Pharmaceutical formulations and dosing regimens for the treatment of amyotrophic lateral sclerosis - Patent Application 20070122999

Abstract

Pharmaceutical formulations and dosing regimens for antibody-based treatment of amyotrophic lateral sclerosis (ALS) are provided.

Description

[Technical Field]

[0001] The present invention relates to therapies for amyotrophic lateral sclerosis (ALS), and in particular to dosing regimens and formulations of antibodies that bind to misfolded forms of superoxide dismutase 1 (SOD1) for use in the treatment of ALS. [Background technology]

[0002] Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that primarily affects motor neurons in the motor cortex and spinal tract, resulting in severe disability and ultimately death. Approximately 90% of ALS cases have a sporadic origin (sporadic ALS, sALS), while the remaining 10% result from inherited genetic mutations (familial ALS, fALS). The clinical symptoms of these two groups of ALS are indistinguishable, suggesting the convergence of various pathways into one distinct outcome: degeneration of motor neurons and deterioration of muscle function, which results in varying amounts of weakness and spasticity in limb, bulbar, and respiratory muscles. In most patients, limb onset, also known as spinal onset, begins with asymmetric, painless limb weakness; however, in approximately 20% of patients, weakness may manifest as bulbar onset, manifesting as dysphagia or dysphagia. Patients with bulbar onset have a worse prognosis. Patients develop progressive disabilities that limit ambulation, communication, nutrition, and independence. Deterioration of respiratory function can lead to respiratory failure and failure, which is often the cause of death, if permanent mechanical ventilation is not selected (Paulukonis et al., PLoS One 10(2015), e0131965.2015, Poulin-Briere et al., Front. Neurosci. 15(2021), 790114).

[0003] Although several efforts have been made to decipher the mechanisms underlying ALS pathogenesis, its etiology remains unknown, and various mechanisms and cellular targets have been suggested. There is currently no cure for ALS. The only currently approved therapies for ALS worldwide are the oral medications riluzole, Rilutek®, and edaravone, Radicava®, which extend survival by 2–3 months (Miller et al., Cochrane Database Syst. Rev. 3 (2012), CD001447). These therapies have limited impact on survival and disease progression, showing only a modest slowing of decline in the ALS Functional Rating Scale (ALSFRS) in a subset of patients. More recently, the combination of sodium phenylbutyrate and tauroursodeoxycholic acid (PB and TUDCA), Relyvrio™, and tofersen, QALSODY™, have been approved for the treatment of ALS. However, no therapies are available that target wt-SOD1 to reverse SOD1 pathology.

[0004] Thus, there is an urgent need for therapies for ALS, preferably therapeutic interventions with disease-modifying effects. Summary of the Invention [Means for solving the problem]

[0005] A solution to the problem of providing a therapy for amyotrophic lateral sclerosis (ALS) is provided by the embodiments characterized in the claims and the description and illustrated in the examples.

[0006] Thus, in a first aspect, the present invention generally relates to a dosing regimen for the treatment of ALS with a recombinant antibody that binds to superoxide dismutase 1 (SOD1), i.e., an anti-SOD1 antibody, in which the antibody is administered to a subject, i.e., a human patient, in high doses, preferably by intravenous administration. In a related aspect, the present invention relates to a pharmaceutical formulation of the antibody that is particularly suitable for such treatment regimens, particularly for intravenous administration. More specifically, the present invention relates to a recombinant antibody that selectively binds to the misfolded, and preferably aggregated, form of SOD1 for use in treating ALS by administering to a patient in need thereof a dose of the antibody of 1000 mg to 10,000 mg, preferably 1000 mg to 5000 mg, and most preferably about 2500 mg. Administration is preferably carried out once every 1 to 4 weeks, more preferably once every 2 to 4 weeks, and most preferably once every 3 weeks. The pharmaceutical formulation of the present invention is a stable liquid aqueous formulation of an anti-SOD1 antibody, characterized by being a liquid aqueous formulation containing the antibody at a concentration of about 10 to 50 mg / mL, preferably about 20 mg / mL, in an L-histidine / L-histidine monohydrochloride buffer solution having a pH of about 6.0.

[0007] Various therapeutic strategies and targets, such as kinase inhibitors, gene therapy, antisense oligonucleotides (ASOs), antibody-based intervention, and gene editing using CRISPR / Cas9 technology, have been tested in animal models with promising results; for a review, see, e.g., Poulin-Briere et al., (2021), supra. In this context, therapeutic interventions based on passive immunization have also attracted attention for ALS, and as summarized by Poulin-Briere et al., approximately 15 antibody targets are currently being investigated.

[0008] The present invention is based on (i) selecting the appropriate target and class of antibody, (ii) developing a drug formulation in which the antibody is stable and can be tolerated by patients even at high antibody doses, and (iii) establishing a treatment regimen that offers the best therapeutic prospects and is at the same time well tolerated by patients, conditions that antibody-based approaches often cannot meet.

[0009] Regarding the first point, a recombinant human monoclonal antibody specific for human misfolded SOD1 (mSOD1), designated AP-101, has been used and investigated, which was originally disclosed in International Application WO 2012 / 080518 A1 and further characterized in Maier et al., Sci. Transl. Med. 10. (2018) doi:10.1126 / scitranslmed.aah3924. In addition to its disease-modifying properties, recent experiments suggest high specificity of anti-SOD1 antibodies for mSOD1 in vivo, as demonstrated by PET imaging.

[0010] Second, pharmaceutical formulations of anti-SOD1 antibodies have been developed that provide sufficient stability of the antibodies and have proven suitable for long-term infusion of high doses of antibodies; see Examples 3-6.

[0011] Third, an appropriate dosing regimen was established based on a multicenter, open-label, single-ascending-dose (SAD) study in patients with ALS using an open-label, oncology-style 3+3 design (ClinicalTrials.gov Identifier: NCT03981536), in which AP-101 was administered to patients in escalating dose steps of 100, 500, or 2500 mg over 1 hour by intravenous (IV) infusion, and specific time points for readout appeared essential to evaluate the safety, tolerability, and pharmacokinetics (PK) of antibody AP-101; see Examples 1 and 2. The feasibility of administering AP-101 at high doses several weeks apart without producing toxicity or undesired effects was evaluated in a 1-month study in cynomolgus monkeys, which consisted of two IV injections, 2 weeks apart (Study 8384444). AP-101 binds to human and monkey SOD-1, making monkeys a relevant toxicology species; AP-101 does not bind to rodent SOD-1. No AP-101-related effects on toxicology or safety pharmacology parameters occurred at or below the highest dose tested, 400 mg / kg (24,000 mg in a 60 kg human). Furthermore, the half-life of AP-101 from monkey toxicity studies was estimated to be 19.5 days. Based on the PK observed in cynomolgus monkeys and humans, the PK profile was utilized to calculate human exposure ratios, resulting in a dosing schedule of 2500 mg every 3 weeks with a 500 mg loading dose, preferably to help achieve the desired steady state more rapidly; see Table 3 in Example 2.

[0012] Thus, in one aspect, the present invention relates to a recombinant antibody that selectively binds to the misfolded, and preferably aggregated, form of SOD1 for use in the treatment of ALS, i.e., by administration of a dosage regimen of about 2500 mg of the antibody to a subject in need thereof, preferably once every three weeks. In a preferred embodiment, the administration is performed intravenously.

[0013] It has further been found to be advantageous to administer a loading dose of 500 mg of antibody one day before administering the initial 2500 mg antibody dose, This type of dosing scheme ensures that the treated subject reaches a steady state as quickly as possible.

[0014] Thus, in one embodiment of the dosing regimen of the invention, the subject being treated receives a loading dose of about 500 mg of the antibody, followed by a dose of about 2500 mg of the antibody on other days before administration of the antibody once every three weeks. This preferred dosing schedule is depicted schematically in Figure 1.

[0015] In one embodiment, treatment will be administered for a period of at least six months. In a particularly preferred embodiment of a dosing regimen of the invention, administration of a 2500 mg dose of antibody will occur once every three weeks for a period of at least six months, preceded by administration of a 500 mg loading dose of antibody one day before administration of the initial 2500 mg maintenance (effective) dose of antibody.

[0016] As mentioned above, there are different forms of ALS, namely, sporadic amyotrophic lateral sclerosis (sALS) and familial amyotrophic lateral sclerosis (fALS). Therefore, the subject to be treated may have fALS, or the subject to be treated has sALS. Diagnosis of fALS and sALS can be performed by the highly sensitive immunoassay disclosed in International Application WO 2021 / 185961 A1, which includes an anti-SOD1 antibody as a capture antibody for determining the presence and level of misfolded SOD1 (mSOD1), respectively, in a body fluid from a subject. The antibody is preferably an antibody further defined below. Therefore, in a preferred embodiment of the present invention, the subject to be treated has been diagnosed by the immunoassay disclosed in WO 2021 / 185961 A1.

[0017] Preferably, the anti-SOD1 antibody used in the dosing regimens and drug formulations of the present invention is a human IgG, most preferably a human IgG1. In a preferred embodiment, the antibody is of the human IgG1m3 allotype.

[0018] In certain preferred embodiments, the anti-SOD1 antibody used in the dosing regimens and drug formulations of the present invention is AP-101 or an equivalent antibody derived from the human antibody NI-204.12G7 characterized in WO 2012 / 080518 A1 and Maier et al., Sci. Transl. Med. 10. (2018) doi:10.1126 / scitranslmed.aah3924. As described further below, antibody AP-101 is a fully human IgG1m3 allotype antibody and, as such, comprises a human constant heavy chain (HC) amino acid sequence present in SEQ ID NO: 12 and a corresponding human constant light chain (LC), herein a lambda light chain, as exemplified in SEQ ID NO: 13. As described further below, IgG antibodies are organized as a tetramer consisting of a HC and two light LC chains linked by disulfide bridges.

[0019] Antibody AP-101 is preferably produced in Chinese hamster ovary (CHO)-K1 cells. CHO cells are the most widely used mammalian cells for the production of recombinant monoclonal antibodies due to their ability to perform post-translational modifications (PTMs) on antibody molecules, which typically occur in humans as well. Through genetic engineering by mutagenesis, various CHO daughter cells with improved qualities have been established. Among these variants are CHO-K1, CHO-S, CHO-DXB11, and CHO-DG44.

[0020] Therefore, in one embodiment, antibodies for use according to the present invention are produced in CHO cells, preferably in the CHO-K1 cell line, and purified from the cell culture medium for further use.

[0021] As shown in Example 7, the major PTMs identified in antibody AP-101 are modification of the N-terminal glutamine to pyroglutamic acid in the HC, loss of the C-terminal lysine, and N-glycosylation. In this context, the N-glycosylation site was identified at position 303 (HC N303).

[0022] Thus, in one embodiment, the anti-SOD1 antibody for use according to the present invention is missing its C-terminal lysine, i.e., the antibody has undergone C-terminal lysine clipping. In particular, a C-terminal lysine has been cleaved from the heavy chain of the antibody, preferably from each heavy chain of the antibody. Additionally or alternatively, the N-terminal glutamine has been modified as pyroglutamic acid, i.e., the antibody has undergone N-terminal glutaminyl cyclization. Additionally or alternatively, the antibody is glycosylated, in particular N-glycosylated. More particularly, the heavy chain of the antibody is glycosylated, and even more particularly, N303 of the heavy chain is glycosylated.

[0023] In a preferred embodiment, the anti-SOD1 antibody for use according to the invention lacks a C-terminal cysteine, has a modified glutamine as pyroglutamic acid at the N-terminus, and contains at least one N-glycosylation site, or when used in a pharmaceutical formulation, preferably the majority of the antibody species present in the formulation have the mentioned modifications.

[0024] To assess the therapeutic efficacy of the antibody and the progression of ALS in human subjects, pharmacodynamic assessments can be performed as described in Example 2. Specifically, pharmacodynamic assessments include determining changes in levels of total SOD1, misfolded SOD1 (mSOD1), phospho-neurofilament heavy chain (pNfH), and / or neurofilament light chain (NfL).

[0025] Thus, in one embodiment of treatment according to the invention, the therapeutic effect of the antibody and the progression of ALS are monitored by assessing one or more biomarkers, preferably any one of those mentioned above, more preferably one or more biomarkers selected from the group consisting of phospho-neurofilament heavy chain (pNfH), neurofilament light chain (NfL) and misfolded SOD1 (mSOD1), or any combination thereof.

[0026] As mentioned above, riluzole and edaravone have been shown to prolong survival in ALS patients, and therefore, in one embodiment, the present invention relates to an antibody for use according to the present invention, wherein the treated subject is taking riluzole or edaravone. In another embodiment of the antibody for use according to the present invention, the treated subject is taking a combination of sodium phenylbutyrate and tauroursodeoxycholic acid (PB and TUDCA) or tofersen.

[0027] In general, concomitant medications should be avoided. However, non-prescription medications, such as acetaminophen, may be administered for the treatment of headaches or for the treatment of infusion reactions if observed. Alternatively, or in addition, antihistamines may be administered for the treatment of infusion reactions if observed.

[0028] Therefore, if headache or infusion reactions are observed during treatment with the present invention, especially if infusion reactions are observed, acetaminophen or a similar analgesic / antipyretic may be administered. Typically, 500 to 1000 mg of acetaminophen may be administered, preferably 30 to 60 minutes prior to the start of antibody administration, particularly prior to the start of antibody infusion for subsequent doses. Acetaminophen may be administered orally or intravenously.

[0029] Additionally or alternatively, if infusion reactions are observed during treatment according to the present invention, an antihistamine may be administered, preferably 30-60 minutes prior to the start of infusion of antibody for subsequent dose administration. Again, administration of the antihistamine can be performed orally and intravenously, respectively.

[0030] In one embodiment of treatment according to the invention, both the antihistamine and acetaminophen in the dosages mentioned above can be administered by the mentioned routes of administration if infusion reactions are observed.

[0031] In particularly preferred embodiments of all of the dosing regimens of the invention disclosed herein, the antibody is administered at a concentration of about 20 mg / mL, and most preferably, the antibody is administered at infusion rates of 500 mg (loading dose) and 2500 mg (maintenance or effective dose) in 60 minutes over a period of at least 60 and at most 120 minutes, respectively.

[0032] If a serious infusion reaction occurs, the infusion rate should preferably be reduced by 50%, and the infusion should preferably be completed at that lower rate. Thus, in one embodiment, administration of antibody at the above-mentioned infusion rate for 60 minutes is followed by an infusion rate that is about 50% lower, i.e., 120 minutes, or a period of about 50% lower infusion rate.

[0033] As highly complex proteins, antibodies are susceptible to various physical and chemical degradation pathways. Antibody aggregation readily occurs during storage in a liquid state, resulting in a decrease in biological activity and increased immunogenicity, which can lead to serious adverse reactions such as anaphylactic shock and other safety issues. To prevent the formation of aggregates that can cause undesirable immunogenicity or altered half-life, pharmaceutical antibody formulations usually contain one or more buffers to maintain a given pH range. Because aggregation is highly dependent on the antibody concentration and the pH of the formulation, selecting a suitable buffer system is a crucial step toward a stable formulation. Furthermore, the desired formulation may depend from and must be suitable for and allow for the intended route of administration, respectively, so that the treatment is also well-tolerated by patients.

[0034] As described in the Examples and demonstrated in the SAD study (ClinicalTrials.gov Identifier: NCT03981536) and monkey study (Study 8384444), a buffer system was established that provided both adequate antibody stability in aqueous solution and good tolerability when infused intravenously into patients over 60 to 120 minutes, allowing for the development of a drug formulation. In particular, for the preferred dosing regimen of the present invention in which the antibody is administered at a concentration of approximately 20 mg / mL, a formulation in an L-histidine / L-histidine monohydrochloride buffer at pH 6.0 was found to be particularly suitable. Buffer system testing was performed to determine the optimal buffer system for the antibody formulation, and based on pH screening tests, an L-histidine / L-histidine monohydrochloride buffer at pH 6.0 was selected as the final buffer system, which ensured that formulations containing approximately 20 mg / mL of antibody had long-term stability; see Examples 3 and 6 and Tables 14-17.

[0035] Additionally, different types of excipients, including disaccharides (sucrose, trehalose, and sorbitol), amino acids (L-arginine-hydrochloride and L-methionine), and salts (NaCl), were evaluated in excipient studies to further enhance the stability of the antibody formulation. Thermal stability, insoluble aggregate formation, and purity were monitored. Overall, sucrose and L-methionine at concentrations of 8% (w / v) and 0.1% (w / v), respectively, were selected as the optimal excipients for the AP-101 formulation because they were shown to minimize the formation of low molecular weight and acidic species, thereby preserving product purity; see Examples 3 and 6 and Tables 14-17.

[0036] In a surfactant type and strength screening study, four different polysorbate 80 (PS 80) concentrations (0.005%, 0.010%, 0.020%, and 0.050% (w / v)) and two different poloxamer 188 concentrations (0.05% and 0.10% (w / v)) were tested. The number and purity of subvisible particles were evaluated. The 0.02% (w / v) polysorbate 80 concentration was selected as the surfactant strength because it effectively suppressed subvisible particle formation and demonstrated acceptable stability; see Examples 3 and 6 and Tables 14-17.

[0037] Thus, in a further aspect, the present invention relates to a liquid, aqueous pharmaceutical formulation (drug formulation) of an anti-SOD1 antibody that selectively binds to misfolded and / or aggregated forms of SOD1, comprising the antibody at a concentration of about 10 to 50 mg / mL in an L-histidine / L-histidine monohydrochloride buffer solution at a pH of about 6.0±1, wherein the antibody remains stable at 5°C±2°C for at least 1 month, and preferably up to 24 months; at 25°C±2°C for at least 1 month, and preferably up to 6 months; and / or at 40°C±2°C for 1 week, as shown in Tables 14, 15, 16, and 17.

[0038] In principle, the antibody can be any anti-SOD1 antibody that recognizes the misfolded and preferably aggregated form of SOD1, preferably preferentially or exclusively, rather than the physiological form of SOD1. To avoid the generation of "anti-drug antibodies" (ADA) by the patient, the antibody is preferably a human antibody, typically human IgG, most preferably human IgG1. In a preferred embodiment, the antibody is of the human IgG1m3 allotype. Thus, as mentioned above, the anti-SOD1 antibody used in the dosing regimen and drug formulation of the present invention is preferably AP-101 or an equivalent antibody derived from the human antibody NI-204.12G7 characterized in WO 2012 / 080518 A1 and Maier et al., Sci. Transl. Med. 10. (2018) doi:10.1126 / scitranslmed.aah3924; see also below.

[0039] As mentioned above, the antibody AP-101 is preferably produced in Chinese hamster ovary (CHO)-K1 cells, and therefore, in one embodiment, the antibody for use in the drug formulation of the present invention is produced in CHO cells, more particularly in a CHO-K1 cell line, and purified from the cell culture medium for formulation.

[0040] As also described above, antibody AP-101 is a fully human IgG1m3 allotype antibody, preferably comprising a heavy chain in which the N-terminal glutamine has been modified as pyroglutamic acid, the C-terminal lysine has been missing, and / or is glycosylated, as described in more detail above.

[0041] Thus, in one embodiment, the formulation of the present invention comprises an anti-SOD1 antibody as defined herein above, wherein the antibody is composed of two heavy chains having SEQ ID NO: 12 and two light chains having SEQ ID NO: 13, in which the N-terminal glutamine in the heavy chain is modified as pyroglutamic acid, the C-terminal lysine is missing, and the heavy chain is N-glycosylated.

[0042] As shown in Example 7, about 99% to 100% of the antibodies present in a sample of a typical antibody formulation have an N-terminal pyroglutamic acid in their heavy chains, and about 94% of the antibodies have a loss of a C-terminal lysine. Thus, in one embodiment, about 99% of the antibodies in a formulation of the invention have heavy chains in which an N-terminal pyroglutamic acid has been modified from an N-terminal glutamine, and / or about 94% of the antibodies have a loss of a C-terminal lysine.

[0043] In one embodiment, the formulation of the invention comprises the antibody at a concentration of 20±5 mg / mL, preferably 20 mg / mL.

[0044] Additionally or alternatively, the concentration of L-histidine / L-histidine monohydrochloride in the formulations of the present invention is 20±10 mM, preferably 20 mM.

[0045] Furthermore, in any one of the preceding embodiments, the formulations of the invention preferably further comprise an antioxidant, preferably L-methionine at a concentration of 0.1%±0.05% (w / v), and most preferably L-methionine at a concentration of 0.1% (w / v).

[0046] Preferably, in addition, or alternatively, any formulation of the present invention further comprises a tonicity adjusting agent, preferably sucrose at a concentration of 8%±1% (w / v), most preferably sucrose at a concentration of 8% (w / v).

[0047] In yet a further preferred embodiment, any formulation of the invention further comprises a surfactant such as polysorbate, preferably polysorbate 80 at a concentration of 0.02%±0.01 (w / v), most preferably polysorbate 80 at a concentration of 0.02% (w / v).

[0048] The formulations of the invention may have any of the compositions described above, provided that the antibody remains stable at 5°C ± 2°C for at least 1 month, and preferably up to 24 months; at 25°C ± 2°C for at least 1 month, and preferably up to 6 months; and / or at 40°C ± 2°C for 1 week.

[0049] In certain preferred embodiments, the formulations of the present invention are substantially free of excipients other than those specifically listed above.

[0050] As mentioned above, the drug formulations of the present invention have been specifically developed for use in the dosage regimens of the present invention for the treatment of ALS. Therefore, the dosage regimens of the present invention for the treatment of ALS are preferably implemented with the drug formulations in the L-histidine / L-histidine monohydrochloride buffer of the present invention described above and disclosed in more detail below and in the Examples. In this context, the drug formulation used in clinical trials and currently in use is the most preferred formulation of the present invention, which comprises 20 mg / mL of an antibody, preferably AP-101, in 20 mM L-histidine / L-histidine monohydrochloride buffer at pH 6.0, 8% (w / v) sucrose, 0.1% (w / v) L-methionine, and 0.02% (w / v) polysorbate 80.

[0051] Additionally, the compatibility of the antibody formulation with clinically used materials was evaluated, particularly with polyvinyl chloride IV bags, polypropylene syringes, and PVC infusion sets. Two concentrations, 20 mg / mL and 5 mg / mL, were evaluated, and the data provided in Tables 11-13 of Example 5 demonstrate that the formulation is compatible with the clinically used materials evaluated.

[0052] The physical and chemical integrity of biopharmaceuticals must be maintained not only during long-term storage but also during administration. While some biopharmaceuticals are formulated and provided ready for clinical administration without further manipulation, many such products require varying degrees of handling by healthcare professionals. The physical and chemical stability of protein drugs must be maintained during handling and administration.

[0053] When delivering biopharmaceuticals via the intravenous route, several factors must be considered, including protein properties, formulation composition, active pharmaceutical ingredient concentration, diluent selection, product contact surfaces, and / or infusion time and rate. A final critical factor to be evaluated is environmental influences (e.g., area lighting and temperature). Contact surfaces are of particular interest because proteins tend to adsorb to interfaces due to their amphiphilic nature. The risk of protein loss due to adsorption is substantial due to the widespread use of various plastic polymers in syringes and intravenous infusion containers and lines. Current pharmaceutical regulations require that intravenously delivered pharmaceuticals always be within 10% of their nominal concentration; see Morar-Mitrica et al., MAbs. 7 (2015), 792-803 and references cited therein.

[0054] Therefore, in an overall preferred embodiment, the formulations of the present invention are suitable for intravenous administration.

[0055] The present invention further relates to a medicament comprising a formulation of the present invention for use in the treatment of ALS, most preferably for use in the dosing regimen of the present invention as defined above.

[0056] The present invention further relates to pharmaceutical containers containing the formulations and medicaments of the present invention, respectively. In a preferred embodiment, the container is a single-use glass vial providing 100 mg of antibody at a concentration of 20 mg / mL.

[0057] In one embodiment, the containers, particularly vials, of the present invention comprise an approximately 10% volume overfill.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples are illustrative only and not limiting.

[0059] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

[0060] For the avoidance of doubt, the expressions "in some embodiments," "in particular embodiments," "in particular cases," "in some cases," "in further embodiments," "in one embodiment," etc. are used and meant to indicate that any of the embodiments described therein should be read in conjunction with each of the features of these embodiments in combination, and it is emphasized that the present disclosure should be treated as if a combination of the features of these embodiments were recited in one embodiment. The same applies to any combination of embodiments and features of the appended claims, illustrated in the Examples, which are also intended to be combined with the features of the corresponding embodiments disclosed in the specification. For consistency and brevity only, the embodiments are characterized by dependencies; however, in practice, each embodiment and combination of features, although interpreted for dependency(s), must be understood as literally disclosed and not as a selection from different alternatives. In this context, those skilled in the art will recognize that the embodiments and features disclosed in the Examples for antibody AP-101 are intended to be generalized to any anti-SOD1 antibody and equivalents having substantially the same properties. [Brief explanation of the drawings]

[0061] [Figure 1]Schematic diagram of one embodiment of the dosing schedule of the present invention. [Figure 2] Pharmacokinetic profile of antibody AP-101 in serum of ALS patients after administration of 100 mg, 500 mg, and 2500 mg of antibody, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0062] The present invention relates to embodiments as characterized in the claims, disclosed in the specification, and further illustrated below in the Examples and drawings, namely, in a first aspect, to a dosing regimen for the treatment of amyotrophic lateral sclerosis (ALS) with a recombinant antibody that binds to SOD1, and in a related aspect, to pharmaceutical formulations of anti-SOD1 antibodies that are particularly suitable for that treatment regimen. In particular, the present invention relates to a recombinant antibody that selectively binds misfolded and / or aggregated forms of SOD1 for use in the treatment of ALS in a dosing regimen by administration of a high dose, e.g., 1000 mg to 10,000 mg, preferably 1000 mg to 5000 mg, and most preferably 2500 mg, of the antibody to a subject in need thereof, preferably once every 1 to 5 weeks, most preferably once every 3 weeks, or by administration of another treatment regimen that delivers the antibody to the subject with substantially the same area under the curve. The pharmaceutical formulation of the present invention is characterized more specifically as a stable liquid aqueous formulation of an anti-SOD1 antibody, comprising the antibody at a concentration of about 10-50 mg / mL in an L-histidine / L-histidine monohydrochloride buffer solution at a pH of about 6.0. More specifically, the present invention relates to the embodiments characterized in the claims, disclosed in the specification, and further illustrated below in the Examples and drawings.

[0063] Unless otherwise specified, terms used herein are given the definitions given in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 0 19 850673 2; Second edition published 2006, ISBN 0-19-852917-1 978-0-19852917-0.

[0064] Furthermore, unless otherwise specified, the terms and expressions used herein to characterize the present invention are given in the definitions provided in WO 2012 / 080518 A1, particularly in subsection "I. Definitions" on pages 10-30, the disclosure of which is expressly incorporated herein by reference. The same applies to the general embodiments disclosed in WO 2012 / 080518 A1, such as with respect to antibodies.

[0065] As used herein, the term "about" refers to a value that is ±10%; preferably ±5% of the recited value.

[0066] As used herein, the term "subject" or "patient" refers to a human patient (e.g., a patient with ALS). As used herein, the terms "subject" and "patient" are interchangeable.

[0067] In one embodiment, administration of an antibody according to the invention is carried out according to a specific clinical dosing regimen (eg, at a specific dosage and / or according to a specific dosing schedule).

[0068] In one embodiment of the dosing regimen of the invention, the anti-SOD1 antibody is administered at a fixed dose, i.e., in an amount that is fixed regardless of the patient's weight. The term "fixed dose" refers to a dose administered to a patient without regard to the patient's weight or body surface area (BSA). Thus, a fixed or uniform dose is not provided as a mg / kg dose, but rather as an absolute amount of antibody. In one embodiment, the antibody is administered to a subject in need thereof at a fixed dose of 1000 mg to 10,000 mg, preferably 1000 mg to 9000 mg, more preferably 1000 mg to 7500 mg, more preferably 1000 mg to 5000 mg, more preferably 2000 mg to 3000 mg, particularly 1000 mg, 1500 mg, 2000 mg, 2500 mg, 3000 mg, 3500 mg, 4000 mg, 4500 mg, 5000 mg, 5500 mg, 6000 mg, 6500 mg, 7000 mg, 7500 mg, 8000 mg, 8500 mg, 9000 mg, 9500 mg, or 10,000 mg, as well as any range therebetween, preferably 2500 mg, regardless of the subject's body weight.

[0069] In one embodiment of the dosing regimen of the invention, the anti-SOD1 antibody is administered in a milligram per kilogram (mg / kg) dosage. In one embodiment, the antibody is administered to a subject in need thereof at a dosage of 17 mg / kg to 167 mg / kg, preferably 17 mg / kg to 150 mg / kg, more preferably 17 mg / kg to 125 mg / kg, more preferably 17 mg / kg to 83 mg / kg, more preferably 33 mg / kg to 50 mg / kg, in particular 17 mg / kg, 25 mg / kg, 33 mg / kg, 42 mg / kg, 50 mg / kg, 58 mg / kg, 67 mg / kg, 75 mg / kg, 83 mg / kg, 92 mg / kg, 100 mg / kg, 108 mg / kg, 117 mg / kg, 125 mg / kg, 133 mg / kg, 142 mg / kg, 150 mg / kg, 158 mg / kg, or 167 mg / kg, and any range therebetween, preferably 42 mg / kg.

[0070] In one embodiment of the dosing regimen of the invention, the antibody is administered to a subject in need thereof once every 1 to 5 weeks, preferably once every 2 to 4 weeks, particularly once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, and any time range therebetween, most preferably once every 3 weeks.

[0071] In particular, in one embodiment of the dosing regimen of the invention, the antibody is administered at a fixed dose of 1000 mg, 1500 mg, 2000 mg, 2500 mg, 3000 mg, 3500 mg, 4000 mg, 4500 mg, 5000 mg, 5500 mg, 6000 mg, 6500 mg, 7000 mg, 7500 mg, 8000 mg, 8500 mg, 9000 mg, 9500 mg, or 10,000 mg, and any range therebetween, preferably 2500 mg, to a subject in need thereof once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, and any time range therebetween, preferably once every three weeks. Therefore, the dosing regimens of the present invention include 1000 mg every week, 1000 mg every 2 weeks, 1000 mg every 3 weeks, 1000 mg every 4 weeks, 1000 mg every 5 weeks, 1500 mg every week, 1500 mg every 2 weeks, 1500 mg every 3 weeks, 1500 mg every 4 weeks, 1500 mg every 5 weeks, 2000 mg every week, 2000 mg every 2 weeks, 2000 mg every 3 weeks, 2000 mg every 4 weeks, 5 2000mg weekly, 2500mg weekly, 2500mg every 2 weeks, 2500mg every 3 weeks, 2500mg every 4 weeks, 2500mg every 5 weeks, 3000mg weekly, 3000mg every 2 weeks, 3000mg every 3 weeks, 3000mg every 4 weeks, 3000mg every 5 weeks, 3500mg weekly, 3500mg every 2 weeks, 3500mg every 3 weeks, 3500mg every 4 weeks, 3500mg every 5 weeks 0mg, 4000mg every week, 4000mg every 2 weeks, 4000mg every 3 weeks, 4000mg every 4 weeks, 4000mg every 5 weeks, 4500mg every week, 4500mg every 2 weeks, 4500mg every 3 weeks, 4500mg every 4 weeks, 4500mg every 5 weeks, 5000mg every week, 5000mg every 2 weeks, 5000mg every 3 weeks, 5000mg every 4 weeks, 5000mg every 5 weeks, 5500mg, 5500mg every 2 weeks, 5500mg every 3 weeks, 5500mg every 4 weeks, 5500mg every 5 weeks, 6000mg every week, 6000mg every 2 weeks, 6000mg every 3 weeks, 6000mg every 4 weeks, 6000mg every 5 weeks, 6500mg every week, 6500mg every 2 weeks, 6500mg every 3 weeks, 6500mg every 4 weeks, 6500mg every 5 weeks, 7000mg every week,7000mg every 2 weeks, 7000mg every 3 weeks, 7000mg every 4 weeks, 7000mg every 5 weeks, 7500mg every week, 7500mg every 2 weeks, 7500mg every 3 weeks, 7500mg every 4 weeks, 7500mg every 5 weeks, 8000mg every week, 8000mg every 2 weeks, 8000mg every 3 weeks, 8000mg every 4 weeks, 8000mg every 5 weeks, 8500mg every week, 8500mg every 2 weeks, 8500mg every 3 weeks, 8500mg every 4 weeks, 8500mg every 5 weeks and preferably 2500 mg every 3 weeks.

[0072] The above-mentioned dosage regime is, in principle, suitable for any subject to be treated, regardless of body weight. As can be seen from Example 1, the body weight of patients treated with 2500 mg of antibody ranged from 60 kg to 120.9 kg. Of course, if the subject to be treated weighs significantly more than 121 kg or less than 60 kg, dosage adjustment may be advantageous for therapeutic effect.

[0073] In one embodiment of the dosing regimen of the invention, the antibody is administered at a dose of 17 mg / kg to 167 mg / kg, preferably 17 mg / kg to 150 mg / kg, more preferably 17 mg / kg to 125 mg / kg, more preferably 17 mg / kg to 83 mg / kg, more preferably 33 mg / kg to 50 mg / kg, particularly 17 mg / kg, 25 mg / kg, 25 mg / kg, 42 mg / kg, 50 mg / kg, 58 mg / kg, 67 mg / kg, 75 mg / kg, 83 mg / kg, A dose of 92 mg / kg, 100 mg / kg, 108 mg / kg, 117 mg / kg, 125 mg / kg, 133 mg / kg, 142 mg / kg, 150 mg / kg, 158 mg / kg, or 167 mg / kg, and any ranges therebetween, preferably 42 mg / kg, is administered to a subject in need thereof once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, and any time ranges therebetween, preferably once every three weeks. Therefore, the dosing regimens of the present invention include 17 mg / kg every week, 17 mg / kg every 2 weeks, 17 mg / kg every 3 weeks, 17 mg / kg every 4 weeks, 17 mg / kg every 5 weeks, 25 mg / kg every week, 25 mg / kg every 2 weeks, 25 mg / kg every 3 weeks, 25 mg / kg every 4 weeks, 25 mg / kg every 5 weeks, 25 mg / kg every week, 25 mg / kg every 2 weeks, 25 mg / kg every 3 weeks, 25 mg / kg every 4 weeks, 25 mg / kg every 5 weeks, 42 mg / kg every week, 42 ​​mg / kg every 2 weeks, 42 mg / kg every 3 weeks, 42 mg / kg every 4 weeks, 42 mg / kg every 5 weeks, 50 mg / kg every week, 50 mg / kg every 2 weeks, 50 mg / kg every 3 weeks 50mg / kg, 50mg / kg every 4 weeks, 50mg / kg every 5 weeks, 58mg / kg every week, 58mg / kg every 2 weeks, 58mg / kg every 3 weeks, 58mg / kg every 4 weeks, 58mg / kg every 5 weeks, 67mg / kg every week, 67mg / kg every 2 weeks, 67mg / kg every 3 weeks, 67mg / kg every 4 weeks, 67mg / kg every 5 weeks, 75mg / kg every 2 weeks, 75mg / kg every 3 weeks, 75mg / kg every 4 weeks, 75mg / kg every 5 weeks, 83mg / kg every week, 83mg / kg every 2 weeks, 83mg / kg every 3 weeks, 83mg / kg every 4 weeks, 83mg / kg every 5 weeks, 92mg / kg every week92mg / kg every 2 weeks, 92mg / kg every 3 weeks, 92mg / kg every 4 weeks, 92mg / kg every 5 weeks, 100mg / kg every week, 100mg / kg every 2 weeks, 100mg / kg every 3 weeks, 100mg / kg every 4 weeks, 100mg / kg every 5 weeks, 108mg / kg every week, 108mg / kg every 2 weeks, 108mg / kg every 3 weeks, 108mg / kg every 4 weeks , 108mg / kg every 5 weeks, 117mg / kg every week, 117mg / kg every 2 weeks, 117mg / kg every 3 weeks, 117mg / kg every 4 weeks, 117mg / kg every 5 weeks, 125mg / kg every week, 125mg / kg every 2 weeks, 125mg / kg every 3 weeks, 125mg / kg every 4 weeks, 125mg / kg every 5 weeks, 133mg / kg every week, 133mg / kg every 2 weeks g, 133 mg / kg every 3 weeks, 133 mg / kg every 4 weeks, 133 mg / kg every 5 weeks, 142 mg / kg every week, 142 mg / kg every 2 weeks, 142 mg / kg every 3 weeks, 142 mg / kg every 4 weeks, 142 mg / kg every 5 weeks, 150 mg / kg every week, 150 mg / kg every 2 weeks, 150 mg / kg every 3 weeks, 150 mg / kg every 4 weeks, 150 mg / kg every 5 weeks, 158 mg / kg every 2 weeks, 158 mg / kg every 3 weeks, 158 mg / kg every 4 weeks, 158 mg / kg every 5 weeks, 167 mg / kg every week, 167 mg / kg every 2 weeks, 167 mg / kg every 3 weeks, 167 mg / kg every 4 weeks, 167 mg / kg every 5 weeks, preferably 42 mg / kg every 3 weeks.

[0074] In one embodiment of the dosing regimen of the invention, the subject being treated receives a loading dose of antibody on day 1, followed by a different maintenance dose on day 2, and every 1 to 5 weeks thereafter, preferably every 2 to 4 weeks, and most preferably every 3 weeks. In a preferred embodiment, the subject being treated receives a loading dose of antibody on day 1, followed by a different maintenance dose on day 2, 22 days ± 3, 43 days ± 3, 64 days ± 3, 85 days ± 3, 106 days ± 3, 127 days ± 3, 148 days ± 3, and 169 days ± 3. The maintenance dose is preferably any one of the doses described above, but is preferably 2500 mg and 42 mg / kg, respectively, and most preferably 2500 mg. The loading doses are preferably 200 to 800 mg and 3 mg / kg to 13 mg / kg, respectively, more preferably 300 to 700 mg and 5 mg / kg to 11 mg / kg, respectively, more preferably 400 to 600 mg, 7 mg / kg and 10 mg / kg, respectively, most preferably 500 mg and 8 mg / kg, respectively, in particular 500 mg.

[0075] In one embodiment of the dosing regimen, treatment is maintained for at least 4 months, preferably at least 5 months, and more preferably at least 6 months (maintenance phase). However, in general, there is no limit to the duration of treatment, and in one embodiment of the dosing regimen of the present invention, treatment is carried out for 1, 2, 3, or several years. In one embodiment, treatment is continued for the patient's lifetime, until cure of the disease, as long as clinical benefit is observed, or until unmanageable toxicity and / or disease progression occurs.

[0076] The term "loading dose" refers to the initial dose administered to a patient. The term "maintenance phase" refers to the second phase of the dosing regimen, which in preferred embodiments begins on day 2. The term "maintenance dose" (or effective dose) refers to the dose administered to a patient after the loading dose and during the maintenance phase.

[0077] In one embodiment of the dosing regimen of the invention, the antibody is administered at a concentration of about 10 mg / mL to 50 mg / mL, preferably about 10 mg / mL to 40 mg / mL, preferably about 20 mg / mL to 30 mg / mL, and most preferably about 20 mg / mL.

[0078] Administration of antibodies can occur in different ways, for example, intravenously, intraperitoneally, subcutaneously, intramuscularly, intranasally, orally, topically or intradermally, or by spinal or brain delivery. Aerosol formulations, such as nasal spray formulations, contain purified aqueous or other solutions of an active agent, a preservative, and an isotonic agent. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucosa. Formulations for rectal or vaginal administration can be presented as suppositories with a suitable carrier. In a preferred embodiment of the present invention, antibodies are administered intravenously.

[0079] In this context, as mentioned above, a drug formulation in L-histidine / L-histidine monohydrochloride buffer at a pH of about 6.0 has been developed that is particularly suitable for intravenous administration of anti-SOD1 antibodies at a concentration of 20 mg / mL, which is described in further aspects and examples of the invention below. Thus, in the most preferred embodiment of the dosing regimen of the invention, the antibody is administered in a drug formulation according to the invention, according to any of the embodiments disclosed herein.

[0080] In one embodiment of the dosing regimen of the present invention, 500 mg (loading dose) and 2500 mg (maintenance or effective dose), respectively, of antibody are administered over a minimum of 60 minutes and a maximum of 120 minutes, preferably over a 60-minute period. In one embodiment, the antibody is administered over at least 60 minutes, where the infusion period can be increased or the infusion can be stopped if deemed necessary or if an infusion reaction is observed. However, intravenous administration should preferably be completed within 120 minutes of the start of the infusion. If higher or lower doses of antibody are administered, the infusion time can be increased or decreased appropriately.

[0081] Hypersensitivity reactions may occur after administration of any monoclonal antibody. Acute hypersensitivity reactions, including infusion reactions or anaphylaxis, may occur during or within several hours of infusion. Subjects should be monitored for hypotension, fever, chills, bronchospasm, angioedema, and other symptoms or signs of anaphylaxis. The infusion should be slowed or stopped, and the reaction should be managed. In particular, if a severe infusion reaction occurs, the infusion rate should be reduced, preferably by 50%. In one embodiment, if the infusion rate is reduced, the infusion is completed at the lower rate. In one embodiment, if the infusion rate is reduced, the infusion rate is increased again to its previous rate (before the reduction), for example, if the infusion reaction subsides. Thus, in one embodiment, administration of antibody at the above-mentioned infusion rates is followed or separated by infusion rates that are about 50% slower, preferably with the desired amount of antibody, e.g., 500 mg (loading dose) and 2500 mg (maintenance or effective dose), administered over a period of a minimum of 120 minutes and a maximum of 240 minutes, respectively.

[0082] Generally, premedication should not be administered prior to administration of an antibody according to the present invention. However, if an infusion reaction occurs, appropriate medication may be administered as determined by the investigator. If an infusion reaction is observed, 500-1000 mg of acetaminophen and / or an antihistamine may be administered orally or intravenously 30-60 minutes before the start of the infusion of the subsequent dose. Additionally, acetaminophen may be administered to treat headaches.

[0083] Therefore, if headache or infusion reactions are observed during treatment according to the present invention, but especially if infusion reactions are observed, acetaminophen, preferably 500 to 1000 mg, can be administered, preferably 30 to 60 minutes before the start of antibody administration, particularly before the start of antibody infusion for subsequent doses. Acetaminophen administration can be performed orally or intravenously. Instead of acetaminophen, other analgesics and antipyretics, such as acidic (nonsteroidal anti-inflammatory drugs, NSAIDs) and non-acidic (paracetamol, pyrazolinone) drugs, and selective cyclooxygenase-2 (COX-2) inhibitors, can also be administered, as reviewed, for example, in Hinz and Brune, Handb. Exp. Pharmacol. 177 (2007), 65-93.

[0084] Additionally, or alternatively, if an infusion reaction is observed during treatment according to the present invention, an antihistamine may be administered, preferably 30 to 60 minutes prior to the start of infusion of antibody for subsequent dose administration, and administration of the antihistamine may be performed orally or intravenously.

[0085] In one embodiment of treatment according to the present invention, both the antihistamine and acetaminophen in the dosages described above can be administered by the routes of administration described above if an infusion reaction is observed.

[0086] In one embodiment, the antibody is administered during treatment according to the present invention with one or more additional pharmaceutical or therapeutic agents useful for treating ALS. The additional agents can be, for example, therapeutic agents art-recognized as useful for treating ALS. The combination can also include two or more additional agents, for example, two or three additional agents. In one embodiment, the patient treated according to the present invention is treated with riluzole (Rilutek®) or edavarone (Radicava®), both of which are approved by the U.S. Food and Drug Administration for the treatment of ALS, or with both riluzole and edaravone. Preferably, the patient is on a stable dose of riluzole and / or edaravone before treatment according to the present invention begins. In one embodiment, treatment with riluzole and / or edaravone is initiated simultaneously with treatment with the antibody according to the present invention.

[0087] In one embodiment, the additional therapeutic agent is a therapeutic agent for non-specific treatment, such as a drug that reduces SOD1 levels, e.g., pyrimethamine (Lange et al. Ann. Neurol. 81 (2017), 837-848), a therapeutic agent used in gene silencing, e.g., morpholino oligonucleotides (MOs), or rapamycin. Furthermore, the additional therapeutic agent may be a drug that targets some of the specific symptoms of ALS, such as an analgesic or muscle relaxant. Therefore, the additional therapeutic agent is preferably baclofen (Gablofen®, Kemstro®, Lioresal®) or diazepam (Diastat®, Valium®), which can help relieve spasms. Accumulation of saliva in the mouth due to difficulty swallowing is also a symptom of ALS and can be treated with a different pharmaceutical agent, which is an additional therapeutic agent according to the present invention. Preferably, the therapeutic agent is Elavil® (amitriptyline), trihexyphenidyl, Scopoderm® (scopolamine patch), or Robinul® (glycopyrrolate).

[0088] In one embodiment, the additional therapeutic agent is another antibody useful in treating ALS, e.g., a C5-antibody such as ravulizumab or eculizumab. Ravulizumab (also known as BNJ441, ALXN1210, or Ultomiris®) is described in WO 2015 / 134894 A1. Eculizumab (also known as Soliris®) is described in WO 2007 / 106585 A1.

[0089] As mentioned above, two forms of ALS are known, namely familial ALS (fALS) and sporadic ALS (sALS), and there is strong evidence that both genetic and non-genetic drivers of SOD1 misfolding result in the formation of toxic conformers that initiate and promote ALS pathogenesis; i.e., fALS and sALS are driven by pathogenic SOD1 accumulation. Importantly, and in support of this hypothesis, recent evidence suggests that misfolded SOD1 aggregates can also be detected in the spinal cord of sALS patients, suggesting common elements of disease pathology between familial and sporadic ALS (Li and Cashman, Prion 8 (2014), 33-41; Bosco et al., Nat. Neurosci. 13 (2010), 1396-403; Forsberg et al. Acta Neuropathol. 121 (2011), 623-34; Forsberg et al., PLos One 5 (2010), e11552).

[0090] Thus, a subject treated according to the present invention may be suffering from fALS or sALS.

[0091] Diagnosis of fALS and sALS can be performed by the highly sensitive immunoassay disclosed in WO 2021 / 185961 A1, which includes an anti-SOD1 antibody as a capture antibody for determining the presence and level, respectively, of misfolded SOD1 (mSOD1) in a body fluid from a subject. The antibody is preferably an antibody as further defined below. This assay is particularly capable of identifying patients with sALS with a high degree of certainty. Therefore, in one embodiment of the present invention, the subject to be treated has been diagnosed by the immunoassay disclosed in WO 2021 / 185961 A1, the contents of which are incorporated herein by reference.

[0092] In one embodiment, ALS is defined in a subject treated according to the present invention by King's ALS clinical staging system (Roche et al., Brain 135(Part 3)(2012), 847-8S2). The King's ALS system classifies the extent to which ALS has progressed in a patient or subject based on the occurrence of distinct milestones defined as the first occurrence of ALS symptoms (e.g., functional involvement of one central nervous system region, defined as the medulla oblongata, upper limbs, lower limbs, or diaphragm, due to weakness, wasting, spasticity, dysphagia, or swallowing problems), diagnosis, functional involvement of a second region, functional involvement of a third region, and the need for gastrostomy and non-invasive ventilation. Based on these observable and measurable events, King's system classifies ALS progression according to four stages: Stage 1 - symptom onset with first zone involvement; Stage 2A - diagnosis; Stage 2B - second zone involvement; Stage 3 - third zone involvement; Stage 4A - need for gastrostomy; and Stage 4B - need for non-invasive ventilation.

[0093] In one embodiment, ALS is defined as meeting possible, laboratory-supported probable, probable, or definite criteria for the diagnosis of ALS according to the revised World Federation of Neurology El Escorial criteria in a subject treated according to the present invention.

[0094] Thus, in one embodiment, a subject treated according to the present invention has possible, clinically probable, clinically probable with laboratory support, or definite fALS or sALS according to the El Escorial criteria, or has been diagnosed with ALS as defined by the Gold Coast criteria: a progressive motor disorder preceded by normal motor development and documented by medical history or repeated clinical examinations, and the presence of upper and lower motor neuron dysfunction in at least one body region or lower motor neuron dysfunction in at least two body regions, and investigations that exclude other pathologies.

[0095] In one embodiment, a subject treated according to the present invention is diagnosed with onset ALS, defined as the time of onset of first muscle weakness (e.g., limb weakness, dysphagia, dysphagia, and / or shortness of breath) within 24-60 months, preferably 48 months or less, and most preferably within the past 24 months, prior to being evaluated for treatment with the antibody.

[0096] In one embodiment, a subject treated according to the invention has a standing vital capacity (SVC) of at least 50% at the time of assessment for treatment with the antibody.

[0097] Because additional neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), are known to be associated with superoxide dismutase (SOD1) accumulation, it is reasonable to predict that these diseases will also be treatable when the dosing regimen of the present invention is applied to patients suffering from AD and PD. Thus, the present invention also relates to a recombinant antibody that selectively binds to misfolded and / or aggregated forms of SOD1 for use in treating neurodegenerative diseases associated with SOD1 accumulation, such as AD or PD, by administration of a dose of 1000 mg to 10,000 mg, preferably 1000 mg to 5000 mg, preferably 2000 mg to 3000 mg, and most preferably 2500 mg of the antibody to a patient in need thereof once every 1 to 5 weeks, preferably once every 2 to 4 weeks, and most preferably once every 3 weeks.

[0098] The antibody for use in the dosing regimen and the pharmaceutical product, i.e., the liquid, aqueous pharmaceutical formulation of the present invention, can in principle be any anti-SOD1 antibody that selectively binds to misfolded and / or aggregated forms of SOD1, preferably preferentially or exclusively over the physiological form of SOD1.

[0099] In principle, the antibody can be in any format that recognizes misfolded SOD1, including, for example, chimeric antibodies, single-chain antibodies, Fab fragments, bispecific antibodies, fusion antibodies, labeled antibodies, or analogs of any one of these. Corresponding methods for producing such variants are known to those skilled in the art and are described, for example, in Harlow and Lane "Antibodies, A Laboratory Manual," CSH Press, Cold Spring Harbor (1988) First Edition; Second Edition by Edward A. Greenfield, Dana-Farber Cancer Institute (Copyright) 2014, ISBN 978-1-936113-81-1. For example, Fab and F(ab')2 fragments can be produced by genetic recombination or by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments contain the variable region, the light chain constant region, and the CH1 domain of the heavy chain.

[0100] In one embodiment, the antibodies used in the dosing regimens and drug formulations of the invention may therefore be provided in a format selected from the group consisting of single-chain Fv fragments (scFv), F(ab') fragments, F(ab) fragments, and F(ab')2 fragments, Fd, Fv, single-chain antibodies, and disulfide-linked Fvs (sdFv), and / or are chimeric, murine-human, or humanized antibodies. Preferably, the anti-SOD1 antibodies utilized comprise human constant domains.

[0101] The antibody AP-101 corresponds to the antibody NI-204.12G7 characterized in WO 2012 / 080518 A1 and Maier et al., Sci. Transl. Med. 10 (2018) doi:10.1126 / scitranslmed.aah3924. It is a particularly preferred fully human IgG1m3 allotype antibody with selective, high-affinity binding to misfolded SOD1 protein. In two mouse models of ALS, intravenous or intrathecal administration of chimeric AP-101 (chα-miSOD1) attenuated spinal motor neuron loss, improved motor function, and prolonged overall survival (Welt et al., Sci. Transl. Med. 10 (2018) doi:10.1126 / scitranslmed.aah3924; WO 2012 / 080518 A1). The therapeutic effect of AP-101 treatment may be mediated by different, non-exclusive mechanisms. These include direct neutralization of extracellular toxic SOD1 conformers (Zhao et al., Glia 58 (2010), 231-243) and interference with the prion-like spread of SOD1 aggregates released from dying or living cells by exosome-dependent or -independent pathways (Basso and Bonetto, Front. Neurosci. 10 (2016), 127; Silverman et al., Cell. Mol. Life Sci 70 (2016), 377-381), interference with transsynaptic propagation (Ayers et al., Acta Neuropathol. 131 (2016), 103-114), and / or interference with macropinocytotic uptake of aggregates (Grad and Cashman, Prion 8 (2014), 33-41; Bidhendi, J Clin. Invest. 126 (2016), 2249-2253; and Sundaramoorthy et al., Cell. Mol. Life Sci. 70 (2013), 4181-4195). Functional and glycosylated fragment crystallizable (Fc) regions of the antibody AP-101 confer full therapeutic efficacy, suggesting that Fc gamma receptor (FcγR)-mediated effects are responsible for in vivo activity.Therefore, microglial recruitment and phagocytosis of toxic SOD1 aggregates may be a plausible underlying mechanism of action (Welt et al., 2018, supra).

[0102] Thus, in one embodiment, the antibody for use according to the invention, i.e. in the dosing regimens and drug formulations of the invention, is an anti-SOD1 antibody that recognizes an epitope comprising the amino acid sequence 73-GGPKDEERHVG-83 (SEQ ID NO: 11).

[0103] In a particular preferred embodiment, the antibody for use according to the invention is AP-101, which is derived from the human antibody NI-204.12G7 and has in its variable region, i.e., binding domain, a variable heavy chain (VH) having the amino acid sequence set out in Figure 1B of WO 2012 / 080518 A1. H ) and variable light chain (V L ), wherein one or more of the CDRs may differ in their amino acid sequence from that set forth in Figure 1B of WO 2012 / 080518 A1 by one, two, three or more amino acids in the case of CDR2 and CDR3, and wherein the antibody exhibits substantially the same or identical immunological properties as the anti-SOD1 antibody NI-204.12G7 shown in the Examples of WO 2012 / 080518 A1. The positions of the CDRs are shown in Figure 1B of WO 2012 / 080518 A1 and explained in the figure legend to Figure 1. The corresponding nucleotide sequence is set forth in Table II on page 54 of WO 2012 / 080518 A1. Additionally or alternatively, the framework regions or the complete V H and / or V L The strand is 80% identical to the framework region set forth in Figure 1B of WO 2012 / 080518 A1, preferably the framework region set forth in Figure 1B of WO 2012 / 080518 A1 as well as the V H and / or V LThe NI-204.B antibody is 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the NI-204.B chains, respectively. Furthermore, the cloning and expression of antibody NI-204.B was performed as described in the "Materials and Methods" section on pages 84-88 of WO 2012 / 080518 A1, and therefore, the methods are incorporated herein by reference.

[0104] In certain preferred embodiments, the antibody is selected from the group consisting of V and VIII as set forth in Figure 1B of WO 2012 / 080518 A1. H and / or V L Characterized by a chain.

[0105] Therefore, the antibody preferably (i) a variable heavy (VH) chain comprising VH complementary determining regions (CDRs) 1, 2, and 3 and / or a variable light (VL) chain comprising VL CDRs 1, 2, and 3; (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and (f) VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 10 or a variant thereof, wherein the variant contains one or two amino acid substitutions; and / or (ii) a VH chain and / or a VL chain, (a) the VH chain comprises the amino acid sequence set forth in SEQ ID NO: 1 or 2 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and (b) the VL chain comprises the amino acid sequence set forth in SEQ ID NO: 6 or 7 or a variant thereof, wherein the variant contains one or more amino acid substitutions; Including, Preferably, the VH and VL chain amino acid sequences are at least 90% identical to SEQ ID NOs: 1 or 2 and 6 or 7, respectively.

[0106] Thus, most preferably, the anti-SOD1 antibody used in the dosing regimens and drug formulations of the present invention is AP-101 or an equivalent antibody derived from the human antibody NI-204.12G7 characterized in WO 2012 / 080518 A1 and Maier et al., Sci. Transl. Med. 10. (2018) doi:10.1126 / scitranslmed.aah3924.

[0107] The five major classes of immunoglobulins are IgG, IgM, IgA, IgD, and IgE. They are distinguished by the type of heavy chain present in the molecule. IgG molecules have heavy chains known as gamma chains; IgM have mu chains; IgA have alpha chains; IgE have epsilon chains; and IgD have delta chains; for a review, see, e.g., Schroeder et al., J. Allergy Clin. Immunol. 125 (2010), S41-S52. Furthermore, different subclasses exist: IgA is further divided into subclasses IgA1 and IgA2, and IgG is further divided into subclasses IgG1, IgG2, IgG3, and IgG4. Furthermore, there are two types of light chains: kappa (κ) and lambda (λ).

[0108] In principle, antibodies used according to the present invention may be of any kind of class and subclass and may comprise any kind of light chain, as long as the antibody binds to misfolded, and preferably aggregated, form of SOD1, preferably as long as its binding specificity for SOD1, as shown in the Examples of WO 2012 / 080518 A1 for the antibody NI-204.12G7, remains in kind and unaffected, and as long as no adverse effects occur when the antibody is administered to a patient, which can be determined as described in Examples 1 and 2. Preferably, however, full IgG antibodies are used, which comprise constant domains. Thus, in one embodiment, the immunoglobulin heavy and / or light chain constant domains present in an antibody used according to the present invention are of the IgG, IgM, IgA, IgD, or IgE type, preferably of the IgG type. In one embodiment, the immunoglobulin heavy and / or light chain constant domains present in the antibodies used according to the invention are of the IgA1, IgA1, IgG1, IgG2, IgG3, or IgG4 subclass, preferably the IgG1, IgG2, IgG3, or IgG4 subclass, most preferably the IgG1 subclass.

[0109] Recombinant expression of fully human IgG1 antibodies with human or mouse constant domains can be performed essentially as described in the Examples of WO 2012 / 080518 A1. Preferably, the antibodies are monoclonal or derived from monoclonal antibodies. As shown in Table V of Example 10 on page 105 of WO 2012 / 080518 A1, which describes IgG germline family classification by aligning the nucleotide sequence of the original antibody with human germline sequences in Vbase (http: / / vbase.mrc-cpe.cam.ac.uk / ), a database maintained by the MRC Centre for Protein Engineering (Cambridge, UK), germlines are specified by the locus of their heavy chain and by their Vbase entry number for the light chain, according to which antibody NI-204.12G7 is classified as 3a.119B4 / V2-11+ and of the L-lambda type (NI-204.12G7L), i.e., containing a lambda chain.

[0110] Human heavy and light chain genes, as well as the four IgG subclasses mentioned above, also exhibit extensive structural polymorphism and are closely linked and therefore inherited as haplotypes. Allotypic variants can be immunogenic and can elicit antibody responses as a result of alloimmunization. Therefore, allotype switching may be particularly interesting for providing non-immunogenic antibody therapeutics. Currently, numerous allotypes (polymorphisms) are known, but serologically defined allotypes are of particular interest. IgG protein allotypes are defined by the expression of unique epitopes recognized by unique serological reagents. Allotypes expressed in the constant region of the IgG heavy chain are designated by the subclass as a Gm (genetic marker), e.g., G1m, and the allotype number (or letter), e.g., G1m1 [or G1m(a)], G3m5 [or G3m(b1)]. Human immunoglobulin allotypes are listed in Table 1 of Jefferis and Lefrance, mAbs 1 (2009), 1-7 and Figure 1A of Irani et al., Molecular Immunology 67 (2015), 171-182, the contents of which are incorporated by reference. Thus, in one embodiment, the antibody used in accordance with the present invention is, but is not limited to, any one of the following allotypes: G1m1, G1m2, G1m3, G1m17, G2m23, G3m21, G3m28, G3m11, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2, A2m3, Em1, Km1, Km2, and Km3, but preferably G1m2, G1m3, or G1m17, most preferably G1m3.

[0111] As explained above, the antibody AP-101 is a fully human IgG1m3 allotype antibody, composed of two identical heavy chains of the IgG1 subclass and the IgG1m3 allotype. Furthermore, as mentioned above, the original human antibody NI-204.12G7 is an L-lambda type, and therefore, AP-101 is composed of two identical light chains of the lambda subclass. The sequences of the variable heavy (VH) chain and variable light (VL) chain of AP-101 are set forth in SEQ ID NOs: 1, 2, 6, and 7, respectively, and the sequences of the corresponding human constant regions are known in the art. For example, each isotype, such as the IgG1m3 isotype, has a unique amino acid sequence in the constant region of its heavy chain; see Jefferis and Lefrance (2009), supra. Thus, in one embodiment, an antibody used according to the present invention is characterized by two heavy chains, each of which comprises the amino acid sequence set forth in SEQ ID NO: 12, and two light chains, each of which comprises the amino acid sequence set forth in SEQ ID NO: 13. Each heavy chain consists of 453 amino acid residues, and each light chain consists of 213 amino acid residues. The four chains are stabilized by 12 intrachain disulfide bonds and four interchain disulfide bonds. Each heavy chain contains a single N-linked glycosylation site at Asn303. The N-linked glycosylation structure is mostly a fucosylated, complex biantennary glycan with either zero galactose residues (G0F) or one galactose residue (G1F).

[0112] Additionally, to a low extent, and preferably in negligible amounts, some antibody species may be found in the formulations of the invention and, when used in the dosing regimens described herein, may have undergone other post-translational modifications (PTMs), such as partial cleavage, oxidation, deamidation, succinimide or pyroglutamate formation, and isomerization. The PTMs identified as present in AP-101 are shown in Example 7 and Table 18.

[0113] In a preferred embodiment, antibodies for use according to the invention have heavy chains that do not include a C-terminal lysine, for example, in such an embodiment, the C-terminal lysine contained in SEQ ID NO:12 is absent.

[0114] Additionally or alternatively, antibodies for use according to the invention have heavy chains in which the N-terminal glutamine is replaced by pyroglutamic acid, this pyroglutamic acid formation also being referred to as N-terminal cyclization.

[0115] Additionally, or alternatively, antibodies for use according to the invention have heavy chains that are N-glycosylated, preferably heavy chains in which the N-linked glycosylation site is Asn303.

[0116] Most preferably, antibodies for use according to the invention have heavy chains that are N-glycosylated, without a C-terminal lysine, i.e., where the C-terminal lysine has been C-terminally lysine clipped, and where the N-terminal glutamine has been replaced with pyroglutamic acid, i.e., where the N-terminal glutaminyl cyclization has been performed.

[0117] The amino acid sequences of the heavy and light chains are shown below: [ka] (SEQ ID NO: 12, AP-101, heavy chain amino acid sequence, constant region amino acids are underlined, C-terminal lysine (K) is optional and / or N-terminal glutamine (Q) undergoes intramolecular cyclization resulting in the formation of pyroglutamic acid) [ka] (SEQ ID NO: 13, AP-101, light chain amino acid sequence, constant region amino acids are underlined)

[0118] In one embodiment, treatment of ALS according to the present invention includes alleviating or ameliorating one or more symptoms associated with ALS, including the progressive loss of motor neurons resulting in varying degrees of weakness and spasticity of limb, bulbar, and respiratory muscles.

[0119] In one embodiment, treating ALS includes ameliorating at least one clinical and biomarker, respectively, of ALS progression. The revised ALSFRS-R is a recognized clinical endpoint of functional impact on disease progression in ALS (FDA 2019 (Guidance for Industry: Amyotrophic lateral sclerosis: developing drugs for treatment. September 2019); EMA 2015 (European Medicines Agency. Committee for medicinal products for human use (CHMP) Guideline on clinical investigation of medicinal products for the treatment of amyotrophic lateral sclerosis (ALS))). Recent data further suggest that some neurofilament proteins may have clinical utility in ALS prognosis and response to therapy. Preclinical models originally suggested that phospho-neurofilament heavy chain (pNfH) and neurofilament light chain (NfL) could act as biomarkers to monitor axonal degeneration during ALS (Brettschneider et al., Neurology 66 (2006), 852-856; Boylan et al. al., J. Neurochem. 111 (2009), 1182-1191; Gaiottino et al., PLoS One 8 (2013), e75091). These studies have been supported more recently in publications describing an increase in neurofilament markers with disease onset and a correlation between the levels of these proteins and the overall rate of ALS progression (Benatar et al., Neurology 95 (2020), e59-e69). Furthermore, evidence is now emerging that neurofilament levels in serum and CSF have the potential as early markers of therapeutic efficacy (Miller et al., N. Engl. J. Med. 383 (2020), 109-119).However, additional markers may be suitable, and non-limiting examples of these markers of ALS progression include: · Change from baseline in ALSFRS-R total score, i.e., a measure of the clinical effect of treatment on the rate of functional decline as measured by the ALSFRS-R; · Change from baseline in percent predicted SVC, i.e., a measure of the clinical effect of treatment on pulmonary function (SVC); · Measuring the clinical effect of treatment on pulse oximetry; Percent change in combined muscle megascore from baseline, assessed by handheld dynamometry (HHD), a measure of the clinical effect of treatment on muscle strength (HHD); Time to earliest occurrence of one of the following ventilator-assisted survival (VAFS) events during the randomized controlled period: all-cause death; first use of noninvasive ventilation (NIV) for approximately 22 hours per day on approximately 10 consecutive days; and first use of permanent assisted ventilation (PAV) for approximately 22 hours per day on approximately 7 consecutive days, i.e., a composite time to event measure including time to death, ventilatory tracheostomy, or noninvasive ventilation use for 22 to 24 hours per day; · Change from baseline in ALSAQ-40 score; Change from baseline in the European Quality of Life Health 5-item questionnaire (EQ 5D 5L); · Change from baseline on the Short Form Health Survey (SF 36); Change from baseline in Treatment Satisfaction Questionnaire for Medication (TSQM) total score; Shift from baseline on the Columbia-suicide severity rating scale (C-SSRS); · Changes from baseline in vital signs, electrocardiogram (EDG) parameters, and laboratory assessments; · Changes from baseline in levels of biomarkers of complement dysregulation, neuroinflammation, and neurodegeneration; ·Measurement of the clinical effect of treatment on disease staging (King's staging tool), i.e., reduction from baseline stage in the King's staging system; Measuring the clinical effect of treatment on quality of life with the Amyotrophic Lateral Sclerosis Specific Quality of Life Instrument-Short Form (ALSSQOL-SF); · Change from baseline in serum, CSF and / or plasma NfL concentrations, i.e., measurement of serum, CSF and / or plasma NfL levels; - Change from baseline in the concentration of pNfH in any body fluid, preferably serum, CSF and / or plasma, i.e. measurement of the level of pNfH in any body fluid, preferably serum, CSF and / or plasma; - Change from baseline in the concentration of total SOD1 in any body fluid, preferably in CSF, i.e., measurement of the level of total SOD1 in any body fluid, preferably in CSF; Change from baseline in mSOD1 concentration in any body fluid, preferably CSF, i.e., measurement of mSOD1 levels in any body fluid, preferably CSF; and / or Measurement of creatinine and other biological markers of disease activity, including transcriptional markers.

[0120] Preferably, assessment of markers is performed at several time points throughout the study, but at least at the endpoint, here 24 weeks (6 months) after initiation of treatment. Of course, longer or shorter time periods can also be observed, for example, up to 50 weeks.

[0121] Therefore, the therapeutic efficacy of the antibodies used in accordance with the present invention and the dosing regimens of the present invention, respectively, and the progression of ALS are preferably monitored by assessing one or more markers selected from the markers listed above.

[0122] In a preferred embodiment of the treatment according to the invention, the therapeutic effect of the antibody and the progression of ALS are monitored by assessing one or more markers selected from the group consisting of: · Measuring the clinical effect of treatment on the rate of functional decline, as measured by the ALSFRS-R; · Measurement of the clinical effect of treatment on pulmonary function (SVC); · Measuring the clinical effect of treatment on pulse oximetry; · Measurement of the clinical effect of treatment on muscle strength (HHD); · Composite time to event measures including time to death, tracheostomy for ventilation, or noninvasive ventilation use for 22–24 hours per day; · Measuring the clinical effect of treatment on disease staging (King's staging tool); ·Measurement of the clinical effect of treatment on quality of life by ALSSQOL-SF; Measurement of NfL levels in any body fluid, preferably in serum, CSF and / or plasma, preferably in CSF and plasma; Measurement of pNfH levels in any body fluid, preferably in serum, CSF and / or plasma, preferably in CSF and plasma; Measurement of total SOD1 levels in any body fluid, preferably CSF; Measurement of mSOD1 levels in any body fluid, preferably in CSF; and Measurement of creatinine and transcriptional markers or other biological markers of disease activity, including any one of those disclosed in Table 2 of Vijayakumar et al., Front. Neurol. 10 (2019), 400, and / or Tables 1-5 disclosed in Vu and Bowser, Neurotherapeutics 14 (2017), 119-134.

[0123] In a further preferred embodiment of the treatment according to the invention, the therapeutic effect of the antibody and the progression of ALS are monitored by assessing one or more markers selected from the group consisting of: Measurement of NfL levels in any body fluid, preferably in serum, CSF and / or plasma, more preferably in CSF and plasma; Measurement of pNfH levels in any body fluid, preferably in serum, CSF and / or plasma, more preferably in CSF and plasma; Measurement of total SOD1 levels in any body fluid, preferably in CSF; and Measurement of mSOD1 levels in any body fluid, preferably CSF.

[0124] In an even more preferred embodiment of the treatment according to the invention, the therapeutic effect of the antibody and the progression of ALS are monitored by assessing one or more markers selected from the group consisting of: Measurement of NfL levels in any body fluid, preferably in serum, CSF and / or plasma, more preferably in CSF and plasma; Measurement of pNfH levels in any body fluid, preferably in serum, CSF and / or plasma, more preferably in CSF and plasma; and Measurement of mSOD1 levels in any body fluid, preferably CSF.

[0125] As already indicated above, the measurement of SOD1 levels, in particular mSOD1 levels, can be carried out by immunoassay as disclosed in WO 2021 / 185961 A1.

[0126] In a further aspect, but in conjunction with the dosing regimens described herein, the present invention relates to a liquid, aqueous pharmaceutical formulation (medicament) of an anti-SOD1 antibody that selectively binds to the misfolded, and preferably aggregated, form of SOD1, characterized by an L-histidine / L-histidine monohydrochloride buffer having a pH of about 6.0±1, preferably about 6.0±0.5. As noted above, such formulations have proven particularly suitable for intravenous administration and for use in the dosing regimens of the present invention.

[0127] In a preferred embodiment, the formulation of the present invention comprises an antibody as defined herein above. In one embodiment, the antibody is characterized in that it comprises in its variable region the six CDRs of the VH and VL chains and a constant region, preferably a human Ig constant region, as described herein above, and / or in that it comprises in its variable region the VH and VL chains and a constant region, preferably a human Ig constant region, as described herein above. The antibody comprised in the formulation of the present invention is preferably characterized in that it binds to an epitope of SOD1 within the amino acid sequence 73-GGPKDEERHVGD-84 set forth in SEQ ID NO: 11. In one embodiment, the antibody comprised in the formulation of the present invention is of the human IgG, preferably human IgG1, more preferably human IgG1m3 allotype. Preferably, the antibody is defined by its heavy and light chains as described herein above, wherein the light chain is preferably a lambda (λ) light chain. Most preferably, the antibody is antibody AP-101, characterized by two heavy chains, each heavy chain comprising the amino acid sequence set forth in SEQ ID NO:12, and two light chains, each light chain comprising the amino acid sequence set forth in SEQ ID NO:13.

[0128] In one embodiment, the antibody in the formulation of the invention has a heavy chain that does not contain a C-terminal lysine. For example, in such an embodiment, the C-terminal lysine of SEQ ID NO: 12 is absent. Thus, in a preferred embodiment, the formulation of the invention comprises an antibody as defined herein above, characterized by two heavy chains, each heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 12, wherein the C-terminal lysine contained in SEQ ID NO: 12 is absent in one of the heavy chains, preferably in both heavy chains, and two light chains, each light chain comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0129] Additionally or alternatively, the antibody in the formulation of the invention has a heavy chain in which the N-terminal glutamine is replaced by pyroglutamic acid. This pyroglutamic acid formation is also referred to as N-terminal cyclization. Thus, in a preferred embodiment, the formulation of the invention comprises an antibody as defined herein above, characterized by two heavy chains, each heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 12, one of the heavy chains, preferably both heavy chains, having the N-terminal glutamine replaced by pyroglutamic acid, and two light chains, each light chain comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0130] Additionally or alternatively, the antibody in the formulation of the invention has a heavy chain that is N-glycosylated, preferably with an N-linked glycosylation site at Asn 303. Thus, in a preferred embodiment, the formulation of the invention comprises an antibody as defined herein above, characterized by two heavy chains, each heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 12, one of the heavy chains, preferably both heavy chains, being N-glycosylated, preferably at Asn position 303 of the heavy chain, and two light chains, each light chain comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0131] Most preferably, the antibody in the formulation of the present invention has a heavy chain that does not contain a C-terminal lysine, i.e., the C-terminal lysine has been C-terminally lysine-clipped, and the N-terminal glutamine has been replaced with pyroglutamic acid, i.e., the heavy chain has been N-terminally glutaminyl cyclized and N-glycosylated.

[0132] As shown in Example 7, approximately 99% to 100% of the antibodies in a sample of an antibody formulation of the present invention have an N-terminal pyroglutamic acid, and approximately 94% of the antibodies have a loss of a C-terminal lysine. Thus, in one embodiment, at least 90% to 100%, preferably 95% to 100%, of the antibodies in the formulation have an N-terminal pyroglutamic acid modified from an N-terminal glutamine. In one embodiment, at least about 90%, preferably 91%, more preferably 92%, more preferably 93%, more preferably 94%, more preferably 95%, more preferably 96%, more preferably 97%, more preferably 98%, more preferably 99%, and most preferably 99.5% of the antibodies in the formulation have an N-terminal pyroglutamic acid modified from an N-terminal glutamine. In one embodiment, at least 85% to 100%, preferably 90% to 97%, of the antibodies in the formulation have a loss of a C-terminal lysine. In one embodiment, at least about 85%, preferably 86%, more preferably 87%, more preferably 88%, more preferably 89%, more preferably 90%, more preferably 91%, more preferably 92%, more preferably 93%, and most preferably 94% of the antibodies in the formulation have a loss of a C-terminal lysine.

[0133] In a preferred embodiment, the formulations of the invention comprise a therapeutically effective amount of an antibody, preferably at a concentration of about 10-50 mg / mL, wherein the antibody remains stable at 5°C ± 2°C for at least 1 month, preferably up to 24 months, more preferably up to 53 months; or at 25°C ± 2°C for at least 1 month, preferably up to 6 months, or at 40°C ± 2°C for 1 week; or at 5°C ± 2°C for at least 1 month, preferably up to 24 months, more preferably up to 53 months and at 25°C ± 2°C for at least 1 month, preferably up to 6 months; or at 5°C ± 2°C for at least 1 month, preferably up to 24 months, more preferably up to 53 months and at 40 ...6 months; or at 5°C ± 2°C for at least 1 month, preferably up to 6 months and at 40°C ± 2°C for at least 1 month, preferably up to 6 months and at 5°C ± 2°C for at least 1 month, preferably up to 24 months, more preferably up to 53 months and at 25°C ± 2°C for at least 1 month, preferably up to 6 months and at 40°C ± 2°C for at least 1 week.

[0134] In particular, the antibody concentration can be 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL, or any range between 10 mg / mL and 50 mg / mL. In a preferred embodiment, the formulation of the invention contains the antibody at a concentration of 20±5 mg / mL, preferably 20 mg / mL. The antibody is as defined in accordance with the invention, and the formulation of the invention is particularly suitable for implementing the dosing regimen of the invention.

[0135] In the context of the present invention, a "therapeutically effective amount" of an antibody refers to an amount that is effective in preventing or treating a disorder for which the antibody is effective, in this case, ALS or a related disease. The "pharmaceutical formulation" referred to in this invention contains only pharmaceutically acceptable excipients, diluents, and other additives, prepared in a form that allows the active ingredient to be effective, deemed safe by regulatory authorities, and containing no additional components that are toxic to the subject to whom the formulation will be administered. As used herein, the phrase "liquid aqueous pharmaceutical formulation" refers to a pharmaceutical formulation, as defined above, in a liquid state that uses water as a solvent and contains a pharmaceutically active drug in combination with one or more excipients. In this context, the term "pharmaceutical formulation," used by the present invention to define a drug formulation or referred to as a "medicine" and a "drug," respectively, may not be confused with a "test formulation," which is proposed as a "pharmaceutical formulation" that is hypothetically considered but has not yet proven human tolerability and / or efficacy in at least an accepted animal model. In one embodiment, such a "test formulation" is excluded from the scope of the preset invention. In a further embodiment, the liquid, aqueous pharmaceutical formulation of the present invention is a pharmaceutical formulation as defined above that maintains stability (e.g. chemical and / or physical stability and / or biological activity) upon storage without the need for lyophilization, spray drying, and / or freezing.

[0136] As used herein, the term "buffer" or "buffer solution" refers to an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid that resists pH changes. It is well known that both the type and concentration of a buffer can affect protein stability. Therefore, the selection of a buffer is crucial for achieving pH control and the desired stabilization of antibody formulations. With regard to antibody aggregation, the selection of a suitable buffer system and pH play an important role, as they determine the net charge of the protein / antibody structure and the subsequent electrostatic interactions as the acidity or basicity of the solution increases (Chi et al., Pharmaceutical Research 20 (2003), 1325-1336). Increased charge repulsion between charged groups within a dissolved antibody can destabilize its folded or native state (Vermeer and Norde, Biophys. J. 76 (2000), 394-404). In the context of the present invention, an L-histidine / L-histidine monohydrochloride buffer at a pH of about 6.0±1 has been found to be particularly suitable for antibody formulations.

[0137] In one embodiment, the concentration of L-histidine / L-histidine monohydrochloride in the formulation of the invention is 20±10 mM, preferably 20 mM.

[0138] In a further embodiment, any of the formulations of the present invention comprises an osmolality of 310±50 mOsmol / kg. In a preferred embodiment, the osmolality of the formulations of the present invention is 290-320 mOsmol / kg, more preferably 300-310 mOsmol / kg, and most preferably about 307 mOsmol / kg.

[0139] The formulations of the invention allow for intravenous, intraperitoneal, subcutaneous, intramuscular, intranasal, oral, topical or intradermal administration or spinal or brain delivery of antibodies. In a preferred embodiment, the formulations of the invention are designed for intravenous administration.

[0140] In one embodiment, the formulations of the present invention comprise additional excipients, such as antioxidants, tonicity modifiers, and / or surfactants, in any combination of two or all three excipients. On the other hand, compared to the most preferred pharmaceutical formulations illustrated in the examples, one or more excipients may be omitted from the solution or replaced by another excipient due to their known properties.

[0141] As used herein, the term "excipient" encompasses therapeutically inactive ingredients of the pharmaceutical formulation of the present invention. Depending on their individual properties, excipients may be included in the pharmaceutical formulation for a variety of purposes, including, for example, buffers to control pH, carbohydrates as bulking agents for lyophilization, polymers to increase solution viscosity, salts or sugars to stabilize proteins and act as tonicity agents to achieve physiological tonicity and osmolality, surfactants to inhibit protein adsorption to interfaces, preservatives, antioxidants, cryoprotectants, or diluents to prevent microbial growth. Various formulation excipients, including sugars such as sucrose, lactose, or dextrose, polyols (also known as sugar alcohols), such as mannitol or sorbitol, salts such as sodium chloride, amino acids such as arginine, histidine, lysine, aspartic acid, or glutamic acid, surfactants, and water as a solvent, have been shown to stabilize antibodies under different processing conditions and during storage (Paborji et al., Pharm. Res. 11 (1994), 764-771).

[0142] An "antioxidant" or free radical scavenger is a molecule capable of reducing or preventing the oxidation of other molecules. As used herein, the term "tonicity agent" or "isotonizing agent" or "tonicity adjuster" or "tonifier" or "tonicity modifier" refers to an agent that functions to make a liquid aqueous formulation similar in osmotic properties to physiological fluids; in other words, an isotonic pharmaceutical formulation of interest therefore has essentially the same osmotic pressure as human blood. Typical isotonic agents include dextrose, mannitol, sodium chloride, potassium chloride, and glycerin. Isotonic or physiological formulations will generally have an osmotic pressure of about 275-325 mOsm. "Surfactants" as used within the present invention can be nonionic and ionic surfactants. These surfactants reduce the surface tension of protein solutions, reducing the driving force for protein adsorption and / or aggregation on hydrophobic surfaces. Nonionic surfactants are generally preferred for protein stabilization. Low concentrations of nonionic surfactants are often sufficient to prevent or reduce protein surface adsorption and / or aggregation due to their relatively low "critical micelle concentration" (CMC) (Bam et al., Pharm Res. 12 (1995), 2-11). Therefore, the term "surfactant" as used herein refers specifically to nonionic surfactants widely used to stabilize proteins, inhibit aggregation, and assist protein refolding (Chi et al. 2003, supra). The surfactant is preferably a polysorbate, an emulsifier derived from PEGylated sorbitan (a derivative of sorbitol) esterified with fatty acids. Polysorbate 80 and polysorbate 20, also known as Tween 80® and Tween 20®, have been widely incorporated into commercially available protein pharmaceuticals at concentrations ranging from 0.0003 to 0.3%, respectively.

[0143] In one embodiment, the formulation of the present invention comprises an antioxidant. In one embodiment, the antioxidant is an amino acid or amino acid derivative such as L-methionine, L-arginine hydrochloride, or N-acetyl-l-cysteine, or a vitamin such as ascorbic acid, or a thiol. In a preferred embodiment, the antioxidant is an amino acid, most preferably L-methionine. Thus, in a preferred embodiment, the formulation of the present invention comprises L-methionine, preferably at a concentration of 0.1%±0.05% (w / v), most preferably at a concentration of 0.1% (w / v).

[0144] In one embodiment, the formulation of the present invention comprises a tonicity adjusting agent. In one embodiment, the tonicity adjusting agent is selected from the group consisting of sucrose, trehalose, sorbitol, dextrose, mannitol, sodium chloride, potassium chloride, and glycerin, or any combination thereof. In a preferred embodiment, the tonicity adjusting agent is selected from sucrose, trehalose, sorbitol, and sodium chloride, or any combination thereof. Most preferably, the formulation of the present invention comprises sucrose, preferably at a concentration of 8%±1% (w / v), most preferably sucrose at a concentration of 8% (w / v).

[0145] In one embodiment, the formulation of the invention comprises a surfactant, preferably a non-ionic surfactant. In one embodiment, the surfactant is poloxamer 188 or a polysorbate, preferably a polysorbate, preferably polysorbate 80 or polysorbate 20. In a preferred embodiment, the surfactant is polysorbate 80. Thus, in a preferred embodiment, the formulation of the invention comprises polysorbate 80 at a concentration of 0.02% ± 0.01 (w / v), most preferably polysorbate 80 at a concentration of 0.02% (w / v).

[0146] Thus, the formulation of the present invention comprises an antibody as defined herein above in an L-histidine / L-histidine monohydrochloride buffer solution, which may further comprise any one of the excipients mentioned. Accordingly, the present invention relates to a formulation comprising an anti-SOD1 antibody as defined herein above in a concentration of about 10-50 mg / mL, preferably about 20±5 mg / mL, most preferably 20 mg / mL, and an L-histidine / L-histidine monohydrochloride buffer solution having a pH of about 6.0±1, preferably wherein the concentration of the L-histidine / L-histidine monohydrochloride buffer solution is 20±10 mM, most preferably 20 mM, and preferably further comprising: an antioxidant, preferably L-methionine, preferably L-methionine at a concentration of 0.1%±0.05% (w / v), most preferably L-methionine at a concentration of 0.1% (w / v); or a tonicity adjusting agent, preferably sucrose, preferably sucrose at a concentration of 8%±1% (w / v), most preferably sucrose at a concentration of 8% (w / v); or a surfactant, preferably a polysorbate, preferably polysorbate 80, preferably polysorbate 80 at a concentration of 0.02% ± 0.01 (w / v), most preferably polysorbate 80 at a concentration of 0.02% (w / v); or an antioxidant, preferably L-methionine, preferably L-methionine at a concentration of 0.1% ± 0.05% (w / v), most preferably L-methionine at a concentration of 0.1% (w / v), and a tonicity adjusting agent, preferably sucrose, preferably sucrose at a concentration of 8% ± 1% (w / v), most preferably sucrose at a concentration of 8% (w / v); or an antioxidant, preferably L-methionine, preferably L-methionine at a concentration of 0.1% ± 0.05% (w / v), most preferably L-methionine at a concentration of 0.1% (w / v), and a surfactant, preferably polysorbate, preferably polysorbate 80, preferably polysorbate 80 at a concentration of 0.02% ± 0.01% (w / v), most preferably polysorbate 80 at a concentration of 0.02% (w / v); or a tonicity adjusting agent, preferably sucrose, preferably sucrose at a concentration of 8%±1% (w / v), most preferably sucrose at a concentration of 8% (w / v), and a surfactant, preferably polysorbate, preferably polysorbate 80, preferably polysorbate 80 at a concentration of 0.02%±0.01% (w / v), most preferably polysorbate 80 at a concentration of 0.02% (w / v); or an antioxidant, preferably L-methionine, preferably L-methionine at a concentration of 0.1% ± 0.05% (w / v), most preferably L-methionine at a concentration of 0.1% (w / v), and a tonicity adjusting agent, preferably sucrose, preferably sucrose at a concentration of 8% ± 1% (w / v), most preferably sucrose at a concentration of 8% (w / v), and a surfactant, preferably polysorbate, preferably polysorbate 80, preferably polysorbate 80 at a concentration of 0.02% ± 0.01 (w / v), most preferably polysorbate 80 at a concentration of 0.02% (w / v).

[0147] In one embodiment, the formulations of the present invention may include one or more additional excipients, and in preferred embodiments, the formulations of the present invention are substantially free of other additional excipients. In certain preferred embodiments, the formulations of the present invention are substantially free of excipients other than those specifically listed above.

[0148] As used throughout this disclosure of the present invention, the phrase "substantially free" refers to the exclusion of a recited element or group of elements and the optional inclusion of other elements, of similar or different nature to the recited elements, that do not materially alter the basic or novel characteristics of the specified dosing regimen, method, or composition.

[0149] In one embodiment, a formulation of the invention may have any of the compositions described above, provided that the antibody remains stable at 5°C ± 2°C for at least 1 month, preferably at least 3 months, more preferably at least 6 months, more preferably at least 12 months, more preferably at least 18 months, more preferably at least 24 months, more preferably at least 30 months, more preferably at least 36 months, more preferably at least 44 months, and most preferably at least 53 months; at 25°C ± 2°C for at least 1 month, preferably at least 3 months, and most preferably at least 6 months; and / or at 40°C ± 2°C for at least 1 week.

[0150] The stability of the formulations of the present invention can be determined by various parameters such as the presence of aggregates (particularly high molecular weight species (HMWS) and additional low molecular weight species (LMWS)) and monomer content, the presence of acidic and basic species, respectively, and the reduction of antibody in HC and LC, as performed in Example 6.

[0151] Intact IgG antibodies are expressed as tetramers consisting of two heavy chains (HC) and two light chains (LC) linked by disulfide bridges. During production and storage, cleavage of either the HC or LC primary structure can occur by different cleavage mechanisms, resulting in the reduction of the antibody to LC and HC, which can have a negative impact on product safety and efficacy. Therefore, such reduction must be avoided.

[0152] Protein alterations such as aggregation must also be minimized in therapeutic antibody formulations, as aggregation can result in reduced in vivo efficacy, increased batch-to-batch variability of the therapeutic product, and perhaps most importantly, immunogenicity in patients.

[0153] Additionally, there are charge variants of antibodies that can be analyzed by charge-based separation techniques. These variants are generally referred to as acidic or basic species relative to the predominant species. Charge variants can significantly affect the in vitro and in vivo properties of antibodies, and therefore, these variants must also be avoided in therapeutic antibody formulations.

[0154] Therefore, in one embodiment, a formulation of the present invention is considered to be stable when, after storage at 5°C±2°C for 1 to 3 months, preferably 1 to 6 months, more preferably 1 to 9 months, more preferably 1 to 12 months, more preferably 1 to 18 months, more preferably 1 to 24 months, more preferably 1 to 30 months, more preferably 1 to 36 months, more preferably 1 to 44 months, and most preferably 1 to 53 months, it has a content of antibody aggregate species (HMWS and LMWS) of 8% or less, preferably 5% or less, more preferably 3% or less, and more preferably 2% or less, and a monomer content of 92% or more, preferably 95% or more, more preferably 97% or more, and more preferably 98% or more, respectively. In other words, a formulation of the invention is considered stable if it has a content of aggregated species (HMWS and LMWS) of the antibody of 8% or less, preferably 5% or less, more preferably 3% or less, more preferably 2% or less, and a monomer content of 92% or more, preferably 95% or more, more preferably 97% or more, more preferably 98% or more, respectively, after storage for at least 1 month, preferably at least 3 months, more preferably at least 6 months, more preferably at least 9 months, more preferably at least 12 months, more preferably at least 18 months, more preferably at least 24 months, more preferably at least 30 months, more preferably at least 36 months, more preferably at least 44 months, and most preferably at least 53 months at 5° C.±2° C. The content of aggregated species is preferably measured by SEC.

[0155] Additionally or alternatively, formulations of the present invention are considered stable when they have a content of aggregated antibody species (HMWS and LMWS) of 8% or less, preferably 5% or less, and more preferably 4% or less, and a monomer content of 92% or more, preferably 95% or more, and more preferably 96% or more, respectively, after storage at 25°C ± 2°C and 60% ± 5% RH for one to three months, preferably one to six months. In other words, formulations of the present invention are considered stable when they have a content of aggregated antibody species (HMWS and LMWS) of 8% or less, preferably 5% or less, and more preferably 4% or less, and a monomer content of 92% or more, preferably 95% or more, and more preferably 96% or more, respectively, after storage at 25°C ± 2°C and 60% ± 5% RH for at least one month, preferably at least three months, and more preferably at least six months. The content of aggregated species is preferably measured by SEC.

[0156] Additionally or alternatively, formulations of the invention are considered stable if they have an aggregated species (HMWS and LMWS) content of the antibody of 8% or less, preferably 5% or less, more preferably 2% or less, and a monomer content of 92% or more, preferably 95% or more, more preferably 98% or more, respectively, after storage for at least one week at 40°C ± 2°C and 75% ± 5% RH. The aggregated species content is preferably measured by SEC.

[0157] Additionally, or alternatively, formulations of the invention are considered stable if they have a content of 90% or greater, preferably 95% or greater, of unreduced antibody (not reduced to its LC and HC) after storage for 1 to 3 months, preferably 1 to 6 months, more preferably 1 to 12 months, more preferably 1 to 18 months, more preferably 1 to 24 months, more preferably 1 to 30 months, more preferably 1 to 36 months, more preferably 1 to 44 months, and most preferably 1 to 53 months at 5° C. ± 2° C. In other words, formulations of the invention are considered stable if they have a content of 90% or greater, preferably 95% or greater, of unreduced antibody (not reduced to its LC and HC) after storage for at least 1 month, preferably at least 3 months, more preferably at least 6 months, more preferably at least 9 months, more preferably at least 12 months, more preferably at least 18 months, more preferably at least 24 months, more preferably at least 30 months, more preferably at least 36 months, more preferably at least 44 months, and most preferably at least 53 months at 5° C. ± 2° C. The content of unreduced antibody is preferably measured by reduced CS-SDS.

[0158] Additionally or alternatively, formulations of the present invention are considered stable if they have a content of 90% or more, preferably 95% or more, of unreduced antibody (unreduced to its LC and HC) after storage for 1 to 3 months, preferably 1 to 6 months. In other words, formulations of the present invention are considered stable if they have a content of 90% or more, preferably 95% or more, of unreduced antibody (unreduced to its LC and HC) after storage for at least 1 month, preferably at least 3 months, more preferably at least 6 months at 25°C ± 2°C and 60% ± 5% RH. Unreduced antibody content is preferably measured by reduced CS-SDS.

[0159] Additionally or alternatively, formulations of the invention are considered stable if they have a content of unreduced antibody (unreduced to its LC and HC) of 90% or more, preferably 95% or more, after at least one week of storage at 40°C ± 2°C and 75% ± 5% RH, preferably as measured by reduced CS-SDS.

[0160] Additionally or alternatively, formulations of the invention are considered stable if they have an antibody acidic species content of 45% or less, preferably 40% or less, more preferably 35% or less, more preferably 30% or less, more preferably 27% or less after storage at 5°C ± 2°C for 1 to 3 months, preferably 1 to 6 months, more preferably 1 to 9 months, more preferably 1 to 12 months, more preferably 1 to 18 months, more preferably 1 to 24 months, more preferably 1 to 30 months, more preferably 1 to 36 months, more preferably 1 to 44 months, and most preferably 1 to 53 months. In other words, a formulation of the invention is considered stable if it has an antibody acidic species content of 45% or less, preferably 40% or less, more preferably 35% or less, more preferably 30% or less, more preferably 27% or less after storage for at least 1 month, preferably at least 3 months, more preferably at least 6 months, more preferably at least 9 months, more preferably at least 12 months, more preferably at least 18 months, more preferably at least 24 months, more preferably at least 30 months, more preferably at least 36 months, more preferably at least 44 months, and most preferably at least 53 months at 5° C.±2° C. The acidic species content is preferably measured by cIEF.

[0161] Additionally, or alternatively, formulations of the invention are considered stable if they have a content of basic antibody species of 15% or less, preferably 10% or less, and more preferably 7% or less after storage for 1 to 3 months, preferably 1 to 6 months, more preferably 1 to 9 months, more preferably 1 to 12 months, more preferably 1 to 18 months, more preferably 1 to 24 months, more preferably 1 to 30 months, more preferably 1 to 36 months, more preferably 1 to 44 months, and most preferably 1 to 53 months at 5° C. ± 2° C. In other words, formulations of the invention are considered stable if they have a content of basic antibody species of 15% or less, preferably 10% or less, and more preferably 7% or less after storage for at least 1 month, preferably at least 3 months, more preferably at least 6 months, more preferably at least 9 months, more preferably at least 12 months, more preferably at least 18 months, more preferably at least 24 months, more preferably at least 30 months, more preferably at least 36 months, more preferably at least 44 months, and most preferably at least 53 months at 5° C. ± 2° C. The content of basic species is preferably measured by cIEF.

[0162] Additionally or alternatively, formulations of the present invention are considered stable if they have an antibody acidic species content of 45% or less, preferably 40% or less, more preferably 35% or less, more preferably 30% or less, and more preferably 27% or less after storage for 1 to 3 months, preferably 1 to 6 months. In other words, formulations of the present invention are considered stable if they have an antibody acidic species content of 45% or less, preferably 40% or less, more preferably 35% or less, more preferably 30% or less, and more preferably 27% or less after storage at 25°C ± 2°C and 60% ± 5% RH for at least 1 month, preferably at least 3 months, more preferably at least 6 months. Acidic species content is preferably measured by cIEF.

[0163] Additionally or alternatively, formulations of the invention are considered stable if they have a basic antibody species content of 15% or less, preferably 10% or less, and more preferably 7% or less after storage for 1 to 3 months, preferably 1 to 6 months. In other words, formulations of the invention are considered stable if they have a basic antibody species content of 15% or less, preferably 10% or less, and more preferably 7% or less after storage at 25°C ± 2°C and 60% ± 5% RH for at least 1 month, preferably at least 3 months, more preferably at least 6 months. The basic species content is preferably measured by cIEF.

[0164] Additionally or alternatively, formulations of the invention are considered stable if they have an antibody acidic species content of 45% or less, preferably 40% or less, more preferably 35% or less, more preferably 30% or less, more preferably 27% or less after at least one week of storage at 40°C ± 2°C and 75% ± 5% RH, where the acidic species content is preferably measured by cIEF.

[0165] Additionally or alternatively, formulations of the invention are considered stable if they have a content of basic species of the antibody of 15% or less, preferably 10% or less, more preferably 7% or less after at least one week of storage at 40°C ± 2°C and 75% ± 5% RH, the content of basic species preferably being measured by cIEF.

[0166] Additionally, the potency of the antibody and the appearance of the formulation, particularly clarity, color and / or visible particles, pH, osmolality, and particulate matter, should be stable during storage.

[0167] Therefore, in one embodiment, the antibody in the formulation of the present invention has a relative potency of 60% to 140%, more preferably 70% to 130%, more preferably 80% to 120%, more preferably 90% to 110%, and most preferably 93% to 110% under all of the above storage conditions. For details, see Tables 14 to 17. Potency is preferably determined by FRET binding.

[0168] Most preferably, the formulations of the invention comprise or consist essentially of 20 mg / mL anti-SOD1 antibody, preferably AP-101, in 20 mM L-histidine / L-histidine monohydrochloride buffer at pH 6.0, 8% (w / v) sucrose, 0.1% (w / v) L-methionine and 0.02% (w / v) polysorbate 80, and preferably exhibit any one or any one combination of the stability criteria described above.

[0169] The present invention also relates to the treatment of ALS and further neurodegenerative diseases, such as AD or PD, associated with SOD1 accumulation, by administering the above-described antibodies according to the dosing regimens of the present invention. Preferably, the antibodies are administered in the form of the formulations and medicaments of the present invention, respectively. The method may further comprise diagnosing the treated subject as described above and / or monitoring the therapeutic effect of the antibodies and the progression of ALS as described above, for example using the highly sensitive immunoassays disclosed in WO 2021 / 185961 A1; see above.

[0170] The present invention further relates to a medicament comprising the formulation of the present invention. In one embodiment, the present invention relates to a medicament of the present invention for use in treating a disease associated with SOD1 accumulation, preferably ALS. In one embodiment, the present invention relates to a medicament of the present invention for use according to the present invention.

[0171] The present invention further relates to a pharmaceutical container containing the formulation and medicament of the present invention, respectively. In one embodiment, the container is a pre-filled syringe, a pen-type injector, an ampoule, a bottle, an auto-injector, a glass vial, or an infusion bag. In a preferred embodiment, the container of the present invention is an infusion bag or an A-class vial, more preferably a glass vial, and most preferably a single-use glass vial. In one embodiment, the container of the present invention is a single-use glass vial sealed with a rubber stopper and a flip-off cap. In one embodiment, the glass and the container are each an 8 ml glass vial.

[0172] In one embodiment, the container of the invention is a glass vial as defined above, providing approximately 100 mg of antibody at a concentration of 20 mg / mL. Thus, the dosage strength of one vial is 100 mg. In one embodiment, the container of the invention, particularly the vial, comprises approximately a 10% volume overfill.

[0173] Several documents are cited throughout the text of this specification. The contents of all cited references (including literature references, issued patents, published patent applications cited throughout this application including the background section, and manufacturer's specifications, instructions, etc.) are expressly incorporated herein by reference, without any admission that any of the cited documents are in fact prior art to the present invention.

[0174] A more complete understanding can be obtained by reference to the following specific examples, which are provided herein for illustrative purposes only and are not intended to limit the scope of the invention. [Example]

[0175] Example 1: Phase 1 trial of AP-101 in ALS patients A multicenter, open-label, single-ascending dose (SAD) study in patients with familial and sporadic ALS was conducted to evaluate the safety, tolerability, and pharmacokinetics of intravenous (IV) administration of AP-101 in humans.

[0176] Briefly, AP-101 is a fully human IgG1 antibody with high affinity and selective binding to misfolded SOD1. A 3+3 study design was utilized, with expansion to n=6 for each cohort possible if safety signals were observed in the first three participants. After a 28-day screening period, AP-101 was administered via IV infusion at SAD levels of 100 mg (Cohort 1), 500 mg (Cohort 2), and 2500 mg (Cohort 3) in three cohorts. Participants were observed in-situ for 24 hours after dosing and attended scheduled clinic visits for safety assessments and pharmacokinetic (PK) sample collection up to 12 weeks after dosing. CSF samples were collected at screening for exploratory measurement of misfolded SOD1. Nine patients, three in each cohort, completed the study. One SAE of lower back pain was experienced at the lowest dose, but was not treatment-related. Overall, AP-101 was found to be safe and well tolerated when administered as a single IV infusion. No toxicity or reactions related to administration of the drug were experienced up to doses of 2500 mg.

[0177] Participants were over 18 years of age, diagnosed with ALS according to the El Escorial criteria (Brooks et al., Amyotroph Lateral Scler Other Motor Neuron Disord. 1 (2000), 293-299), and genetic testing was performed to determine whether participants had a pathogenic SOD1 mutation. ALS symptom onset, specifically muscle weakness, occurred within the last 48 months prior to study enrollment. A standing vital capacity (SVC) of 60% or greater predicted for age, height, sex, and ethnicity at screening was required for inclusion. Concomitant use of both riluzole and edaravone was permitted if participants had been on a stable dose for 30 days or more or completed two or more cycles of each prior to screening. Participants using noninvasive ventilation (NIV) for more than 4 hours per day were excluded from the study, as were participants taking other investigational drugs for ALS or who had previously been exposed to stem cell treatment within the 30 days prior to enrollment.

[0178] Definitions and Methods Adverse events (AEs) A clinical trial AE was defined as any untoward medical event associated with the use of an investigational drug, whether or not considered drug-related, for the studies described below. Any injection / infusion site reaction experienced was captured as an AE, and the time of day, time relative to injection / infusion, size, amount of erythema, induration, and pruritus were recorded. The severity of all AEs was graded according to the National Cancer Institute-Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 5.0. For AEs not specifically graded by these criteria, the following criteria were applied: For each episode, the highest severity grade obtained was reported.

[0179] Grade 1: Mild; asymptomatic or mildly symptomatic; clinical or diagnostic observation only; no intervention indicated. Grade 2: Moderate; minor, local or non-invasive interventions are applicable; limitations in age-appropriate instrumental activities of daily living (ADL). Grade 3: Severe or medically significant, but not immediately life-threatening; hospitalization or extended hospitalization is indicated; disabling; limiting personal ADL. Grade 4: Life-threatening consequences; urgent intervention is indicated. Grade 5: Death related to an AE.

[0180] Instrumental ADLs include preparing meals, shopping for groceries or clothes, using the telephone, managing money, etc. Personal ADLs include bathing, dressing, feeding oneself, using the toilet, taking medication, and not being bedridden.

[0181] Clinical laboratory evaluation If a study patient experienced an elevation of alanine aminotransferase (ALT) at least 3 times and / or more than 3 times (≥ 3X) the upper limit of normal (ULN), alkaline phosphatase (ALP) ≥ 2X ULN, or an elevation of total bilirubin (TBL) ≥ 2X ULN, liver tests (within 3-5 days, including ALT, aspartate aminotransferase (AST), ALP, TBL, direct bilirubin, gamma-glutamyltransferase (GGT)) along with creatinine kinase were repeated to confirm the abnormality, determine whether it was associated with liver toxicity, and determine whether it was increasing or decreasing. If the abnormality persisted or worsened, clinical and laboratory monitoring was initiated by the investigator in consultation with the sponsor. Monitoring continued until levels normalized and / or returned to approximately baseline levels. Patient follow-up in consultation with the sponsor was required in addition to these selected safety studies in case of clinical indications of liver abnormalities emerging during treatment.

[0182] Vital signs measurement Vital signs (blood pressure [BP], heart rate, respiratory rate, and temperature) were measured at predetermined time points. Single measurements were performed at screening and at study site entry. Three supine BP and pulse rate measurements were collected at all other time points. Temperature was obtained as a single measurement. Pre-dose vital signs were measured approximately 1 hour before scheduled dosing. At the time the upright measurement was obtained, the patient had been supine for at least 5 minutes and had been standing for approximately 2 minutes. If three BP or pulse rate measurements preceded the upright measurement, the last supine BP or pulse rate measurement was used for the upright calculation. If the patient felt unable to stand, only supine vital signs were recorded. If CS worsening was observed, the change was documented as an AE in the eCRF.

[0183] Physical examination Physical examinations were performed based on the investigator's discretion. If physical examination CS findings were observed at baseline, they were recorded as medical history. Otherwise, they were recorded as an AE. Subjects' height was measured during the initial physical examination performed during the screening visit. Weight was measured at predetermined time points. If CS worsening was observed, the change was documented as an AE in the eCRF. The investigator continued to monitor the subject until the parameters returned to their baseline state or until an agreement was reached between the investigator and the sponsor.

[0184] Neurological examination Directed neurological examinations were performed by the investigator or designee at predetermined time points. If abnormalities were noted at these time points, additional testing was performed until the patient returned to baseline. Mandatory examination components included cranial nerve examination, tremor, extraocular movements, brachial and patellar deep tendon reflexes, finger-to-nose test, and Romberg's sign.

[0185] electro-cardiogram A standard 12-lead ECG was recorded. The ECG was interpreted at the site by a qualified physician (investigator or qualified designee) as soon as possible after the time of ECG collection. The investigator or qualified designee was responsible for documenting whether the ECG was normal or abnormal; if abnormal, whether the ECG tracing was NCS or CS. CS worsening was to be reported as an AE.

[0186] C-SSRS C-SSRS (Mental Health Assessed by Suicidal Tendencies) was collected at predetermined time points. The C-SSRS questionnaire assesses suicidal ideation, its potential intensity, and the behaviors intended to implement these tendencies. Each patient completed the C-SSRS twice at screening, once relating to their entire life, and once relating to the past month / year. Each patient completed the questionnaire again at EOS, and changes in responses to each question were recorded.

[0187] AP-101 concentration measurement AP-101 concentrations in human serum and CSF were determined using an immunoassay with electrochemiluminescent (ECL) detection at Nexelis (Seattle, WA) and confirmed in the 100.0–3200.0 ng / mL range (serum) and 10.0–1280.0 ng / mL range (CSF).

[0188] ALSFRS-R Each patient's responses to the ALSFRS-R (functional in life skills for ALS patients) questionnaire were collected over the course of the study. The ALSFRS-R is a clinically validated assessment tool for monitoring the progression of disability in ALS patients in terms of life skill function. Its scores correlate significantly with the results of tests using the Sickness Impact Profile (Cedarbaum et al., Neurol. Sci. 169 (1999), 13-21; Damiano et al., Med. Care 37 (1999), 15-26), which is commonly used to measure health and quality of life. The ALSFRS-R consists of 12 categories of life skills, each scored from 0 to 4. A score of zero (0) represents complete loss of function, and a score of 4 represents full function (Cedarbaum et al. 1999, supra). The overall ALSFRS-R score was calculated as the sum of the responses to the 12 questions. The ALSFRS-R was completed by each patient at screening, pre-treatment (day 1), follow-up (day 28), and EOS (day 84). Change from baseline in the ALSFRS-R was calculated and presented for each patient.

[0189] SVC Changes in respiratory function, measured as SVC%, were assessed for each patient over the study time course. SVC is the volume of air expelled during slow, unforced expiration and is a standard means of assessing airflow limitation. Exhalation results in part from the elastic recoil of the respiratory system, in addition to the activity of expiratory muscles. Elastic recoil is typically lost in ALS due to poorly understood degeneration of respiratory motor neurons and associated declines in respiratory muscle control and plasticity. SVC correlates with clinically significant events in ALS, such as the use of assisted ventilation and tracheostomy, and ALS patients with slower declines in SVC have been shown to live longer. Based on these findings, SVC has been used as a clinical prognostic marker for disease progression in ALS (Andrews et al., JAMA Neurol 75(2018),58-64). SVC percent (%) is a measure of respiratory function and is predicted to decline over time as the disease progresses in ALS patients. Upright SVC% was determined by performing 3-5 measurements at screening, pre-dose (Day 1), follow-up (Day 28), and EOS (Day 84). Changes from baseline to Day 28 and to EOS were calculated and listed for each patient.

[0190] Safety Endpoint Methods Safety analyses were performed according to SAP for all enrolled patients, regardless of whether they completed all protocol requirements. For continuous variables, summary statistics included number of patients, mean, median, standard deviation (SD), minimum, and maximum. Categorical endpoints were summarized using number of patients, frequency, and percentage.

[0191] All treatment and protocol procedural AEs were recorded and listed. Safety data were summarized using descriptive methodology when the frequency of events allowed.

[0192] Pharmacokinetic Endpoint Methods PK analysis was performed on all patients who received the study drug and had evaluable PK. Both noncompartmental analysis (NCA) and a two-compartment model were utilized to elucidate the PK of AP101. In NCA, serum concentrations of AP-101 and actual sample collection times were used to determine individual PK parameters using standard noncompartmental procedures in R 4.0.4 (Vienna, Austria). The primary PK parameters calculated included C max (maximum observed drug concentration), t max (time to maximum observed drug concentration), AUC 0-t , AUC 0-inf (area under the drug concentration-time curve), t 1 / 2 (terminal elimination half-life), and λz (first-order terminal elimination rate constant). A two-compartment model was developed using NONMEM, version 7.4 (San Francisco, CA). CL and volume of distribution (V d ) were predicted from the model rather than from NCA, assuming a two-compartment PK profile exhibited by AP-101. Individual serum AP-101 PK parameters were calculated using NCA. Actual sampling times were used for parameter derivation.

[0193] Clinical trial design and evaluation This was a non-randomized, open-label, first-in-human, single-ascending-dose (SAD) trial. A "3+3" study design was utilized, allowing for expansion of each cohort to n=6 if safety signals were observed in the first three participants in that cohort. After a 28-day screening period, participants received a single dose of AP-101 intravenously (IV) at a dose level determined by the participant's cohort: 100 mg in Cohort 1, 500 mg in Cohort 2, or 2500 mg in Cohort 3.

[0194] Specifically, AP-101 was administered via IV infusion over a period of at least 60 minutes and up to 120 minutes. AP-101 drug product was formulated for IV administration according to Examples 3 and 4. AP-101 for injection was supplied as a solution in single-use glass vials. Vials were manufactured to provide 100 mg of AP-101 at a concentration of 20 mg / mL. To ensure complete withdrawal and delivery of the labeled amount of 100 mg of AP-101, the vials contained an approximately 10% volume overfill. The drug product was filled into Type I glass vials with rubber stoppers and flip-off caps. The investigational product was administered as an IV infusion over a period of at least 60 minutes and up to 120 minutes. The infusion volumes were 5 ml, 25 ml, and 125 ml to administer doses of 100 mg, 500 mg, and 2500 mg of AP-101.

[0195] Using a 21-day sentinel dosing period in Cohort 1 and a 7-day sentinel dosing period in Cohorts 2 and 3, safety data were reviewed by a Safety Review Committee (SRC) prior to dosing of subsequent participants in that cohort. Participants were housed for a minimum of 24 hours after dosing and returned to the clinical trial site for safety assessments and PK sample collection for up to 12 weeks after dosing.

[0196] Primary endpoints were the incidence of treatment-emergent adverse events (TEAEs) and serious adverse events (SAEs) and the incidence of abnormalities in clinical safety measures (vital signs, laboratory assessments, physical examination, neurological examination, and electrocardiogram (ECG)). Suicidal ideation, as is typical for investigational drugs for neurological indications, was assessed using the Columbia-Suicide Severity Rating Scale (C-SSRS). Secondary endpoints included pharmacokinetic measures in both serum and cerebrospinal fluid (CSF). Exploratory endpoints included changes in quality of life and function, as assessed by the ALS Functional Rating Scale-Revised (ALSFRS-R), and changes in respiratory function, as assessed using the SVC. CSF levels of misfolded SOD1 were also included as an exploratory measure.

[0197] statistical analysis The full analysis set (FAS) consisted of all enrolled participants, consisting of nine participants who received one dose of AP-101. All treatment and protocol-related adverse events (AEs) and their frequency were recorded, categorized, and summarized using descriptive methods. Summary statistics for each cohort were compiled by dose level. Safety parameters for this study were summarized using standard descriptive statistics. Results of individual question responses on the C-SSRS assessment were listed by participant, visit, time point, and dose. No further analysis was performed on the C-SSRS results.

[0198] Serum AP-101 PK parameters (e.g., C max and AUC) were calculated using noncompartmental analysis (NCA) and two-compartment modeling. Actual sampling times were used for parameter derivation. PK parameters were summarized by dose level using descriptive statistics, and mean and individual AP-101 serum concentration-time curves were derived. CSF PK, as a secondary objective, was obtained at unique time points for each participant in Cohorts 2 and 3.

[0199] Numerical responses to each ALSFRS-R question were listed by time point, participant, and cohort. Changes in participants' total ALSFRS-R scores from baseline to day 28 and to the end of study (EOS) were reported. Results of individual SVC tests, measured as SVC%, were listed according to time point, participant, and cohort. Changes in SVC% from baseline to day 28 and to EOS were calculated and reported.

[0200] result Nine participants were screened and enrolled in AP-101-01. All participants met El Escorial criteria for a diagnosis of ALS, including eight with definite or probable sALS and one with fALS due to a pathogenic variant in the SOD1 gene. Three participants were enrolled in each dosing cohort, and all completed the study.

[0201] Participant characteristics are detailed in Table 1. Due to the small sample size of this safety study, variability in baseline characteristics is observed.

[0202] [Table 1]

[0203] safety Overall, AP-101 was found to be safe and tolerable following a single IV infusion. Forty-seven treatment-emergent adverse events (TEAEs), defined as AEs occurring after administration of the study drug, were reported: 20 in the 100 mg dose phase, 6 in the 500 mg dose phase, and 21 in the 2500 mg dose phase. Of treated patients, eight (89%) experienced a TEAE. All patients in Cohorts 1 and 3 (100%) and Cohort 2 (67%) experienced an AE. The most reported TEAEs were falls (7 events), headache (6 events), and fatigue (3 events). Two grade 2 non-ALS TEAEs were recorded: one fatigue event and one back pain event, but none of the TEAEs were treatment-related. No dose-limiting toxicities, treatment-related TEAEs, or reactions related to the administration of the study drug were observed.

[0204] No abnormal laboratory results were recorded in this study. No clinically significant (CS) abnormalities in vital signs were observed. All but one patient (89%) experienced at least one abnormal physical examination, and all patients (100%) experienced at least one abnormal neurological examination across all time points. Six of nine patients (67%) experienced abnormal ECG recordings across all time points: one patient in Cohort 1 (33% of Cohort 1, N=1), two patients in Cohort 2 (67%, N=2), and three patients in Cohort 3 (100%, N=3). All but one patient experienced abnormal readings pre-dose.

[0205] Most baseline physical and neurological examination screening results were abnormal, and the majority of these results were attributed to ALS. The majority of participants had no shifts from baseline in physical and neurological examination results. Non-ALS-related abnormal findings at later time points after screening included musculoskeletal injuries and neuropathy attributed to clinical laboratory chemistry sampling. Rash, gingival abscess, and wheezing were also recorded. All abnormal laboratory results were not clinically significant (NCS). ECG abnormalities recorded as shifts from baseline were common, but none were associated with AEs and all were considered NCS. No correlation between study drug administration and patients' physical condition or cardiac function was evident.

[0206] Changes in mental health status were measured using the C-SSRS. One participant indicated having had suicidal ideation during their lifetime, but no participants had suicidal ideation or behavior either before or recent to screening or at the end of the study.

[0207] Pharmacokinetics (PK) Blood samples for PK analysis were collected pre-dose and at the following time points after IV administration of AP-101: 5 minutes and 0.25, 0.5, 1, 4, 8, 12, 24, 48, and 72 hours post-dose. AP-101 median t max The time to the end of the IV infusion was 0.0833-7.63 hours (min-max) for all three dose levels. Of note, AP-101 concentrations showed unusual behavior toward the end of the IV infusion, with t max did not occur at the first time point after infusion. AP-101 exposure generally increased proportionally with dose. Variability (CV%) was observed for the exposure (C max and AUC 0-inf ) is moderate to high, and C max 25.9-35.1 and AUC 0-inf In the 500 mg dose group, two patients had AUC values ​​greater than 20% of the extrapolated values. 0-inf One patient in the 2500 mg dose group also had an AUC 0-infThe geometric mean elimination half-life (t 1 / 2 ) values ​​appeared similar at the 100 mg and 500 mg doses and were lower at the 2500 mg dose. 1 / 2 The PK model predicted value for was approximately 244 hours.

[0208] efficacy Assessment of change from baseline in ALSFRS-R score and change from baseline in SVC% were included as exploratory efficacy endpoints. Baseline misfolded SOD1 levels in CSF were performed as an exploratory analysis.

[0209] The ALSFRS-R was collected at screening, pre-treatment (baseline), day 28, and EOS. The ALSFRS-R was assessed as a change from baseline (pre-treatment) in functional status loss or gain at EOS on a summed score. A decline in overall functional status was calculated for five participants, with declines ranging from 1 to 8 points on the ALSFRS-R total score. Two participants showed no functional change, and two participants showed a gain in functional status, with a 2- to 4-point gain on the ALSFRS-R.

[0210] SVC was planned to be collected at screening, pre-dose (baseline), day 28, and EOS; however, due to staffing constraints and COVID-19 restrictions, five SVC time points were unsuccessful for four participants. Additionally, SVC could not be assessed at EOS for three participants and at baseline for one additional participant. Recognizing these variables in the data, overall SVC% declined from baseline to last observed SVC (day 28 for three participants), with declines ranging from 2% to 32%. One participant experienced no change in SVC%, and two participants experienced an increase in SVC% of 3% to 5% from baseline to EOS.

[0211] summary In summary, considering the data surrounding misfolded SOD1 pathogenesis in both sALS and SOD1-fALS, AP-101 is a fully human IgG1 antibody with high affinity and selective binding to misfolded SOD1 protein. This was a first-in-human, single-ascending-dose, non-randomized, open-label study of AP-101 conducted at multiple centers in Canada. Overall, AP-101 was found to be safe and tolerable following a single IV infusion. There was no correlation between TEAEs, SAEs, or other abnormal findings and study drug administration. No dose-limiting toxicities or IV infusion-related reactions were observed.

[0212] PK analysis indicates that AP-101 exposure is generally dose-proportional, with biexponential clearance (see Figure 2), although as explained above, the PK profile also varies. The time versus concentration profile indicates that AP-101 is slowly eliminated from the body. In the lowest dose group (100 mg), the mean concentration was 731 ng / mL at Day 84 (2016 hours). Analysis and further modeling support the conclusion that AP-101 has a PK profile consistent with every-3-week dosing and that a 500 mg loading dose may help rapidly achieve a desirable steady state. These analyses suggest that AP-101 elimination is slow and that every-3-week dosing will provide sufficiently consistent levels of drug.

[0213] Changes from baseline in both ALSFRS-R and SVC were included as exploratory measures, and an overall greater decline in function than gain was experienced. Because there was no control group or enrollment strategy that accounted for heterogeneity in disease status, no conclusions can be drawn regarding the effect of AP-101 on function or respiratory status.

[0214] Example 2: Phase 2a Study of AP-101 in ALS Patients A Phase 2a, multicenter, randomized, double-blind, placebo-controlled study of AP-101 in patients with familial amyotrophic lateral sclerosis (fALS) and sporadic amyotrophic lateral sclerosis (sALS) is being conducted to evaluate safety, tolerability, pharmacodynamic (PD) markers, and pharmacokinetics (PK); see also ClinicalTrials.gov Identifier: NCT05039099. AP-101 has been administered to humans in a single-ascending dose study and was found to be safe and tolerable up to doses including 2500 mg (see Example 1).

[0215] Two participant cohorts will participate in this study: patients with fALS and those with sALS who have confirmed mutations in SOD1. Patients in each cohort will be randomized in a 2:1 ratio to receive either AP-101 treatment or placebo. Patients will be permitted to continue taking riluzole or edaravone for the duration of the study, depending on the inclusion / exclusion criteria. After completing six months of treatment, participants will be invited to participate in a six-month open-label extension (OLE), which will continue to evaluate the long-term safety of AP-101. Along with safety and tolerability, the goal of this study is to determine the impact of AP-101 on measurable levels of several ALS-related biomarkers. The revised ALSFRS-R is a recognized clinical endpoint of functional impact on disease progression in ALS (FDA 2019; EMA 2015). Based on these lines of evidence, the study will examine the effects of AP-101 on a number of potential biomarkers of disease, including SOD1, misfolded SOD1, pNfH, and NfL. The primary and secondary objectives of the study are outlined in the study details for this Phase 2a study, published at ClinicalTrials.gov Identifier: NCT05039099.

[0216] Potential participants who have had symptom onset within 24 months of screening will be screened within 28 days prior to baseline (Day 1). Study eligibility for each participant will be reviewed based on all enrollment criteria outlined below. The screening procedures are displayed in the SoE set forth in Table 2.

[0217] [Table 2] JPEG2025527710000005.jpg245170

[0218] Abbreviations: ALS = amyotrophic lateral sclerosis; ALSFRS R = Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised; ALSSQOL-SF = Amyotrophic Lateral Sclerosis-Specific Quality of Life Scale-Short Form; CSF = cerebrospinal fluid; CSSR S = Columbia-Suicide Severity Rating Scale; ECG = electrocardiogram; HBsAg = hepatitis B virus surface antigen; HCV = hepatitis C virus; HHD = handheld dynamometry; IV = intravenous; PK = pharmacokinetics; SVC = vital capacity; W = week. Participants who discontinue the study at any time point (end of treatment period, end of 6-month OLE, or early discontinuation for any reason) must return to the site 28 days after their last dose of treatment and undergo all assessments scheduled for the 28-day visit after their last dose, and then return to the site 8 and 12 weeks after their last dose of treatment and undergo all assessments scheduled per the SOE. A ±3-day visit allowance period allows flexibility in scheduling (i.e., if dosing occurs on a Friday, the next visit can be scheduled for Monday). Note: If multiple procedures occur at the same time point, the following order of procedures should be used: ECG, vital signs, and venipuncture. Unless otherwise specified, pre-dose samples may be collected any time prior to dosing based on the study site activity schedule. After randomization, all participants will receive either placebo or AP-101 according to the schedule of events. On Day 1, participants will receive 500 mg of AP-101 or placebo, and on Day 2, participants will receive 2500 mg of AP-101 or placebo. Thereafter, the dosing schedule will follow the SoE with 2500 mg of AP-101 or placebo every 3 weeks starting on Day 22. The observation period after IV infusion will be 1 hour or can be extended to 3-6 hours according to local facility procedures if an infusion-related reaction occurs. b The call will be conducted approximately 24 hours after the baseline dose. c Pulse oximetry can be performed at any time prior to administration and can be performed between other assessments. d Survival endpoints will include time to event measures including time to death, tracheotomy for ventilation, or noninvasive ventilation use for 22–24 hours per day. e A single ECG will be collected at screening. Three ECGs will be collected at all other time points. A minimum of 1 minute will be allowed between each ECG. f A single serum PK sample will be collected immediately after injection pre-dose and during all visits except Week 24, where PK sampling will be performed pre-dose and 4 (± 2) hours post-dose. At Day 28, Week 8, and Week 12 after the last dose, only a single PK sample will be collected and may be performed at any time between visits. The time of PK sampling is intended to be a guideline and may be modified to accommodate clinical procedures. The actual date and time of the sample must be collected. For participants who discontinue early, samples should be obtained at the ED visit. g CSF samples for PK and biomarkers: Lumbar punctures (LP) at weeks 12 and 24 may be performed 1-3 days prior to dosing.

[0219] Participants will receive a 500 mg AP-101 loading dose on Day 1, followed by the regularly scheduled dose of 2500 mg beginning on Day 2. After Day 2, dosing will occur once every 3 weeks for a period of 6 months, according to the SoE (Table 2), beginning on Day 22. After the last dose or early discontinuation for any reason at any time during the study, participants will be followed for safety and PK for 3 months after their last dose of study treatment. Safety / tolerability, PK, and exploratory assessments will be conducted at the stated time points throughout the study. Cerebrospinal fluid (CSF) and other biological samples for biomarker analysis will also be collected from each participant according to the SoE. Efficacy measures (Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised [ALSFRS-R], vital capacity [SVC], pulse oximetry, handheld dynamometry [HHD]) and King's Staging Tool will be assessed according to the SoE.

[0220] After completing 6 months of treatment, participants will be invited to participate in a 6-month open-label extension (OLE) to evaluate the long-term safety of AP-101. If participating in the OLE, patients will complete planned safety and PK follow-up after their final dose of AP-101.

[0221] A maximum of 63 participants (21 with fALS and 42 with sALS) will be randomized to the study intervention, such that approximately 18 evaluable participants with fALS and 36 evaluable participants with sALS will complete the study. Participants who discontinue the study before completion of all study activities may be replaced at the sponsor's discretion. Participants will complete the study when all planned procedures for the 24-week dosing and 28-day follow-up visit are completed as indicated in the SoE.

[0222] It is expected that participants' total study duration will be approximately 10 months, including screening, the 6-month treatment period, and the 3-month safety follow-up period. For participants who subsequently enter the 6-month OLE, the 3-month safety follow-up period will occur at the end of the OLE or after early discontinuation of the study for any reason. Participants will be screened within 28 days prior to enrollment in each group and randomized to either AP-101 2500 mg IV or placebo, administered according to the SoE, during the 6-month treatment period. After completion of the treatment period, participants may continue in the study as part of the OLE if they complete all study visits during the treatment period. All study treatment will be administered at the study site. Drug PK results (exposure) and total body clearance calculated after intravenous (IV) administration [CL], if available, will be used as supporting data for dose modifications.

[0223] Participants were considered to have completed the study when all scheduled procedures for the 24-week dosing and 28-day follow-up visits were completed, as indicated in the SoE (Table 2). The end of the study was defined as the day after the last participant completed 6 months of study treatment and any applicable follow-up visits.

[0224] Dosage rationale The proposed dose of 2500 mg AP-101 was selected based on a dose-ranging first-in-human (FIH) study in ALS patients (NCT03981536) and a toxicity study in cynomolgus monkeys (Study 8384444). This dose level was confirmed to have an acceptable safety and tolerability profile after a single IV administration. The study is designed with two loading doses on consecutive days, followed by repeat dosing once every three weeks for six months. Based on in silico modeling of pharmacokinetic (PK) data from the FIH study, the dosing schedule was designed to allow patients to reach steady state as quickly as possible. The dosing frequency is based on maintaining steady state throughout the treatment phase of the study.

[0225] Based on available preclinical, toxicological, and clinical data, a loading dose of 500 mg and 2500 mg given on consecutive days, followed by 2500 mg IV dosing once every three weeks, is proposed. The dose escalation is based on preclinical efficacy studies conducted in a mouse transgenic model of ALS, which suggested that weekly intraperitoneal dosing of mouse-chimeric α-miSOD1 at 3, 10, or 30 mg / kg (180–1800 mg dose equivalent; FDA 2005) demonstrated efficacy at specific endpoints. Furthermore, no overt toxicity (weight loss) was observed in these animals.

[0226] The potential for AP-101 to produce toxic or undesired effects was evaluated in a 1-month study in cynomolgus monkeys (Study 8384444), which consisted of two IV injections, 2 weeks apart, each followed by an approximately 2-week post-dose observation period. AP-101 binds to human and monkey SOD-1, making monkeys a relevant toxicological species; AP-101 does not bind to rodent SOD-1. No AP-101-related effects on toxicological or safety pharmacology parameters occurred at doses up to 400 mg / kg (24,000 mg in a 60 kg human), including the highest dose tested. Therefore, 400 mg / kg is considered a no-observed-adverse-effect level (NOAEL).

[0227] In this 1-month study, monkeys received bolus IV injections of 10, 60, or 400 mg / kg AP-101 on study days 1 and 15, with subsequent necropsies performed on study day 29. The highest dose was selected to provide a multiple of the proposed human dose. Toxicity assessment was based on mortality, clinical observations, body weights, ophthalmologic observations, clinical pathology (hematology, coagulation, clinical chemistry, and urinalysis parameters), and anatomic pathology (organ weights, macroscopic and microscopic pathology).

[0228] No AP-101-related effects were observed in any of the study parameters evaluated. Serum AP-101 was measured at several time points, and CSF AP-101 was measured at a single time point collected at necropsy 2 weeks after the second dose of AP-101. Serum exposure increased dose-proportionally from 10 to 400 mg / kg, with no evidence of accumulation over this dosing interval. AP-101 was detectable in several 60- and 400-mg / kg monkey CSF samples collected at necropsy (approximately 2 weeks after the second IV dose).

[0229] The relevant safety margins based on the NOAEL from the 1-month monkey study are presented in Table 3. The proposed clinical starting dose of 100 mg is 235 times lower than the NOAEL established in the 1-month monkey study and slightly lower than the lowest efficacy dose in the transgenic mouse efficacy study, while the proposed highest clinical dose, 2500 mg, is approximately 10 times lower than the toxicology study NOAEL and slightly higher than the highest efficacy dose in the transgenic mouse efficacy study (as noted above, the chimeric molecule was tested in the transgenic mouse study). The proposed clinical trial will utilize conservative dose escalation and appropriate safety monitoring. The results of the 1-month monkey study, along with the clinical trial design, indicate that AP-101 has a safety profile that supports initiation of clinical development. Based on the PK observed in cynomolgus monkeys and humans, the PK profile was utilized to calculate the human exposure ratio; see Table 3.

[0230] [Table 3]

[0231] Abbreviation: AUC 0-336 = area under the plasma concentration-time curve from time 0 to 336 hours; NOAEL = no observed adverse effect level. a Dose multiples are animal dose / human dose on a mg / kg basis. Exposure ratios are calculated as observed AUC0-336 in animals / predicted AUC0-336 in humans. bNOAEL determined in a 1-month repeated dose toxicity study (Study 8384444).

[0232] In a 6-month repeated-dose study (Study 8384445), monkeys received IV injections of 60 or 400 mg / kg AP-101 every 2 weeks for 26 weeks (a total of 13 doses). Toxicity assessment was based on mortality, clinical observations, body weight, ophthalmologic observations, clinical pathology (hematology, coagulation, clinical chemistry, and urinalysis parameters), and anatomic pathology (organ weights, macroscopic and microscopic pathology). Since no AP-101-related effects were observed in any of the study parameters evaluated, the NOAEL was considered to be 400 mg / kg. Consistent with the lack of adverse effects in the 1-month and 6-month monkey toxicology studies, there were also no drug-related adverse events during the FIH study (AP101-01).

[0233] Dosage Preparation Description of AP-101 for Injection, 100 mg per vial (5 mL at 20 mg / mL) AP-101 drug product is administered as a slow intravenous (IV) infusion using a syringe pump for the 500 mg dose and an IV bag for the 2500 mg dose. AP-101 for injection is supplied for clinical trial use as a solution in single-use glass vials. Further details are outlined in Examples 3-6.

[0234] Target population Eligibility for study participants will be based on the results of assessments including screening, medical and disease history, physical examination, vital signs, laboratory tests, Columbia-Suicide Severity Rating Scale (CSSR-S), ALS-Specific Quality of Life-Revised (ALSSQOL-SF), and ECG as described in the System of Examination (SEX). Detailed inclusion and exclusion criteria are listed in the study details for the Phase 2a study published under ClinicalTrials.gov Identifier: NCT05039099.

[0235] Study intervention A study intervention is defined as any investigational intervention, commercial product, placebo, or medical device intended to be administered / used to study participants according to the study protocol.

[0236] Study intervention to be administered After randomization on Day 1, participants will receive either a 500 mg dose of AP-101 or a placebo, and on Day 2, another dose of either a 2500 mg dose of AP-101 or a placebo. At each visit during the treatment period, all participants will receive either a 2500 mg dose of AP-101 or a matching placebo via IV infusion over at least 60 minutes. The infusion duration may be increased or stopped if deemed necessary based on the study site's standard operating procedures, or if an infusion reaction is observed. However, IV administration must be completed within 2 hours (120 minutes) of the start of the infusion. During the infusion and throughout the patient visit, the study site must have resuscitation equipment, emergency medications, and appropriately trained staff available for the duration of the infusion and for up to 6 hours after participants complete their infusion. The actual infusion start and stop times will be recorded in the electronic data capture (EDC). AP-101 drug product is a monoclonal antibody formulated for IV administration. AP-101 for injection is supplied as a solution in single-use glass vials for clinical trial use. Vials are manufactured to provide 100 mg of AP-101 at a 20 mg / mL concentration. To ensure complete withdrawal and delivery of the labeled amount of 100 mg of AP-101, the vials contain an approximately 10% volume overfill. The drug product is filled into single-use Type I glass vials.

[0237] Preparation / Handling / Storage / Accountability AP-101 vials are stable and must be stored under refrigerated conditions (2°C to 8°C). Pharmacy personnel must be trained and instructed in the preparation of each dose of AP-101 IV solution. Further guidelines for preparation, handling, storage, and medication administration are as follows: 1. The investigator or designee must ensure that appropriate storage conditions are maintained during shipment of all study interventions received and that any discrepancies are reported and resolved prior to use of the study intervention. 2. Only participants enrolled in the study may receive the study intervention. Only study personnel may supply, prepare, or administer the study intervention. All study interventions must be stored in a secure, environmentally controlled, and monitored (manual or automated) location with access limited to the investigator and authorized study personnel, in accordance with the storage conditions written on the label. 3. The investigator or authorized study personnel is responsible for study intervention accountability, reconciliation, and record-keeping (i.e., receipt, medication reconciliation, and final disposition). 4. The investigator or designee is responsible for returning all unused drug products to the sponsor or its designee at the end of the study. In some cases, the site may dispose of materials if the assessor verifies and documents during site selection that the site has adequate facilities and written procedures for disposing of clinical materials.

[0238] Strategies to minimize bias: randomization and blinding This is a double-blind study. On Day 1, all participants will be centrally assigned to their randomized study intervention using an Interactive Web-Based Response System (IWRS). The study intervention will be distributed at study visits as outlined in the System of Experience (SoE). If the investigator, site personnel performing the assessments, or the participant is not blinded, the participant must discontinue the study but will be given the opportunity to enroll in the OLE. If there are ethical reasons to keep the participant in the study, the investigator must obtain specific authorization from the sponsor clinical research physician for the participant to continue in the study.

[0239] The IWRS will be programmed with unblinding instructions. In emergency cases, the investigator has sole responsibility for deciding whether unblinding of a participant's intervention assignment is authorized. Participant safety should always be the primary consideration when making such a decision. If a participant's intervention assignment is unblinded, the sponsor should be notified immediately after unblinding. The date and reason for unblinding must be recorded in the source documents and case report form, if applicable.

[0240] Participants will be randomly assigned in a 2:1 ratio to receive the study intervention. Investigators will remain blinded to each participant's assigned study intervention throughout the course of the study. To maintain this blinding, an otherwise uninvolved, unblinded third party will be responsible for preparing all blinded study interventions according to each participant's treatment assignment. Unblinded third-party personnel will receive training and instruction for preparing each dose of study intervention, i.e., AP-101 or matching placebo solution for IV administration. The blinded study intervention will be administered to participants by blinded staff after randomization.

[0241] In the case of a quality assurance audit, auditors will be allowed access to open-label study intervention records at the site to verify that randomization / dispensing was performed correctly.

[0242] Study intervention compliance The study intervention will be administered under medical supervision by the investigator or designee. Documentation of treatment administration will occur at the site. The date and time of each dose administered will be recorded on the source document and electronic case report form (eCRF).

[0243] Combination therapy Treatment with riluzole and / or edaravone is permitted by authorization as standard of care. Participants are required to be on a stable dose for at least 30 days prior to screening and to remain on a stable dose throughout the study. Riluzole or edaravone cannot be initiated during the placebo-controlled portion of the study; however, either can be initiated if the participant is recruited into the planned open-label extension. Concomitant medications should generally be avoided; however, non-prescription medications may be administered at the investigator's discretion (e.g., acetaminophen for headache treatment). If a need for concomitant medications (other than non-prescription medications) arises, subject enrollment or continuation may be at the investigator's discretion after consultation with the sponsor. All medications used during the course of the study must be documented. Medically prescribed cannabis use is permitted during the study, per the investigator's discretion.

[0244] Dose change Dose levels, sampling schedules, and timing of procedures (e.g., PK sample collection, time of ECG) may be adjusted in light of emerging safety or PK data during the study. This is the first repeat-dose study of AP-101, and therefore safety data will be the primary basis for dose modifications. Safety data, particularly AEs, SAEs, and adverse laboratory abnormalities, will be independently evaluated by the investigator, sponsor, and SRC and will be considered related to the study drug unless there is clear evidence that the event is unrelated. Pharmacokinetic results (exposure) and total body clearance of the drug calculated after IV administration [CL] will be used as supporting data for dose modifications. After review of these data, consensus regarding appropriate dose modifications will be reached by the investigator, sponsor, and SRC. Dose modifications will be made only by consensus of the investigator, sponsor, and SRC.

[0245] Premedication for injection No premedication is planned for the infusion. However, if an infusion reaction occurs, appropriate medications may be used as determined by the investigator. If an infusion reaction is observed, acetaminophen, 500-1000 mg, and / or an antihistamine may be administered orally or IV 30-60 minutes before the start of the infusion of the subsequent dose. The decision to administer premedication to a particular participant who exhibits an infusion reaction will be made by the investigator, sponsor, and SRC and will be recorded in the study documentation. Any premedication given will be documented as concomitant therapy.

[0246] Managing Infusion Reactions There is a risk of infusion reactions with any biologic agent; therefore, all participants must be closely monitored. Symptoms and signs that may occur as part of an infusion reaction include, but are not limited to: fever, chills, nausea, headache, bronchospasm, hypotension, angioedema, throat irritation, rash, pruritus, muscle pain, and dizziness. If a significant infusion reaction occurs, the following guidelines should be followed: - The investigational drug infusion should be slowed (e.g., infusion rate reduced by 50% [e.g., 12 mL / hour infusion rate to 6 mL / hour or less]) or stopped depending on the symptoms / signs present: -If slowed, the infusion should be completed at the slower rate as tolerated - If it is determined by the investigator that the infusion should no longer be continued, no further attempts should be made to administer the participant -Supportive care should be utilized according to symptoms / signs.

[0247] Discontinuation of study intervention and participant withdrawal / withdrawal Participants who discontinue treatment early for any reason should complete AE and other follow-up procedures, see SoE.

[0248] Discontinuation of study intervention If the sponsor or investigator identifies a participant who was inadvertently enrolled because they did not meet the entry criteria, a discussion between the sponsor and investigator must determine whether the participant may continue in the trial. If both parties agree that continuation is medically appropriate, the investigator must obtain written authorization from the sponsor to allow the inadvertently enrolled participant to continue in the trial, with or without continued treatment with the investigational drug.

[0249] Participants will discontinue the study in the following circumstances: - Enrollment in any other clinical trial involving an investigational drug or any other type of medical research that is deemed scientifically or medically incompatible with this study - Participation in the study must be suspended for medical, safety, regulatory, or other reasons consistent with applicable law, regulation, and Good Clinical Practice (GCP). - If the investigator, after consultation with the sponsor-designated medical monitor, confirms that a systemic hypersensitivity reaction occurred in association with study drug administration, the participant should permanently discontinue the study drug. -Investigator's decision The investigator decides that the participant should discontinue the study - If the participant, for any reason, requires treatment with another therapeutic agent that has been demonstrated to be effective in treating the study indication, discontinuation of the study will occur prior to the introduction of the new agent. -Participant's decision - The participant or their legal representative requests to withdraw from the study - Termination of the trial by the sponsor or regulatory authority

[0250] Participants who withdraw from the study before completing all study activities may be replaced at the sponsor's discretion. If a clinically significant finding is identified after enrollment (including, but not limited to, a change from baseline in the QT interval corrected using the Bazett formula [QTcB] or Fridericia formula [QTcF]), the investigator or qualified designee will determine whether the participant can continue in the study and whether a change in participant management is required. This review of the printed ECG at the time of collection must be documented. New clinically significant findings should be reported as an AE. Please refer to the SoE regarding data to be collected at the time of intervention discontinuation and follow-up and any further evaluations that need to be completed. Temporary discontinuations are not permitted for this study.

[0251] Participant withdrawal / withdrawal from the study Participants may withdraw from the study: -At any time, at his / her own request -At the request of his / her designee (e.g., parent or legal guardian) - For safety, behavioral, compliance, or administrative reasons, at the discretion of the investigator - If the participant becomes pregnant during the study - Enrollment in another clinical trial involving an investigational drug or other type of medical research that is scientifically or medically incompatible with this study - If the participant, for any reason, requires treatment with another therapeutic agent that has been demonstrated to be effective in treating the study indication, discontinuation of the study will occur prior to the introduction of the new agent.

[0252] Discontinuation should be rare. At the time of study discontinuation, an early discontinuation visit should be conducted, if possible, as indicated in the SoE. Please refer to the SoE regarding data to be collected at the time of study discontinuation and follow-up, as well as any further assessments that need to be completed. The participant will permanently discontinue both the study intervention and the current study. If the participant withdraws consent for future disclosure of information, the sponsor may continue to retain and use any data collected prior to such withdrawal of consent. If a participant withdraws from the study, he / she may request the destruction of any samples collected but not tested, and the investigator must document this in the site study records.

[0253] If the sponsor or investigator identifies a participant who was inadvertently enrolled because they did not meet the entry criteria, the participant should discontinue study treatment unless there are extenuating circumstances that medically require the participant to continue study treatment. If the investigator and the sponsor clinical research investigator agree that continuation is medically appropriate, the investigator must obtain written authorization from the sponsor clinical research investigator to allow the inadvertently enrolled participant to continue in the study with or without treatment with the investigational product. Safety follow-up should be conducted as outlined in the SoE and the sections on safety evaluations and AEs, see below.

[0254] Untraceable A participant will be considered lost to follow-up if he or she repeatedly fails to return for scheduled visits and cannot be contacted by the study site. Site personnel or designees are expected to make diligent attempts to contact participants who fail to return for scheduled visits and cannot otherwise be followed up by the site. Site personnel or an independent third party will attempt to collect participant vital status within legal and ethical boundaries for all randomized participants, including those who did not receive the study drug. Public sources may be consulted for vital status information. If a vital status is confirmed as deceased, this will be documented and the participant will not be considered lost to follow-up. Sponsor personnel will not be involved in any attempts to collect vital status information.

[0255] Test Evaluation and Procedures - The test procedures and their timing are outlined in the SoE. - Any imminent safety concern will be discussed with the sponsor as soon as it arises or is recognized, and a decision will be made as to whether the participant should continue or discontinue the study intervention. - Compliance with the study design requirements, including those specified in the SoE, is mandatory and required for study conduct. -All screening assessments must be completed and reviewed to ensure that potential participants meet all eligibility criteria. The investigator must maintain a screening record to record the details of all participants screened and confirm eligibility or record the reason for screening withdrawal, if applicable.

[0256] Please see the section on Laboratory Tests below, which lists the laboratory tests that will be performed in this study.

[0257] Table 4 provides a summary of the maximum number and volume of invasive samples for all sampling during the study.

[0258] [Table 4]

[0259] In addition to the blood sampling volumes listed in Table 4, three post-screening CSF samples of 5 mL each will be collected for a total of 15 mL. A 5 mL cleaning CSF sample will also be collected. Unless otherwise specified in this protocol, all samples collected for specified clinical tests will be disposed of within 60 days of receipt of confirmed test results. Certain samples may be retained for longer periods if required to comply with applicable laws, regulations, or clinical laboratory certification standards.

[0260] Efficacy evaluation Efficacy in this study will be measured as a reduction in the rate of disease progression as measured by ALSFRS-R, pulmonary function by spirometric vital capacity (SVC) and / or pulse oximetry, muscle strength by handheld dynamometry (HHD), quality of life by ALSSQOL-SF, and disease staging by the King's staging tool.

[0261] Survival data can be complicated by the use of ventilation strategies, so a composite measure of time to death, tracheostomy, or permanent ventilator use may be used.

[0262] The ALSFRS-R will be assessed at visits and times specified in the System of Examination (SEX). The ALSFRS-R is a validated assessment instrument for monitoring disability progression in patients with ALS. ALSFRS-R scores significantly correlate with quality of life (qualify) as indicated by the Disease Impact Profile, indicating that functional quality is a strong determinant of quality of life in ALS. The ALSFRS-R has been demonstrated to predict survival. It measures four functional domains, including respiration, bulbar function, gross motor skills, and fine motor skills. There are 12 questions, each scored from 0 to 4 for a total possible score of 48, with higher scores indicating better function (Cedarbaum et al., J. Neurol. Sci. 169 (1999), 13-21).

[0263] The upright SVC will be determined by performing 3 to 5 measurements at visits and times specified in the SoE according to the criteria established by the American Thoracic Society and the European Respiratory Society (Goncalves de Barros et al., J. Bras. Pneumol 39 (2013), 317-322).

[0264] SpO2 represents peripheral capillary oxygen saturation and is an estimate of the amount of oxygen in the blood. A low SPO2 is associated with poor respiratory function. SpO2 measurements will be performed according to the State of Emergency.

[0265] Muscle strength will be assessed using the HHD device at visits and times specified in the System of Examination (SEX). Muscle strength is an important determinant of both function and ultimate survival in ALS. Quantitative muscle strength will be assessed using the HHD, which tests the isometric strength of multiple muscles using standard positioning. Approximately eight muscle groups will be investigated bilaterally in both the upper and lower limbs. Statistical analysis of four muscle groups (HHD0) can be used to accurately predict outcome.

[0266] Participants' ALS will be staged according to the King's staging tool (Roche et al., Brain 135 (Part 3) (2012), 847-852), measured by visits and time specified in the SoE. This tool uses the following classification of ALS progression: -Stage 1: Symptom onset (first area of ​​invasion) -Stage 2A: Diagnosis -Stage 2B: Second zone invasion -Stage 3: Invasion of the Third Realm -Stage 4A: Requires gastrostomy -Stage 4B: Non-invasive ventilation required This functional staging system ranks patients based on their level of independence (Stages 1 and 2) and need for significant intervention (feeding tube, respiratory support).

[0267] Safety evaluation Safety assessments will include clinical laboratory evaluations (hematology, clinical chemistry, endocrinology, and urinalysis), coagulation assessments, physical and neurological examinations, ECG, vital signs, pregnancy test, CSSR-S, quality of life, AE documentation, and AE grading according to the National Cancer Institute Common Terminology Criteria for Adverse Events v.5.0 (NCI CTCAE). In the event of treatment-emergent liver abnormalities, additional selected clinical trials may be obtained, which are detailed below in the section on liver monitoring studies. Planned time points for all safety assessments are provided in the SoE.

[0268] A complete physical examination will, at a minimum, include evaluation of the cardiovascular, respiratory, gastrointestinal, and neurological systems. Height (screening only) and weight will also be measured and recorded. A brief physical examination will, at a minimum, include evaluation of the skin, lungs, cardiovascular system, and abdomen (liver and spleen). The investigator should pay particular attention to clinical signs of previous serious illness.

[0269] Mandated neurological examinations will be performed by the investigator or designee. If abnormalities are noted at these time points, additional testing should be performed until the participant returns to baseline. The investigator should be familiar with the participant's baseline examination. Mandated examination components include cranial nerve workup, tremor, extraocular movements, brachial and patellar deep tendon reflexes, finger-to-nose test, and Romberg's sign. Participants with clinically significant changes on the neurological examination should be considered for further testing.

[0270] Vital signs will be recorded for each participant, including blood pressure, pulse rate, and temperature measurements. Single measurements will be taken at screening and baseline visits. Three supine blood pressures and pulse rates will be collected at all other time points. Temperature will be obtained as a single measurement. Pre-dose vital signs should be taken approximately 1 hour before scheduled dosing. At the time the upright measurement is obtained, participants should remain supine for at least 5 minutes and stand for approximately 2 minutes. If three blood pressure or pulse rate measurements precede the upright measurement, the last supine blood pressure or pulse rate measurement will be used for the upright calculation. If the participant feels unable to stand, only supine vital signs will be recorded. During AEs of dizziness or position-induced symptoms, unplanned upright vital signs should be assessed, if possible. Additional vital signs may be measured during each study period if warranted.

[0271] A 12-lead digital ECG will be collected for each participant. Sites are encouraged to perform the ECG prior to blood sample collection. Participants must be supine for approximately 5-10 minutes before ECG collection and must remain supine but awake during ECG collection. ECGs may be obtained at additional times if deemed clinically necessary. All recorded ECGs should be kept at the study site. ECGs will be read by a qualified physician (investigator or qualified designee) at the site as soon as possible after the ECG collection time, and ideally while the participant is still present. If clinically significant findings are identified, a determination will be made as to whether the participant meets the inclusion criteria for the relevant visit and immediate participant management. If clinically significant quantitative or qualitative changes from baseline are identified after enrollment, the investigator will evaluate the participant for symptoms (e.g., palpitations, presyncope, syncope) to determine whether the participant can continue in the study. The investigator or qualified designee is responsible for determining if changes in participant management are needed and must document his / her review of the printed ECG at the time of assessment form with at least one of the repeated ECGs at each time point. Machine-read ECG intervals and heart rates may be used for data analysis and report writing purposes unless a cardiologist overread of the ECG is performed prior to completion of the final study report (in which case the overread data will be used).

[0272] A list of laboratory tests to be performed and the SoE regarding timing and frequency are given below in the corresponding sections. -The investigator must review laboratory test results, document this review, and report any clinically significant changes that occur during the study as AEs. Laboratory test results must be retained with the source documents unless a Source Document Agreement or equivalent document cites an electronic location that accommodates the expected retention duration. Clinically significant abnormal laboratory findings are not related to the underlying disease unless judged by the investigator to be more severe than expected for the participant's condition. - All laboratory tests with values ​​considered to be clinically significantly abnormal during study participation or within 12 weeks after the last dose of study intervention should be repeated until the values ​​return to normal or baseline or are no longer considered clinically significant by the investigator or medical monitor. If such values ​​do not return to normal / baseline within a period of time deemed reasonable by the investigator, the etiology should be identified and the sponsor notified. -All protocol-required laboratory evaluations must be performed in accordance with the SoE, standard collection requirements, and the Laboratory Manual. If a laboratory value from a clinical laboratory evaluation not specified in the protocol, performed at a local clinical laboratory designated by the investigator, calls for a change in participant management or is considered clinically significant by the investigator (e.g., an SAE or AE or dose change), report the information as an AE.

[0273] With regard to clotting, the prothrombin time (PT) w / INR test measures the length of time it takes for a clot to form in a sample of blood.

[0274] Regarding quality of life, measuring health-related quality of life (HLQOL-SF) is a useful measure of treatment efficacy and will utilize the ALSSQOL-SF questionnaire. This assessment will be administered at the time indicated in the System of Experience (SoE). The ALSSQOL-SF is a 20-item instrument measuring overall quality of life (QOL) and six specific domains in individuals with ALS (Felgoise et al., Muscle Nerve 58 (2018), 646-654).

[0275] Suicidal ideation and behavioral risk monitoring will be assessed using the C-SSRS, which is a semi-structured clinical interview that assesses suicidal ideation severity, suicidal ideation intensity, and suicidal behavior. The C-SSRS will be assessed at the visits and times specified in the SoE.

[0276] Adverse events, serious adverse events, and investigational product complaints Definitions of the following events can be found below in the section on adverse events: -AE -SAE - Investigational Product Complaints (IPC)

[0277] These events will be reported by the participant (or by the caregiver, representative, or legal representative of the participant, as appropriate). The investigator and qualified designee are responsible for detecting, documenting, and recording events that meet these definitions and remain responsible for following up on events that are considered serious, related to the study intervention or procedures, or that cause the participant to discontinue the study. Care will be taken not to introduce bias in detecting events. Open-ended, non-leading verbal questioning of participants is the preferred method for inquiring about event occurrence. After initial reporting, the investigator is expected to actively monitor each participant at subsequent visits / contacts. All SAEs and AEs of particular interest will be followed until they resolve, stabilize, the event is otherwise explained, or the participant is lost to follow-up (as defined above). For investigational drug complaints, the investigator is responsible for ensuring that follow-up includes follow-up investigations as indicated to clarify the nature and / or causality. Table 5 below lists the timing, deadlines, and mechanisms for collecting events.

[0278] [Table 5] JPEG2025527710000009.jpg117170

[0279] Notable adverse events Lumbar punctures (LP) to obtain CSF will be performed at several time points during the study. Headaches can occur in up to 60% of patients undergoing LP, and back pain can occur in up to 40% of patients. More serious adverse events are rare and include cerebral herniation, intracranial subdural hemorrhage, spinal epidural hemorrhage, and infection.

[0280] Hypersensitivity reactions can occur after administration of any monoclonal antibody. Acute hypersensitivity reactions, including infusion reactions or anaphylaxis, can occur during or within hours of infusion. Sites should have appropriately trained medical staff and appropriate medical equipment available when participants are taking the investigational product. It is recommended that participants who experience a systemic hypersensitivity reaction be treated in accordance with national and international guidelines. In the event of systemic urticaria or anaphylaxis, additional blood samples should be collected as described below in the section on recommended laboratory tests for hypersensitivity events. Laboratory test results will be provided to the sponsor via a central laboratory.

[0281] Subjects should be monitored for hypotension, fever, chills, bronchospasm, angioedema, and other symptoms or signs of anaphylaxis. The infusion should be slowed or stopped, and reactions should be managed as indicated in the study protocol. If such reactions occur, additional data describing each symptom should be provided to the sponsor in the eCRF. Delayed hypersensitivity reactions may occur several days or weeks after the infusion. Subjects should be monitored for fever, rash, arthralgia, myalgia, hematuria, hematological changes, liver, or kidney tests. Delayed hypersensitivity reactions are managed symptomatically and may be treated with corticosteroids. Subjects should not receive additional doses of AP-101 in the event of any confirmed / suspected delayed hypersensitivity reaction to AP-101.

[0282] Overdose treatment For this study, a dose of AP-101 higher than 2500 mg within a 24-hour period (± 4 hours) would be considered an overdose. Because the study drug will be administered by trained personnel, an overdose is not expected. In the event of an overdose, the investigator will 1. The medical monitor should be contacted immediately. 2. Participants should be closely monitored for AEs / SAEs and laboratory abnormalities until AP-101 is no longer detectable systemically (at least 100 days). 3. The amount of overdose and the duration of the overdose should be documented on the eCRF.

[0283] Decisions regarding dose interruptions or changes will be made by the investigator based on the participant's clinical evaluation in consultation with the medical monitor.

[0284] Pharmacokinetics (PK) At the visits and times specified in the SoE, venous samples will be collected to measure serum concentrations of AP-101. CSF sampling will also be collected at baseline and at defined time points during the study in accordance with the SoE. Sampling times may be modified, at the sponsor's discretion, based on review of interim PK data as they become available. Actual PK sample collection dates and times will be recorded.

[0285] Samples will be analyzed by a sponsor-approved clinical laboratory and stored at a facility designated by the sponsor. AP-101 concentrations will be assayed from serum and CSF using a validated immunoassay. Bioanalytical samples collected to measure study drug concentrations will be retained for up to one year after the last participant visit for the study. Table 4 presents a sampling summary for this study.

[0286] Pharmacodynamics (PD) Pharmacodynamic assessments in this study include changes in total SOD1, mSOD1, pNfH, and NfL, as well as additional exploratory assessments. mSOD1 levels will be measured retrospectively from participants' CSF at baseline and at least one time point after treatment with AP-101. This is an exploratory endpoint that will be used to investigate whether mSOD1 levels correlate with response and whether treatment with AP-101 can reduce detectable mSOD1 levels in participants.

[0287] SOD1 and mSOD1 assays Total SOD1 and misfolded SOD1 levels will be measured from participants' CSF at baseline and retrospectively after at least one time point after treatment with AP-101. This is an exploratory endpoint that will be used to investigate whether SOD1 levels correlate with response and whether treatment with AP-101 can reduce detectable SOD1 levels in participants. Correlations with other exploratory biomarkers and endpoints will also be performed.

[0288] Neurofilament assay Levels of pNfH and NfL will be measured in both CSF and serum at various time points during the 6-month study. This endpoint will be used to determine the effect of AP-101 on pNfH and NfL levels. pNfH and NfL will also be investigated for correlation with other exploratory biomarkers and endpoints.

[0289] Genetics Mandatory genetic testing of all participants to identify common variants associated with ALS will be performed by an accredited clinical genetic laboratory. Blood samples for DNA isolation will be collected from participants.

[0290] Biomarkers Additional biomarkers will include creatinine and transcriptional and microRNA profiles from various body fluids. These are exploratory biological markers of disease activity and will be investigated at various time points during the study as indicated in the System of Experiments. -This study will analyze biomarkers related to the mechanism of action of AP-101. Samples collected for these analyses may also be used to develop related research methods or to validate diagnostic tools or assays. Additional samples may be collected during the study if warranted and agreed upon by the investigator and sponsor.

[0291] Immunogenicity assessment Antibodies to AP-101 will be assessed in serum samples collected from all participants according to the SoE. Additionally, serum samples should be collected at the final visit from participants who discontinue the study intervention or withdraw from the study. These samples will be tested by the sponsor or its designee. Serum samples will be screened for antibody binding to AP-101, and titers of confirmed positive samples will be reported. Other analyses may be performed to confirm the stability of antibodies to AP-101 and / or further characterize the immunogenicity of AP-101. Detection and characterization of antibodies to AP-101 will be performed with or under the supervision of the sponsor using validated assay methods. All samples collected for detection of antibodies to the study intervention will also be assessed for AP-101 serum concentration to enable interpretation of antibody data. Antibodies may be further characterized and / or assessed for their ability to neutralize the activity of the study intervention. Samples may be stored at a facility selected by the sponsor for up to two years (or according to each facility's regulations) after the last participant's last study visit to allow for further analysis of the immune response to AP-101.

[0292] Statistical considerations Descriptive statistics, instead of hypothesis testing, will be used for the statistical evaluation of efficacy, safety, and tolerability.

[0293] A maximum of 63 participants (21 with fALS and 42 with sALS) will be randomized to study intervention or placebo in a 2:1 randomization ratio such that approximately 18 evaluable participants with fALS and 36 evaluable participants with sALS will complete the study. This sample size is based on clinical considerations and is not powered for hypothesis testing.

[0294] The populations described in Table 6 will be defined.

[0295] [Table 6]

[0296] All PK and PD (including neurofilament) analyses will be performed on the safety population.

[0297] Statistical analysis of this study will be the responsibility of the sponsor or its designee. All CIs will be given at the two-sided 90% level. Changes to the data analysis methods described in the protocol will require modification only if they alter key features of the protocol. All other changes to the data analysis methods described in the protocol and the reasons for making the changes will be described in the statistical analysis plan and clinical trial report. Additional exploratory analyses of the data will be conducted as deemed appropriate. The statistical analysis plan will be completed prior to unblinding and will include a more technical and detailed description of the statistical analyses described in this section. This section summarizes the planned statistical analyses of the most important endpoints, including the primary and key secondary endpoints.

[0298] Statistical analysis of this study will be the responsibility of the sponsor or its designee. Statistical analysis will be detailed in the statistical analysis plan. Safety analyses will be conducted on all dosed participants according to the safety population, regardless of whether they completed all protocol requirements. For continuous variables, summary statistics will include number of participants, mean, median, standard deviation, minimum, and maximum. Categorical endpoints will be summarized using number of participants, frequency, and percentage. A detailed description of participant disposition will be given at the end of the study. All participants who discontinue the study will be identified, and their level of study participation will be summarized. Reasons for their discontinuation, if known, will be provided. Participant demographics and baseline characteristics (age, sex, race, ethnicity, height, weight, and body mass index [BMI]) will be summarized by treatment. A safety summary will also be provided.

[0299] The primary endpoints of this study are the incidence of AEs and SAEs, including immunogenicity, and the incidence of abnormalities in vital signs, clinical laboratory assessments, physical and neurological examinations, electrocardiograms, and weight change.

[0300] All treatment and protocol-related AEs will be listed. Summary statistics of AEs and SAEs will be provided by treatment arm and categorized by system organ class and preferred terminology. A summary will also be provided by relationship to study drug and maximum severity. AEs leading to study drug or study discontinuation will be summarized. AEs will be summarized with respect to the safety population. Abnormal vital signs, clinical laboratory assessments, physical and neurological examinations, electrocardiograms, and weight changes will be tabulated. The number of participants experiencing an abnormality and the number of abnormalities will be summarized using the safety population.

[0301] All secondary endpoints will be performed on the PK population. PK analyses will be performed only on participants who received at least one full dose of study treatment and had a baseline PK sample and at least three post-baseline evaluable PK samples. All serum PK samples will be utilized in a compartmental model to describe the PK of these patients. PK parameters will be reported by descriptive statistics, including arithmetic mean, geometric mean, standard deviation, coefficient of variation (CV)%, geometric CV%, median, minimum, and maximum (Table 7).

[0302] [Table 7]

[0303] During the 6-month treatment period, levels of AP-101 in CSF will be summarized by treatment at each available treatment visit and by change from baseline at each visit using the safety population. Data will be summarized by treatment.

[0304] Both pNfH and NfL will be summarized by type (either CSF or plasma) at each available treatment visit and by change from baseline at each visit for both the ITT and per-protocol (PP) populations. Additional summaries by fALS status will be provided for the PP population. Statistical models will be used to estimate the mean change per month in pNfH and NfL for treatment and placebo participants. The estimated change per month will be included in the summary, along with a two-sided 90% confidence interval. Plots showing baseline levels of pNfH and NfL and their mean change per month will be provided.

[0305] Total ALSFRS-R scores and change from baseline will be summarized at visits according to the System of Examination (SEX). Statistical models will be used to estimate the mean ALSFRS-R change per month for treatment and placebo participants. This estimated change per month and two-sided 90% confidence intervals will be provided. Pulmonary function (SVC) or equivalent alternative, muscle strength (HHD), and disease staging (King's Staging Tool) scores will be summarized by visit and by change from baseline. Change from baseline in King's Staging Tool will be summarized as the number of stages decreased since baseline. Kaplan-Meier analyses will be performed comparing treatment and placebo groups. First, second (median), and third quartiles will be presented along with their 90% confidence intervals, as well as survival curve plots. ALSSQOL-SF will be summarized by treatment visit. CSF-derived total and misfolded SOD1 will be summarized at each available visit and by change from baseline. Additional summaries by fALS status will be provided. Two-sided 90% confidence intervals will be included in the summaries. Plots showing baseline levels of SOD1 and mSOD1 and their change over time will be provided. All other biological markers of disease activity will be summarized at each available visit and by change from baseline. Two-sided 90% confidence intervals will be provided. All safety analyses will be performed on the safety population and will be provided as tables. Subgroup analyses by fALS status (participants will have either fALS or sALS) will be performed on various endpoints, including measures of neurofilaments (NfL and pNfH) and mSOD1 and SOD1. Subgroup analyses will be similar to standard analyses in that they will also be stratified by treatment. Optional statistical modeling in subgroup analyses will include covariate terms for fALS status and interaction terms between fALS and treatment. Additional exploratory subgroup analyses may be performed.

[0306] Laboratory tests Laboratory tests are detailed in Table 8.

[0307] [Table 8]

[0308] Recommended Laboratory Tests for Hypersensitivity Events Laboratory testing should be performed at the time of a systemic hypersensitivity event. Management of the adverse event may justify laboratory testing beyond those described below and should be performed if clinically indicated. Laboratory testing during a systemic hypersensitivity event is not performed for diagnostic purposes. The intent is multiple: -To help characterize and classify systemic hypersensitivity reactions -To meet regulatory expectations -To help differentiate between the various mechanistic bases of anaphylaxis and thereby improve subsequent clinical management.

[0309] Laboratory tests should be obtained in the presence of generalized urticaria or if anaphylaxis is suspected. Obtain samples within 1 to 2 hours of the event after the subject is stabilized; however, samples can be obtained as early as 12 hours after the event, as analytes can remain altered for extended periods. Record the time the sample is collected. Obtain follow-up samples at the next regularly scheduled visit or after 4 weeks.

[0310] The clinical examination should include the following: Tryptase (If a tryptase sample is obtained more than 2 hours after the event (i.e., within 2-12 hours) or is not available because more than 12 hours have passed since the event, obtain a urine for N-methylhistamine (NMH) testing. Note that for tryptase serum samples obtained within 2-12 hours of the event, a urine NMH test is performed in addition to the tryptase test. Collect the first urine after the event. Obtain a follow-up urine for NMH testing at the next regularly scheduled visit or after 4 weeks, whichever is later.) -ADA and AP-101 concentrations (PK) Complement: C3, C3a and C5a -Cytokines: IL-6, IL-1β, IL-10 (or any cytokine panel that includes these three cytokines)

[0311] Liver monitoring tests for abnormalities that develop during treatment Selected tests shown in Table 9 may be obtained in the event of liver abnormalities emerging during treatment and may be required for follow-up by the subject.

[0312] [Table 9]

[0313] Adverse events (AEs) An AE is any untoward medical occurrence in a participant receiving a medicinal product that does not necessarily have a causal relationship to the study intervention. Thus, an AE can be any untoward and unintended sign (including abnormal clinical laboratory findings), symptom, or disease (new or worsening) temporally associated with the use of a medicinal product, whether or not related to the medicinal product.

[0314] Events that meet the AE definition: Any abnormal laboratory result (hematology, clinical chemistry, or urinalysis) or other safety assessment (e.g., ECG, radiology scan, vital sign measurement) that, in the medical and scientific judgment of the investigator, is considered clinically significant (i.e., not associated with progression of the underlying disease), including any deterioration from baseline. - Exacerbation of a chronic or intermittent pre-existing condition, including either an increase in the frequency and / or intensity of the condition. - New medical conditions that may have been present before the start of the study but that are detected or diagnosed after administration of the study intervention. - Signs, symptoms, or clinical sequelae of a suspected drug-drug interaction. - Signs, symptoms, or clinical sequelae of suspected overdose of either the study intervention or concomitant medication. Overdose itself would not be reported as an AE / SAE unless it was an intentional overdose with possible suicidal / self-harming intent. Such overdoses should be reported regardless of sequelae. - "Lack of efficacy" or "failure of expected pharmacological action" itself would not be reported as an AE or SAE. Such cases would be recorded in the efficacy assessment. However, signs, symptoms, and / or clinical sequelae resulting from the lack of efficacy would be reported as an AE or SAE if they meet the definition of an AE or SAE.

[0315] Events that do not meet the AE definition - Any clinically significant abnormal laboratory finding or other abnormal safety assessment related to the underlying disease, unless judged by the investigator to be more severe than expected for the participant's condition. - The disease / disorder being studied or the predicted progression, signs, or symptoms of the disease / disorder being studied, if less severe than would be predicted given the participant's condition. Medical or surgical procedure (e.g., endoscopy, appendectomy): A condition leading to a procedure is an AE. - Situations in which no adverse medical events occurred (social and / or convenience hospitalization). - Expected day-to-day fluctuations of pre-existing diseases or conditions present or detected at the start of the study that do not worsen.

[0316] SAE If the event is not an AE according to the above definition, it cannot be an SAE, even if the serious condition is met (e.g., hospitalization due to signs / symptoms of disease during the study, death due to disease progression).

[0317] An SAE is defined as any untoward medical occurrence at any dose that: a.Causing death; b. Life-threatening. The term "life-threatening" in the definition of "serious" refers to an event in which the participant was in danger of dying at the time of the event. It does not refer to an event that, if more severe, could hypothetically have caused death. c. Requiring inpatient hospitalization or extension of existing hospitalization - Generally, hospitalization means that the participant was admitted to a hospital for observation and / or treatment that would not have been appropriate in a physician's office or outpatient setting. A complication that occurs during hospitalization is an AE. An event is serious if the complication prolongs the hospital stay or meets other serious criteria. If there is any doubt as to whether a "hospitalization" occurred or was necessary, the AE should be considered serious. - Hospitalization for elective treatment of a pre-existing condition that did not worsen from baseline is not considered an AE. d. Causes permanent disability / incapacity -The term disability means a significant disruption of a person's ability to perform normal life functions. -This definition is not intended to include experiences of relatively minor medical significance, such as uncomplicated headache, nausea, vomiting, diarrhea, flu, and accidental trauma (e.g., sprained ankle), which may interfere with or hinder daily life functioning but do not constitute a significant disruption. e. Congenital anomalies / birth defects -Any abnormal pregnancy outcome (e.g., spontaneous abortion, fetal death, stillbirth, congenital anomaly, ectopic pregnancy) is considered an SAE. f.Other situations: - Medical or scientific judgment should be exercised in determining whether SAE reporting is appropriate in other situations, such as serious medical events that may not be immediately life-threatening or result in death or hospitalization, but which may endanger the participant or require medical or surgical intervention to prevent one of the other outcomes listed in the definition above. These events should generally be considered serious. Examples of such events include invasive or malignant cancer, intensive emergency room or home treatment for allergic bronchospasm, blood dyscrasia or convulsions that do not result in hospitalization, or the development of drug dependence or drug abuse.

[0318] Strength evaluation The investigator will perform an intensity assessment of each AE and SAE reported during the study and assign it to one of the NCI-CTCAE Version 5.0 classifications outlined in Example 1.

[0319] Causal assessment The investigator is obligated to assess the relationship between the study intervention and each occurrence of each AE / SAE. A "reasonable possibility" of relationship conveys that there are facts, evidence, and / or arguments suggesting a causal relationship, rather than that a relationship cannot be excluded. The investigator will use clinical judgment to determine the relationship. Alternative causes, such as underlying disease, concomitant therapy, and other risk factors, as well as the temporal relationship of the event to the administration of the study intervention, will be considered and investigated. The investigator will also consult the IB in his / her assessment. For each AE / SAE, the investigator must document in the medical record that he / she reviewed the AE / SAE and provided an assessment of causality. There may be situations in which an SAE occurs and the investigator has minimal information to include in the initial report to the sponsor or designee. However, it is very important that the investigator always conducts a causality assessment for all events prior to the initial communication of SAE data to the sponsor or designee. The investigator may change his / her opinion of causality in light of follow-up information and send an SAE follow-up report with an updated causality assessment, which is one of the criteria used in determining regulatory reporting requirements.

[0320] The investigator has an obligation to perform or arrange for the performance of supplementary measurements and / or evaluations, if medically necessary or requested by the sponsor or designee, to clarify as completely as possible the nature and / or causality of the AE or SAE, which may include additional laboratory tests or investigations, histopathological examination, or consultation with other medical professionals.

[0321] Examples 3-6 Related to AP-101 Formulations method In certain methods, reference is made to the European Pharmacopoeia, Ph.Eur.

[0322] Appearance (transparency, color and visible particles) Visual inspection is performed to determine transparency, color, and visible particles. Transparency inspection complies with Ph.Eur. <2.2.1. Clarity and Degree of Opalescence of Liquids>. A turbidity meter is used to measure transparency. Color inspection complies with Ph.Eur. <2.2.2. Degree of Coloration of Liquids>. Observation methods are used to measure color. Visible particle inspection complies with Ph.Eur. <2.9.20. Particulate Contamination: Visible Particles>. Observation methods are used to detect visible particles.

[0323] Osmolality The test for osmolality complies with Ph.Eur.2.2.35. Osmolality is determined indirectly by measuring the depression of the freezing point of the solution.

[0324] Collection capacity The collection volume test complies with Ph.Eur.2.9.17. The volumetric method is used for the collection volume test.

[0325] particulate matter Particulate matter testing complies with Ph.Eur.2.9.19. Subvisible particles are determined based on the light obscuration method. When particles in a liquid pass through a narrow detection channel, incident light perpendicular to the direction of liquid flow is attenuated due to obstruction by the particles, leading to a decrease in the signal output by the sensor. The change in signal is related to the cross-sectional area.

[0326] sterile Sterility testing of pharmaceuticals is established in accordance with Ph.Eur.2.6.1. A membrane filtration assay is used, in which the contents of the container to be tested are filtered through two 0.45 μm membranes; any microorganisms present are retained on the membranes. Each membrane is flushed with a defined volume of rinse solution. Two culture media are used for membrane incubation: fluid thioglycollate medium (FTM) and soybean casein digest (TSB). FTM is transferred to one canister and TSB to another. The FTM and TSB canisters are incubated for 14 days (FTM at 30-35°C and TSB at 20-25°C). The absence of microbial growth indicates that the tested sample is sterile.

[0327] Further analytical procedures used to control pharmaceutical parameters, such as the measurement of pH and protein concentration, for example, by using UV-visible spectrophotometry, size exclusion chromatography (SEC), reduced capillary electrophoresis with sodium dodecyl sulfate (reduced CE-SDS), capillary isoelectric focusing (cIEF), and fluorescence resonance energy transfer (FRET)-coupling, are described in Ph.Eur. and in Mikkelsen and Corton "Bioanalytical Chemistry", Second Edition (2016), Hoboken, New Jersey: John Wiley & Sons, Inc., ISBN 9781119057703 (pdf); Schiel et al., "State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume 2. Biopharmaceutical Characterization: The NIST mAb Case Study", ACS Symposium Series 1201 (2015), ISBN 978-0-8412-3029-3, e.g., Chapter 5 by Michels et al.; Kulkarni et al., "Essential chemistry for formulators of semisolid and liquid dosages", Academic Press (2016), ISBN 978-0-12-801024-2, Chapter 11 - Miscellaneous Physical, Chemical, and Microbiological Test Methods, pages 193-221; or according to the official quality control standards described on the Protagen Protein Services homepage (https: / / www.protagenproteinservices.com / biopharmaceuticals / antibodies).

[0328] Example 3: Development of a stable antibody formulation for antibody AP-101 Formulation development for AP-101 (referred to herein as the drug substance) included studies designed to select buffer systems, excipients, and surfactants to stabilize the protein. The formulation was developed to prevent product loss and minimize loss of purity and biological activity in response to stresses encountered during manufacturing, storage, shipping, and handling.

[0329] Buffer system studies were performed to determine the optimal buffer system for the antibody formulation. Based on pH screening studies, 20 mM L-histidine / L-histidine monohydrochloride buffer, pH 6.0 was selected as the final buffer system.

[0330] Different types of excipients, including disaccharides (sucrose, trehalose, and sorbitol), amino acids (L-arginine hydrochloride and L-methionine), and salts (NaCl), were evaluated in the excipient study. Samples were incubated at 25°C and 40°C for up to 4 weeks. Thermal stability, insoluble aggregate formation, and purity were monitored. Overall, sucrose and L-methionine at concentrations of 8% (w / v) and 0.1% (w / v), respectively, were selected as the optimal excipients for the AP-101 formulation because they were shown to minimize the formation of low molecular weight and acidic species, thereby preserving product purity.

[0331] In a surfactant type and strength screening study, four different polysorbate 80 (PS 80) concentrations (0.005%, 0.010%, 0.020%, and 0.050% (w / v)) and two different poloxamer 188 concentrations (0.05% and 0.10% (w / v)) were tested. The number and purity of subvisible particles were evaluated. The 0.02% (w / v) PS 80 concentration was selected as the surfactant strength because it effectively suppressed subvisible particle formation and demonstrated acceptable stability.

[0332] The final formulation developed is AP-101 at a target concentration of 20 mg / mL in 20 mM L-histidine / L-histidine monohydrochloride buffer at pH 6.0, 8% (w / v) sucrose, 0.1% (w / v) L-methionine, and 0.02% (w / v) PS 80. The excipients used in the formulation of AP-101 and their stabilizing role are summarized in Table 10.

[0333] [Table 10]

[0334] Example 4: Production of a final pharmaceutical product containing antibody AP-101 Drug product manufacturing was performed by thawing the drug substance (bulk product), pooling and blending, sterile filtration, aseptic filling, stoppering, and capping. Optionally, visual inspection, UV printing, bulk packaging, and storage at 2-8°C were performed. Specifically, frozen drug substance stored in 10 L polycarbonate bottles was thawed at room temperature (18-24°C) in a room protected from light. After complete thawing, the drug substance was pooled into a 100 L blending bag and stirred at an appropriate speed of 250 rpm or less to observe movement without foam generation. The blending time was controlled to be more than 15 minutes. Prior to sterile filtration, samples were taken for pH, osmolality, protein concentration, endotoxin, and bioburden.

[0335] Sterile filtration was selected as the method to obtain a sterile drug product and was performed by aseptically filtering the bulk drug substance using two series-connected sterile filters (0.22, polyvinylidene fluoride membrane) into a sterile 50 L single-use bag with a peristaltic pump under laminar airflow (LAF) with a grade C background. Filter integrity tests were performed on both filters before and after sterile filtration.

[0336] The drug product was then aseptically filled into 8 mL sterile glass vials using a filler / sealer coupled to a peristaltic pump system. All filling elements were autoclaved and aseptically assembled. The compatibility of the drug product with contact elements of the filling line, the effect of shear stress induced by the peristaltic pump, and stability under light exposure were evaluated to mitigate potential adverse effects on product quality attributes during manufacturing. Based on shear stress testing and previous manufacturing knowledge, the drug product was filled with a peristaltic pump at a speed of 350 rpm or less. The filling pump with the automated filling and stoppering unit was enclosed within a restricted access barrier system (RABS) to enclose the aseptic process and provide a Grade A environment.

[0337] In the next step, the vials were automatically stoppered with 20 mm rubber stoppers inside the RABS unit. The stoppers were steam sterilized at 122°C for 30 minutes. Capping was performed by transferring the stoppered vials to a capping machine via a conveyor belt under laminar flow protection. The stoppered vials were capped with 20 mm plastic-aluminum flip-off caps. The caps were steam sterilized at 122°C for 30 minutes. Thus, the container closure system for the pharmaceutical product is an 8 mL Type I glass vial sealed with a 20 mm rubber stopper and a 20 mm flip-off cap.

[0338] A manual 100% visual inspection was then performed on the filled vials by manufacturing personnel, followed by a statistically based AQL review by Quality Assurance. Release and stability samples were taken after the visual inspection. A portion of the lot number was printed with UV ink on the side of the cap after the visual inspection process. A manual 100% check was performed on the printed number.

[0339] The filled drug vials were then bulk packaged and labeled. The bulk packaged drug vials were stored at 2-8°C.

[0340] Example 5: Compatibility of AP-101 formulations with clinically used materials Stability data were collected to confirm the stability of the drug administration solution in a clinical trial setting. Compatibility with polyvinyl chloride IV lines and bags, polypropylene syringes, PVC infusion sets, polyolefin IV bags, and fluorinated ethylene propylene (FEP)-indwelling catheters was evaluated. Two concentrations were evaluated: 20 mg / mL (undiluted drug) and 5 mg / mL. Normal saline was used as the diluent for the 5 mg / mL condition. The solution was held in the infusion container (syringe or IV bag) at 2-8°C for 24 hours, then held at room temperature for 6 hours, and finally infused over a 2-hour period (defined as T30H). Samples were tested at T0 and T30H. Some of the results are shown in Tables 11-13.

[0341] [Table 11]

[0342] [Table 12]

[0343] [Table 13]

[0344] The data demonstrate that AP-101 is compatible with the clinically used materials evaluated. The product diluted in saline at concentrations of 5 mg / mL and 20 mg / mL was stable for 24 hours at 2-8°C, followed by up to 6 hours at room temperature and 2 hours post-infusion. No significant changes in appearance, protein concentration, pH, osmolality, SEC, non-reduced CE-SDS purity, or cIEF purity were observed. The results indicate that after preparation for dosing, the drug product solution is stable for at least 24 hours at refrigerated temperatures and 6 hours at room temperature.

[0345] Example 6: Stability of AP-101 formulations Representative batches of drug product were evaluated for stability to ensure pharmaceutical performance and strength for the duration of the clinical trials. Long-term stability studies (real-time studies) were conducted at 5°C ± 3°C, with sample analyses conducted at TO, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, and 24 months. Additionally, accelerated stability studies were conducted at TO, 1 month, 3 months, and 6 months at 25°C ± 2°C and 60%RH ± 5%RH (relative humidity). Additionally, stability was analyzed under stress conditions at 40°C ± 2°C and 75%RH ± 5%RH.

[0346] In this context, 3-month real-time data supports 12-month shelf life 6 months of real-time data supports 18-month shelf life 9 months of real-time data supports 21-month shelf life 12-month real-time data supports 24-month shelf life 24-month real-time data supports 36-month shelf life 36-month real-time data supports 48-month shelf life 44-month real-time data supports 56-month shelf life 53 months of real-time data supports 65 months shelf life Please note:

[0347] The results are summarized in Tables 14-17. The data demonstrated that the AP-101 formulation is long-term stable. There is no evidence of significant physical or chemical changes to the drug product at any of the storage conditions utilized. Because the formulation was shown to be stable for 24 months, the shelf life is expected to be at least 36 months. Stability was further confirmed for 53 months, so the shelf life is expected to be up to 65 months.

[0348] [Table 14] JPEG2025527710000019.jpg242170

[0349] [Table 15] JPEG2025527710000021.jpg237170

[0350] [Table 16]

[0351] [Table 17]

[0352] Example 7: N-linked glycosylation of the heavy chain, N-terminal glutamine modified to N-terminal pyroglutamic acid, and C-terminal lysine clipping of the heavy chain are the major post-translational modifications of AP-101 The antibody AP-101 is produced in the CHO-K1 cell line (ATCC No. CCL 61) and is obtained from the cell culture after cultivation in a large-scale production bioreactor.

[0353] Liquid chromatography with tandem mass spectrometry (LC-MS / MS) analysis of fragments of AP-101 obtained by sequential Lys-C and trypsin digestion, as well as free sulfhydryl analysis, were used to identify post-translational modifications of AP-101. Characterization of antibody-based therapeutics by LC-MS analysis is a standard procedure and can be performed by those skilled in the art; see, for example, Robotham and Kelly, Approaches to the Purification, Analysis, and Characterization of Antibody-Based Therapeutics (2020), 1-33.

[0354] N-terminal glutamine modified as pyroglutamic acid and C-terminal lysine clipping of the heavy chain were identified as the major post-translational modifications, with N303 identified as the N-glycosylation site. Additionally, minor modifications such as methionine oxidation, asparagine deamidation, and asparagine succinimide formation were observed. The results of the LC-MS / MS analysis are summarized in Table 18.

[0355] [Table 18]

Claims

1. A recombinant antibody that selectively binds to misfolded and / or aggregated forms of superoxide dismutase (SOD1) for use in the treatment of amyotrophic lateral sclerosis (ALS), wherein the treatment comprises administering to a patient in need thereof a dose of about 2500 mg of the antibody.

2. The antibody for use according to claim 1, wherein the antibody is administered once every three weeks.

3. 3. The antibody for use according to claim 1 or 2, wherein the antibody is administered by intravenous (IV) infusion.

4. 4. The antibody for use of any one of claims 1 to 3, wherein the patient receives a loading dose of about 500 mg of the antibody, followed by 2500 mg doses of the antibody on other days prior to administration of the antibody once every three weeks.

5. The antibody for use according to any one of claims 1 to 4, wherein the administration of the antibody is carried out once every three weeks for a period of at least six months.

6. The antibody for use according to any one of claims 1 to 5, wherein the antibody is administered at a concentration of about 20 mg / mL.

7. 7. The antibody for use according to claim 6, wherein the antibody is administered over a period of a minimum of 60 minutes and a maximum of 120 minutes.

8. The antibody for use according to any one of claims 1 to 7, wherein the patient is suffering from familial amyotrophic lateral sclerosis (fALS).

9. The antibody for use according to any one of claims 1 to 7, wherein the patient is suffering from sporadic amyotrophic lateral sclerosis (sALS).

10. The antibody for use according to any one of claims 1 to 9, wherein the patient is taking riluzole, edaravone, a combination of sodium phenylbutyrate and tauroursodeoxycholic acid (PB and TUDCA), or tofersen.

11. 11. The antibody for use according to any one of claims 1 to 10, wherein if an infusion reaction is observed, 500-1000 mg acetaminophen and / or an antihistamine may be administered orally or IV 30-60 minutes before the start of the infusion for administration of the subsequent dose.

12. The antibody for use according to any one of claims 1 to 11, wherein the therapeutic effect of the antibody and the progression of ALS are monitored by assessing one or more biomarkers.

13. The antibody for use according to claim 12, wherein the biomarker is selected from the group consisting of phospho-neurofilament heavy chain (pNfH), neurofilament light chain (NfL) and misfolded SOD1 (mSOD1).

14. The antibody for use according to any one of claims 1 to 13, wherein said antibody binds to an epitope of SOD1 within the amino acid sequence 73-GGPKDEERHVGD-84 set forth in SEQ ID NO:

11.

15. The antibody is characterized in that it comprises six CDRs of a variable heavy (VH) chain and a variable light (VL) chain in its variable region, i.e., binding domain, (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof, wherein said variant comprises one or two amino acid substitutions; (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof, wherein said variant comprises one or two amino acid substitutions; (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and (f) The antibody for use according to any one of claims 1 to 14, wherein VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 10 or a variant thereof, wherein said variant comprises one or two amino acid substitutions.

16. the antibody is characterized in that it comprises a VH chain and a VL chain in its variable region, i.e., binding domain, (a) the VH chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 or 2; and (b) the VL chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 6 or 7.

17. The antibody for use according to any one of claims 1 to 16, wherein the antibody comprises a human Ig constant region.

18. The antibody for use according to any one of claims 1 to 17, wherein the antibody is a human IgG.

19. The antibody for use according to any one of claims 1 to 18, wherein the antibody is human IgG1.

20. The antibody for use according to any one of claims 1 to 19, wherein the antibody is of the human IgG1m3 allotype.

21. The antibody for use according to any one of claims 18 to 20, wherein the antibody comprises a kappa (κ) or lambda (λ) light chain.

22. The antibody for use according to any one of claims 1 to 21, wherein the antibody is AP-101 and is of the human IgG1m3 allotype.

23. 23. The antibody for use according to claim 22, wherein each heavy chain is composed of 453 amino acid residues having SEQ ID NO: 12 and each light chain is composed of 213 amino acid residues having SEQ ID NO:

13.

24. The antibody for use according to any one of claims 1 to 23, produced in CHO-K1 host cells and purified from the cell culture.

25. The antibody for use according to any one of claims 16 to 24, wherein the heavy chain of said antibody has undergone N-terminal glutaminyl cyclization.

26. 11. The antibody for use according to claim 9 or 10, wherein the heavy chain has undergone C-terminal lysine clipping.

27. The antibody for use according to any one of claims 16 to 26, which is N-glycosylated.

28. The antibody for use according to any one of claims 1 to 27, administered in a formulation according to any one of claims 29 to 47.

29. 29. A liquid, aqueous formulation of the recombinant antibody of any one of claims 1 to 28, which selectively binds to the misfolded and / or aggregated form of superoxide dismutase (SOD1), the liquid formulation being suitable for intravenous administration, comprising the antibody at a concentration of about 10-50 mg / mL in an L-histidine / L-histidine monohydrochloride buffer at a pH of about 6.0±1, wherein the antibody remains stable at 5°C±2°C for at least one month, at 25°C±2°C for at least one month, and / or at 40°C±2°C for one week.

30. The antibody is characterized in that it comprises six CDRs of a variable heavy (VH) chain and a variable light (VL) chain in its variable region, i.e., binding domain, (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof, wherein said variant comprises one or two amino acid substitutions; (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof, wherein said variant comprises one or two amino acid substitutions; (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and (f) VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 10 or a variant thereof, wherein the variant contains one or two amino acid substitutions; and 30. The formulation of claim 29, wherein the antibody comprises a human Ig constant region.

31. the antibody is characterized in that it comprises a VH chain and a VL chain in its variable region, i.e., binding domain, (a) the VH chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 or 2; and 31. The formulation of claim 29 or 30, wherein (b) the VL chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 6 or 7.

32. 32. The formulation of any one of claims 29 to 31, wherein the antibody binds to an epitope of SOD1 within the amino acid sequence 73-GGPKDEERHVGD-84 set forth in SEQ ID NO:

11.

33. The formulation of any one of claims 29 to 32, wherein the antibody is a human IgG.

34. The formulation of any one of claims 29 to 33, wherein the antibody is human IgG1.

35. The formulation of any one of claims 29 to 34, wherein the antibody is of the human IgG1m3 allotype.

36. 36. The formulation of any one of claims 31 to 35, wherein the antibody comprises a kappa (κ) or lambda (λ) light chain.

37. 37. The formulation of claim 35 or 36, wherein each heavy chain is composed of 453 amino acid residues having SEQ ID NO: 12 and each light chain is composed of 213 amino acid residues having SEQ ID NO:

13.

38. 38. The formulation of any one of claims 29 to 37, produced in CHO-K1 host cells and purified from the cell culture.

39. The formulation of any one of claims 29 to 38, wherein the antibody is AP-101.

40. 40. The formulation of any one of claims 31 to 39, wherein the antibody is composed of two heavy chains having SEQ ID NO: 12 and two light chains having SEQ ID NO: 13, and optionally, in the heavy chains, the N-terminal glutamine is modified as pyroglutamic acid, the C-terminal lysine is missing, and the heavy chains are N-glycosylated.

41. 41. The formulation of any one of claims 29 to 40, wherein the antibody is present at a concentration of 20±5 mg / mL.

42. 42. The formulation according to any one of claims 29 to 41, wherein the concentration of L-histidine / L-histidine monohydrochloride is 20±10 mM.

43. A formulation according to any one of claims 29 to 42, wherein the formulation further comprises a tonicity adjusting agent, preferably sucrose, in a concentration of 8%±1% (w / v).

44. A formulation according to any one of claims 29 to 43, wherein the formulation further comprises a surfactant, preferably polysorbate 80, in a concentration of 0.02% ± 0.01 (w / v).

45. A formulation according to any one of claims 29 to 44, wherein the formulation further comprises an antioxidant, preferably L-methionine, in a concentration of 0.1%±0.05% (w / v).

46. 46. ​​The formulation of any one of claims 29 to 45, which is substantially free of other additional excipients.

47. 47. The formulation of any one of claims 43 to 46, comprising or consisting essentially of 20 mg / mL antibody in 20 mM L-histidine / L-histidine monohydrochloride buffer at pH 6.0, 8% (w / v) sucrose, 0.1% (w / v) L-methionine and 0.02% (w / v) PS 80; preferably, the antibody is AP-101.

48. A medicament comprising a formulation according to any one of claims 29 to 47, preferably for use in the treatment of amyotrophic lateral sclerosis (ALS), most preferably for use in a treatment as defined in any one of claims 1 to 47.

49. 49. A sterile pharmaceutical container comprising the formulation of any one of claims 29 to 47 or the medicament of claim 48, preferably a single-use glass vial to provide 100 mg of the antibody at a concentration of 20 mg / mL, optionally wherein the vial comprises approximately a 10% volume overfill.

50. 50. A method for the treatment of ALS, wherein the treatment is performed according to any one of claims 1 to 28, or comprises administering a formulation according to any one of claims 29 to 49 or a medicament according to claim 48.

51. 50. Use of an antibody according to any one of the preceding claims or a formulation according to any one of claims 29 to 49 for the manufacture of a medicament for the treatment of ALS, wherein said treatment is carried out according to any one of claims 1 to 28.

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