Autologous hematopoietic stem cell transplantation (HSCT) is the only strategy proven to cure T1D so far (Voltarelli et al., 2007). For the last 12 years, autologous HSCT has been used as a therapeutic option for autoimmune diseases (AD) such as multiple sclerosis, systemic sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease and others (Swart et al., 2017). A more powerful and broader immunological approach consists in the "immune reset" performed with high-dose immunosuppression, which includes nonspecific suppression of autoreactive T- and B-cell responses, followed by hematopoietic stem cell transplantation to reconstitute a tolerant immune system. Remarkably, in clinical trials, this approach has allowed up to 80% of T1D patients to experience a period of insulin independence and, in parallel, an increase in C-peptide levels in a mixed meal challenge (Couri et al., 2018). However, the adoption of immune resetting as a therapeutic approach for T1D is hampered by serious concerns.

[0013] The risks associated with HSCT procedures outweigh the positive benefits they provide for T1D. HSCT remains associated with significant toxicity and mortality rates up to 3% (Alexander et al., 2018; Pallera et al., 2004; Henig et al., 2014). Moreover, current protocols for immune reset are based on cytotoxic immunosuppressive regimens (e.g. chemotherapy, radiotherapy), which expose patients to a series of safety issues, including short-term risks of infection, acute organ failure and death, as well as long-term risks of malignancies and secondary autoimmune diseases (Daikeler et al., 2012).

[0014] Almost all T1D patients treated with HSCT resumed exogenous insulin use as an effect of incomplete removal of the autoimmune pathophysiological substrate after preconditioning (Loh et al., 2007), with a subsequent decrease in C-peptide levels (Magdalena et al., 2018). Increasing the intensity of transplant conditioning regimens or repeating the procedure to improve outcomes would expose patients to excessive risks and toxicities (Couri et al., 2018).

[0015] HSCT is associated with high costs, ranging from approximately US$80,000 to US$300,000, depending on the conditioning regimen administered prior to HSCT, the type of transplant, and the hospital costs associated with inpatient care (Broder et al., 2017). *** Summary of the Invention [Problem to be solved by the invention]

[0016] There is a need for additional treatments for autoimmune disorders and other lymphocyte mediated diseases. Additional treatments that are simpler and less costly than HSCT would be desirable. [Means for solving the problem]

[0017] The present invention is based on the surprising discovery that high doses of glucocorticoids can act to cause lymphodepletion of peripheral blood lymphocytes without substantially affecting the cell count of other cells. Additional effects such as ablation of germinal centers also support certain aspects of the invention. The present invention provides a medical application of these effects of high dose glucocorticoid agonists for use in the treatment of lymphocyte-mediated diseases.

[0018] Thus, in a first aspect, the present invention provides a pharmaceutical composition comprising a glucocorticoid for use in treating a lymphocyte-mediated disease in a subject, the treatment comprising administering a dose of the pharmaceutical composition that delivers the glucocorticoid to the patient at a dose equivalent to about 3-26 mg / kg human equivalent dose (HED) of dexamethasone base. This dose of glucocorticoid may be referred to as an "acute high dose". In some embodiments, the dose of the pharmaceutical composition to the patient delivers the glucocorticoid at a dose equivalent to about 10-26 mg / kg or about 12-26 mg / kg human equivalent dose (HED) of dexamethasone base. The pharmaceutical composition may (or may not) comprise a pharmaceutically acceptable carrier, as defined herein. The pharmaceutical composition may (or may not) comprise a pharmaceutically acceptable preservative, as defined herein. The pharmaceutical composition may or may not include a pharma- ceutically acceptable chelating agent, as defined herein. However, in all embodiments of this aspect, the pharmaceutical composition includes one or more components selected from the group consisting of a pharma- ceutically acceptable carrier, a preservative, and / or a chelating agent. The pharmaceutical composition may also include an excipient in some embodiments. In some embodiments, the pharmaceutical composition includes more than one pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition includes more than one pharma- ceutically acceptable preservative. In some embodiments, the pharmaceutical composition includes more than one pharma- ceutically acceptable chelating agent. An embodiment of the invention may be defined as acting to achieve systemic lymphodepletion in a subject.

[0019] In some embodiments, the lymphocyte-mediated disease is an autoimmune disease, such as an autoimmune disease selected from the group consisting of type 1 diabetes, multiple sclerosis, amyotrophic lateral sclerosis, scleroderma, pemphigus, and lupus. The lymphodepleting effect of the present invention underpins the efficacy of these embodiments.

[0020] Although glucocorticoids have established uses in many autoimmune conditions (Flammer et al., 2011), they have never been considered for immune resetting. Furthermore, studies based on the use of low pharmaceutical doses to precondition patients prior to autologous cell transplantation have shown that this approach is ineffective (Medicines Agency; 2017). The complex mode of action of the pharmaceutical composition of the present invention, based on multiple in vivo effects, provides the first effective alternative to chemotherapy that can be used as a safe immune resetting regimen for the treatment of autoimmune conditions such as diabetes.

[0021] Several advantages are associated with the present invention with respect to the action of the pharmaceutical composition, including: (i) Non-myeloablative immune reset: The pharmaceutical composition can deplete all types of peripheral blood lymphocytes, including, for example, islet-specific autoreactive T cells responsible for diabetic autoimmunity, but spares neutrophils, platelets, RBCs, and stem cells (both HSCs and MSCs) based on a specific receptor-mediated mode of action. The present invention thus reduces the risk of infection and eliminates the need for HSCT to restore blood cells after immune reset. The result is a non-myeloablative regimen that can perform a safe immune reset with efficacy comparable to chemotherapy. (ii) Reduction of germinal centers (GCs) and marginal zones in secondary lymphoid tissues. The pharmaceutical composition temporarily eliminates germinal centers (DeFranco et al., 2016) in secondary lymphoid organs that generate anti-affinity antibodies and long-lived plasma cells that increase efficacy against autoimmune pathophysiological substrates. (iii) Simple mode of administration. The pharmaceutical composition can be formulated for oral or intravenous administration routes and is effective within hours, with lymphocyte and GC recovery occurring within 7-14 days. This is the first time that complete lymphodepletion does not require hospitalization. (iv) Reduced chance of relapse. Unlike chemotherapy or radiation, the pharmaceutical composition of the present invention can be safely administered in a complete lymphodepleting dose to eliminate memory T and B cells that cause relapse. (v) Tolerability of re-administration. The safety profile of high dose glucocorticoids allows repeated administration of the pharmaceutical composition of the present invention in the event of relapse of the autoimmune pathophysiological substrate.

[0022] These effects and advantages associated with the present invention as disclosed herein mean that one of skill in the art will appreciate that the present invention provides an effective strategy for treating autoimmune diseases and other lymphocyte-mediated diseases discussed herein.

[0023] In some embodiments, the lymphocyte-mediated disease is residual HIV disease. In these embodiments, as described herein, the reduction in the number of germinal centers in lymphoid organs of the subject pushes the residual HIV-infected T cells that bind to the niches in these centers into the circulation, where they can be eliminated by the immune system or standard therapy. In the context of this disclosure, those skilled in the art will understand that HIV is a lymphocyte-mediated disease in the sense that the virus infects T lymphocytes. The lymphocyte-depleting effect of the present invention also contributes to the efficacy of these embodiments.

[0024] In other embodiments, the lymphocyte-mediated disease is a lymphoma, such as germinal center lymphoma (GC lymphoma) or marginal zone lymphoma. In these embodiments, as described herein, the reduction in the number of germinal centers in the lymphoid organs of the subject pushes cancer cells that bind to the niche in these centers (e.g., germinal center lymphoma) into the circulation, where they can be eliminated by the immune system or standard therapy. Those skilled in the art will recognize that standard cancer therapy includes, for example, chemotherapy. Thus, the glucocorticoid-based therapy described herein may be used in combination with chemotherapy, preferably with reduced intensity cytotoxic chemotherapy (wherein the effective dose of chemotherapy is less when used in combination with the high-dose glucocorticoid-based therapy described herein than the effective dose of the same chemotherapy not combined with the high-dose glucocorticoid described herein). The lymphodepleting effect of the present invention also contributes to the efficacy of these embodiments. In certain embodiments, the treatment of Burkitt's Lymphoma (BL) is specifically contemplated. In Africa, the treatment of BL is centered on a combination of three chemotherapy drugs (systemic and intrathecal): cyclophosphamide, vincristine, and methotrexate. This combination is repeated at two-week intervals for a total of six cycles of 12 weeks (Burkitt's Lymphoma National Treatment Guidelines. 2009). Lower doses of dexamethasone are currently on the WHO list of essential medicines; however, existing WHO-listed dexamethasone products are not suitable for BL treatment because the high doses of the present invention require compounding vials that can lead to contamination and serious or fatal infections in patients, and compounding can also result in excipients such as benzyl alcohol or parabens reaching toxic levels.

[0025] In yet another embodiment, the lymphocyte-mediated disease is graft versus host disease (GvHD). GvHD is a medical complication after receiving transplanted tissue from a genetically different individual. GvHD can occur even in the case of autologous grafts, but it is more likely caused by the processes and storage of autologous cells that cause the transplanted cells to recognize the body as foreign. In GvHD, the white blood cells of the donor's immune system that remain in the donated tissue (graft) recognize the recipient (host) as foreign (non-self). The white blood cells present in the transplanted tissue then attack the recipient's body's cells, resulting in the disease state. GvHD is commonly associated with stem cell transplants, such as those that occur in bone marrow transplants. GvHD also applies to other forms of transplanted tissue, such as solid organ transplants. The lymphodepleting effect of the present invention also contributes to the efficacy of these embodiments.

[0026] In another embodiment, the lymphocyte-mediated disease is allergic disorder.This includes chronic allergy and acute allergy.For example, the pharmaceutical composition of the present invention can be used in the treatment of asthma.The lymphocyte depletion effect of the present invention also contributes to the efficacy of these embodiments.

[0027] In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a preservative and / or a chelating agent. In some embodiments, the pharmaceutical composition comprises a preservative. Preferably, the preservative is a sulfite. In some embodiments, the pharmaceutical composition comprises a chelating agent, which may be EDTA.

[0028] In a preferred embodiment, the glucocorticoid of the pharmaceutical composition comprises dexamethasone. This may be in the form of dexamethasone base, dexamethasone sodium phosphate, or dexamethasone acetate. Most preferably, the glucocorticoid is dexamethasone sodium phosphate.

[0029] As described above and as defined by the claims, the pharmaceutical composition of the present invention is for use in the treatment of lymphocyte-mediated diseases. The treatment may comprise administering a dose of the pharmaceutical composition as a single acute dose. Alternatively, the treatment comprises administering a dose of the pharmaceutical composition as a total dose administered over a period of about 72 hours.

[0030] Treatment of lymphocyte-mediated diseases includes administration of the composition to a patient in need of anti-inflammation, immunosuppression, lymphocyte depletion, germinal center depletion, elevation of IL-2, IL-7, IL-12 and / or IL-15, elevation of mesenchymal stem cells, elevation of G-CSF, or neutrophilia. Additionally, treatment of lymphocyte-mediated diseases may result in a detectable change in expression of PD-1 or PD-L1 or CTLA-4.

[0031] As described above and as defined by the claims, the pharmaceutical composition of the present invention is for use in the treatment of lymphocyte-mediated diseases, the treatment comprising administering a dose of the pharmaceutical composition to a patient. The pharmaceutical composition may be administered intravenously (IV) or orally. When administered intravenously, preferably the dose is administered as a single IV infusion over 0.25 to 2 hours. The composition to be infused may be in saline or half saline, or lactated Ringer's solution or 5% dextrose or other standard IV solutions. For oral administration, the composition may be provided as a single oral dose mixed with a small amount of juice or sweetener.

[0032] In preferred embodiments, the pharmaceutical compositions are provided as aqueous solutions of the glucocorticoid, which one of skill in the art will understand means that the pharmaceutical compositions of these embodiments use water as a solvent.

[0033] Pharmaceutical compositions for use according to the present invention are administered to deliver a dose of glucocorticoid equivalent to a human equivalent dose (HED) of dexamethasone base of at least about 3 mg / kg, at least about 4 mg / kg, at least about 5 mg / kg, at least about 6 mg / kg, at least about 7 mg / kg, at least about 8 mg / kg, at least about 9 mg / kg, at least about 10 mg / kg, at least about 11 mg / kg, at least about 12 mg / kg, or at least about 13 mg / kg, or at least about 14 mg / kg, or at least about 15 mg / kg, or at least about 16 mg / kg, or at least about 17 mg / kg, or at least about 18 mg / kg, or at least about 19 mg / kg, or at least about 20 mg / kg, or at least about 21 mg / kg, or at least about 22 mg / kg, or at least about 23 mg / kg, or at least about 24 mg / kg, or at least about 25 mg / kg, or at least about 26 mg / kg. A dose of the pharmaceutical composition may be defined as delivering a glucocorticoid at a dose equivalent to a value taken from a "range" of doses equivalent to the HED of dexamethasone base, where the range is defined by an endpoint selected from the list of values ​​above, e.g., about 10 mg / kg to 26 mg / kg, or about 15 mg / kg to 25 mg / kg (or any two values ​​from the list above). In a preferred embodiment, the subject is a human, the glucocorticoid comprises dexamethasone base, and the pharmaceutical composition is administered to the human subject at a dose of about 3.0 to about 18.0 mg / kg of dexamethasone base.

[0034] Those skilled in the art understand that the lymphodepletion achieved by the present invention can be measured using conventional techniques.For example, after the pharmaceutical composition is administered (e.g., 48 hours after administration), the population of CD4+, CD8+, Treg and / or B cells can be measured.Flow cytometry is one typical method that can be used to perform cell counting.

[0035] Those skilled in the art will appreciate that the present invention may be used with other therapeutic approaches as described herein, for example, with chemotherapy and / or cell-based therapy. In these embodiments, the subject may be administered chemotherapy. In these embodiments, the subject may be administered cell-based therapy. However, most embodiments of the present invention do not involve chemotherapy or cell-based therapy. That is, in some embodiments, the subject is not administered chemotherapy. In some embodiments, the subject is not administered cell-based therapy.

[0036] The mechanisms of action of the present invention are discussed in detail herein and in some instances these mechanisms may form part of the distinguishing features of the invention, particularly where they open up new clinical situations (e.g. by allowing the targeting of subgroups of patients).

[0037] BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present invention will now be discussed with reference to the accompanying drawings, in which: FIG. [Brief description of the drawings]

[0038] [Figure 1] Acute high dose dexamethasone eliminates connective niches in mouse spleen and secondary lymphoid tissues. Black and white scale bright field (top) and immunofluorescence (bottom) images of fresh spleen thick sections stained with FITC-PNA to quantify germinal centers from mice administered IP human equivalent dose (HED) 9.3 mg kg dexamethasone base 96 hours prior to splenectomy are shown. Graph shows a bar graph of the mean number of germinal centers per spleen area versus standard error of the mean (SEM) for IP placebo control and mice administered IP HED 9.3 mg kg dexamethasone base (Dex) 96 hours prior to splenectomy. Control mice have significant FITC-PNA immunofluorescence, while mice injected with dexamethasone have almost no immunofluorescent signal.

[0039] [Diagram 2]Acute high-dose dexamethasone dose-dependently ablates the connective niche in mouse spleen. Bar graphs of mean germinal center staining intensity measured using immunofluorescence staining of fresh spleen thick sections stained with FITC-PNA are shown. Immunofluorescence intensity was calculated using thresholding and MetaMorph image analysis. Bar graphs are mean plus SEM. Mice were administered placebo or 3 mg / kg HED, 6 mg / kg HED, 9 mg / kg HED, or 12 mg / kg HED of dexamethasone base 48 hours prior to splenectomy. Reduction of germinal centers is evident at 6 mg / kg HED, with marked reduction at 9 and 12 mg / kg HED doses.

[0040] [Diagram 3] Acute high-dose dexamethasone eliminates the connective niche in the rat spleen (MZ: marginal zone). Bar fluff of marginal zone width measured in 5 micron spleen sections from rats treated IV or PO with placebo, or 20 mg / kg (HED 3.23 mg / kg), 40 mg / kg (HED 6.45 mg / kg), or 80 mg / kg (HED 12.9 mg / kg) dexamethasone base 48 hours prior to splenectomy is shown. Marginal zone area was reduced at all dexamethasone doses, with maximal inhibition achieved at 12.9 mg / kg HED. n = 5 per group. *p<0.05 ANOVA (Dunnett's post-hoc test) vs. vehicle IV; †p<0.05 ANOVA (Dunnett's post-hoc test) vs. vehicle PO; ‡p<0.05 Student's t-test vs. vehicle IV.

[0041] [Figure 4]Acute high-dose dexamethasone eliminates the connective niche in rat spleens. A bar graph of the area per spleen of 5 micron fixed spleen sections stained for BCL-6 as a measure of germinal center number given as the average per section is shown. Rats were treated IV or PO with placebo or 20 mg / kg (HED 3.23 mg / kg), 40 mg / kg (HED 6.45) or 80 mg / kg (HED 12.9 mg / kg) dexamethasone base 48 hours prior to splenectomy. Germinal center area was reduced at all dexamethasone doses, with maximal inhibition obtained at 12.9 mg / kg HED. Groups 1-4 IV: 1 = 20 mg / kg (HED 3.23 mg / kg), 2 = 40 mg / kg (HED 6.45 mg / kg), 3 = 80 mg / kg (HED 12.9 mg / kg), 4 = placebo. Groups 5-9 PO: 5 = 20 mg / kg (HED 3.23 mg / kg), 6 = 40 mg / kg (HED 6.45 mg / kg), 7 = 80 mg / kg (HED 12.9 mg / kg), 8 = placebo.

[0042] [Diagram 5] Acute high dose dexamethasone reduces thymus mass. The photographs show the size of the thymus from a placebo-treated mouse subject (top photograph) and a mouse subject treated with a 6 mg / kg HED dose of the pharmaceutical composition of the present invention (bottom photograph). The bottom panel shows the percentage of thymus weight to body weight for the placebo-treated subject (control) and subjects treated with the pharmaceutical composition of the present invention at 3 mg / kg HED, 6 mg / kg HED, 9 mg / kg HED and 12 mg / kg HED.

[0043] [Figure 6]Acute high-dose dexamethasone reduces lymphocyte counts in rats. Graphs of individual absolute and mean lymphocyte counts measured by complete blood count 48 hours after rats were treated IV (right) or PO (left) with placebo or 20 mg / kg (HED 3.23 mg / kg), 40 mg / kg (HED 6.45) or 80 mg / kg (HED 12.9 mg / kg) dexamethasone base. Dexamethasone was administered 48 hours prior to blood collection. Significant lymphodepletion was observed in rats at all doses compared to controls, regardless of whether they were administered IV (right) or orally (left). Doses are shown as HEDs (human equivalent doses).

[0044] [Figure 7] Acute high-dose dexamethasone does not reduce neutrophil counts in rats. Graphs of individual absolute and mean neutrophil counts measured by complete blood count 48 hours after rats were treated IV (right) or PO (left) with placebo or 20 mg / kg (HED 3.23 mg / kg), 40 mg / kg (HED 6.45) or 80 mg / kg (HED 12.9 mg / kg) dexamethasone base are shown. Data in Figures 3, 46 are from the same rats. Acute high-dose dexamethasone has a lymphodepleting profile that spares neutrophils. Oral (left) and IV (right) doses were administered once 48 hours prior to blood collection. Doses are shown as HED (human equivalent dose).

[0045] [Figure 8]CD3 and CD4 positive lymphocytes. Graphs of individual CD3+ (left) and CD4+ (right) lymphocytes and mean numbers measured as relative counts by flow cytometry and normalized to relative absolute counts using complete blood counts 48 hours after mice were treated PO with placebo or HED 3 mg / kg, HED 6 mg / kg, HED 9 mg / kg, or HED 12.mg / kg dexamethasone base. Relative counts / ul = combined flow cytometry and complete blood count. Compared to controls, there was a 65% reduction in CD3+ cells and a 75% reduction in CD4+ cells in the 12 mg / kg group. Doses are shown as HED (human equivalent dose). One-way ANOVA followed by Tukey's test was incorporated to determine statistically significant differences between treatment groups; * p<0.05, ** p<0.01, *** p<0.001.

[0046] [Figure 9] Acute high-dose dexamethasone reduces CD8 positive lymphocytes and Tregs in mice. Graphs of individual CD8+ (left) and Treg (right) lymphocytes and mean numbers measured as relative counts by flow cytometry and normalized to relative absolute counts using complete blood counts 48 hours after mice were treated PO with placebo or HED 3 mg / kg, HED 6 mg / kg, HED 9 mg / kg, or HED 12.mg / kg dexamethasone base. Treg lymphocytes were identified by being CD3+CD4+CD25+FoxP3+. Relative counts / ul = combined flow cytometry and complete blood count. There was a 56% reduction in CD8+ cells and a 78% reduction in mouse Tregs in the 12 mg / kg group compared to controls. Doses are shown as HED (human equivalent dose). One-way ANOVA followed by Tukey's post-hoc test was used to determine statistically significant differences between treatment groups; * p<0.05, ** p<0.01, *** p<0.001.

[0047] [Figure 10]Acute high-dose dexamethasone reduces NK cells and B lymphocytes in mice. Graphs of individual natural killer (NK) cells (left) and B lymphocytes (right) and mean numbers measured as relative counts by flow cytometry and normalized to relative absolute counts using complete blood counts 48 hours after mice were treated PO with placebo or HED 3 mg / kg, HED 6 mg / kg, HED 9 mg / kg, or HED 12.mg / kg dexamethasone base. NK cells were identified by being CD3-CD49b+. B lymphocytes were identified by being CD3-B220+. Relative counts / ul = flow cytometry and complete blood count combined. There was an 87% reduction in NK cells and an 83% reduction in B lymphocytes in the 12 mg / kg group compared to controls. Doses are shown as HED (human equivalent dose). One-way ANOVA followed by Tukey's post-hoc test was used to determine statistically significant differences between treatment groups; * p<0.05, ** p<0.01, *** p<0.001.

[0048] [Figure 11] Acute high-dose dexamethasone reduces absolute lymphocyte counts but spares neutrophils in mice. Graphs of individual absolute neutrophil counts (left) and total lymphocyte counts (right) and mean counts measured by complete blood count 24-48 hours after mice were treated PO with placebo or HED 3 mg / kg, HED 6 mg / kg, HED 9 mg / kg, HED 12 mg / kg, or HED 17.5 mg / kg dexamethasone base are shown. Cells / ul = absolute counts obtained from complete blood counts (CBC). Acute high-dose dexamethasone causes near complete lymphodepletion at HED doses above 12 mg / kg, but does not affect neutrophils. Acute high-dose dexamethasone thus eliminates the need for transfusions and offers a safer, non-toxic alternative to chemotherapy regimens. Doses are shown as HED (human equivalent dose).

[0049] [Figure 12]Acute high-dose dexamethasone spares RBCs and platelets in mice. Graphs of individual RBC (left) and platelet (right) absolute and mean counts measured by complete blood count 24-48 hours after mice were treated PO with placebo or HED 3 mg / kg, HED 6 mg / kg, HED 9 mg / kg, HED 12.mg / kg, or HED 17.5 mg / kg dexamethasone base are shown. Cells / ul = absolute counts obtained from CBC. Acute high-dose dexamethasone does not affect RBCs and platelets and eliminates the need for transfusions, thus providing a safer, non-toxic alternative to chemotherapy regimens. Doses are shown as HED (human equivalent dose).

[0050] [Figure 13] The number of viable hematopoietic stem cells is measured 48 hours after naive mice are treated with placebo (vehicle) or acute high-dose dexamethasone at low or high doses.Even high doses of acute high-dose dexamethasone do not significantly change the number of viable hematopoietic stem cells.This non-myeloablative regimen represented by acute high-dose dexamethasone can therefore eliminate the need for stem cell infusion for hematopoietic recovery after immune reset.

[0051] [Figure 14] Fifty percent (2 out of 4) of human patients treated with 3 mg / kg dexamethasone base were depleted of CD3, CD4, and CD8 positive lymphocytes. Shown are the individual values ​​and line plots of CD3+, CD4+, and CD8+ lymphocytes measured by flow cytometry pre-treatment and 48 hours after oral administration of 3 mg / kg dexamethasone base to 4 human patients. Pre-treatment values ​​for each patient are connected to post-treatment values ​​by a connecting line. CD4+ cells are also CD3+. CD8+ cells are also CD3+.

[0052] [Figure 15]Twenty-five percent (1 in 4) of human patients treated with 3 mg / kg dexamethasone base were depleted of Tregs and B lymphocytes. Lines are individual values ​​and line plots of Tregs and B lymphocytes measured by flow cytometry before and 48 hours after oral administration of 3 mg / kg dexamethasone base to four human patients. Pre-treatment values ​​for each patient are linked to post-treatment values ​​by connecting lines. Tregs are identified by being CD3+CD4+CD25+FoxP3+. B lymphocytes are identified by being CD3-CD19+.

[0053] [Figure 16] Seventy-five percent (3 of 4) of human patients treated with 3 mg / kg dexamethasone base depleted NK cells while sparing hematopoietic stem cells. Lines are individual values ​​and line plots of NK cells and hematopoietic stem cells (HSCs) measured by flow cytometry pre-treatment and 48 hours post-treatment of 4 human patients with oral administration of 3 mg / kg dexamethasone base. Pre-treatment values ​​for each patient are linked to post-treatment values ​​by connecting lines. NK cells are identified by being CD3-CD16 / 56+. HSCs are identified by being CD34+CD38-.

[0054] [Figure 17] One hundred percent of human patients treated with 3 mg / kg dexamethasone base showed increased serum IL-2 and / or IL-15 levels, but no elevation of IL-6. Column plots for each patient of plasma levels of interleukin-2 and interleukin-15 measured by ProCartaPlex-9plx Luminex assay before (Pre) and 48 hours (Post) oral administration of 3 mg / kg dexamethasone base to four human patients. Figures 14, 15, 16 and 17 show data from the same four human patients.

[0055] [Figure 18] Oral administration of 3 mg / kg dexamethasone base increased bone marrow MSC numbers after 48 hours. Column plots and standard deviations of data from 31 historical untreated control humans and from 2 human patients treated with 3 mg / kg dexamethasone base 48 hours prior to aspirating concentrated bone marrow from the iliac crest using a MarrowCellution™ needle. Plots show bone marrow CFU / ml + / - standard deviation. Bone marrow was added directly to colony forming unit assay fibroblast (CFU-F) medium without further manipulation 24 hours after collection and shipping at controlled room temperature. CFU-F colony counts are a measure of mesenchymal stem cell (MSC) numbers in the starting material. 48 hours after oral administration of 3 mg / kg dexamethasone base, ileac crest bone marrow MSC numbers appear to be approximately 2-fold higher than 31 historical controls. Oral dexamethasone base at 3 mg / kg increases human bone marrow CFU-F per ml after 48 hours compared with 31 historical controls whose aspirates were performed using a MarrowCellution™ needle in the same manner as patients M and P.

[0056] [Figure 19]FIG. 1 compares oral doses of 12 mg / kg and 17-18 mg / kg dexamethasone base on day -2 with a single dose of cyclophosphamide 166 mg / kg (HED 500 mg / m2) and fludarabine 10 mg / kg on day -5 combined with 12 mg / kg or 17-18 mg / kg dexamethasone base on day -2, and 2 days of repeat cyclophosphamide 166 mg / kg on days -5 and -4 and 4 days of fludarabine 10 mg / kg (HED 30 mg / m2) on days -5, -4, -3, -2. Mice were treated IP with PBS (vehicle), or with repeated IP cyclophosphamide 166 mg / kg on days -5 and -4 and IP fludarabine 10 mg / kg (HED 30 mg / m2) on days -5, -4, -3, -2 (Flu+Cy), or with a single IP dose of cyclophosphamide 166 mg / kg (HED 500 mg / m2) and IP fludarabine 10 mg / kg, both on day -5, followed by oral dexamethasone base at 12 mg / kg or 17-18 mg / kg on day -2 (Flu+Cy + AVM0703(12 mg / kg); Flu+Cy + AVM0703(17 mg / kg)), or with oral dexamethasone base at 12 mg / kg or 17-18 mg / kg (AVM0703(12 mg / kg)). Absolute and mean individual lymphocyte counts (left) as measured by complete blood count 48 hours after treatment with either 100 mg / kg AVM0703 (17 mg / kg); or 17 mg / kg AVM0703 (17 mg / kg). Also shown is a representation of the dosing schedule in mice for these regimens (right).

[0057] [Figure 20]A single dose of cyclophosphamide 166 mg / kg (HED 500 mg / m2) and fludarabine 10 mg / kg on day -5 combined with 12 mg / kg or 17 to 18 mg / kg dexamethasone base on day -2 lymphodepleted CD3+ and CD4+ lymphocytes comparably compared with 2 days of repeat cyclophosphamide 166 mg / kg on days -5 and -4 and 4 days of fludarabine 10 mg / kg (HED 30 mg / m2) on days -5, -4, -3, and -2. Mice were treated IP with PBS (vehicle), or with repeated IP cyclophosphamide 166 mg / kg on days -5 and -4 and 4 days of IP fludarabine 10 mg / kg (HED 30 mg / m2) on days -5, -4, -3, -2 (Flu+Cy), or with a single IP dose of cyclophosphamide 166 mg / kg (HED 500 mg / m2) and IP fludarabine 10 mg / kg, both on day -5, followed by oral dexamethasone base at 12 mg / kg or 17-18 mg / kg on day -2 (Flu+Cy + AVM0703(12 mg / kg); Flu+Cy + AVM0703(17 mg / kg)), or with oral dexamethasone base at 12 mg / kg or 17-18 mg / kg (AVM0703(12 mg / kg)). Graphs of individual CD3+ (left) and CD4+ (right) lymphocyte counts and mean counts measured as relative counts by flow cytometry 48 hours after treatment with AVM0703 (17 mg / kg); AVM0703 (17 mg / kg) and normalized to relative absolute counts using complete blood counts are shown. In both the CD3+ plot (left) and CD4+ plot (right), data for 12 mg / kg or 17-18 mg / kg dexamethasone base are shown in the respective right-hand columns (relative counts of 92, 71, 37, and 25).

[0058] [Figure 21]A single dose of cyclophosphamide 166 mg / kg (HED 500 mg / m2) and fludarabine 10 mg / kg on day -5 combined with 12 mg / kg or 17-18 mg / kg dexamethasone base on day -2 lymphodepleted CD8+ lymphocytes and Tregs comparably compared with 2 days of repeat cyclophosphamide 166 mg / kg on days -5 and -4 and 4 days of fludarabine 10 mg / kg (HED 30 mg / m2) on days -5, -4, -3, and -2. Mice were treated IP with PBS (vehicle), or with repeated IP cyclophosphamide 166 mg / kg on days -5 and -4 and 4 days of IP fludarabine 10 mg / kg (HED 30 mg / m2) on days -5, -4, -3, -2 (Flu+Cy), or with a single IP dose of cyclophosphamide 166 mg / kg (HED 500 mg / m2) and IP fludarabine 10 mg / kg, both on day -5, followed by oral dexamethasone base at 12 mg / kg or 17-18 mg / kg on day -2 (Flu+Cy + AVM0703(12 mg / kg); Flu+Cy + AVM0703(17 mg / kg)), or with oral dexamethasone base at 12 mg / kg or 17-18 mg / kg (AVM0703(12 mg / kg)). Graphs of individual Treg (right) and CD8+ lymphocyte (left) counts and mean counts measured as relative counts by flow cytometry 48 hours after treatment with AVM0703 (17 mg / kg); AVM0703 (17 mg / kg) and normalized to relative absolute counts using complete blood counts are shown. For both the CD8+ plot (left) and the CD4+ plot (right), data for 12 mg / kg or 17-18 mg / kg dexamethasone base are shown in the right hand columns of each (relative counts of "33", "1.4", "0.2" and "0.5").

[0059] [Figure 22]A single dose of cyclophosphamide 166 mg / kg (HED 500 mg / m2) and fludarabine 10 mg / kg on day -5 combined with 12 mg / kg or 17 to 18 mg / kg dexamethasone base on day -2 lymphodepleted NK cells and B lymphocytes comparably compared with 2 days of repeat cyclophosphamide 166 mg / kg on days -5 and -4 and 4 days of fludarabine 10 mg / kg (HED 30 mg / m2) on days -5, -4, -3, and -2. Mice were treated IP with PBS (vehicle), or with repeated IP cyclophosphamide 166 mg / kg on days -5 and -4 and 4 days of IP fludarabine 10 mg / kg (HED 30 mg / m2) on days -5, -4, -3, -2 (Flu+Cy), or with a single IP dose of cyclophosphamide 166 mg / kg (HED 500 mg / m2) and IP fludarabine 10 mg / kg, both on day -5, followed by oral dexamethasone base at 12 mg / kg or 17-18 mg / kg on day -2 (Flu+Cy + AVM0703(12 mg / kg); Flu+Cy + AVM0703(17 mg / kg)), or with oral dexamethasone base at 12 mg / kg or 17-18 mg / kg (AVM0703(12 mg / kg)). Figure 1 shows graphs of individual B lymphocyte (left) and NK cell (right) counts and mean counts measured as relative counts by flow cytometry 48 hours after treatment with AVM0703 (17 mg / kg); AVM0703 (17 mg / kg) and normalized to relative absolute counts using complete blood counts. In both the B cell plot (left) and the NK cell plot (right), data for 12 mg / kg or 17-18 mg / kg dexamethasone base are shown in the right columns of each (relative counts are 111 and 58 for B cells; not shown for NK cells).

[0060] [Diagram 23]Cyclophosphamide 166 mg / kg (HED 500 mg / m2) and a single dose of fludarabine 10 mg / kg on day -5 combined with dexamethasone base at 12 mg / kg or 17 to 18 mg / kg on day -2 produced equivalent lymphodepletion of absolute lymphocyte counts but spared neutrophils compared with 2 days of repeat cyclophosphamide 166 mg / kg on days -5 and -4 and 4 days of fludarabine 10 mg / kg (HED 30 mg / m2) on days -5, -4, -3, and -2. Mice were treated IP with PBS (vehicle), or with repeated IP cyclophosphamide 166 mg / kg on days -5 and -4 and 4 days of IP fludarabine 10 mg / kg (HED 30 mg / m2) on days -5, -4, -3, -2 (Flu+Cy), or with a single IP dose of cyclophosphamide 166 mg / kg (HED 500 mg / m2) and IP fludarabine 10 mg / kg, both on day -5, followed by oral dexamethasone base at 12 mg / kg or 17-18 mg / kg on day -2 (Flu+Cy + AVM0703(12 mg / kg); Flu+Cy + AVM0703(17 mg / kg)), or with oral dexamethasone base at 12 mg / kg or 17-18 mg / kg (AVM0703(12 mg / kg)). Graphs of individual absolute neutrophil (left) and lymphocyte (right) counts and mean counts measured by complete blood count 48 hours after treatment with AVM0703 (17 mg / kg); AVM0703 (17 mg / kg). In both the neutrophil plot (left) and lymphocyte plot (right), data for 12 mg / kg or 17-18 mg / kg dexamethasone base are shown in the right hand columns of each (relative counts of 321, 605, 521, and 88, respectively).

[0061] [Figure 24]A single dose of cyclophosphamide 166 mg / kg (HED 500 mg / m2) and fludarabine 10 mg / kg (HED 30 mg / m2) on day -5 combined with 12 mg / kg or 17 to 18 mg / kg dexamethasone base on day -2 spared red blood cells (RBCs) and platelets. Mice were treated IP with PBS (vehicle), or with repeated IP cyclophosphamide 166 mg / kg on days -5 and -4 and 4 days of IP fludarabine 10 mg / kg (HED 30 mg / m2) on days -5, -4, -3, -2 (Flu+Cy), or with a single IP dose of cyclophosphamide 166 mg / kg (HED 500 mg / m2) and IP fludarabine 10 mg / kg, both on day -5, followed by oral dexamethasone base at 12 mg / kg or 17-18 mg / kg on day -2 (Flu+Cy + AVM0703(12 mg / kg); Flu+Cy + AVM0703(17 mg / kg)), or with oral dexamethasone base at 12 mg / kg or 17-18 mg / kg (AVM0703(12 mg / kg)). Graphs of individual absolute platelet (left) and absolute RBC (right) counts and mean counts measured by complete blood count 48 hours after treatment with AVM0703 (17 mg / kg); AVM0703 (17 mg / kg). In both the RBC plot (left) and platelet plot (right), data for 12 mg / kg or 17-18 mg / kg dexamethasone base are shown in the right-hand columns of each (relative counts of "10", "10", "348", and "373").

[0062] [Diagram 25]A single dose of cyclophosphamide 166 mg / kg (HED 500 mg / m2) and fludarabine 10 mg / kg on day -5 combined with 12 mg / kg or 17 to 18 mg / kg dexamethasone base on day -2 spared body weight, a measure of toxicity, compared with 2 days of repeat cyclophosphamide 166 mg / kg on days -5 and -4 and 4 days of fludarabine 10 mg / kg on days -5, -4, -3, and -2. Mice were treated IP with PBS (vehicle), or with repeated IP cyclophosphamide 166 mg / kg on days -5 and -4 and 4 days of IP fludarabine 10 mg / kg (HED 30 mg / m2) on days -5, -4, -3, -2 (Flu+Cy), or with a single IP dose of cyclophosphamide 166 mg / kg (HED 500 mg / m2) and IP fludarabine 10 mg / kg, both on day -5, followed by oral dexamethasone base at 12 mg / kg or 17-18 mg / kg on day -2 (Flu+Cy + AVM0703(12 mg / kg); Flu+Cy + AVM0703(17 mg / kg)), or with oral dexamethasone base at 12 mg / kg or 17-18 mg / kg (AVM0703(12 mg / kg)). Graphs of individual and average body weight changes calculated by subtracting body weight 48 hours after treatment with either 100 mg / kg or 17 mg / kg AVM0703 from body weight before treatment are shown (left). The acute high-dose dexamethasone group did not experience weight loss, unlike the chemotherapy group. Thus, acute high-dose dexamethasone provides a lymphodepleting effect similar to chemotherapy but without the toxicity. The dosing schedules for these regimens are also shown (right).

[0063] [Figure 26]Comparison of 15 mg / kg dexamethasone-based HED (AVM0703) with standard chemotherapy regimens: Antitumor efficacy. Twenty B-cell lymphoma mice (8-10 weeks old) were treated with PBS (control), 15 mg / kg dexamethasone-based HED (AVM0703), or one or two cycles of cyclophosphamide 100 mg / kg ip, doxorubicin 6 mg / kg ip, vincristine 0.1 mg / kg ip, and dexamethasone 0.2 mg / kg ip (CHOP). Dosing for one cycle CHOP was on day 0, two cycle CHOP mice were dosing on days 0 and 10, and dexamethasone dosing was on days 7, 10, 18, 23, 24, 28, 35, and 42 (indicated by arrows). Mice were followed for tumor growth by measuring tumor volume (mm3) every 2-3 days. The efficacy of 15 mg / kg dexamethasone base HED was greater than one cycle of CHOP in terms of controlling tumor volume, but not as effective as two cycles of CHOP. However, 15 mg / kg dexamethasone base HED was associated with a much more favorable toxicity profile compared to two cycles of CHOP.

[0064] [Figure 27] Comparison of 15 mg / kg dexamethasone base HED (AVM0703) with standard chemotherapy regimens: Toxicity. Panel A shows the percent body weight change for 15 mg / kg dexamethasone base HED (AVM0703) compared to PBS control. Panel B shows the percent body weight change for one or two cycles of cyclophosphamide 100 mg / kg ip, doxorubicin 6 mg / kg ip, vincristine 0.1 mg / kg ip, and dexamethasone 0.2 mg / kg ip (CHOP) compared to PBS control. The weight loss seen in mice treated with two cycles of CHOP (B) is much greater than that seen in mice treated with 15 mg / kg dexamethasone base HED (A). Additionally, 18% of mice treated with two cycles of CHOP died from CHOP treatment, whereas no mice died from dexamethasone treatment.

[0065] [Figure 28] Statistical comparison of 15 mg / kg dexamethasone base HED (AVM0703) with PBS control. Twenty B cell lymphoma mice (8-10 weeks old) were treated with PBS (control) or 15 mg / kg dexamethasone base HED (AVM0703). Dexamethasone dosing was administered on days 7, 10, 18, 23, and 24 (indicated by arrows). Mice were followed for tumor growth by measuring tumor volume (mm3) every 2-3 days. The efficacy of 15 mg / kg dexamethasone base HED (filled squares) was greater than the PBS control (filled circles) as indicated by reduced tumor growth. Statistically significant differences in tumor volume were seen on days 15, 17, and 20.

[0066] [Figure 29] Glucocorticoid therapy reduces the required dose for effective chemotherapy.Tumor-bearing subjects treated with PBS alone ("Control") or dexamethasone base ("AVM0703") showed continued tumor growth, with generally high growth rates after 20 days.Tumor-bearing subjects treated with AVM0703 on day 11 followed by one dose of Cy / Flu chemotherapy on day 14 ("Combination") showed a steady and sustained reduction in tumor volume, in a similar manner to tumors treated with two doses of Cy / Flu chemotherapy ("Cy / Flu") on days 11 and 14.

[0067] [Diagram 30] High dose glucocorticoid therapy reduces tumor density without significantly affecting body weight. After tumor establishment, subjects were measured for tumor density following administration of weekly doses of glucocorticoid AVM0703 at 6 mg / Kg HED per week, 15 mg / Kg HED per week, or 21 mg / Kg HED per week (left panel). Mouse body weights over the study period are also shown (right panel). The dotted line represents the 20% loss of average mouse weight at the start of the study. No significant weight loss due to toxicity was observed and no mice died. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] Cytotoxic chemotherapeutic agents cause cell death through mechanisms or means that are not receptor-mediated. Cytotoxic chemotherapeutic agents cause cell death by interfering with cell division, metabolism, or functions necessary for cell survival. Due to this mechanism of action, cells that are growing fast (meaning they are proliferating or dividing) or are metabolically active are killed preferentially over cells that are not. The state of different cells in the body when they are dividing or using energy (which is the metabolic activity that supports the function of the cells) determines the dose of chemotherapeutic agent that causes cell death. Those skilled in the art will understand that the glucocorticoids utilized in the present invention are not cytotoxic chemotherapeutic agents. Cytotoxic chemotherapy non-exclusively refers to alkylating agents, antimetabolites, plant alkaloids, topoisomerase inhibitors, antineoplastic agents and arsenic trioxide, carmustine, fludarabine, IDA ara-C, Mylotarg, GO, mustergen, cyclophosphamide, gemcitabine, bendamustine, total body irradiation, cytarabine, etoposide, melphalan, pentostatin, and radiation.

[0069] The present invention relates to a pharmaceutical composition comprising a glucocorticoid for use in the treatment of diseases by immunodepletion. In particular, the composition of the present invention may be for use in the treatment of diseases mediated by immune cells such as lymphocytes. Treatment involves administering a dose of the pharmaceutical composition to a patient to deliver a dose of glucocorticoid equivalent to about 3-26 mg / kg human equivalent dose (HED) of dexamethasone base.

[0070] As used herein, the term glucocorticoid includes glucocorticoid receptor agonists and any compound that binds to glucocorticoid receptor. Such compounds include, but are not limited to, dexamethasone, dexamethasone-containing agents, hydrocortisone, methylprednisone, prednisone, corticone, budesonide, betamethasone, and beclomethasone. Other glucocorticoids include prednisolone, mometasone furoate, triamcinolone acetonide, and methylprednisolone. Glucocorticoids further include glucocorticoid receptor modulating agonists. In addition, selective glucocorticoid receptor agonists can be used in the pharmaceutical compositions disclosed herein. Such agonists or modulators include, for example, selective glucocorticoid receptor modulators (SEGRMs) and selective glucocorticoid receptor agonists (SEGRAs). Glucocorticoids, glucocorticoid receptor modulators, and selective glucocorticoid receptor agonists (SEGRAs) that can be utilized in the methods and compositions disclosed herein are well known to those skilled in the art.

[0071] Glucocorticoids and glucocorticoid receptor (GR) modulators exert their effects through both membrane glucocorticoid receptors and cytoplasmic GR, which activate or repress gene expression. Some of the desirable lymphodepleting effects of glucocorticoids and GR modulators appear to be mediated through membrane GR or other non-genomic effects in addition to genomic effects. Interestingly, it has been shown that co-treatment with dexamethasone can reduce glucocorticoid resistance (Serafin et al., 2017).

[0072] The effects of glucocorticoids are complex and depend on the affinity of each particular glucocorticoid for the GR and mineralocorticoid receptor (MR). In addition, there are currently nine known isoforms of cytoplasmic GR, and additional membrane-expressed GR receptors that have been identified but not fully characterized. Glucocorticoids have been reported to have different effects on lymphocyte levels depending on the concentration of glucocorticoid administered and the length of treatment. In general, at low doses, typically used for chronic therapy, glucocorticoids are reported to redistribute lymphocytes from peripheral blood to the bone marrow, at medium doses glucocorticoids are reported to cause leukocytosis, which is believed to be a redistribution of leukocytes from the bone marrow, spleen, and thymus to the peripheral blood, and at high doses glucocorticoids have lymphotoxic effects on lymphocytes by inducing apoptosis and necroptosis. The length of effect also depends on the dose level, for example, Fauci et al (1976) reported that a single oral 0.24 mg / kg dexamethasone dose suppressed peripheral blood T and B lymphocytes by 80%, with recovery beginning at 12 hours and reaching normal levels by 24 hours. However, the present invention demonstrates that acute oral doses of 3 mg / kg or greater are required to reduce peripheral blood T and B cells 24-48 hours after administration, with recovery to baseline levels occurring approximately 5-14 days after dosing.

[0073] Desired in vivo effects of exemplary glucocorticoids include, but are not limited to, reduction of germinal centers and marginal zones in secondary lymphoid tissues, direct tumor killing of certain cancers (particularly multiple myeloma, renal cell carcinoma, leukemia and lymphoma, non-small cell lung cancer (NSCLC), prostate cancer and breast cancer), depletion of all peripheral blood lymphocyte types, lack of lymphocyte redistribution to the BM and other organs, and increase in plasma cytokines, including IL-2, and / or IL-7, and / or IL-12, and / or IL-15, preferably to levels of 20 pg / ml or greater. Exemplary glucocorticoids do not increase plasma levels of IL-6, one of the major contributors to ACT-induced cytokine release syndrome (CRS). Exemplary glucocorticoids do not increase plasma levels of GM-CSF, one of the major contributors to ACT-induced neuroedema. Acute doses of dexamethasone at about HED 6 mg / kg or more reduce germinal centers and marginal zones in secondary lymphoid tissues. Acute doses of dexamethasone at about 1.6 mg / kg HED within a 48 hour period have about 50% direct tumor killing potential against multiple myeloma and other cancer cell lines, which is maintained but not increased at doses up to about 12 mg / kg HED. Acute doses of dexamethasone above about HED 3 mg / kg are required for lymphodepletion, as illustrated by the observation that 50% of patients treated with 3 mg / kg HED exhibited lymphocytosis (Figure 14). Plasma IL-2 and IL-15 cytokine increases are observed at doses of dexamethasone base at about HED 3 mg / kg or more (Figure 17). Based on the desired in vivo effects in the indications disclosed herein, the most preferred acute dexamethasone base dose (which can be converted to equivalent doses of other glucocorticoids based on known calculations or as disclosed herein) will most likely be about HED 9 mg / kg or more.

[0074] A single high dose of glucocorticoid can be given orally or as an IV infusion over about 1 hour. The total dose may be given as repeated IV or oral doses in any amount to provide a total dose of about 3 mg / kg to about 26 mg / kg (e.g., of dexamethasone) within a period of about 24 to about 72 hours.

[0075] The equivalent dose of another glucocorticoid or glucocorticoid receptor modulator can be conveniently and easily calculated using a publicly available corticoid conversion algorithm, preferably at http: / / www.medcalc.com. For example, 3-12 mg / kg dexamethasone is converted to 19-75 mg / kg prednisone. The biological half-life of prednisone is about 20 hours, while the biological half-life of dexamethasone is about 36-54 hours. Thus, for equivalent biological dosing, prednisone is dosed at 19-75 mg / kg every 24 hours. More specifically, a dose of 12 mg / kg dexamethasone is equivalent to 1) a dose of 75 mg / kg prednisolone, which requires repeated dosing of about 2 to about 3 doses every 24 hours. A dose of 10 mg / kg betamethasone is approximately 12 mg / kg dexamethasone and has a similar pharmacodynamic (biological) half-life to dexamethasone, however betamethasone reduces RBCs at a dose of approximately 24 mg / 50 kg (Gaur 2017).

[0076] DEX (dexamethasone base) doses in the examples of this application are listed as human equivalent doses (HED). AVM0703 (also referred to as AugmenStem™ or PlenaStem™) in the examples described is Dex (dexamethasone base) as dexamethasone sodium phosphate in a proprietary buffer.

[0077] Methods for calculating human equivalent dose (HED) are known in the art. For example, the FDA's Center for Drug Evaluation and Research (CDER) published a commonly cited guidance document in 2005 (US Department of Health CDER, 2005), which provides an established algorithm for converting animal dose to HED based on body surface area in Table 1 on page 7 (this is a commonly accepted method for extrapolating dose between species). Table 1 is reproduced below for reference. Those skilled in the art will understand that the animal dose, HED, in mg / kg described below is easily calculated using the standard conversion factors in the right column of Table 1.

[0078] [Table 1] a Based on a 60 kg human. For species not listed, or weights outside the standard range, the HED can be calculated from the following formula: HED = Animal Dose in mg / kg x (Animal weight in kg / Human weight in kg) 0.33 b Because healthy children rarely volunteer for Phase 1 trials, m Values ​​are provided for reference only. c For example, cynomolgus macaques, rhesus macaques, and stump-tailed macaques.

[0079] Doses described herein may be expressed as "weight-based doses" or as "body surface area (BSA)-based doses." A weight-based dose is a dose administered to a patient that is calculated based on the patient's weight, e.g., mg / kg. A BSA-based dose is a dose administered to a patient that is calculated based on the patient's surface area, e.g., mg / m 2The dose measurements for these two forms can be converted within the context of human dosing by multiplying the weight-based dose by 37 or dividing the BSA-based dose by 37, as shown in Table 1 above.

[0080] The terms "subject" and "patient" are used interchangeably herein and refer to a human or animal.

[0081] Dexamethasone, like other glucocorticoid steroids at equivalent doses, inhibits the formation and proliferation of germinal centers in lymphoid tissues and lymphodepletes peripheral blood.The dose of glucocorticoid, especially dexamethasone, preferably achieves lymphodepletion of more than 75%.More preferably, the dose of glucocorticoid, especially dexamethasone, achieves lymphodepletion of more than 80%.Most preferably, the dose of glucocorticoid, especially dexamethasone, achieves lymphodepletion of more than 95%.Those skilled in the art will understand that lymphodepletion can be easily measured by measuring complete blood count (CBC).

[0082] Dexamethasone and other preferred glucocorticoids spare neutrophils, do not inhibit neutrophil function (Schleimer RP, J Pharmacol Exp Ther 1989;250:598-605), and spare red blood cells (RBCs), platelets, mesenchymal stem cells (MSCs), and hematopoietic stem cells (HSCs). Neutrophil sparing in humans is associated with mm 3The absolute neutrophil count (ANC) is greater than 500 per 1000. By sparing neutrophils, RBCs and platelets, lymphocyte-depleting glucocorticoids reduce or eliminate the need for transfusions. Lymphocyte-depleting glucocorticoids also spare bone marrow mesenchymal stem cells (MSCs) and do not affect the ability of bone marrow MSCs to differentiate into chondrocytes, osteocytes, or adipocytes. Lymphocyte-depleting glucocorticoids also increase the number of endogenous BM MSCs or their ex vivo viability in both humans and horses. Lymphocyte-depleting glucocorticoids increase plasma levels of IL-2, IL-7, IL-12 and IL-15, but not IL-6 or GM-CSF. In some embodiments of the invention, subjects are selected prior to treatment and / or evaluated following treatment based on measurements of plasma levels of one or more of these cytokines.

[0083] Dexamethasone is approved for use with initial doses of dexamethasone sodium phosphate injection ranging from 0.5 to 9 mg per day depending on the disease being treated, which is a daily dose of 0.01 to 0.18 mg / kg based on 50 kg BW. In less severe disease, doses of less than 0.5 mg may be sufficient, while in severe disease doses greater than 9 mg may be required. Current medical practice tends to use (pharmacologically) higher doses of corticosteroids for the treatment of unresponsive shock. For cerebral edema, dexamethasone sodium phosphate injection is typically administered intravenously at an initial dose of 10 mg, followed by 4 mg intramuscularly every 6 hours until the symptoms of cerebral edema subside. This total dose corresponds to a total 24 hour dose of about 0.34-0.48 mg / kg, and a total 72 hour dose of 0.8-1.12 mg / kg over 72 hours, which is not an effective dose according to the present invention (which uses doses of about 3 mg / kg to about 26 mg / kg).

[0084] For acute allergic disorders, Dexamethasone Sodium Phosphate Injection USP 4 mg / mL is recommended as follows: Day 1, 1 or 2 mL (4 or 8 mg) intramuscularly followed by Dexamethasone Sodium Phosphate Tablets 0.75 mg; Days 2 and 3, 4 tablets in 2 divided doses each day; Day 4, 2 tablets in 2 divided doses; Days 5 and 6, 1 tablet each day; Day 7, no treatment; Day 8, follow-up visit. In the emergency room, Dexamethasone has been used at 2 mg / kg for severe acute childhood asthma, which is a lower dose than the glucocorticoid dose defined in this invention.

[0085] Conventional formulations of glucocorticoids such as dexamethasone may not be suitable for use in the therapeutic applications of the present invention. For example, dexamethasone sodium phosphate (DSP) is currently available in low dose (2-4 mg / ml) and low volume formulations containing antimicrobial preservatives such as benzyl alcohol (BA) and propyl paraben (PP) (e.g., APP Pharmaceuticals, Mylan). The target dose of DSP required to achieve complete lymphocyte depletion would result in the use of multiple vials, resulting in excipient overload. Exceeding the WHO acceptable daily intake (ADI) of both benzyl alcohol and propylparaben has been associated with genotoxicity and increased cancer risk (Darbre et al., 2014), reproductive toxicity (Aker et al., 2016), increased risk of allergic diseases (Savage et al., 2012; Spanier et al., 2014), and neonatal CNS dysfunction (Medicines Agency, 2017). Furthermore, according to the package inserts of commercially available DSPs, serious neuropsychiatric effects occur in approximately 6% of patients receiving steroids (Malmegrim et al., 2017). As the present invention involves the administration of high doses of glucocorticoids, formulations with low levels of potentially toxic preservatives or no toxic preservatives should be used. The preservatives are preferably antioxidants.

[0086] The pharmaceutical compositions of the present invention may include a preservative (e.g., antioxidant) additive, such as sodium sulfite, to maintain the stability of the composition. Sulfite salts are also widely used as preservatives and antioxidant additives in the pharmaceutical industry. Exposure to such sulfite salts has been reported to induce a wide range of adverse clinical effects in susceptible individuals, ranging from dermatitis, hives, flushing, hypotension, and abdominal pain to life-threatening anaphylactic and asthmatic reactions. Sulfite-induced symptoms may be mild in some individuals but severe in others, and in some individuals the reaction may become life-threatening. In a preferred embodiment, when sodium sulfite is included as an antioxidant, its concentration is 0-70 ppm sodium sulfite (anhydrous).

[0087] Antioxidants may be added in amounts reduced from levels normally utilized in glucocorticoid-containing compositions, thereby reducing the toxicity and adverse side effects associated with the use of such antioxidants. In some instances, the formulations of the present invention may lack the addition of antioxidants.

[0088] As used herein, antioxidant is an excipient that increases the stability of composition by retarding or inhibiting the oxidation process of molecules.The antioxidant that can be used includes, for example, ascorbic acid, acetylcysteine, butyl hydroxyanisole, cysteine ​​hydrochloride, sodium dithionite, gentisic acid, monosodium glutamate, glutathione, sodium formaldehyde sulfoxylate, methionine, monothioglycerol, propyl gallate, sulfite, sodium thioglycolate, α-thioglycerol, tocopherol alpha, alpha tocopherol hydrogen succinate, and sodium thioglycolate.

[0089] In addition to the active glucocorticoids and antioxidants, the pharmaceutical compositions disclosed herein may include additional ingredients well known to those skilled in the art. Pharmaceutical compositions may be prepared using pharma- ceutically acceptable "carriers" that are composed of materials that are considered safe and effective. "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe," e.g., physiologically acceptable when administered to humans and do not normally produce allergic or similar adverse reactions, such as stomach upset. In some embodiments, the term refers to molecular entities and compositions that have been approved by a U.S. federal or state government regulatory agency, e.g., the GRAS list or similar list under sections 204(s) and 409 of the Federal Food, Drug, and Cosmetic Act, subject to premarket review and approval by the FDA, the United States Pharmacopeia, or other pharmacopoeias that are generally recognized for use in animals (more specifically, humans).

[0090] The term "carrier" refers to diluents, binders, lubricants, and disintegrants. Those skilled in the art are familiar with such pharmaceutical carriers and how to formulate pharmaceutical compositions using such carriers.

[0091] The pharmaceutical compositions provided herein may include one or more excipients, such as, for example, solvents, solubility enhancers, suspending agents, buffers, isotonicity agents, antioxidants, and antimicrobial preservatives. When used, the excipients of the composition will not adversely affect the stability, bioavailability, safety, and / or efficacy of the active ingredient (i.e., the glucocorticoid used in the composition). That is, those skilled in the art will understand that a composition is provided in which there is no incompatibility between any of the components of the dosage form. The excipients may be selected from the group consisting of buffers, stabilizing agents, osmotic agents, chelating agents, antioxidants, antimicrobial agents, and preservatives.

[0092] The pharmaceutical composition of the present invention may contain a chelating agent used to capture metal ions that may be present in the composition and reduce their activity. Calcium disodium EDTA 0.01-0.1% (EDTA=ethylenediaminetetraacetic acid or edetic acid), disodium EDTA 0.01-0.11%, sodium EDTA 0.20%, calcium vertamide sodium 2.84%, cartelidol 0.023%, DTPA 0.04-1.2% (diethylenetriaminepentaacetic acid) are possible chelating agents. In a preferred embodiment, the concentration of disodium EDTA (edetic acid) is 0-500 ppm. ***

[0093] As described in WO2018 / 183927, glucocorticoids may also be used as preconditioning agents in conjunction with adoptive cell therapies (ACT). Glucocorticoids, particularly dexamethasone at a single acute dose of about 3 mg / kg to about 26 mg / kg administered about 12 to about 72 hours prior to administration of cellular immunotherapy or a total dose of about 3 mg / kg to about 26 mg / kg administered about 12 to about 72 hours prior to administration of cellular therapy, increase plasma levels of IL-2 and IL-15.

[0094] Glucocorticoids, especially dexamethasone, at a single acute dose of about 3 mg / kg to about 26 mg / kg or a total dose of about 3 mg / kg to about 26 mg / kg over a period of about 72 hours, administered alone or in combination with reduced intensity cytotoxic preconditioning, can be useful for the treatment of autoimmune diseases. For the treatment of autoimmune diseases, ACT can be targeted to disease-causing immune cells in an effort to wipe out autoimmune recognition cells. In addition, for autoimmune diseases, ACT can be Tregs targeted by CAR or TCR or expressed antibodies against antigens specifically or selectively expressed by the area or organ of the body where the autoimmune attack is occurring. Tregs (plural Tregs) include, but are not limited to, CD4+ Tregs, CD4+CD45RA+ Tregs, CD4+CD25+CD45RA+ Tregs, FoxP3+ Tregs, CD4+CD25+FoxP3+CD152+ Tregs, CD4+CD25+CD152+ Tregs, CD8+ Tregs, CD8+CD28- Tregs, CD4+CD25int / high, CD127low, CTLA4+, GITR+, FoxP3+, CD127low, CD4+CD25- - They may be inducible Tregs, or type IT regs.

[0095] "Natural" regulatory T cells, originally recognized by constitutive expression of CD4 and CD25, can be further defined by expression of the transcription factor foxP3 and surface CD152. The generation of these cells and some of their suppressive activity are dependent on TGF-beta, and it has been shown that they can induce IDO in appropriate DCs by CD152-mediated ligation of CD80 / 86. Anergic CD4+ T cells generated by antigen stimulation in the absence of costimulation appear to be characterized by an intrinsic elevated tolerance threshold for antigenic stimulation that can be maintained by expression of E3 ubiquitin ligases such as GRAIL, c-cbl, and Itch. Anergic cells can behave as regulatory T cells by competing for and adsorbing stimulatory cytokines such as IL-2 at the site of antigen presentation. Tr1 cells represent an induced subset of CD4 helper T cells and are dependent on IL-10 for their differentiation and for some of their regulatory properties. They do not express foxP3, but may express markers associated with Th2 cells and repressor of GATA (ROG). Like natural Tregs, they express high levels of surface CD152 and can induce IDO and tryptophan catabolism in appropriate DCs. CD8+CD28- suppressor T (Ts) cells were first analyzed in humans, but have recently been demonstrated in rodents. Like Tr1 cells, they can be induced in the absence of IL-10, which may be involved in the downregulation of dendritic cell costimulation and the upregulation of ILT-3 and ILT-4 (in the case of human DCs) that appear to play an important role in presenting antigen and tolerizing further cohorts of T cells.

[0096] Regulatory T cells (Tregs) play an important role in maintaining immune homeostasis. Tregs suppress the function of other T cells to limit immune responses. Alterations in the number and function of Tregs have been implicated in several autoimmune diseases, including multiple sclerosis, active rheumatoid arthritis, and type 1 diabetes. High levels of Tregs have been found in many malignant disorders, including lung, pancreatic, and breast cancers. Tregs may also impede antitumor immune responses, which leads to increased mortality.

[0097] To date, two major classes of Tregs have been identified: CD4 and CD8 Tregs. CD4 Tregs consist of two subtypes: "natural" Tregs (nTregs), which constitutively express CD25 and FoxP3, and so-called adaptive or inducible Tregs (iTregs).

[0098] Natural Tregs (nTregs) are CD4 T cells that express high levels of the transcription factor FoxP3 (which is also a lineage marker) along with CD25. + nTregs originate in the thymus as all CD4 + They account for approximately 5-10% of the T cell population and can first be found at the single positive stage of T lymphocyte development. These are positively selected thymocytes with relatively high avidity for self-antigens. (Fehervari Z, Sakaguchi S. Development and function of CD25 + CD4 + regulatory T cells. Curr Opin Immunol. 2004;16:203-208.)

[0099] The signal for the development of Treg cells is thought to come from the interaction between the T cell receptor and a complex of MHC II with a self-peptide expressed in the thymic stroma. nTregs are essentially cytokine-independent.

[0100] Adaptive or inducible Tregs originate in the thymus as single positive CD4 cells. They differentiate into Tregs (iTregs) expressing CD25 and FoxP3 after sufficient antigenic stimulation in the presence of the corresponding antigen and specialized immunoregulatory cytokines such as TGF-β, IL-10, and IL-4. (Chatenoud L, Bach JF. Adaptive human regulatory T cells: myth or reality? J Clin Invest. 2006;116:2325-2327.)

[0101] FoxP3 - Although minor populations of Tregs have been reported, FoxP3 is currently the most accepted Treg marker. The discovery of the transcription factor FoxP3 as a marker for Tregs has allowed scientists to better define Treg populations and has led to the discovery of additional Treg markers, including CD127.

[0102] Glucocorticoids, particularly dexamethasone at a single acute dose of about 3 mg / kg to about 26 mg / kg or a total dose of about 3 mg / kg to about 26 mg / kg over a period of about 72 hours, administered alone or in combination with reduced intensity cytotoxic chemotherapy or radiation, may be useful for the treatment of residual HIV disease and for the treatment of germinal center lymphomas such as Burkitt's lymphoma.

[0103] Follicular helper CD4 T cells, or T cells, are present in B cell follicles in secondary lymphoid tissues. FHThe B-cell follicles are susceptible to infection by the AIDS virus and represent a major source of virus that persists despite relative control of viral replication. This persistence is due, at least in part, to the relative exclusion of effective antiviral CD8 T cells from the B-cell follicles. AIDS viral persistence in individuals on effective drug therapy or who spontaneously control viremia continues to be a barrier to definitive treatment. Infected follicular helper CD4 T cells, T FH represent the major source of this residual virus. Effective CD8 T cell responses can control viral replication, either in conjunction with drug therapy or, in rare cases, spontaneously, but most antiviral CD8 T cells do not enter B cell follicles, and even when they do, they are secreted into T FH HIV-1 is unable to robustly control viral replication in the population; therefore, these sites become sanctuaries and reservoirs for the replicating AIDS virus. Lymphodepletion and reduction of germinal centers and marginal zones in the spleen pushes residual HIV-infected cells into the bloodstream, where they can be killed by existing therapies. Latently infected, resting CD4 T cells have been detected in the peripheral blood, gastrointestinal (GI) tract, and lymph nodes of HIV-1-infected individuals, and are likely present in other organs that contain lymphoid tissue.

[0104] Highly active antiretroviral therapy (HAART) can long-term suppress plasma HIV-1 loads in infected individuals, but low levels of virus persist and rebound after cessation of treatment. During HAART, the virus resides in latently infected cells, such as resting CD4 T cells, and in other cell types that can support residual viral replication. Therapeutic eradication requires removal of the virus from all reservoirs.

[0105] Burkitt's Lymphoma (BL) is a germinal center lymphoma that originates and grows within the secondary lymphatic system and is always associated with c-Myc activating chromosomal translocations. It is one of the fastest growing cancers and can double in size every 14-18 hours. BL is an aggressive B-cell lymphoma found in the germinal centers of the spleen and secondary lymphatic tissues. BL is named after Dr. Denis Parsons Burkitt, a surgeon who first described the disease in 1958 while working in equatorial Africa (Burket, D., 1958). BL is most common in children living in sub-Saharan Africa, with the highest incidence and mortality rates found in East Africa (Orem, J., et al.). Boys are more likely to develop BL than girls. Outside of Africa, BL is most likely to occur in people with weakened immune systems.

[0106] Among B-cell malignancies, CLL is the most responsive to ibrutinib, and unfortunately, ibrutinib is unlikely to be particularly beneficial for people with Burkitt's lymphoma and other germinal center lymphomas.However, the same results can be achieved for germinal center lymphomas, such as Burkitt's lymphoma, by using agents that remove secondary lymphoid tissue germinal centers, redistributing B-cell cancers into the circulation, where they are more chemosensitive and less proliferative.Thus, in some embodiments, the present invention provides combination therapy that increases the sensitivity of lymphomas to chemotherapy and / or involves reduced-intensity cytotoxic chemotherapy in addition to glucocorticoids, such as dexamethasone.Suitable various chemotherapy methods are disclosed herein.

[0107] Clinical observations of the ability of the Bruton's tyrosine kinase inhibitor ibrutinib for the treatment of chronic lymphocytic leukemia (CLL) have demonstrated that redistribution of CLL cells from lymphoid tissues to the bloodstream is the mechanism of action that contributes to its beneficial effects in CLL. Circulating CLL cells are not proliferative, and clonal expansion is restricted to the lymphoid tissue microenvironment. Thus, redistribution to the bloodstream reduces cancer proliferation. Similarly, redistribution of ALL from the bone marrow to the bloodstream has also been reported to enhance sensitivity to standard chemotherapy (Chang BY, Blood 2013 122 : 2412-24;).

[0108] Glucocorticoids have been reported to have multiple, opposing actions on lymphocytes, depending on the dose, duration of administration, and species investigated. Glucocorticoids have been studied since 1943 as lymphocytosis inducers, i.e., agents that increase the number of lymphocytes in circulation (for review, see Burger et al., 2013), typically with the use of 0.5–1 mg / kg prednisone, equivalent to a dexamethasone dose of 0.1–0.2 mg / kg. High-dose methylprednisone (HDMP), used for refractory CLL, in contrast, does not appear to induce lymphocytosis at the 0.5–1.0 mg / kg methylprednisone-equivalent doses where prednisone induced lymphocytosis. Lymphotoxic high-dose steroids are typically considered to be approximately 100 mg prednisone equivalent per day, which we have shown is not an effective preconditioning dose, as this is a 16 mg dexamethasone equivalent dose of approximately 0.23-0.32 mg / kg. Dexamethasone does not reduce germinal centers in mice until a HED of approximately 3 mg / kg or more is administered. Prednisone does not significantly affect spleen weight or germinal centers until it is used at doses of >2.5 mg / kg po per day for 13 weeks in mice (Yan et al., 2015). Since a dose of 30 mg per day (approximately 0.48-0.72 mg / kg) is considered high in human lupus patients, this human dose would have unacceptable mineralocorticoid activity.

[0109] For the treatment of Burkitt lymphoma (BL) with standard chemotherapy regimens such as COPADM, prednisone is typically administered at 60 mg / m 2 Dexamethasone is also included in various cycles at 0.05 mg / kg prednisone and 0.3 mg / kg dexamethasone equivalent doses, which translates to an equivalent dose of 1.62 mg / kg prednisone and 0.3 mg / kg dexamethasone, which are not effective preconditioning doses. Dexamethasone is also typically included in an oral regimen of 40 mg daily for 4-5 days or 6 mg / m over 5 days. 2It is used clinically for the treatment of B-cell malignancies. In certain indications such as ALL, dexamethasone is administered daily for several weeks and can be associated with osteonecrosis, especially in postpubertal boys. The risk of osteonecrosis can be virtually eliminated by dosing dexamethasone every other week, but may be present especially in ALL, where asparaginase regimens are part of the treatment (Chang BY, Blood 2013 122 : 2412-24).

[0110] Epstein-Barr virus (EBV) infection is found in almost all African BL patients, and chronic malaria is thought to reduce resistance to EBV and allow it to take hold. The disease is characterized by involvement of the mandible or other facial bones, distal ileum, cecum, ovaries, kidneys, or breasts. In addition, BL affects immunocompromised people, such as those with HIV.

[0111] BL is classified into three main clinical variants: endemic, sporadic, and immunodeficiency-associated; the endemic variant (also called "African variant") occurs most commonly in children living in malaria-endemic regions of the world.

[0112] One advantage of the present invention may be to ablate germinal centers and / or marginal zones and selectively drive BL and other germinal center or marginal zone cancer cells out of the germinal centers or marginal zones and into the circulation where the cancer cells can be more easily killed by chemotherapy or other drugs. This may dramatically, safely, and cost-effectively advance BL treatment outcomes. Asthma is a chronic inflammation characterized by increased numbers of CD8+ type 1 T lymphocytes and macrophages in lung tissue and neutrophils in the airway lumen. Lymphocytes, which are significantly different in the two inflammatory conditions, play a key role in the pathogenesis of asthma and COPD. There is now overwhelming evidence supporting a major role for T cells in asthma, especially the involvement of T helper type 2 (Th2) cells in atopic allergic asthma as well as nonatopic and occupational asthma. There may also be a minor contribution from T cytotoxic type 2 CD8+ T cells. Several Th2 cytokines have the potential to modulate airway inflammation, and interleukin-13 in particular induces IgE-independent airway hyperresponsiveness and eosinophilia in animal models. Asthma and chronic obstructive pulmonary disease (COPD) are two distinct inflammatory disorders of the lung that share a common functional abnormality, namely airflow limitation (Baraldo et al., 2007).

[0113] In asthma, airflow limitation is largely reversible, either spontaneously or with treatment, and in most cases does not progress. In contrast, airflow limitation in COPD is usually progressive and poorly reversible. In asthma, chronic inflammation causes a concomitant increase in airway responsiveness to various stimuli, resulting in recurrent episodes of wheezing, shortness of breath, chest tightness, and coughing, especially at night and early in the morning. Many cells are involved in the inflammatory response in asthma, among which CD4+ type 2 lymphocytes, mast cells, and eosinophils are thought to play important roles. In COPD, poorly reversible airflow limitation is associated with an abnormal pulmonary inflammatory response to noxious particles and gases. This chronic inflammation is characterized by increased numbers of CD8+ type 1 T lymphocytes and macrophages in the lung tissue and neutrophils in the airway lumen. Lymphocytes, which are significantly different in these two inflammatory conditions, play a key role in the pathogenesis of asthma and COPD ( Baraldo et al., 2007 ).

[0114] [Definition] Below are definitions used to describe embodiments of the present invention.

[0115] The biological mechanism of lymphocyte depletion implies the induction of programmed cell death via apoptosis or necroptosis or pyroptosis or autophagy or necrosis. Various stimuli can initiate a non-apoptotic form of cell death called necroptosis, which occurs when caspases required for apoptosis are inhibited. Pyroptosis is a caspase-dependent form of programmed cell death, but differs from apoptosis in many ways. Unlike apoptosis, pyroptosis depends on the activation of caspase 1 or caspase 11 (caspase 5 in humans). Autophagy is a lysosome-dependent process.

[0116] Apoptosis: a form of cell death in which a programmed series of events leads to the removal of a cell without releasing harmful substances into the surrounding area. Apoptosis plays an important role in the development and maintenance of bodily health by removing old, unnecessary, and unhealthy cells.

[0117] The term "and / or" as used herein should be interpreted as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0118] The term "about," when referring to a measurable value, such as an amount, length of time, etc., indicates a variation of + / - 20%, or + / - 10%.

[0119] "Administering" refers to physically introducing an agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary administration routes for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the formulation is administered by a route other than parenteral, such as orally. Non-parenteral routes Other routes include topical, epithelial, or mucosal routes, such as intranasal, vaginal, rectal, sublingual, or topical.

[0120] A pharmacological dose is one that far exceeds normal levels in the body.

[0121] As used herein, "anti-tumor effect" refers to a biological effect that may be manifested as a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall survival or progression-free survival, an increase in life expectancy, or an alleviation of various physiological symptoms associated with tumors.Anti-tumor effect may also refer to the prevention of tumor development (e.g., a vaccine).

[0122] A therapeutic agent is an agent that enhances the efficacy of cellular immunotherapy compared to the non-therapeutic cellular immunotherapy.

[0123] "Autologous" refers to material derived from the same individual, which may be a human or other animal, into which it is later reintroduced.

[0124] "Allogeneic" refers to material derived from one individual and introduced into another individual of the same species, which may be a human or other animal.

[0125] The term dexamethasone (also referred to as Dex) non-exclusively refers to any formulation, which may be a liquid solution, a liquid suspension, an oral solution, a tablet form, a tablet form dissolved in a liquid containing the active ingredient dexamethasone, an injectable form, a gel formulation, a patch formulation, or any formulation containing the active ingredient dexamethasone.

[0126] The term glucocorticoid receptor modulator non-exclusively relates to glucocorticoid receptor agonists or glucocorticoid receptor modulators, including, but not limited to, Compound A [CpdA; (2-((4-acetophenyl)-2-chloro-N-methyl)ethylammonium chloride)] and N-(4-methyl-1-oxo-1H-2,3-benzoxazin-6-yl)-4-(2,3-dihydrobenzofuran-7-yl)-2-hydroxy-2-(trifluoromethyl)-4-methylpentanamide (ZK216348), AL-438, maplacorat, LGD-5552, RU-24858, fosdagrocholat, PF-802, Compound 10, MK5932, C108297, LGD5552, and ORG 214007-0.

[0127] Immunotoxins are proteins that contain toxins along with antibodies or growth factors that specifically bind to target cells. Immunotoxins are created by chemically conjugating antibodies to full-length protein toxins that lack natural binding domains. Immune-related proteins smaller than monoclonal antibodies (MoAbs), such as growth factors and cytokines, have also been chemically conjugated and genetically fused to protein toxins. Toxins used in immunotoxin constructs are derived from bacteria, fungi, and plants, and most function by inhibiting protein synthesis. Bacterial toxins commonly used in immunotoxins include Diphtheria toxin (DT), and toxins from Pseudomonas exotoxin (PE). Plant toxins utilized in immunotoxins include the A chain of ricin (RTA), and ribosome inactivating protein (RIP) gelonin, pokeweed antiviral protein, and dodecandrone. Being enzymes, one toxin molecule can act on many substrate molecules, with devastating effects on the cell. Toxins such as diphtheria toxin (DT) and Pseudomonas exotoxin (PE) block protein synthesis by their action on elongation factor 2 (EF-2).

[0128] The term systemic injection as used herein non-exclusively refers to routes of administration that rapidly (within seconds or 2-3 hours) result in circulating levels of the cellular immunotherapy, including non-exclusively intravenous, intraperitoneal, subcutaneous, nasal submucosal, lingual, bronchoscopically mediated, intravenous, intraarterial, intramuscular, intraocular, intrastriatal, subcutaneous, intradermal, via transdermal patch, via skin patch, via patch, into cerebrospinal fluid, into the portal vein, into the brain, into the lymphatic system, intrapleural, retroorbital, intradermal, intrasplenic, intralymphatic, etc.

[0129] The term "injection site" as used herein non-exclusively relates to intratumor, or intraorgans such as kidney or liver or pancreas or heart or lung or brain or spleen or eye, intramuscular, intraocular, intrastriatal, intradermal, via a transdermal patch, via a skin patch, via a patch, into the cerebrospinal fluid, into the brain, etc.

[0130] The term lymphodepletion as used herein non-exclusively relates to reducing the number of lymphocytes in the peripheral blood without redistribution of lymphocytes to other organs such as the bone marrow, thymus, lymph nodes, lungs or spleen or other organs.

[0131] The term lymphodepletion as used herein non-exclusively relates to reducing the lymphocyte count in peripheral blood to less than 200 per microliter, preferably less than 100 per microliter, without redistribution of lymphocytes to another organ such as bone marrow, thymus, lymph nodes, lungs or spleen or other organs.

[0132] The term cytotoxic lymphodepletion as used herein relates to the reduction of lymphocyte numbers in the peripheral blood by ADCC, cell-mediated cytotoxicity or mechanisms of direct lysis or cytotoxic removal of lymphocytes, chemotherapy, or radiation.

[0133] Antibody-dependent cell-mediated cytotoxicity (ADCC), also known as antibody-dependent cellular cytotoxicity, is a cell-mediated immune defense mechanism in which effector cells of the immune system actively lyse target cells whose surface antigens are bound by specific antibodies.

[0134] As used herein, the terms "cellular immunotherapy", "adoptive cellular immunotherapy", "ACT" or cellular immunotherapy or cell therapy non-exclusively refer to treatments containing cells used to assist the immune system in fighting disease or immune lineage-derived cells that directly fight disease such as cancer, autoimmune diseases, and infections by certain viruses. Cellular immunotherapy can be from either autologous or allogeneic sources. In a preferred embodiment, the adoptive immunotherapy used in the methods disclosed herein can be adoptive T cell immunotherapy or "T cell therapy".

[0135] The term preconditioning as used herein relates to preparing a patient with a cytotoxic or non-toxic lymphodepleting agent prior to ACT.

[0136] The term immunotherapy as used herein, also referred to as biological therapy, non-exclusively refers to a type of treatment for cancer, autoimmune disease, or infectious disease treatment designed to boost the body's natural defenses to fight the cancer, autoimmune disease, or infection. It uses substances made by the body or in a laboratory to improve or restore immune system function. The term "immunotherapy" refers to treating subjects with a disease or at risk of developing a disease or experiencing a relapse of a disease by methods that include inducing, enhancing, suppressing, or otherwise modulating an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy may include adoptive T cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation. However, one skilled in the art will recognize that the conditioning method disclosed herein will enhance the effectiveness of any transplanted T cell therapy. Examples of T cell therapy are described in U.S. Patent Publication Nos. 2014 / 0154228 and 2002 / 0006409, U.S. Patent No. 5,728,388, and International Publication No. WO 2008 / 081035.

[0137] The term "immunomodulation" as used herein non-exclusively refers to a broad range of treatments aimed at harnessing a patient's immune system to achieve control, stabilization, and potentially disease eradication of tumors, autoimmune causing cells, or viruses in cancer, autoimmune disease, or infectious diseases.

[0138] The term immunomodulatory agent as used herein non-exclusively relates to chemical agents (e.g., dexamethasone) or biologic agents (e.g., Humira® and rituximab) that modify the immune response or function of the immune system (such as stimulating antibody formation or inhibiting white blood cell activity). Traditional immunomodulatory agents that are immunosuppressants non-exclusively relate to glucocorticoids, calcineurin inhibitors, antimetabolites, and alkylating agents. Antimetabolites non-exclusively relate to purine analogs (e.g., azathioprine and mycophenolate mofetil), and antifolates (e.g., methotrexate and dapsone).

[0139] An immune suppressant (also called an immunosuppressant) can be a chemical or biological agent that can suppress or block surface responses. For example, antagonists to CD26 and dexamethasone are immune suppressants. The NTLA used in the present invention can be an NTLA immune suppressant.

[0140] The terms "conditioning" and "preconditioning" are used interchangeably herein to refer to preparing a patient or animal that requires T cell therapy for the appropriate conditions. Conditioning as used herein can include, but is not limited to, reducing the number of germinal centers and marginal zones, reducing the number of endogenous lymphocytes, removing cytokine sinks, increasing the serum level of one or more of homeostatic cytokines or proinflammatory factors, enhancing the effector function of T cells administered after conditioning, enhancing the activation and / or availability of antigen-presenting cells, or any combination thereof, prior to T cell therapy.

[0141] The term "adoptive immunotherapy" or "cellular adoptive immunotherapy" as used herein non-exclusively refers to immune cells taken from a patient (autologous or autogenous) or from a related or unrelated donor (allogeneic) and grown in a laboratory. This increases the number of immune cells that can kill cancer cells, cells that cause autoimmune disease, or fight infection. These immune cells are then returned to the patient to help the immune system fight disease. This is also called cellular adoptive immunotherapy. The immune cells can be T cells and / or other immune system cells, non-exclusively referring to macrophages, monocytes, dendritic cells, neutrophils, granulocytes, phagocytes, mast cells, basophils, thymocytes, or innate lymphoid cells, or any combination of these.

[0142] The term agonist as used herein non-exclusively refers to an entity that activates a particular receptor or activates downstream signaling pathways essential to mediate the action(s) of the receptor. Agonists may non-exclusively refer to, but are not limited to, antibodies, antibody fragments, soluble ligands, small molecules, cyclic peptides, cross-linking agents.

[0143] The term antagonist as used herein non-exclusively refers to an entity that interferes with the binding of a receptor to its partner structure(s) or interferes with the activation of downstream signaling pathways that are essential for mediating the activation of a particular receptor or the action(s) of a receptor.Antagonists may non-exclusively refer to, but are not limited to, antibodies, antibody fragments, soluble ligands, Fc fusion receptors, chimeric receptors, small molecules, cyclic peptides, peptides.

[0144] The term inhibitor as used herein non-exclusively refers to an entity that reduces the targeting action of a particular receptor. Inhibitors can be small molecules, antisense agents, nucleic acids including siRNA and microRNA.

[0145] The term "lymphocyte" as used herein includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell-toxic) lymphocyte that is a major component of the innate immune system. NK cells reject tumor and virus-infected cells. They act through the process of apoptosis, or programmed cell death. They are named "natural killer" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (not involving antibodies). Their T-cell receptors (TCR) differentiate from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells. These include helper T cells (e.g., CD4+ cells), cytotoxic T cells (TCs, also known as cytotoxic T lymphocytes, CTLs, T-killer cells, cytolytic T cells, CDS+ T cells, or killer T cells), memory T cells ((i) stem cell memory T scM cells, which, like naive cells, are CD45RO-, CCR 7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Ra+, but also express high amounts of CD95, IL-2R~, CXCR3, and LFA-1, and display many of the functional attributes characteristic of memory cells; (ii) central memory TcM cells, which express L-selectin and CCR7 and secrete IL-2, but not IFNy or IL-4; (iii) effector memory T EM cells are: Tregs (which do not express L-selectin or CCR7 but produce effector cytokines such as IFNy and IL-4), regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. B cells, on the other hand, play a major role in humoral immunity (involving antibodies). They make antibodies and antigens and act as antigen presenting cells (APCs), and after activation by antigen interaction, transform into memory B cells. In mammals, immature B cells are formed in the bone marrow, hence the name.

[0146] The term "cancer" refers to a disease characterized by uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid tumors and liquid tumors, e.g., diffuse or circulating tumors. The term "cancer" or "tumor" as used herein includes pre-malignant and malignant cancers and tumors.

[0147] A particular cancer may be responsive to chemotherapy or radiation therapy, or the cancer may be refractory, which refers to a cancer that is not amenable to surgical intervention, either because the cancer is initially refractory to chemotherapy or radiation therapy or because the cancer becomes refractory over time.

[0148] As used herein, "anti-tumor effect" refers to a biological effect that may be manifested as a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall survival or progression-free survival, an increase in life expectancy, or an alleviation of various physiological symptoms associated with tumors.Anti-tumor effect may also refer to the prevention of tumor development (e.g., a vaccine).

[0149] As used herein, the term "progression-free survival", which may be abbreviated as PFS, refers to the time from the date of treatment to the date of disease progression (per the revised IWG Lymphoma Response Criteria) or to the date of death from any cause.

[0150] "Disease progression" is assessed by radiographic or other measurement of malignant lesions and should not be reported as an adverse event. Deaths due to disease progression in the absence of signs and symptoms should be reported as the primary tumor type (e.g., DLBCL).

[0151] As used herein, "duration of response," which may be abbreviated as DOR, refers to the time from a subject's first objective response to the date of confirmed disease progression (per the revised IWG Lymphoma Response Criteria) or to the date of death.

[0152] The term "overall survival", which may be abbreviated as OS, is defined as the time from the date of treatment to the date of death.

[0153] In this specification, the terms "reduction" and "depletion" are used interchangeably and refer to any change that is less than the original. "Reduction" and "depletion" are relative terms, requiring a comparison between pre- and post-measurements. "Reduction" and "depletion" also include complete depletion.

[0154] "Treatment" or "treating" of a subject refers to any type of intervention or procedure or administration of an active agent to a subject for the purpose of reversing, ameliorating, improving, inhibiting, delaying, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, or condition, or biochemical manifestation associated with a disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission.

[0155] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of those alternatives. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of the described or listed components.

[0156] The term "about" or "substantially composed of" refers to a value or composition within an acceptable error range as determined by one of ordinary skill in the art for that particular value or composition, which will depend in part on how that value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "substantially composed of" may mean within one standard deviation or more than one standard deviation, as per the practice in the art. Alternatively, "about" or "substantially composed of" may mean within a range of up to 20% (i.e., ±20%). For example, about 3 mg may include any number between 2.3 mg and 3.6 mg (about 20%). Furthermore, particularly with respect to biological systems or processes, these terms may mean up to an order of magnitude or up to 5 times the numerical value. When a particular value or composition is presented in the application and claims, unless otherwise stated, the meaning of "about" or "substantially composed of" should be presumed to be within an acceptable error range for that particular value or composition.

[0157] Concentration ranges, percentage ranges, ratio ranges, or integer ranges described herein should be understood to include every integer value within the described range, and, where appropriate, fractions thereof (e.g., integer tenths and percentiles), unless otherwise indicated.

[0158] Ranges: Various aspects of the invention are presented in a range format. The description in range format is for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered as specifically disclosing all possible subranges and individual numerical values ​​within that range. For example, a range of 3 to 12 includes 3.1, 3.2, 3.3, etc.

[0159] Autoimmune disorders and other diseases that are mediated by lymphocytes and require simpler and less expensive treatments than HSCT include, but are not limited to, allergies, asthma, residual HIV, germinal center lymphomas such as Burkitt's lymphoma and diffuse large B-cell lymphoma, marginal zone lymphoma, graft versus host disease (GvHD), steroid-resistant GvHD, achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), and autoimmune encephalomyelitis. disease), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuropathic neuropathy (AMAN), Baro's disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS), Syndrome) or Eosinophilic Granulomatosis (EGPA), Cicatricial Pemphigoid, Cogan's Syndrome, Cold Agglutinin Disease, Congenital Heart Block, Coxsackie Myocarditis, CREST Syndrome, Crohn's Disease, Dermatitis Herpetiformis, Dermatomyositis, Devic's Disease (Neuromyelitis Optica), Discoid Lupus, Dressler's Syndrome, Endometriosis, Eosinophilic Esophagitis (EoE)esophagitis), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura (HSP), herpes gestationis or pemphigoid gestationis (PG), hidradenitis suppurativa (HS) (acne contralateral), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), Purpura), Inclusion body myositis (IBM), Interstitial cystitis (IC), Juvenile arthritis, Juvenile diabetes (type 1 diabetes), Juvenile myositis (JM) myositis), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD) disease), Mooren's ulcer, Much-Habermann disease, multifocal motor neuropathy (MMN). Neuropathy) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH),hemoglobinuria, Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome type I, II, III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS) syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes mellitus, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, leukoplakia, and Vogt-Koyanagi-Harada disease.

[0160] In certain embodiments of the invention, one may wish to exclude diseases such as: allergies, asthma, residual HIV, germinal center lymphomas such as Burkitt's lymphoma and diffuse large B-cell lymphoma, marginal zone lymphoma, graft versus host disease (GvHD), steroid-resistant GvHD, achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), and / or autoimmune encephalomyelitis. disease), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuropathic neuropathy (AMAN), Baro's disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS), Syndrome) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura (HSP)purpura), herpes gestationis or pemphigoid gestationis (PG), hidradenitis suppurativa (HS) (acne contralateralis), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignoconjunctivitis, linear immunoglobulin A disease (LAD), lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA). polyangiitis), mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), hemoglobinuria, Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome type I, II, III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA)aplasia, pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS) transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, leukoplakia, Vogt-Koyanagi-Harada disease.

[0161] Further considerations of the immune settings of the present invention The spleen contains both white and red pulp. The red pulp of the spleen holds aged or defective red blood cells (RBCs) and macrophages that normally filter and remove antibody-coated bacteria or RBCs from the circulation. The white pulp of the spleen contains the lymphoid compartment and is important for immune surveillance and response. The white pulp synthesizes antibodies against invading pathogens and releases platelets and neutrophils in response to bleeding or infection. During development, the spleen is thought to have multiple roles, including being the primary site of hematopoiesis (at 6 weeks of gestation). Preclinical and clinical trials have shown that without cytotoxic chemotherapy preconditioning, cellular immunotherapies are largely cleared from the circulation within 1 hour of administration and accumulate in the spleen. In order to maintain the cellular immunotherapies in the circulation, cytotoxic chemotherapy preconditioning must be administered in close proximity to the administration of the cellular immunotherapies (typically 48 hours prior to administration of the cellular immunotherapies). If cytotoxic chemotherapy preconditioning is administered 4 weeks prior or at a pretreatment time that allows bone marrow recovery, cellular immunotherapies cannot be effectively maintained in the circulation (Ritchie DS et al. Mol Ther. Nov;21(11):2122-9 (2013)).

[0162] The periarterial lymphoid sheath (PALS) of the white pulp of the spleen is populated mainly by T cells, whereas the lymphoid portion is populated mainly by B cells. Germinal centers (GCs) are sites within lymph nodes or lymphoid nodules in peripheral lymphoid tissues and in the white pulp of the spleen where prominent mature B lymphocytes, also known as central cells, rapidly proliferate, differentiate, and mutate through somatic hypermutation and class switching during antibody responses. Germinal centers are an important part of the B cell humoral immune response. They develop dynamically after activation of B cells by T-dependent antigens. Histologically, GCs describe microscopically identifiable areas in lymphoid tissues. Activated B cells migrate from primary reactive foci into the follicular system of primary follicles and begin monoclonal proliferation in the environment of follicular dendritic cells (FDCs).

[0163] After several days of proliferation, B cells mutate their antibody-encoding DNA, thereby generating clonal diversity in the germinal center. This involves random substitutions, deletions, and insertions by somatic hypermutation. Upon some unidentified stimulus from the FDC, maturing B cells (centroblasts) migrate from the dark to the light zone and begin to expose antibodies on their surface; at this stage they are called centroblasts. Centroblasts are in a state of activated apoptosis and compete for survival signals from the antigen-presenting FDC. This rescue process is thought to depend on the affinity of the antibody for the antigen. To obtain the final differentiation signal, functional B cells must then interact with helper T cells. This involves isotype switching, for example from IgM to IgG. It is thought that the interaction with T cells prevents the generation of autoreactive antibodies. B cells can either become plasma cells that shed antibodies or become memory B cells that are activated on subsequent contact with the same antigen. According to the recycling hypothesis, they can also start the whole process of proliferation, mutation, and selection anew.

[0164] B cells contained within the white pulp region of the spleen can be further divided into specific sections identified by staining with specific molecular markers. The marginal zone of the spleen contains non-circulating mature B cells that border the white pulp, forming a separation between the white and red pulp, and expresses high levels of CD21, IgM, CD24, and CD79a, as well as measurable levels of CD9 and CD22. The mantle zone surrounds normal germinal center follicles and expresses CD21, CD23, and CD38. The follicular zone is contained within the germinal center, expresses high levels of IgD and CD23, intermediate levels of CD21 and CD24, and can also be identified by PNA staining. Germinal centers are best identified by PNA binding and express higher levels of CD54 than the follicular zone. Germinal centers harbor a special population of helper T cells that appear to be evenly distributed throughout all germinal centers. Germinal centers are traditionally associated with immune responses that require T helper cells, but this is not absolute. Germinal centers are where hypervariable gene mutations occur to generate high affinity IgG-producing B cells. Active germinal centers have prominent macrophages and CD21-expressing dendritic cells. Follicular centers can also be identified by expression of CD45R (B220) (Cytotoxicologic Pathology, 35:366-375, 2007). CD45R follicular centers are found surrounding germinal centers and express Bcl6 and Bcl2. BioEssays 29:166-177, 2007;Cytotoxicol Pathol 34(5): 648-655, (2006)]

[0165] The response to pathogens or cancer cells is orchestrated by the complex interactions and activities of a large number of diverse cell types involved in the immune response. The innate immune response is the first line of defense and occurs soon after exposure to a pathogen. It is carried out by phagocytes such as neutrophils and macrophages, cytotoxic natural killer (NK) cells, and granulocytes. The subsequent adaptive immune response induces antigen-specific defense mechanisms and can take several days to develop. Cell types with a crucial role in adaptive immunity are antigen-presenting cells, including macrophages and dendritic cells. Antigen-dependent stimulation of various cell types, including subsets of T cells, B cells, and macrophages, all play crucial roles in host defense. Immune cells non-exclusively refer to B cells, dendritic cells, granulocytes, innate lymphoid cells (ILCs), megakaryocytes, monocytes / macrophages, myeloid-derived suppressor cells (MDSCs), natural killer (NK) cells, platelets, red blood cells (RBCs), T cells, and thymocytes.

[0166] Zwang et al (2014) showed that after lymphodepletion, lymphocytes repopulate the immune space both through enhanced thymopoiesis and proliferation of residual non-depleted peripheral lymphocytes. The term homeostatic proliferation (or homeostatic expansion or lymphopenia-induced proliferation) refers to the latter. Homeostatic prolifi- ration is particularly relevant for reconstitution of the lymphoid compartment after immune depletion therapy in transplantation. Repopulating lymphocytes can be biased towards effector memory types that can cause graft rejection, autoimmunity, or, in the case of allogeneic bone marrow transplantation, graft-versus-host disease.

[0167] Two immune depleting agents, alemtuzumab and rabbit antithymocyte globulin, have been extensively characterized for their ability to induce an effector memory phenotype in rehoming lymphocytes.

[0168] Early studies on homeostatic proliferation showed that T cells surviving lymphodepletion divided, developed memory phenotypes and functions, and then acted in a dominant manner to render animals resistant to cardiac or renal allograft tolerance via costimulatory blockade. 1,2 Consistent with these findings, recent studies have shown that lymphopenia per se is sufficient to break peripheral tolerance based on stable costimulatory blockade. 3 In a mouse model of MHC-mismatched cardiac transplantation, lymphopenia (achieved either by irradiation or anti-CD4+ / CD8+ monoclonal antibodies) induced T- and B-cell-mediated acute rejection, which was accompanied by a T-cell shift to a CD44hi effector memory (EM) phenotype and the appearance of host-specific antibodies. The process of homeostatic proliferation can be divided into “slow” (one cell division per 24–36 h) or “fast” (one cell division per 6–8 h) kinetics. Slow proliferation occurs in response to "empty space sensing," whereas fast proliferation is a process driven primarily by intestinal antigens.4 In homeostatic proliferation after lymphodepletion in mouse models, slow homeostatic proliferation predominates. Furthermore, both T cells and B cells can undergo homeostatic proliferation.

[0169] Alemtuzumab (anti-CD52) is a potent lymphodepleting agent and has been used as induction therapy for transplantation and multiple sclerosis treatment. CD4+ cells, and to a lesser extent naive CD8+ cells, are most susceptible to alemtuzumab-induced lymphodepletion. 5,6,7,8 However, a larger population of naive T cells may escape depletion, as peripheral lymph nodes may become a reservoir for these cells after induction with alemtuzumab. 9 Alemtuzumab therapy results in a bias towards memory CD4+ and CD8+ phenotypes in kidney transplant recipients. Recipients who show evidence of rejection (by biopsy, de novo or donor-specific antibodies) after alemtuzumab therapy have an increased proportion of CD8+ effector memory cells (CD45RO-CD62L-). 10 These same patients further have a decreased frequency of regulatory T cells (Treg) among their CD4+ cells. Other studies, in contrast, suggested an increase in the frequency of Foxp3+ cells after alemtuzumab induction.11 Foxp3 expression in this instance may only be a transient marker of T cell activation.12,13,14 In patients with multiple sclerosis, homeostatic expansion after alemtuzumab therapy results in the recovery of populations of highly activated, proliferative, oligoclonal and memory-like CD4+ and CD8+ cells.15 In particular, the CD8 pool is dominated by a terminally differentiated effector memory CD28-CD57+CD8 population expressing perforin and granzyme B. Such populations are known to be associated with autoimmunity, and indeed in this study of 87 patients, two-thirds developed autoimmunity (mainly thyroid).

[0170] A recent investigation into the dynamics of lymphodepletion after rATG administered as induction therapy in kidney transplantation found that rATG persistently depleted the T cell compartment to counts below 250 CD3+ cells / uL at 6 months compared with only limited T cell depletion after basiliximab or no induction therapy. 19 In contrast to previous studies, this recent investigation did not find an increase in thymopoiesis (i.e., CD31+ cells among CD4+ or CD8+ cells) 1 month after rATG induction. Rather, there was an increase in peripheral cytokine-mediated signaling by IL-7 and IL-15 via Stat5 (particularly among memory T cell subsets) during the first month after rATG therapy. These studies indicate that T cell recovery after ATG comes from the peripheral T cell pool rather than increased thymopoiesis.

[0171] In humans, unlike mice, the majority of proliferating T cells originate from the periphery rather than the thymus. 20 Therefore, peripheral cytokine signaling is essential to maintain lymphoid repletion and rehoming of the T cell compartment in lymphocytopenia. IL-7 is the major cytokine responsible for T cell homeostatic proliferation. In young thymectomized subjects and older adults, circulating IL-7 levels are higher than in healthy controls. 21 In these patients with absent or low thymic function, IL-7 appears to stimulate T cell proliferation via STAT5 signaling. IL-7 itself has been described as a “rheostat” to maintain the T cell compartment. 22 In lymphocytopenia, excess IL-7 stimulates T cell proliferation. Proliferating T cells consume IL-7, and levels fall to basal levels as the T cell compartment rehomes. This mechanism prevents excessive proliferation and preserves T cell homeostasis. Recent studies have found that IL-7-induced proliferation requires intermittent (rather than continuous) signaling, and that TCR engagement provides this intermittency. 23 T cells with insufficient affinity for peripheral (self) TCR ligands die after prolonged IL-7 signaling. This mechanism maintains a population of T cells with the appropriate affinity for the self-ligand. In addition to IL-7, IL-15 signaling is important for CD8+ T cell survival and proliferation. 24,25,26 IL-15 enhances homeostatic proliferation of memory CD8+ cells, but IL-15 alone is insufficient for homeostatic proliferation of naive CD8 T cells. 27 Naive CD8+ cells also require MHC I engagement for homeostatic proliferation. 28 Emerging data indicate that memory CD4+ can also respond to IL-15. 29,30,31 Finally, TGB-β may act as a brake on homeostatic proliferation-induced autoimmunity by attenuating IL-15 signaling.32,33,34,35,36,37

[0172] PTPN2, a protein tyrosine phosphatase gene product that attenuates TCR signaling in CD4+ and CD8+ cells, has been implicated in autoimmunity in humans. 38,39 T cell knockout of PTPN2 in a mouse model resulted in faster lymphopenia-induced CD8+ proliferation compared to control animals. Adoptive transfer of PTPN2-deficient CD8+ cells into congenic hosts resulted in effector / memory differentiation and autoimmunity compared to adoptive transfer of control CD8+ cells. 40 This response was IL-7 independent. miRNA-181a enhances TCR signaling, in part by suppressing the expression of other protein phosphatases. 41 Thus, miRNA-181 or other miRNAs may inhibit PTPN2 expression and thereby attenuate lymphopenia-induced proliferation. It has been suggested that transcription factors may control the ability of hematopoietic stem cells to rehome the lymphoid compartment. For example, Hoxb4 signaling can promote a CD4+ central memory (CD44hiCD62L+) phenotype in hematopoietic stem cells in response to lymphopenia. 42 In competitive adoptive transfer experiments, Hoxb4-overexpressing central memory cells contributed less to lymphoid organ reconstitution than wild-type central memory cells. Finally, the integrin CD18 (lymphocyte function-associated antigen 1, or LFA-1) functions in the trafficking of naive T cells between the intestine and secondary lymphoid organs43,44 and has been implicated in intestinal autoimmunity. 45 Adoptive transfer of CD4+ CD18- / - cells into Rag- / - hosts demonstrated that CD18 was required for both fast and slow lymphopenia-induced proliferation. 46 The above studies illustrate the importance of non-cytokine regulators of homeostatic proliferation that bias effector memory phenotypes.

[0173] Another potential approach to overcome homeostatic proliferation as a barrier to transplantation is to specifically delete potentially pathological CD8+ cells in transplant recipients. Yamada et al. utilized this approach by using anti-CD8 mAbs during lymphodepletion in a mixed chimerism model of MHC-mismatched kidney transplantation in nonhuman primates. 54 Their finding of reduced Tmem responses in CD8-depleted animals is encouraging. This same group subsequently studied Alefacept, a fusion protein of the extracellular CD2-binding portion of the human leukocyte function antigen-3 (LFA-3) adhesion molecule. 55 This agent is thought to interfere with cytotoxic effector memory T cell proliferation by blocking the interaction between effector memory CD2+ cells and LFA-3. Alefacept therapy for psoriasis preferentially depleted CD4+CD45RO+ effector memory cells, which correlated with clinical improvement of skin lesions. 56 Alefacept preferentially and reversibly depleted CD8+ effector memory (CD28-CD95+) cells in a non-human primate transplantation model.157 The CD28- cells in this model were CD2hi, helping to explain the ability of Alefacept to preferentially deplete CD8+ cells.

[0174] Posttransplant cyclophosphamide is an attractive approach to prevent GVHD by depleting alloreactive CD8+ cells that might otherwise survive induction therapy.58,59 Recent data suggest that posttransplant cyclophosphamide primarily targets rapidly dividing allospecific cells, relatively sparing naive cells essential for maintaining immune competence after HSCT.60 CD4+Foxp3+ Tregs appear to be resistant to cyclophosphamide and are rapidly restored after cyclophosphamide induction for allogeneic bone marrow transplantation.61 Preservation of Tregs may be a partial mechanism by which cyclophosphamide prevents GVHD.

[0175] Thangavelu et al. (2005) demonstrated prolonged and severe CD4+ T-lymphopenia in rheumatoid arthritis (RA) patients after lymphodepletion therapy. The poor reconstitution could result from either reduced new T cell production through the thymus or poor peripheral expansion of residual T cells. Interleukin 7 (IL-7) is known to stimulate the thymus to produce new T cells and expand circulating mature T cells, thereby playing a crucial role in T cell homeostasis. In this study, we demonstrated reduced levels of circulating IL-7 in a cross-section of RA patients. IL-7 production by bone marrow stromal cell cultures was also impaired in RA. To investigate whether such IL-7 deficiency could explain the long-term lymphopenia observed in RA after therapeutic lymphodepletion, we compared RA and solid tumor patients treated with high-dose chemotherapy and autologous progenitor cell rescue. Chemotherapy rendered all patients similarly lymphopenic, but whereas the cancer patients experienced a decline by 3–4 months, this was maintained at 12 months in the RA patients. Both cohorts produced naive T cells containing T cell receptor excision circles. The main distinguishing feature between the groups was the failure to expand peripheral T cells in the RA, particularly memory T cells during the first 3 months after treatment. Most importantly, there was a four-fold increase in serum IL-7 levels in non-RA control individuals during lymphopenia, whereas there was no increase in serum IL-7 levels in the RA patients. Thus, our data suggest that RA patients are relatively IL-7 deficient, and that this deficiency is likely to be an important contributor to the poor initial T cell reconstitution in RA after therapeutic lymphodepletion. Furthermore, in patients with RA with stable and well-controlled disease, IL-7 levels positively correlated with T cell receptor excision circle content in CD4+ T cells, demonstrating a direct effect of IL-7 on thymic activity in this cohort. EXAMPLES

[0176] The following examples demonstrate that high doses of glucocorticoid receptor agonists can cause near complete lymphodepletion of peripheral blood lymphocytes, and can further reduce the number of germinal centers in lymphoid organs and deplete thymic lymphocytes. These effects are achieved without substantially affecting cell counts of neutrophils, platelets, RBCs, and stem cells (both HSCs and MSCs).

[0177] These examples also show that this lymphodepletion profile of high-dose glucocorticoid agonists is similar to that of standard chemotherapy regimens (based on cyclophosphamide (Cy) and fludarabine (Flu)), but does not induce concomitant weight loss, a common measure of toxicity of such chemotherapy regimens.

[0178] High-dose glucocorticoid agonists thus represent a non-myeloablative regimen that can produce an "immune reset" with efficacy comparable to chemotherapy but without the attendant toxicity. High-dose glucocorticoid receptor agonists therefore represent a promising therapy for use in the treatment of diseases mediated by immune cells such as lymphocytes.

[0179] Example 1 - Reduction of secondary and primary lymphatic sites with immunosuppressants Acute high dose dexamethasone may also be referred to herein as Dex, AugmenStem™, PlenaStem™, or AVM0703.

[0180] Mice were injected intraperitoneally on day 0 with 114.6 mg / kg dexamethasone base (HED 9.32 mg / kg) in male mice and sacrificed 96 hours after dexamethasone injection. Mice were sacrificed by exsanguination, followed by flushing of residual blood cells into the thoracic jugular vein via retrograde perfusion with 5 U heparin / ml PBS. Spleens were removed, wet weighted, and fixed in 10% formalin. Spleens were then sectioned by proprietary methods, incubated with FITC-PNA for 24 hours at 4°C, washed, mounted on slides, and immunofluorescence images were acquired. Immunofluorescence signals were quantified using Metamorph software. Sample images and results normalized to spleen area are shown in Figure 1.

[0181] Control mice have significant FITC-PNA immunofluorescence, while mice injected with dexamethasone sodium phosphate have almost no immunofluorescence signal. FITC-PNA labels germinal centers (non-exclusively, in spleen and lymph nodes). This example demonstrates the ability of high-dose dexamethasone to reduce the number of germinal centers (GCs) in lymphoid organs, which can eliminate autoreactive immune memory. Reduction of the number of germinal centers in lymphoid organs can also push cancer cells (e.g., germinal center lymphoma) or residual HIV-infected T cells that bind to the niche of these centers into the circulation, where they can be eliminated by the immune system or standard therapy.

[0182] Figure 2 shows the dose response of acute high dose dexamethasone (HED) effects on germinal center numbers in mouse spleens. Germinal center reduction is seen at 6 mg / kg HED, but is not significant until 9 and 12 mg / kg HED doses.

[0183] Rats were administered (IV or PO) between 3.23, 6.45, and 12.9 mg / kg dexamethasone HED (rat doses 20, 40, and 80 mg / kg) to determine GC and marginal zone suppression after 48 hours. In rats, the 12.9 mg / kg HED Dex dose maximally suppressed both GC and marginal zone number and area, as shown in Figures 3 and 4. Formalin-fixed spleens were cross-sectioned into 5 pieces, trimmed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Measurements of perivascular lymphatic sheath (PAL) diameter and marginal zone (MZ) width in the area of ​​the white pulp where the PAL diameter is largest were taken using an ocular micrometer. BCL-6 immunohistochemical staining in rat spleens was evaluated to determine GC area using automated image analysis.

[0184] Acute high-dose dexamethasone also reduces thymus mass and volume (Figure 5). Male mice were orally administered vehicle or 3, 6, 9, and 12 mg / kg HED dexamethasone base dexamethasone sodium phosphate on day 0 and sacrificed 48 hours after dexamethasone treatment. Mice were sacrificed by exsanguination and then flushed with residual blood cells via retrograde perfusion into the thoracic jugular vein with 5U heparin / ml PBS. Thymuses were removed from each mouse, wet weights were measured, and graphed as thymus weight / body weight.

[0185] Example 2 - Immunosuppressant lymphodepletion in mice and rats 24-48 hours after acute administration of dexamethasone with neutrophil-, RBC-, platelet-, and stem cell-sparing properties. Preliminary dose-escalation experiments performed in naïve mouse and rat models showed that administration of high-dose dexamethasone resulted in complete lymphodepletion (Figure 11, right side). High doses of dexamethasone were able to induce ~98% reduction in CD4+, CD8+, Tregs, and B cell populations measured 48 hours after administration, supporting rapid elimination of autoimmune pathophysiological substrates. Early stage validation showed that acute high-dose dexamethasone has a pharmacokinetic half-life of 2-3 hours and 4-5 days, which rules out prolonged immunosuppression. In addition, oral dosing of acute high-dose dexamethasone had comparable efficacy to IV dosing, supporting the use of acute high-dose dexamethasone as a single oral treatment.

[0186] As shown in Figure 6, IV or PO administration of dexamethasone at 20 (3.2 HED), 40 (6.5 HED) or 80 (12.9 HED) mg / kg to male Lewis rats weighing 250-300 grams significantly reduced lymphocyte counts at all doses compared to placebo 48 hours after administration. In contrast, neutrophils were not reduced by acute high-dose dexamethasone, as shown in Figure 7. In fact, neutrophil counts increased with all doses of dexamethasone, likely due to a demargination effect. RBC, platelets, Hct, and HgB were not affected by dexamethasone treatment.

[0187] Acute oral administration of dexamethasone to C57Bl male mice at HED of 3 mg / kg (n=4), HED of 6 mg / kg (n=6), 9 mg / kg (n=4), 12 mg / kg (n=4), 15 mg / kg (n=4), or 17.5 mg / kg (n=4) reduced CD3+ T lymphocytes by 65% ​​and CD4+ T lymphocytes by 75% (Figure 8), CD8+ T lymphocytes by 56% and Tregs by 78% (Figure 9), natural killer cells (NK) by 87% and B lymphocytes by 83% (Figure 10), and reduced absolute lymphocyte counts by 84%, but not neutrophils (Figure 11), RBCs (Figure 12), or platelets (Figure 13). 12 ) were spared. Blood was collected for complete blood chemistry (CBC) and flow cytometry 24-48 hours after oral dexamethasone gavage. Near-complete lymphodepletion was observed in normal mice at HED doses above 12 mg / kg. In tumor-bearing mice, near-complete lymphodepletion was observed at HED doses above 6 mg / kg.

[0188] Acute high-dose dexamethasone activates receptor-mediated apoptosis via the caspase pathway, resulting in lymphodepletion or lymphocytoplasty of all lymphocytes, depending on the dose used. As expected from its receptor-mediated mechanism of action, dexamethasone induces lymphodepletion, but neutrophils, platelets, and red blood cells (RBC) cells are spared due to the lack or difference of glucocorticoid receptors. With high doses of dexamethasone, a trend towards elevated neutrophil counts above placebo in both peripheral blood and bone marrow was observed, supporting the possibility of protection against infection during lymphodepletion treatment.

[0189] Notably, high-dose dexamethasone did not significantly alter the amount of hematopoietic stem cells in mice (Figure 13). Thus, the non-myeloablative regimen represented by acute high-dose dexamethasone may eliminate the need for stem cell transfusion for hematopoietic recovery after immune reset.

[0190] Example 3 - Immunosuppressant lymphodepletion in humans 36-48 hours after acute administration of dexamethasone with neutrophil-, RBC-, platelet-, and stem cell-sparing properties. Four human patients (three with osteoarthritis of the knee and one with an aortic aneurysm) were given an acute oral dose of 3 mg / kg dexamethasone base equivalent (all doses described in these examples are dexamethasone base equivalent). Blood was drawn before and 48 hours after drug treatment for CBC analysis and flow cytometry to determine lymphocyte and other blood cell populations. Serum was analyzed for cytokine levels. For one patient, a pretreatment CBC was not obtained, so normalized flow cytometry data is shown for only three patients. According to the unnormalized flow cytometry data, only two of four patients responded to dexamethasone with lymphodepletion (Figures 14, 15, and 16), while two of four patients showed a lymphocytosis response in CD3 and CD4 lymphocytes and one of four patients showed a lymphocytosis response in CD8, B lymphocytes, and NK cells to this dose of dexamethasone. 48 hours after acute oral dexamethasone base (3 mg / kg), 3 of 4 patients showed elevated levels of IL-2 and 4 of 4 patients showed elevated levels of IL-15 (Figure 17). IL-6, a cytokine known to be a major driver of the potentially fatal cytokine release syndrome (CRS), was not elevated in any patient. Based on the lymphocytosis response observed in 2 of 4 non-cancer patients at the 3 mg / kg dose, the preferred lymphodepleting dose would be 3 mg / kg or higher based on the increased sensitivity of tumor-bearing mice to dexamethasone (minimum lethal dose was HED 43 mg / kg in tumor-bearing mice compared to HED 114 mg / kg in healthy mice (Scorza Barcellona, ​​1984)).

[0191] Bone marrow was harvested 48 hours after dexamethasone administration and mesenchymal stem cell (MSC) numbers were determined by colony forming assay fibroblast (CFU-F). Oral administration of 3 mg / kg dexamethasone base increased iliac crest bone marrow (BM) MSCs by approximately two-fold (Figure 18). The tri-lineage differentiation capacity of BM MSCs was also determined in equine studies. 6 mg / kg HED doubled sternal BM MSC stem cell numbers 48 hours after a one-hour IV infusion in horses, but did not alter the tri-lineage differentiation capacity of MSCs into osteocytes, chondrocytes, and adipocytes.

[0192] Example 4 - Comparison of acute 12 mg / kg and 17.5 mg / kg dexamethasone base HED with standard Cy (cyclophosphamide) Flu (fludarabine) chemotherapy regimens. Adult male mice were gavaged with dexamethasone base at 12 mg / kg or 17.5 mg / kg HED on day -2. Another group of mice received Cy at 166 mg / kg (HED 500 mg / m2) on days -5 and -4, and fludarabine 10 mg / kg (HED 30 mg / m2) IP on days -5, -4, -3, and -2. A third group of mice received Cy at 166 mg / kg (HED 500 mg / m2) on day -5, fludarabine 10 mg / kg (HED 30 mg / m2) IP on day -5, and then dexamethasone base at 12 mg / kg or 17.5 mg / kg HED orally on day -2. CBC and flow cytometry results are shown in Figures 19-24, and body weights are shown in Figure 25.

[0193] Dexamethasone base at 12 mg / kg or 17.5 mg / kg HED administered 12-72 hours prior to blood collection produced lymphodepletion profiles comparable to standard 2-day Cy and 4-day Flu, as did single Cy on day -5 and single Flu on day -5 in combination with 12 mg / kg dexamethasone HED on day -2 (Figure 23). Single Cy and single Flu doses were equally effective when administered on days -6, -4, or -3. The lymphodepletion profile of dexamethasone alone may be preferable as it does not deplete absolute lymphocyte counts as dramatically as CyFlu, and the degree of this lymphodepletion may be associated with neuroedema if adoptive cell therapy is administered after CyFlu.

[0194] The standard repeated CyFlu regimen significantly reduced body weight as a general measure of toxicity, whereas 12 mg / kg or 17.5 mg / kg dexamethasone base HED did not affect body weight. The combination of one Cy dose and one Flu dose on day -5 with 12 mg / kg dexamethasone HED had significantly less effect on body weight than the standard CyFlu regimen, whereas the combination of one Cy dose and one Flu dose on day -5 with 17.5 mg / kg dexamethasone HED did not affect body weight (Figure 25). This demonstrates that acute high-dose dexamethasone has a lymphodepletion profile comparable to standard chemotherapy based on cyclophosphamide (Cy) and fludarabine (Flu) but without the associated weight loss, confirming the safety of dexamethasone formulations compared to chemotherapy.

[0195] Additionally, in a double-blind, controlled equine clinical trial with acute high-dose dexamethasone, no adverse side effects were observed up to 70 days.

[0196] Data collected to date suggest that acute high-dose dexamethasone offers a safety profile consistent with that of licensed DSP products. The proposed doses of acute high-dose dexamethasone (HED 3–18 mg / kg) are comparable to or less than the cumulative doses of DSP used safely and effectively in daily pulse therapy for up to 5 days for a variety of conditions, and DSP has been well tolerated in clinical use of physician-directed high-dose pulse therapy (Han et al., 2014; Annane et al., 2004; Ayache et al., 2014). Preliminary studies conducted in a small number of human osteoarthritis patients revealed that acute high-dose dexamethasone increased plasma IL-2 and IL-15 cytokine levels but did not affect concentrations of proinflammatory cytokines (e.g., IL-6) as seen following chemotherapy regimens (US9855298B2). A full analysis of clinical chemistry in mice treated with acute high-dose dexamethasone at increasing HED doses (6-12 mg / kg) showed that the acute oral dose was safe and did not cause elevations in clinical chemistry levels outside the normal range, including cholesterol and total protein. Furthermore, although chronic low doses of DSP have been shown to cause undesirable side effects including weight gain and glucose elevation (Ferris & Kahn, 2012), glucose levels after acute high-dose dexamethasone were not found to be elevated above the normal range. Overall, the lymphodepleting activity of acute high-dose dexamethasone and its safety profile strongly support its use as an immune resetting treatment with efficacy comparable to chemotherapy for autoimmune diseases.

[0197] Other standard chemotherapy regimens that may be administered as a single dose(s) on day -1 or -2 or -3 or -4 or -5 and combined with about 3 to about 12 mg / kg dexamethasone on day -2 include: Cy 120 mg / kg and Flu 75 mg / m2; 30 mg / m2 flu and 50 mg / kg Cy and 200 cGy TBI; Cy 1500 mg / m2 and bendamustine 120 mg / m2; between about 300 mg / m2 and about 2300 mg / m2 of Cy; between about 10 mg / m2 and about 900 mg / m2 of Flu; Cy 600 mg / m2 and Flu 30 mg / m2; busulfan and melphalan and Flu; busulfan (dose adjusted according to body weight) and thiotepa (10 mg / kg) and fludarabine (160 mg / m2); Flu 30 mg / m2 and Cy 300 mg / m2 and Mensa 300 mg / m2; Flu 30 mg / m2 and Cy 60 mg / m2 and Alemtuzumab 0.2 mg / kg.

[0198] Example 5 - Treatment of patients with autoimmune diseases For example, patients with autoimmune diseases, including but not limited to SLE, psoriasis, rheumatoid arthritis, psoriatic arthritis, type I diabetes, multiple sclerosis, Sjogren's syndrome, scleroderma, Graves' disease, Hashimoto's thyroiditis, celiac disease, Addison's disease, myasthenia gravis, autoimmune hepatitis, antiphospholipid syndrome, and biliary cholangitis, can be treated with doses of glucocorticoid immunosuppressants, or dexamethasone. Doses of acute high-dose dexamethasone (as base) range from about 3 mg / kg to about 24 mg / kg, with doses of about 9 mg / kg to about 18 mg / kg being preferred.

[0199] A dose of acute high-dose dexamethasone reduces the number of B lymphocytes by more than 90%, and since memory B cells make up approximately 50% of the B cell compartment in people over 20 years of age, the memory B cell population is also reduced by more than 90%. The patient's autoimmune aggressive B cells undergo apoptosis and the patient no longer has an active autoimmune attack. The patient's physical symptoms improve or disappear. In most patients, remission from the autoimmune disease lasts indefinitely, but if relapse occurs, repeat doses of glucocorticoid immunosuppressants, dexamethasone base, or CD26 antagonists may be administered. Repeat treatments can be administered as frequently as once a month if necessary, but preferably no more than once a year, and most preferably no more than once every five years.

[0200] Example 6 - Treatment of residual HIV Patients with residual HIV are treated with glucocorticoid immunosuppressants, or dexamethasone. Acute high-dose dexamethasone (as base) doses range from about 3 mg / kg to about 24 mg / kg, with doses of about 9 mg / kg to about 18 mg / kg being preferred. This treatment eliminates the niche in the spleen where HIV harbors and releases infected T cells into the circulation where they can be killed by standard HIV therapy, including antiretroviral drugs (including, but not limited to, nucleoside reverse transcriptase inhibitors (NTRIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), fusion and entry inhibitors, pharmacokinetic enhancers, and integrase strand transfer inhibitors (INSTIs)).

[0201] Example 7 - Treatment of germinal center lymphoma (e.g., Burkitt's lymphoma) For example, patients with germinal center lymphoma, such as, but not limited to, Burkitt's lymphoma or diffuse large B-cell lymphoma (DLBCL), are treated with glucocorticoid immunosuppressants, or dexamethasone. Acute high-dose dexamethasone (as base) doses range from about 3 mg / kg to about 24 mg / kg, with doses of about 9 mg / kg to about 18 mg / kg being preferred. This treatment eliminates the niche in the spleen where germinal center lymphoma binds, sending the cells into the circulation where they can be eliminated more completely or at lower doses by standard chemotherapy such as R-CHOP, or antibodies against CD20 such as Rituxan, Bexar, or Zevalin, or antibodies against CD22 or CD70 such as Lymphoside or Borsetuzumab Mahodotin, or Bcl-2 inhibitors such as oblimersen sodium, ABT-737 (oral form navitoclax, ABT-263), or fenretinide, or Syk inhibitors such as fostamatinib or tamatinib, or proteasome inhibitors such as bortezomib (Velcade), or COMPADME, CODOX-M / IVAC. Recurrence rates are reduced and disease-free survival rates are increased.

[0202] Example 8 - Conversion of dexamethasone doses to equivalent doses of other glucocorticoids To calculate the equivalent dosage of another glucocorticoid, the dose of dexamethasone is entered into a publicly available glucocorticoid conversion calculator, preferably http: / / www.medcalc.com. The total dosage is then determined based on the half-life of the glucocorticoid. For example, 3-12 mg / kg of dexamethasone is converted to 19-75 mg / kg of prednisone. The biological half-life of prednisone is about 20 hours, and the biological half-life of dexamethasone is about 36-54 hours. Thus, prednisone is dosed between 19-75 mg / kg every 24 hours for the equivalent biological dosage.

[0203] Example 9 - Treatment of patients with autoimmune disease with prednisone Patients with autoimmune diseases, such as, but not limited to, SLE, psoriasis, rheumatoid arthritis, psoriatic arthritis, type I diabetes, multiple sclerosis, Sjogren's syndrome, scleroderma, Graves' disease, Hashimoto's thyroiditis, celiac disease, Addison's disease, myasthenia gravis, autoimmune hepatitis, antiphospholipid syndrome, and biliary cholangitis, can be treated with acute high-dose prednisone. The dose of acute high-dose prednisone ranges from about 19 mg / kg to about 150 mg / kg, with a dose of about 56 mg / kg to about 112 mg / kg being preferred, with a repeat (second) dose of this dose 24 hours later and an optional repeat (third) dose of this dose 48-72 hours after the first dose.

[0204] A dose of acute high-dose prednisone reduces the number of B lymphocytes by more than 90%, and since memory B cells make up approximately 50% of the B cell compartment in people over 20 years of age, the memory B cell population also reduces by more than 90%. The patient's autoimmune aggressive B cells undergo apoptosis and the patient no longer has an active autoimmune attack. The patient's physical symptoms improve or disappear. In most patients, remission from the autoimmune disease lasts indefinitely, but if relapse occurs, repeat doses of prednisone can be administered. Repeat treatments can be as frequent as once a month if necessary, but preferably no more than once a year, and most preferably no more than once every five years.

[0205] Example 10 - Comparison of 15 mg / kg dexamethasone base HED with standard chemotherapy regimens Previous studies have shown that standard chemotherapy regimens for aggressive non-Hodgkin lymphoma have significant toxicity in the A20 B cell lymphoma mouse model (Bascus et al., 2016).

[0206] The standard chemotherapy regimen for aggressive NHL in patients, as well as the regimen most commonly used for indolent NHL, is a combination of cyclophosphamide, doxorubicin, vincristine, and prednisone / steroids (CHOP) administered every 21 days for 6–8 cycles. Bascus et al. (2016) evaluated the efficacy and toxicity of CHOP in an A20 B-cell lymphoma mouse model.

[0207] In the study by Bascus et al. (2016), 8-10 week old female BALB / c mice were used for in vitro experiments. Animals were housed in racks with filtered air and food and water ad libitum with a 12:12 h light / dark cycle. The A20 cell line was derived from B lymphocytes of a spontaneous reticulum cell sarcoma from an aged BALB / cAnN mouse and was obtained from the American Type Culture Collection (Manassas, VA, USA). The following doses were used in each chemotherapy cycle: cyclophosphamide 100 mg / kg ip, doxorubicin 6 mg / kg ip, vincristine 0.1 mg / kg ip, and dexamethasone 0.2 mg / kg ip. At 25 days post-tumor implantation (pti), groups of mice (n = 9) inoculated with the A20 cell line were treated with one cycle of chemotherapy (CHOP × 1), two cycles of chemotherapy (CHOP × 2), or PBS as a control and followed for tumor growth. Tumor volumes (mm3) were measured every 2–3 days. 3 To assess in vivo toxicity, body weight was measured before and after CHOP administration.

[0208] In this study, mice were housed, inoculated, and treated in the same manner as reported in the study by Bascus et al. (2016). Mice were treated with 15 mg / kg dexamethasone basic HED or PBS as a control and followed for tumor growth. Dexamethasone dosing was performed at 15 mg / kg HED on days 7, 10, 18, 23, 24, 28, 35, and 42 after A20 2M tumor cell inoculation. Tumor volume (mm 3 To assess in vivo toxicity, body weight was measured before and after dexamethasone administration.

[0209] The efficacy of 15 mg / kg dexamethasone base HED compared to CHOP and PBS controls is shown in Figure 26. As can be seen in Figure 26, the efficacy of 15 mg / kg dexamethasone base HED was greater than one cycle of CHOP, but was not as effective as two cycles of CHOP in terms of controlling tumor volume.

[0210] However, Figure 27 demonstrates that 15 mg / kg dexamethasone base HED has a favorable toxicity profile compared to two cycles of CHOP. The weight loss seen in mice treated with two cycles of CHOP (Figure 27B) was much greater than that seen for mice treated with 15 mg / kg dexamethasone base HED (Figure 27A). In addition, 18% of mice treated with two cycles of CHOP died from CHOP treatment, whereas no mice died from dexamethasone treatment. Thus, it can be concluded that dexamethasone can be as effective as traditional chemotherapy treatments without the associated toxicity.

[0211] The results of the statistical analysis of the 15 mg / kg dexamethasone base HED compared to the PBS control are shown in Figure 28A, demonstrating significant differences in tumor volume at multiple time points during the study, with dexamethasone-treated mice having significantly reduced tumor volumes.

[0212] Example 11 - Sensitization to chemotherapy This example shows that glucocorticoid therapy reduces the dose required for effective chemotherapy.

[0213] The A20 B cell lymphoma mouse model was used essentially as described in Example 10, except that male rather than female mice were used. Mice were inoculated on day 0. Mice treated with PBS only ("Control") or with HED 15 mg / kg dexamethasone base on days 11 and 14 ("AVM0703") showed continued tumor growth, with a high growth rate from day 20 onwards (see Figure 29). "Combination therapy," which consisted of administration of HED 15 mg / kg dexamethasone base on day 11 followed by Cy / Flu therapy (13.5 mg / kg HED cyclophosphamide and 0.8 mg / kg HED fludarabine) on day 14, showed a steady reduction in tumor volume, which after 24 and 26 days was reduced to a similar extent to tumor size in mice treated with two doses of Cy / Flu chemotherapy (13.5 mg / kg HED cyclophosphamide and 0.8 mg / kg HED fludarabine) on days 11 and 14 (Figure 29). Subjects treated with either combination therapy or a double dose of Cy / Flu show a reduction in tumor volume after 14 days.

[0214] Example 12 - Tumor selectivity of glucocorticoids in lymphoma. This example demonstrates that administration of high dose dexamethasone preferentially affects cancer cells.

[0215] The A20 B cell lymphoma mouse model was used essentially as described in Example 10, except that male mice were used instead of female mice. Mice were inoculated on day 0 and treated with either PBS (placebo) or 15 mg / kg HED dexamethasone on day 28. Cell counts were measured by using a Complete Blood Count analyzer (CBC) and the results are shown in Table 2 below. [Table 2] The data presented in TIFF0007671125000003.tif221165 Table 2 support the finding that tumor-bearing mice have increased sensitivity to dexamethasone (described herein; see, e.g., Example 3). Peripheral lymphodepletion is not observed in tumor-bearing mice, likely because dexamethasone is absorbed by tumors. In contrast, healthy mice show lymphodepletion. This indicates that dexamethasone has a direct effect on tumors, suggesting the possibility of deep-seated lymphodepletion in tumors and thus a superior tumor targeting profile for high-dose glucocorticoid therapy as described herein. Dexamethasone is more specific for tumor infiltrating lymphocytes (TILs) than peripheral lymphocytes when tested in tumor-bearing mice.

[0216] Example 13 - Tumor response to increasing doses of glucocorticoid treatment. The purpose of this study was to evaluate the effect of different doses of dexamethasone on tumors. After establishing tumors in 10-week-old BALB / c mice, mice were randomized by excel into four groups with approximately equal mean tumor volumes. Mice were dosed with 6 mg / Kg HED dexamethasone weekly, 15 mg / Kg HED dexamethasone weekly, or 21 mg / Kg HED dexamethasone weekly for four cycles (five mice per treatment group). Tumors were measured using the published formula V = L x W 2 x 0.5 to 1500 mm 3 Once the tumor volume reached 0.1 mg / kg, the mice were considered to be at endpoint and removed. As shown in Figure 30, increasing doses of dexamethasone decreased the density as mean cell number per tumor.

[0217] References A number of publications have been cited above in order to more fully describe and disclose the invention and the state of the art to which it pertains. Full citations for these references are provided below. TIFF0007671125000004.tif217169TIFF0007671125000005.tif248167TIFF0007671125000006.tif250167TIFF0007671125000007.tif163167

[0218] Numbered paragraphs The following numbered paragraphs describe different aspects of the inventors' propositions and form part of the detailed description. 1. A pharmaceutical composition comprising a glucocorticoid for use in treating a lymphocyte-mediated disease in a subject, said treatment comprising administering a dose of said pharmaceutical composition to a patient to deliver a dose of said glucocorticoid equivalent to about 3-26 mg / kg human equivalent dose (HED) of dexamethasone base; The pharmaceutical composition comprises one or more pharma- ceutically acceptable carriers, preservatives, and / or chelating agents. Pharmaceutical compositions. 2. The pharmaceutical composition of paragraph 1, wherein the lymphocyte-mediated disease is an autoimmune disease. 3. The pharmaceutical composition according to paragraph 1, wherein said lymphocyte mediated disease is residual HIV disease. 4. The pharmaceutical composition of paragraph 1, wherein the lymphocyte mediated disease is germinal center lymphoma. 5. The pharmaceutical composition of paragraph 1, wherein the lymphocyte-mediated disease is graft-versus-host disease. 6. The pharmaceutical composition of paragraph 1, wherein the lymphocyte mediated disease is an allergic disorder, and optionally the allergic disorder is asthma. 7. The pharmaceutical composition of paragraph 2, wherein the autoimmune disease is selected from the group consisting of type 1 diabetes, multiple sclerosis, amyotrophic lateral sclerosis, scleroderma, pemphigus, and lupus. 8. The pharmaceutical composition according to any of paragraphs 1 to 7, comprising a preservative, said preservative being a sulfite. 9. The pharmaceutical composition according to any of paragraphs 1 to 8, comprising a chelating agent, said chelating agent being EDTA. 10. The pharmaceutical composition according to any of paragraphs 1 to 9, wherein the glucocorticoid comprises dexamethasone, and optionally the dexamethasone is selected from the group consisting of dexamethasone base, dexamethasone sodium phosphate, and dexamethasone acetate. 11. The pharmaceutical composition according to paragraph 10, wherein the dexamethasone is dexamethasone sodium phosphate. 12. The pharmaceutical composition according to any of paragraphs 1 to 11, wherein the dose of said pharmaceutical composition is a single acute dose or a total dose administered over a period of about 72 hours. 13. The pharmaceutical composition of any of paragraphs 1-12, wherein the pharmaceutical composition is administered as an intravenous (IV) or oral dose, optionally wherein the IV or oral dose is administered as a single IV or oral dose. 14. The pharmaceutical composition according to any of paragraphs 1 to 13, wherein the pharmaceutical composition is an aqueous glucocorticoid solution. 15. The pharmaceutical composition of any of paragraphs 1-14, wherein the pharmaceutical composition is administered at a dose equivalent to a human equivalent dose (HED) of at least about 4 mg / kg, at least about 5 mg / kg, at least about 6 mg / kg, at least about 7 mg / kg, at least about 8 mg / kg, at least about 9 mg / kg, at least about 10 mg / kg, at least about 11 mg / kg, at least about 12 mg / kg, at least about 15 mg / kg, at least about 18 mg / kg, or at least about 24 mg / kg of dexamethasone base.

Claims

1. 1. A pharmaceutical composition comprising a glucocorticoid for use in treating a lymphocyte-mediated disease in a subject, said treatment comprising administering a single acute dose of said pharmaceutical composition to a patient to deliver a dose of said glucocorticoid equivalent to a human equivalent dose (HED) of 9-26 mg / kg dexamethasone base; The pharmaceutical composition is administered as a single intravenous (IV) or oral dose; the glucocorticoid is any one of dexamethasone, dexamethasone base, dexamethasone sodium phosphate, dexamethasone acetate, or betamethasone; The pharmaceutical composition comprises one or more pharma- ceutically acceptable carriers, preservatives, and / or chelating agents; The lymphocyte-mediated disease is selected from autoimmune diseases, cancer, residual HIV disease, graft-versus-host disease, and allergic disorders; Pharmaceutical compositions.

2. 2. The pharmaceutical composition of claim 1, wherein the lymphocyte-mediated disease is an allergic disorder, and the allergic disorder is asthma.

3. 2. The pharmaceutical composition of claim 1, wherein the lymphocyte mediated disease is cancer, and the cancer is germinal center lymphoma.

4. 2. The pharmaceutical composition of claim 1, wherein the lymphocyte-mediated disease is cancer, and the cancer is leukemia, lymphoma, or multiple myeloma.

5. 2. The pharmaceutical composition of claim 1, wherein the lymphocyte-mediated disease is an autoimmune disease selected from the group consisting of type 1 diabetes, multiple sclerosis, amyotrophic lateral sclerosis, scleroderma, pemphigus, and lupus.

6. The pharmaceutical composition according to any one of claims 1 to 5, comprising a preservative, said preservative being a sulfite.

7. The pharmaceutical composition of any one of claims 1 to 6, comprising a chelating agent, said chelating agent being EDTA.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the glucocorticoid comprises dexamethasone.

9. The pharmaceutical composition according to any one of claims 1 to 7, wherein the glucocorticoid is selected from the group consisting of dexamethasone base, dexamethasone sodium phosphate, and dexamethasone acetate.

10. The pharmaceutical composition according to any one of claims 1 to 7, wherein the glucocorticoid is dexamethasone sodium phosphate.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein the dose of said pharmaceutical composition is a single acute dose administered as a single IV dose over 0.25 to 2 hours.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the pharmaceutical composition is an aqueous glucocorticoid solution.

13. 13. The pharmaceutical composition of any one of claims 1 to 12, wherein the pharmaceutical composition is administered at a dose equivalent to a human equivalent dose (HED) of at least 9 mg / kg, at least 10 mg / kg, at least 11 mg / kg, at least 12 mg / kg, at least 15 mg / kg, at least 18 mg / kg, or at least 24 mg / kg of dexamethasone base.

14. 1. A pharmaceutical composition comprising a glucocorticoid for use in treating a lymphocyte-mediated disease in a subject, said treatment comprising administering a dose of said pharmaceutical composition to a patient to deliver a single acute dose of said glucocorticoid equivalent to a human equivalent dose (HED) of 9-26 mg / kg of dexamethasone base; the glucocorticoid is any one of dexamethasone, dexamethasone base, dexamethasone sodium phosphate, dexamethasone acetate, or betamethasone; The pharmaceutical composition comprises one or more pharma- ceutically acceptable carriers, preservatives, and / or chelating agents; The lymphocyte-mediated disease is selected from an autoimmune disease, a cancer, a residual HIV disease, and an allergic disorder. Pharmaceutical compositions.

15. said lymphocyte mediated disease is an allergic disorder and said allergic disorder is asthma; said lymphocyte mediated disease is cancer and said cancer is germinal center lymphoma; the lymphocyte mediated disease is cancer, and the cancer is leukemia, lymphoma, or multiple myeloma; or The lymphocyte-mediated disease is an autoimmune disease selected from the group consisting of type 1 diabetes, multiple sclerosis, amyotrophic lateral sclerosis, scleroderma, pemphigus, and lupus; 15. The pharmaceutical composition of claim 14.

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