Low-dose human interleukin-2 for the treatment of amyotrophic lateral sclerosis (ALS)

Abstract

The present invention is in the field of amyotrophic lateral sclerosis (ALS) and relates to human interleukin-2 (IL-2) for use in treating amyotrophic lateral sclerosis in a human subject, wherein each dose of human IL-2 administered to the subject is 0.1 x 10 6 ~3×10 6 Human IL-2 is preferably 0.1 x 10 6 ~3×10 6 The treatment is administered in a 3-7 day cycle of subcutaneous administration of 1U of human IL-2. This treatment preferably does not include administration of regulatory T cells to a subject who is also receiving riluzole treatment. The administered human IL-2 preferably does not form complexes with anti-hIL-2 antibodies, and this treatment also preferably does not include administration of rapamycin or any other inhibitor of effector T cells (Teff) to the subject. This treatment reduces plasma CCL2 concentrations and allows for a shift in the polarization of blood macrophages from an M1 inflammatory phenotype to an anti-inflammatory M2 phenotype involved in tissue repair.

Description

[Technical Field]

[0001] Technical field to which the invention belongs This invention relates to the field of amyotrophic lateral sclerosis (ALS). This invention relates to human interleukin-2 (IL-2) for use in the treatment of amyotrophic lateral sclerosis in human subjects, wherein each dose of human IL-2 administered to the subject is 0.1 × 10⁻⁶ 6 ~3×10 6 It is an International Unit (IU). Human IL-2 is preferably 0.1 × 10⁻¹⁶ 6 ~3×10 6 It is administered as a subcutaneous injection of IU of human IL-2. This treatment preferably does not involve the administration of regulatory T cells to subjects who are also receiving riluzole treatment. The administered human IL-2 preferably does not form complexes with anti-hIL-2 antibodies, and this treatment preferably also does not involve the administration of rapamycin or any other effector T cell (Teff) inhibitor to the subject. Low-dose IL2 treatment in ALS subjects allows for (i) increased regulatory T cell suppressive control compared to Teff, (ii) a decrease in plasma CCL2 concentration, (iii) a shift in circulating macrophage polarization from the M1 inflammatory phenotype to the anti-inflammatory M2 phenotype involved in tissue repair, and (iv) a reduction in overall ALS-related cytopathic activity. [Background technology]

[0002] Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease characterized by relentlessly progressing weakness, muscle wasting, and loss of motor function. Despite the introduction of riluzole 20 years ago (Bensimon G et al N Engl J Med 1994; 330: 585-91), subsequent clinical trials have failed to provide a more effective disease-modifying therapy.

[0003] Many drug discovery failures in ALS are primarily related to the fact that ALS is a complex disease and there are no validated predictive animal models. In particular, transgenic mutant SOD1 (mSOD1) mice are often used as an animal model of ALS, but all positive results obtained from this model have not translated into efficient treatments for human subjects with ALS (DiBernardo AB et al. Biochimica et Biophysica Acta 1762 (2006) 1139-1149; van den Berg LH et al. Neurology 2019;92:e1610-e1623).

[0004] Neuroinflammatory processes are a prominent pathological feature of ALS patients. Microglial cell activation is associated with the pathology of ALS at all stages (Troost D et al. Neuropathol Appl Neurobiol 1990; 16: 401-10; Kawamata T et al. Am J Pathol 1992; 140: 691; Engelhardt JI et al. Arch Neurol 1993; 50: 30-6; Thonhoff JR et al. Curr Opin Neurol 2018; 31: 635-9), and also with transgenic SOD1 ALS mice (Engelhardt JI et al. Arch Neurol 1993; 50: 30-6; McGeer PL et al. Muscle Nerve 2002; 26: 459-70; Hall ED, Oostveen JA, Gurney ME. Glia 1998; 23: It has been proven that the expression of cytokines typical of macrophages precedes clinical symptoms (Alexianu ME et al. Neurology 2001; 57: 1282-9; Hensley K et al. J Neurochem 2002; 82: 365-74). Furthermore, biomarkers of neuroinflammation are elevated in ALS patients, correlate with disease severity, and have been shown to predict disease progression (Gille B et al. J Neurol Neurosurg Psychiatry. 2019 Dec;90(12):1338-1346; Tateishi T et al. J Neuroimmunol. 2010 May;222(1-2):76-81).

[0005] While the evidence for the contribution of neuroinflammation to the pathogenesis of ALS is compelling (Evans MC et al. Mol Cell Neurosci 2013; 53: 34-41; Zhao W et al. J Neuroimmune Pharmacol Off J Soc NeuroImmune Pharmacol 2013; 8: 888-99), all attempts to modify the neuroinflammatory response in the clinical context of ALS have failed to date (Appel SH et al. Arch Neurol 1988; 45: 381; Drachman DB et al. Ann Neurol 1994; 35: 142-50; Tan E et al. Arch Neurol 1994; 51: 194; Beghi E et al. Neurology 2000; 54: 469-469; Cudkowicz ME et al. Ann Neurol 2006; 60: 22-31; (Gordon PH et al. Lancet Neurol 2007; 6: 1045-53; Stommel EW et al. Amyotroph Lateral Scler 2009; 10: 393-404). However, most of these clinical trials targeted nonspecific suppression of neuroinflammation. Such an approach carries a high risk of harming ALS patients, first and foremost, because the toxicity may easily outweigh the beneficial drug effects.

[0006] In this regard, a new approach is needed to enhance the dominance of physiological tolerance within the neuroimmune inflammatory system without suppressing all immunity.

[0007] CD4+FOXP3+ regulatory T cells (Tregs) physiologically regulate the immune response, contributing to the induction and maintenance of tolerance, and ultimately preventing the development of autoimmune and inflammatory diseases (Sakaguchi S et al. Cell 2008; 133: 775-87).

[0008] Previous studies have shown that in ALS patients, decreased Treg levels correlate with increased disease severity and predict disease progression and survival (Mantovani S et al. J Neuroimmunol 2009; 210: 73-9; Rentzos M et al. Acta Neurol Scand 2012; 125: 260-4; Henkel JS et al. EMBO Mol Med 2013; 5: 64-79). In addition to decreased levels, Tregs in ALS patients exhibit low FOXP3 expression levels (Henkel JS et al. EMBO Mol Med 2013; 5: 64-79) and dysfunction, and this dysfunction has been shown to correlate with increased disease severity (Beers DR et al. JCI Insight. 2017;2(5):e89530; Thonhoff JR et al. Curr Opin Neurol 2018; 31: 635-9). Therefore, in ALS patients, Tregs not only decrease in number but also show significant dysfunction correlated with impaired FOXP3 expression levels, and this dysfunction correlates with disease severity and progression, suggesting that the inhibitory function of Tregs may be a meaningful indicator of clinical status (Thonhoff JR et al. Curr Opin Neurol 2018; 31: 635-9).

[0009] Treg cells rely almost entirely on the cytokine interleukin-2 (IL-2) for their generation, activation, and survival (Malek TR, Bayer AL. Nat Rev Immunol 2004; 4: 665-74). Furthermore, in contrast to human effector T cells (Teff), human Treg constitutively express high levels of CD25 and form high-affinity receptors for IL-2, thus responding to low concentrations of IL-2 that are insufficient to stimulate Teff (Dupont G. et al. Cytokine. 2014 Sep;69(1):146-9). Based on this, low-dose (ld) IL-2 administration was tested and shown to induce selective amplification of Tregs in mice and humans in healthy volunteers or those with type 1 diabetes (Hartemann A et al. Lancet Diabetes Endocrinol 2013; 1: 295-305 Ito S et al. Mol Ther J Am Soc Gene Ther 2014. 22: 1388-95).

[0010] Based on this, the use of ld-IL-2 in the treatment of various autoimmune and inflammatory conditions has been proposed (WO2012123381A1, WO2014023752A1, WO2016025385A1, WO2016164937A2), and is currently being used for graft-versus-host disease (Koreth J et al. N Engl J Med 2011; 365: 2055-66), HCV-induced vasculitis (Saadoun D et al. N Engl J Med 2011; 365: 2067-77), type 1 diabetes (Hartemann A et al. Lancet Diabetes Endocrinol 2013; 1: 295-305), and alopecia areata (Castela E et al. JAMA Dermatol 2014; 150: Several clinical trials have been reported to investigate the potential of ld-IL-2 as a treatment in 748-51).

[0011] Regarding animal models of ALS, it has been shown that administering a combination of a mouse IL-2 immune complex (a complex of IL-2 and an anti-IL-2 antibody) and rapamycin to transgenic SOD1 ALS mice resulted in a significant delay in disease progression accompanied by an extension of survival time (Sheean RK et al. JAMA Neurol. 2018;75(6):681-689). However, as explained above, many of the positive results obtained in this model using new candidate drugs, including vitamin E (Desnuelle C. et al, Amyotroph. Lateral Scler. Other Motor Neuron Disord. 2 (2001) 9-18), gabapentin (Miller RG, et al, Neurology 56 (2001) 843-848), topiramate (Cudkowicz M., et al, Neurology 61 (2003) 456-464), celecoxib (Cudkowicz M., 15th International Symposium on ALS / MND, Philadelphia, 2004), and creatine (Shefner J. et al, Neurology 63 (2004) 1656-1661), did not translate into efficient treatments for human ALS patients (DiBernardo AB et al. Biochimica et Biophysica Acta). 1762 (2006) 1139-1149; van den Berg LH et al. Neurology 2019;92:e1610-e1623).

[0012] Furthermore, it has been argued that Tregs in human ALS patients have impaired intrinsic responsiveness to IL-2 (Thonhoff JR et al. Neurol Neuroimmunol Neuroinflammation 2018; 5: e465), potentially leading to a lack of response to ld IL-2 treatment, meaning the same strategy may not be effective in ALS patients. In particular, based on an initial pilot study (unpublished data) in five ALS patients, Thonhoff et al. showed that ld IL-2 did not alter clinical outcomes or increase the number of endogenous Tregs (see Discussion §2 in Thonhoff JR et al. Neurol Neuroimmunol Neuroinflammation 2018; 5: e465). Based on this, although it was only in three ALS patients, an alternative autologous cell therapy treatment consisting of ex-vivo isolation of Treg cells from ALS patients, followed by ex-vivo amplification of the Treg cells using both IL-2 and rapamycin, and then reinfusion of the amplified Treg cells into the patients, along with ldIL-2 administration, was proposed and found to be effective in reducing the rate of functional decline (Thonhoff JR et al. Neurol Neuroimmunol Neuroinflammation 2018; 5: e465).

[0013] However, the results observed in an open-label, non-placebo-controlled trial involving only three patients are clearly insufficient to demonstrate a therapeutic effect on ALS. Furthermore, such a treatment would be extremely expensive and impractical for many hospitals, as it requires isolating the patient's Tregs and amplifying them in vitro before reinjecting them into the patient. Therefore, there is still a need for an alternative ALS treatment, which would be not only more efficient but also less expensive and more accessible for most ALS patients due to its much simpler protocol. [Overview of the Initiative]

[0014] With regard to the present invention, the inventors unexpectedly found that simply injecting a low dose of human interleukin-2 (ldIL-2) was sufficient to induce a significant improvement not only in the number of Tregs but, most importantly, in their inhibitory function, without the need for Treg isolation and ex vivo amplification (see Example 1). Furthermore, the inventors found that ldIL-2 not only increases the number and function of Tregs but also leads to a significant decrease in the plasma concentration of the inflammatory chemokine CCL2 (see Example 1), a small chemokine belonging to the CC subfamily also known as MCP1, which transmits signals via chemokine receptor 2 (CCR2) and guides circulating leukocytes to the site of neuroinflammation. Importantly, CCL2 plasma or CSF levels correlate with ALS disease scores (Nagata T, et al. Neurol Res. 2007 Dec;29(8):772-6) and, above all, with the survival rate of ALS patients (Gille B et al. J Neurol Neurosurg Psychiatry. 2019 Dec;90(12):1338-1346), thus making it a useful biomarker for ALS disease activity. The inventors also found that ld IL-2 can further upregulate the expression of chemokines (CCL17, CCL18), indicating a shift in the macrophage phenotype from the M1 inflammatory polarization to the anti-inflammatory M2 phenotype involved in tissue repair (see Mantovani A et al. J Pathol 2013; 229: 176-85; Mammana S et al. Int. J. Mol. Sci. 2018, 19(3), 831, Example 1).

[0015] Finally, the inventors found that an overall reduction in cytopathic processes leads to a reduction in plasma accumulation of NFL, a nonspecific marker of axonal injury. Thus, in contrast to the conclusions of Thonhoff et al., which are based on a very small number of subjects (only 5) and possibly unrepresentative numbers of subjects, the inventors demonstrated that in a three-group randomized (1:1:1), double-blind, single-center trial of two doses of ldIL-2 in a placebo-controlled parallel clinical trial involving 36 subjects (12 in each of the three treatment groups), ldIL-2 injection in ALS patients (i) was safe and well-tolerated, (ii) upmodulated the number and inhibitory function of Tregs above Teffs, (iii) downmodulated the inflammatory marker of disease progression (CCL2), (iv) shifted monocyte polarization from an M1 pro-inflammatory phenotype to an M2 anti-inflammatory and tissue-repairing phenotype, and (v) reduced overall ALS-related cytopathic activity, as evidenced by the plasma NFL response to treatment, which is an indicator of reduced axonal injury.

[0016] Therefore, in the first aspect, the present invention relates to human interleukin-2 (IL-2) for use in the treatment of amyotrophic lateral sclerosis in human subjects, wherein each dose of human IL-2 administered to the subject is 0.1 × 10⁻⁶ 6 ~3×10 6 This is an International Unit (IU), and the treatment does not include the administration of regulatory T cells to the subject. [Brief explanation of the drawing]

[0017] [Figure 1] Trial profile. ALSFRS-R: Amyotrophic Lateral Sclerosis Functional Assessment Score - Revised; BT: Regular blood tests; Cyt: Fresh blood cytometry; SVC: Slow vital capacity; PBMC: Peripheral blood mononuclear cells; Inj: Subcutaneous injection; D: Day; ECG: Electrocardiogram; ITT: Treatment intention. *Time slots correspond to hospital visit time. [Figure 2]Effect of IL-2 treatment on the number and frequency of Tregs. A-D Variation in Treg frequency (A-B) and absolute number (C-D) across all trials in three groups (white squares, placebo; black triangles, 1 MIU of IL-2; white circles, 2 MIU of IL-2). Data points A and C show the mean and error bars of their associated SEMs. B and D Changes in Treg number and frequency between baseline and 3 days after the final injection of one treatment cycle (d8) or three treatment cycles (d64). Data points represent patient-specific changes in Treg frequency (B) and number (D). E-F iAUC of trough levels of Tregs during the trial. Data points show the mean and error bars of the associated SEMs for Treg number (E) and frequency (F). [Figure 3] Effect of IL-2 treatment on Treg frequency and effector T cell phenotype as measured at baseline (d1) and 3 days after completion of 3 treatment cycles (d64). A-C Frequency of Tregs isolated from cryopreserved PBMCs for use in suppression assays of individuals treated with (A) 2 MIU, (B) 1 MIU, and (C) placebo. D-F CD25 expression in effector T cells with (D) 2 MIU, (E) 1 MIU, and (F) placebo. G-I Proliferation of effector T cells in the absence of Tregs in individuals treated with (G) 2 MIU, (H) 1 MIU, and (I) placebo. [Figure 4]Effect of IL-2 treatment on Treg phenotype and inhibitory function. A-C CD25 expression in Tregs at baseline (d1) and 3 days after completion of 3 treatment cycles (d64) in all three treatment groups: (A) 2 MIU, (B) 1 MIU, and (C) placebo. D-F Self-inhibitory function of Tregs measured by in vitro co-culture assay at baseline (d1) and 3 days after completion of 3 treatment cycles (d64) in individuals treated with (D) 2 MIU, (E) 1 MIU, and (F) placebo. (G) Change in Treg inhibitory function after 3 cycles of treatment compared to baseline levels in all three groups. Bars represent mean values, and error bars represent their associated SEMs. (H~I) Relationship between the relative changes in Treg frequency (H) and Treg CD35 mfi (I), as measured by clinical cytometry (x axis) and Treg inhibitory function (Y axis) after 3 cycles of treatment (values ​​at d64 versus d1). White squares indicate placebo, black triangles indicate 1 MIU of IL2, and white circles indicate individuals administered 2 MIU of IL2. [Figure 5] Transcriptome analysis of Treg activation markers FOXP3, CTLA4, IKZF2, and IL2RA (CD25) at D64. This plot shows increased gene expression of Treg activation markers FOXP3, CTLA4, IKZF2, and IL2RA in patients treated with 1 MIU and 2 MIU ldIL-2 compared to placebo. Each box represents the distribution of expression values ​​between each treatment group (the maximum, minimum, and median of the distribution are shown in each box and are visible as three horizontal lines). Multiple t-tests were performed to identify significant differences between sample groups. Legend: *=p value < 0.05, **=p value < 0.01, ***=p value < 0.001, ****=p value < 0.0001. [Figure 6]Effect of IL-2 therapy on plasma cytokine concentrations. A-C Changes in plasma cytokine levels during the study for CCL2(A), CCL17(B), and CCL18(C). Concentrations are expressed as a percentage of each individual's baseline value, and points show the mean, error bars, and associated SEM. White squares indicate placebo, black triangles indicate individuals who received 1 MIU of IL-2, and white circles indicate individuals who received 2 MIU of IL-2. [Figure 7] Percentage change from baseline in plasma NFL levels at D85 due to treatment. Bars represent the mean ± sem of the percentage change from baseline. Gray fill = placebo; dot = 1 MIU IL-2; white = 2 MIU IL-2. [Modes for carrying out the invention]

[0018] With regard to the present invention, the inventors unexpectedly found that simply injecting a low dose of human interleukin-2 (ldIL-2) was sufficient to induce a significant improvement not only in the number of Tregs but, most importantly, in their inhibitory function, without the need for ex vivo selection and amplification of Tregs. Furthermore, the inventors also found that ldIL-2 not only increases the number and function of Tregs but also leads to a significant decrease in the plasma concentration of the inflammatory chemokine CCL2, a small chemokine belonging to the CC subfamily, also known as MCP1. This CCL2 signals via chemokine receptor 2 (CCR2), guiding circulating leukocytes to the site of neuroinflammation and correlating with ALS disease scores (Nagata T, et al. Neurol Res. 2007 Dec;29(8):772-6) and with the survival rate of ALS patients (Gille B et al. J Neurol Neurosurg Psychiatry. 2019 Dec;90(12):1338-1346), thus making it a useful biomarker for ALS disease activity. The inventors also found that ld IL-2 can further upregulate the expression of chemokines (CCL17, CCL18), indicating a shift in the macrophage phenotype from the M1 inflammatory polarization to the anti-inflammatory M2 phenotype involved in tissue repair (Mantovani A et al. J Pathol 2013; 229: 176-85; Mammana S et al. Int. J. Mol. Sci. 2018, 19(3), 831). Furthermore, all these changes, in contrast to those observed in the placebo group, were paralleled in the treatment group with a sustained cessation of NFL plasma increase, indicating a sustained positive effect on overall cytopathic ALS-related activity.Thus, in contrast to the conclusions of Thonhoff et al. based on a very small number (only 5 subjects) and probably non-representative number of patients, the inventors have shown in a placebo-controlled parallel clinical trial including 36 subjects (12 in each of the 3 treatment groups), a three-arm randomized (1:1:1) double-blind single-site trial of two doses of ldIL-2, that injection of ldIL-2 into ALS patients is safe and well tolerated and enables (i) upregulation of the number and suppressive function of Tregs, (ii) transition of monocytes to an anti-inflammatory phenotype, and (iii) downregulation of markers of disease progression (CCL2, NFL).

[0019] Use of low-dose human IL-2 in the treatment of amyotrophic lateral sclerosis in humans Thus, the present invention relates to human interleukin-2 (IL-2) for use in the treatment of amyotrophic lateral sclerosis in a human subject, wherein each dose of human IL-2 administered to the subject is from 0.1×10 6 to 3×10 6 international units (IU), and the treatment does not include administration of regulatory T cells to the subject.

[0020] The present invention also relates to the use of human interleukin-2 (IL-2) for the manufacture of a medicament for use in the treatment of amyotrophic lateral sclerosis in a human subject, wherein each dose of human IL-2 administered to the subject during the treatment is from 0.1×10 6 to 3×10 6 international units (IU), and the treatment does not include administration of regulatory T cells to the subject.

[0021] The present invention also relates to the use of human interleukin-2 (IL-2) in the treatment of amyotrophic lateral sclerosis in a human subject, wherein each dose of human IL-2 administered to the subject is from 0.1×10 6 to 3×10 6 international units (IU), and the treatment does not include administration of regulatory T cells to the subject.

[0022] The present invention also relates to a pharmaceutical composition comprising human interleukin-2 (IL-2) for use in the treatment of amyotrophic lateral sclerosis in human subjects, wherein each dose of human IL-2 administered to the subject is 0.1 × 10⁻⁶ 6 ~3×10 6 This is an International Unit (IU), and the treatment does not include the administration of regulatory T cells to the subject.

[0023] The present invention also relates to a method for treating amyotrophic lateral sclerosis in a human subject requiring it, comprising administering human interleukin-2 (IL-2) to the human subject, wherein each dose of human IL-2 administered to the subject is 0.1 × 10⁻⁶ 6 ~3×10 6 This is an International Unit (IU), and the treatment does not include the administration of regulatory T cells to the subject.

[0024] Human IL-2 The claimed treatment relies on the administration of low doses of human IL-2 to human ALS patients.

[0025] In this specification, “human interleukin 2” or “human IL-2” refers to any source of human IL-2, including naturally occurring human IL-2 or human IL-2 obtained by recombinant or synthetic techniques, including recombinant IL-2 polypeptides produced by a microbial host. The nucleotide and amino acid sequences of naturally occurring human IL-2 are disclosed, for example, in the description of the human IL-2 gene in Pubmed Entrez Gene reference 3558. The reference sequence for naturally occurring human IL-2 protein can be found in NCBI Reference Sequence NP_000577.2 (updated January 5, 2020), and the reference sequence for naturally occurring human IL-2 mRNA can be found in NCBI Reference Sequence NM_000586.4 (updated January 5, 2020).

[0026] Human IL-2 may consist of, contain, or be an active variant of a natural human IL-2 polypeptide sequence. Preferably, recombinant human IL-2, in particular recombinant human IL-2 produced by a microbial host, is used.

[0027] Active variants of IL-2 are disclosed in the literature. Variants of natural IL-2 may be fragments, analogs, and derivatives thereof. A “fragment” is intended to be a polypeptide containing only a portion of the complete polypeptide sequence. An “analog” represents a polypeptide containing the natural polypeptide sequence having one or more amino acid substitutions, insertions, or deletions. Mutant proteins and pseudopeptides are specific examples of analogs. A “derivative” includes any modified natural IL-2 polypeptide or its fragments or analogs, such as those that have been glycosylated, phosphorylated, fused to another polypeptide or molecule, polymerized, or modified or added to improve the properties of IL-2 (e.g., stability, specificity, etc.). Active variants of natural human IL-2 polypeptide generally have at least 75%, preferably at least 85%, and more preferably at least 90% amino acid sequence identity with the amino acid sequence of the natural human IL-2 polypeptide. Methods for determining whether a mutant IL-2 polypeptide is active are available in the art, and examples of IL-2 mutants are disclosed, for example, in EP109748, EP136489, US4,752,585;EP200280, or EP118617, which are incorporated herein by reference. The active mutant is most preferably a mutant that activates Treg.

[0028] Preferably, recombinant human IL-2, i.e., human IL-2 produced by recombinant DNA technology, is used. The host organism used to express the recombinant DNA encoding human IL-2 may be a prokaryote (bacteria such as Escherichia coli) or a eukaryote (e.g., yeast, fungi, plant, or mammalian cells). Processes for producing recombinant IL-2 are described, for example, in US4,656,132;US4,748,234;US4,530,787; or US4,748,234, which are incorporated herein by reference.

[0029] The human IL-2 used in the present invention must be in a pharmaceutically acceptable form, in particular in an essentially pure form, for example, with a purity of 95% or higher, more preferably 96%, 97%, 98%, or 99%.

[0030] Human IL-2 is commercially available, including for pharmaceutical use, and is permitted for use in humans. For example, Aldesleukin (trademark Proleukin®) is an analogue of the human interleukin-2 gene produced by recombinant DNA technology using genetically modified E. coli strains approved by the FDA for cancer treatment. Aldesleukin differs from natural human interleukin-2 in the following ways: a) Aldesleukin is derived from E. coli and is therefore not glycosylated; b) This molecule lacks an N-terminal alanine; the codon of this amino acid was deleted during the genetic engineering procedure; c) This molecule has a serine substitution at amino acid position 125, replacing cysteine.

[0031] Aldesleukin is preferably used in the present invention.

[0032] however, • Yeast Saccharomyces cerevisiae containing the human IL-2 gene ( Saccharomyces cerevisiae Roncoleukin® is a pharmaceutical form of recombinant human IL-2 isolated and purified from the cells of [unspecified organism]. Interking, a recombinant IL-2 with serine at residue 125, is sold by Shenzhen Neptunus. Albuleukin is recombinant human interleukin-2 (rIL-2) that is genetically fused to recombinant human serum albumin (rHSA). Other recombinant human IL-2 strains are also available.

[0033] Human IL-2 dose, administration scheme, and route As explained above, human Tregs constitutively express high levels of CD25 and form high-affinity receptors for IL-2, which is not present in resting Teffs, and therefore respond to low concentrations of IL-2 that are insufficient to stimulate Teffs. The claimed treatment is intended to enlarge the number and function of Tregs, but not to enlarge or stimulate Teffs, and each dose of human IL-2 administered to ALS patients is 0.1 × 10⁻⁶ 6 ~3×10 6 International Units (IU) are kept low. Furthermore, in the case of type 1 diabetes, 3 x 10 6 Doses up to IU have been shown to be safe, but 3 × 10 6 At the highest dose of IU, less severe adverse events occurred (Hartemann A et al. Lancet Diabetes Endocrinol 2013; 1: 295-305). Each dose of human IL-2 was also limited to a maximum of 3 × 10⁶ to limit potential toxicity. 6 IU needs to be kept.

[0034] As explained above, Tregs depend entirely on IL-2 for their generation, activation, and survival (Malek TR, Bayer AL. Nat Rev Immunol 2004; 4: 665-74). Furthermore, the half-life of aldesleukin administered to human patients is generally about 2-3 hours (see, e.g., Proleukin® label). Therefore, human IL-2 is commonly administered repeatedly to ALS patients to obtain a sustained expansion of Treg numbers and immunosuppressive function.

[0035] In a preferred embodiment, human IL-2 is 0.1 × 10⁻⁶ 6 ~3×10 6 IU of human IL-2, preferably 0.2 × 10⁻¹⁶. 6 ~3×10 6 Human IL-2 in IU, 0.3 × 10⁻¹⁶ units 6 ~3×10 6 Human IL-2 in IU, 0.4 × 10⁻¹⁴ 6 ~3×10 6 IU of human IL-2, more preferably 0.5 × 10⁻¹⁶. 6 ~3×10 6 Human IL-2 in IU, 0.6 × 10⁻⁶ 6 ~3×10 6 Human IL-2 in IU, 0.7 × 10⁻⁶ 6 ~3×10 6 Human IL-2 in IU, 0.8 × 10⁻⁶ 6 ~3×10 6 Human IL-2 in IU, 0.9 × 10⁻⁶ 6 ~3×10 6 IU of human IL-2, 1 × 10⁻¹⁶ 6 ~3×10 6 IU of human IL-2, 1 × 10⁻¹⁶ 6 ~2×10 6 IU of human IL-2, especially 0.5 × 10⁻⁶ 6 IU of human IL-2, 1 × 10⁻¹⁶ 6 IU of human IL-2, or 2 × 10⁶ 6 IU of human IL-2 is administered repeatedly, preferably by subcutaneous injection.

[0036] The injection is • Dosage schemes based on repeated cycles of once-daily or several-times-daily injections of human IL-2 with periods of human IL-2 injection-free intervals; and • Continuous metronome administration, i.e., injection of human IL-2 once or several times a day. This can be done according to various administration schemes, including those listed below.

[0037] In contrast to what Thonhoff et al. suggested regarding the IMODALS clinical trial (see Example 1), 1 × 10⁻¹⁰ 6 or 2 × 10 6 The initial 5-day cycle, initiated with IU / day of human IL-2 subcutaneously once daily, was sufficient to significantly increase the number and function of Tregs up to D29 (see Figure 2). Furthermore, the same 5-day cycle was administered at week 5 (cycle 2, initiated at D29) and week 9 (cycle 3, initiated at D57) (see Figure 1), and the peaks in Treg number and frequency during cycle 3 were higher than those observed during cycle 1, suggesting a potential cumulative residual effect from consecutive treatment cycles. This suggestion is further supported by significantly higher iAUC trough Treg levels (measuring the change in residual Tregs before initiating a new cycle) in the IL2 group compared to placebo (Figures 2E-F, Tables 3 and 4 above).

[0038] Therefore, 0.1 × 10 6 ~3×10 6 IU of human IL-2, preferably 0.2 × 10⁻¹⁶ 6 ~3×10 6 Human IL-2 in IU, 0.3 × 10⁻¹⁶ units 6 ~3×10 6 Human IL-2 in IU, 0.4 × 10⁻¹⁴ 6 ~3×10 6 IU of human IL-2, more preferably 0.5 × 10⁻⁶ 6 ~3×10 6 Human IL-2 in IU, 0.6 × 10⁻⁶ 6 ~3×10 6 Human IL-2 in IU, 0.7 × 10⁻⁶ 6 ~3×10 6 Human IL-2 in IU, 0.8 × 10⁻⁶ 6 ~3×10 6 Human IL-2 in IU, 0.9 × 10⁻⁶ 6 ~3×10 6 IU of human IL-2, 1 × 10⁻¹⁶ 6 ~3×106 IU of human IL-2, 0.5×10 6 ~2×10 6 IU of human IL-2, 1×10 6 ~2×10 6 IU of human IL-2, particularly, 0.5×10 6 IU of human IL-2, 1×10 6 IU of human IL-2, or 2×10 6 IU of human IL-2 for once-daily subcutaneous administration for 3 to 7 consecutive days is thought to significantly increase the number of Tregs during that cycle and for at least an additional maximum of 3 weeks.

[0039] Therefore, in the therapeutic use or method according to the present invention, 0.1×10 6 ~3×10 6 IU of human IL-2, preferably, 0.2×10 6 ~3×10 6 IU of human IL-2, 0.3×10 6 ~3×10 6 IU of human IL-2, 0.4×10 6 ~3×10 6 IU of human IL-2, more preferably, 0.5×10 6 ~3×10 6 IU of human IL-2, 0.6×10 6 ~3×10 6 IU of human IL-2, 0.7×10 6 ~3×10 6 IU of human IL-2, 0.8×10 6 ~3×10 6 IU of human IL-2, 0.9×10 6 ~3×10 6 IU of human IL-2, 1×10 6 ~3×10 6 IU of human IL-2, 0.5×10 6 ~2×10 6 IU of human IL-2, 1×10 6 ~2×10[[ID=6)) 6 IU of human IL-2, particularly, 0.5×10 6 IU of human IL-2, 1×10 6 IU of human IL-2, or 2×10 6The target is preferably administered several cycles of IU of human IL-2 subcutaneously once daily for 3 to 7 consecutive days. More preferably, each cycle consists of 0.1 × 10⁻⁶ doses. 6 ~3×10 6 IU of human IL-2, preferably 0.2 × 10⁻¹⁶ 6 ~3×10 6 Human IL-2 in IU, 0.3 × 10⁻¹⁶ units 6 ~3×10 6 Human IL-2 in IU, 0.4 × 10⁻¹⁴ 6 ~3×10 6 IU of human IL-2, more preferably 0.5 × 10⁻⁶ 6 ~3×10 6 Human IL-2 in IU, 0.6 × 10⁻⁶ 6 ~3×10 6 Human IL-2 in IU, 0.7 × 10⁻⁶ 6 ~3×10 6 Human IL-2 in IU, 0.8 × 10⁻⁶ 6 ~3×10 6 Human IL-2 in IU, 0.9 × 10⁻⁶ 6 ~3×10 6 Human IL-2 in IU, 0.5 × 10⁻¹⁶ units 6 ~2×10 6 IU of human IL-2, 1 × 10⁻¹⁶ 6 ~2×10 6 IU of human IL-2, especially 0.5 × 10⁻⁶ 6 IU of human IL-2, 1 × 10⁻¹⁶ 6 ~3×10 6 IU's IL-2, or 2×10 6 This treatment consists of subcutaneous administration of IU of human IL-2 once daily for five consecutive days.

[0040] Since each cycle is expected to significantly increase the number and function of Tregs during that cycle and for at least about three weeks thereafter, the cycle is preferably administered every two to six weeks, preferably every two to five weeks, more preferably every two to four weeks, and especially every two, three, or four weeks.

[0041] However, while the above administration scheme is preferred, other administration schemes that can significantly increase the number and function of Tregs without unacceptable toxicity can also be defined by those skilled in the art, based on their knowledge of low-dose IL-2 administration in humans in other contexts.

[0042] Indeed, there is teaching in the art suggesting that ALS patients have dysfunctional Tregs (Beers DR et al. JCI Insight. 2017;2(5):e89530; Thonhoff JR et al. Curr Opin Neurol 2018; 31: 635-9), and unlike other human patients, they did not respond to low-dose IL-2 administration (Thonhoff JR et al. Neurol Neuroimmunol Neuroinflammation 2018; 5: e465). Now, a placebo-controlled, parallel, three-group, randomized (1:1:1), double-blind, single-center trial of two doses of ldIL-2 has shown that ALS patients can indeed respond favorably to ld-IL-2, resulting in a significant increase in the number and function of Tregs, and, most importantly, a decrease in CCL2 plasma concentration, a marker of ALS disease activity. Based on this unexpected discovery by the inventors, knowledge regarding the effects of low-dose IL-2 in other human subjects can now be used to design other appropriate dosing schemes.

[0043] For example, while subcutaneous administration was used in the IMODALS clinical trial, intravenous administration of IL-2 has been shown to significantly increase Treg count in human cancer patients (Ahmadzadeh M, Rosenberg SA. Blood 2006;107:2409-14). Therefore, in the therapeutic use and methods according to the present invention, human IL-2 is preferably administered via subcutaneous or intravenous route. Subcutaneous administration is still preferred because it is easier, better tolerated, and has been shown to be efficient in the IMODALS clinical trial.

[0044] Regarding dosage, in order to limit potential toxicity, each single dose is 3 × 10⁻⁶ 6It is necessary to avoid exceeding IU, but the results obtained in the IMODALS clinical trial showed that the effect of low doses of human IL-2 on the number and function of Tregs in ALS patients was dose-dependent, administered once daily at 2 × 10¹⁶ times per 5-day cycle. 6 The highest dose of human IL-2, IU, is the most effective. Therefore, when using a dosing scheme that includes repeated and isolated cycles of low-dose human IL-2 administration, 1 × 10⁻¹⁶ doses are obtained during the cycle. 6 IU~2×10 6 IU, preferably 2 × 10 6 A daily dose of IU is preferred. However, the daily dose can be administered either once a day or in several divided low doses. For example, 2 × 10 6 To achieve the daily dose of IU, 2 x 10 6 Administer a single dose of IU once daily, or 2 x 10 6 The daily dose of IU can be reduced to two or more lower doses, for example, 1 x 10⁶ doses of two doses. 6 IU (for example, once in the morning and once in the evening), 0.67 × 10 times the dose for 3 doses 6 IU (for example, once in the morning, once during the day, and a third time in the evening), or 0.5 × 10 times four doses. 6 It can be divided into IU. Such divisions of the daily dose would be 2-3 x 10 6 This may be particularly useful in ALS patients experiencing adverse events when administered as a single daily dose of IU.

[0045] Furthermore, while the dosing scheme used in the IMODALS clinical trial is based on a 5-day cycle every 4 weeks, other dosing schemes may also be considered. For example, alternative dosing schemes include: A longer cycle where the interval between two cycles is longer; A shorter cycle is one in which the interval between two cycles is shorter; • Continuous administration of low-dose human IL-2 (without cycling) It can be based on this.

[0046] For example, when using dosing cycles, each cycle can vary significantly between 3 and 7 days, but can be shorter (e.g., 2 days) or longer (e.g., 8, 9, 10, 11, 12, 13, 14 days, or even 3 or 4 weeks). * Cycles are available. When using shorter cycles (e.g., 2 days), it is preferable to repeat the cycles more frequently than the 4-week schedule used in IMODALS, such as every 3 weeks, every 2 weeks, every 10 days, or weekly. When using longer cycles (e.g., 8, 9, 10, 11, 12, 13, 14 days, or even 3 or 4 weeks), it is preferable to repeat the cycles at the same frequency as the 4-week schedule used in IMODALS, or slightly less frequently, for example, every 4 weeks, every 5 weeks, or every 6 weeks. However, since the duration of effect of human IL-2 is relatively short, the interval between cycles should not be too long.

[0047] Furthermore, continuous metronome administration (without cycles) of low-dose human IL-2 may also be considered. While administration schemes based on repeated cycles of low-dose human IL-2 have been used as a commercial product for patients in IMODALS and other autoimmune diseases based on prior knowledge derived from cancer treatment, continuous metronome administration of low-dose human IL-2 may still be considered, particularly when a pump that enables continuous administration of low-dose human IL-2 (similar to those used to deliver insulin to diabetic patients) is used. In this case, considering the tendency for Treg to accumulate after 3 cycles, as seen in IMODALS (see Example 1 and Figure 2), 0.1 × 10⁻¹⁶ 6 ~2×10 6 IU, preferably 0.1 × 10⁻⁶ 6 ~1.5×10 6 IU, 0.1 × 10 6 ~1 × 10 6 IU, or even 0.1 × 10⁻⁶ 6 ~0.5 × 10 6 Lower cumulative daily doses of human IL-2, such as IU, may be considered. This type of treatment may be particularly considered in ALS patients who exhibit adverse effects from higher daily doses of human IL-2.

[0048] More generally, each single dose is 0.1 × 10⁻⁶ 6 ~3×10 6 While it should be included between IUs, clinicians know how to adapt the dosing scheme to observe efficiency without unacceptable toxicity. In particular, starting with a given dosing scheme (such as one of the schemes selected in the IMODALS and MIROCALS clinical trials), clinicians can monitor the number or frequency of Tregs, their immunosuppressive function, and / or serum, plasma, or cerebrospinal fluid (CSF) concentrations of CCL2 and / or CCL17 and / or CCL18, as well as potential adverse events and the dosing scheme, to optimize the benefit / risk ratio (improving efficiency and / or limiting drug-related adverse events based on the number or frequency of Tregs, their immunosuppressive function, and / or serum, plasma, or cerebrospinal fluid (CSF) concentrations of CCL2 markers and / or CCL17 and / or CCL18).

[0049] In this regard, in one aspect of the present invention, the treatment is a) Measure the number or frequency (in the blood) of Tregs at baseline (i.e., the date of initiation of low-dose human IL-2 treatment) from the biological sample of interest, and / or the immunosuppressive function of the Tregs, and / or the serum or plasma concentration or CSF concentration of CCL2, and / or the serum or plasma concentration or CSF concentration of CCL17 and / or CCL18. b) Administering human IL-2 to the subject according to a first administration scheme according to the present invention, which includes either repeated isolation cycles of human IL-2 administration or continuous metronome human IL-2 administration. c) Monitor for drug-related adverse events and measure the same parameters as at baseline from biological samples of the subject taken 1–3 days after the end of the treatment cycle or at least 7 days after continuous metronome human IL-2 administration, and d) Continue with the first dosing scheme if the results are acceptable, or plan a second dosing scheme depending on the results of step c). Includes.

[0050] In step d), if an ALS patient experiences a poorly tolerated adverse event with compliance issues, the cyclic or continuous daily dose may be reduced or divided into several lower single doses (instead of a once-daily dose). If both the first and second dosing schemes are cycle-based and the daily dose of human IL-2 is reduced during a cycle, the duration of each cycle may be extended to compensate. Alternatively, if continuous dosing is used instead of cycles, the second dosing scheme may be based on continuous dosing rather than cycles, as a lower daily dose is expected to be sufficient.

[0051] Conversely, if the therapeutic effect is insufficient, as measured by Treg count, frequency or immunosuppressive function (markers negatively correlated with disease progression) or plasma or CSF concentrations of CCL2, CCL17 and / or CCL18 (CCL2 is positively correlated with disease progression, and CCL17 and / or CCL18 are indicators of macrophage polarization from pro-inflammatory M1 phenotype to anti-inflammatory M2 phenotype), the daily dose of the cycle or continuous dose may be increased to enhance the likelihood that the ALS patient will respond to treatment, with each single dose administered to the subject being up to 3 × 10⁶ 6 It can be increased on the condition that it is IU. The number, frequency, or immunosuppressive function of Tregs, and CCL2 plasma concentration or CSF concentration are particularly correlated with disease progression, so it is preferable to use at least one of these markers. Plasma or serum concentration of CCL2 is even more preferred because it is easier and more reliable to measure.

[0052] CCL2 concentration can be measured in plasma, serum, or CSF. Measurements can be performed in fresh or frozen (-20°C) plasma, serum, or CSF samples. CCL2 concentration is measured in fresh or frozen plasma, serum, or CSF using solid-phase immunoassays such as enzyme-linked immunosorbent assay (ELISA) (as performed in the MIROCALS test) or cytometry bead assay (as performed in the IMODALS test). An increase in IL2 unit dose is considered when the CCL2 concentration exceeds 80% of the baseline concentration before treatment (i.e., shows a decrease of less than 20% at the time of treatment).

[0053] Whatever the chosen administration scheme (which may be modified during treatment as described above), treatment is preferably continued for the lifetime of the subject or until an unacceptable drug-related serious adverse event occurs.

[0054] Other treatments not administered to human subjects In the therapeutic use and method of the present invention, the treatment does not involve the administration of regulatory T cells to the subject. In fact, contrary to what was suggested by Thonhoff et al. (Thonhoff JR et al. Neurol Neuroimmunol Neuroinflammation 2018; 5: e465), the inventors unexpectedly found that injection of low-dose human interleukin-2 (ldIL-2) alone, without the need for isolation and ex vivo amplification of Tregs prior to reinjection, was sufficient to induce a significant improvement not only in the number of Tregs but, most importantly, in the inhibitory function of all ALS patients. Thus, the claimed treatment, which does not require ex vivo selection, amplification, and reinjection of Tregs, is far simpler, far less expensive, and therefore more accessible to a wider range of ALS patients.

[0055] Regulatory T cells, or Tregs, are T lymphocytes that possess immunosuppressive activity. Natural Tregs are CD4 + CD25 + Foxp3 +They are characterized by their phenotypic features. Tregs are also characterized by their functional ability to inhibit the proliferation of T effector cells.

[0056] Human IL-2 may be administered to ALS patients in combination with other treatments (see below), but in addition to the lack of combination therapy with Treg, the claimed treatment is also preferably not combined with one or more of the following treatments.

[0057] In the first embodiment, in addition to the absence of combination therapy with Treg, the human IL-2 administered to the subject does not form a complex with the anti-human IL-2 antibody.

[0058] Because the IL-2 / IL-2 monoclonal antibody conjugate was thought to enhance the biological activity of IL-2, and because the selected anti-IL-2 monoclonal antibody clone was able to confer specificity to cells expressing high-affinity αβγ IL-2R (CD25hiCD4+Foxp3+Treg and activated effector T cells) rather than cells expressing low-affinity βγ IL-2R (memory CD8+ cells or natural killer cells) (see Sheean RK et al. JAMA Neurol. 2018;75(6):681-689, Methods, section “Data Collection From Animal Participants”, paragraph 3), the IL-2 and anti-IL-2 antibody conjugate was used instead of IL-2 purified by Sheean et al. in transgenic SOD1 ALS mice.

[0059] However, we have now shown that administration of low doses of non-complexing human IL-2 is sufficient to specifically increase the number, proportion, and inhibitory function of Tregs in ALS patients (see Example 1).

[0060] As a result, with respect to the present invention, it is preferable that the human IL-2 administered to the subject does not form a complex with the anti-human IL-2 antibody.

[0061] In the second embodiment, in addition to the absence of combination therapy with Tregs, the claimed treatment is preferably not used in combination with rapamycin or any other agent that suppresses effector T cells (Teffs).

[0062] In transgenic SOD1 ALS mice, Sheean et al. not only used IL-2 conjugated with an anti-IL-2 antibody, but also combined the conjugated IL-2 with rapamycin therapy. Rapamycin is an immunosuppressant known to particularly inhibit the expansion of effector T cells (Teff), and Sheean et al. used it in combination with the IL-2 / anti-IL-2 conjugate to specifically expand Treg cells with an activated phenotype and to exert its immunosuppressive function. This combination is considered essential because it inhibits the proliferation of T effector cells, enabling selective amplification of Tregs (see Sheean RK et al. JAMA Neurol. 2018;75(6):681-689, Methods, Data Collection From Animal Participants section, paragraph 3).

[0063] However, we have now shown that administering low doses of non-complexed human IL-2 to ALS patients is sufficient to specifically increase the number, proportion, and function of Tregs, without the concomitant administration of Teff immunosuppressants such as rapamycin (see Example 1).

[0064] As a result, in addition to the absence of combination therapy with Tregs in relation to the present invention, the claimed treatment is preferably not used in combination with rapamycin or any other agent that suppresses effector T cells (Teff).

[0065] "Effector T cells" or "Teff" include all CD4 cells other than Treg cells. In particular, Teff cells do not constitutively express FOXP3.

[0066] In a preferred embodiment, in addition to the absence of combination therapy with Treg, the human IL-2 administered to the subject does not form a complex with the anti-human IL-2 antibody, and the claimed treatment is not combined with rapamycin or any other agent that inhibits Teff (as disclosed above).

[0067] Other treatments that are preferably administered to human subjects Currently, the only therapeutic agent available for the treatment of ALS is riluzole (2-amino-6-(trifluoromethoxy)benzothiazole, CAS number 1744-22-5, trademark Rilutek®), which is the compound shown below. [ka]

[0068] Riluzole, approved by the FDA in 1995 as a treatment for ALS, has been shown to be associated with a short median survival benefit of 2-3 months, which corresponds to an absolute increase of 9% in the one-year survival rate (Miller RG, et al. Cochrane Database of Systematic Reviews 2012, Issue 3. Art. No.: CD001447).

[0069] While this short survival benefit is not satisfactory, it is still better than receiving no treatment, and therefore, most ALS patients are treated with riluzole.

[0070] Therefore, in a preferred embodiment of the present invention, the treatment of the present invention using a low dose of human IL-2 preferably further includes administering riluzole to the subject.

[0071] In ALS, riluzole is generally administered orally at a daily dose of 100 mg / day, taken in two equal doses of 50 mg approximately 12 hours apart. If toxicity occurs, a lower daily dose, such as 50 mg / day taken orally in two equal doses of 25 mg approximately 12 hours apart, may be used.

[0072] Therefore, when the treatment of the present invention is combined with riluzole therapy, riluzole is preferably administered orally in two equal doses of 25 mg to 50 mg each, with an interval of approximately 12 hours between doses, in a daily dose of 50 mg to 100 mg.

[0073] Further optional treatments commonly administered to ALS patients, including antidepressants (if the ALS patient is suffering from depressive symptoms), analgesics (to limit pain), anticholinergics (in cases of excessive salivation), and antibiotics (in cases of bacterial infection), may be further administered to ALS patients in accordance with the present invention.

[0074] Biological effects of treatment As shown in Example 1, the present inventors have now demonstrated that administering a low dose of human IL-2 to human ALS patients can improve their condition. • Increasing the number / frequency of Tregs and improving their inhibitory function (see Figures 2-5 and Tables 4 and 5 in particular), • Changing the polarization of macrophages from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype. It is possible.

[0075] In particular, the IMODALS clinical trial shows that administering low doses of human IL-2 to human ALS patients can achieve both of the following: • Reduce the plasma concentration of the inflammatory chemokine CCL2 (see Figure 6A in particular). CCL2, also known as MCP1, is a small inflammatory chemokine belonging to the CC subfamily. It transmits signals via chemokine receptor-2 (CCR2), inducing circulating leukocytes to the site of neuroinflammation, and has been shown to correlate with disease score (Nagata T, et al. Neurol Res. 2007 Dec;29(8):772-6) and survival rate (Gille B et al. J Neurol Neurosurg Psychiatry. 2019 Dec;90(12):1338-1346). Therefore, CCL2 concentration can serve as an indicator of the effect on disease progression in ALS patients. • Increase the plasma concentrations of CCL17 and / or CCL18 (see Figures 6A and 6B in particular). CCL17 and CCL18 are chemokines primarily expressed by anti-inflammatory M2 polarized macrophages (Katakura T, et al. J Immunol. 2004 Feb 1;172(3):1407-13; Schraufstatter IU, et al. Immunology. 2012 Apr;135(4):287-98). Therefore, elevated CCL17 and / or CCL18 concentrations are indicators of a shift in macrophage polarization from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype.

[0076] Therefore, with respect to the present invention, the treatment is preferably performed for the following purposes: • Induces an increase in the number of Tregs and improves the immunosuppressive function of Tregs; • Induces a decrease in CCL2 plasma, serum, or cerebrospinal fluid (CSF) concentration; and / or Preferably, it induces a shift in monocyte polarization in the blood or central nervous system (CNS) to the M2 phenotype, which is involved in repair function, evidenced by elevated CCL17 and / or CCL18 plasma or serum or CSF concentrations.

[0077] This can be achieved by: Continue treatment as long as at least one of the above biological effects is achieved. • Adapting the administration scheme for human IL-2 to maintain at least one of the above biological effects (within the low-dose scheme used throughout the invention) (see the above section on dose and administration scheme), and / or • Selection of low-dose human IL-2 administration (within the low-dose scheme used throughout the invention) to treat ALS patients resulting in at least one of the above-described biological effects.

[0078] In this case, the number of Tregs and / or immunosuppressive function, or plasma or CSF concentrations of CCL2, CCL17, or CCL18, or any combination thereof, are used as biomarkers to select ALS patients who would benefit from low-dose human IL-2 treatment, and then treat them with low-dose human IL-2.

[0079] The following examples are intended solely to illustrate the present invention. [Examples]

[0080] Example 1: Phase II study (IMODALS) on the safety and activity of immunomodulation-low-dose interleukin-2 in amyotrophic lateral sclerosis (ALS) Low-dose human IL-2 was tested in ALS patients in the IMODALS Phase 2 clinical trial (clinicaltrials.gov NCT02059759).

[0081] Patients and Methods Study plan and participants This placebo-controlled, parallel, three-group, randomized (1:1:1), double-blind, single-center trial of two doses of ld-IL-2 included 36 patients with ALS. The trial protocol was approved by an independent ethics committee (Le Comite de Protection des Personnes Sud Mediterranee III; reference number: 2014.09.01 ter) and declared on clinicaltrials.gov NCT02059759, and was designed for adults under 75 years of age with probable, probable and laboratory-supported, or definite ALS as defined by the revised El Escorial diagnostic criteria for ALS (Brooks BR, et al. Amyotroph Lateral Scler Other Motor Neuron Disord 2000; 1: 293-9). The primary inclusion criteria consisted of disease duration less than 5 years, stable for at least 3 months on riluzole treatment, and lung capacity ≥ 70% of normal. Patients with severe heart or lung disease, cancer, other life-threatening illnesses, respiratory or feeding difficulties, clinical signs of infection, positive serology (cytomegalovirus, Epstein-Barr virus, or human immunodeficiency virus), autoimmune diseases (excluding asymptomatic Hashimoto's thyroiditis), clinically significant laboratory abnormalities (excluding cholesterol, triglycerides, and glucose), or other conditions that would interfere with functional assessment were excluded, as were patients who had received vaccination 8 weeks prior to the first experimental dose. All patients submitted signed informed consent before participating in the study.

[0082] Randomization and masking Assignments were made via a web-based inclusion and randomization (with blocking) application, and blinding was ensured (otherwise, a statistician not involved in the trial would have created the randomization list). The size of block(3) was not disclosed to all participants until blinding was de-blinded. Preparation and labeling of clinical treatment units (CTUs) were performed by a pharmacist, the only open-label trial participant. All laboratory assays were fully blinded. Samples were identified only by a randomization number, trial time (number of sampling points = 7), or a barcode corresponding to the treatment group (placebo, 1 MIU IL-2, 2 MIU IL-2), which was not disclosed to the laboratories creating the data.

[0083] procedure Upon selection by the investigating physician, a baseline assessment was performed, including routine hematology and biochemistry, slow vital capacity, the Modified ALS Functional Rating Scale (ALSFRS-R), chest X-ray, electrocardiogram (ECG), and thyroid function (Figure 1). After randomization (up to two weeks before the first dose on day 1), patients began a 5-day cycle of once-daily subcutaneous injections, repeated twice at weeks 5 and 9, for a total of three treatment cycles per patient (Figure 1). After the final treatment cycle, all patients were followed for an additional three months for safety monitoring.

[0084] Proleukin® (Aldesleukin) 22 MIU vials were purchased from Novartis-pharma France. The drug formulations for the clinical trial units consisted of 1 ml polypropylene syringes, visually indistinguishable, containing either 0.5 ml of placebo (5% glucose D solution for injection) or 1 MIU or 2 MIU of IL-2, according to randomization.

[0085] The assessments conducted during the 6-month trial period are shown in Figure 1. Vital signs, concomitant medications, and adverse events were assessed at each visit. Slow vital capacity was assessed according to current recommendations (https: / / www.encals.eu / outcome-measures).

[0086] Clinical immunophenotyping Clinical flow cytometry was performed on fresh blood within two hours of collection. Peripheral blood was collected in EDTA tubes and stained with two panels of monoclonal antibodies to determine CD3 + CD4 + CD8 + and regulatory T cells (Treg;CD4 + CD25 + CD127 low / - FoxP3 + ), NK cells (CD16 / 56 + ), B lymphocytes (CD19 + ) and monocytes (CD14 + ) were identified. B cells, NK cells, and CD3 + T cells are expressed as a percentage of all lymphocytes, and CD4 + and CD8 + T cells are CD3 + Expressed as a percentage of cells. Treg and effector T cells (Teff) (CD4 + (and calculated as the difference between Treg) is CD4 + It is expressed as a percentage of cells. Monocytes are expressed as a percentage of CD45+ white blood cells.

[0087] Mechanical immunophenotyping During each trial visit, 20 ml of blood was collected in a heparin sodium tube, and peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved. For Treg function analysis, the cryopreserved PBMCs were thawed, stained with a cocktail of monoclonal antibodies (see below), and 500 selected CD4 cells were identified. + CD25 - / lo CD127 + Effector T cells (Teff) are CD4 + CD25 high CD127 low In the presence or absence of Treg, and in various ratios (Treg:Teff 0:1, 1:2, and 1:1), 1 × 10⁻¹⁰ 3 CD19 +A suppression assay was established in a V-bottom 96-well plate by co-culturing with B cells. Cells were stimulated with PHA (4 μg / ml; Alere) and incubated at 37°C and 5% CO2 for 6 days. Growth was suppressed in the last 20 hours of co-culturing with 0.5 μCi / well [ 3 Growth was evaluated by adding thymidine (PerkinElmer) [H]. The conditions were repeated six times, and the average growth measurement (counts per minute [CPM]) was taken. Any samples with an average growth of less than 3,000 CPM from the Teff well alone were excluded. The inhibition rate in each culture was calculated using the following formula: inhibition rate = 100 - [(CPM in the presence of Treg ÷ CPM in the absence of Treg) × 100]. All time points from each individual were analyzed simultaneously.

[0088] Details of flow cytometry antibodies used in clinical and mechanistic immunophenotypic analysis. The antibodies used for clinical and mechanical immunophenotypic analysis are listed in Table 1 below.

[0089] [Table 1]

[0090] Transcriptome analysis of Treg activation markers in leukocytes RNA was isolated from patient blood using the LeukoLOCK Total RNA Isolation System (ThermoFisher). Whole blood was filtered through a LeukoLOCK filter to capture leukocytes, which were cryopreserved in RNAlater (-80°C). RNA was then extracted from these cells and its quality and quantity were measured. Subsequently, the RNA was converted to single-stranded DNA according to a standard protocol and applied to a Clariom D microarray (Affymetrix). All arrays were normalized, and changes in transcription levels (gene expression levels) were determined using the Transcriptome Analysis Console (TAC) (Affymetrix). Transcripts of treated samples were defined as significantly differentially expressed compared to placebo if the plicatus change was greater than 1.2 and increased or decreased by p = < 0.05. Some of the differentially expressed genes were associated with Treg regulation and development.

[0091] Determination of plasma cytokines Plasma cytokine analysis was performed on frozen plasma samples at -80°C. CCL2 and CCL17 plasma cytokine levels were measured using a Multiplex bead assay (Luminex Human HS Cytokine Panel-R&D Systems), and CCL18 was measured using ELISA (Quantikine ELISA kit (DCL180B, R&D Systems)).

[0092] Determination of plasma NfL Plasma concentrations of neurofilament light chains (NFLs) were assessed using enzyme-linked immunosorbent assay (ELISA). More precisely, quantitative measurement of NF-light in human serum was performed by Meso Scale Discovery (MSD). MSD GOLD plates (L45SA-1-MSD) were coated with 30 μl of supplement antibody (27016-UmanDiagnostics, 1:880 dilution) in 0.05 M carbonate buffer, pH 9.5 (w / v, 2 / 10000) overnight at 4°C. These plates were rinsed three times with 0.1% Tween 20 / 1×TBS washing buffer and blocked with 100 μl of 3% Milk / 1×TBS at room temperature (RT) for 1 hour. Before loading, standards and samples were mixed with Heteroblock (1:34 SD300 and 1:17 SD600, respectively). After washing all 25 μl of sample, standards and quality controls were loaded in two replicates. The plates were then incubated on a shaker for 1 hour (RT). After washing (3 × 5 mins), 25 μl of detection antibody (27017-UmanDiagnostics, 1:1282 dilution diluted in 1% milk washing buffer) was added to each well and incubated on a shaker (RT) for 1 hour. Next, the plates were washed (3 × 5 mins) before incubating 25 μl of streptavidin SULFO-TAG (R32AD-5, MSD) per well on a shaker (RT) for 1 hour. After washing (3 × 5 mins), 150 μl of 2x read buffer was added to each well. These plates were read on an MSD instrument. Each plate contained a calibrator (0–1000 pg / mL) and a quality control. The inter-assay variance was mostly less than 10%, and the mean intra-assay variance was less than 10%.

[0093] Plasma NFL was also analyzed using the Simmore method. Plasma NfL concentrations were measured using in-house ELISA on a single-molecule array platform (Quanterix, Lexington, MA) as previously described in detail (Gisslen M, et al. EBioMedicine. 2015 Nov 22;3:135-140). Samples were performed once using a 4-fold dilution, and this was corrected for the final output. Two QC samples were performed in two replicates at the beginning and end of each run. For QC samples with a concentration of 10.9 pg / mL, the reproducibility was 3.7% and the intermediate precision was 5.4%. For QC samples with a concentration of 168 pg / mL, the reproducibility was 2.9% and the intermediate precision was 3.4%. Measurements were performed by committee-certified laboratory technicians who were not informed of clinical data.

[0094] Outcome The primary pharmacodynamic outcome was CD4 on day 8, as measured by clinical flow cytometry. + The primary pharmacodynamic measure was the change in Tregs as a percentage of T lymphocytes. Secondary pharmacodynamics included the number and percentage of Tregs at all time points, expressed as increasing area under the curve (iAUC), and plasma levels of CCL2 and neurofilamentous light chains (NFLs) as markers of disease activity. Exploratory analyses included measurement of leukocyte population number and frequency by flow cytometry, as well as Treg cell function tests. Monocyte polarization in response to treatment was investigated through analysis of chemokine production profiles (CCL17 and CCL18). Safety was assessed through a systematic check of predetermined events (injection site reactions, flu-like symptoms, malaise, gastrointestinal symptoms, allergic reactions), abnormal vital signs, ECG results, chest X-rays, laboratory tests, and all adverse events reported during the study. As a secondary clinical outcome, changes in clinical function (ALSFRS-R and slow vital capacity - SVC) over time were evaluated throughout the study.

[0095] Sample size Previous data (Thonhoff JR et al. Neurol Neuroimmunol Neuroinflammation 2018; 5: e465) showed that 6 patients in each group achieved 88% power to detect a 60% increase in Tregs with pα=0.05 (Mann-Whitney test). Since the impact on disease activity is also a major concern, and based on previous ALS trials suggesting that this figure may be sufficient to observe the impact on plasma NFLs (Gaiottino J, et al. PloS One 2013; 8: e75091), we retained 12 patients in each group to achieve 80% power with pα(two-sided)=0.05 to detect a 40% decrease in plasma NFLs at 3 and 6 months (Mann-Whitney test).

[0096] Preliminary safety data included the first 12 patients and was assessed once after the completion of the first treatment cycle and then on day 8 (primary efficacy). This report was kept confidential until the end of the trial.

[0097] statistical analysis Categorical variables are described as absolute and relative frequencies. Quantitative variables are summarized by mean, median, standard deviation, and range. Flow cytometry parameters were analyzed as changes from baseline at D8 (primary criterion) and D64, i.e., the absolute difference between each time point and baseline D1. The overall change in immune cells for the time-series measurements in the first cycle (D1, D8, D29) and the third cycle (D57, D64, D85) was summarized as the area under the incremental time-normalized curve (iAUC, using the trapezoidal rule) minus the D1 or D57 value, respectively. The trough value iAUCt was calculated using the values ​​measured at D1, D29, D57, and D85 minus D1. Eosinophil counts were analyzed in the same way as the cytometry parameters. ALSFRS-R measurements were summarized by the regression gradient from D1 to D85. For SVC and NFL, the absolute difference between D85 and baseline D1 was analyzed. For CCL2, CCL17, and CCL18, baseline normalized values ​​at D64 were analyzed.

[0098] The Kruskal-Wallis test was used to test for differences between the three test groups. If a significant difference was detected (p<0.05), the Mann-Whitney test was used for pairwise comparisons to identify the detected differences.

[0099] We analyzed the dose-response relationship of the summary measure using a one-way ANOVA (linear trend test) to assess whether the change in the outcome variable consistently increased across the entire dose level.

[0100] result Implementation of the test Between September 21 and December 4, 2015, 39 patients were screened. Three were excluded, and 36 were randomized (Figure 1). After the selection of 12 patients and one cycle of treatment, an independent data safety monitoring committee found no safety concerns and continued selection. With one exception (see Figure 1), all randomized patients completed three cycles of treatment over three months and underwent a three-month post-treatment follow-up. All 36 randomized patients were included in both the treatment-targeted population and the safety population (Figure 1). Of the maximum 252 possible clinical and laboratory measures of the trial's primary and secondary outcomes, all but one were available for analysis (Figure 1).

[0101] Patient characteristics and medical history Although baseline differences were not statistically significant, the 2MIU group had a higher proportion of women compared to men and exhibited slightly more severe disease characteristics (see Table 2 below for clinical parameters, Table 4 (column D1) for immune cell parameters expressed as absolute numbers at baseline, Table 5 for immune cell parameters expressed as frequencies, and Table 6 for neurofilaments). No significant imbalances affecting the outcomes were observed between the groups.

[0102] [Table 2]

[0103] Safety and tolerability Clinical tolerability was satisfactory for both doses of IL-2. No serious drug-related adverse events (SAEs) occurred throughout the entire follow-up period (D1-D169), and most non-serious drug-related adverse events (NSAEs) were transient and mild to moderate (see Table 3 below).

[0104] [Table 3]

[0105] During the treatment period (D1-D85), the frequency of patients experiencing NSAEs during the cycle was higher in the IL-2 group compared to the placebo group (n=3, 25.0%), with n=11 (91.7%) and n=12 (100%) for the 1 MIU / day doses, respectively (Table 3). Local reactions at the injection site (erythema, pain) were the most common NSAEs, occurring at a similar frequency in both active treatment groups (all patients except one who experienced an injection site reaction), although only one patient in the placebo group reported such an event. Influenza-like symptoms (including muscle pain, chills, fever, and arthralgia), characteristic of IL-2 treatment (Hartemann A et al. Lancet Diabetes Endocrinol 2013; 1: 295-305), were reported only at the 2 MIU / day dose (25%). One patient in the 2 MIU / day group discontinued treatment after 2 days of treatment in the third cycle due to severe flu-like symptoms that did not respond to orthodontic treatment (see Figure 1).

[0106] Outside of the treatment cycle, only one case of nausea / vomiting was attributed to the treatment, among other adverse events. One patient with a history of prostatic adenoma (1 MIU / day group) developed severe urinary retention 10 days after the last dose and required hospitalization for prostate surgery. Other events were associated with ALS disease or other pre-existing conditions.

[0107] In the 2 MIU / day group, no abnormalities were observed in routine laboratory parameters, except for one patient with influenza-like symptoms who showed elevated CRP on day 8, and another patient who showed elevated CRP on day 57 in connection with a viral infection. Regarding hematological parameters, no significant changes were observed in the 2 MIU / day group, except for a significant increase in eosinophil count on days 8 and 64 compared to placebo (see Tables 4 and 5 below). Less significant changes were observed in the 1 MIU / day group, and were only significant on day 64. In the 2 MIU / day group, three patients showed an increase of 1.5 × 10⁶. 9 Although there was an increase in eosinophils exceeding 1 / l, the patient remained asymptomatic, and all counts were close to the baseline values ​​for D169 (no significant difference between groups), and all were within the normal range.

[0108] Table 4

[0109] Table 5

[0110] Effects of Ld IL-2 on peripheral blood mononuclear cells The a priori-defined primary pharmacodynamic outcome of increased frequency of Tregs as a percentage of CD4+ T lymphocytes on day 8 was significantly higher in both the 2MIU and 1MIU groups (p<0.0001 by Mann-Whitney U test) (compared to placebo (mean [SD]: -0.5% [1.2]), 2MIU: mean [SD]: +6.2% [2.2]; 1MIU: mean [SD]: +3.9% [1.2], see Table 4 and Figures 2A-B above). The effect size was large in both IL-2 groups: 2MIU ES=3.7 (IC95%: 2.3-4.9); 1MIU ES=3.5 (IC95%: 2.1-4.6). Furthermore, when examining the change in Treg frequency from baseline, clear differences were observed in all IL-2 recipients (increase range 23–139%), with no overlap with the placebo group (change range -51–9%) (Figure 2B). Secondary outcomes for Tregs revealed a significant increase in frequency and absolute number during subsequent treatment cycles compared to baseline and placebo (Figures 2A–D, Tables 4 and 5 above). In addition, the peak during cycle 3 was higher than that observed during cycle 1, suggesting that there may be a cumulative residual effect from consecutive treatment cycles. This suggestion is further supported by significantly higher iAUC trough Treg levels (measurement of residual Treg changes before starting a new cycle) in the IL2 group compared to placebo (Figures 2E–F, Tables 4 and 5 above). In general, the 2MIU group showed higher Treg peak and trough levels than the 1MIU group. Ld IL2 also resulted in moderate increases in NK cell frequency and number in both IL-2 groups (up to a 1.7-fold increase in the 2MIU group on D64), an increase in CD8 T cell number in both IL-2 groups (up to a 1.4-fold increase in the 2MIU group on D64), an increase in CD4 Teff number in both IL-2 groups (up to a 1.6-fold increase in the 2MIU group on D64), and a decrease in monocyte frequency in the 2MIU group on D64 (all data are shown in Tables 4 and 5 above).

[0111] Exploratory analyses of the phenotype and function of Tregs were performed using cryopreserved PBMCs, focusing primarily on baseline and post-treatment (day 1 and day 64) responses. The inventors found a good correlation between the frequency of Tregs defined in blood by clinical cytometry and the selection of Tregs from cryopreserved PBMCs (R 2(=0.91, p<0.0001). Similar to the results with fresh blood, analysis of cryopreserved PBMCs revealed a significant increase in the frequency of Tregs after 3 cycles of IL-2 treatment (Figure 3A-C). Furthermore, batch analysis across all time points from a single individual on the same day allowed for a direct comparison of CD25 expression before and after treatment, revealing a dose-dependent increase in expression on Tregs in response to treatment (median and range of CD25 MFI for D1 vs. D64): 2 MIU = 4651, range 2892-5886 and 9015, range 4220-14446, p=0.002; 1 MIU = 4230, range 3388-5423 and 7778, range 5564-9037, p=0.001; placebo = 4105, range 2992-5656 and 3867, range 1923-5669, p=0.83; Figures 4A-C). A small but significant increase in CD25 expression was also observed in effector T cells (median and range of CD25 MFI for D1 vs. D64: 2 MIU = 426, range 116–897 pairs, 528, range 122–948, p = 0.02; 1 MIU = 336, range 132–478 pairs, 362, range 124–577, p = 0.001; placebo = 251, range 195–919 pairs, 263, range 158–881, p = 0.24, Figures 3D–F). Treg function was evaluated by in vitro co-culture assays using effector T cells from the corresponding time points as responding cells. In cultures lacking Tregs, no effect of IL-2 administration on the proliferation of responding T cells was observed (Figures 3G–I). However, the inventors observed an increase in Treg inhibitory function after three cycles of IL-2 treatment, reaching statistical significance at a dose of 1 MIU (median % inhibition rate between D1 and D64, range: 2 MIU = 53%, range 18-88% vs. 76%, range 19-91%, p=0.06; 1 MIU = 65%, range 23-84% vs. 80%, range 36-97%, p=0.001; Figures 4D-E). In contrast, a slight decrease in Treg function was observed in the placebo group (median % inhibition rate between d0 and d64, range: placebo = 73%, range 53-95%, range 59%, range 32-97%, p=0.07; Figure 4F).When comparing the percentage change in Treg inhibitory function over the treatment period (compared to baseline inhibition), a significant difference was observed between the two groups treated with IL-2 compared to placebo (2 MIU vs. placebo, p=0.008; 1 MIU vs. placebo, p=0.005; Figure 4G). Furthermore, the inventors evaluated the relationship between changes in Treg frequency and CD25 expression in response to each individual's treatment and changes in Treg inhibitory function. The inventors found a very significant correlation between these measurements and individual clustering based on treatment group (Treg frequency, p=0.0001, R. 2 =0.42;Treg CD25 mfi, p=0.001, R 2 =0.33 (Figure 4H~I).

[0112] Effect of Ld IL-2 on the leukocyte transcriptome Figure 5 shows transcriptome data obtained from leukocytes on day 64, demonstrating increased gene expression of the following Treg activation markers (FOXP3, CTLA4, IKZF2, and IL2RA) in leukocytes. When patients were treated with 1 MIU or 2 MIU, dose-dependent increases in the expression of each of these genes were observed after 3 cycles of low-dose IL-2 therapy on day 65, compared to the untreated (placebo) group.

[0113] Effect of Ld IL-2 on plasma cytokine concentration The inventors evaluated plasma levels of cytokines / chemokines previously reported to be elevated in individuals with ALS (CCL2) or those associated with macrophage / microglia polarization (CCL17 and CCL18). After the third treatment cycle (D64), the inventors observed differences in plasma levels of CCL2 among the three groups (p=0.005, Figure 6A), with both effective dose groups showing dose-dependent changes in CCL2 levels, which were significantly reduced at the 2 MIU dose compared to placebo (p=0.005), but not statistically significant at the 1 MIU dose (p=0.06). The inventors also observed differences between the treatment groups in D64 in CCL17 and CCL18 (p=0.00001 and 0.0028, respectively, Figures 6B-C), with increases observed in both treatment groups compared to placebo (CCL17: 2MIU p=0.0001 and 1MIU p=0.0138; CCL18: 2MIU p=0.0012 and 1MIU p=0.0094). These results suggest that ld IL-2 treatment was associated with a decrease in the ALS-related inflammatory marker CCL2 and the subsequent shift of monocytes to the M2 phenotype.

[0114] The effect of Ld IL-2 on disease progression, as assessed by changes in ALSFRS-R, slow vital capacity, and plasma NFL over time (D1-D85). There were no significant differences among the three groups in terms of changes over time in ALSFRS-R score (Kruskal-Wallis = 4.25, p=NS), slow vital capacity (Kruskal-Wallis = 1.07, p=NS), or plasma NFL levels (Kruskal-Wallis = 0.34, p=NS). Similar results were obtained for NFL by reanalysis of plasma samples using the SIMOA approach (Kruskal-Wallis = 2.44, p=NS). However, in the entire population, none of these parameters showed a statistically significant change over the 3-month treatment period - ALSFRS-R (points / month) mean gradient [95% CI] = -0.8 [-2.4, +0.8]; SVC (predicted percent) mean change from d1 at d85 [95% CI] = -2.2 [-23.4, +19.0]; NFL-Elisa change (pg / ml) mean change at d85 from d1 [95% CI] = +0.63 [-62.5, +63.7]; NFL-SIMOA change (pg / ml) mean change at d85 from d1 [95% CI] = -1.64 [-26.25, +22.97] - these parameters were less sensitive to change over the 3-month period.

[0115] Furthermore, the large variability in baseline plasma NFL levels across all treatment groups (see Table 6 below) is likely to hinder significant observations regarding the selected sample size.

[0116] [Table 6]

[0117] Despite no significant observations, there is a trend in changes in plasma levels at D85, with plasma NFL levels tending to increase in the placebo group (suggesting some disease progression), but not in ALS patients treated with 1 or 2 MIU doses (see Figure 7).

[0118] conclusion First, our results show that two doses of ld IL-2 were clinically well tolerated in ALS patients over three cycles, and no further safety issues were detected after discontinuation of treatment. In line with previous reports (Koreth J et al. N Engl J Med 2011; 365: 2055-66; Saadoun D et al. N Engl J Med 2011; 365: 2067-77; Hartemann A et al. Lancet Diabetes Endocrinol 2013; 1: 295-305; Castela E et al. JAMA Dermatol 2014; 150: 748-51), our findings clearly demonstrate that ALS patients, who are particularly vulnerable to therapeutic toxicity, can tolerate repeated treatment cycles with ld IL-2. The safety of ld IL-2 is further supported by the absence of significant worsening of ALSFRS-R or SVC over the treatment period, both among all groups and within the group including placebo.

[0119] Secondly, the inventors observed a significant dose-dependent increase in both the absolute number and relative frequency of Treg cells in both the 2MIU and 1MIU groups. Comparing these results with those obtained in a double-blind, randomized clinical trial in patients with type 1 diabetes (T1D; Hartemann A et al. Lancet Diabetes Endocrinol 2013; 1: 295-305), the inventors found that the magnitude of the Treg response to ld IL-2 was similar, with the percentage of Treg cells (Treg frequency) in CD4 cells increasing by approximately 1.5 times after treatment with 1MIU for 5 days.

[0120] Notably, all individuals in both groups undergoing active treatment showed an increase in the number and frequency of Tregs. This suggests that Tregs in ALS patients may have impaired endogenous responsiveness to IL-2 (Thonhoff JR et al. Neurol Neuroimmunol Neuroinflammation 2018), potentially leading to their unresponsiveness to ld IL-2 treatment. However, in this cohort of ALS patients, the inventors found no evidence of an essential impairment in Treg responsiveness to ld IL-2. This is highly significant because, in contrast to what was suggested by Thonhoff et al., it means that Tregs in ALS patients can be significantly enlarged simply by administering ld IL-2 without requiring prior isolation, ex vivo amplification, and reinfusion of patient Tregs. Due to its simplicity and much lower cost, the proposed treatment based on simple administration of ld IL-2 could be made available to a much larger number of ALS patients.

[0121] Thirdly, we demonstrated that the increase in Treg response persisted for four weeks after a five-day treatment cycle. This is important because a sustained increase in Treg levels is likely necessary for optimal clinical efficacy. We selected a treatment schedule based on a T1D study (Hartemann A et al. Lancet Diabetes Endocrinol 2013; 1: 295-305) and confirmed in this study that the number and frequency of Tregs were significantly amplified at trough levels (i.e., before the start of the next treatment cycle), and that this amplification increased with repeated cycles. However, it remains unclear whether our treatment schedule is most effective in controlling neuroinflammation in ALS, or whether a different treatment schedule (e.g., more frequent administration of ld IL-2) would be more therapeutically beneficial.

[0122] In summary, our findings suggest that there was no loss of sensitivity to ld IL-2 with repeated administration in this ALS cohort. Consistent with this, and in line with other reports (Todd JA, et al. PLoS Med. 2016 Oct 11;13(10):e1002139; Hirakawa M, et al. JCI Insight. 2016 Nov 3;1(18):e89278), we found that ld IL-2 leads to a preferential increase in CD25 expression on Treg cells. This may increase the sensitivity of Treg cells to both administered IL-2 and endogenous IL-2, potentially enhancing and sustaining the therapeutic effect.

[0123] To understand the relationship between changes in Treg number and therapeutic effect, the inventors evaluated Treg function before and after administration of ld IL-2. In fact, in ALS patients, Tregs not only decreased in number but also showed decreased FOXP3 expression levels (Henkel JS et al. EMBO Mol Med 2013; 5: 64-79) and dysfunction (decreased immunosuppression, Beers DR et al. JCI Insight. 2017; 2(5): e89530), and both decreased FOXP3 levels and decreased immunosuppressive function correlated with disease progression (Beers DR et al. JCI Insight. 2017; 2(5): e89530; Thonhoff JR et al. Curr Opin Neurol 2018; 31: 635-9). The inventors used an autologous co-culture assay to measure the ability of Tregs isolated by FACS to suppress the proliferation of CD4 effector T cells. Our results, overall, indicate that treatment with ld IL-2 leads to an increase in both Treg frequency and Treg inhibitory function. This improvement in Treg function was highly significant in the 1 MIU group, with all individuals showing increased function. In the 2 MIU group, only a significant trend (p=0.06) was observed due to increased variability in responses between individuals (Figure 4). The dual efficacy of ld IL-2 is demonstrated by the highly significant correlation between increased Treg frequency and function, although we also observed individual variability in treatment responses, with some treated individuals showing significant changes in either Treg frequency or Treg function, and others showing increases in both. Conversely, individuals in the placebo group tended to lose either Treg frequency or function within the same timeframe.

[0124] Furthermore, transcriptome data obtained from D64 leukocytes show a dose-dependent increase in the gene expression of the following Treg activation markers (FOXP3, CTLA4, IKZF2, and IL2RA) in leukocytes. These results further reinforce our findings that ld IL2 administration enhances Treg immunosuppressive function in ALS patients. Moreover, since Treg FOXP3 expression levels have been shown to correlate with disease progression (Beers DR et al. JCI Insight. 2017;2(5):e89530; Thonhoff JR et al. Curr Opin Neurol 2018; 31: 635-9), these results further support the therapeutic effect of low-dose human IL-2 administration in ALS patients.

[0125] Regarding the effect of ld IL-2 on blood markers of ALS disease activity, we found a significant and dose-dependent decrease in plasma CCL2 concentration. CCL2 is a small chemokine belonging to the CC subfamily that transmits signals via chemokine receptor-2 (CCR2) and guides circulating leukocytes to neuroinflammatory sites. CCL2 knockout mice showed reduced circulating leukocyte infiltration into neuroinflammation and disease resistance in autoimmune and inflammatory models, suggesting that this pathway plays a role in promoting pathogenesis. Furthermore, elevated CCL2 expression levels have been observed in the nerve tissue of ALS patients, and expression is associated with macrophage and microglia infiltration and activation (Henkel JS, et al. Ann Neurol. 2004 Feb;55(2):221-35; Baron P, et al. Muscle Nerve. 2005 Oct;32(4):541-4). CCL2 levels in body fluids are elevated in ALS patients (Martinez HR, et al. Neurologia. 2017 Oct 10. pii: S0213-4853(17)30280-3; Gille B et al. J Neurol Neurosurg Psychiatry. 2019 Dec; 90(12):1338-1346, Gupta PK, et al. J Neuroinflammation. 2011 May 13;8:47; Kuhle J, et al. Eur J Neurol. 2009 Jun;16(6):771-4; Baron P, et al. Muscle Nerve. 2005 Oct;32(4):541-4), disease score (Nagata T, et al. Neurol Res. 2007 Dec;29(8):772-6), and survival (Gille B et al. J A correlation has been shown with Neurol Neurosurg Psychiatry. 2019 Dec;90(12):1338-1346), indicating that CCL2 is a useful biomarker for disease activity. Therefore, the finding of a significant and dose-dependent decrease in plasma CCL2 concentration in IMODALS further supports the therapeutic effect of low-dose human IL-2 administration in ALS patients.

[0126] Interestingly, ld IL-2 treatment was also associated with significant increases in plasma levels of CCL17 and CCL18, coinciding with a shift in macrophage / microglia polarization to an anti-inflammatory M2-like phenotype (Katakura T, et al. J Immunol. 2004 Feb 1;172(3):1407-13; Schraufstatter IU, et al. Immunology. 2012 Apr;135(4):287-98). Overall, the changes in macrophage activation inflammatory biomarkers and polarization are consistent with the role of ld IL-2 in regulating the activation of cytopathic microglia associated with ALS progression.

[0127] Tregs are known to affect macrophage activation and polarization (primarily leading to an M2-like phenotype) (Tiemessen MM, et al. Proc Natl Acad Sci US A. 2007 Dec 4;104(49):19446-51), and it has emerged that these changes may be a direct result of the increased number or functional capacity of Tregs induced by ld IL-2 therapy. However, since monocyte-macrophage cells express functional IL-2 receptors (Ohashi Y, et al. J Immunol. 1989 Dec 1;143(11):3548-55; Wahl SM, et al. J Immunol. 1987 Aug 15;139(4):1342-7), and CD25 (IL2RA) expression increases in inflammatory states (Espinoza-Delgado I, et al. J Immunol. 1992 Nov 1;149(9):2961-8; Dendrou CA, et al. Nat Genet. 2009 Sep;41(9):1011-5), the possibility has emerged that macrophage / microglia polarization may also occur as a direct result of ld IL-2 acting directly on these cells.

[0128] Finally, no statistically significant change in plasma neurofilament light chain (NFL) levels was observed with respect to treatment. Based on previously published data (Gaiottino J, et al. PloS One 2013; 8: e75091), the inventors estimated that a treatment effect should be detectable in a relatively small number of patients in each group. However, for this randomized (and strictly blinded) trial, post-hoc power analysis suggests that this was significantly underestimated, and a much larger sample size was needed to demonstrate the changes observed in plasma NFL levels, especially since there was significant variability in plasma NFL levels in all treatment groups at baseline (Table 6 above). Nevertheless, despite the lack of statistical significance, there is a clear trend suggesting that plasma NFL levels increased over time in the placebo group compared to the treatment group, which is consistent with the reduction in disease activity associated with ld IL2 treatment.

[0129] In conclusion, this study demonstrates that ld-IL-2 is safe in ALS patients over a 3-month cycle. Furthermore, the inventors provide clear evidence of in vivo amplification of Treg number, frequency, and function by ld-IL-2. Since Treg function correlates with disease progression (Beers DR et al. JCI Insight. 2017;2(5):e89530; Thonhoff JR et al. Curr Opin Neurol 2018; 31: 635-9), these results support the therapeutic effect of ld-IL-2 in ALS patients. Importantly, given previous findings regarding the correlation between elevated CCL2 in plasma and CSF of ALS patients and disease scores (Nagata T, et al. Neurol Res. 2007 Dec;29(8):772-6) and survival rates (Gille B et al. J Neurol Neurosurg Psychiatry. 2019 Dec;90(12):1338-1346), our findings that plasma CCL2 decreases in a dose-dependent manner in response to ld IL-2 treatment further support the therapeutic effect of ld IL-2 in ALS patients. As a result, a Phase 2 / 3 trial based on these findings is underway (MIROCALS, ClinicalTrials.gov NCT03039673, see Example 2).

[0130] Example 2: MIROCALS: Modification of the immune response and ALS outcomes (MIROCALS) Following the extremely positive data obtained in the IMODALS trial, particularly regarding a significant decrease in plasma concentrations of CCL2, a marker of disease progression, and a shift in macrophage polarization from an inflammatory M1 phenotype to an anti-inflammatory and repairing M2 phenotype, a novel Phase II trial called "MIROCALS" was initiated to confirm the positive effects of low-dose human IL-2 on ALS.

[0131] Patients and Methods The "Modifying Immune Response and Outcomes in ALS" project (MIROCALS) will test, through a proof-of-concept / proof-of-mechanism (PoC / PoM) trial, the hypothesis that ld IL-2 therapy leads to a reduction in the rate of neuronal damage in ALS patients, as measured by early changes in CSF neurofilament and CCL2 levels, and that these early changes predict long-term clinical effects, as assessed by survival over an 18-month treatment period.

[0132] design: This is a double-blind, randomized, stratified (country and site of onset) placebo-controlled, parallel-group trial of low-dose IL-2 at 2 MIU / day for 5 days every 4 weeks for 18 months. Prior to the trial, there is a 3-month induction period to establish the safety and stability of riluzole treatment.

[0133] Treatment: All treatment packages, consisting of five 1 ml pre-filled polypropylene syringes containing either 0.6 ml of 2 MIU aldezleukin solution or 0.6 ml of 5% glucose solution for injection, are prepared, labeled, and packaged in a central pharmacy under sterile and temperature-controlled conditions. In cases of poor tolerability, the PI may prescribe flexible dose reductions of 1 MIU (0.3 ml) or 0.5 MIU (0.15 ml) to manage patient compliance.

[0134] Primary evaluation criteria: Long-term survival over an 18-month treatment period, and early changes in CSF-pNFH and CCL2 levels (after 4 months of treatment).

[0135] Secondary evaluation items: Long-term safety of ld IL-2 therapy over 8 months of treatment, efficacy against functional decline (ALSFRS, vital capacity), chemokine markers of immune inflammation (CSF and blood), immunocytoassay (blood), and pNFH levels (CSF and blood).

[0136] Supplementary examination:Novel biomolecular targets for drug intervention based on genomics and transcriptomics profiling of ld IL-2 responsive and non-responsive patients.

[0137] measurement: Major laboratory measurements (CSF, blood pNFH and CCL2, and hematopoietic immunoassays) are performed in the central laboratory under GCLP conditions. Additional supporting immunoinflammatory markers and omics studies are conducted at the academic research institute.

[0138] Patient selection criteria: Selection of induction period: De novo patients diagnosed with El Escorial, likely or likely and supported by laboratory findings, or definite ALS, disease duration ≤ 24 months, lung capacity ≥ 70% of normal, no prior or current riluzole treatment, and signed informed consent.

[0139] RCT duration: The criteria are the same except that the disease duration ≤ 27 months and "no previous or current riluzole treatment" has been replaced with "stable after 3 months of riluzole treatment."

[0140] Patient exclusion criteria: Contraindications include: lumbar puncture; other life-threatening conditions; other conditions that interfere with functional assessment; cancer within the past five years (excluding stable non-metastatic basal cell carcinoma or cervical carcinoma in situ); severe heart or lung disease; documented autoimmune disorders (excluding asymptomatic Hashimoto's thyroiditis); unprotected women of childbearing age who are pregnant or lactating; and clinically significant laboratory abnormalities (excluding cholesterol, triglycerides, and glucose).

[0141] Statistical analysis:In the primary efficacy analysis, (i) stratified log-rank tests will be used to compare treatment groups for survival according to (ii) adjustments for candidate prognostic factors including age, vital capacity, and ALSFRS score (Cox model analysis). Changes in pNFH and CCL2 from randomization to M4, and immunocytometric parameters will be analyzed using analysis of variance / covariance (ANOVA / ANCOVA). Analysis of repeated measures of functions (ALSFRS and SVC) will be performed using co-rank analysis of beneficially censored data.

[0142] Number of patients: To achieve a power of >0.80 with a pα (two-sided) of 0.05, 216 patients were randomized into groups of 108, detecting an absolute difference in mortality of 17% at 18 months (placebo expected survival rate: 0.65 vs. 0.82 in the ld IL-2 group), representing a 54% reduction in mortality risk (RR ld IL-2 / PLA = 0.46). Considering a 10% reduction from eligible selection to randomization, approximately 240 patients should be screened.

[0143] Expected patient or public health benefits Based on the results obtained in IMODALS, particularly the positive effect of ldIL-2 on plasma concentrations of disease activity markers (i.e., reductions in CCL2 and NFL), as well as changes in immunoinflammatory parameters, including improved Treg suppression function and a shift from an inflammatory M1 phenotype to an anti-inflammatory and repairing M2 phenotype of macrophage polarization, treatment including repeated cycles of low-dose human IL-2 subcutaneous injection is expected to significantly improve survival rates and reduce the rate of functional decline in ALS patients.

[0144] Due to limited knowledge of the etiology of ALS and the lack of predictive preclinical models, drug trials for ALS pursue two objectives: (i) testing clinical efficacy, and (ii) testing pathogenicity hypotheses that justify drug evaluation. However, “standard” trials have no way of knowing whether drug failure is due to a flawed hypothesis, insufficient drug involvement in the target pathway, or an ineffective dose. By including biomarkers that quantify both pharmacodynamic and disease activity responses to drugs in the design, and examining their surrogates in survival rates for hard clinical endpoints, a high level of evidence and supporting PoC / PoM evidence for the clinical efficacy of ld IL-2 should be achieved. Therefore, combined with the high quality settings of GCP / GCLP, this trial should provide optimal regulatory approval for clinical outcomes. Furthermore, while this design determines the strength of the evidence for the pathogenicity hypothesis being tested, the omics approach provides a deeper understanding of drug effects, the pathways involved, and their interaction with the disease process, ultimately paving the way for new drug development and even advances in treatment.

Claims

1. A pharmaceutical composition comprising human interleukin-2 (IL-2) for use in the treatment of amyotrophic lateral sclerosis in human subjects, wherein each dose of human IL-2 administered to the subject is 0.1 × 10⁻⁶ ６ ~3 x 10 ６ A pharmaceutical composition comprising international units (IU), wherein the treatment does not involve the administration of regulatory T cells (Treg) to the subject, and the human subject is administered by repeated cycles of once-daily subcutaneous injections with periods without IL-2 injection, or by continuous once-daily subcutaneous administration of IL-2.

2. 0.1 × 10 ６ ~3 x 10 ６ The pharmaceutical composition according to claim 1, wherein the subject is administered a repeated cycle of once-daily injections of IU of human IL-2 for 3 to 7 consecutive days.

3. Each cycle is 0.1 × 10 ６ ~3 x 10 ６ The pharmaceutical composition according to claim 2, comprising subcutaneous injection of IU of IL-2 once daily for five consecutive days.

4. The aforementioned injection 1 × 10 ６ ~3 x 10 ６ The pharmaceutical composition according to claim 3, wherein IU is IL-2.

5. The injection is 2 × 10 ６ IU of human IL-2, and the pharmaceutical composition according to claim 4.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the cycle is administered every 2 to 6 weeks.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the cycle is administered every two, three, or four weeks.

8. The pharmaceutical composition according to claim 1, wherein IL-2 is administered continuously according to a metronome schedule.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the treatment is administered over the lifetime of the subject or until an unacceptable drug-related serious adverse event occurs.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the subject is not administered an anti-human IL-2 antibody.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the treatment does not involve the administration of rapamycin to the subject or the administration of any other effector T cell (Teff) inhibitor.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the treatment further comprises administering riluzole to the subject.

13. The pharmaceutical composition according to claim 12, wherein riluzole is administered orally in a daily dose of 50 mg to 100 mg, and taken in two equal doses of 25 mg to 50 mg at 12-hour intervals.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the human IL-2 is recombinant human IL-2.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the human IL-2 is aldesleukin.

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