RU2795201C2 - Biomarkers of methotrexate-induced immune tolerance - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to therapy and immunology, and is intended for the treatment of a subject requiring treatment with acidic human α-glucosidase. A method of treating a subject comprises administering to the subject methotrexate and acidic human α-glucosidase; and detection of an erythropoiesis biomarker in a sample obtained from a subject over a period of time from at least one day to about 30 days after methotrexate administration. Further treatment with acid human α-glucosidase is continued with or without additional therapy to induce immune tolerance or immunosuppressive therapy based on the level of the erythropoiesis biomarker compared to that in the control. If the level of the erythropoiesis biomarker is approximately equal to or lower than the control level, the specified additional therapy is administered simultaneously with further treatment with acidic human α-glucosidase; or if the level of the biomarker of erythropoiesis is increased compared with the control, continue further treatment with acidic human α-glucosidase without the introduction of the said additional therapy. In other embodiments, a method for assessing the level of immune tolerance in a subject with Pompe disease and a method for assessing the level of immune tolerance to acidic human α-glucosidase is provided.

EFFECT: use of a group of inventions makes it possible to increase the effectiveness of treating a subject with acidic human α-glucosidase through a strategy to reduce an unwanted immune response.

16 cl, 1 tbl, 11 dwg, 3 ex

Description

[1] This application claims priority in US Provisional Application No. 62/609986, filed December 22, 2017, which is incorporated herein by reference in its entirety.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[2] The present invention relates generally to immunology, and in particular to methods of treating a subject with a therapeutic agent and evaluating methotrexate-induced immune tolerance to a therapeutic agent based on detection of erythropoiesis biomarkers.

BACKGROUND OF THE INVENTION

[3] Methotrexate has a long history of use in the treatment of malignant and autoimmune diseases (see, e.g. Kremer 2004). It was first described for the treatment of cancer in the 1940s, and high-dose courses of methotrexate continue to be used to treat a number of neoplastic conditions. In autoimmune conditions, among others, long-term weekly low-dose methotrexate is used to treat diseases such as rheumatoid arthritis and psoriasis (reviewed in Cronstein 2005; Taylor et al., 2008). More recent studies, however, have identified an alternative use of methotrexate to induce immune tolerance. A short-term, low-dose regimen of methotrexate to induce immune tolerance (ITI) has been shown to induce immune tolerance to enzyme replacement therapies (ERTs), such as recombinant human acid α-glucosidase (rhGAA, alglucosidase alfa) (Garman, Munroe et al. , 2004; Joseph, Munroe et al., 2008). Co-administration of low-dose short-term treatment with methotrexate and a therapeutic biologic has also been shown to induce long-term immune tolerance to the biologic, as demonstrated with mouse antithymocyte globulin used to treat graft rejection in allogeneic heart transplantation (Joseph, Neff et al., 2012). In a clinical observation in patients with high-risk classic infantile Pompe disease, an immune tolerance protocol in which short-term, low-dose methotrexate was co-administered with rituximab (a monoclonal antibody that depletes B cells) and optionally with intravenous immunoglobulin (IVIG), demonstrated success in providing long-term reduction in rhGAA anti-drug antibody (ADA) levels (Mendelsohn, Messinger et al. 2009, Messinger, Mendelsohn et al. 2012, Banugaria, Prater et al. 2013). Since rituximab alone has generally not been able to reproducibly induce long-term immune tolerance, methotrexate is thought to promote successful immune tolerance in this clinical regimen (Franchini, Mengoli et al., 2008, Collins, Mathias et al. 2009).

[4] Pompe disease is caused by a deficiency in the lysosomal acid α-glucosidase (GAA) gene. Patients with high-risk classic infantile Pompe disease lack endogenous GAA, the enzyme that breaks down glycogen in the lysosome. The absence of functional GAA leads to neuromuscular pathology, which occurs due to excessive accumulation of glycogen in muscle tissue. If left untreated, disease progression in these patients is rapid, with muscle damage (myopathy), respiratory weakness, eventually requiring invasive ventilation, heart failure, and death, usually within the first year of life. Enzyme replacement therapy with rhGAA is effective in the treatment of Pompe disease (Kishnani, Corzo et al., 2007). However, rhGAA monotherapy in high-risk patients with classic infantile Pompe disease usually induces high titers of anti-rhGAA ADA, which affects the effectiveness of treatment. To date, long-term immune tolerance to rhGAA has been successfully induced in clinical trials in several patients with classic infantile Pompe disease by co-administration of short-term, low-dose methotrexate with rituximab and IVIG. In these studies, patients were characterized by the absence of ADA for more than 35 months with ongoing treatment (Mendelsohn, Messinger et al., 2009; Messinger, Mendelsohn et al., 2012; Banugaria, Prater et al., 2013). However, the degree of immune modulation required to attenuate ADA responses varied among patients depending on prior enzyme replacement therapy: patients who had previously received ERT prior to treatment required additional immune modulation to ensure immune tolerance (Messinger, Mendelsohn et al., 2012 ). Although clinical observations are still limited, it is clear that successful ERT in high-risk patients with infantile Pompe disease will be more successful the sooner ERT immune tolerance is achieved to limit the development and potential impairment of the ADA immune response in relation to rhGAA (Banugaria, Prater et al., 2013). However, the tools available to monitor immune tolerance for the purpose of guiding immunomodulatory therapy may be of limited utility in eligible patients: drug antibodies are major markers of the degree of tolerance (or lack thereof), but such antibodies generally do not appear over a period of several weeks to months after starting treatment with a therapeutic biologic. At this point, critical time may have already been lost to effectively stimulate and maintain immune tolerance. The lack of immune tolerance markers that occur early in therapy is of particular concern in patients such as those with high-risk infantile Pompe disease, who deteriorate rapidly in the absence of effective therapy.

[5] Accordingly, there is a need for improved methods for assessing immune tolerance (e.g., early induction of immune tolerance) to guide strategies to reduce an unwanted immune response in patients receiving therapeutic agents capable of inducing immune responses, including therapeutic biologics such as rhGAA, with Pompe disease.

[6] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are incorporated herein by reference in their entirety.

BRIEF DESCRIPTION OF THE INVENTION

[7] The present invention provides methods of treatment and methods for assessing the level of immune tolerance to a therapeutic agent based on the detection of a biomarker of erythropoiesis.

[8] Accordingly, in one aspect of the present application, a method of treating a subject in need of treatment with a therapeutic agent is provided, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of the therapeutic agent; (c) detecting an erythropoiesis biomarker in a sample obtained from the subject over a period of time from at least one day to about 30 days after methotrexate administration; and (d) continuing further treatment with a therapeutic agent with or without additional therapy to induce immune tolerance or immunosuppressive therapy based on the level of the biomarker of erythropoiesis compared to that level in the control. In some embodiments, the change in the level of the erythropoiesis biomarker is associated with the induction of erythropoiesis. In some embodiments, where if the level of the erythropoiesis biomarker is approximately equal to or below that level in the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or where if the level of the erythropoiesis biomarker is elevated compared to that level in the control, then step (d) involves continuing further treatment with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, where if the level of the erythropoiesis biomarker is substantially elevated compared to that level in the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or where if the level of the biomarker of erythropoiesis is equal to or below that level in the control, then step (d) involves continuing further treatment with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy.

[9] In some embodiments, a method of treating a subject in need of treatment with a therapeutic agent is provided, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of the therapeutic agent; (c) detecting an erythropoiesis biomarker in a sample obtained from the subject over a period of time from at least one day to about 30 days after methotrexate administration; (d) if the level of the biomarker of erythropoiesis is approximately equal to or below that level in the control, conducting additional therapy to induce immune tolerance or immunosuppressive therapy simultaneously with further treatment with a therapeutic agent; or (e) if the level of the biomarker of erythropoiesis is elevated compared to that level in the control, continuing further treatment with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is a level of immature nucleated erythrocytes, an immature reticulocyte fraction, or cellular nucleic acids. In some embodiments, if the level of immature nucleated erythrocytes initially decreases before the level of immature nucleated erythrocytes increases, further treatment with a therapeutic agent is continued without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, where methotrexate is administered in a single cycle, the level of immature nucleated erythrocytes decreases about one, about two, about three, about four, about five, about six, or about seven days before the level of immature nuclear forms erythrocytes becomes elevated. In some embodiments, where methotrexate is administered in two or more cycles, the level of immature nucleated erythrocytes decreases over a period of time from about one to about 14 days before the level of immature nucleated erythrocytes becomes elevated. In some embodiments, where methotrexate is administered in two, three, four, five or more cycles, the level of immature nucleated erythrocytes decreases over a period of time from about one to about 14 days before the level of immature nucleated erythrocytes becomes elevated.

[10] In some embodiments, a method of treating a subject in need of treatment with a therapeutic agent is provided, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of the therapeutic agent; (c) detecting an erythropoiesis biomarker in a sample obtained from the subject over a period of time from at least one day to about 30 days after methotrexate administration; (d) if the level of the biomarker of erythropoiesis is practically elevated compared to that level in the control, the introduction of additional therapy to induce immune tolerance or immunosuppressive therapy simultaneously with further treatment with a therapeutic agent; or (e) if the level of the erythropoiesis biomarker is equal to or below that level in the control, continuing further treatment with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is hematocrit.

[11] In some embodiments according to any of the methods described above, detection of an erythropoiesis biomarker is detection of erythropoiesis-associated gene expression, wherein the erythropoiesis-associated gene is a gene associated with one or more of hematopoiesis, iron ion homeostasis , regulation of erythrocyte differentiation and heme metabolism. In some embodiments, the erythropoiesis-associated gene is a gene encoding transferrin receptor, transferrin, Ly76 (antigen for Ter-119), CD44, CD235a, ALAS2, or GATA-1. In some embodiments, the transferrin receptor is the transferrin receptor 1 (CD71). In some embodiments, detection of an erythropoiesis-associated gene includes detection of an RNA transcript of an erythropoiesis-associated gene or detection of a protein product of an erythropoiesis-associated gene. In some embodiments, the protein product is hemoglobin.

[12] In some embodiments according to any of the methods described above, the erythropoiesis biomarker is an erythropoiesis-associated cofactor. In some embodiments, the erythropoiesis-associated cofactor is a porphyrin, a porphyrin-containing compound, or tetrapyrrole.

[13] In some embodiments according to any of the methods described above, the therapeutic agent is a polypeptide, nucleic acid, virus, gene therapy agent, lipid, liposome, or carbohydrate. In some embodiments, the therapeutic agent is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a lymphocyte depleting agent. In some embodiments, the antibody is alemtuzumab. In some embodiments, the therapeutic polypeptide is an enzyme. In some embodiments, the enzyme is human alpha-galactosidase A. In some embodiments, the enzyme is human acid α-glucosidase.

[14] In some embodiments according to any of the methods described above, the subject has a lysosomal storage disease. In some embodiments, the subject has Pompe disease. In some embodiments, the subject has an infantile form of Pompe disease, such as classic infantile Pompe disease. In some embodiments, Pompe disease is cross-reactive immunological material (CRIM) negative. In some embodiments, the method further comprises administering initial therapy to induce immune tolerance concurrently with administering a therapeutic agent, wherein the therapeutic agent is human acid α-glucosidase. In some embodiments, initial therapy to induce immune tolerance involves the administration of one or more of rituximab and IVIG.

[15] In some embodiments, a method is provided for treating Pompe disease in a subject in need (e.g., in humans) comprising: (a) administering to a subject an effective amount of methotrexate; (b) administering to the subject an effective amount of human acid α-glucosidase; (c) detecting an erythropoiesis biomarker in a sample obtained from the subject over a period of time from at least one day to about 30 days after methotrexate administration; (d) if the level of the biomarker of erythropoiesis is approximately equal to or below that level in the control, the introduction of additional therapy to induce immune tolerance or immunosuppressive therapy simultaneously with further treatment with human acid α-glucosidase; or (e) if the level of the biomarker of erythropoiesis is elevated compared to that level in the control, continuing further treatment with human acid α-glucosidase without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is a level of immature nucleated erythrocytes, an immature reticulocyte fraction, or cellular nucleic acids. In some embodiments, if the level of immature nucleated erythrocytes initially decreases before the level of immature nucleated erythrocytes increases, further treatment with a therapeutic agent is continued without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, where methotrexate is administered in a single cycle, the level of immature nucleated erythrocytes decreases about one, about two, about three, about four, about five, about six, or about seven days before the level of immature nuclear forms erythrocytes becomes elevated. In some embodiments, where methotrexate is administered in two or more cycles, the level of immature nucleated erythrocytes decreases over a period of time from about one to about 14 days before the level of immature nucleated erythrocytes becomes elevated. In some embodiments, where methotrexate is administered in two, three, four, five or more cycles, the level of immature nucleated erythrocytes decreases over a period of time from about one to about 14 days before the level of immature nucleated erythrocytes becomes elevated.

[16] In some embodiments, a method is provided for treating Pompe disease in a subject in need (e.g., in humans) comprising: (a) administering to a subject an effective amount of methotrexate; (b) administering to the subject an effective amount of human acid α-glucosidase; (c) detecting an erythropoiesis biomarker in a sample obtained from the subject over a period of time from at least one day to about 30 days after methotrexate administration; (d) if the level of the biomarker of erythropoiesis is practically elevated compared to that level in the control, conducting additional therapy to induce immune tolerance or immunosuppressive therapy simultaneously with further treatment with human acid α-glucosidase; or (e) if the level of the erythropoiesis biomarker is equal to or below that level in the control, continuing further treatment with human acid α-glucosidase without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is hematocrit. In some embodiments, detection of an erythropoiesis biomarker is detection of erythropoiesis-associated gene expression, wherein the erythropoiesis-associated gene is a gene associated with one or more of hematopoiesis, iron ion homeostasis, regulation of erythrocyte differentiation, and heme metabolism. In some embodiments, detection of an erythropoiesis-associated gene includes detection of an RNA transcript of an erythropoiesis-associated gene or detection of a protein product of an erythropoiesis-associated gene. In some embodiments, the protein product is hemoglobin.

[17] In some embodiments, a method for treating Pompe disease in a subject in need (e.g., in humans) comprising: (a) administering to a subject an effective amount of methotrexate; (b) administering to the subject an effective amount of human acid α-glucosidase; (c) detecting an erythropoiesis biomarker in a sample obtained from the subject over a period of time from at least one day to about 30 days after methotrexate administration; (d) if the level of the biomarker of erythropoiesis is approximately equal to or below that level in the control, the introduction of additional therapy to induce immune tolerance or immunosuppressive therapy simultaneously with further treatment with human acid α-glucosidase; or (e) if the level of the biomarker of erythropoiesis is elevated compared to that level in the control, continuing further treatment with human acid α-glucosidase without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, detection of an erythropoiesis biomarker is detection of erythropoiesis-associated gene expression, wherein the erythropoiesis-associated gene is a gene associated with one or more of hematopoiesis, iron ion homeostasis, regulation of erythrocyte differentiation, and heme metabolism. In some embodiments, the erythropoiesis-associated gene is a gene encoding transferrin receptor, transferrin, Ly76 (antigen for Ter-119), CD44, CD235a, ALAS2, or GATA-1. In some embodiments, the transferrin receptor is the transferrin receptor 1 (CD71). In some embodiments, detection of an erythropoiesis-associated gene includes detection of an RNA transcript of an erythropoiesis-associated gene or detection of a protein product of an erythropoiesis-associated gene. In some embodiments, the erythropoiesis biomarker is an erythropoiesis-associated cofactor. In some embodiments, the erythropoiesis-associated cofactor is a porphyrin, a porphyrin-containing compound, or tetrapyrrole.

[18] In some embodiments, according to any of the treatments for Pompe disease described above, Pompe disease is an infantile form of Pompe disease. In some embodiments, Pompe disease is the classic infantile form of Pompe disease. In some embodiments, the subject is CRIM negative.

[19] In some embodiments, in accordance with any of the methods for treating Pompe disease, the method further comprises monitoring for one or more of human acid α-glucosidase antibody level, CD19 level, or disease progression.

[20] In some embodiments, according to any of the methods described above, the sample is a blood sample. In some embodiments, the blood sample is a blood fraction containing peripheral blood mononuclear cells (PBMCs). In some embodiments, the blood sample is a blood serum sample or a blood plasma sample, and the detection of the biomarker is the detection of the expression of a gene associated with erythropoiesis.

[21] In some embodiments, according to any of the methods described above, the control is a historical control or a placebo control.

[22] In some embodiments, in accordance with any of the methods described above, the subject is a human.

[23] In some embodiments, in accordance with any of the methods described above, an effective amount of methotrexate is administered in one cycle or in three cycles. In some embodiments, methotrexate is administered in a single cycle. In some embodiments, methotrexate is administered in two, three, four, five or more cycles. In some embodiments, a methotrexate cycle consists of 1 day of methotrexate administration, or 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 consecutive days of methotrexate administration.

[24] In some embodiments, in accordance with any of the methods described above, methotrexate is administered to the subject at a time selected from one or more of the times before, during, and after administration of the therapeutic agent. In some embodiments, methotrexate is administered from 48 hours prior to treatment with a therapeutic agent to 48 hours thereafter. In some embodiments, the implementation of methotrexate is administered simultaneously with the introduction of a therapeutic agent and about 24 hours and about 48 hours after the administration of a therapeutic agent.

[25] In some embodiments, according to any of the methods described above, methotrexate is administered at a dose of from about 0.1 mg/kg to about 5 mg/kg.

[26] In some embodiments, in accordance with any of the methods described above, the sample is obtained from the subject at least one day to about 30 days after the last administration of methotrexate. In some embodiments, a sample is obtained from a subject between about 7 days and about 14 days after the last administration of methotrexate. In some embodiments, samples are obtained within a few days of the last administration of methotrexate. In some embodiments, the sample is obtained at one or more time points from day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 14, day 21, or day 28 after the last methotrexate administration.

[27] In some embodiments, according to any of the methods described above, step (d) further comprises administering an agent that depletes T cells, B cells, or plasma cells. In some embodiments, the plasma cell depleting agent is bortezomib. In some embodiments, step (d) comprises one or more of the following: (a) administering an effective amount of one or more of rituximab, intravenous immunoglobulin (IVIG), cyclophosphamide, bortezomib, azathioprine, cyclosporine, mycophenolate, and a corticosteroid, and (b) administering a strategy formation of antigen-specific tolerance. In some embodiments, the antigen-specific tolerance building strategy involves more frequent dosing of the therapeutic agent, or a higher dose of the therapeutic agent, or both. In some embodiments, a higher dose of a therapeutic agent is administered concurrently with IVIG.

[28] In one aspect, the present application provides a method for assessing the level of immune tolerance in a subject with Pompe disease, comprising: (a) obtaining a sample from a subject where the subject has previously received at least one cycle of treatment with methotrexate and at least one dose of a therapeutic agent; and (b) detecting an erythropoiesis biomarker in the sample, wherein the level of the erythropoiesis biomarker in the sample compared to that in the control is indicative of immune tolerance induction. In some embodiments, step (b) involves contacting the sample with an agent that binds to an erythropoiesis biomarker.

[29] In one aspect of the present application, a method is provided for assessing the level of immune tolerance to a therapeutic agent in a subject in need of a therapeutic agent, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of the therapeutic agent; and (c) detecting an erythropoiesis biomarker in a sample obtained from the subject, wherein the level of the erythropoiesis biomarker in the sample compared to that in the control is indicative of induction of immune tolerance in the subject to the therapeutic agent.

[30] In one aspect of the present application, a method for assessing the level of immune tolerance to a therapeutic agent in a subject in need of a therapeutic agent is provided, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of the therapeutic agent; (c) detecting an erythropoiesis biomarker in a sample obtained from the subject over a period of time from at least one day to about 30 days after methotrexate administration; and (d) determining the level of immune tolerance to the therapeutic agent in the subject based on the level of the biomarker detected in step c).

[31] In some embodiments, according to any of the scoring methods described above, a change in the level of an erythropoiesis biomarker is associated with the induction of erythropoiesis. In some embodiments, if the level of the erythropoiesis biomarker is approximately equal to or below that level in the control, then the subject needs additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or if the level of the biomarker of erythropoiesis is elevated compared to that level in the control, then the subject does not need additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, if the level of the erythropoiesis biomarker is substantially elevated compared to that level in the control, then the subject is in need of additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or where if the level of the biomarker of erythropoiesis is equal to or below that level in the control, then the subject does not need additional therapy to induce immune tolerance or immunosuppressive therapy.

[32] Also provided are compositions, kits, and articles of manufacture for use in any of the methods described herein.

[33] These and other aspects and advantages of the present invention will become apparent from the following detailed description and the appended claims. It should be understood that one, more or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention.

BRIEF DESCRIPTION OF GRAPHICS

[34] The file with this patent or this application contains at least one graphic material, made in color. Copies of this patent or patent application publication with color(s) graphic(s) material(s) will be provided by the Patent Office upon request and upon payment of the required fee.

[35] FIG. 1A shows a heat map of a subset of erythropoiesis-associated gene expression levels in mouse spleen samples from different experimental groups.

[36] FIG. 1B shows a heat map of a subset of erythropoiesis-associated gene expression levels in blood samples from mice in different experimental groups. Four to nine groups of C57BL/6 mice/group were treated with rhGAA with and without low dose methotrexate as described in Example 1. Experimental control groups of mice treated with FB (formulation buffer) with and without methotrexate were included. him. On day 7 from the start of rhGAA administration, spleen (A) and blood (B) samples were examined by RNA sequencing on an Illumina HISEQ2000® after RNA amplification. The genes shown are a subset of the total set of genes that are associated with erythropoiesis. RNA sequencing datasets were screened for differential expression with fold change (FC) ≥2 and p-value ≤0.05. Red denotes highly upregulated genes, while blue denotes downregulated genes. The results demonstrate that an exemplary low-dose, short-term regimen of methotrexate enhances transcriptional signatures in erythropoiesis in the spleen and blood.

[37] FIG. 1C shows a schematic of the study design described in Examples 1-4.

[38] FIG. 2 shows the upregulation of transferrin and transferrin 1 receptor in the spleen in response to an exemplary low dose short course regimen of methotrexate. Eight groups of C57BL/6 mice/group were treated with rhGAA in the absence or presence of an exemplary short-term low-dose methotrexate regimen as described in Example 1. -spectrometric analysis of protein. Differentially expressed genes were screened with FC ≥2 and p-value ≤0.05.

[39] FIG. 3A shows significant accumulation of B10 regulatory B cells in the rhGAA+MTX group. In FIG. 3B shows a significant uptake of activated follicular (FO) B cells in the rhGAA+MTX group. In FIG. 3C shows significant accumulation of total activated B cells in the rhGAA+MTX group. In FIG. 3D shows the gating strategy in flow cytometry for selection for live CD19 ⁺ . Regulatory B cells selected by B10 sequential gating (top: CD19 ⁺ , CD1d ^high CD5 ⁺ ), activated follicular B cells (second row: CD19 ⁺ , IgM ^low/high CD23 ⁺ , CD86 ⁺ ), and total activated B cells (bottom: CD19 ⁺ CD86 ⁺ ). Four to ten C57BL/6 or C57BL/6NTac mice/group were treated with rhGAA alone or together with one round of low-dose methotrexate as described in Example 1. Seven days after the start of rhGAA administration, spleens were removed post-mortem, homogenized in single cell suspension, lysed with RBC and examined by flow cytometry as described in Example 2. These data were obtained from three or more separate experiments. Analyzes were performed using Student's t -test and significance is reported as *≤0.05. Enriched B cell subpopulations showed patterns of immune tolerance following treatment with low-dose methotrexate treatment.

[40] FIG. 4A shows representative images of spleen samples (black bar=1 cm) from mice in different experimental groups. In FIG. 4B is a graphical representation of mouse spleen weight on day 7 from the start of rhGAA administration. In FIG. 4C shows the total number of cells in the spleen samples measured on days 5, 6, 7 and 8 by flow cytometry. Four to six C57BL/6NTac mice/group were treated with 20 mg/kg IV rhGAA alone or in combination with one round of low-dose methotrexate (three consecutive daily and/or 5 mg/kg injections within 15 minutes of processing by rhGAA). The spleens were homogenized in a single cell suspension and examined by flow cytometry as described in Example 2.

[41] FIG. 4D shows the gating strategy in flow cytometry of mouse spleen samples. Subpopulations of living common RBCs (Ter-119 ⁺ ) and nuclear forms of RBCs (Ter-119 ⁺ CD71 ⁺ ) were selected. In FIG. 4E shows the amount of total RBCs in the spleens of mice on days 2, 6, 7 and 9 from the start of rhGAA administration. In FIG. 4F shows the numbers of immature nuclear RBCs in the spleens of mice on days 2, 6, 7 and 9 from the start of rhGAA administration. Mice were dosed with either rhGAA alone (rhGAA) or rhGAA in combination with one cycle of low dose methotrexate (rhGAA+1 cycle MTX). In FIG. 4G shows the amount of total RBC in the spleens of mice on days 2, 7 and 9 from the start of rhGAA administration. In FIG. 4H shows the numbers of immature nuclear forms of RBC in the spleens of mice on days 2, 7 and 9 from the start of rhGAA administration. Experimental control groups of mice were included, which were treated with vehicle control FB (formulation buffer) with and without methotrexate. These data come from three or more separate experiments. Analyzes were performed using Student's t -test and significance is reported as *≤0.05. The vertical bars represent the standard deviation. The results demonstrate that an exemplary low-dose, short-term regimen of methotrexate induced an early transient decline followed by accumulation of total and immature nuclear RBCs and spleen enlargement.

[42] FIG. 5A shows the amount of total RBC in the blood of mice on days 2, 4, 7, 9, 14 and 28 from the start of rhGAA administration according to flow cytometry data. In FIG. 5B shows the numbers of immature nuclear forms of RBC in the blood of mice on days 2, 4, 7, 9, 14 and 28 from the start of rhGAA administration. Five to ten C57BL/6NTac mice/group were treated with rhGAA with or without one round of low dose methotrexate. The total number of erythroid cells was obtained from a 25 μl blood aliquot collected and analyzed using flow cytometry as described in example 2 . Mice were dosed with either rhGAA alone (rhGAA) or rhGAA simultaneously with one cycle of low dose methotrexate (rhGAA+1 cycle MTX). In FIG. 5C shows the amounts of total RBCs in the blood of mice on day 2. FIG. 5D shows the amounts of total RBCs in the blood of mice on day 7. FIG. 5E shows the amounts of total RBCs in the blood of mice on day 9. FIG. 5F shows the numbers of immature nuclear forms of RBC in the blood of mice on day 2. FIG. 5G shows the amounts of immature nuclear forms of RBC in the blood of mice on day 7. FIG. 5H shows the amounts of immature nuclear forms of RBC in the blood of mice on day 9. Included are experimental control groups of mice treated with FB with and without an exemplary short-term low-dose methotrexate regimen. These data come from three or more separate experiments. Analyzes were performed using Student's t -test and significance is reported as *≤0.05. The vertical bars represent the standard deviation. The results demonstrated that an exemplary low-dose, short-term regimen of methotrexate elicited a similar but delayed RBC response in the systemic circulation compartment compared to that in the spleen.

[43] FIG. 6A shows the total RBC count in the systemic circulation compartment of mice on day 7 from the start of rhGAA administration. In FIG. 6B shows the hemoglobin level in the systemic circulation compartment of mice on day 7 from the start of rhGAA administration. In FIG. 6C shows the percentage hematocrit in the systemic circulation compartment of mice on day 7 from the start of rhGAA administration. In FIG. 6D shows the number of mature reticulocytes in the systemic circulation compartment of mice on day 7 from the start of rhGAA administration. In FIG. 6E shows the frequency of detection of mature reticulocytes in the systemic circulation compartment of mice on day 7 from the start of rhGAA administration. In FIG. 6F shows the fraction of immature reticulocytes in the systemic circulation compartment of mice on day 7 from the start of rhGAA administration. Six C57BL/6NTac mice/group were treated with rhGAA with or without one round of low dose methotrexate. Experimental control groups of mice treated with FB with and without an exemplary short-term low-dose methotrexate regimen were included. Samples were taken from the retroorbital sinus into microtubes for samples with EDTA anticoagulant and stored at 2-8°C for no more than 72 hours. Differential analysis of CBC, WBC and reticulocyte count was performed on Sysmex XT2000iV. Analyzes were performed using Student's t -test and significance is reported as *≤0.05. The vertical bars represent the standard deviation. The results demonstrate that an exemplary low-dose, short-term regimen of methotrexate provides a regenerative erythroid response on day 7 in the systemic circulation compartment.

[44] FIG. 7A shows the amounts of total RBCs at days 4, 5, 7 and 9 in bone marrow samples from mice from the start of rhGAA administration according to flow cytometry data. In FIG. 7B shows numbers of immature nuclear RBCs at days 4, 5, 7, and 9 in mouse bone marrow samples from the start of rhGAA administration, as measured by flow cytometry. Six C57BL/6NTac mice/group were treated with rhGAA in the presence or absence of one round of low dose methotrexate. The total number of erythroid cells was obtained from one femur and examined using flow cytometry, as described in example 2 . Mice were dosed with either rhGAA alone (rhGAA) or rhGAA simultaneously with one cycle of low-dose methotrexate (rhGAA+1 cycle MTX). These data were obtained in two separate experiments. Analyzes were performed using Student's t -test and significance is reported as *≤0.05. The vertical bars represent the standard deviation. The results demonstrate that an exemplary low-dose, short-term regimen of methotrexate results in early short-term depletion of total and immature nuclear RBCs, but RBCs soon return to homeostatic levels in the bone marrow.

[45] FIG. 8A shows a gating strategy in flow cytometry to select erythroid cells at different stages of development. By differential expression of CD44 and FSC (forward scatter) in living Ter-119 ⁺ identified: (I) proerythroblasts (Ter-119 ^intermediate , CD44 ^high ), (II) basophilic erythroblasts (Ter-119 ⁺ , CD44 ^high FSC ^high ), ( III) polychromatophilic erythroblasts (Ter-119 ⁺ , CD44 ^intermediate FSC ^intermediate ), (IV) orthochromatophilic erythroblasts and reticulocytes (Ter-119 ⁺ , CD44 ^intermediate FSC ^low ) and (V) mature erythrocytes (Ter-119 ⁺ , CD44 ^low FSC ^low ). In FIG. 8B shows erythroid cell counts in mouse spleen samples on day 2 from the start of rhGAA administration. In FIG. 8C shows erythroid cell counts in mouse spleen samples on day 7 from the start of rhGAA administration. Each spleen sample was obtained from one homogenized spleen. In FIG. 8D shows the numbers of erythroid cells in mouse spleen samples on day 2 from the start of rhGAA administration. In FIG. 8E shows erythroid cell counts in mouse spleen samples on day 7 from the start of rhGAA administration. Each blood sample was obtained from a 25 µl collection from the systemic circulation compartment. In FIG. 8F shows the numbers of erythroid cells in bone marrow samples from mice on day 4 from the start of rhGAA administration. In FIG. 8G shows the numbers of erythroid cells in bone marrow samples from mice on day 5 from the start of rhGAA administration. In FIG. 8H shows the numbers of erythroid cells in bone marrow samples from mice on day 7 from the start of rhGAA administration. In FIG. 8I shows the numbers of erythroid cells in bone marrow samples from mice on day 9 from the start of rhGAA administration. Each bone marrow sample was obtained from one femur. Five to ten C57BL/6NTac mice/group were treated with rhGAA no later than fifteen minutes prior to one cycle of low dose methotrexate. Mice were dosed with either rhGAA alone (rhGAA) or rhGAA together with one cycle of low dose methotrexate (rhGAA+1 cycle MTX). These data are obtained in two or more separate experiments. Analyzes were performed using Student's t -test and significance is reported as *≤0.05. The vertical bars represent the standard deviation. The results showed that erythropoiesis initiated by an exemplary short-term low-dose methotrexate regimen predominantly occurs in the spleen of mice.

[46] FIG. 9A shows that an exemplary low-dose, short-term regimen of methotrexate increases the incidence of nucleated cells (including hematopoietic progenitors) in the red pulp of the spleen. Four to six C57BL/6NTac mice/group were treated with rhGAA with or without one round of low dose methotrexate. Experimental control groups of mice treated with FB with and without an exemplary short-term low-dose methotrexate regimen were included. Spleen samples were taken 7 days after the start of rhGAA administration and processed for immunofluorescence. Spleen samples were taken 7 days after the start of rhGAA administration and were cryosectioned in OCT medium, immunofluorescence stained for CD71 and Ter-119, and analyzed on an AxioScan slide scanner (Carl Zeiss; Peabody, MA) configured for multichannel fluorescence imaging and equipped with PlanApo 20x 0.8NA objective. (A) Shown are representative full slide scans in which CD71 immunofluorescent staining is shown in red (PE) and TER119 in green (FITC). Samples were also stained with DAPI (blue) to visualize the nuclei. Areas lacking CD71 or Ter-119 staining represent white pulp. Areas lacking CD71 and/or Ter-119 staining represent white pulp. The scale mark is presented with a dimension of 500 µm.

[47] FIG. 9B shows a graphical representation of the results of morphometric quantification of red/white pulp area ratio (top) and total tissue area (bottom). Vertical bars represent standard error of the mean (SEM) with Bonferroni-adjusted p-values, where * ≤ 0.05.

[48] FIG. 10 shows that the levels of the proteins, erythroid ankyrin and glycophorin C, are reduced in the spleen of mice treated with an exemplary low dose short course regimen of methotrexate. Four to six C57BL/6NTac mice/group were treated with rhGAA with or without one round of low dose methotrexate. Experimental control groups of mice treated with FB with and without an exemplary short-term low-dose methotrexate regimen were included. Spleen samples were taken 7 days after the start of rhGAA administration and processed for tissue mass imaging analysis. It was shown that spleen sections 10 µm thick demonstrate a signal for erythroid ankyrin and glycophorin C. The spatial resolution is 30 µm.

[49] FIG. 11A-11C show serum anti-rhGAA antibody titers in mice receiving 200 million total erythrocytes from donor mice. Immature nuclear forms of splenic RBC isolated from mice treated with an ITI short-term low-dose methotrexate regimen 7 days after the start of rhGAA administration appear to show a tendency to induce immune tolerance in naive mice. Total erythroid cells were isolated from rhGAA-treated mice with (tolerant-induced mice) and without (tolerance-induced mice) ITI short-term low-dose methotrexate regimen and administered to naive recipient mice. After transfusion and weekly thereafter, recipient mice received rhGAA intravenously. Subsequent doses of rhGAA were followed by a prophylactic dose of diphenhydramine. Serum was collected to assess the titers of antibodies to rhGAA using ELISA. (FIG. 11A) 200 million total erythrocytes from the blood of donor mice 9 days after the start of rhGAA administration were purified by leukoreduction. Donor erythrocytes isolated from blood had a purity of ≥99.9%. (Fig. 11B). 75 million total erythrocytes from the spleens of donor mice 7 days after the start of rhGAA administration were purified by positive selection using anti-Ter-119 antibody microbeads. Donor erythrocytes isolated from the spleen had a purity of ≥93.3%. (Fig. 11C). Control mice treated with rhGAA in the presence and absence of an ITI short-term low-dose methotrexate regimen. The highest titer value has been removed. These data were obtained in one experiment. Analyzes were performed using Student's t-tests and significance is given as *≤0.05. The vertical bars represent the standard error of the mean. AUC: area under the curve.

DETAILED DESCRIPTION OF THE INVENTION

[50] This application provides methods for treating a disease (such as Pompe disease) in a subject with a therapeutic agent and methods for assessing the level of immune tolerance to a therapeutic agent in a subject, involving the detection of a biomarker of erythropoiesis after the administration of methotrexate and therapeutic agent.

[51] Immune responses in the form of the formation of antibodies to the drug (ADA) can reduce the effectiveness of the drug and adversely affect patient safety. To reduce ADA accumulation, an immune tolerance induction ("ITI") regimen was developed that involves treatment with a short-term, low-dose methotrexate regimen along with treatment with a therapeutic biologic and during the first week (Garman, Munroe et al., 2004; Joseph , Munroe et al., 2008). Long-term immune tolerance to enzyme replacement therapies (eg, alglucosidase alfa) and antibody therapies (eg, antithymocyte globulin) has been demonstrated with short-term, low-dose methotrexate ( Id; Joseph, Neff et al., 2012).

[52] Aspects of the present invention relate to the discovery that erythropoiesis initiated by short-term low-dose administration of methotrexate is associated with the induction of immune tolerance. The methods and kits described herein are useful in reducing unwanted immune responses in patients receiving therapeutic agents, such as enzyme replacement therapies, that result in the production of anti-drug antibodies (ADA).

[53] The present invention describes molecular and cellular responses in the spleen, blood, and bone marrow to short-term, low-dose methotrexate (exemplary ITI regimen) in the context of treatment with a therapeutic biologic, then There are treatments with human recombinant acid α-glucosidase (rhGAA, alglucosidase alfa), an enzyme replacement therapy used to treat Pompe disease. Transcriptional and proteomic analyzes described herein revealed upregulation of transcriptional and protein signatures in blood and spleen indicative of erythropoiesis following treatment with short-term, low-dose methotrexate (ITI exemplary scheme). Cellular Studies Confirm Significant Replication of Ter-119⁺ CD71⁺ immature nuclear forms of erythrocytes (RBC) in the spleen and blood when treated with short-term low-dose methotrexate. In addition, immunofluorescent analysis confirmed an increase in the proportion of nuclear forms of cells, including hematopoietic progenitors, in the red pulp of the spleen. Conversely, tissue mass spectrometric imaging (tMSI) showed reduced detection of proteins associated with mature RBCs in the spleen. Remarkably, the time of expansion of these RBCs coincided with a significant accumulation of regulatory B cells, which had previously shown the ability to confer immune tolerance in naive animals. Further analysis described herein shows that transfusion of erythroid cells derived from animals treated with short-term, low-dose methotrexate administration provided immune tolerance in naive recipients. Thus, the biomarkers of erythropoiesis described herein can be used to assess immune tolerance to a therapeutic agent and/or as criteria for additional therapy to induce immune tolerance or immunosuppressive therapy in combination with further administration of a therapeutic agent to patients who received methotrexate and therapeutic agent.

[54] Accordingly, in one aspect, the present invention provides a method of treating a subject in need of treatment with a therapeutic agent (e.g., a subject with Pompe disease in need of treatment with human acid α-glucosidase) comprising: (a) administering to the subject an effective amount of methotrexate ; (b) administering to the subject an effective amount of the therapeutic agent; (c) detecting an erythropoiesis biomarker in a sample obtained from the subject over a period of time from at least one day to about 30 days after methotrexate administration; and (d) continuing further treatment with a therapeutic agent with or without additional therapy to induce immune tolerance or immunosuppressive therapy based on the level of the biomarker of erythropoiesis compared to that level in the control. In some embodiments, the change in the level of the erythropoiesis biomarker is associated with the induction of erythropoiesis. In some embodiments, if the level of the erythropoiesis biomarker is approximately equal to or below that level in the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or if the level of the biomarker of erythropoiesis is elevated compared to that level in the control, then stage (d) involves continuing further treatment with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, if the level of the erythropoiesis biomarker is substantially elevated compared to that level in the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or if the level of the erythropoiesis biomarker is equal to or below that level in the control, then step (d) involves continuing further treatment with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy.

[55] In another aspect of the present application, a method is provided for assessing the level of immune tolerance to a therapeutic agent in a subject in need of a therapeutic agent (for example, in a subject with Pompe disease in need of treatment with human acid α-glucosidase), comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject a therapeutically effective amount of the therapeutic agent; and (c) detecting an erythropoiesis biomarker in a sample obtained from the subject, wherein the level of the erythropoiesis biomarker in the sample compared to that in the control is indicative of induction of immune tolerance in the subject to the therapeutic agent.

[56] Kits and articles applicable in the methods described herein are also provided.

I. Definitions

[57] The term "detection" is used herein in its broadest sense and includes both qualitative and quantitative measurements of the target molecule. Detection includes identifying only the presence of the target molecule in the sample, as well as determining whether the target molecule is present in the sample at detectable levels.

[58] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be separated by non-amino acid compounds. The terms also cover a polymer of amino acids that has been modified naturally or through intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as conjugation with a labeling moiety. Also included within the scope of this definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids etc.), as well as other modifications known in the art. As used herein, the terms "polypeptide" and "protein" specifically encompass antibodies. In some examples, the protein contains a plurality of polypeptides or polymer chains.

[59] As used herein, the term "biomarker" refers to an indicator, such as predictive, diagnostic and/or prognostic, which can be detected in the sample. A biomarker can serve as an indicator of a specific disease subtype, condition, or biological response (eg, an unwanted immune response to a therapeutic agent) characterized by certain molecular, pathological, histological and/or clinical features. In some embodiments, the biomarker is a gene. In some embodiments, the implementation of the biomarker is a population or subpopulation of cells. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and/or RNA), changes in the copy number of polynucleotides (for example, DNA copy number), polypeptides, modifications of polypeptides and polynucleotides (for example, post-translational modifications), hydrocarbons, glycolipid-based molecular markers, cells and/or cell populations.

[60] The "amount" or "level" of a biomarker associated with increased clinical benefit for an individual is the detectable level in a biological sample. They can be measured using methods known to the person skilled in the art, as well as disclosed in this document. The expression level or amount of the biomarker being assessed can be used to determine response to treatment.

[61] The term "household biomarker" refers to a biomarker or group of biomarkers (e.g., polynucleotides and/or polypeptides), which are generally equally present in all cell types. In some embodiments, the housekeeping biomarker is a "housekeeping gene". The term "housekeeping gene" as used herein refers to a gene or group of genes that encode proteins whose activity is essential for maintaining cellular function and which are generally equally present in all cell types.

[62] "Polynucleotide" or "nucleic acid", used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be inserted into a polymer by DNA or RNA polymerase or by synthetic reaction. The polynucleotide may contain modified nucleotides such as methylated nucleotides and their analogs. If present, the modification of the nucleotide structure may be included before or after the assembly of the polymer. The sequence of nucleotides may be interrupted by components other than nucleotides. The polynucleotide can be further modified after synthesis, for example by conjugation with a label. Other types of modifications include, for example, "caps", the replacement of one or more naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, modifications with uncharged bonds (for example, methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged bonds (for example, phosphorothioate, phosphorodithioate etc.) containing side fragments, such as, for example, proteins (for example, nucleases, toxins, antibodies, signal peptides, poly-L-lysine etc.) containing intercalators (for example, acridine, psoralen etc.) containing chelators (for example, metals, radioactive metals, boron, oxidizing metals etc.) containing alkylators containing modified bonds (for example, alpha anomeric nucleic acids etc.), as well as unmodified forms of the polynucleotide(s). In addition, any of the hydroxyl groups normally present on the sugar may be replaced, for example, with phosphonate groups, phosphate groups, protected with standard protecting groups or activated to provide additional bonds to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5' and 3' OH may be phosphorylated or substituted with amines or organic capping moieties of 1-20 carbons. Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides may also contain similar forms of ribose or deoxyribose sugars that are commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose , carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and nucleoside analogs with nitrogenous bases removed, for example, methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, without limitation, those in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), "(O)NR₂ ("amidate"), P(O)R, P(O)OR', CO or CH₂ ("formacetal") in which each R or R' is independently H or substituted or unsubstituted alkyl (C1-20), optionally containing an ether (-O-) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all bonds in a polynucleotide need to be identical. The previous description applies to all polynucleotides specified in this document, including RNA and DNA.

[63] As used herein, the term "oligonucleotide" generally refers to short, single-stranded polynucleotides that are, but need not be, less than about 250 nucleotides in length. The oligonucleotides may be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description for polynucleotides is equally and fully applicable to oligonucleotides.

[64] The term "primer" refers to a single-stranded polynucleotide that is capable of hybridizing to a nucleic acid and thereafter promotes the polymerization of a complementary nucleic acid, typically by providing a free 3'-OH group.

[65] The term "small molecule" refers to any molecule with a molecular weight of about 2000 daltons or less, preferably about 500 daltons or less.

[66] The term "sample" as used herein refers to a composition that is derived from or derived from a subject and/or individual of interest that contains a cellular and/or other molecular entity to be characterized and/or identified, for example, based on physical, biochemical, chemical and/or physiological characteristics. Samples include, without limitation, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, cells blood, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates and culture media, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

[67] By "tissue sample" or "cell sample" is meant a set of similar cells obtained from a tissue or compartment of a subject or individual. The source of the tissue or cell sample may be solid tissue, such as from a fresh, frozen and/or fixed organ, tissue sample, biopsy and/or aspirate; blood or any blood components such as blood plasma; physiological fluids, for example, cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells selected at any time during the gestation or development of the subject. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from diseased tissue/organ. The tissue sample may contain compounds that do not naturally mix with tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

[68] As used herein, the term “reference sample,” “reference cell,” “control tissue,” “control sample,” “control cell,” or “control tissue” refers to a sample, cell, tissue, standard, or level that used for comparison.

[69] For the purposes of the present invention, a "section" of a tissue sample means a separate part or piece of a tissue sample, for example, a thin piece of tissue or cells cut from a tissue sample. It is understood that multiple sections of tissue samples may be taken and analyzed, provided that the same section of tissue sample can be analyzed at both the morphological and molecular levels, or analyzed for both polypeptides and polynucleotides.

[70] By “correlating” or “correlating” is meant comparing, in any way, the performance and/or results of the first assay or protocol with the performance and/or results of the second assay or protocol. For example, the results of the first assay or protocol may be used when performing the second protocol, and/or the results of the first analysis or protocol may be used to determine whether the second analysis or protocol should be performed. With respect to the embodiment of the assay or polypeptide protocol, the results of the assay or polypeptide expression protocol can be used to determine whether a particular treatment regimen should be pursued. With respect to the embodiment of the assay or polynucleotide protocol, the results of the assay or polynucleotide expression protocol can be used to determine whether a particular treatment regimen should be pursued.

[71] The term "substantially the same" as used herein means a sufficiently high degree of similarity between two numerical values that one skilled in the art would consider that the difference between the two values is small or non-biological and/or statistically significant. significance in the context of a biological characteristic as measured by specified values (e.g., K values_d or expression). The difference between the two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10%, depending on the reference/comparison value.

[72] The phrase "significantly different" as used herein refers to a sufficiently high degree of difference between two numerical values that one skilled in the art would consider the difference between the two values to be statistically significant in the context of a biological characteristic measured by specified values (for example, K values_d). The difference between these two values is, for example, more than about 10%, more than about 20%, more than about 30%, more than about 40% and/or more than about 50% depending on the value for the reference/comparison molecule.

[73] "Effective amount" refers to the amount of the agent, effective in doses and for periods of time necessary to achieve the desired physiological result.

[74] "A therapeutically effective amount" refers to the amount of a therapeutic agent for the treatment or prevention of a disease or condition in a subject. In some embodiments, a therapeutically effective amount of a therapeutic agent can prolong survival (including overall survival and progression-free survival); lead to an objective answer (including a full answer or a partial answer); alleviate at least partially one or more signs or symptoms of the disease or condition; and/or improve the subject's quality of life.

[75] The term "pharmaceutical composition" refers to a preparation that is presented in a form that provides the biological activity of the active ingredient present in it, and which does not contain additional components that have unacceptable toxicity to the subject to whom the composition will be administered.

[76] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is non-toxic to the subject. A pharmaceutically acceptable carrier includes, without limitation, a buffer, excipient, stabilizer, or preservative.

[77] As used herein, the term "treatment" (and its grammatical variations such as "treat" or "administer treatment") refers to clinical intervention in an attempt to alter the natural course of a disease in an individual being treated, and may be carried out in the course of course of clinically expressed pathology. Desired effects of treatment include, without limitation, prevention of disease recurrence, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, reduction in the rate of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis.

[78] As used herein, the term "prophylactic treatment" refers to treatment in which an individual is known or suspected to have, or is at risk of having, a disorder, but no symptoms or minimal symptoms of the disorder. A subject undergoing prophylactic treatment may be treated before the onset of symptoms.

[79] "Individual" or "subject" is a mammal. Mammals include, without limitation, domesticated animals (e.g., cows, sheep, cats, dogs and horses), primates (for example, humans and non-human primates such as monkeys), rabbits and rodents (for example, mice and rats). In some embodiments, the individual or subject is a human.

[80] The term "simultaneously" is used herein to refer to the administration of two or more therapeutic agents when at least part of the administration overlaps in time, or when the administration does not strictly overlap in time, but when one agent remains at a systemic level sufficient to continue providing a physiological effect, while the second agent is administered at least partially. Accordingly, co-administration includes a dosing regimen in which administration of one or more agents is continued after administration of one or more other agents has ceased, or in which one or more agents are administered after one or more other agents have been administered but remain at the systemic level, sufficient to continue to provide a physiological effect.

[81] By "reduction or inhibition" is meant the ability to cause an overall reduction of approximately any percentage of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80% , 85%, 90%, 95% or more.

[82] By "increase or increase" is meant the ability to cause an overall increase of approximately any percentage of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80 %, 85%, 90%, 95% or more.

[83] The term "package leaflet" is used to refer to instructions commonly included in commercial packages of therapeutic products that contain information about the indications, use, dosage, administration, combination therapy, contraindications and/or warnings regarding the use of such therapeutic products.

[84] A "finished product" is any product (for example, package or container) or kit containing at least one reagent, for example medicine for the treatment of a disease or condition (for example, cancer), or a probe for the specific detection of a biomarker described herein. In certain embodiments, an article or kit is advertised, distributed, or sold as a unit to carry out the methods described herein.

[85] The phrase "based on" as used herein means that information about one or more biomarkers is used to inform a treatment decision, for information provided on a package leaflet, or for marketing/advertising guidance. etc.

[86] An antibody "which binds" an antigen of interest e.g., a host cell protein, is a protein that binds an antigen with sufficient affinity to enable the antibody to be used as an assay reagent, e.g., as a capture antibody or as a detection antibody. As a rule, such an antibody cross-reacts with other polypeptides (for example, with other antigens) to a small extent.

[87] With regard to the binding of a polypeptide to a target molecule, the term "specific binding" or "specifically binds to" or "specific to" for a particular polypeptide or epitope on a particular target polypeptide means a binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining the binding of the target molecule compared to the binding of a reference molecule, which is typically a molecule composed of similar building blocks with no binding activity (e.g., a control protein that is not identical to the target protein).

[88] "Affinity" generally refers to the strength of the overall non-covalent interactions between one binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to true binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y as a whole can be represented by the dissociation constant (Kd). Affinity can be measured using conventional methods known in the art, including the methods described herein.

[89] The term "antibody" is used herein in its broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, semi-antibodies, polyspecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so far as long as they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used herein interchangeably with the term antibody.

[90] It is understood that the embodiments of the present invention described herein include the embodiments "comprising", "consisting of", and/or "consisting essentially of".

[91] Reference to "approximately" in relation to a value or parameter in this document includes (and describes) options that, in fact, are directed to a given value or parameter. For example, a description referring to "about X" includes a description of "X".

[92] As used herein, reference to "not" in relation to a value or parameter generally means and describes "other than" a value or parameter. For example, a method not used to treat type X cancer means that the method is used to treat cancer other than type X cancer.

[93] As used herein, the term "about X-Y" has the same meaning as "from about X to about Y".

[94] As used herein and in the appended claims, the singular forms include references to the plural unless the context clearly indicates otherwise.

II. Methods of treatment and evaluation

[95] Provided herein are methods of treating a subject in need of treatment with a therapeutic agent based on the detection of an erythropoiesis biomarker in a sample from the subject following administration of the therapeutic agent and methotrexate.

[96] In some embodiments, a method of treating a subject (eg, a human subject) in need of treatment with a therapeutic agent is provided, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of the therapeutic agent; (c) detecting a biomarker of erythropoiesis in a sample obtained from a subject over a period of at least one day to about 30 days (in particular, from about 7 days to about 14 days) after methotrexate administration; and (d) continuing further treatment with a therapeutic agent with or without additional therapy to induce immune tolerance or immunosuppressive therapy based on the level of the biomarker of erythropoiesis compared to that level in the control. In some embodiments, the change in the level of the erythropoiesis biomarker is associated with the induction of erythropoiesis. In some embodiments, if the level of the erythropoiesis biomarker is approximately equal to or below that level in the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or if the level of the biomarker of erythropoiesis is elevated compared to that level in the control, then stage (d) involves continuing further treatment with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is a level of immature nucleated erythrocytes, an immature reticulocyte fraction, or cellular nucleic acids. In some embodiments, if the level of the erythropoiesis biomarker is substantially elevated compared to that level in the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or if the level of the erythropoiesis biomarker is equal to or below that level in the control, then step (d) involves continuing further treatment with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is hematocrit. In some embodiments, said detection of an erythropoiesis biomarker comprises detection of expression of an erythropoiesis-associated gene, wherein the gene is associated with one or more of hematopoiesis, iron ion homeostasis, regulation of erythrocyte differentiation, and heme metabolism. In some embodiments, an erythropoiesis-associated gene is a gene encoding a transferrin receptor (such as CD71), transferrin, Ly76, CD44, CD235a, ALAS2, or GATA-1. In some embodiments, the erythropoiesis biomarker is an erythropoiesis-associated cofactor, such as a porphyrin, a porphyrin-containing compound, or tetrapyrrole. In some embodiments, an effective amount of methotrexate is administered in one cycle or in three cycles. In some embodiments, an effective amount of methotrexate is administered in one, two, three, four, five, or more cycles. In some embodiments, methotrexate is administered from 48 hours before treatment with a therapeutic agent to 48 hours (in particular, at about 24 hours and about 48 hours) thereafter. In some embodiments, methotrexate is administered at a dose of 0.1 mg/kg to 5 mg/kg. In some embodiments, adjunctive therapy to induce immune tolerance or immunosuppressive therapy comprises administering one or more of rituximab, intravenous immunoglobulin (IVIG), cyclophosphamide, bortezomib, azathioprine, cyclosporine, mycophenolate, a corticosteroid, and applying an antigen-specific tolerance building strategy (e.g., more frequent dosing and/or higher dose of therapeutic drug, optionally with concomitant IVIG).

[97] In some embodiments, a method for treating a subject in need of treatment with a therapeutic polypeptide (eg, a monoclonal antibody or enzyme) is provided, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of the therapeutic polypeptide; (c) detecting a biomarker of erythropoiesis in a sample obtained from a subject over a period of at least one day to about 30 days (in particular, from about 7 days to about 14 days) after methotrexate administration; and (d) continuing further treatment with the therapeutic polypeptide with or without additional immune tolerance inducing therapy or immunosuppressive therapy based on the level of the erythropoiesis biomarker compared to that in the control. In some embodiments, the change in the level of the erythropoiesis biomarker is associated with the induction of erythropoiesis. In some embodiments, if the level of the erythropoiesis biomarker is approximately equal to or below that level in the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic polypeptide; or if the level of the erythropoiesis biomarker is elevated compared to that level in the control, then step (d) involves continuing further treatment with a therapeutic polypeptide without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is a level of immature nucleated erythrocytes, an immature reticulocyte fraction, or cellular nucleic acids. In some embodiments, if the level of the erythropoiesis biomarker is substantially elevated compared to that level in the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic polypeptide; or if the level of the erythropoiesis biomarker is equal to or below that level in the control, then step (d) involves continuing further treatment with a therapeutic polypeptide without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is hematocrit. In some embodiments, said detection of an erythropoiesis biomarker comprises detection of expression of an erythropoiesis-associated gene, wherein the gene is associated with one or more of hematopoiesis, iron ion homeostasis, regulation of erythrocyte differentiation, and heme metabolism. In some embodiments, an erythropoiesis-associated gene is a gene encoding a transferrin receptor (such as CD71), transferrin, Ly76, CD44, CD235a, ALAS2, or GATA-1. In some embodiments, the erythropoiesis biomarker is an erythropoiesis-associated cofactor, such as a porphyrin, a porphyrin-containing compound, or tetrapyrrole. In some embodiments, an effective amount of methotrexate is administered in one cycle or in three cycles. In some embodiments, an effective amount of methotrexate is administered in one, two, three, four, five, or more cycles. In some embodiments, methotrexate is administered between 48 hours prior to and 48 hours (in particular, at about 24 hours and about 48 hours) after treatment with a therapeutic polypeptide. In some embodiments, methotrexate is administered at a dose of 0.1 mg/kg to 5 mg/kg. In some embodiments, adjunctive therapy to induce immune tolerance or immunosuppressive therapy comprises administering one or more of rituximab, intravenous immunoglobulin (IVIG), cyclophosphamide, bortezomib, azathioprine, cyclosporine, mycophenolate, a corticosteroid, and applying an antigen-specific tolerance building strategy (e.g., more frequent dosing and/or higher dose of therapeutic agent, optionally with concomitant IVIG). In some embodiments, the therapeutic polypeptide is a lymphocyte depleting agent, such as alemtuzumab. In some embodiments, the therapeutic polypeptide is an enzyme, such as human alpha-galactosidase A or human acidic alpha-glucosidase.

[98] In some embodiments, a method of treating a subject (such as a human subject) in need of treatment with a therapeutic polypeptide (such as a monoclonal antibody or enzyme) is provided, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of the therapeutic polypeptide; (c) detection of immature nuclear forms of erythrocytes in a sample obtained from the subject in the period of time from at least one day to about 30 days after administration of methotrexate; and (d) if the level of immature nucleated erythrocytes is approximately equal to or below that level in the control, conducting additional therapy to induce immune tolerance or immunosuppressive therapy simultaneously with further treatment with a therapeutic polypeptide; or if the level of immature nuclear forms of erythrocytes is increased compared with that level in the control, continuing further treatment with a therapeutic polypeptide without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the sample is a blood sample or a sample of a blood fraction containing peripheral blood mononuclear cells. In some embodiments, the sample is a blood plasma sample. In some embodiments, the control is a historical control or a placebo control. In some embodiments, an effective amount of methotrexate is administered in one cycle or in three cycles. In some embodiments, an effective amount of methotrexate is administered in one, two, three, four, five, or more cycles. In some embodiments, methotrexate is administered between 48 hours prior to and 48 hours (in particular, at about 24 hours and about 48 hours) after treatment with a therapeutic polypeptide. In some embodiments, methotrexate is administered at a dose of 0.1 mg/kg to 5 mg/kg. In some embodiments, adjunctive therapy to induce immune tolerance or immunosuppressive therapy comprises administering one or more of rituximab, intravenous immunoglobulin (IVIG), cyclophosphamide, bortezomib, azathioprine, cyclosporine, mycophenolate, a corticosteroid, and applying an antigen-specific tolerance building strategy (e.g., more frequent dosing and/or higher dose of therapeutic agent, optionally with concomitant IVIG).

[99] In some embodiments, a method of treating a subject (such as a human subject) in need of treatment with a therapeutic polypeptide (such as a monoclonal antibody or enzyme) is provided, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of the therapeutic polypeptide; (c) detecting a gene (such as an RNA transcript or protein) associated with erythropoiesis in a sample obtained from a subject from at least one day to about 30 days after methotrexate administration; and (d) if the level of the erythropoiesis-associated gene is about or below that level in the control, administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with the therapeutic polypeptide; or if the level of the gene associated with erythropoiesis is elevated compared to that level in the control, continuing further treatment with a therapeutic polypeptide without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, an erythropoiesis-associated gene is associated with one or more of hematopoiesis, iron ion homeostasis, regulation of erythrocyte differentiation, and heme metabolism. In some embodiments, an erythropoiesis-associated gene is a gene encoding a transferrin receptor (such as CD71), transferrin, Ly76, CD44, CD235a, ALAS2, or GATA-1. In some embodiments, the sample is a blood sample or a sample of a blood fraction containing peripheral blood mononuclear cells. In some embodiments, the sample is a blood plasma sample. In some embodiments, the control is a historical control or a placebo control. In some embodiments, an effective amount of methotrexate is administered in one cycle or in three cycles. In some embodiments, an effective amount of methotrexate is administered in one, two, three, four, five, or more cycles. In some embodiments, methotrexate is administered between 48 hours prior to and 48 hours (in particular, at about 24 hours and about 48 hours) after treatment with a therapeutic polypeptide. In some embodiments, methotrexate is administered at a dose of 0.1 mg/kg to 5 mg/kg. In some embodiments, adjunctive therapy to induce immune tolerance or immunosuppressive therapy comprises administering one or more of rituximab, intravenous immunoglobulin (IVIG), cyclophosphamide, bortezomib, azathioprine, cyclosporine, mycophenolate, a corticosteroid, and applying an antigen-specific tolerance building strategy (e.g., more frequent dosing and/or higher dose of therapeutic drug, optionally with concomitant IVIG).

[100] In some embodiments, a method of treating a subject (such as a human subject) in need of treatment with a therapeutic polypeptide (such as a monoclonal antibody or enzyme) is provided, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of the therapeutic polypeptide; (c) detecting an erythropoiesis-associated cofactor in a sample obtained from the subject from at least one day to about 30 days after methotrexate administration; and (d) if the level of the erythropoiesis-associated cofactor is approximately equal to or lower than that of the control, administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with the therapeutic polypeptide; or if the level of the erythropoiesis-associated cofactor is elevated compared to that in the control, continuing further treatment with the therapeutic polypeptide without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the erythropoiesis-associated cofactor is a porphyrin, a porphyrin-containing compound, or tetrapyrrole. In some embodiments, the sample is a blood sample or a sample of a blood fraction containing peripheral blood mononuclear cells. In some embodiments, the sample is a blood plasma sample. In some embodiments, the control is a historical control or a placebo control. In some embodiments, an effective amount of methotrexate is administered in one cycle or in three cycles. In some embodiments, an effective amount of methotrexate is administered in one, two, three, four, five, or more cycles. In some embodiments, methotrexate is administered between 48 hours prior to and 48 hours (in particular, at about 24 hours and about 48 hours) after treatment with a therapeutic polypeptide. In some embodiments, methotrexate is administered at a dose of 0.1 mg/kg to 5 mg/kg. In some embodiments, adjunctive therapy to induce immune tolerance or immunosuppressive therapy comprises administering one or more of rituximab, intravenous immunoglobulin (IVIG), cyclophosphamide, bortezomib, azathioprine, cyclosporine, mycophenolate, a corticosteroid, and applying an antigen-specific tolerance building strategy (e.g., more frequent dosing and/or higher dose of therapeutic drug, optionally with concomitant IVIG).

[101] In some embodiments, a method of treating a subject with lysosomal storage disease in need of treatment with a therapeutic agent (such as an enzyme) is provided, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of the therapeutic agent; (c) detecting a biomarker of erythropoiesis in a sample obtained from a subject over a period of at least one day to about 30 days (in particular, from about 7 days to about 14 days) after methotrexate administration; and (d) continuing further treatment with a therapeutic agent with or without additional therapy to induce immune tolerance or immunosuppressive therapy based on the level of the biomarker of erythropoiesis compared to that level in the control. In some embodiments, the change in the level of the erythropoiesis biomarker is associated with the induction of erythropoiesis. In some embodiments, if the level of the erythropoiesis biomarker is approximately equal to or below that level in the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or if the level of the biomarker of erythropoiesis is elevated compared to that level in the control, then stage (d) involves continuing further treatment with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is a level of immature nucleated erythrocytes, an immature reticulocyte fraction, or cellular nucleic acids. In some embodiments, if the level of the erythropoiesis biomarker is substantially elevated compared to that level in the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or if the level of the erythropoiesis biomarker is equal to or below that level in the control, then step (d) involves continuing further treatment with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is hematocrit. In some embodiments, said detection of an erythropoiesis biomarker comprises detection of expression of an erythropoiesis-associated gene, wherein the gene is associated with one or more of hematopoiesis, iron ion homeostasis, regulation of erythrocyte differentiation, and heme metabolism. In some embodiments, an erythropoiesis-associated gene is a gene encoding a transferrin receptor (such as CD71), transferrin, Ly76, CD44, CD235a, ALAS2, or GATA-1. In some embodiments, the erythropoiesis biomarker is an erythropoiesis-associated cofactor, such as a porphyrin, a porphyrin-containing compound, or tetrapyrrole. In some embodiments, the sample is a blood sample or a sample of a blood fraction containing peripheral blood mononuclear cells. In some embodiments, the sample is a blood plasma sample. In some embodiments, the control is a historical control or a placebo control. In some embodiments, an effective amount of methotrexate is administered in one cycle or in three cycles. In some embodiments, an effective amount of methotrexate is administered in one, two, three, four, five, or more cycles. In some embodiments, methotrexate is administered from 48 hours before treatment with a therapeutic agent to 48 hours (in particular, at about 24 hours and about 48 hours) thereafter. In some embodiments, methotrexate is administered at a dose of 0.1 mg/kg to 5 mg/kg. In some embodiments, adjunctive therapy to induce immune tolerance or immunosuppressive therapy comprises administering one or more of rituximab, intravenous immunoglobulin (IVIG), cyclophosphamide, bortezomib, azathioprine, cyclosporine, mycophenolate, a corticosteroid, and applying an antigen-specific tolerance building strategy (e.g., more frequent dosing and/or higher dose of therapeutic agent, optionally with concomitant IVIG). In some embodiments, the therapeutic polypeptide is human alpha-galactosidase A or human acidic α-glucosidase. In some embodiments, the subject has Gaucher disease and is in need of treatment with human alpha-galactosidase A.

[102] In some embodiments, a method for treating Pompe disease (eg, infantile and/or CRIM-negative Pompe disease) in a subject (such as a human) is provided, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of a therapeutic agent (such as human acid α-glucosidase); (c) detecting a biomarker of erythropoiesis in a sample obtained from a subject over a period of at least one day to about 30 days (in particular, from about 7 days to about 14 days) after methotrexate administration; and (d) continuing further treatment with a therapeutic agent with or without additional therapy to induce immune tolerance or immunosuppressive therapy based on the level of the biomarker of erythropoiesis compared to that level in the control. In some embodiments, the change in the level of the erythropoiesis biomarker is associated with the induction of erythropoiesis. In some embodiments, if the level of the erythropoiesis biomarker is approximately equal to or below that level in the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or if the level of the biomarker of erythropoiesis is elevated compared to that level in the control, then stage (d) involves continuing further treatment with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is a level of immature nucleated erythrocytes, an immature reticulocyte fraction, or cellular nucleic acids. In some embodiments, if the level of the erythropoiesis biomarker is substantially elevated compared to that level in the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or if the level of the erythropoiesis biomarker is equal to or below that level in the control, then step (d) involves continuing further treatment with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is hematocrit. In some embodiments, said detection of an erythropoiesis biomarker comprises detection of expression of an erythropoiesis-associated gene, wherein the gene is associated with one or more of hematopoiesis, iron ion homeostasis, regulation of erythrocyte differentiation, and heme metabolism. In some embodiments, an erythropoiesis-associated gene is a gene encoding a transferrin receptor (such as CD71), transferrin, Ly76, CD44, CD235a, ALAS2, or GATA-1. In some embodiments, the erythropoiesis biomarker is an erythropoiesis-associated cofactor, such as a porphyrin, a porphyrin-containing compound, or tetrapyrrole. In some embodiments, the sample is a blood sample or a sample of a blood fraction containing peripheral blood mononuclear cells. In some embodiments, the sample is a blood plasma sample. In some embodiments, the control is a historical control or a placebo control. In some embodiments, an effective amount of methotrexate is administered in one cycle or in three cycles. In some embodiments, an effective amount of methotrexate is administered in one, two, three, four, five, or more cycles. In some embodiments, methotrexate is administered from 48 hours before treatment with a therapeutic agent to 48 hours (in particular, at about 24 hours and about 48 hours) thereafter. In some embodiments, methotrexate is administered at a dose of 0.1 mg/kg to 5 mg/kg. In some embodiments, adjunctive therapy to induce immune tolerance or immunosuppressive therapy comprises administering one or more of rituximab, intravenous immunoglobulin (IVIG), cyclophosphamide, bortezomib, azathioprine, cyclosporine, mycophenolate, a corticosteroid, and applying an antigen-specific tolerance building strategy (e.g., more frequent dosing and/or higher dose of therapeutic agent, optionally with concomitant IVIG). In some embodiments, step (d) further comprises administering an agent that depletes T cells, B cells, or plasma cells, such as bortezomib. In some embodiments, the method further comprises monitoring for one or more of anti-therapeutic agent levels, CD19 levels, and disease progression.

[103] In some embodiments, a method is provided for treating infantile Pompe disease (such as classic infantile Pompe disease) in a subject (such as a human) comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of human acid α-glucosidase concomitantly with initial therapy to induce immune tolerance (such as rituximab and/or IVIG); (c) detecting a biomarker of erythropoiesis in a sample obtained from a subject over a period of at least one day to about 30 days (in particular, from about 7 days to about 14 days) after methotrexate administration; and (d) continuing further treatment with human acid α-glucosidase with or without additional therapy to induce immune tolerance or immunosuppressive therapy based on the level of the erythropoiesis biomarker compared to the control level. In some embodiments, the change in the level of the erythropoiesis biomarker is associated with the induction of erythropoiesis. In some embodiments, if the level of the erythropoiesis biomarker is approximately equal to or below that level in the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with human acid α-glucosidase; or if the level of the biomarker of erythropoiesis is elevated compared to that level in the control, then stage (d) involves continuing further treatment with human acid α-glucosidase without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is a level of immature nucleated erythrocytes, an immature reticulocyte fraction, or cellular nucleic acids. In some embodiments, if the level of the erythropoiesis biomarker is substantially elevated compared to that level in the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with human acid α-glucosidase; or if the level of the biomarker of erythropoiesis is equal to or below that level in the control, then step (d) involves continuing further treatment with human acid α-glucosidase without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is hematocrit. In some embodiments, said detection of an erythropoiesis biomarker comprises detection of expression of an erythropoiesis-associated gene, wherein the gene is associated with one or more of hematopoiesis, iron ion homeostasis, regulation of erythrocyte differentiation, and heme metabolism. In some embodiments, an erythropoiesis-associated gene is a gene encoding a transferrin receptor (such as CD71), transferrin, Ly76, CD44, CD235a, ALAS2, or GATA-1. In some embodiments, the erythropoiesis biomarker is an erythropoiesis-associated cofactor, such as a porphyrin, a porphyrin-containing compound, or tetrapyrrole. In some embodiments, the sample is a blood sample or a sample of a blood fraction containing peripheral blood mononuclear cells. In some embodiments, the sample is a blood plasma sample. In some embodiments, the control is a historical control or a placebo control. In some embodiments, an effective amount of methotrexate is administered in one cycle or in three cycles. In some embodiments, an effective amount of methotrexate is administered in one, two, three, four, five, or more cycles. In some embodiments, methotrexate is administered between 48 hours before and 48 hours (eg, at about 24 hours and about 48 hours) after treatment with human acid α-glucosidase. In some embodiments, methotrexate is administered at a dose of 0.1 mg/kg to 5 mg/kg. In some embodiments, adjunctive therapy to induce immune tolerance or immunosuppressive therapy comprises administering one or more of rituximab, intravenous immunoglobulin (IVIG), cyclophosphamide, bortezomib, azathioprine, cyclosporine, mycophenolate, a corticosteroid, and applying an antigen-specific tolerance building strategy (e.g., more frequent dosing and/or higher dose of therapeutic agent, optionally with concomitant IVIG). In some embodiments, step (d) further comprises administering an agent that depletes T cells, B cells, or plasma cells, such as bortezomib. In some embodiments, the method further comprises monitoring one or more of an anti-human α-glucosidase antibody level (eg, an anti-rhGAA IgG antibody) and a CD19 level, as well as disease progression. In some embodiments, the infantile form of Pompe disease is CRIM-negative Pompe disease.

[104] In some embodiments, a method is provided for treating a subject with Pompe disease (such as infantile and/or CRIM-negative Pompe disease) comprising: (a) obtaining a sample from a subject where the subject has previously received at least one cycle of administration of methotrexate and administered at least one dose of a therapeutic agent (such as human acid α-glucosidase); (b) detecting an erythropoiesis biomarker in the sample from at least one day to about 30 days (in particular, from about 7 days to about 14 days) after methotrexate administration; and (c) continuing further treatment with a therapeutic agent with or without additional therapy to induce immune tolerance or immunosuppressive therapy based on the level of the erythropoiesis biomarker compared to the control level. In some embodiments, the change in the level of the erythropoiesis biomarker is associated with the induction of erythropoiesis. In some embodiments, if the level of the erythropoiesis biomarker is approximately equal to or below that level in the control, then step (c) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or if the level of the biomarker of erythropoiesis is elevated compared to that level in the control, then stage (c) involves continuing further treatment with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is a level of immature nucleated erythrocytes, an immature reticulocyte fraction, or cellular nucleic acids. In some embodiments, if the level of the erythropoiesis biomarker is substantially elevated relative to that level in the control, then step (c) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or if the level of the biomarker of erythropoiesis is equal to or below that level in the control, continuing further treatment with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is hematocrit. In some embodiments, said detection of an erythropoiesis biomarker comprises detection of expression of an erythropoiesis-associated gene, wherein the gene is associated with one or more of hematopoiesis, iron ion homeostasis, regulation of erythrocyte differentiation, and heme metabolism. In some embodiments, an erythropoiesis-associated gene is a gene encoding a transferrin receptor (such as CD71), transferrin, Ly76, CD44, CD235a, ALAS2, or GATA-1. In some embodiments, the erythropoiesis biomarker is an erythropoiesis-associated cofactor, such as a porphyrin, a porphyrin-containing compound, or tetrapyrrole. In some embodiments, the sample is a blood sample or a sample of a blood fraction containing peripheral blood mononuclear cells. In some embodiments, the sample is a blood plasma sample. In some embodiments, the control is a historical control or a placebo control. In some embodiments, an effective amount of methotrexate is administered in one cycle or in three cycles. In some embodiments, an effective amount of methotrexate is administered in one, two, three, four, five, or more cycles. In some embodiments, methotrexate is administered from 48 hours before treatment with a therapeutic agent to 48 hours (in particular, at about 24 hours and about 48 hours) thereafter. In some embodiments, methotrexate is administered at a dose of 0.1 mg/kg to 5 mg/kg. In some embodiments, adjunctive therapy to induce immune tolerance or immunosuppressive therapy comprises administering one or more of rituximab, intravenous immunoglobulin (IVIG), cyclophosphamide, bortezomib, azathioprine, cyclosporine, mycophenolate, a corticosteroid, and applying an antigen-specific tolerance building strategy (e.g., more frequent dosing and/or higher dose of therapeutic agent, optionally with concomitant IVIG). In some embodiments, step (d) further comprises administering an agent that depletes T cells, B cells, or plasma cells, such as bortezomib. In some embodiments, the method further comprises monitoring for one or more of anti-therapeutic agent levels, CD19 levels, and disease progression.

[105] In one aspect of the present application, a method is provided for assessing the level of immune tolerance to a therapeutic agent in a subject in need of a therapeutic agent, comprising (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject a therapeutically effective amount of the therapeutic agent; and (c) detecting an erythropoiesis biomarker in a sample obtained from the subject, wherein the level of the erythropoiesis biomarker in the sample compared to that in the control is indicative of induction of immune tolerance in the subject to the therapeutic agent. In some embodiments, the change in the level of the erythropoiesis biomarker is associated with the induction of erythropoiesis. In some embodiments, if the level of the erythropoiesis biomarker is approximately equal to or below that level in the control, then the subject needs additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or if the level of the biomarker of erythropoiesis is elevated compared to that level in the control, then the subject does not need additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is a level of immature nucleated erythrocytes, an immature reticulocyte fraction, or cellular nucleic acids. In some embodiments, the erythropoiesis biomarker is an erythropoiesis-associated gene, wherein the gene is associated with one or more of hematopoiesis, iron ion homeostasis, regulation of erythrocyte differentiation, and heme metabolism. In some embodiments, an erythropoiesis-associated gene is a gene encoding a transferrin receptor (such as CD71), transferrin, Ly76, CD44, CD235a, ALAS2, or GATA-1. In some embodiments, the erythropoiesis biomarker is an erythropoiesis-associated cofactor, such as a porphyrin, a porphyrin-containing compound, or tetrapyrrole. In some embodiments, if the level of the erythropoiesis biomarker is substantially elevated relative to that level in the control, then the subject is in need of additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or where if the level of the biomarker of erythropoiesis is equal to or below that level in the control, then the subject does not need additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is hematocrit. In some embodiments, the sample is a blood sample or a sample of a blood fraction containing peripheral blood mononuclear cells. In some embodiments, the sample is a blood plasma sample. In some embodiments, the control is a historical control or a placebo control.

[106] In some embodiments, a method for assessing the level of immune tolerance to a therapeutic agent in a subject in need of a therapeutic agent is provided, comprising (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject a therapeutically effective amount of the therapeutic agent; and (c) detecting an erythropoiesis biomarker at least one day to about 30 days (e.g., about 7 days to about 14 days) after methotrexate administration, wherein detecting an elevated level of the erythropoiesis biomarker is indicative of inducing immune tolerance in the subject to therapeutic agent. In some embodiments, the change in the level of the erythropoiesis biomarker is associated with the induction of erythropoiesis. In some embodiments, if the level of the erythropoiesis biomarker is approximately equal to or below that level in the control, then the subject needs additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or if the level of the biomarker of erythropoiesis is elevated compared to that level in the control, then the subject does not need additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is a level of immature nucleated erythrocytes, an immature reticulocyte fraction, or cellular nucleic acids. In some embodiments, the erythropoiesis biomarker is an erythropoiesis-associated gene, wherein the gene is associated with one or more of hematopoiesis, iron ion homeostasis, regulation of erythrocyte differentiation, and heme metabolism. In some embodiments, an erythropoiesis-associated gene is a gene encoding a transferrin receptor (such as CD71), transferrin, Ly76, CD44, CD235a, ALAS2, or GATA-1. In some embodiments, the erythropoiesis biomarker is an erythropoiesis-associated cofactor, such as a porphyrin, a porphyrin-containing compound, or tetrapyrrole. In some embodiments, if the level of the erythropoiesis biomarker is substantially elevated compared to that level in the control, then the subject is in need of additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or where if the level of the biomarker of erythropoiesis is equal to or below that level in the control, then the subject does not need additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is hematocrit. In some embodiments, the sample is a blood sample or a sample of a blood fraction containing peripheral blood mononuclear cells. In some embodiments, the sample is a blood plasma sample. In some embodiments, the control is a historical control or a placebo control.

[107] In some embodiments, a method for assessing immune tolerance in a subject with Pompe disease is provided, comprising: (a) obtaining a sample from a subject where the subject has previously received at least one cycle of treatment with methotrexate and at least one dose of a therapeutic agent; and (b) contacting the sample with an agent that binds to an erythropoiesis biomarker, wherein detecting an increased level of erythropoiesis compared to a control is indicative of induction of immune tolerance. In some embodiments, the change in the level of the erythropoiesis biomarker is associated with the induction of erythropoiesis. In some embodiments, if the level of the erythropoiesis biomarker is approximately equal to or below that level in the control, then the subject needs additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or if the level of the biomarker of erythropoiesis is elevated compared to that level in the control, then the subject does not need additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is a level of immature nucleated erythrocytes, an immature reticulocyte fraction, or cellular nucleic acids. In some embodiments, the erythropoiesis biomarker is an erythropoiesis-associated gene, wherein the gene is associated with one or more of hematopoiesis, iron ion homeostasis, regulation of erythrocyte differentiation, and heme metabolism. In some embodiments, an erythropoiesis-associated gene is a gene encoding a transferrin receptor (such as CD71), transferrin, Ly76, CD44, CD235a, ALAS2, or GATA-1. In some embodiments, the erythropoiesis biomarker is an erythropoiesis-associated cofactor, such as a porphyrin, a porphyrin-containing compound, or tetrapyrrole. In some embodiments, if the level of the erythropoiesis biomarker is substantially elevated relative to that level in the control, then the subject is in need of additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or where if the level of the biomarker of erythropoiesis is equal to or below that level in the control, then the subject does not need additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, the biomarker for erythropoiesis is hematocrit. In some embodiments, the sample is a blood sample or a sample of a blood fraction containing peripheral blood mononuclear cells. In some embodiments, the sample is a blood plasma sample. In some embodiments, the control is a historical control or a placebo control.

A. Biomarkers of erythropoiesis

[108] This application provides methods for detecting one or more biomarkers of erythropoiesis in a sample. The erythropoiesis biomarkers described herein may be used alone or in combination at any appropriate time during treatment to monitor immune tolerance induction. In some embodiments, detection of an erythropoiesis biomarker at a level above that level in a control is indicative of immune tolerance to the therapeutic agent induced by the administration of methotrexate in the subject. In some embodiments, the subject is more likely to respond to further treatment with a therapeutic agent without concomitantly receiving additional therapy to induce immune tolerance or immunosuppressive therapy if an erythropoiesis biomarker is detected at an elevated level. In some embodiments, the subject is likely to benefit from concurrent administration of additional immune tolerance inducing or immunosuppressive therapy and further treatment with a therapeutic agent if the erythropoiesis biomarker is detected at or below that of a control.

[109] In some embodiments, the level of the erythropoiesis biomarker in the control is predetermined. In some embodiments, the level of the erythropoiesis biomarker in the control is based on the mean or median level of the erythropoiesis biomarker in the individual prior to receiving methotrexate and/or the therapeutic agent. In some embodiments, the control erythropoiesis biomarker level is based on the mean or median erythropoiesis biomarker level in one or more healthy individuals. In some embodiments, the control erythropoiesis biomarker level is based on the mean or median erythropoiesis biomarker level in one or more individuals having the same disease or condition (such as Pompe disease) being the individual receiving the therapeutic agent. In some embodiments, the control erythropoiesis biomarker level is based on the mean or median erythropoiesis biomarker level in one or more individuals in need of a therapeutic before or without receiving methotrexate and/or a therapeutic. In some embodiments, the control erythropoiesis biomarker level is based on the mean or median erythropoiesis biomarker level in one or more individuals who have been induced immune tolerance to a therapeutic agent by treatment with methotrexate. In some embodiments, the control erythropoiesis biomarker level is based on the mean or median erythropoiesis biomarker level in one or more individuals who were treated with a therapeutic agent but who did not develop an immune response (e.g., ADA) to the therapeutic agent. In some embodiments, the level of the erythropoiesis biomarker in the control is determined from a control sample.

[110] This application partially discloses further evaluation of the mechanism of methotrexate-induced immune tolerance by transcriptional, proteomic and cellular analysis of samples isolated at day 7, when immune tolerance can be transferred adoptively. Through this assessment, various erythropoiesis-associated genes were identified as biomarkers for inducing immune tolerance. The present disclosure also relates to the discovery that methotrexate temporarily depletes erythrocytes (RBCs), thereby inducing erythropoiesis, as demonstrated by the greater incidence and appearance of immature nuclear forms of RBCs in the red pulp of the spleen.

[111] In some embodiments, the implementation of the biomarker of erythropoiesis is detected in the period of time from at least one day to about 30 days after administration of methotrexate. In some embodiments, the detection of the biomarker of erythropoiesis is carried out several times after the administration of methotrexate. In some embodiments, the erythropoiesis biomarker is detected on one or more of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 after methotrexate. In some embodiments, methotrexate is administered during a cycle. For example, one cycle of methotrexate may consist of one dose of methotrexate, or approximately any 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 consecutive daily doses of methotrexate. In some embodiments, the erythropoiesis biomarker is detected from one day to about 30 days after the last methotrexate administration in the cycle. In some embodiments, the erythropoiesis biomarker is detected from one day to about 30 days after the first administration of methotrexate in a cycle. In some embodiments, methotrexate is administered in one cycle, two cycles, or three cycles. In some embodiments, an effective amount of methotrexate is administered in one, two, three, four, five, or more cycles. In some embodiments, the erythropoiesis biomarker is detected from one day to about 30 days after the last methotrexate administration in one cycle or in the first cycle of multiple cycles. In some embodiments, the erythropoiesis biomarker is detected from one day to about 30 days after the first administration of methotrexate in a single cycle or in the first cycle of multiple cycles. In some embodiments, the erythropoiesis biomarker is detected from one day to about 30 days after the last administration of methotrexate in the third cycle. In some embodiments, the erythropoiesis biomarker is detected from one day to about 30 days after the first administration of methotrexate in the first or third cycle.

[112] In some embodiments, the implementation of the biomarker of erythropoiesis is the level of immature nuclear forms of erythrocytes. In some embodiments, the level of immature nucleated erythrocytes decreases after administration of methotrexate, but then increases over time, so that the level of immature nucleated erythrocytes is elevated compared to a reference sample. In some embodiments, the level of immature erythrocytes decreases after about any number of days out of one, two, three, four, five, six, or seven days, or after any period of time from about one to seven days, after administration of methotrexate. In some embodiments, the level of immature red blood cells decreases after about any number of days out of one, two, three, four, five, six, or seven days, or for any period of time from about one to seven days after the last methotrexate administration in a methotrexate administration cycle. In some embodiments, the immature red blood cell level decreases after about any number of days out of one, two, three, four, five, six, or seven days, or for any period of time from about one to seven days after the first methotrexate administration in a methotrexate administration cycle. In some embodiments, the level of immature red blood cells rises more than about any of one, two, three, four, five, six, seven, 14, 21, 28, or 30 days, or for any period of time from about one to 30 days after administration of methotrexate. In some embodiments, the level of immature red blood cells rises more than about any of one, two, three, four, five, six, seven, 14, 21, 28, or 30 days, or for any period of time from about one to 30 days after the last methotrexate injection in the methotrexate administration cycle. In some embodiments, the level of immature red blood cells rises more than about any of one, two, three, four, five, six, seven, 14, 21, 28, or 30 days, or for any period of time from about one to 30 days after the first injection of methotrexate in a cycle of methotrexate administration.

[113] Erythropoiesis is the process by which immature nucleated erythrocytes, also known as RBC progenitors, develop into mature nucleated erythrocytes in the form of discoids (Nandakumar et al., 2016). Immature nuclear forms of RBC can be identified by the cell surface transferrin receptor CD71 (Pan and Johnstone, 1983). The earliest immature nuclear form of RBC or erythroblast in hematopoietic tissue is the proerythroblast. Through successive stages of mitosis, proerythroblasts, which further differentiate into morphologically distinguishable in several stages of mitosis, gradually transform into basophilic, polychromic, then orthochromatic erythroblasts, then turn into reticulocytes (denucleated), and finally become mature erythrocytes (Nandakumar et al. , 2016). The erythroid-specific monoclonal antibody Ter-119 reacts with erythroid cells at various stages of differentiation from nuclear pro-erythroblast to non-nuclear mature erythrocytes (Kina et al., 2000). Early nucleated erythroblasts and reticulocytes, unlike mature erythrocytes or erythrocytes, express the CD71 transferrin receptor (Pan and Johnstone 1983). The proerythroblast and individual successive stages of erythropoiesis can be uniquely identified by CD71 expression or similarly by differential expression of the CD44 adhesion molecule (Chen et al., 2009, Koulnis et al., 2011). In mice, mature red blood cells can circulate in the body for up to 40 days, after which they age due to age-related changes and natural deterioration (Van Putten, 1958). Old RBCs undergo a characteristic form of apoptosis called eryptosis and are cleared from the circulation (Daugas et al., 2001, Testa 2004). It is important to note that mature RBCs and immature nuclear RBCs have the ability to immunomodulate through independent mechanisms (Schakel et al., 2006; Cremel et al., 2013; Getts et al., 2013; Cremel et al., 2015; Grimm et al. ., 2015; Lorentz et al., 2015; Yi et al., 2015).

[114] Excretion of mature RBCs is believed to provide sustained and persistent immune tolerance to self-antigens (Cremelet al., 2013; Gettset al., 2013). Theoretically, the excretion of old RBCs undergoing eryptosis occurs tolerogenically, thereby inducing tolerance to RBC and its entire antigenic load. Taking advantage of this natural phenomenon, a number of groups have been able to achieve immune tolerance to biological therapies in mice by encapsulating or binding a target protein sequence to mature red blood cells (Kontoset al., 2013; Cremelet al., 2015; Grimmet al., 2015; Lorentzet al., 2015). Alternatively, it has been suggested that immature nuclear RBCs that express CD71 directly mediate innate immunosuppression in newborns (Elahiet al., 2013). In addition, recent literature shows that RBCs can suppress the ability of dendritic cells to stimulate T cell activation and antibody responses through the interaction of CD47 on erythrocytes with signal regulatory protein α (SIRPα) on dendritic cells (Schakelet al., 2006; Yiet al., 2015).

[115] Through the mechanisms discussed above and/or other mechanisms, without being bound by any theory, methotrexate-induced erythropoiesis, as described herein, may further stimulate the induction of immune tolerance to biological therapies. Thus, the biomarkers of erythropoiesis described herein can provide an accurate and convenient early assessment of the level of immune tolerance induced by short-term low-dose methotrexate treatment, which can be used as criteria for choosing therapies for inducing immune tolerance and/or immunosuppression with for the purpose of supplementing further cycles of treatment with a therapeutic agent.

[116] In some embodiments, the implementation of the biomarker of erythropoiesis is a total population of erythroid cells or a subpopulation of erythroid cells. The level of a given population of erythroid cells can be expressed in several ways, as will be understood by a person skilled in the art. For example, the population level can be expressed as a comparative percentage, proportion, or frequency of detection (eg, total blood cells or peripheral blood mononuclear cells), absolute number, or concentration (eg, cells/mL). Erythroid cells are usually obtained from whole blood or cellular blood fractions, although erythroid cells can also be obtained from the spleen, bone marrow, or other tissues. In some embodiments, the biomarker for erythropoiesis is hematocrit. As used herein, the term "hematocrit" refers to the ratio of erythrocyte volume to total blood volume. In some embodiments, the biomarker for erythropoiesis is the level of a subpopulation of erythroid cells. As used herein, the terms "erythroid cells" and "erythrocytes" (i.e. "RBC") are used interchangeably to refer to all cell types in the erythroid lineage that are characterized by the expression of Ly76 (ie, the antigen for the Ter-119 antibody) on the cell surface of mouse RBCs or CD235a on the cell surface of human PBMCs. Erythroid cells include proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts, orthochromatophilic erythroblasts, reticulocytes, including immature reticulocytes, and erythrocytes (i.e., mature erythrocytes). In some embodiments, the biomarker for erythropoiesis is the level of erythroid progenitor cells. In some embodiments, the biomarker for erythropoiesis is the level of immature nucleated erythrocytes (RBCs). An "immature nuclear erythrocyte" is an erythrocyte precursor having a nucleus characterized by the expression of Ly76 (an antigen for Ter-119) and the CD71 transferrin receptor on the cell surface. Immature nuclear forms of RBC include RBC progenitors that include a nucleus from proerythroblasts and differentiate into immature reticulocytes. In some embodiments, the biomarker for erythropoiesis is the immature reticulocyte fraction (also known as "IRF"). In some embodiments, the biomarker for erythropoiesis is the level of mature erythrocytes. Mature reticulocytes are reticulocytes that lose their nuclei and turn into mature erythrocytes.

[117] In some embodiments, the implementation of the biomarker of erythropoiesis is the content of cellular nucleic acids. In some embodiments, the biomarker for erythropoiesis is the content of cellular RNA, such as the content of RNA in the total number of blood cells in a blood sample. In some embodiments, the biomarker for erythropoiesis is cellular DNA content, such as the DNA content of total blood cells in a blood sample. The level of reticulocytes in a blood sample correlates with the content of RNA and/or DNA in blood cells in a blood sample.

[118] Different levels of subpopulations of erythroid cells and the content of nucleic acids in the total number of blood cells can be measured using methods known in this field. Commercial automated cell counting systems, such as the SYSMEX ^® hematology analyzers, are available for determining blood parameters, including immature fetal RBC, immature reticulocyte fraction, hematocrit, and RNA/DNA content in total blood cells.

[119] Erythropoiesis biomarkers identified herein include genes associated with erythropoiesis and/or one or more erythroid cell subpopulations, which are referred to herein as "erythropoiesis biomarker genes". In some embodiments, the erythropoiesis biomarker is a gene associated with one or more erythropoiesis biochemical pathways, including, for example, hematopoiesis, iron ion homeostasis, regulation of erythrocyte differentiation, and heme metabolism. In some embodiments, the biomarker for erythropoiesis is a gene selected from the group of upregulated genes in the rhGAA+MTX group shown in FIG. 1A and 1B. Illustrative genes include, without limitation, the transferrin receptor, transferrin, Ly76, CD44, CD235a, ALAS2, and GATA-1 genes. In some embodiments, the erythropoiesis biomarker is a gene encoding a cell surface marker expressed by erythroid cells or a subpopulation of erythroid cells. In some embodiments, the erythropoiesis biomarker is Ly76 (antigen for Ter-119) or CD235a. CD235a is also known as glycophorin A, GYPA, MN, MNS, GPSAT, PAS-2, GPErik, HgpMiv, HgpMiXI, or HgpSta(C). In some embodiments, the biomarker for erythropoiesis is the transferrin receptor. In some embodiments, the biomarker for erythropoiesis is the transferrin 1 receptor. The transferrin 1 receptor is also known as TfR1, differentiation cluster 71, CD71, T9, TR, TFR, p90, TRFR, or IMD46. In some embodiments, the biomarker for erythropoiesis is transferrin. Transferrin is also known as TF, TFQTL1, PRO1557, PRO2086 or HEL-S-71p. In some embodiments, the biomarker for erythropoiesis is CD44. CD44 is also known as IN, LHR, MC56, MDU2, MDU3, MIC4, Pgp1, CDW44, CSPG8, HCELL, HUTCH-1, or ECMR-III. In some embodiments, the biomarker for erythropoiesis is a gene expressed by erythroid cells or a subset of erythroid cells. In some embodiments, the biomarker for erythropoiesis is hemoglobin. In some embodiments, the biomarker for erythropoiesis is a gene expressed by mature erythrocytes, such as erythroid ankyrin or glycophorin C.

[120] Expression levels of one or more genes associated with erythropoiesis or encoding cell surface markers of erythroid cells can be determined qualitatively and/or quantitatively based on any suitable criterion known in the art, including, but not limited to, DNA, mRNA, cDNA, proteins, protein fragments, and/or gene copy number. "Expression level" and "amount" of a biomarker gene are used interchangeably herein. In some embodiments, the level of expression of the biomarker gene in the first sample is increased or elevated compared to that level of expression in the second sample. In some embodiments, the level of expression of the biomarker gene in the first sample is reduced or reduced compared to that level of expression in the second sample. In some embodiments, the second sample is a reference (eg, a reference sample, a reference cell, or a reference tissue) or a control (eg, a control sample, a control cell, or a control tissue). In some embodiments, the control is a historical control or a placebo control. "Historical control" refers to the control obtained in one or more previous experiments. In some embodiments, the historical control is a control based on one or more clinical trials. Placebo control refers to a control obtained from a subject who is not receiving treatment with methotrexate or treatment with a therapeutic agent, or who is receiving treatment with a therapeutic agent without treatment with methotrexate. The detection of an "increased" or "increased" level of expression of the biomarker gene in the first sample compared to that level of expression in the control sample may indicate the "presence" of the biomarker gene in the first sample. The detection of a "reduced" or "reduced" level of expression of the biomarker gene in the first sample compared to that level of expression in the control sample may indicate the "absence" of the biomarker gene in the first sample. Additional disclosures for determining the level of expression of a biomarker gene are described in this document. In some embodiments, the erythropoiesis biomarker is a transferrin receptor (such as CD71), transferrin, Ly76, CD44, CD235a, ALAS2, and/or GATA-1. In some embodiments, the erythropoiesis biomarker is hemoglobin, erythroid ankyrin, and/or glycophorin C.

[121] In some embodiments, the erythropoiesis biomarker is an erythropoiesis-associated cofactor. Illustrative cofactors associated with erythropoiesis include, without limitation, porphyrin, porphyrin-containing compounds, or tetrapyrroles. Cofactor levels can be measured using any of the methods known in the art, including, for example, mass spectroscopy and HPLC.

[122] In some embodiments, the biomarker for erythropoiesis is the presence of immature nuclear forms of erythrocytes. In some embodiments, the level of immature nucleated erythrocytes decreases after the administration of methotrexate, but then increases with time, so the level of immature nucleated erythrocytes is increased compared to the reference sample. In some embodiments, the level of immature erythrocytes decreases after about any number of days out of one, two, three, four, five, six, or seven days, or after any period of time from about one to seven days, after administration of methotrexate. In some embodiments, the level of immature red blood cells decreases after about any number of days out of one, two, three, four, five, six, or seven days, or for any period of time from about one to seven days after the last methotrexate injection in a methotrexate cycle. In some embodiments, the immature red blood cell level decreases after about any number of days out of one, two, three, four, five, six, or seven days, or for any period of time from about one to seven days after the first methotrexate administration in a methotrexate cycle. In some embodiments, where methotrexate is administered in a single cycle, the level of immature nucleated erythrocytes decreases about one, about two, about three, about four, about five, about six, or about seven days before the level of immature nuclear forms erythrocytes becomes elevated. In some embodiments, where methotrexate is administered in two or more cycles, the level of immature nucleated erythrocytes decreases over a period of time from about one to about 14 days before the level of immature nucleated erythrocytes becomes elevated. In some embodiments, the level of immature red blood cells rises more than about any of one, two, three, four, five, six, seven, 14, 21, 28, or 30 days, or for any period of time from about one to 30 days after administration of methotrexate. In some embodiments, the level of immature red blood cells rises more than about any of one, two, three, four, five, six, seven, 14, 21, 28, or 30 days, or for any period of time from about one to 30 days after the last methotrexate injection in the methotrexate administration cycle. In some embodiments, the level of immature red blood cells rises more than about any of one, two, three, four, five, six, seven, 14, 21, 28, or 30 days, or for any period of time from about one to 30 days after the first injection of methotrexate in a cycle of methotrexate administration.

[123] The levels of one or more erythroid cell subpopulations can be determined qualitatively and/or quantitatively based on any suitable criterion known in the art, including, for example, the expression of suitable cell surface markers, histological markers, and morphology (e.g., cell volume and the presence or absence of nuclei). As used herein, the "level" of an erythroid cell subpopulation refers to the number of cells, the fraction of an erythroid cell subpopulation among all erythroid cells, or the frequency of detection of erythroid cells in total blood cells or PBMCs. In some embodiments, the level of the erythroid cell subpopulation in the first sample is increased or increased compared to that level in the second sample. In some embodiments, the level of the erythroid cell subpopulation in the first sample is reduced or decreased compared to that level in the second sample. In some embodiments, the second sample is a reference (eg, a reference sample or a reference tissue) or a control (eg, a control sample or a control tissue). In some embodiments, the control sample/tissue is a historical control or a placebo control. The detection of an "elevated" or "increased" level of an erythroid cell subpopulation in the first sample compared to that level in a control sample may indicate the "presence" of an erythroid cell subpopulation in the first sample. The detection of a "reduced" or "reduced" level of the erythroid cell subpopulation in the first sample compared to that level in the control sample may indicate the "absence" of the erythroid cell subpopulation in the first sample. Additional disclosures for determining the level of a subpopulation of erythroid cells are described in this document. In some embodiments, the biomarker for erythropoiesis is the level (e.g., the number, fraction or frequency of occurrence) of immature nuclear forms of RBC. In some embodiments, the biomarker for erythropoiesis is the fraction of immature reticulocytes. In some embodiments, the biomarker for erythropoiesis is hematocrit.

[124] In some embodiments of any of the methods, an elevated level of an erythropoiesis biomarker means an overall increase of at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 %, 95%, 96%, 97%, 98%, 99% or more of an erythropoiesis biomarker level, such as an expression level (e.g., protein or mRNA) an erythropoiesis-associated gene or a gene encoding an erythroid cell surface marker or level (e.g., number, fraction, or frequency of occurrence) of a subpopulation of erythroid cells, as determined by standard methods known in the art, such as those described herein, compared to a reference or control. In some embodiments, an elevated level of an erythropoiesis biomarker means an increase in the level of an erythropoiesis biomarker in a sample, where the increase is at least about any of 1.5X, 1.75X, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X , 25X, 50X, 75X, or 100X compared to that level of the corresponding erythropoiesis biomarker in the reference or control. In some embodiments, an elevated erythropoiesis biomarker level means an overall increase of more than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3 .0 times or approximately 3.25 times compared to the reference or control. In some embodiments, the erythropoiesis biomarker level is compared to a control, which is the average of the biomarker level in a particular sample or group of samples. For example, in some embodiments, the control is the average level of an erythropoiesis biomarker in a sample from a subject in whom immunotolerance to a therapeutic agent is induced by methotrexate treatment, such as an ITI short-term low-dose methotrexate regimen. In some embodiments, the average level of the erythropoiesis biomarker is obtained from a database of patients with a specific disease in which immunotolerance to a therapeutic agent has been induced by methotrexate treatment. In some embodiments, the average level of the erythropoiesis biomarker is obtained from a database of patients in need of treatment with a particular therapeutic agent in whom methotrexate has induced immune tolerance to the therapeutic agent. For example, the level of expression of an erythropoiesis biomarker gene in a patient with Pompe disease requiring rhGAA treatment can be compared with the median level of expression of an erythropoiesis biomarker gene based on data in a database of patients with Pompe disease who developed immune tolerance to rhGAA due to treatment. methotrexate or did not develop an immune response to rhGAA. In some embodiments, the erythropoiesis biomarker is a transferrin receptor (such as CD71), transferrin, Ly76, CD44, CD235a, ALAS2, or GATA-1. In some embodiments, the biomarker for erythropoiesis is the level (e.g., the number, fraction or frequency of occurrence) of immature nuclear forms of RBC.

[125] In some embodiments of any of the methods, a reduced level of an erythropoiesis biomarker means an overall decrease of approximately any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95 %, 96%, 97%, 98%, 99% or more of an erythropoiesis biomarker level, such as an expression level (e.g., protein or mRNA) an erythropoiesis-associated gene or a gene encoding an erythroid cell surface marker or level (e.g., number, fraction, or frequency of occurrence) of a subpopulation of erythroid cells, as determined by standard methods known in the art, such as those described herein, compared to a reference or control. In some embodiments, a reduced level of an erythropoiesis biomarker means a decrease in the level of an erythropoiesis biomarker in a sample, where the decrease is at least about any of 0.9X, 0.8X, 0.7X, 0.6X, 0.5X, 0.4X, 0 .3X, 0.2X, 0.1X, 0.05X, or 0.01X of the level of the corresponding erythropoiesis biomarker in the standard or control. In some embodiments, the erythropoiesis biomarker is hemoglobin, erythroid ankyrin, or glycophorin C. In some embodiments, the erythropoiesis biomarker is the level of total erythroid cells or mature RBCs. In some embodiments, the biomarker for erythropoiesis is the fraction of immature reticulocytes. In some embodiments, the biomarker for erythropoiesis is hematocrit.

[126] In some embodiments, the erythropoiesis biomarker is elevated on approximately any of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 24, or 28 after initial administration of methotrexate and/or a therapeutic agent. In some embodiments, the erythropoiesis biomarker is decreased on approximately any of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 24, or 28 after initial administration. methotrexate and/or a therapeutic agent. In some embodiments, the erythropoiesis biomarker is elevated on approximately any of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 24, or 28 after the last administration. methotrexate. In some embodiments, the erythropoiesis biomarker is decreased on approximately any of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 24, or 28 after the last administration. methotrexate. In some embodiments, methotrexate is administered in a single cycle. In some embodiments, methotrexate is administered in three cycles. In some embodiments, an effective amount of methotrexate is administered in one, two, three, four, five, or more cycles. In some embodiments, the erythropoiesis biomarker is elevated on approximately any of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 24, or 28 after the last administration. methotrexate in the first cycle or third cycle. In some embodiments, the erythropoiesis biomarker is elevated on approximately any of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 24, or 28 after the last administration. methotrexate in the first, second, third, fourth or fifth cycle. In some embodiments, the erythropoiesis biomarker is decreased on approximately any of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 24, or 28 after the last administration. methotrexate in the first cycle or third cycle. In some embodiments, the erythropoiesis biomarker is decreased on approximately any of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 24, or 28 after the last administration. methotrexate in the first, second, third, fourth or fifth cycle. In some embodiments, the level of the erythropoiesis biomarker first decreases and then increases after initial administration of methotrexate and/or therapeutic agent. In some embodiments, a biomarker for erythropoiesis, such as levels of immature nuclear RBCs, immature reticulocyte fractions, cellular nucleic acids, a gene selected from the gene for transferrin receptor, transferrin, Ly76 (an antigen for Ter-119), CD44, CD235a, ALAS2, and GATA -1, or an erythropoiesis-associated cofactor, is characterized by elevated levels in a blood sample (e.g., PBMC or plasma sample) of a subject between day 6 and day 16 (e.g., any of day 7, day 8, day 9, day 10, day 11, day 12, day 13 and day 14) from the start of treatment with a therapeutic agent.

[127] Expression levels of various erythropoiesis biomarker genes in a sample can be analyzed using a number of techniques, many of which are known in the art and understood by one of skill in the art, including, but not limited to, immunohistochemical analysis ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence-activated cell sorting ("FACS"), MASSARRAY ^® , proteomics, mass spectrometry, tissue mass spectrometric imaging (tMSI), quantitative blood tests (as e.g., serum ELISA), enzyme assay, in situ hybridization, southern assay, northern assay, whole genome sequencing, polymerase chain reaction (“PCR”), including real-time quantitative PCR (“qRT-PCR "), and other types of detection methods based on amplification, such as, for example, using branched DNA, SISBA, TMA, and the like), RNA-Seq, FISH, microarray-based analysis, gene expression profiling, and/or sequential expression analysis genes ("SAGE"), as well as any of the wide range of analyzes that can be performed using the panel analysis of proteins, genes and/or tissues. Typical protocols for assessing the status of genes and gene products can be found, for example, in Ausubel et al ., eds., 1995, Current Protocols In Molecular Biology, sections 2 (Northern blot), 4 (Southern blot), 15 (Immunoblot) and 18 (PCR analysis). Multiplex immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery ("MSD") may also be used.

[128] In some embodiments, expression levels of one or more erythropoiesis biomarker genes are determined using a method comprising: (a) performing gene expression profiling, PCR (such as rtPCR or qRT-PCR), RNA-seq, analysis based on microarray, SAGE, ^MASSARRAY® or FISH techniques on a sample (such as a PBMC or a blood plasma sample from a subject); and b) determining the expression levels of one or more erythropoiesis biomarker gene products in the sample. In some embodiments, the microarray method comprises using a microarray with one or more nucleic acid molecules that can hybridize under stringent conditions to a nucleic acid molecule encoding a gene mentioned above, or to one or more polypeptides (e.g., peptides or antibodies) which can bind to one or more of the proteins encoded by the genes mentioned above. In some embodiments, gene expression is measured using a microarray. In some embodiments, gene expression is measured using qRT-PCR. In some embodiments, gene expression is measured using multiplex PCR. In some embodiments, the erythropoiesis biomarker is a transferrin receptor (such as CD71), transferrin, Ly76, CD44, CD235a, ALAS2, or GATA-1.

[129] Methods for assessing mRNA in cells are well known and include, for example, hybridization assays using complementary DNA probes (e.g., in situ hybridization using labeled riboprobes specific for one or more genes, Northern blotting, and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for one or more genes, and other types of amplification-based detection methods such as, for example, branched DNA, SISBA, TMA, and the like ). To evaluate the expression of the relevant gene(s), the gene product can be quantified prior to translation (e.g., spliced, unspliced, or partially spliced mRNA) Such methods may include one or more steps that determine the levels of the target mRNA in a biological sample (e.g., by simultaneously evaluating the levels of a comparative control mRNA sequence of a "housekeeping" gene, for example, a member of the actin family). Additionally, the sequence of the amplified target cDNA can be determined.

[130] Additional methods include protocols by which mRNAs, such as target mRNAs, are examined or detected in a tissue or cell sample using microarray technologies. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. Then the probes are hybridized with a chip based on nucleic acids immobilized on a solid support. The chip is configured so that the sequence and position of each element on the chip is known. For example, a set of genes associated with erythropoiesis may be arranged on a solid support. Hybridization of a labeled probe with a specific element of the chip indicates that the sample from which the probe was obtained expresses this gene.

[131] The expression level of one or more erythropoiesis biomarker genes can also be determined at the protein level, using the advantages of immunoagglutination, immunoprecipitation (e.g., immunodiffusion, immunoelectrophoresis, immune fixation), immunostaining and/or immunobinding detection methods (including, for example, Western blotting, in situ immunohistochemistry, in situ immunocytochemistry, immunofluorescence, immunoaffinity chromatography, enzyme immunoassay), and the like. These and other methods for quantifying a particular polypeptide in a mixture may be based on specific binding, for example antibodies, and can be performed in a quantitative manner (for example, by quantitative histomorphometry of tissue and/or cell samples). The amounts of purified polypeptide can also be determined using physical methods, for example, photometry or mass spectrometric methods (for example, mass spectrometric imaging of tissues).

[132] In some embodiments, the expression level of the erythropoiesis biomarker protein in the sample is examined using IHC and staining protocols. IHC staining of tissue sections has been shown to be a reliable way to detect or detect the presence of proteins in a sample. In some embodiments, the erythropoiesis biomarker is detected by immunohistochemical analysis. In some embodiments, an increased level of expression of an erythropoiesis biomarker in a sample from an individual is an increased level of protein expression, and in additional embodiments, it is determined using IHC. In some embodiments, the expression level of an erythropoiesis biomarker is determined using a method comprising: (a) performing an IHC analysis of a sample (e.g., a PBMC or a blood plasma sample from a subject) with an antibody, and b) determining the expression level of an erythropoiesis biomarker gene in the sample. In some embodiments, the intensity of IHC staining is determined by comparison with a reference. In some embodiments, the reference is a reference value (for example, from the database of full responders and partial responders for immunotherapy). In some embodiments, the erythropoiesis biomarker is a transferrin receptor (such as CD71), transferrin, Ly76, CD44, CD235a, ALAS2, or GATA-1. In some embodiments, the biomarker for erythropoiesis is hemoglobin, erythroid ankyrin, or glycophorin C.

[133] IHC can be performed in combination with additional techniques, such as morphological staining and/or fluorescent in-situ hybridization. Two main IHC methods are available: direct and indirect assays. According to the first analysis, the binding of the antibody to the target antigen is directly determined. This direct assay uses a labeled reagent, such as a fluorescent label or an enzymatically labeled primary antibody, which can be assayed visually without further antibody interaction. In a typical indirect assay, an unconjugated primary antibody binds to an antigen and then a labeled secondary antibody binds to the primary antibody. When the secondary antibody is conjugated to an enzymatic label, a chromogenic or fluorogenic substrate is added to allow visualization of the antigen. Signal amplification occurs because multiple secondary antibodies may react with different epitopes on the primary antibody.

[134] For use in the detection methods described herein, a person skilled in the art is able to label the polypeptides or oligonucleotides covered by the present invention. As is commonly practiced in the art, hybridization probes for use in detecting mRNA levels and/or antibodies or antibody fragments for use in IHC methods can be labeled and visualized according to standard methods known in the art. Non-limiting examples of commonly used systems include the use of radioactive labels, enzyme labels, fluorescent labels, biotin-avidin complexes, chemiluminescence, and the like.

[135] Primary and/or secondary antibodies used for IHC will typically be labeled with a detectable fragment. Numerous labels are available and can generally be grouped into the following categories: (a) radioisotopes such as³⁵S,¹⁴c,¹²⁵I³H and¹³¹I; (b) particles of colloidal gold; (c) fluorescent labels, including but not limited to rare earth chelates (Europium chelates), TEXAS RED^®, rhodamine, fluorescein, dansyl, lyssamine, umbelliferone, phycocriterin, phycocyanin or commercially available fluorophores such as SPECTRUM ORANGE^TM and SPECTRUM GREEN^TM or derivatives of any one or more of the foregoing; (d) enzyme-substrate labels, including but not limited to those described in US Pat. No. 4,275,149; and (e) enzyme labels, including, for example, luciferases (for example, firefly luciferase and bacterial luciferase; cm., for example, US patent No. 4737456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase, such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (for example, glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (eg uricase and xanthine oxidase), lacteroperoxidase, microperoxidase and the like.

[136] Examples of enzyme-substrate combinations include, for example, horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate; alkaline phosphatase (AP) with para-nitrophenyl phosphate as chromogenic substrate and β-D-galactosidase (β-D-Gal) with chromogenic substrate (e.g., p-nitrophenyl-D-galactosidase) or a fluorogenic substrate (for example, 4-methylumbelliferyl-D-galactosidase). For a general overview of these, see US Pat. Nos. 4,275,149 and 4,318,980.

[137] In some embodiments of any of the methods, the expression of one or more erythropoiesis biomarker genes is detected by immunohistochemical analysis using a diagnostic antibody (i.e., primary antibody) to the expression product of the erythropoiesis biomarker gene. In some embodiments, the diagnostic antibody specifically binds a human erythropoiesis biomarker gene product. In some embodiments, the diagnostic antibody is a non-human antibody. In some embodiments, the diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the diagnostic antibody is a monoclonal antibody. In some embodiments, the implementation of the diagnostic antibody is directly labeled. In some embodiments, the erythropoiesis biomarker is a transferrin receptor (such as CD71), transferrin, Ly76, CD44, CD235a, ALAS2, or GATA-1. In some embodiments, the diagnostic antibody is Ter-119.

[138] Samples for IHC can be obtained by bringing the sample into contact with any of the diagnostic antibodies described above, which can be enclosed in a medium and applied under a coverslip. Then the evaluation is carried out on glass, for example, using a microscope, the staining intensity criteria commonly used in the art can be used. In some embodiments, the presence of an erythropoiesis biomarker is detected by IHC in >0% of samples, in at least 1% of samples, in at least 5% of samples, in at least 10% of samples.

[139] In alternative methods, the sample can be contacted with an antibody specific for said biomarker under conditions sufficient to form an antibody-biomarker complex and then subjected to detection of said complex. The presence of a biomarker can be detected using a number of methods, such as Western blotting and ELISA procedures to analyze a wide range of tissues and samples, including blood plasma or serum. A wide range of immunoassay techniques are available using this assay format, see such as US patents. No. 4016043; 4,424,279 and 4,018,653. These include both single-site and two-site or "sandwich" assays of non-competitive types, as well as conventional competitive binding assays. These assays also include direct binding of the labeled antibody to the target biomarker.

[140] The expression level of the erythropoiesis biomarker gene in a tissue or cell sample can also be examined using functional or activity-based assays. For example, if the biomarker is an enzyme, assays known in the art can be performed to determine or detect the presence of that enzyme activity in a tissue or cell sample.

[141] In some embodiments, the level of a subpopulation of erythroid cells or total erythroid cells is determined using flow cytometry (eg, FACS), using methods known in the art or methods described herein. Each subgroup of erythroid cells is characterized by its unique morphology (for example, cell volume and morphological complexity) and the expression of specific cell surface markers such as CD44 and CD71. Cell volume can be measured using direct scattered light (then is "FSC") using flow cytometry. Erythroid cells and their subpopulations can be identified and isolated by flow cytometry using the following gating strategy: total RBC (Ter-119⁺ for mice or CD235a⁺ for humans), immature nuclear forms of RBC (Ter-119⁺, CD71⁺), mature RBCs (Ter-119⁺/CD235a⁺, CD71^- or Ter-119⁺/CD235a⁺, CD44^lowFSC^low), proerythroblasts (Ter-119^intermediate/ CD235a^intermediate, CD44^high), basophilic erythroblasts (Ter-119⁺/CD235a⁺, CD44^highFSC^high), polychromatophilic erythroblasts (Ter-119⁺/CD235a⁺, CD44^intermediateFSC^intermediate), orthochromatophilic erythroblasts and reticulocytes (Ter-119⁺/CD235a⁺, CD44^intermediateFSC^low). See Chen, Liu et al., 2009 and Koulnis, Pop et al., 2011.

[142] In certain embodiments, samples are normalized both for differences in the level of the erythropoiesis biomarker being analyzed, and for variability in the quality of the samples used, as well as for variability between assay replicates. Such normalization can be achieved by identifying and inserting certain normalizing biomarkers, including well-known "housekeeping" genes. Alternatively, normalization can be performed based on the mean or median signal value of all analyzed genes or a large subset of them (global normalization approach). Based on the order for each of the genes, the measured normalized amount of mRNA or protein in the subject is compared with the amount determined in the reference set or historical control. Normalized expression levels for each mRNA or protein per test sample per subject can be expressed as a percentage of the expression level measured in a reference set or historical control. The level measured in a particular test sample of a subject will fall by some percentile in a given range, which can be determined using methods well known in the art.

[143] The level of the biomarker of erythropoiesis is detected in the sample. Typically, the sample contains a cell, or a population of cells, or some tissue or fluid from a subject. The specimens contemplated herein include tissue sections, such as those obtained by biopsy and autopsy, and frozen sections taken for the purposes of histological examination. Biological samples also include blood and blood fractions or products (eg, blood serum, blood plasma, platelets, erythrocytes, and the like). "Biopsy" refers to a method of removing a tissue sample for diagnostic or prognostic evaluation and to the tissue sample itself. Any biopsy technique known in the art can be used in relation to the diagnostic and prognostic methods of the present invention. The biopsy technique used will depend on the type of tissue being assessed (eg, spleen, bone marrow, blood cell, etc.) among other factors. Exemplary biopsy techniques include, without limitation, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy. Biopsy techniques are discussed, for example, in Harrison's Principles of Internal Medicine, Kasper, et al., eds., ^16th ed., 2005, Chapter 70, and throughout Part V.

[144] In some embodiments, the sample is a clinical sample. In some embodiments, the sample is obtained from the subject's spleen. In some embodiments, the sample is obtained from the subject's bone marrow. In some embodiments, the tissue sample is formalin-fixed and paraffin-embedded, archived, fresh, or frozen. In some embodiments, the sample is whole blood. In some embodiments, the sample is a blood fraction containing peripheral blood mononuclear cells (PBMCs). In some embodiments, the sample is a blood plasma sample or a blood serum sample.

[145] In some embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or a combined sample from the same subject or individual that is obtained at one or more time points other than from the time of receipt of the test sample. For example, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a historical control obtained at an earlier point in time than when the test sample was obtained from the same subject or individual. Such a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be useful if the reference sample is obtained prior to administration of methotrexate and therapeutic agent to a subject to provide a baseline biomarker level in the subject.

[146] In some embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined sample from one or more healthy individuals who are not the subject or individual being treated. In some embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined sample from one or more individuals with a disease or condition (e.g., Pompe disease) who are not the subject or individual being treated. In some embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue are pooled samples (e.g., RNA or protein samples) from pooled PBMCs or blood plasma samples from one or more individuals who are not the subject. or the individual to be treated. In some embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination sample from one or more individuals who are not the subject or individual being treated, but which are characterized by induced immune tolerance to a therapeutic agent achieved by methotrexate treatment. In some embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue are pooled samples (e.g., RNA or protein samples) from pooled plasma or PBMC samples from one or more individuals who are not the subject or an individual to be treated, but who is characterized by induced immune tolerance to a therapeutic agent achieved by treatment with methotrexate (for example, positive control). In some embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue are pooled samples (e.g., RNA or protein samples) from pooled blood plasma or PBMC samples from one or more individuals who are not the subject. or an individual and do not show immune tolerance to a therapeutic agent (for example, negative control). In some embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined sample from one or more individuals who are not receiving treatment with a therapeutic agent and methotrexate ("placebo control"). In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue are pooled samples (e.g., RNA or protein samples) from pooled PBMCs or blood plasma samples from one or more individuals who are not receiving treatment. therapeutic agent and methotrexate ("placebo control"). In some embodiments, placebo control samples from one or more individuals have the same disease or disorder (eg, Pompe disease) as the subject being treated.

[147] In some embodiments, the sample is obtained from the subject at least about one day to about 30 days, including about any of days 1, 2, 4, 7, 9, 14, or 28 after the last injection. methotrexate. In some embodiments, a sample is obtained from a subject within a period of time from about 7 days to about 14 days after the last administration of methotrexate. In some embodiments, the sample is obtained from the subject from at least about one day to about 30 days, including about any of days 1, 2, 4, 7, 9, 14, or 28 after the initial administration of the therapeutic agent. In some embodiments, the sample is obtained from the subject within a period of time from about 7 days to about 14 days from the start of administration of the therapeutic agent. In some embodiments, the sample is obtained from the subject at least about one day to about 30 days, such as about 7, 9, and/or 14 days after initial methotrexate and therapeutic agent administration. In some embodiments, a sample is obtained from a subject within a period of time from about 7 days to about 14 days from the start of methotrexate and therapeutic agent administration. In some embodiments, the sample is obtained from the subject more than 1 time (eg, any number of 2, 3, 4 or more times). In some embodiments, the sample is received on one or more of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 after methotrexate treatment (eg, last methotrexate injection in a methotrexate treatment cycle). In some embodiments, the sample is obtained from the subject prior to the subject receiving additional therapy to induce immune tolerance and/or immunosuppressive therapy.

B. Therapeutic agents

[148] The methods described herein are useful for monitoring, evaluating and/or controlling an undesirable immune response to a therapeutic agent in a subject. In many cases, unwanted immune responses to a therapeutic agent can have a different impact on patient outcome. Such responses arise because biological therapeutics, for example, often contain sequences and conformations that constitute epitopes that are foreign to humans. For example, anti-drug antibodies (ADA) may adversely affect therapeutic efficacy and/or increase safety risks. ADA response can cause hypersensitivity reactions, anaphylaxis, serum sickness, immune complex disease, and acute renal failure. The clinician can monitor for ADA in patients receiving protein therapy using well known methods, including ELISA and immunohistochemistry.

[149] Various therapeutic agents are known to induce unwanted immune responses, which may include anti-drug antibodies (ADA) and other unwanted immune responses mediated by T and/or B cells. In some embodiments, the therapeutic agent is a biological agent, such as a protein, nucleic acid, virus, gene therapy agent, lipid, liposome, carbohydrate, metabolite, or a combination thereof.

[150] In some embodiments, the therapeutic agent is a protein therapy. Protein therapy refers to therapy in which the therapeutic agent is a protein-like substance, including peptides or proteins. Protein therapy can be, for example, enzymes, cytokines, growth factors, immunomodulators, thrombolytics, antibodies (including polyclonal and monoclonal antibodies), antibody fragments, or modified antibodies (for example, Fab, F(ab') ₂ , Fv, Fd , scFv and dAb).

[151] In some embodiments, the therapeutic agent is a therapeutic polypeptide. In some embodiments, the therapeutic agent is an enzyme. For example, many enzyme replacement therapies have been developed for patients with certain genetic diseases, including ^FABRAZYME® (recombinant human alpha-galactosidase) for Fabry disease, ^CEREZYME® (imiglucerase) for Gaucher disease, ^ALDURAZYME® (laronidase) for mucopolysaccharidosis I (MPS I) and MYOZYME ^® and LUMIZYME ^® (alglucosidase alfa) for Pompe disease. In some embodiments, the therapeutic agent is human alpha-galactosidase A. In some embodiments, the enzyme is human acid α-glucosidase (variously referred to as acid maltase, alpha-glucosidase, alpha-1,4-glucosidase, or acid alpha-1,4-glucosidase). In some embodiments, the enzyme is an enzyme-oligosaccharide conjugate, including an acid alpha-glucosidase-oligosaccharide conjugate or another enzyme-oligosaccharide conjugate, as described in US Pat. No. 7,001,994; US patent No. 7723296; US patent No. 7786277; US patent No. 8399657; US patent No. 8841427; US patent No. 9687531; US Pat. No. 8,759,501 and US Pat. No. 9,469,850.

[152] In some embodiments, the therapeutic agent is an enzyme, a neurotrophic factor, a polypeptide deficient or mutated in an individual with a CNS-related disorder, an antioxidant, an anti-apoptotic factor, an anti-angiogenic factor and an anti-inflammatory factor, alpha-synuclein, acid beta- glucosidase (GBA), beta-galactosidase-1 (GLB1), iduronate-2-sulfatase (IDS), galactosylceramidase (GALC), mannosidase, alpha-D-mannosidase (MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA ), N-acetylglucosamine-1-phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), protein associated with Niemann-Pick type C disease (NPC1), acid alpha-1,4-glucosidase (GAA), hexosaminidase beta subunit, HEXB, N-sulfoglucosamine sulfohydrolase (MPS3A), N-alpha-acetylglucosaminidase (NAGLU), heparin-acetyl-CoA-alpha-glucosaminide-N-acetyltransferase (MPS3C), N-acetylglucosamine-6-sulfatase (GNS), alpha-N- acetylgalactosaminidase (NAGA), beta-glucuronidase (GUSB), hexosaminidase alpha subunit (HEXA), huntingtin (HTT), lysosomal acid lipase (LIPA), aspartylglucosaminidase, alpha-galactosidase A, palmitoylprotein thioesterase, tripeptidyl peptidase, lysosomal transmembrane protein, cysteine transporter, acid ceramidase, acid alpha-L-fucosidase, cathepsin A, alpha-L-iduronidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulphate sulfatase, acid beta-galactosidase or alpha-neuraminidase. In some embodiments, the therapeutic agent is a polypeptide selected from the group consisting of neuronal apoptosis inhibitory protein (NAIP), nerve growth factor (NGF), glial growth factor (GDNF), brain growth factor (BDNF), ciliary neurotrophic factor ( CNTF), tyrosine hydroxylase (TH), GTP cyclohydrolase (GTPCH), amino acid decarboxylase (AADC), antioxidant, anti-angiogenic polypeptide, anti-inflammatory polypeptide and aspartoacylase (ASPA).

[153] In some embodiments, the therapeutic agent is factor VIII (FVIII), factor IX (FIX), dystrophin, dysferlin, and cystic fibrosis transmembrane conductance regulator (CFTR).

[154] In some embodiments, the therapeutic agent is an antibody or antigen-binding fragment thereof. Examples of antibody based therapeutics include ^CAMPATH® (alemtuzumab), ^{THYMOGLOBULIN®} , ^AVASTIN® (bevacizumab), ^LUCENTIS® (ranibizumab), ^REMICADE® (infliximab), ^HUMIRA® (adalimumab), ^RITUXAN® (rituximab), ^TYSABRI® (natalizumab ), ^SIMULECT® (basiliximab), ^ZENAPAX® (daclizumab), ^OKT3® (muromonab-CD3), ^ERBITUX® (cetuximab), ^MYLOTARG® (gemtuzumab), ^HERCEPTIN® (trastuzumab), and ^BENLYSTA® (belimumab). Examples of other protein-based therapeutics include ^ENBREL® (etanercept) and other fusion proteins.

[155] In some embodiments, the therapeutic agent is a lymphocyte depleting agent. Lymphocyte depletion is a type of immunosuppression by reducing the number of circulating lymphocytes, such as T cells and/or B cells, resulting in lymphopenia. Lymphocyte depleting agents include, for example, THYMOGLOBULIN ^® , humanized anti-CD52 monoclonal antibody CAMPATH-1H ^® (alemtuzumab) and rituximab. Lymphocyte depletion is desirable in the treatment of a number of autoimmune conditions, including multiple sclerosis (Coles et al, Ann. Neurol. 46, 296-304 (1999); Coles et al, 2008), rheumatoid arthritis, vasculitis, and lupus. Therapy through lymphocyte depletion can induce secondary autoimmunity. Autoimmunity is referred to herein as "secondary autoimmunity" when it occurs after the onset of a first ("primary") disease, such as a "primary" autoimmune disease. Secondary autoimmunity occasionally occurs in MS patients who have or have experienced lymphopenia following, for example, lymphocyte depletion therapy. In some people, secondary autoimmunity occurs shortly after lymphocyte depletion therapy (eg, treatment with alemtuzumab). In other individuals, secondary autoimmunity may not develop until months or years after lymphocyte depletion therapy; in some of these individuals, by the time they develop secondary immunity, there may be a significant recovery of lymphocytes (total lymphocyte count) so that they no longer have lymphopenia. Depletion of lymphocytes may occur in the context of treatment with antibodies or small molecule agents.

[156] In some cases, the therapeutic agent is a viral therapy, where a viral vector is used to deliver a nucleic acid therapeutic drug. Illustrative viruses used in such therapeutics include, without limitation, adenoviruses, adeno-associated viruses, and retroviruses. Antibodies can be synthesized against the capsid proteins of the virus, reducing the effectiveness and increasing the safety risks of such therapeutic drugs. The methods in this application are useful in controlling unwanted immune responses (eg, ADA responses and other unwanted immune responses mediated by T and/or B cells) as well as in viral therapy. In some embodiments, the viral vector encodes any of the therapeutic agents described herein. In some embodiments, the viral vector encodes an enzyme, a neurotrophic factor, a polypeptide deficient or mutated in an individual with a CNS-related disorder, an antioxidant, an anti-apoptotic factor, an anti-angiogenic factor, an anti-inflammatory factor, alpha-synuclein, acid beta-glucosidase (GBA) , beta-galactosidase-1 (GLB1), iduronate-2-sulfatase (IDS), galactosylceramidase (GALC), mannosidase, alpha-D-mannosidase (MAN2B1), beta-mannosidase (MANBA), pseudoarylsulfatase A (ARSA), N- acetylglucosamine-1-phosphotransferase (GNPTAB), acid sphingomyelinase (ASM), protein associated with Niemann-Pick disease type C (NPC1), acid alpha-1,4-glucosidase (GAA such as alglucosidase alfa), hexosaminidase beta subunit , HEXB, N-sulphoglucosamine sulfohydrolase (MPS3A), N-alpha-acetylglucosaminidase (NAGLU), heparin-acetyl-CoA-alpha-glucosaminide-N-acetyltransferase (MPS3C), N-acetylglucosamine-6-sulfatase (GNS), alpha-N -acetylgalactosaminidase (NAGA), beta-glucuronidase (GUSB), hexosaminidase alpha subunit (HEXA), huntingtin (HTT), lysosomal acid lipase (LIPA), aspartylglucosaminidase, alpha-galactosidase A, palmitoylprotein thioesterase, tripeptidyl peptidase, lysosomal transmembrane protein, cysteine transporter , acid ceramidase, acid alpha-L-fucosidase, cathepsin A, alpha-L-iduronidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate, acid beta-galactosidase, or alpha-neuraminidase. In some embodiments, the viral vector encodes a polypeptide selected from the group consisting of neuronal apoptosis inhibitory protein (NAIP), nerve growth factor (NGF), glial growth factor (GDNF), brain growth factor (BDNF), ciliary neurotrophic factor (CNTF). ), tyrosine hydroxylase (TH), GTP-cyclohydrolase (GTPCH), amino acid decarboxylase (AADC), antioxidant, anti-angiogenic polypeptide, anti-inflammatory polypeptide and aspartoacylase (ASPA). In some embodiments, the viral vector encodes factor VIII (FVIII), factor IX (FIX), dystrophin, dysferlin, or cystic fibrosis transmembrane conductance regulator (CFTR). In some embodiments, the viral vector encodes an antibody or an antigen-binding fragment thereof. Examples of antibody based therapeutics include ^CAMPATH® (alemtuzumab), ^{THYMOGLOBULIN®} , ^AVASTIN® (bevacizumab), ^LUCENTIS® (ranibizumab), ^REMICADE® (infliximab), ^HUMIRA® (adalimumab), ^RITUXAN® (rituximab), ^TYSABRI® (natalizumab ), ^SIMULECT® (basiliximab), ^ZENAPAX® (daclizumab), ^OKT3® (muromonab-CD3), ^ERBITUX® (cetuximab), ^MYLOTARG® (gemtuzumab), ^HERCEPTIN® (trastuzumab), and ^BENLYSTA® (belimumab). In some embodiments, the viral vector encodes a fusion protein; eg ^ENBREL® (etanercept).

[157] The methods in this application can also be used to control unwanted immune responses to biotherapies other than protein therapy. Illustrative biotherapies other than protein therapies include, but are not limited to, nucleic acid therapies (eg, antisense therapies, siRNA based therapies, and miRNA based therapies).

C. Methotrexate, other therapies to induce immune tolerance, and immunosuppressive therapies

[158] The methods described herein include administering to the subject an effective amount of methotrexate. In some embodiments, methotrexate is used to induce immune tolerance to a therapeutic agent in a subject. Any of the available dosing regimens can be used to induce immune tolerance, including long-term weekly repeat dosing regimens (Garman et al. Clin Exp Immunol, 137(3):496-502 (2004); Joseph et al., Clin Exp Immunol, 152(1): 138-46 (2008); Mendelsohn et al., N Engl J Med, 360(2): 194-5 (2009)); U.S. Patent Publication No. 20140135337). In some embodiments, an effective amount of methotrexate is administered in three cycles. In some embodiments, methotrexate is administered in a single cycle. In some embodiments, methotrexate is administered in one, two, three, four, five or more cycles.

[159] The use of methotrexate via low-dose short-term administration to induce immune tolerance is unique due to its well-recognized role as a chemotherapeutic agent or long-term immunosuppressive agent in rheumatic diseases (Kremer 2004). Methotrexate was first described for the treatment of cancer in the 1940s, but high-dose courses of methotrexate continue to be prescribed to treat a number of cancers and neoplastic conditions. In autoimmune disorders, inter alia, long-term weekly low-dose methotrexate is used to treat rheumatoid arthritis and psoriasis (Cronstein 2005; Taylor et al ., 2008). In short-term, low-dose methotrexate regimens, low-dose methotrexate is administered in one cycle or three cycles simultaneously with the start of biologic therapy. In some embodiments, one cycle is equivalent to 3 consecutive daily doses of low dose methotrexate administered at time points 0, 24, and 48 hours. Although originally designed as a three-cycle regimen, one cycle of low-dose methotrexate has been shown to be equally effective in inducing long-term immune tolerance in mice (Joseph, Neff et al., 2012, Joly, Martin et al. ., 2014).

[160] Methotrexate acts as a folate antagonist that competitively inhibits dihydrofolate reductase, which is critical for de novo thymidine nucleoside synthesis and purine and pyrimidine biosynthesis. Because DNA synthesis is critical to the cell cycle, high doses of methotrexate are believed to provide cancer treatment by killing highly proliferative cells. Conversely, the mechanism by which long-term low-dose weekly methotrexate controls autoimmune disorders is thought to be through an effect on purine metabolism, resulting in T-cell depletion or suppression of antigen-dependent T-cell proliferation (Cronstein 2005). It has also been shown that methotrexate induces the accumulation of the anti-inflammatory mediator adenosine, which can act as a mediator of T-regulatory cells in inflammatory diseases (Cronstein 2005, Ernst, Garrison et al., 2010, Han, Thomas et al., 2013).

[161] The mechanism by which immune tolerance is achieved by short-term low-dose methotrexate treatment in mice appears to be different from that of immunosuppression by prolonged weekly repeated doses of methotrexate. Weekly repeat dosing of methotrexate may result in different immune outcomes compared to a short dosing regimen.

[162] As used herein, the term "single cycle" refers to a treatment regimen or treatment unit of consecutive or non-consecutive days, and begins preferably no more than five (e.g., no more than three) days after the administration of a dose of a therapeutic agent. One methotrexate cycle may consist of a single dose of methotrexate, or approximately any 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 consecutive daily doses of methotrexate. If the therapeutic agent is administered over multiple periods, one cycle of methotrexate treatment is preferably not continued beyond the first therapeutic dosing period. For example, for a weekly, monthly, or yearly dose of a therapeutic agent, one methotrexate cycle consists of three consecutive days of methotrexate (eg, oral) administration, starting on day 0, the day the therapeutic agent is first administered to the patient. The patient then receives a single dose of methotrexate on day 1 (approximately 24 hours later) and on day 2 (approximately 48 hours later). In some embodiments, one cycle of methotrexate does not last longer than about 8 days.

[163] The methotrexate and therapeutic agent may be administered in any order deemed appropriate. For example, methotrexate can be administered to a subject before, during, and/or after administration of a therapeutic agent. In some embodiments, methotrexate is administered between 48 hours before and 48 hours after treatment with a therapeutic agent. For example, methotrexate may be administered at approximately one of the following times: 48 hours before, 36 hours before, 24 hours before, 12 hours before, concurrent with, 12 hours after, 24 hours after, 36 hours after, or 48 hours after treatment with a therapeutic agent. In some embodiments, methotrexate is administered simultaneously with a therapeutic agent. In some embodiments, the implementation of methotrexate is administered simultaneously with the introduction of a therapeutic agent and about 24 hours and about 48 hours after the administration of a therapeutic agent. In some embodiments, the therapeutic agent is alglucosidase alfa, wherein the methotrexate is administered concurrently with the administration of the therapeutic agent and approximately 24 hours and approximately 48 hours after the administration of the therapeutic agent.

[164] Methotrexate can be administered at any dose effective to reduce unwanted immune responses, such as humoral or cellular responses. An effective amount of methotrexate in human patients may range from about 0.05 mg/kg to about 10 mg/kg, for example, not more than about any of 0.1 mg/kg, 0.2 mg/kg, 0, 3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3, 5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 6 mg/kg, 7.5 mg/kg or 10 mg/kg. In some embodiments, the effective amount is any from about 0.1 mg/kg to about 1.5 mg/kg, from about 0.12 mg/kg to about 1.28 mg/kg, from about 1 mg/kg to about 2.5 mg/kg, or about 2.5 mg/kg to about 5 mg/kg, or about 0.1 mg/kg to about 5 mg/kg. In some embodiments, the dosage of methotrexate may pose minimal safety risk because the dosing regimen includes only a short course of methotrexate at dose levels that are more in line with the doses used in rheumatoid arthritis rather than the low doses used in neoplastic conditions. Patients with rheumatoid arthritis can receive up to 25 mg of methotrexate per week without significant toxicity. A low dose of methotrexate for neoplastic conditions is considered to be 30 mg/m ² .

[165] In some embodiments, methotrexate is administered in more than one cycle, but at a low total dosage. For example, methotrexate can be administered in two or more (eg, 3, 4, 5, 6, etc.) cycles, but with a total combined patient dose of no more than 5 mg/kg.

[166] Low-dose methotrexate regimens appear to be well tolerated by adults. The exact dose and regimen of methotrexate should be determined by the physician, taking into account the patient's physical condition, age, weight, sex, concomitant medications he/she is taking and their known side effects, and any other relevant factors.

[167] In some embodiments, in addition to methotrexate, the subject is given initial therapy to induce immune tolerance simultaneously with the administration of a therapeutic agent. As used herein, "initial immune tolerance induction therapy" refers to an immune tolerance induction regimen administered in a subject from the start of administration of a therapeutic agent, as opposed to immune tolerance induction therapy administered in subsequent cycles of treatment with a therapeutic agent. In some embodiments, initial therapy to induce immune tolerance is administered simultaneously with methotrexate. In some embodiments, initial therapy to induce immune tolerance is administered after methotrexate administration, eg, 1, 2, 3, 4, 5, 6, 7 or more days after methotrexate administration. In some embodiments, initial therapy to induce immune tolerance is given prior to methotrexate administration, eg, 1, 2, 3, 4, 5, 6, 7 or more days prior to methotrexate administration. In some embodiments, the subject has Pompe disease, e.g., CRIM-negative Pompe disease and/or infantile Pompe disease (e.g., classic infantile form of Pompe disease). In some embodiments, methotrexate is administered orally. In some embodiments, methotrexate is administered at a dose of about 0.5 mg/kg. In some embodiments, methotrexate is administered weekly. In some embodiments, methotrexate is administered subcutaneously. In some embodiments, methotrexate is administered at a dose of about 0.4 mg/kg. In some embodiments, methotrexate is administered at three doses per week for three weeks.

[168] In some embodiments, the initial therapy for inducing immune tolerance comprises rituximab, IVIG, or a combination thereof. In some embodiments, initial therapy to induce immune tolerance comprises administering rituximab. In some embodiments, rituximab is administered at a dose of approximately 375 mg/m ² . In some embodiments, for example, if the body surface area (BSA) of the subject is less than 0.5 m ² , rituximab is administered at a dose of approximately 12.5 mg/kg. In some embodiments, rituximab is administered intravenously. In some embodiments, rituximab is administered weekly. In some embodiments, the initial therapy for inducing immune tolerance comprises the administration of IVIG. In some embodiments, IVIG is administered at a dose of about 0.5 mg/kg. In some embodiments, IVIG is administered at a dose of about 400-500 mg/kg. In some embodiments, IVIG is administered approximately every four weeks. In some embodiments, IVIG is administered monthly. In some embodiments, the initial therapy to induce immune tolerance comprises the administration of both rituximab and IVIG.

[169] In some embodiments, the subject is further treated with a therapeutic agent based on the level of one or more biomarkers of erythropoiesis. In some embodiments, where the levels of one or more erythropoiesis biomarkers are elevated relative to such levels in controls, the subject is further treated with a therapeutic agent without additional immune tolerance inducing or immunosuppressive therapy. In some embodiments, where the levels of one or more erythropoiesis biomarkers are reduced compared to such levels in controls, the subject is further treated with a therapeutic agent concurrently with additional therapy to induce immune tolerance and/or immunosuppressive therapy. In some embodiments, one or more biomarkers of erythropoiesis include immature nucleated erythrocytes. In some embodiments, one or more biomarkers of erythropoiesis include an erythropoiesis-associated gene, wherein the gene is associated with one or more of hematopoiesis, iron ion homeostasis, regulation of erythrocyte differentiation, and heme metabolism. In some embodiments, one or more biomarkers of erythropoiesis include a gene encoding a transferrin receptor (such as CD71), transferrin, CD44, CD235a, ALAS2, or GATA-1. In some embodiments, detection of one or more biomarkers of erythropoiesis is performed on a sample obtained from a subject between about 7 days and about 14 days after the last administration of methotrexate. In some embodiments, the sample is a blood sample, a PBMC sample, or a blood plasma sample.

[170] In some embodiments, where the levels of one or more biomarkers of erythropoiesis are about equal (as, for example, at least about one of 90%, 95%, 98%, 99%, or more) or less (i.e., reduced compared to such levels in the control, the subject is further treated with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, where the levels of one or more erythropoiesis biomarkers are elevated compared to such levels in controls, the subject is further treated with a therapeutic agent concurrently with additional therapy to induce immune tolerance and/or immunosuppressive therapy. In some embodiments, one or more biomarkers of erythropoiesis include mature erythrocytes. In some embodiments, the implementation of one or more biomarkers of erythropoiesis include the total number of erythroid cells. In some embodiments, one or more biomarkers of erythropoiesis include hemoglobin, erythroid ankyrin, or glycophorin C. In some embodiments, one or more biomarkers of erythropoiesis are detected in a sample obtained from a subject over a period of about one day to 30 days (from about 7 days to approximately 14 days) after the last administration of methotrexate. In some embodiments, the sample is a blood sample, a PBMC sample, or a blood plasma sample.

[171] Any of the therapies known in the art for inducing immune tolerance and/or immunosuppressive therapy can be administered to a subject in combination with further treatment with a therapeutic agent. In some embodiments, an additional immune tolerance inducing or immunosuppressive therapy involves the administration of a small molecule therapeutic agent, a protein therapeutic agent, or a combination thereof. Illustrative immunosuppressive agents include, without limitation, an anti-CD20 antibody (e.g., rituximab), anti-BAFF antibody (for example, belimumab), anti-CD3 antibody (eg. muromonab or telixizumab), anti-CD19 antibody, anti-CD22 antibody, anti-IgE antibody (eg. omalizumab), a corticosteroid (eg. prednisolone), rapamycin, methotrexate, WIG, cyclophosphamide, cyclosporine A, azathioprine, mycophenolate mofetil, and a proteasome inhibitor (eg, bortezomib). These additional immunomodulatory agents or derivatives thereof include agents that target antigen/alter antigen presentation and/or humoral or cellular immune response. In some embodiments, additional therapy for inducing immune tolerance or immunosuppressive therapy involves the administration of intravenous gamma globulin (IVIG). In some embodiments, the immunosuppressive therapy comprises a lymphocyte depleting agent that depletes T cells, B cells, and/or plasma cells. In some embodiments, the plasma cell depleting agent is bortezomib.

[172] In some embodiments, adjunctive therapy to induce immune tolerance or immunosuppressive therapy involves the use of an antigen-specific tolerance building strategy. Various therapies have been developed to generate antigen-specific tolerance using, for example, antigenic peptides, antigen- or peptide-linked cells, altered peptide ligands (APL) and derivatives thereof. Types of therapy for the formation of antigen-specific tolerance are described, for example, in Miller et al. Nature Reviews Immunology 7.9 (2007): 665-677. In some embodiments, the antigen-specific tolerance therapy involves more frequent doses of the therapeutic agent, or a higher dose of the therapeutic agent, or both. For example, the therapeutic agent may be administered at a dose that is at least about 1.5X, 2X, 3X, 5X, 10X or more of the initial dose. In some embodiments, the therapeutic agent is administered at a frequency that is at least about any of 1.5X, 2X, 3X, 5X, or higher of the initial frequency. In some embodiments, a higher dose of a therapeutic agent is administered concurrently with IVIG.

[173] In some embodiments, the implementation of additional therapy for inducing immune tolerance or immunosuppressive therapy involves the administration of metrotrexate. In some embodiments, the implementation of additional therapy to induce immune tolerance or immunosuppressive therapy does not involve the introduction of metrotrexate. In some embodiments, an adjunct therapy to induce immune tolerance or immunosuppressive therapy comprises one or more of the following: administering an effective amount of one or more of rituximab, intravenous immunoglobulin (IVIG), cyclophosphamide, bortezomib, azathioprine, cyclosporine, mycophenolate, and a corticosteroid, and applying a shaping strategy antigen specific tolerance.

[174] Additional therapy to induce immune tolerance or immunosuppressive therapy can be administered to a subject prior to further administration of a therapeutic agent or concurrently with further administration of a therapeutic agent. In some embodiments, further treatment with a therapeutic agent lasts at least about any of 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years, or more. Additional therapy to induce immune tolerance or immunosuppressive therapy can be carried out during the entire duration of further treatment with a therapeutic agent or during part of the period (for example, just the beginning) of further treatment. Various types and/or regimens of additional therapy for inducing immune tolerance and/or immunosuppressive therapy can be carried out in each cycle of treatment with a therapeutic agent. The dosing regimen and duration of treatment with adjunctive immune tolerance inducing therapy or immunosuppressive therapy may be selected by the clinician based on disease progression, drug antibody levels, and/or levels of one or more erythropoiesis biomarkers.

[175] The effect of methotrexate and optionally other therapies for inducing immune tolerance and immunosuppressive therapy in managing unwanted humoral responses in a patient can be monitored using well-known methods, including clinical examination of the patient, symptoms, blood tests that determine antibody titers to the drug , and immunohistochemical assays (eg, C4 precipitation assays and other solid-phase antibody detection methods such as enzyme-linked immunosorbent assay (ELISA) and bead-based fluorimetric assays). Effects can also be monitored by measuring levels of biomarkers such as MCP-1, IL-13, IL-6, and IL-12, which have been shown to decrease with methotrexate treatment, and transitional 2 B cells, transitional 3 B β-cells, follicular B-cells, marginal zone B-cells, B10 B-cells and B1 B-cells, which have been shown to increase with methotrexate treatment. In addition, TGF-beta, FoxP3, IL-5, IL-10, IL-15, and GM-CSF can be used as biomarkers to monitor the effect of methotrexate on unwanted immune responses as needed. Biomarker levels can also be used to monitor the effect of methotrexate on T cell sensitivity to a therapeutic agent (eg, a therapeutic polypeptide). Biomarkers of T cell activation, such as IL-2, interferon-γ, and TNF-α, can also be monitored as recordable measures to assess the effect of methotrexate on T cell responses. In some embodiments, CD19 levels are additionally determined to monitor an unwanted immune response. In some embodiments, the subject is monitored throughout treatment for the presence of one or more of anti-drug antibody levels (eg, anti-rhGAA IgG antibody levels), CD19 levels, and disease progression.

D. Diseases and conditions

[176] The methods described herein are applicable to the treatment of subjects who have a disease or condition that can be treated with a therapeutic agent, for example, a protein therapeutic agent. For example, enzyme replacement therapy has been developed to treat many diseases and conditions, but subjects receiving enzyme replacement therapy may develop an undesirable immune response, such as developing antibodies to the drug.

[177] In some embodiments, the subject has a lysosomal storage disease. Lysosomal storage disease describes a class of more than 40 genetic disorders (see, e.g., Holton, JB, THE INHERITED METABOLIC DISEASES 205-242 ( ^2nd ed. 1994); Scriver et al., 2 THE METABOLIC BASIS OF INHERITED DISEASE ( 7 ^th ed. 1995)), each of which is the result of a deficiency of a certain lysosomal enzyme, usually resulting from a genetic mutation. Lysosomal enzymes are essential for the breakdown of protein, glycolipid, and carbohydrate metabolites in cell lysosomes. When one or more of these enzymes are defective in affected individuals due to hereditary mutations, the lysosomes in the cells of the affected individual accumulate a subset of undegraded substrates, mainly liposaccharides and carbohydrates, as storage substances that cannot be degraded by the defective enzymes. For example, in Gaucher disease, beta-glucocerebrosidase deficiency causes accumulation of glucosylceramide; in Fabry disease, defective alpha-galactosidase A leads to accumulation of globotriazylceremide; in Pompe disease, lack of acid alpha-glucosidase causes accumulation of alpha-1-4-linked glycogen oligosaccharides; and in Tay-Sachs disease, beta-N-acetylhexosaminidase deficiency leads to accumulation of GM2 ganglioside. Clinically, patients with these syndromes manifest various symptoms associated with the accumulation of these reserve substances in lysosomes, which ultimately affect the normal function of cells or tissues, which leads to organ dysfunction in the human body. The severity of the disease depends on the residual activity of the enzyme; in severe cases, when there is little or no enzyme activity, death can occur at an early age.

[178] In some embodiments, the subject has Gaucher disease and is in need of treatment with beta-glucocerebrosidase. In some embodiments, the subject has Fabry disease and is in need of treatment with alpha-galactosidase A. In some embodiments, the subject has Tay-Sachs disease and is in need of treatment with beta-N-acetylhexosaminidase. In some embodiments, the subject has Pompe disease and is in need of acid α-glucosidase treatment.

[179] In some embodiments, the subject has Pompe disease (variously known as acid maltase deficiency and type II glycogen storage disease). The methods described herein are effective in subjects with early (infantile) and late (juvenile and adult) Pompe disease. In some embodiments, the subject has genetically inherited Pompe disease. In some embodiments, the subject has an infantile form of Pompe disease. In some embodiments, the subject has non-classic infantile Pompe disease. In some embodiments, the subject has late-onset Pompe disease, such as childhood, juvenile, or adult Pompe disease. In some embodiments, the subject has adult Pompe disease.

[180] The classic infantile form of Pompe disease begins within a few months of birth. Children with this disorder typically present with muscle weakness (myopathy), weak muscle tone (hypotension), liver enlargement (hepatomegaly), and heart defects, including cardiac hypertrophy, due to the accumulation of glycogen in all organs. Affected children may also not gain weight and grow at the expected rate (developmental failure) and may have breathing problems. On clinical examination, there is generalized hypotension with muscle wasting, tachypnea with retraction of the sternum, moderate enlargement of the liver, and protrusion of the tongue. Cardiac ultrasound shows progressive hypertrophic cardiomyopathy, eventually leading to cardiac output deficiency. The ECG is characterized by marked left axis deviation, short PR interval, large QRS complexes, inverted T waves, and ST depression. The disease is characterized by a rapidly progressive course leading to cardiorespiratory failure during the first year of life. On histological examination at autopsy, the accumulation of lysosomal glycogen is noted in various tissues, and it is most pronounced in the heart muscle and skeletal muscles. Left untreated, this form of Pompe disease usually results in death from heart failure within the first year of life.

[181] The nonclassical infantile form of Pompe disease usually appears at 1 year of age. It is characterized by a delay in motor skills (such as rolling over and sitting) and progressive muscle weakness. The heart may be abnormally large (cardiomegaly), but affected individuals generally do not experience heart failure. The muscle weakness associated with this disorder leads to severe breathing problems, and if left untreated, most children with non-classic infantile Pompe disease survive only into early childhood.

[182] Late-onset Pompe disease may not become apparent later than childhood, adolescence, or adulthood. Late-onset Pompe disease is generally milder than the infantile forms of the disorder and less likely to affect the heart. Most people with late-onset Pompe disease experience progressive muscle weakness, especially in the legs and trunk, including the muscles that control breathing. As the disorder progresses, breathing problems can lead to respiratory failure.

[183] Treatment with human acid alpha-glucosidase may prolong the life of such patients (eg, to more than 1, 2, or 5 years of normal life expectancy). Treatment can also eliminate or reduce the clinical and biochemical manifestations of Pompe disease, as discussed above. Treatment is given shortly after birth or in the antenatal period if parents are known to carry variant alpha-glucosidase alleles that put their offspring at risk.

[184] Patients with late-onset adult Pompe disease may not experience symptoms for the first two decades of life. In this clinical subtype, effects on skeletal muscles predominate, in particular the muscles of the shoulder and pelvic girdle, trunk and diaphragm. Difficulty climbing stairs is often the initial complaint. Respiratory impairment varies greatly. It may dominate the clinical picture, or the patient may not experience this problem until later in life. Most late-onset patients eventually die due to respiratory failure. In patients with the juvenile subtype, symptoms usually become apparent in the first decade of life. As with adult Pompe disease, skeletal muscle weakness is the main problem; cardiomegaly, hepatomegaly, and macroglossia are seen but are rare. In many cases, nocturnal assisted ventilation is ultimately required. Pulmonary infections in combination with wasting of the respiratory muscles are life-threatening and are usually fatal before the third decade of life. Treatment with the present methods extends the life of patients with juvenile or adult late-onset Pompe disease to normal life expectancy. With the help of treatment, the clinical and biochemical symptoms of the disease are also eliminated or significantly reduced.

[185] In some embodiments, the subject suffering from Pompe disease is CRIM negative. As used herein in the context of Pompe disease, the term "CRIM" refers to the immunoreactivity of endogenous GAA protein in a patient. For example, patients with two detrimental mutations and no GAA at all, as assessed by Western blotting using an antibody to wild-type human GAA protein, are classified as negative for cross-reactive immunological material (CRIM). Patients with GAA protein determined by Western blotting are classified as CRIM-positive. While the majority of CRIM-positive patients show sustained therapeutic responses to ERT, CRIM-negative patients do almost equally poorly, experiencing rapid clinical deterioration due to the production of high-titre resistant anti-rhGAA antibodies. Cm., Kishnani PS, et al. Mol Genet Metab. 2010;99:26-33. Such ADA has also been observed in some CRIM-positive patients (Banugaria et al., 2011). In some embodiments, CRIM status is identified by GAA mutational analysis with or without confirmation by Western blot analysis of skin fibroblast cells.

[186] In some embodiments, the subject has an autoimmune disease, such as multiple sclerosis (MS). In some embodiments, the subject is in need of treatment with an anti-CD52 antibody. In some embodiments, such an antibody is alemtuzumab.

[187] In some embodiments, the subject has a genetic disorder. In some embodiments, the subject has a disease or disorder that is being treated by administering a biologic (eg, polypeptide, nucleic acid, etc.). In some instances, the subject has a disease or disorder that is being treated with a compound (eg, biologic) that can induce anti-drug antibodies (ADA) or an immune response. In some embodiments, the subject has hemophilia A, hemophilia B, muscular dystrophy, cystic fibrosis, Huntington's disease, Parkinson's disease, or Alzheimer's disease. In some embodiments, the subject has a disease or disorder that is being treated with viral therapy, where a viral vector is used to deliver the nucleic acid. In some embodiments, the viral vector is derived from a retrovirus, adenovirus, lentivirus, herpes simplex virus, vaccinia virus, or adeno-associated virus.

III. Kits, assays and products

[188] In addition, this document provides kits containing one or more reagents for detecting a biomarker of erythropoiesis in a sample obtained from a subject after administration of methotrexate and a therapeutic agent. In some embodiments, the kit further comprises a therapeutic agent and/or methotrexate. In some embodiments, the kit further comprises an adjuvant therapy for inducing immune tolerance or immunosuppressive therapy. In some embodiments, the kit further comprises instructions for using the kit to select a drug (eg, for adjunctive therapy to induce immune tolerance or immunosuppressive therapy) to treat a disease or disorder if the individual has a reduced level of an erythropoiesis biomarker. In some embodiments, the kit contains reagents for detecting two or more erythropoiesis biomarkers.

[189] In some embodiments, an assay is provided to assess the level of immune tolerance to a therapeutic agent (e.g., human acid α-glucosidase) in a subject with Pompe disease, the method comprising obtaining a sample from a subject where the subject has previously received at least one cycle treatment with methotrexate and at least one dose of a therapeutic agent; and contacting the sample with an agent that binds to an erythropoiesis biomarker, wherein detecting an elevated level of the erythropoiesis biomarker compared to a control level is indicative of induction of immune tolerance.

[190] In some embodiments, an assay is provided to assess the level of immune tolerance to a therapeutic agent in a subject in need of a therapeutic agent, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject a therapeutically effective amount of the therapeutic agent; and (c) detecting an erythropoiesis biomarker in a sample obtained from the subject, wherein the level of the erythropoiesis biomarker in the sample compared to that in the control is indicative of induction of immune tolerance in the subject to the therapeutic agent. In some embodiments, a biomarker of erythropoiesis is detected in a sample obtained from a subject from at least one day to about 30 days after methotrexate administration. In some embodiments, the change in the level of the erythropoiesis biomarker is associated with the induction of erythropoiesis, where the change in the level of the erythropoiesis biomarker is associated with the induction of erythropoiesis. In some embodiments, if the level of the erythropoiesis biomarker is approximately equal to or below that level in the control, then the subject needs additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or if the level of the biomarker of erythropoiesis is elevated compared to that level in the control, then the subject does not need additional therapy to induce immune tolerance or immunosuppressive therapy. In some embodiments, if the level of the erythropoiesis biomarker is substantially elevated relative to that level in the control, then the subject is in need of additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or where if the level of the biomarker of erythropoiesis is equal to or below that level in the control, then the subject does not need additional therapy to induce immune tolerance or immunosuppressive therapy.

[191] This document also provides articles containing packaged together a pharmaceutical composition containing a therapeutic agent (e.g., human acid α-glucosidase), a pharmaceutical composition containing methotrexate, and a package leaflet indicating that the therapeutic agent is intended to treat a patient with a disease or condition based on the level of the biomarker of erythropoiesis after administration of a therapeutic agent and methotrexate. In some embodiments, the product further comprises an additional immune tolerance inducing therapy and/or an immunosuppressive therapy. Methods of treatment include any of the methods of treatment disclosed in this document.

[192] The product includes a container and a label or leaflet on or with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers can be made from a number of materials such as glass or plastic. The container holds or contains the composition containing the therapeutic agent as the active agent and may have a sterile entry port (for example, the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle).

[193] The article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The product may additionally include other substances necessary from a commercial and consumer point of view, including other buffers, diluents, fillers, needles and syringes.

[194] The article of the present invention also includes information, for example in the form of a package leaflet, indicating that the composition is used to treat a disease or condition based on the level of one or more of the erythropoiesis biomarkers described herein. The insert or label may be in any form, such as paper or electronic media, such as a storage device that includes flash memory (such as a USB flash drive or the like), magnetic recording media (such as diskette) or CD-ROM. The label or insert may also include other information regarding the pharmaceutical compositions and dosage forms in the kit or article.

[195] The present invention also relates to a method of manufacturing an article containing combined in a package a pharmaceutical composition containing a therapeutic agent (for example, human acid α-glucosidase), a pharmaceutical composition containing methotrexate, and a package leaflet indicating that the therapeutic agent is intended for the treatment of a patient with a disease or condition (for example, Pompe disease) based on the level of an erythropoiesis biomarker after administration of a therapeutic agent and methotrexate. In some embodiments, the product further comprises an additional immune tolerance inducing therapy and/or an immunosuppressive therapy.

IV. Illustrative Embodiments

[196] The present invention provides the following embodiments:

[197] Embodiment 1. A method of treating a subject in need of treatment with a therapeutic agent, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of the therapeutic agent; (c) detecting an erythropoiesis biomarker in a sample obtained from the subject over a period of time from at least one day to about 30 days after methotrexate administration; and (d) continuing further treatment with a therapeutic agent with or without additional therapy to induce immune tolerance or immunosuppressive therapy based on the level of the biomarker of erythropoiesis compared to that level in the control.

[198] Embodiment 2. The method of Embodiment 1, wherein the change in the level of an erythropoiesis biomarker is associated with the induction of erythropoiesis.

[199] Embodiment 3. The method of Embodiment 2, wherein if the level of the erythropoiesis biomarker is approximately equal to or lower than that of the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy simultaneously with further treatment with therapeutic agent; or where if the level of the erythropoiesis biomarker is elevated compared to that level in the control, then step (d) involves continuing further treatment with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy.

[200] Embodiment 4. The method of Embodiment 2, wherein if the level of the erythropoiesis biomarker is substantially elevated relative to that of the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy simultaneously with further treatment with therapeutic agent; or where if the level of the biomarker of erythropoiesis is equal to or below that level in the control, then step (d) involves continuing further treatment with a therapeutic agent without additional therapy to induce immune tolerance or immunosuppressive therapy.

[201] Embodiment 5. The method of Embodiment 3, wherein the erythropoiesis biomarker is the level of immature nucleated erythrocytes, immature reticulocyte fraction, or cellular nucleic acids.

[202] Embodiment 6. The method of Embodiment 4, wherein the erythropoiesis biomarker is hematocrit.

[203] Embodiment 7. The method of Embodiment 5, wherein if the level of immature nucleated erythrocytes initially decreases before the level of immature nucleated erythrocytes rises, further treatment with a therapeutic agent is continued without additional therapy to induce immune tolerance or immunosuppressive therapy.

[204] Embodiment 8. The method of Embodiment 7, wherein the methotrexate is administered in one cycle, the level of immature nucleated erythrocytes decreases in about one, about two, about three, about four, about five, about six, or about seven days. before the level of immature nuclear forms of erythrocytes becomes elevated.

[205] Embodiment 9. The method of Embodiment 7, wherein methotrexate is administered in two or more cycles, the level of immature nucleated erythrocytes decreases over a period of time from about one to about 14 days before the level of immature nucleated erythrocytes becomes elevated.

[206] Embodiment 10. The method of embodiment 3 or 4, wherein the detection of the erythropoiesis biomarker is detection of expression of an erythropoiesis-associated gene, wherein the erythropoiesis-associated gene is a gene associated with one or more of hematopoiesis, ion homeostasis iron, regulation of erythrocyte differentiation and heme metabolism.

[207] Embodiment 11. The method of Embodiment 10, wherein the erythropoiesis-associated gene is a gene encoding transferrin receptor, transferrin, Ly76 (antigen for Ter-119), CD44, CD235a, ALAS2, or GATA-1.

[208] Embodiment 12. The method of Embodiment 11, wherein the transferrin receptor is transferrin receptor 1 (CD71).

[209] Embodiment 13. The method of any one of embodiments 10-12, wherein detecting an erythropoiesis-associated gene comprises detecting an RNA transcript of an erythropoiesis-associated gene or detecting a protein product of an erythropoiesis-associated gene.

[210] Embodiment 14. The method of Embodiment 13, wherein the protein product is hemoglobin.

[211] Embodiment 15. The method of Embodiment 3, wherein the erythropoiesis biomarker is an erythropoiesis-associated cofactor.

[212] Embodiment 16. The method of Embodiment 15, wherein the erythropoiesis-associated cofactor is a porphyrin, a porphyrin-containing compound, or tetrapyrrole.

[213] Embodiment 17. The method of any one of Embodiments 1-16, wherein the sample is a blood sample.

[214] Embodiment 18. The method of Embodiment 17, wherein the blood sample is a blood fraction containing peripheral blood mononuclear cells (PBMCs).

[215] Embodiment 19. The method of Embodiment 18, wherein the blood sample is a blood serum sample or a blood plasma sample, and wherein the biomarker detection is detection of erythropoiesis-associated gene expression.

[216] Embodiment 20. The method of any one of Embodiments 1-19, wherein the control is a historical control or a placebo control.

[217] Embodiment 21. The method of any one of Embodiments 1-20, wherein the subject is a human.

[218] Embodiment 22. The method of any one of Embodiments 1-21, wherein the effective amount of methotrexate is administered in one cycle or in three cycles.

[219] Embodiment 23. The method of Embodiment 22 wherein the methotrexate is administered in one cycle.

[220] Embodiment 24. The method of Embodiment 22 or Embodiment 23, wherein the methotrexate cycle consists of 1 day of methotrexate administration or 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 consecutive days of methotrexate administration .

[221] Embodiment 25. The method of any one of Embodiments 1-24, wherein the methotrexate is administered to the subject at a time selected from one or more of the times before, during, and after administration of the therapeutic agent.

[222] Embodiment 26. The method of Embodiment 25, wherein the methotrexate is administered from 48 hours prior to administration of the therapeutic agent to 48 hours thereafter.

[223] Embodiment 27. The method of Embodiment 26 wherein the methotrexate is administered concurrently with administration of the therapeutic agent and approximately 24 hours and approximately 48 hours after administration of the therapeutic agent.

[224] Embodiment 28. The method of any one of items 1-27, wherein methotrexate is administered at a dose of about 0.1 mg/kg to about 5 mg/kg.

[225] Embodiment 29. The method of any one of Embodiments 1-28, wherein the sample is obtained from the subject at least about one day to about 30 days after the last methotrexate administration.

[226] Embodiment 30. The method of any one of Embodiments 1-29, wherein the sample is obtained from the subject between about 7 days and about 14 days after the last administration of methotrexate.

[227] Embodiment 31. The method of any one of Embodiments 1-30, wherein samples are obtained within a few days of the last administration of methotrexate.

[228] Embodiment 32. The method of Embodiment 30, wherein the sample is obtained at one or more of day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 14, day 21, or days 28 after the last administration of methotrexate.

[229] Embodiment 33. The method of any one of embodiments 1-32, wherein step (d) comprises administering an agent that depletes T cells, B cells, or plasma cells.

[230] Embodiment 34. The method of any one of embodiments 1-33, wherein step (d) comprises one or more of the following: (a) administering an effective amount of one or more of rituximab, intravenous immunoglobulin (IVIG), cyclophosphamide, bortezomib , azathioprine, cyclosporine, mycophenolate, and a corticosteroid; and (b) applying an antigen-specific tolerance building strategy.

[231] Embodiment 35. The method of Embodiment 34, wherein the antigen-specific tolerance building strategy involves more frequent doses of the therapeutic agent, or a higher dose of the therapeutic agent, or both.

[232] Embodiment 36. The method of Embodiment 35, wherein the higher dose of therapeutic agent is administered simultaneously with IVIG.

[233] Embodiment 37. The method of Embodiment 1-36, wherein the therapeutic agent is a polypeptide, nucleic acid, virus, gene therapy agent, lipid, liposome, or carbohydrate.

[234] Embodiment 38. The method of any one of Embodiments 1-37, wherein the therapeutic agent is a therapeutic polypeptide.

[235] Embodiment 39. The method of Embodiment 38, wherein the therapeutic polypeptide is an antibody or an antigen-binding fragment thereof.

[236] Embodiment 40. The method of Embodiment 39, wherein the antibody is a monoclonal antibody.

[237] Embodiment 41. The method of Embodiment 39 or Embodiment 40, wherein the antibody is a lymphocyte depleting agent.

[238] Embodiment 42. The method of Embodiment 40 or Embodiment 41, wherein the antibody is alemtuzumab.

[239] Embodiment 43. The method of Embodiment 38, wherein the therapeutic polypeptide is an enzyme.

[240] Embodiment 44. The method of Embodiment 42, wherein the enzyme is human alpha-galactosidase A.

[241] Embodiment 45. The method of Embodiment 42, wherein the enzyme is human acid α-glucosidase.

[242] Embodiment 46. The method of any of embodiments 1-38 and 43-45, wherein the subject has lysosomal storage disease.

[243] Embodiment 47. The method of Embodiment 46 wherein the subject has Pompe disease.

[244] Embodiment 48. The method of Embodiment 47 wherein Pompe disease is an infantile form of Pompe disease.

[245] Embodiment 49. The method of Embodiment 46, wherein Pompe disease is the classic infantile form of Pompe disease.

[246] Embodiment 50. The method of any of embodiments 46-49, wherein the subject is negative for cross-reactive immunological material (CRIM).

[247] Embodiment 51. The method of Embodiment 50, wherein the method further comprises administering the initial therapy to induce immune tolerance concurrently with administering the therapeutic agent, wherein the therapeutic agent is human acid α-glucosidase.

[248] Embodiment 52. The method of Embodiment 51, wherein the initial therapy for inducing immune tolerance comprises administering one or more of rituximab and IVIG.

[249] Embodiment 53. A method of treating Pompe disease in a subject in need thereof, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of human acid α-glucosidase; (c) detecting an erythropoiesis biomarker in a sample obtained from the subject over a period of time from at least one day to about 30 days after methotrexate administration; and (d) continuing further treatment with human acid α-glucosidase with or without additional therapy to induce immune tolerance or immunosuppressive therapy based on the level of the erythropoiesis biomarker compared to that level in the control.

[250] Embodiment 54. The method of Embodiment 53, wherein the change in the level of an erythropoiesis biomarker is associated with the induction of erythropoiesis.

[251] Embodiment 55. The method of Embodiment 54, wherein if the level of the erythropoiesis biomarker is approximately equal to or lower than that of the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with human acid α-glucosidase; or if the level of the biomarker of erythropoiesis is elevated compared to that level in the control, then stage (d) involves continuing further treatment with human acid α-glucosidase without additional therapy to induce immune tolerance or immunosuppressive therapy.

[252] Embodiment 56. The method of Embodiment 54, wherein if the level of the erythropoiesis biomarker is substantially elevated compared to that level in the control, then step (d) involves administering additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with human acid α-glucosidase; or if the level of the biomarker of erythropoiesis is equal to or below that level in the control, then step (d) involves continuing further treatment with human acid α-glucosidase without additional therapy to induce immune tolerance or immunosuppressive therapy.

[253] Embodiment 57. The method of Embodiment 55, wherein the erythropoiesis biomarker is the level of immature nucleated erythrocytes, immature reticulocyte fraction, or cellular nucleic acids.

[254] Embodiment 58. The method of Embodiment 56, wherein the erythropoiesis biomarker is hematocrit.

[255] Embodiment 59. The method of Embodiment 57, wherein if the level of immature nucleated erythrocytes initially decreases before the level of immature nucleated erythrocytes rises, further treatment with a therapeutic agent is continued without additional therapy to induce immune tolerance or immunosuppressive therapy.

[256] Embodiment 60. The method of Embodiment 59, wherein the methotrexate is administered in a single cycle, the level of immature nucleated erythrocytes decreases in about one, about two, about three, about four, about five, about six, or about seven days. before the level of immature nuclear forms of erythrocytes becomes elevated.

[257] Embodiment 61. The method of Embodiment 59, wherein the methotrexate is administered in two or more cycles, the level of immature nucleated erythrocytes decreases over a period of time from about one to about 14 days before the level of immature nucleated erythrocytes becomes elevated.

[258] Embodiment 62. The method of embodiment 55 or 56, wherein the detection of the erythropoiesis biomarker is detection of expression of an erythropoiesis-associated gene, wherein the erythropoiesis-associated gene is a gene associated with one or more of hematopoiesis, ion homeostasis iron, regulation of erythrocyte differentiation and heme metabolism.

[259] Embodiment 63. The method of Embodiment 62, wherein the erythropoiesis-associated gene is a gene encoding transferrin receptor, transferrin, Ly76 (antigen for Ter-119), CD44, CD235a, ALAS2, or GATA-1.

[260] Embodiment 64. The method of Embodiment 63, wherein the transferrin receptor is transferrin receptor 1 (CD71).

[261] Embodiment 65. The method of any one of embodiments 62-64, wherein detecting an erythropoiesis-associated gene comprises detecting an RNA transcript of an erythropoiesis-associated gene or detecting a protein product of an erythropoiesis-associated gene.

[262] Embodiment 66. The method of Embodiment 65, wherein the protein product is hemoglobin.

[263] Embodiment 67. The method of Embodiment 55, wherein the erythropoiesis biomarker is an erythropoiesis-associated cofactor.

[264] Embodiment 68. The method of Embodiment 67, wherein the erythropoiesis-associated cofactor is a porphyrin, a porphyrin-containing compound, or tetrapyrrole.

[265] Embodiment 69. The method of any one of Embodiments 53-68, wherein the sample is a blood sample.

[266] Embodiment 70. The method of Embodiment 69, wherein the blood sample is a blood fraction containing peripheral blood mononuclear cells (PBMCs).

[267] Embodiment 71. The method of any one of Embodiments 53-70, wherein the subject is a human.

[268] Embodiment 72. The method of any of embodiments 53-71, wherein Pompe disease is an infantile form of Pompe disease.

[269] Embodiment 73. The method of Embodiment 72 wherein Pompe disease is the classic infantile form of Pompe disease.

[270] Embodiment 74. The method of Embodiment 72 or 73, wherein the subject is CRIM negative.

[271] Embodiment 75. The method of any one of Embodiments 53-74, wherein the control is a historical control or a placebo control.

[272] Embodiment 76. The method of any one of Embodiments 53-75, wherein the effective amount of methotrexate is administered in one cycle or in three cycles.

[273] Embodiment 77. The method of Embodiment 76, wherein the effective amount of methotrexate is administered in one cycle.

[274] Embodiment 78. The method of Embodiment 76 or Embodiment 77, wherein the methotrexate cycle consists of 1 day of methotrexate administration or 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 consecutive days of methotrexate administration .

[275] Embodiment 79. The method of any one of embodiments 53-78, wherein an effective amount of methotrexate is administered to the subject at a time selected from one or more of the times before, during, and after administration of the therapeutic agent.

[276] Embodiment 80. The method of Embodiment 79, wherein an effective amount of methotrexate is administered from 48 hours prior to treatment with a therapeutic agent to 48 hours thereafter.

[277] Embodiment 81. The method of Embodiment 80 wherein the methotrexate is administered concurrently with administration of the therapeutic agent and approximately 24 hours and approximately 48 hours after administration of the therapeutic agent.

[278] Embodiment 82. The method of any one of items 53-81, wherein the effective amount of methotrexate is from about 0.1 mg/kg to about 5 mg/kg.

[279] Embodiment 83. The method of any one of embodiments 53-82, wherein the detection of an erythropoiesis biomarker in the sample is performed from at least about one day to about 30 days after the last methotrexate administration.

[280] Embodiment 84. The method of any one of embodiments 53-83, wherein the detection of the erythropoiesis biomarker in the sample is performed from about 7 days to about 14 days after the last administration of methotrexate.

[281] Embodiment 85. The method of any one of Embodiments 53-84, wherein the samples are obtained within a few days of the last administration of methotrexate.

[282] Embodiment 86. The method of Embodiment 85, wherein the sample is obtained at one or more of day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 14, day 21, or days 28 after the last administration of methotrexate.

[283] Embodiment 87. The method of any one of embodiments 53-86, wherein step (d) further comprises administering an agent that depletes T cells, B cells, or plasma cells.

[284] Embodiment 88. The method of Embodiment 87, wherein the plasma cell depleting agent is bortezomib.

[285] Embodiment 89. The method of any one of embodiments 53-88, wherein step (d) comprises one or more of the following: (a) administering an effective amount of one or more of rituximab, intravenous immunoglobulin (IVIG), cyclophosphamide, bortezomib , azathioprine, cyclosporine, mycophenolate, and a corticosteroid; and (b) applying an antigen-specific tolerance building strategy.

[286] Embodiment 90. The method of Embodiment 89, wherein the antigen-specific tolerance building strategy involves more frequent doses of the therapeutic agent, or a higher dose of the therapeutic agent, or both.

[287] Embodiment 91. The method of Embodiment 90, wherein the higher dose of therapeutic agent is administered concurrently with IVIG.

[288] Embodiment 92. The method of any one of embodiments 53-91, further comprising monitoring for one or more of anti-human acid α-glucosidase antibody level, CD19 level, or disease progression.

[289] Embodiment 93. A method for assessing the level of immune tolerance in a subject with Pompe disease, comprising: (a) obtaining a sample from a subject where the subject has previously received at least one cycle of methotrexate treatment and administered at least one dose of a therapeutic agent; and (b) detecting an erythropoiesis biomarker in the sample, wherein the level of the erythropoiesis biomarker in the sample compared to that in the control is indicative of immune tolerance induction.

[290] Embodiment 94. The method of Embodiment 93, wherein step (b) comprises contacting the sample with an agent that binds to an erythropoiesis biomarker.

[291] Embodiment 95. A method for assessing the level of immune tolerance to a therapeutic agent in a subject in need of a therapeutic agent, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of the therapeutic agent; and (c) detecting an erythropoiesis biomarker in a sample obtained from the subject, wherein the level of the erythropoiesis biomarker in the sample compared to that in the control is indicative of induction of immune tolerance in the subject to the therapeutic agent.

[292] Embodiment 96. A method for assessing the level of immune tolerance to a therapeutic agent in a subject in need of a therapeutic agent, comprising: (a) administering to the subject an effective amount of methotrexate; (b) administering to the subject an effective amount of the therapeutic agent; (c) detecting an erythropoiesis biomarker in a sample obtained from the subject over a period of time from at least one day to about 30 days after methotrexate administration; and (d) determining the level of immune tolerance to the therapeutic agent in the subject based on the level of the biomarker detected in step (c).

[293] Embodiment 97. The method of any one of embodiments 93-96, wherein the change in the level of an erythropoiesis biomarker is associated with the induction of erythropoiesis.

[294] Embodiment 98. The method of Embodiment 97, wherein if the level of the erythropoiesis biomarker is approximately equal to or below that level in the control, then the subject needs additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or if the level of the biomarker of erythropoiesis is elevated compared to that level in the control, then the subject does not need additional therapy to induce immune tolerance or immunosuppressive therapy.

[295] Embodiment 99. The method of Embodiment 97, wherein if the level of the erythropoiesis biomarker is substantially elevated compared to that level in the control, then the subject needs additional therapy to induce immune tolerance or immunosuppressive therapy concurrently with further treatment with a therapeutic agent; or where if the level of the biomarker of erythropoiesis is equal to or below that level in the control, then the subject does not need additional therapy to induce immune tolerance or immunosuppressive therapy.

[296] All features disclosed in this detailed description may be combined in any combination. Each feature disclosed in this detailed description may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is merely an example of a general sequence of equivalent or similar features.

[297] Additional details of the present invention are illustrated in the following non-limiting examples. The disclosure of all references in this detailed description is expressly incorporated herein by reference.

EXAMPLES

[298] The following examples are intended solely to illustrate the present invention and therefore should not be construed as limiting the present invention in any way. The following examples and detailed description are presented for illustrative purposes only and not for the purpose of limitation.

Example 1 Transcriptional and Protein Response in Spleen and Blood of Mice Treated in a Low Dose Methotrexate Short Course to Induce Immune Tolerance

Introduction

[299] In order to understand the mechanism of action of inducing immune tolerance with an ITI short-term, low-dose methotrexate regimen, next-generation sequencing and protein mass spectrometry studies were performed to study spleen cells and peripheral blood mononuclear cells (PBMCs) from mice treated with rhGAA with methotrexate in low doses or without it, and mice from control groups. A schematic of the study design is shown in Fig. 1C.

Materials and methods

Mice, rhGAA and low-dose methotrexate induction scheme

[300] Female C57BL/6 (Jackson Laboratory) or C57BL/6NTac (Taconic Biosciences, Inc.) mice between 8 and 12 weeks of age were housed and maintained in accordance with the American Association for the Accreditation of Laboratory Animal Care Guidelines for the Care and Use of Laboratory Animals. . The Institutional Animal Care and Use Committee has approved all animal procedures. Mice were treated with a single cycle of a low-dose methotrexate induction regimen simultaneously with rhGAA injection (Genzyme, Sanofi) as previously described (Joly et al ., 2014). Briefly, age- and sex-matched mice were treated with a single cycle (3 consecutive daily doses at 0, 24, and 48 hours) of a low-dose methotrexate induction schedule, 5 mg/kg, by intraperitoneal injection for 15 minutes prior to tail vein injection of rhGAA. 20 mg/kg. Subsequent weekly doses of rhGAA (up to 16 weeks) were administered 15 minutes after prophylactic intraperitoneal administration of 30 mg/kg diphenhydramine (West-Ward Pharmaceutical). Vehicle control or formulation buffer (FB) for rhGAA was administered similarly to reconstituted rhGAA. Blood was collected by sampling from the retroorbital sinus of anesthetized mice or by terminal bleeds into CAPIJECT® micro sample collection tubes with anticoagulant EDTA (Terumo) on water ice. From spleens, single cell suspensions were prepared between glass slides with or without RBC lysis. Bone marrow was collected from one femur on water ice.

Transcriptional analysis

[301] Age-matched, sex-matched mice were treated with rhGAA or formulation buffer (FB) 15 minutes prior to injection in a low-dose methotrexate induction regimen with or without a single cycle as described above. Animals were sacrificed on day 7 from the start of rhGAA administration. Terminal blood sampling was performed from the retroorbital sinuses as described above. Whole blood diluted 1 to 2 with PBS was layered over ficoll to separate peripheral blood mononuclear cells (PBMCs). Suspensions of individual cells were obtained from spleens between glass slides. Samples were treated for RBC lysis (BD Pharm Lyse). Samples were flash frozen in TRIZOL ^™ (phenol guanidine isothiocyanate solution, ThermoFisher Scientific) and stored at -80°C before being sent to BGI (bgi.com/us/ worldwide) for next generation sequencing analysis. The RNA integrity number (RIN) was assessed on an Agilent 2100 bioanalyzer using RNA 6000 nano kit (Agilent) or RNA 6000 pico kit (Agilent). RNA amplification was performed using the SMARTER ^™ PCR cDNA Synthesis Kit (Clonetech) for cDNA synthesis. Sequencing fragment detection was performed on an Illumina HiSeq2000 with Illumina sequencing kits. RNA-seq datasets were filtered to remove adapters and low quality reads and evaluated for differential expression with a fold change of 2 and p-value ≤0.05. Differentially expressed genes were annotated and examined using NextBio biological data collection software (Illumina).

Protein mass spectrometry

[302] Age-matched, sex-matched mice were treated with or without a single cycle of low-dose methotrexate induction regimen concomitant with rhGAA or FB injection, and the animals were sacrificed 7 days from the start of rhGAA administration as described above. Terminal blood sampling was carried out from the retroorbital sinuses, as described above, followed by separation of blood plasma and PBMCs by centrifugation. Single cell suspensions were prepared from spleens as described above. Spleen and blood samples were flash frozen and sent to MS Bioworks (on the worldwide web msbioworks.com) for LC/MS protein mass spectrometry. At MS Bioworks, plasma was depleted of albumin, IgG, and transferrin using a polyaffinity spin cartridge 3 to remove proteins from mouse blood plasma (MARS3). Cells were lysed in modified RIPA buffer containing protease and phosphatase inhibitors. A single LC/MS/MS analysis was performed on one cleaved peptide sample on an Orbitrap (Velos Pro) tandem mass spectrometer (ThermoFisher) coupled to HPLC NanoAcquity (Waters). Data were analyzed using Progenesis QI for proteomics (non-linear dynamics) to identify peaks. Protein identification and characterization was performed using the Mascot database (Matrix Science version 2.4) with a threshold of 10 ppm precursor and 0.05 Da products (Q-Exactive). False negative and false positive results were filtered through Scaffold (Proteome Software version 4.0). Protein quantification was performed using Progenesis QI for proteomics. The fold change and p-values were calculated based on the data of the average normalized protein content for each experimental group. Differentially expressed proteins were determined at a fold change of 2 and p-value ≤0.05. The data was analyzed to assess accumulation in GO processes and other biological categories in MetaCore (Thomson Reuters).

results

Low-Dose Methotrexate Induction Scheme Positively Regulates Transcriptional Signatures in Erythropoiesis

[303] Mice were treated with rhGAA with or without low dose methotrexate as described above. Experimental controls were included where mice were treated with vehicle control (formulation buffer, FB) in the presence or absence of methotrexate. Seven days after the start of rhGAA administration, spleen and blood were collected and processed to obtain splenocytes and PBMC, respectively. Differences between treatment groups were examined in these tissue preparations by next generation sequencing (NGS). RNA sequencing data showed upregulation of transcriptional signatures of erythropoiesis in spleen (Figure 1A) and PBMC (Figure 1B) in mice treated with rhGAA in combination with low doses of methotrexate. In FIG. 1A and 1B show a representative subset of differentially expressed genes that are associated with erythropoiesis from the overall data set. The upregulation of erythropoiesis-associated gene expression in the spleen and PBMC in mice treated with rhGAA and low dose methotrexate differs markedly from other groups, even from mice treated with methotrexate alone.

[304] Transcriptional module analysis showed cell cycle pathways affected in the spleen of mice treated with rhGAA and a low dose of methotrexate compared to mice treated with rhGAA alone. In addition to changes in the cell cycle, analysis of transcriptional modules showed an increase in pathways associated with erythrocyte proliferation, including hematopoiesis, iron ion homeostasis, regulation of erythrocyte differentiation, and heme metabolism (Table 1 below). Conversely, blood samples showed signs of a cellular stress response (data not shown) but also showed increased heme metabolism (Table 1). Overall, spleen and blood transcriptional analysis showed upregulation of erythropoiesis-related pathways in the rhGAA and methotrexate treated group seven days after the start of rhGAA administration.

Low-Dose Methotrexate Induction Scheme Positively Regulates Protein Signatures in Erythropoiesis

[305] Proteomic analysis was also performed to identify pathways at the protein level that can be modulated using low-dose methotrexate treatment. The study design was similar to the transcription study described above, but the study was performed using protein mass spectrometry. The proteomic dataset showed enhancement of hematopoietic pathways and pathways associated with iron ion homeostasis (Table 1). Associated proteins include transferrin receptor 1 (TfR1, also referred to as CD71, highly expressed), a marker of RBC immature nuclear forms [Dong et al., 2011]) and its transferrin ligand (Fig. 2) (TfR1 also shows activation in the transcription dataset) . The transferrin 1 receptor is required for the transport of iron from transferrin into cells. These proteins provide iron absorption and play an important role in heme biosynthesis in developing erythroid cells. The upregulation of these proteins suggests that active hemoglobin biosynthesis may occur in developing RBCs. An increase in the levels of genes and proteins involved in erythrocyte production and function was generally observed in each of the experimental datasets showing differential expression in the spleen between rhGAA and low dose methotrexate treated groups compared to rhGAA alone treated groups. Therefore, spleen proteomic analysis showed upregulation of erythropoiesis-related pathways in rhGAA and methotrexate treated groups seven days after rhGAA initiation.

Table 1. Increase in the level of differentially regulated objects by functional categories associated with hematological effects.

Functional category mRNA Protein p Ratio p Ratio Biological processes GO Hematopoiesis 2.02E-11: 52/889 0.060 7/153 Homeostasis of iron ions 3.35E-16: 13/130 0.001 5/36 Regulation of erythrocyte differentiation 7.74E-6: 9/65 NS 1/9 MetaCore signaling pathway maps Heme metabolism 4.25E-5: 10/103 NS 1/16

mRNA: Differentially expressed mRNA species from the RNA-Seq dataset (FC ≥2, p ≤0.01). Protein: differentially expressed proteins from the MS Bioworks dataset (FC ≥ 2, p ≤ 0.05). p: hypergeometric p-value distribution for differentially expressed features from each experiment. Ratio: the number of differentially expressed objects within a functional category relative to the number of category objects analyzed in the experiment. NS: not significant.

Example 2 Effect on erythroid cells in the spleen, blood, and bone marrow of mice treated with a low-dose methotrexate short-term regimen to induce immune tolerance

Introduction

[306] To evaluate and understand the effect of inducing immune tolerance with the ITI short-term low-dose methotrexate regimen in spleen, blood, and bone marrow, flow cytometry studies were performed to evaluate spleen, blood, and bone marrow samples from mice treated with rhGAA with the ITI regimen. low-dose administration of methotrexate with or without a short course, or mice from control groups. In addition, to further evaluate erythropoiesis in spleen samples from mice treated with rhGAA with an ITI low-dose methotrexate regimen with or without a short course and control mice, immunohistochemistry and tissue mass spectrometric imaging were performed.

Materials and methods

flow cytometry

[307] Mouse antibodies used for flow cytometry include BioLegend PE-Ter-119 (Cat. No. 116208), PerCP-Cy5.5-CD71 (Cat. No. 113816), PE-Cy7-IgM (Cat. No. 406516). ), Pacific Blue (PB)-CD86 (cat. no. 105022) and allophycocyanin-CD45 (cat. no. 103116). Antibodies from BD Biosciences include FITC-CD44 (cat. no. 553133), allophycocyanin-CD5 (cat. no. 550035), allophycocyanin-CD19 or allophycocyanin-Cy7-CD19 (cat. no. 557655 and 550992), FITC-CD1d (cat. no. 553845), FITC-CD23 (cat. no. 553138) and allophycocyanin-Cy7-CD3 _ε (cat. no. 557596). Live/dead differentiation was performed by staining with the BioLegend Zombie Aqua Fixable Viability Kit. Samples were run through BD FACS Canto II (BD Biosciences) and results were obtained using BD FACSDiva software. Cytometry data was analyzed using FlowJo software version 10 (FlowJo, LLC). Cell population absolute numbers were calculated from CountBright Absolute Counting Beads (ThermoFisher Scientific). Cell types assessed include total RBCs (Ter-119 ⁺ ), immature nuclear RBCs (Ter-119 ⁺ , CD71 ⁺ ), mature RBCs (Ter-119 ⁺ , CD71 ^- ), proerythroblasts (Ter-119 ^intermediate , CD44 ^high ), basophilic erythroblasts (Ter-119 ⁺ , CD44 ^high FSC ^high ), polychromatophilic erythroblasts (Ter-119 ⁺ , CD44 ^intermediate FSC ^intermediate ), orthochromatophilic erythroblasts and reticulocytes (Ter-119 ⁺ , CD44 ^intermediate FSC ^low ), mature erythrocytes (Ter-119 ⁺ , CD44 ^low FSC ^low ), total activated B cells (CD19 ⁺ CD86 ⁺ ), activated follicular B cells (CD19 ⁺ , IgM ^low/high CD23 ⁺ , CD86 ⁺ ), activated marginal zone B cells (CD19 ⁺ , IgM ^high CD23 ^- , CD86 ⁺ ) and B10 regulatory B cells (CD19 ⁺ , CD1d ^high CD5 ⁺ ) (Allman et al ., 2004; Chen et al ., 2009; Koulnis et al ., 2011; Joly et al . , 2014).

Hematological research

[308] Samples were taken from the retroorbital sinus into CAPIJECT® microtubes for samples with EDTA anticoagulant and stored at 2-8°C for no more than 72 hours. Differential analysis of CBC, WBC and reticulocyte count was performed on Sysmex XT2000iV (Sysmex).

Histological, immunohistochemical analysis (IHC)

[309] Spleen samples were fixed in 4% paraformaldehyde in PBS, soaked in 30% sucrose, placed in optimum cutting temperature (OCT) medium, and flash frozen for immunofluorescence or histological analysis. Alternatively, the samples were processed in paraffin for histology. Hematoxylin and eosin staining was performed as previously described and examined by light microscopy (Burrow, Sun et al. 2015). For immunofluorescent analysis, samples were incubated with BioLegend's rat anti-mouse Ter-119 (Cat. No. 116202) or CD71 (Cat. No. 113802) monoclonal antibody and detected with a biotinylated rabbit anti-rat IgG antibody. Immunofluorescent analysis of sections stained with Ter-119 or CD71 antibody was performed on a Discovery XT Staining Module on an IHC/ISH BenchMark XT automated slide staining system (Ventana Medical System). Ventana IHC DAB Map Kit was used for detection. Immunofluorescent slides were scanned using a 20x objective in a Mirax scanner (Carl Zeiss). Tissue sections were counterstained with hematoxylin and analyzed by light microscopy. Representative 100x digital images were subjected to quantitative histomorphometry for the Ter-119 or CD71 positive region using HALO image analysis software (Indica Labs).

Mass Spectrometric Tissue Imaging (tMSI)

[310] Fresh spleen samples for tMSI were harvested in 10% gelatin (Alfa Aesar, Cat # J62699), frozen in one block, cut into 10 μM sections, and plated on indium tin oxide (ITO) glass slides. ITO slides were matrix-coated using matrix-assisted laser desorption/ionization (MALDI) (α-cyano-4-hydroxycinnamic acid) for tMSI. Mass spectrometric imaging was performed using AB Sciex 4800 MALDI-TOF/TOF, Autoflex MALDI-TOF/TOF and 7T SolariX MALDI-FTICR mass spectrometers. Image datasets were analyzed with TissueView, flexImaging or Multimaging 1.1 (ImaBiotech).

results

Proliferation of Activated B Cell Subpopulations in Response to Short-Term Low-Dose Methotrexate Treatment

[311] Regulatory B cells have previously been shown to be important in inducing immune tolerance with short-term low-dose methotrexate treatment (Joly et al ., 2014). In all of the experiments described herein, including the transcription studies described in Example 1, flow cytometry demonstrated patterns of immune tolerance induction, in particular the expansion of activated B cell subpopulations previously associated with immune tolerance induction (FIGS. 3A-3D).

Short-term treatment with low-dose methotrexate leads to short-term depletion of erythroid cells, followed by enrichment of immature nuclear forms of RBC Ter-119 ⁺ CD71 ⁺ in the spleen

[312] Mice treated with rhGAA concomitantly with one cycle of low-dose methotrexate treatment significantly increased spleen mass (FIGS. 4A-4B) and showed significantly greater spleen cell saturation as determined by flow cytometry, day 7 from onset. administration of rhGAA (Fig. 4C). The observed splenomegaly, together with the upregulation in erythropoiesis signatures shown in the proteomics data, prompted the RBC cell assay during short-term low-dose methotrexate induction treatment.

[313] Age-matched, sex-matched mice were treated with rhGAA with and without a single cycle of low-dose methotrexate, and the animals were sacrificed over time for flow cytometry analysis. Erythroid-specific monoclonal antibody Ter-119 was used to study the total amount of live RBC (FIG. 4D). Immature nuclear RBCs were differentiated from mature RBCs by the transferrin receptor CD71 (Pan and Johnstone 1983). On day 2 after the start of rhGAA administration, but before the second administration of methotrexate, a significant decrease in the total number of erythroid cells (Ter-119⁺) and an almost complete decrease in the number of immature nuclear forms of RBC (Ter-119⁺ CD71⁺) in the spleen of mice from the low-dose methotrexate group (Fig. 4E). On day 6, no differences were observed between the groups. The large and significant accumulation of total erythroid cells that was observed on day 7 was also marked by a simultaneous and significant increase in the number of immature nuclear forms of RBC (Fig. 4F). A smaller but significant accumulation of total erythroid cells and immature nuclear forms of RBC was observed at day 9. In the short-term low-dose methotrexate treated groups, the erythroid response mimicked that of the short-term low-dose methotrexate-treated group along with rhGAA (Fig. 4G, 4H) .

[314] Thus, a temporary but significant decrease in the number of immature nuclear forms of RBC and mature RBC can be observed in the spleen within 24 hours after the first administration of low-dose methotrexate. This is followed by a response where there was a significant accumulation of immature nuclear forms of RBC in the spleen of mice with induced tolerance. Accumulation of immature nuclear forms of RBC peaks on day 7, which may explain the significant enlargement of the spleen seen on this day. When comparing groups treated with rhGAA along with the ITI short-term low-dose methotrexate regimen and groups treated with the ITI short-term low-dose methotrexate regimen alone, the erythroid response was similar. This indicates that the short-term ITI low-dose methotrexate regimen is the major contributor to the observed erythroid response.

Late erythroid response in the bloodstream after a low-dose methotrexate induction regimen

[315] The blood compartment study also showed a significant depletion of total (FIG. 5A) and immature nuclear forms of RBC (FIG. 5B) on day 2 in the single cycle low dose methotrexate treatment group. By day 4, immature nuclear forms of RBC were virtually absent in the blood of the group treated with one cycle of low-dose methotrexate. By day 7, the total RBC level was significantly reduced, while the accumulation of immature nuclear forms of RBC was significantly increased. On day 9, the blood was significantly enriched in total and immature nuclear forms of RBC. By day 14, a small but significant accumulation of immature nuclear forms of RBC was observed in the blood of the group receiving one cycle of low-dose methotrexate, which disappeared by day 28.

[316] Blood was also examined for the effect of low-dose short-term methotrexate administration alone. When examining total RBCs, no differences were observed between the methotrexate-only group and the vehicle control group (FIGS. 5C-5E). However, the response due to immature nuclear forms of RBC in mice treated with only one cycle of low-dose methotrexate was similar to the response in mice treated with one cycle of low-dose methotrexate together with the rhGAA biologic (Fig. 5F - 5H).

[317] To confirm our results, blood from mice 7 days after the start of rhGAA administration was examined on a Sysmex XT2000iV for complete clinical blood count (CBC) and reticulocyte count. The total number of RBCs was significantly reduced in the short-term low-dose methotrexate treatment groups (FIG. 6A). Hemoglobin levels (FIG. 6B) and hematocrit (FIG. 6C) also decreased significantly in the short-term, low-dose methotrexate treatment groups. No differences were observed in detection rate and number of mature reticulocytes (FIGS. 6D - 6E). However, the detection rate of the immature reticulocyte fraction (IRF) was significantly increased in the short-term low-dose methotrexate treatment groups (FIG. 6F).

[318] Thus, the erythroid response in the blood was similar, although delayed. A complete blood count on day 7 from the start of rhGAA administration showed a decrease in total RBC, hematocrit, and hemoglobin, combined with an increased frequency of detecting an immature reticulocyte fraction. This pattern is consistent with a regenerative erythroid response after RBC loss.

Erythroid cells are depleted but return to homeostatic levels in the bone marrow after using a low-dose methotrexate induction regimen

[319] Bone marrow erythroid cell responses to one cycle of low-dose methotrexate treatment showed no difference in total RBC on day 4 compared to mice treated with rhGAA alone (FIG. 7A). However, immature nuclear forms of RBC were significantly reduced on day 4 in the tolerance-induced group (Fig. 7B). On day 5, both the total number of RBCs and the number of immature nuclear RBCs were significantly reduced in the tolerance-induced group. By day 7 and 9, the total number of RBCs and the number of immature nuclear forms of RBC showed no difference between mice with induced tolerance and mice treated with rhGAA alone. Thus, there is also an early significant decrease in RBCs in the bone marrow, but by day 7, erythroid levels return to homeostatic levels.

Low-dose short-term treatment with methotrexate induces erythropoiesis in the spleen

[320] In FIG. 8A shows that differential expression of the CD44 adhesion molecule was used to assess various stages of erythropoiesis, from proerythroblasts to mature erythrocytes, during an ITI short-term low-dose methotrexate regimen. On day 2 from the start of rhGAA administration, erythroid cell levels of all early stages, including proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts, orthochromatophilic erythroblasts, and reticulocytes, are significantly reduced in the spleen in the low-dose methotrexate single cycle group (Fig. 8B). Mature RBCs were also significantly reduced in the tolerance-induced groups on day 2. On day 7 from the start of rhGAA administration, a significant accumulation of proerythroblasts, basophilic, polychromatophilic, and orthochromatophilic erythroblasts, and reticulocytes can be observed in the group receiving one cycle of low-dose methotrexate administration (Fig. 8C) . On day 7, the spleen showed no difference in mature RBC levels. Accumulation of immature nuclear forms of RBC in the spleen on day 7 from the start of rhGAA administration coincides with the accumulation of multiple populations of B cells capable of conferring immune tolerance in naive recipient mice, namely B10 regulatory B cells, follicular B cells, and B cells that express Foxp3, TGF-β, and IL-10 (Joly et al ., 2014).

[321] In blood, a small but significant decrease in basophilic erythroblasts, orthochromatophilic erythroblasts, reticulocytes, and mature erythrocytes can be observed on day 2 from the start of rhGAA administration in the low-dose methotrexate single cycle group (Fig. 8D). However, no change in blood levels of immature nuclear forms of RBC was observed on day 7 from the start of rhGAA administration (FIG. 8E). On day 7, a significant decrease in the level of mature RBCs was observed in the blood of mice with induced tolerance.

[322] Significant depletion of erythroid cells of all early stages, including proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts, orthochromatophilic erythroblasts, and reticulocytes, was observed in the bone marrow on day 4 from the start of rhGAA in the short-term low-dose methotrexate group, but no statistically significant difference with levels of mature RBCs (Fig. 8F). Depletion of erythroid cells of all stages, including early and mature RBC, could be observed on day 5 in the induced tolerance group (Fig. 8G). By day 7, a small but significant accumulation of proerythroblasts and polychromatophilic erythroblasts could be observed in the tolerance-induced group (Fig. 8H). However, the levels of mature RBCs were significantly reduced in the tolerance-induced group. On day 9, a small but significant accumulation of proerythroblasts and polychromatophilic erythroblasts could be observed in the tolerance-induced group, but no difference in the levels of mature RBCs in the bone marrow among the groups (Fig. 8I).

The frequency of detection of nuclear forms of cells increased in the red pulp of the spleen in mice with induced tolerance

[323] An enlarged spleen in mice treated with short-term, low-dose methotrexate indicates a possible change in spleen morphology. To assess this, mice were injected with rhGAA with a short-term low-dose methotrexate (e.g., ITI regimen) and without it, and spleens were collected 7 days after the start of rhGAA administration. Microscopic analysis of the spleen showed a significantly increased red pulp area relative to the white pulp area in mice treated with short-term, low-dose methotrexate compared to mice treated with rhGAA alone (FIGS. 9A-9B). Mice treated with low dose methotrexate in a short course with FB also showed significantly increased red pulp area relative to white pulp area compared to mice treated with FB alone. The nuclear forms of the cells are likely to be hematopoietic progenitors, as evidenced by cellular studies using flow cytometry.

The appearance of proteins associated with mature RBCs was reduced in the spleen of mice with induced tolerance

[324] The spatial distribution of proteins in the spleen was examined using tissue mass spectrometric imaging. Mice were treated with rhGAA in the presence and absence of methotrexate as described above. Included experimental control groups of mice, which were subjected to treatment with buffer FB with and without methotrexate. Spleens were collected 7 days after the start of rhGAA administration and frozen en bloc. Analysis of 10 μM spleen sections showed that the protein erythroid ankyrin and glycophorin C were uniformly distributed in the spleen in the rhGAA or FB treated groups alone (Fig. 10). In groups treated with rhGAA or FB along with short-term ITI low-dose methotrexate, erythroid ankyrin and glycophorin C signal decreased. Erythroid ankyrin and glycophorin C are erythroid-specific proteins that are highly expressed in mature RBCs (Chen et al., 2009). Decreased levels of these proteins are consistent with a temporary loss of mature RBCs followed by production of new RBCs in the spleen due to the ITI short-term low-dose methotrexate regimen.

Example 3 Transfusion of Erythroid Cells from Tolerant Mice to Naive Mice

Introduction

[325] To determine whether total RBCs, which include immature nuclear RBCs and mature RBCs from tolerant mice, could confer immune tolerance, RBC transfusion studies were conducted to transfuse RBCs from mice treated with rhGAA with and without low-dose short-term methotrexate administration. , naive mice. Immune tolerance was assessed by measuring the titers of antibodies to rhGAA in the blood serum of naive mice, which underwent erythrocyte transfusion.

Materials and methods

Serum antibody titers to rhGAA

[326] Quantification of anti-rhGAA antibody titers in mouse serum was measured by ELISA as described previously (Joly, Martin et al., 2014). Briefly, 96-well plates (Corning Cat # 29442-322) were coated overnight (5 mg/mL rhGAA in sodium acetate buffer, pH 5.0), treated with blocking reagent (0.1% BSA in PBS) and incubated with serial dilutions of mouse serum in duplicate (1 hour at 37°C). After washing, samples were treated with HRP-conjugated secondary goat anti-mouse IgG (Southern Biotechnology Associates cat. no. 1030-05), washed and developed with the addition of TMB substrate (3,3',5,5'-tetramethylbenzidine) (BioFX Laboratories cat. no. TMBW-1000-01) for 15 minutes at room temperature. The reaction was stopped by adding 1 N. HCl and immediately measured the absorbance at 450/650 nm on a SpectraMax M2 (Molecular Devices). Endpoint titers were obtained from the reciprocal of the sample dilution, giving an absorbance of 0.2.

Transfusion of erythroid cells

[327] Harvest of erythroid cells from age-matched, sex-matched mice was performed on day 7 (isolation of erythroid cells from spleen) and day 9 (isolation of erythroid cells from blood) after treatment with rhGAA with the ITI regimen with one cycle of methotrexate short-term course in low doses or without it, as described above. Blood and spleen were collected under aseptic conditions and processed as described above. Blood was collected in sterile Monoject EDTA blood collection tubes (Covidien cat. no. 8881311149). The pooled blood was passed through a 25 mm Acrodisk PSF WBC filter adapted for mice with leukosorption medium (Pall cat. no. AP-4851) for leukoreduction. Erythroid cells from pooled spleen samples were isolated using mouse anti-Ter-119 MicroBeads (Miltenyi Biotec, cat no. 130-049-901) microbeads by positive selection according to the manufacturer's instructions. Purified erythroid cells from blood (≥99.9% confirmed by flow cytometry) and from spleen (≥93.3% confirmed by flow cytometry) were washed in sterile PBS. Purified erythroid cells were intravenously administered to recipient mice via the tail vein. Mice received weekly doses of rhGAA following diphenhydramine prophylaxis as described above. Once every two weeks, blood was collected from the retroorbital sinuses into Capiject microtubes for collecting samples with the anticoagulant EDTA, as described above. Serum samples were collected to assess anti-rhGAA antibody titers by ELISA.

results

Immature nuclear forms of spleen RBC isolated from methotrexate-induced rhGAA tolerance mice conferred immune tolerance on naïve mice

[328] Transfusion of erythroid cells from mice treated with rhGAA with an ITI regimen with one cycle of short-term low-dose methotrexate with or without it was performed in naive mice to determine whether total erythroid cells, which include immature nuclear forms of RBC and mature RBC confer immune tolerance. Naïve recipient mice received 75 million erythrocyte cells (≥93.3%) from the spleens of donor mice 7 days after initiation of rhGAA or 200 million donated erythrocytes isolated from blood (≥99.9%) 9 days after initiation introduction of rhGAA. After transfusion and weekly thereafter, recipient mice received rhGAA intravenously. Serum was collected biweekly up to and including the twentieth week to assess anti-rhGAA IgG antibody titers by ELISA (FIGS. 11A - 11C). In dosed controls (Fig. 11C), titers at weeks 14, 16, 18, and 20 and area under the curve (AUC; 38% reduction in AUC) decreased significantly in the rhGAA treated group with the short-term ITI low-dose methotrexate regimen (rhGAA+MTX ) compared to the rhGAA-only group. A trend towards decreasing titers at 14, 16, 18 and 20 weeks and towards decreasing AUC was observed in mice that received total erythrocytes from blood (tolerance-induced blood, 38% reduction in AUC) (Fig. 11A) or spleen (spleen with induced tolerance, 36% reduction in AUC) (FIG. 11B) from donor mice treated with rhGAA in combination with an ITI short-term low-dose methotrexate regimen. Statistical significance was assessed by removing the outliers (highest rhGAA titer value) from each group and comparing the difference in AUC between respective groups.

[329] To determine whether total erythroid cells from mice treated with rhGAA in combination with an ITI short-term low-dose methotrexate regimen could confer immune tolerance, transfusion studies were conducted in naive recipient mice. Historical data showed that rhGAA-treated groups with an ITI short-term low-dose methotrexate regimen had significantly reduced rhGAA titers and AUC compared with rhGAA-only groups. However, there were no differences between groups in the dosed control groups, possibly due to large outliers (data not shown). Significant differences between groups were observed when outliers were removed. Large outliers were removed from each group to avoid error. Transfusion studies suggest that total isolated erythroid cells from donor mice treated with rhGAA in combination with an ITI short-term low-dose methotrexate regimen appear to tend to confer immune tolerance in naïve recipient mice, regardless of whether they are isolated from blood or spleen. The study using transfusion was performed once.

Literary sources

[330] Allman, D., B. Srivastava and R. C. Lindsley (2004). "Alternative routes to maturity: branch points and pathways for generating follicular and marginal zone B cells." Immunol Rev 197 : 147-160.

[331] Banugaria, SG, SN Prater, TT Patel, SM Dearmey, C. Milleson, KB Sheets, DS Bali, CW Rehder, JA Raiman, RA Wang, F. Labarthe, J. Charrow, P. Harmatz, P. Chakraborty , AS Rosenberg and PS Kishnani (2013). "Algorithm for the early diagnosis and treatment of patients with cross reactive immunological material-negative classic infantile Pompe disease: a step towards improving the efficacy of ERT." PLoS One 8 (6): e67052.

[332] Burrow, T.A., Y. Sun, C.E. Prada, L. Bailey, W. Zhang, A. Brewer, S.W. Wu, K.D. Setchell, D. Witte, M.B. Cohen and G.A. Grabowski (2015). "CNS, lung, and lymph node involvement in Gaucher disease type 3 after 11 years of therapy: clinical, histopathologic, and biochemical findings." Mol Genet Metab 114 (2): 233-241.

[333] Chen, K., J. Liu, S. Heck, J. A. Chasis, X. An and N. Mohandas (2009). "Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis." Proc Natl Acad Sci USA 106 (41): 17413-17418.

[334] Collins, PW, M. Mathias, J. Hanley, D. Keeling, R. Keenan, M. Laffan, D. Perry, R. Liesner and UKHCD Organization (2009). "Rituximab and immune tolerance in severe hemophilia A: a consecutive national cohort." J Thromb Haemost 7 (5): 787-794.

[335] Cremel, M., N. Guerin, G. Campello, Q. Barthe, W. Berlier, F. Horand and Y. Godfrin (2015). "Innovative approach in Pompe disease therapy: Induction of immune tolerance by antigen-encapsulated red blood cells." Int J Pharm 491 (1-2): 69-77.

[336] Cremel, M., N. Guerin, F. Horand, A. Banz and Y. Godfrin (2013). "Red blood cells as an innovative antigen carrier to induce specific immune tolerance." Int J Pharm 443 (1-2): 39-49.

[337] Cronstein, BN (2005). "Low-dose methotrexate: a mainstay in the treatment of rheumatoid arthritis." Pharmacol Rev 57 (2): 163-172.

[338] Daugas, E., C. Cande and G. Kroemer (2001). "Erythrocytes: death of a mummy." Cell Death Differ 8 (12): 1131-1133.

[339] Dong, HY, S. Wilkes and H. Yang (2011). "CD71 is selectively and ubiquitously expressed at high levels in erythroid precursors of all maturation stages: a comparative immunochemical study with glycophorin A and hemoglobin A." Am J Surg Pathol 35 (5): 723-732.

[340] Elahi, S., JM Ertelt, JM Kinder, TT Jiang, X. Zhang, L. Xin, V. Chaturvedi, BS Strong, JE Qualls, KA Steinbrecher, TA Kalfa, AF Shaaban and SS Way (2013). "Immunosuppressive CD71+ erythroid cells compromise neonatal host defense against infection." Nature 504 (7478): 158-162.

[341] Ernst, PB, JC Garrison and L.F. Thompson (2010). "Much ado about adenosine: adenosine synthesis and function in regulatory T cell biology." J Immunol 185 (4): 1993-1998.

[342] Franchini, M., C. Mengoli, G. Lippi, G. Targher, M. Montagnana, G. L. Salvagno, M. Zaffanello and M. Cruciani (2008). "Immune tolerance with rituximab in congenital haemophilia with inhibitors: a systematic literature review based on individual patients'analysis." Haemophilia 14 (5): 903-912.

[343] Garman, R.D., K. Munroe and S. M. Richards (2004). "Methotrexate reduces antibody responses to recombinant human alpha-galactosidase A therapy in a mouse model of Fabry disease." Clin Exp Immunol 137 (3): 496-502.

[344] Getts, DR, DP McCarthy and SD Miller (2013). "Exploiting apoptosis for therapeutic tolerance induction." J Immunol 191 (11): 5341-5346.

[345] Grimm, AJ, S. Kontos, G. Diaceri, X. Quaglia-Thermes and JA Hubbell (2015). "Memory of tolerance and induction of regulatory T cells by erythrocyte-targeted antigens." Sci Rep 5 : 15907.

[346] Han, KL, SV Thomas, SM Koontz, CM Changpriroa, SK Ha, HL Malech and EM Kang (2013). "Adenosine A(2)A receptor agonist-mediated increase in donor-derived regulatory T cells suppresses development of graft-versus-host disease." J Immunol 190 (1): 458-468.

[347] Joly, MS, RP Martin, S. Mitra-Kaushik, L. Phillips, A. D'Angona, SM Richards and AM Joseph (2014). "Transient low-dose methotrexate generates B regulatory cells that mediate antigen-specific tolerance to alglucosidase alfa." J Immunol 193 (8): 3947-3958.

[348] Joseph, A., K. Munroe, M. Housman, R. Garman and S. Richards (2008). "Immune tolerance induction to enzyme-replacement therapy by co-administration of short-term, low-dose methotrexate in a murine Pompe disease model." Clin Exp Immunol 152 (1): 138-146.

[349] Joseph, A., K. Neff, J. Richard, L. Gao, D. Bangari, M. Joly, K. Culm-Merdek, R. Garman, J. Williams, S. Richards and M. Ruzek ( 2012). "Transient low-dose methotrexate induces tolerance to murine anti-thymocyte globulin and together they promote long-term allograft survival." J Immunol 189 (2): 732-743.

[350] Kina, T., K. Ikuta, E. Takayama, K. Wada, AS Majumdar, I. L. Weissman and Y. Katsura (2000). "The monoclonal antibody TER-119 recognizes a molecule associated with glycophorin A and specifically marks the late stages of murine erythroid lineage." Br J Haematol 109 (2): 280-287.

[351] Kishnani, PS, D. Corzo, M. Nicolino, B. Byrne, H. Mandel, WL Hwu, N. Leslie, J. Levine, C. Spencer, M. McDonald, J. Li, J. Dumontier, M. Halberthal, YH Chien, R. Hopkin, S. Vijayaraghavan, D. Gruskin, D. Bartholomew, A. van der Ploeg, JP Clancy, R. Parini, G. Morin, M. Beck, GS De la Gastine, M Jokic, B. Thurberg, S. Richards, D. Bali, M. Davison, M. A. Worden, Y. T. Chen and J. E. Wraith (2007). "Recombinant human acid [alpha]-glucosidase: major clinical benefits in infantile-onset Pompe disease." Neurology 68 (2): 99-109.

[352] Kontos, S., IC Kourtis, KY Dane and JA Hubbell (2013). "Engineering antigens for in situ erythrocyte binding induces T-cell deletion." Proc Natl Acad Sci USA 110 (1): E60-68.

[353] Koulnis, M., R. Pop, E. Porpiglia, JR Shearstone, D. Hidalgo and M. Socolovsky (2011). "Identification and analysis of mouse erythroid progenitors using the CD71/TER119 flow-cytometric assay." J Vis Exp (54).

[354] Kremer, JM (2004). "Toward a better understanding of methotrexate." Arthritis Rheum 50 (5): 1370-1382.

[355] Lorentz, KM, S. Kontos, G. Diaceri, H. Henry and JA Hubbell (2015). "Engineered binding to erythrocytes induces immunological tolerance to E. coli asparaginase." Sci Adv 1 (6): e1500112.

[356] Mendelsohn, NJ, YH Messinger, AS Rosenberg and PS Kishnani (2009). "Elimination of antibodies to recombinant enzyme in Pompe disease." N Engl J Med 360 (2): 194-195.

[357] Messinger, YH, NJ Mendelsohn, W. Rhead, D. Dimmock, E. Hershkovitz, M. Champion, SA Jones, R. Olson, A. White, C. Wells, D. Bali, LE Case, SP Young , AS Rosenberg and PS Kishnani (2012). "Successful immune tolerance induction to enzyme replacement therapy in CRIM-negative infantile Pompe disease." Genet Med 14 (1): 135-142.

[358] Nandakumar, SK, JC Ulirsch and VG Sankaran (2016). "Advances in understanding erythropoiesis: evolving perspectives." Br J Haematol 173 (2): 206-218.

[359] Pan, BT and R.M. Johnstone (1983). "Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor." Cell 33 (3): 967-978.

[360] Schakel, K., M. von Kietzell, A. Hansel, A. Ebling, L. Schulze, M. Haase, C. Semmler, M. Sarfati, AN Barclay, GJ Randolph, M. Meurer and E. P. Rieber ( 2006). "Human 6-sulfo LacNAc-expressing dendritic cells are principal producers of early interleukin-12 and are controlled by erythrocytes." Immunity 24 (6): 767-777.

[361] Taylor, WJ, E. Korendowych, P. Nash, PS Helliwell, E. Choy, GG Krueger, ER Soriano, NJ McHugh and CF Rosen (2008). "Drug use and toxicity in psoriatic disease: focus on methotrexate." J Rheumatol 35 (7): 1454-1457.

[362] Testa, U. (2004). "Apoptotic mechanisms in the control of erythropoiesis." Leukemia 18 (7): 1176-1199.

[363] Van Putten, L.M. (1958). "The life span of red cells in the rat and the mouse as determined by labeling with DFP32 in vivo." Blood 13 (8): 789-794.

[364] Yi, T., J. Li, H. Chen, J. Wu, J. An, Y. Xu, Y. Hu, C. A. Lowell and J. G. Cyster (2015). "Splenic Dendritic Cells Survey Red Blood Cells for Missing Self-CD47 to Trigger Adaptive Immune Responses." Immunity 43 (4): 764-775.

Claims (52)

1. A method of treating a subject in need of treatment with a therapeutic agent, comprising: (a) administering to the subject about 0.1 mg/kg to about 5 mg/kg methotrexate; (b) administering to the subject an effective amount of a therapeutic agent, wherein the therapeutic agent is human acid α-glucosidase; (c) detecting an erythropoiesis biomarker in a sample obtained from the subject over a period of time from at least one day to about 30 days after methotrexate administration; And (d) continuing further treatment with human acid α-glucosidase with or without additional therapy to induce immune tolerance or immunosuppressive therapy based on the level of the biomarker of erythropoiesis compared with that level in the control, Where: 1) if the level of the biomarker of erythropoiesis is approximately equal to or below the control level, stage (d) includes the introduction of additional therapy to induce immune tolerance or immunosuppressive therapy simultaneously with further treatment with human acid α-glucosidase; or 2) if the erythropoiesis biomarker level is elevated compared to control, step (d) involves continuing further treatment with human acid α-glucosidase without administering additional therapy to induce immune tolerance or immunosuppressive therapy. 2. The method of claim. 1, where the change in the level of the biomarker of erythropoiesis is associated with the induction of erythropoiesis. 3. The method according to claim 2, where: (i) the erythropoiesis biomarker is the level of immature nucleated erythrocytes, immature reticulocyte fraction, or cellular nucleic acids; (ii) the biomarker for erythropoiesis is the hematocrit or hemoglobin level in the blood; (iii) detection of an erythropoiesis biomarker is detection of expression of an erythropoiesis-associated gene, wherein the erythropoiesis-associated gene is a gene associated with one or more of hematopoiesis, iron ion homeostasis, regulation of erythrocyte differentiation, and heme metabolism, where optional: 1) the erythropoiesis associated gene is a gene encoding transferrin receptor, transferrin, Ly76 (antigen for Ter-119), CD44, CD235a, ALAS2 or GATA-1, where optionally the transferrin receptor is transferrin receptor 1 (CD71); and/or 2) detecting an erythropoiesis-associated gene involves detecting an RNA transcript of an erythropoiesis-associated gene or detecting a protein product of an erythropoiesis-associated gene, where the protein product is optionally hemoglobin; or (iv) the erythropoiesis biomarker is an erythropoiesis-associated cofactor, where optionally the erythropoiesis-associated cofactor is a porphyrin, a porphyrin-containing compound, or tetrapyrrole. 4. The method according to any one of paragraphs. 1-3 where the sample is a blood sample, where optional: (i) the blood sample is a blood fraction containing peripheral blood mononuclear cells (PBMCs); and/or (ii) the blood sample is a blood serum sample or a blood plasma sample, and wherein the detection of the biomarker is the detection of expression of a gene associated with erythropoiesis. 5. The method according to any one of paragraphs. 1-4 where the control is historical control or placebo control. 6. The method according to any one of paragraphs. 1-5 where: (i) from about 0.1 mg/kg to about 5 mg/kg of methotrexate is administered in one cycle or in three cycles, where optionally the methotrexate cycle consists of 1 day of methotrexate administration or 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 consecutive days of methotrexate administration; and/or (ii) methotrexate is administered to the subject at a time selected from one or more of the times before, during, and after administration of human acid α-glucosidase, where optionally methotrexate is administered from 48 hours prior to administration of human acid α-glucosidase up to 48 hours after that. 7. The method according to any one of paragraphs. 1-6 where: (i) the sample is obtained from the subject at least about one day to about 30 days after the last methotrexate administration, where optionally the sample is obtained from the subject from about 7 days to about 14 days after the last methotrexate administration; and/or (ii) samples are taken within a few days of the last methotrexate administration, where optionally a sample is taken at one or more of the times from day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 14, day 21 or 28 days after the last injection of methotrexate. 8. The method according to any one of paragraphs. 1-7 where: (i) step (d) involves administering an agent that depletes T cells, B cells, or plasma cells; and/or (ii) step (d) involves one or more of the following: (a) administering an effective amount of one or more of rituximab, intravenous immunoglobulin (IVIG), cyclophosphamide, bortezomib, azathioprine, cyclosporine, mycophenolate, and a corticosteroid, and (b) administering an antigen-specific tolerance building strategy, where optionally the antigen-specific tolerance building strategy involves more frequent doses of human acid α-glucosidase or a higher dose of human acid α-glucosidase, or both. 9. The method of claim 8 wherein the subject has Pompe disease. 10. The method according to claim 9, where: (i) Pompe disease is an infantile form of Pompe disease, such as classic infantile Pompe disease; and/or (ii) the subject is negative for cross-reactive immunological material (CRIM). 11. The method of claim 10, wherein the method further comprises administering the initial therapy to induce immune tolerance concurrently with the administration of human acid α-glucosidase, where optionally the initial therapy to induce immune tolerance comprises administering one or more of rituximab and IVIG. 12. The method according to any one of paragraphs. 9-11, further comprising monitoring for one or more of human acid α-glucosidase antibody level, CD19 level, or disease progression. 13. A method for assessing the level of immune tolerance in a subject with Pompe disease, comprising: (a) obtaining a sample from a subject, where the subject has previously received at least one cycle of methotrexate treatment and administered at least one dose of a therapeutic agent, where the therapeutic agent is human acid α-glucosidase; And (b) detecting an erythropoiesis biomarker in the sample, wherein the level of the erythropoiesis biomarker in the sample compared to that in the control is indicative of immune tolerance induction, Where: 1) if the level of the erythropoiesis biomarker is approximately equal to or less than the control, the subject requires additional therapy to induce immunological tolerance or immunosuppressive therapy concomitantly with further treatment with human acid α-glucosidase; or 2) if the level of the erythropoiesis biomarker is elevated compared to the control, the subject does not need additional therapy to induce immunological tolerance or immunosuppressive therapy. 14. A method for assessing the level of immune tolerance to a therapeutic agent in a subject in need of a therapeutic agent, comprising: (a) obtaining a sample from a subject, where the subject has previously received at least one cycle of methotrexate treatment and administered at least one dose of a therapeutic agent, where the therapeutic agent is human acid α-glucosidase; And (b) detecting an erythropoiesis biomarker in a sample obtained from the subject, where the level of the biomarker of erythropoiesis in the sample compared with that level in the control indicates the induction of immune tolerance in the subject to human acid α-glucosidase, Where: 1) if the level of the erythropoiesis biomarker is approximately equal to or less than the control, the subject requires additional therapy to induce immunological tolerance or immunosuppressive therapy concomitantly with further treatment with human acid α-glucosidase; or 2) if the level of the erythropoiesis biomarker is elevated compared to the control, the subject does not need additional therapy to induce immunological tolerance or immunosuppressive therapy. 15. The method of claim 14, wherein the sample is obtained from the subject over a period of time from at least one day to about 30 days after methotrexate administration. 16. The method according to any one of paragraphs. 13-15, where the change in the level of the biomarker of erythropoiesis is associated with the induction of erythropoiesis.

Images