ANTI-ABETA VACCINE THERAPY
20220226447 · 2022-07-21
Inventors
Cpc classification
A61P25/28
HUMAN NECESSITIES
A61K39/39
HUMAN NECESSITIES
A61K2039/55555
HUMAN NECESSITIES
A61K2039/55572
HUMAN NECESSITIES
International classification
A61K39/00
HUMAN NECESSITIES
A61K39/39
HUMAN NECESSITIES
Abstract
A liposomal vaccine composition comprising: a. A β-amyloid (Aβ)-derived peptide antigen displayed on the surface of the liposome that comprises, consists essentially of or consists of amino acids 1-15 of Aβ, b. An adjuvant comprising monophosphoryl lipid A (MPLA) is used for inducing an anti-Aβ immune response in a human subject without inducing a serious adverse event. The β-amyloid (Aβ)-derived peptide antigen (SEQ ID NO: 1) is administered in an amount of 300-2000 μg, preferably around 1000 μg. The MPLA is administered in an amount of 15-600 μg, preferably around 175 μg. The liposomal vaccine composition is administered intramuscularly or subcutaneously.
Claims
1. A liposomal vaccine composition comprising: a. A β-amyloid (Aβ)-derived peptide antigen displayed on the surface of the liposome that comprises, consists essentially of or consists of amino acids 1-15 of Aβ; and b. An adjuvant comprising monophosphoryl lipid A (MPLA) for use in inducing an anti-Aβ immune response in a human subject without inducing a serious adverse event, wherein the β-amyloid (Aβ)-derived peptide antigen is administered in an amount of 300-2000 μg.
2. The liposomal vaccine composition for use of claim 1 wherein the β-amyloid (Aβ)-derived peptide antigen is administered in an amount of 500-2000 μg, preferably 1000-1500 μg, more preferably in an amount of 1000 μg.
3. The liposomal vaccine composition for use of claim 1 or 2 wherein the MPLA is administered in an amount of 15-600 μg, such as 50-600 μg, preferably 150-450 μg and more preferably 175 or 225 μg.
4. The liposomal vaccine composition for use of any one of claims 1 to 3 wherein the β-amyloid (Aβ)-derived peptide antigen is administered in an amount between 850 and 1150 μg, preferably 1000 μg and the MPLA adjuvant is administered in an amount of between 50 and 300 μg, preferably 175 or 225 μg.
5. The liposomal vaccine composition for use of any one of claims 1 to 3 wherein the β-amyloid (Aβ)-derived peptide antigen is administered in an amount between 255 and 345 μg, preferably 300 μg and the MPLA adjuvant is administered in an amount of between 15 and 90 μg, preferably 52.5 or 67.5 μg.
6. The liposomal vaccine composition for use of any one of claims 1 to 5 wherein the β-amyloid (Aβ)-derived peptide antigen is lipidated.
7. The liposomal vaccine composition for use of any one of claims 1 to 6 wherein the β-amyloid (Aβ)-derived peptide antigen is tetrapalmitoylated.
8. The liposomal vaccine composition for use of any one of claims 1 to 7 wherein the adjuvant forms part of the outer layer of the liposome, optionally wherein the adjuvant is, at least in part, displayed on the surface of the liposome.
9. The liposomal vaccine composition for use of any one of claims 1 to 8 wherein the monophosphoryl lipid A (MPLA) comprises synthetic monophosphoryl lipid A (MPLA).
10. The liposomal vaccine composition for use of claim 9 wherein the monophosphoryl lipid A (MPLA) comprises monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (Synthetic) (3D-(6-acyl) PHAD®) and/or Phosphorylated HexaAcyl Disaccharide (PHAD®).
11. The liposomal vaccine composition for use of any one of claims 1 to 10 wherein the liposome comprises phospholipids.
12. The liposomal vaccine composition for use of any one of claims 1 to 11 wherein the phospholipids comprise dimyrsitoylphosphatidyl-choline (DMPC) and dimyrsitoylphosphatidyl-glycerol (DMPG).
13. The liposomal vaccine composition for use of any one of claims 1 to 12 wherein the liposome comprises cholesterol.
14. The liposomal vaccine composition for use of claim 13 wherein the molar ratio of dimyrsitoylphosphatidyl-choline (DMPC):dimyrsitoylphosphatidyl-glycerol (DMPG):cholesterol is 9:1:7.
15. The liposomal vaccine composition for use of claim 14 wherein the molar ratio of dimyrsitoylphosphatidyl-choline (DMPC):dimyrsitoylphosphatidyl-glycerol (DMPG):cholesterol:MPLA is 9:1:7:0.05.
16. The liposomal vaccine composition for use of any one of claims 1 to 15 wherein the liposomal vaccine composition is administered by injection.
17. The liposomal vaccine composition for use of any one of claims 1 to 16 wherein the liposomal vaccine composition is administered intramuscularly or subcutaneously.
18. The liposomal vaccine composition for use of claim 17 wherein the liposomal vaccine composition is administered intramuscularly.
19. The liposomal vaccine composition for use of claim 17 wherein the liposomal vaccine composition is administered subcutaneously.
20. The liposomal vaccine composition for use of any one of claims 1 to 19 wherein the liposomal vaccine composition is administered at a first time and is administered at a second time 1 to 4 weeks later.
21. The liposomal vaccine composition for use of any one of claims 1 to 20 wherein the liposomal vaccine composition is administered every 4-12 weeks for a period of at least 48 weeks, preferably wherein the liposomal vaccine composition is administered every 4 weeks for a period of 12 weeks and every 12 weeks for a further period of at least 36 weeks.
22. The liposomal vaccine composition for use of any one of claims 1 to 21 further comprising a booster administration at a subsequent time point.
23. The liposomal vaccine composition for use of any one of claims 1 to 22 wherein the induced anti-Aβ immune response is for treatment, prevention, induction of a protective immune response against or alleviating the symptoms associated with an amyloid-beta associated disease or condition in the human subject.
24. The liposomal vaccine composition for use of claim 23 wherein the amyloid-beta associated disease or condition is selected from Alzheimer's Disease, mild cognitive impairment (MCI), Down syndrome (DS), including Down syndrome-related Alzheimer's disease, cardiac amyloidosis, cerebral amyloid angiopathy (CAA), multiple sclerosis, Parkinson's disease, Lewy body dementia, ALS (amyotrophic lateral sclerosis), Adult Onset Diabetes, inclusion body myositis (IBM), ocular amyloidosis, glaucoma, macular degeneration, lattice dystrophy and optic neuritis.
25. The liposomal vaccine composition for use of claim 24 wherein the amyloid-beta associated disease or condition is Alzheimer's Disease.
26. The liposomal vaccine composition for use of claim 25 wherein the Alzheimer's Disease is early Alzheimer's Disease.
27. The liposomal vaccine composition for use of claim 26 wherein the early Alzheimer's Disease includes mild cognitive impairment due to Alzheimer's Disease and mild Alzheimer's Disease.
28. The liposomal vaccine composition for use of claim 25 wherein the Alzheimer's Disease is mild Alzheimer's Disease.
29. The liposomal vaccine composition for use of claim 25 wherein the Alzheimer's Disease is mild-to-moderate Alzheimer's Disease.
30. The liposomal vaccine composition for use of claim 25 wherein the Alzheimer's Disease is moderate Alzheimer's Disease.
31. The liposomal vaccine composition for use of claim 25 wherein the Alzheimer's Disease is not severe Alzheimer's Disease.
32. The liposomal vaccine composition for use of claim 24 wherein the amyloid-beta associated disease or condition is Down Syndrome.
33. The liposomal vaccine composition for use of claim 24 or 32 wherein the amyloid-beta associated disease or condition is Down syndrome-related Alzheimer's disease.
34. The liposomal vaccine composition for use of any one of claims 1 to 33 wherein the human subject, prior to treatment, displays cognitive function consistent with a Mini Mental State Examination (MMSE) score of at least 18, such as 18-28, or at least 20, such as 20-28.
35. The liposomal vaccine composition for use of any one of claims 1 to 34 wherein the β-amyloid (Aβ)-derived peptide antigen is tetrapalmitoylated Abeta 1-15 as set forth in SEQ ID NO: 1.
36. The liposomal vaccine composition for use of any one of claims 1 to 35 wherein the administered amount of Abeta 1-15 as set forth in SEQ ID NO: 2 is 152-1016 μg.
Description
DESCRIPTION OF THE FIGURES
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[0080]
[0081]
TABLE-US-00003 Table of abbreviations AD Alzheimer's Disease ARIA-E Amyloid-related imaging abnormalities-vasogenic edema ARIA-H Amyloid-related imaging abnormalities-microhemorrhages, superficial siderosis Aβ Amyloid beta (abeta) BPT Brief Praxis Test CANTAB Cambridge Neuropsychological Test Automated Battery CDR Clinical Dementia Rating scale CDR-SB Clinical Dementia Rating scale-Sum of Boxes CGIC Clinical Global Impression of Change CNS Central Nervous System CSF Cerebrospinal Fluid DMPC 1,2-Dimyristoyl-sn-glycero-3-phosphocholine DMPG 1,2-Dimyristoyl-sn-glycero-3-phosphorylglycerol DSMB Data and Safety Monitoring Board ECG Electrocardiogram ELISA Enzyme-linked immunosorbent assay ELISPOT Enzyme-linked immune absorbent spot GABA Gamma-Aminobutyric acid GLP Good Laboratory Practice HE Hematoxylin and eosin IFN Interferon IL interleukin i.m. intramuscular Ig Immunoglobulin MMSE Mini-Mental State Examination MPLA Monophosphoryl Lipid A MRI Magnetic Resonance Imaging NIA-AA National Institute on Aging-Alzheimer's Association NINCDS- National Institute of Neurological and Communicative ADRDA Diseases and Stroke-Alzheimer's Disease and Related Disorders Association NOAEL No Observed Adverse Effet Level NPI Neuropsychiatric Inventory Pal1-15 Tetrapalmitoylated Aβ1-15 PBS Phosphate buffered saline PET Positron Emission Tomography s.c. Subcutaneous SAE Serious Adverse Event sAPPα Soluble amyloid precursor protein alpha sAPPβ Soluble amyloid precursor protein beta SSRI/SNRI Selective serotonin reuptake inhibitor/Serotonin- norepinephrine reuptake inhibitor SGOT serum glutamic-oxaloacetic transaminase SGPT Serum glutamic pyruvic transaminase TLR4 Toll-like receptor 4 TSH Thyroid-stimulating hormone VABS Vineland Adaptive Behavior Scale
[0082] The invention will be further understood with reference to the following non-limiting examples:
Definitions
[0083] The MMSE (Folstein 1975) is a widely used test of overall cognitive function, assessing memory, orientation and praxis in a short series of tests. The score is from 0 to 30 with 30 being the best possible and 0 being the worst possible score.
[0084] The Clinical Dementia Rating Scale (Hughes et al 1982) is a global rating of the function (it is not only purely functioning since cognition is also being checked with memory) of Alzheimer patients assessed in six categories: memory, orientation, judgement and problem solving, community affairs, home and hobbies and personal care. It is based on a semi-structured interview conducted with the patient and caregiver, by a rater without access to the results of the cognitive tests described above. Each category has scores from 0 (no symptoms) to 3 (severe) and the sum of these items (Sum of Boxes) may therefore range from 0 to 18 points.
[0085] Early AD patients include Mild Cognitive Impairment (MCI) due to AD and mild AD.
[0086] According to the National Institute on Aging—Alzheimer Association (NIA-AA) criteria, Mild Cognitive Impairment due to Alzheimer's Disease requires evidence of intra-individual decline, manifested by a change in cognition from previously attained levels, as noted by self- or informant report and/or the judgment of a clinician, impaired cognition in at least one domain (but not necessarily episodic memory) relative to age- and education-matched normative values (impairment in more than one cognitive domain is permissible), a preserved independence in functional abilities, no dementia, and a clinical presentation consistent with the phenotype of AD in the absence of other potentially dementing disorders.
[0087] Probable AD dementia according to NIA-AA criteria meets criteria for dementia and in addition, has the following main characteristics: insidious onset (symptoms have a gradual onset over months to years, not sudden over hours or days), clear-cut history of worsening of cognition by report or observation; and the initial and most prominent cognitive deficits are evident on history and examination in one of the following categories: Amnestic presentation (it is the most common syndromic presentation of AD dementia. The deficits should include impairment in learning and recall of recently learned information). There should also be evidence of cognitive dysfunction in at least one other cognitive domain); Non-amnestic presentations: Language presentation (the most prominent deficits are in word-finding, but deficits in other cognitive domains should be present); Visuospatial presentation: (the most prominent deficits are in spatial cognition, including object agnosia, impaired face recognition, simultanagnosia, and alexia; deficits in other cognitive domains should be present); Executive dysfunction (the most prominent deficits are impaired reasoning, judgment, and problem solving. Deficits in other cognitive domains should be present).
[0088] Early AD patients are patients with the MMSE score of at least 20 (equal or above 20).
[0089] They include patients with Mild Cognitive Impairment due to AD and patients with mild AD.
[0090] Mild AD patients are patients with the MMSE score of 20 to 28.
[0091] Mild-to moderate AD patients are patients with the MMSE score of 12 to 28.
[0092] Moderate AD patients are patients with the MMSE score of 12 to 19.
Example 1. Safety and Efficacy in Humans in Phase I/II AD Trial
Study Objective:
[0093] The overall study objective was to assess the safety, immunogenicity and efficacy of repeated doses of ACI-24 at 4 different dose levels administered to patients with mild to moderate Alzheimer's disease (AD) as diagnosed by the criteria of the National Institute of Neurological and Communicative Diseases and Stroke—Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) and with a score at initial screening of 18-28 on the Mini-Mental State Examination (MMSE).
Primary Objectives:
[0094] To assess the safety and tolerability of ACI-24 in patients with mild to moderate Alzheimer's Disease. [0095] To assess the effect of different doses of ACI-24 on induction of anti-Aβ1-42 IgG titer in serum.
Secondary Objectives:
[0096] To explore the efficacy of ACI-24 in reducing Aβ level in the brain of patients with mild to moderate Alzheimer's Disease. [0097] To explore the effect of ACI-24 on T cell activation. [0098] To explore the effects of ACI-24 on putative biomarkers of the progression of Alzheimer's Disease like total tau and phosphorylated tau protein (phosphotau) and Aβ levels (Aβ1-42 and Aβ1-40) in blood and CSF. [0099] To explore the efficacy of ACI-24 on clinical/cognitive endpoints in patients with mild to moderate Alzheimer's Disease. [0100] To explore the induction of immune response in serum and/or CSF including, but not limited to, anti-Aβ1-42 IgM titer in blood. [0101] To explore the induction of inflammatory cytokines in blood.
[0102] 48 Patients were randomized with a ratio of 3:1 active (ACI-24) versus placebo (normal saline) into 4 dose-cohorts. Patients were administered the study medication 7 times, once every 4 weeks for the first 4 administrations, then once every 12 weeks for the last 3 administrations. The administration schedule of subcutaneous injections was at weeks 0, 4, 8, 12, 24, 36 and 48 with optional booster injections. One additional boosting dose of 300 μg or placebo was administered in 4 patients of cohort 3 (3 were on ACI-24 and 1 was on placebo) who were willing and able to give consent, 6-15 months after the 2 years safety follow-up that is 2.5-3.25 years after the last injection received at visit 16 (V16, week 48 during which the 7th injection was to be administered). An additional boosting dose of ACI-24 1000 μg or placebo was administered to patients of cohort 4, 18 months (week 74) after the first dose. The dose-cohorts were studied sequentially as follows: [0103] Dose-Cohort 1: 10 μg antigen or placebo [0104] Dose-Cohort 2: 100 μg antigen or placebo [0105] Dose-Cohort 3: 300 μg antigen or placebo [0106] Dose-Cohort 4: 1000 μg antigen or placebo
[0107] Antigen dose refers to tetrapalmitoylated Aβ1-15 acetate salt. The pharmaceutical form of the vaccine is a suspension for injection (liposomal suspension in PBS). The dose-cohorts were studied in a sequential manner, each cohort having to complete 4 immunizations and safety data including data 2 weeks after the fourth injection (i.e. at visit 8, week 14) being reviewed by the Data and Safety Monitoring Board (DSMB) before the start of enrolment into the next cohort. To further enhance safety an interval of at least one week was planned between first dose administration in the first 4 subjects in each cohort.
Inclusion Criteria:
[0108] Probable AD according to NINCDS-ADRDA criteria. [0109] Florbetaben-PET scan at screening consistent with the presence of amyloid pathology. [0110] Mini-Mental Status Examination (MMSE) 18-28 points*. [0111] Age over 40 and less than 90 years**. [0112] Patients receiving a stable dose of an acetylcholinesterase inhibitor within 4 months prior to baseline. [0113] Patients cared for by a reliable spouse or caregiver to assure compliance, assist with clinical assessments and report safety issues. [0114] Women must be post-menopausal for at least one year, surgically sterilised or using reliable contraceptive measures. [0115] Patient who in the opinion of the investigator are able to understand and provide written informed consent. [0116] Patients and caregivers must be fluent in the language of the study and able to comply with all study procedures. [0117] The patient is lucid and clear and oriented ×4 and is able to provide their written informed consent (applicable only in some countries).
* For cohort 3 booster injection, the previous lower limit of 18 points for the MMSE was not required but in all cases patients were to be oriented in time, place, awareness person and current activities and able to give informed consent in the opinion of the investigator in order to take part.
** For cohort 3 booster injection, no upper age limit applied.
Exclusion Criteria:
[0118] Patients whose MRI scan within the last 6 months shows alternative pathology including severe vascular encephalopathy and/or more than 5 micro-hemorrhages. [0119] Patients with other medical conditions which may influence cognitive performance e.g. Parkinson's disease. [0120] Patients with any unstable medical condition (e.g. epilepsy, uncontrolled hypertension) which would hamper safety assessments. [0121] Patients receiving memantine within 3 months prior to baseline (for cohort 3 booster injection, memantine is allowed). [0122] Patients receiving any anticoagulant drug. [0123] Patients with a history of hemorrhagic stroke. [0124] Patients with a history of non-hemorrhagic stroke or myocardial infarction within the last year. [0125] Patients with a history of major psychiatric disorder within the past 2 years. [0126] Patients with a history of inflammatory neurology disorders including meningoencephalitis. [0127] Clinically significant abnormalities of clinical hematology or biochemistry including, but not limited to, elevations greater than 1.5 times the upper limit of normal of SGOT, SGPT, or creatinine. [0128] Patients with a history of autoimmune disease. [0129] Patients with a history of cancer other than skin cancer within the past 5 years. [0130] Patients who have received any vaccine within the 2 months before baseline. [0131] Patients who have previously received AD immune therapeutic agents or vaccines. [0132] Patients anticipated to receive any vaccination other than flu vaccine during the study. [0133] Patients unable to undergo MRI examination for any reason, including metal implants and claustrophobia. [0134] Patients with a positive HIV test at screening. [0135] Patients with positive syphilis serology. [0136] Women who are pregnant or planning to be pregnant, or who are lactating.
Results/Conclusions:
[0137] 48 mild to moderate AD patients were randomized and were exposed to ACI-24 at different dose levels (10 μg, 100 μg, 300 μg and 1000 μg per administration) or placebo with up to seven subcutaneous administrations each, over 12 months. Some patients from the 2 highest dose-cohorts received an additional late booster administration (i.e., a total of 8 subcutaneous injections).
[0138] No anti-abeta IgG response was observed in placebo treated patients and in patients treated with the two lowest doses tested (10 and 100 ug of antigen, cohorts 1 and 2). The vaccine was able to induce an anti-abeta antibody response in human subjects in a need thereof at the highest doses tested (300 and 1000 ug of antigen, cohorts 3 and 4) and a dose-dependent anti-Aβ IgG response was observed at the two highest doses. A dose-related late-onset IgG response was observed. Safety was considered good in the study at all doses tested (from 10 μg to 1000 μg of antigen). No SAE related to the study treatment, no signal of CNS Inflammation or other unwanted reactions to the vaccine, no ARIA-E, no ARIA-H (1 tiny lesion with low signal on hemosequence suspicious for a microbleed was noticed at the ACI-24 dose of 100 μg (possible artefact)), no indication of the development of meningoencephalitis and no T-cell activation and induction of inflammatory cytokines were observed.
[0139] A dose-dependent trend in reduction of brain amyloid accumulation was observed at the two highest doses in both cohorts 3 and 4 at week 52 (
Example 2. Safety and Efficacy in Humans in Phase II AD Trial
Study Objective:
[0140] The overall study objective is to assess the safety, immunogenicity and efficacy/target engagement of ACI-24 administered to patients with mild Alzheimer's disease (AD) as diagnosed by the criteria of the National Institute on Aging—Alzheimer's Association (NIA-AA) and with a score at initial screening of 20-28 on the Mini-Mental State Examination (MMSE).
Primary Objectives:
[0141] To assess the safety and tolerability of the ACI-24 in patients with mild Alzheimer's disease. [0142] To assess the effects of ACI-24 on induction of anti-Aβ antibody responses in serum. [0143] To assess the effects of ACI-24 on brain amyloid load in patients with mild Alzheimer's disease, assessed by florbetaben-PET imaging at 52 weeks (12 months) and 76 weeks (18 months).
Secondary Objectives:
[0144] To explore the effects of ACI-24 on putative biomarkers of the progression of Alzheimer's disease including concentrations of total tau and phosphorylated tau protein (phosphotau) and Aβ in blood and/or CSF. [0145] To explore the effects of ACI-24 on T cell activation in blood. [0146] To explore the effects of ACI-24 on whole brain and hippocampal volume by volumetric MRI. [0147] To explore the effects of ACI-24 on clinical and cognitive endpoints in patients with mild Alzheimer's disease. [0148] To explore the influence of ACI-24 on blood inflammatory cytokines.
Inclusion Criteria:
[0149] Patients meeting all of the following inclusion criteria at screening should be considered as eligible to participate to the study:
[0150] 1. Probable AD dementia according to NIA-AA core clinical criteria
[0151] 2. Florbetaben-PET scan at screening consistent with the presence of amyloid pathology
[0152] 3. Mini-Mental Status Examination (MMSE) score 20-28 points
[0153] 4. Age greater than or equal to 50 and less than or equal to 85 years
[0154] 5. Patients receiving a stable dose of an acetylcholinesterase inhibitor for at least 3 months prior to screening
[0155] 6. Patients cared for by a reliable spouse or other caregiver to assure compliance, assist with clinical assessments and report safety issues, and spouse or caregiver consents to serve in this role
7. Women must be post-menopausal for at least one year, surgically sterilized or using reliable contraceptive measures
[0156] 8. Patients who in the opinion of the investigator are able to understand and provide written informed consent
[0157] 9. Patients and caregivers must be fluent in the official language(s) of the country they are living in and able to comply with all study procedures
[0158] 10. Patients are lucid and clear and oriented ×4 (awareness of person, knowledge of place, time/date and event) [applicable in some countries only]
[0159] In a first cohort, ACI-24 given intramuscularly will be investigated. This study is a multicenter prospective placebo-controlled, double-blind and randomized study to assess treatment with ACI-24 formulations versus placebo over 76 weeks (18 months) in patients with mild Alzheimer's disease. Antigen dose refers to tetrapalmitoylated Aβ1-15 acetate salt. The pharmaceutical form of the vaccine is a suspension for injection (liposomal suspension in PBS).
Cohort 1 with ACI-24:
[0160] One dose of ACI-24 at 1000 μg/dose given by the intramuscular route will be tested.
[0161] Patients will be randomized with a ratio of 2:1 active (ACI-24) versus placebo.
[0162] For patients participating in cohort 1, the treatment period will last 76 weeks with the treatment/placebo being administered 8 times (each time the dose of study treatment will be administered in two separate concomitant intramuscular injections); 4 times with 4 weeks' intervals, 3 times with 12 weeks' intervals and 1 time 26 weeks after the preceding 7th dose. The treatment period is followed by a 24-week period of safety follow-up starting 2 weeks after the last administration. Patients who for some reason receive less than 8 administrations will be followed at least for the same duration after their last administration. Free, total and immune complexed IgG titers will be measured.
Example 3. Safety and Efficacy in Humans in Phase Ib DS Trial
Primary Objectives:
[0163] To assess the safety and tolerability of ACI-24 in adults with Down Syndrome. [0164] To assess the effect of different doses ACI-24 on induction of anti-Aβ Ig titer in serum.
Secondary Objectives:
[0165] To explore the efficacy of ACI-24 on Clinical Global Impression of Change (CGIC) in adults with Down Syndrome. [0166] To explore the effect of ACI-24 on cognitive (CANTAB motor control, reaction time, paired associative learning; BPT) and behavioral (VABS, NPI) endpoints in adults with Down Syndrome. [0167] To explore the effect of ACI-24 on whole brain, ventricle and hippocampal volume by MRI. [0168] To explore the effect of ACI-24 on peripheral T cell activation. [0169] To explore the effect of ACI-24 on putative biomarkers of Alzheimer pathology in Down Syndrome including Aβ levels, total tau, phosphorylated tau protein (phospho-tau), sAPPα, sAPPβ, Orexin-A, inflammatory cytokines, angiogenic proteins, TLR-4 expression and vascular injury markers in plasma and/or in CSF* (*in subgroup) as applicable. [0170] To assess the effect of different doses ACI-24 on induction of anti-Aβ Ig titer in CSF* (*in subgroup).
Method:
[0171] This is a prospective multi-center, placebo controlled, double-blind and randomized study of 2 doses of ACI-24 treatment versus placebo over 24 months.
[0172] The study consists of 2 dose-cohorts of 8 subjects each (6 subjects on ACI-24 300 μg, 6 subjects on ACI-24 1000 μg and 2 subjects on placebo in each dose-cohort) with s.c. injections at month 0, 1, 2, 3, 6, 9 and 12 (or more precisely weeks 0, 4, 8, 12, 24, 36 and 48) with 12 months treatment free safety follow-up. The dose-cohorts are studied sequentially in ascending dose order. The 2nd dose-cohort was started once safety and tolerability data up through visit 8 [week 14] of the last subject of the preceding cohort were reviewed by the Data Safety Monitoring Board (DSMB). Antigen dose refers to tetrapalmitoylated Aβ1-15 acetate salt. The pharmaceutical form of the vaccine is a suspension for injection (liposomal suspension in PBS).
[0173] An interim analysis was conducted in this study after visit 8 [week 14] of the last subject of cohort 1 as a basis to allow the dose escalation. The analysis focused on safety and tolerability. The interim analysis was conducted in an unblinded fashion and the unblinded data were presented to the DSMB.
[0174] Additional interim analyses are planned to be conducted after visit 9 [week 16], visit 12 [week 28], visit 15 [week 40] and visit 18 [week 52] of the last subject in cohort 1 and in cohort 2 respectively. These analyses focus on safety, tolerability, antibody titer and inflammatory cytokines data (part of biomarkers). Interim analyses at visit 12 [week 28] and visit 18 [week 52] additionally include biomarkers, as well as CGIC, NPI and Vineland data (part of clinical rating scales and cognitive tests).
Inclusion Criteria:
[0175] Males or females with Down Syndrome aged ≥25 to ≤45 years, with a cytogenetic diagnosis being either Trisomy 21 or Complete Unbalanced Translocation of the Chromosome 21. [0176] Subjects and their study partner/legal representative in the opinion of the investigator able to understand and to provide written informed consent. [0177] Written informed consent obtained from subjects and their study partner/legal representative before any trial-related activities. [0178] In the opinion of the investigator able to fully participate in the trial and sufficiently proficient in English to be capable of reliably completing study assessments. [0179] Subjects have a study partner/legal representative who have direct contact with the subjects at least 10 hours per week and who can be asked questions about the subjects.
Exclusion Criteria:
[0180] Subjects weighing less than 40 kg. [0181] IQ less than 40 (as assessed by Kaufman Brief Intelligence Test, Second Edition (KBIT-2). [0182] In the investigators' opinion, any clinically significant current psychiatric or neurologic illness, including a past illness with a risk of recurrence, other than Down syndrome. [0183] Any medical condition likely to significantly hamper the evaluation of safety of the study drug. [0184] DSM-IV criteria for drug or alcohol abuse or dependence currently met within the past five years. [0185] History or presence of uncontrolled seizures. If history of seizures, they must be well controlled with no occurrence of seizures in the past 2 years prior to study screening. The use of antiepileptic medications is permitted. [0186] History of meningitis or meningoencephalitis. [0187] History of malignant neoplasms within 3 years prior to study screening or where there is current evidence of recurrent or metastatic disease. [0188] History of persistent cognitive deficits immediately following head trauma. [0189] History of inflammatory neurology disorders. [0190] History of autoimmune disease with potential for CNS involvement. [0191] MRI scan at screening showing a single area of cerebral vasogenic edema, superficial siderosis, or evidence of a prior macro-hemorrhage, or showing more than four cerebral microhemorrhages (regardless of their anatomical location or diagnostic characterization as “possible” or “definite”). [0192] MRI examination cannot be done for any reason, including metal implants contraindicated for MRI studies and/or severe claustrophobia. [0193] Significant hearing or visual impairment or other issues judged relevant by the investigator preventing to comply with the protocol and to perform the outcome measures. [0194] Severe infections or a major surgical operation within 3 months prior to screening. [0195] History of chronic or recurrent infections judged to be clinically significant by the investigator. [0196] History or presence of immunological or inflammatory conditions which are judged to be clinically significant by the investigator. [0197] Celiac disease not on a gluten free diet for at least 3 months prior to study screening. [0198] Chronic benign skin pathologies, unless viewed as clinically insignificant in the investigator's opinion. [0199] Any vaccine received within the past 2 months before baseline, except influenza vaccine which, if indicated, must be given at least 2 weeks prior to baseline. [0200] Clinically significant arrhythmias or other abnormalities on ECG at screening. (Minor abnormalities documented as clinically insignificant by the investigator will be allowed). [0201] Clinically significant abnormal vital signs including sustained sitting blood pressure greater than 160/90 mmHg. [0202] In the opinion of the site investigator, deviations from normal values for hematologic parameters, liver function tests, and other biochemical measures, that are judged to be clinically significant. [0203] Subjects with treated hypothyroidism not on a stable dose of medication for at least 3 months prior to screening and having clinically significant abnormal serum T-4 and TSH at screening. [0204] Subjects with diabetes mellitus with an HbA1c of ≥8.0%. [0205] Subjects who have been receiving any experimental drug for Down Syndrome with a washout less than 30 days or less than five half-lives of the drug, whichever is longer. [0206] Female subjects being pregnant as confirmed by serum testing at screening or planning to be pregnant or lactating. [0207] Female subjects not using a reliable method of contraception (unless abstaining). [0208] Patient receiving any anticoagulant drug, or aspirin at doses greater than 100 mg daily in the 7 days prior to lumbar puncture (in order to avoid risk of bleeding during scheduled or unscheduled lumbar puncture) [0209] Use of antidepressants other than SSRI/SNRIs at stable dose, antipsychotics (typical or atypical), GABA agonists (e.g. gabapentin), or stimulants (e.g. methylphenidate, modafinil). In exceptional cases, low doses of atypical antipsychotics (e.g. risperidone up to 0.5 mg/day or quetiapine up to 50 mg/day) or benzodiazepines are only allowed after review by the site principal investigator, in consultation with the project director and/or medical monitor. [0210] Current use of immunosuppressant or immunomodulating drugs or their use within the past 6 months prior to study screening. Current use of oral steroids or their use within the past 3 months prior to study screening. [0211] Use of Cholinesterase Inhibitor or use of Glutamatergic drugs (Topiramate, Memantine, Lamotrigine) if not on stable dose for at least 3 months prior to screening. [0212] Subjects who have donated blood or blood products during the 30 days prior to screening who plan to donate blood while participating in the study or within four weeks after completion of the study.
Results
[0213] The trial is a fully enrolled, placebo-controlled, Phase Ib study of the ACI-24 anti-Abeta vaccine. Sixteen subjects have been randomized in the study. The vaccine was able to induce an anti-Abeta antibody response in human subjects in a need thereof at the both doses tested (300 and 1000 ug of antigen). An early-onset IgG response was observed with a first increase in titers at 4 weeks. According to MSD data, a boosting effect could be observed over time, and the anti-Abeta antibody response was consistent in the majority of patients at the highest dose. The vaccine was well tolerated in DS subjects, demonstrating a favourable safety profile at all doses tested. Safety was considered good in the study at both doses tested. There were no subject withdrawals during the treatment period. No SAE related to the study treatment, no signal of CNS inflammation or other important unwanted reactions to the vaccine, no ARIA-E, no ARIA-H, no indication of the development of meningoencephalitis and no T-cell activation and induction of inflammatory cytokines were observed.
[0214] The subsequent DS clinical development plan (Example 5) will focus on prevention therapy notably using biomarker endpoints (such as Abeta, Neurofilament, and Tau). The vaccine will be administered at the highest dose (1000 μg) via the intramuscular route to boost immunogenicity further. Two of the selected readouts will be PET-scan imaging and measure of free, total and immune complexed IgG titers generated by the vaccine.
Example 4. Toxicology Studies
4.1 Single Dose Toxicity
[0215] Single dose toxicity of ACI-24 was evaluated in two non-clinical models (mice and monkeys). ACI-24 was well tolerated and was not associated with organ toxicity. These two studies are summarized below.
4.1.1 Evaluation of Single-Dose Toxicity following Subcutaneous or Intramuscular Administrations in Mice
Objective
[0216] The potential toxicity, local tolerance and immunogenicity of a single s.c. or i.m. injection of ACI-24 in mice was evaluated.
Design
[0217] The study was conducted under GLP standards. The number of animals, dosage form administered, route of administration and dose-level for each group are summarized in Table 1. Animals were kept for a 14-day observation period to evaluate a possible delayed toxicity and/or the reversibility of observed changes. Satellite groups were added to evaluate the immune response at Day 14 for both routes of administration (s.c. and i.m.) and at Days 1, 3 or 7 for the s.c. route of administration only.
TABLE-US-00004 TABLE 1 Group distribution of study Dose- Dose- Volume level level of [μg of [μg of Number of animals Dosage form injection Route of peptide/ MPLA/ Group Total Male Female administered [mL] administration injection] injection] 1 Principal = 6 6 PBS 0.8 s.c. 0 0 12 2 Principal = 6 6 ACI-24-250 0.2 s.c. 0 30 12 (empty) 3 Principal = 6 6 ACI-24-1000 0.8 s.c. 0 30 12 (empty) Satellite1 = 5 5 10 4 Principal = 7 6 ACI-24-250 2 × 0.1 i.m. 0 11 (empty) 5 Principal = 6 6 ACI-24-250 0.2 s.c. 65 30 12 6 Principal = 6 6 ACI-24-1000 0.8 s.c. 260 30 12 Satellite1 = 3 3 6 Satellite2 = 9 9 18 7 Principal = 6 6 ACI-24-1000-A 0.8 s.c. 385 30 12 8 Principal = 6 6 ACI-24-250 2 × 0.1 i.m. 2 × 32.5 30 12 Satellite1 = 5 5 10 [0218] Dose administered once at Day 0. [0219] Blood sample for s.c. administration were collected on Days: 1, 3 or 7 and 14. [0220] Blood sample for i.m. administration were collected on Day 14 [0221] ACI-24-250 and ACI-24-1000 corresponds to the targeted dose of the abeta1-15 antigen; 250 μg and 1000 μg respectively.
[0222] The animals were checked at least once daily for mortality and at least twice daily (three times on Day 1) for clinical signs. Skin reactions at injection site were recorded before injection, then 6, 24 and 48 hours, and then three and seven days after injection. The rectal temperatures were recorded before injection, then 6, 24 and 48 hours after injection and at the end of the observation period. Body weight and food consumption were recorded at least three times a week. Hematological and blood biochemical investigations were performed on, respectively, the three first principal animals and the three last principal animals, at the end of the observation period. Aβ1-42-specific IgG and IgM antibodies were determined by ELISA.
[0223] At the end of the observation period, all surviving animals were sacrificed and submitted to a full macroscopic post-mortem examination. The spleens of all satellite animals were sampled for separation of lymphocyte cells. Designated organs were weighed and selected tissue specimens were preserved for principal animals. Microscopic examination was conducted on subcutaneous injection sites of two satellite mice from Group 6 (total of nine male and nine female mice killed at 1, 3 and 7 days post-injection), stained with hematoxylin and eosin (HE) or with polyclonal rabbit anti-Aβ1-40 precursor protein termed thereafter Aβ.
[0224] Subsequent microscopic examination was performed on intramuscular injection sites (formalin-fixed muscle samples) of mice from Group 8 (6 males and 6 females), stained with hematoxylin-eosin.
Results
[0225] The administration of ACI-24 once by s.c. (at the dose-levels of 65, 260 or 385 μg/injection) or i.m. route (at the dose-level of 65 μg/injection) to mice followed by an observation period of 14 days, was well tolerated. No deaths attributed to the treatment with vehicle or test item formulations were observed during the study period. No toxicologically relevant clinical signs and/or differences of rectal temperatures were attributed to the treatment with the test item.
[0226] No treatment-related skin reactions were noted.
[0227] The body weight and the food consumption were unaffected by the treatment with the test item. At laboratory investigations, no toxicologically relevant differences among hematological or biochemical parameters were observed in animals receiving the empty liposomes or the test item.
[0228] The microscopic examination of i.m. injection site showed that administration of ACI-24 (2×32.5 μg/injection) in the gastrocnemius muscle yielded in all treated mice minimal to slight non-adverse granulomatous inflammation after 2 weeks, characterized by mononuclear cell infiltrates associated with minimal fibrosis. These findings were considered to be non-adverse as the severity was of low magnitude.
Conclusion
[0229] Under the experimental conditions of the study, the no observed adverse effect level (NOAEL) was established at 65 μg/injection by i.m. route and 385 μg/injection by s.c. route.
4.1.2 Evaluation of the Toxicity of ACI-24 Following Single-Dose Subcutaneous Administration in Monkeys
Objective
[0230] The toxicity and local tolerance of a single subcutaneous injection of ACI-24 in cynomolgus monkeys was evaluated in this GLP study.
Design
[0231] The study design is explained in Table 2.
TABLE-US-00005 TABLE 2 Group distribution of study Dose-level Dose-level Number of Volume of [μg of [μg of animals Dosage form injection Route of peptide/ MPLA/ Group Male Female administered [mL] administration injection] injection] 1 3 3 PBS 0.8 s.c. 0 0 2 3 3 ACI-24-250 0.8 s.c. 0 128 (empty) 3 3 3 ACI-24-250 0.2 s.c. 96 9 4 3 3 ACI-24-1000 0.8 s.c. 385 36 [0232] Dose administered once on Day 1. [0233] Local tolerance evaluated after 6, 24, 48 hours and 7 days. [0234] Rectal temperature recorded after 6, 24, 48 hours and 14 days. [0235] ACI-24-250 and ACI-24-1000 corresponds to the targeted dose of the abeta1-15 antigen; 250 μg and 1000 μg respectively.
[0236] The dosage forms were administered once on Day 1. Clinical signs were evaluated, at least three times a day during the study and additionally approximately six hours after treatment on the day of treatment. The local tolerance at the injection site was evaluated on the day of treatment, before injection and 6, 24, and 48 hours and seven days after treatment. Rectal temperature was recorded on the day of treatment, before injection, 6, 24, and 48 hours after treatment and at the end of a 14-day observation period. The body weight of each animal was recorded at designated intervals and food consumption was estimated during the study. Electrocardiography examinations, blood pressure measurements and laboratory investigations (including hematology, blood biochemistry, urinalysis, blood lymphocyte subset analysis and seric immune response quantification) were performed during the pre-treatment period, after treatment and during the observation period. Ophthalmology examinations were performed during the pre-treatment period and once at the end of the 14-day observation period. On completion of the observation period, the animals were sacrificed for organ weight recording, macroscopic post-mortem examination and microscopic examination of selected tissues.
Results
[0237] The administration of ACI-24 or empty liposomes once by s.c. injection to cynomolgus monkeys, was well tolerated. No unscheduled deaths occurred during the study. No systemic clinical signs were noted after treatment or during the observation period in any animal. There were no statistical differences in the body temperatures recorded between control and treated animals, at any time-point. The values recorded were within the range of normal values recorded in healthy animals of this strain and age. Body weight and food consumption were considered to be unaffected by the test item treatment.
[0238] Electrocardiography parameters, including PQ and QT intervals, QRS-complex duration and heart rate were unaffected by the test-item treatment. Systolic and diastolic blood pressure measurements were unaffected by the test item treatment at all time-points. No relevant ophthalmological findings were observed in any group during the pre-treatment period or at the end of the treatment period. Hematology parameters, including lymphocyte subset populations, blood biochemistry and urinalysis were not affected by the test item treatment at any time-points.
[0239] At necropsy, organ weights were not affected by the test item treatment and no systemic treatment-related macroscopic lesions were observed.
Conclusion
[0240] The NOAEL following systemic single-dose administration of ACI-24 was considered to be 385 μg of peptide/injection under the experimental conditions of this study.
4.2 Repeated Dose Toxicity
[0241] 4.2.1 Study to Assess the Potential Cross Reactivity of Cynomolgus Monkey Antibodies Against ACI-24 with a Selected Panel of Normal Human Tissues
Objective
[0242] The objective of this GLP study was to assess the potential cross-reactivity of the serum antibodies from cynomolgus monkey treated with ACI-24 on histological cryostat sections of human tissues using immunohistochemical techniques.
Design
[0243] The test material was a serum preparation from a cynomolgus monkey previously immunized with ACI-24 (Animal 6529, Day 31) injected at days 2 and 24 (bleeding at day 31, that was used for the immunostaining) with the vaccine ACI-24-250-another vaccine batch (Pal 1-15 antigen: 80 ug/dose target, MPLA: 30 ug/dose target). This serum contained anti-Amyloid (Aβ) IgG antibodies at an approximate concentration of 4 μg/mL. Serum from an empty liposome immunized monkey was used as negative control serum (Animal 6613, Day 49).
[0244] The test system used cryostat sections (5 μm thick) of human Alzheimer's brain tissue (Cortex) identified as being positive for the antibodies raised in Animal 6529, Day 31 (ACI-24 immunized monkey sera). Healthy human brain tissue (same region) was used as negative control. The system was validated by selecting tissue with a large number of small, distinct Amyloid plaques that were positive for Aβ screened with a mouse anti-Aβ antibody.
[0245] The detection method was validated by using serial dilutions of the test serum and negative control serum in order to determine the optimal dilution that yielded specific positive immunohistochemical staining with minimal non-specific background staining on human Alzheimer's and healthy brain tissues.
[0246] Cyrosections from a selected panel of human tissues (Table 3) were used for the assessment of potential tissue crossreactivity.
TABLE-US-00006 TABLE 3 Human tissue titration Adrenal Duodenum Pituitary Urinary Bladder Ileum Placenta Blood Cells Colon Prostate Bone Marrow Heart Skin Breast Kidney (glomerulus, Spinal Cord tubule) Brain-Cerebellum Liver Spleen Brain-Cortex Lung Striated Muscle Endothelium Lymph Node Testis Eye Ovary Thymus Fallopian Tube Pancreas Thyroid Oesophagus Parathyroid Tonsil Gastric Antrum Parotid Ureter Gastric body Peripheral Nerve Uterus (Cervix, Endometrium) For each human tissue, three samples (from three donors) were used except lymph node and pituitary (2 samples) and parathyroid (1 sample).
Results
[0247] Tissue viability was confirmed using anti-human antibodies against Vimentin, Von Willebrand Factor (Endothelial Marker), Cytokeratin and Transferrin Receptor (CD71).
[0248] In addition, a cryo-section from all tissues stained with Haematoxylin and Eosin indicated that there was no marked autolysis.
[0249] The titration results indicated that a 1:2000 dilution of serum 6529, Day 31 (ACI-24 immunized monkey sera) was optimal since there was specific staining seen in the Amyloid plaques and minimal nonspecific background staining of the surrounding tissue in human Alzheimer's brain tissue. No corresponding positive staining was seen in the human brain—Cortex negative control tissue. For the human tissue titration, the 1:2000 dilution and one lower (1:1000) and one higher (1:4000) dilution was used.
[0250] No specific positive staining was seen for serum 6529, Day 31 (ACI-24 immunized monkey sera) in any of the human tissues examined. Throughout the majority of tissues, this serum non-specifically stained smooth muscle cells (blood vessels, muscularis mucosae, and muscle layers), myoepithelial cells and other occasional stromal cells. Variable non-specific staining was seen in most of the tissues examined which was considered to be due to the use of the goat anti-monkey IgG antibody interacting with both the cynomolgus serum 6529, Day 31 (ACI-24 immunized monkey sera) and the negative control serum (Empty liposome immunized monkey sera). Although the intensity was higher with serum 6529, Day 31 (ACI-24 immunized monkey sera) than the negative control (Empty liposome immunized monkey sera), the location and distribution of the staining in serum 6613, Day 49 (Empty liposome immunized monkey sera) requires that it should be considered to be non-specific.
[0251] A minimal amount of non-specific staining was also seen in the buffer substitute negative control and is considered to be attributable to inadequate quenching of endogenous peroxidase in smooth muscle, connective tissue and macrophages. This minimal non-specific staining, considered to be endogenous peroxidase adds to that seen as a result of incubation with serum 6529, Day 31 (ACI-24 immunized monkey sera) and the negative control serum (Empty liposome immunized monkey sera).
Conclusion
[0252] The results indicated that there was no specific positive staining attributable to the anti-ACI-24 antibodies in serum 6529, Day 31. It can therefore be concluded that cynomolgus monkey antibodies against ACI-24 do not cross react with human tissues.
4.2.2 Repeated-Dose Toxicity Following Subcutaneous Administration of ACI-24 in Cynomolgus Monkeys
Objective
[0253] The objective of this study was to evaluate the potential toxicity of the test item, ACI-24, when administered to cynomolgus monkeys by the subcutaneous route every four weeks for a period of 21 weeks.
[0254] Upon completion of the treatment period, designated animals were held for a two week withdrawal period in order to evaluate the reversibility of any observed signs of toxicity.
[0255] Another objective of this study was to analyze the T-cell response induced by ACI-24 in the monkeys.
Design
[0256] Two groups of three males and three females cynomolgus monkeys were treated once every four weeks, by the s.c. route, with the test item, ACI-24, at the dose levels of 28 μg (Group 3) or 78 μg (Group 4) of peptide/injection, with a total of six injections (21 weeks).
[0257] Five male and five female cynomolgus monkeys were treated at the dose-level of 311 μg (Group 5) of peptide/injection according to the same treatment design. Three males and three females (Group 2) were treated with ACI-24-empty and five males and five females (Group 1) were treated with PBS; both acting as control groups. Two animals/sex from Groups 1 and 5 were kept for a two-week recovery period.
TABLE-US-00007 TABLE 4 Group distribution of study Dose-level Dose-level Number of Volume of [μg of Peptide [μg of animals Dosage form injection/ peptide/ concentration MPLA/ Group Male Female administered animal injection] [μg/mL] injection] 1 5 5 PBS 0.8 mL 0 0 0 2 3 3 ACI-24- 0.8 mL 0 0 84 (empty) 3 3 3 ACI-24-30 0.2 mL 28 142 9 4 3 3 ACI-24-125 0.2 mL 78 388 22 5 5 5 ACI-24-500 0.8 mL 311 388 88 [0258] Dose administered six times at following intervals: Week 1, 5, 9, 13, 17 and 21. [0259] Blood sample for immunotoxicology withdrawn at following intervals: Week 15, 19 and 21. [0260] Blood sample for immune response withdrawn every week (except week 1). [0261] ACI-24-30, ACI-24-125 and ACI-24-500 corresponds to the targeted dose of the abeta1-15 antigen; namely 30 μg, 125 μg and 500 μg respectively
[0262] Blood samples for immunotoxicology were taken during the pre-treatment period, in Week 15, Week 19 and at the end of the treatment period. Blood samples for immune response analysis were taken weekly (except Week 1) from all the animals during the treatment period, and from the remaining animals of Groups 1 and 5 during the observation period. The animals were checked twice daily for mortality and clinical signs. Body weights were recorded twice during the pre-treatment period, on the first day of treatment and then once a week until the end of the study. Rectal temperature was taken before treatment (on the days of treatment) and 6, 24 and 48 hours after treatment. Additional measurements were taken at the end of the two-week observation period for the remaining animals in Groups 1 and 5. Rectal temperature was recorded on Day 15 for all animals. The food consumption was estimated daily throughout the study. Ophthalmological examinations were performed on all animals pretrial and on one occasion at the end of the treatment period. Electrocardiography examinations and blood pressure measurements were performed on all animals pretrial then at least two hours after the first dosing and on one occasion at the end of the treatment period.
[0263] Hematological investigations were performed on all animals pretrial then in Weeks 9, 15, 19, 21, 22 and at the end of the recovery period. Blood biochemistry analysis were performed on all animals pretrial then in Weeks 9 and 22 (end of treatment period) and at the end of the observation period. Urinalysis was performed pretrial and at the end of the treatment period. These examinations were also performed at the end of the observation period for the remaining of Group 1 and 5 animals.
[0264] Animals were submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on designated tissues from all animals sacrificed at the end of the treatment period.
[0265] To investigate the T-cell response, Peripheral Blood Mononuclear Cell (PBMCs) from monkeys treated with PBS, ACI-24-empty, ACI-24-30, ACI-24-125 or ACI-24-500 were pooled from Day 113 to Day 148 after the first immunization, corresponding to time points where antibody responses were observed. PBMCs were re-stimulated with Concanavalin A (positive control), Aβ1-42, Aβ1-15 or cell culture medium (negative control). The cells were pre-incubated with the stimulant for three hours and then transferred onto ELISPOT plates, where they were incubated for 48 h. The detection of IFN-γ, IL-4 and IL-5 producing cells was performed by an alkaline phosphatase-based detection system using an ELISPOT reader.
Results
[0266] No unscheduled deaths or premature sacrifices occurred during the study. Thickening, edemas and nodules were observed with a dose-related severity at the injection sites and lasted for between 1-2 days and 1-2 weeks after administration of the dosage forms. Nodules were observed for one month in some animals, with no relationship to the dose-level administered. No local reactions were observed in control animals treated with PBS or animals treated with ACI-24-empty. Animals treated with active levels of test item showed slight to moderate local reactions at the injection sites.
[0267] The body weights and body weight gains were considered to be similar in control and treated animals during the treatment and observation periods. Food consumption was considered to be unaffected by the test item treatment. No ophthalmological alterations or electrocardiography findings were noted during the study in control or treated animals. Hematological and blood biochemistry parameters and urinalysis were considered to be unchanged at the different time-points evaluated.
[0268] The ACI-24 vaccine injected s.c. induced robust Aβ-specific IgG responses in five monkeys. The responding monkeys had been treated with ACI-24-30 (one monkey) ACI-24-125 (one monkey) or ACI-24-500 (three monkeys). Sustained anti-Aβ IgG titers were observed from Day 120 and onwards in three monkeys, suggesting that five immunizations were required to elicit an anti-Aβ IgG response in monkeys. Monkey treated with PBS or empty liposomes did not show any detectable anti-Aβ IgG antibodies as expected. Similar results were obtained when the Aβ-specific IgG response was measured in the plasma instead of the sera. ACI-24 induced anti-Aβ IgM titers in one of the monkeys receiving the highest dose (ACI-24-500). ACI-24 induced anti-MPLA IgG titers in two monkeys following ACI-24-30.
[0269] Complete reversibility was noted at the end of the observation period. At the injection sites, nodules and thickening of subcutaneous tissue correlated with s.c. granulomatous inflammation in all treated groups, including the vehicle control group (empty liposomes). Lesions in the vehicle control group were all of minimal severity. Minimal lesions in animals receiving active test item were similar in nature.
Conclusion
[0270] Under the experimental conditions of the study, the NOAEL was established at 311 μg peptide/injection after six injections in cynomolgus monkeys, considering that the local reactions observed at the injection sites did not have an impact on the clinical status of the animals and were consistent with a normal granulomatous inflammatory reaction after s.c. injection of a foreign body.
[0271] This study also demonstrates that ACI-24 is capable of overcoming the immune tolerance to Aβ1-15 in monkeys.
[0272] The IL-4 results and the lack of correlation between IFN-γ secretion by PBMCs from monkeys immunized with ACI-24 and re-stimulation with Aβ1-15 together with the very low T-cell response indicate a preferential Th2 response for ACI-24 vaccine and thus a positive safety profile of ACI-24.
4.2.3 13-Week Toxicity Study by Subcutaneous Route in hAPP V717I Transgenic Mice
Objective
[0273] The objective of this GLP compliant study was to evaluate the potential toxicity of ACI-24 in human Amyloid Precursor Protein over-expressing transgenic mice (hAPP V717I). The transgenic mouse model hAPP V71 was selected because it reflects the pathophysiology of patients with Aβ plaque deposits in the brain and is therefore, from a biological perspective, the most relevant model for the safety evaluation of ACI-24.
Design
[0274] The hAPP V717I mice were immunized by subcutaneous administration of ACI-24 every two weeks for a total treatment period of 13 weeks. hAPP V717I mice were allocated to five different groups including three different doses of the peptide per injection (80, 160 and 400 μg; n=28) whereas PBS and empty liposomes (lacking the peptide antigen) served as negative controls (n=24). The study also examined the toxicity of MPLA integrated in liposomes in a dose of 100 μg MPLA per injection.
[0275] The study design is summarized in Table 5:
TABLE-US-00008 TABLE 5 Group distribution of study Number Peptide Volume of μg Peptide MPLA (ug) of Test or Concentration Injection per per Group Animals Control Items [mg/mL] [mL] Injection injection 1 24 PBS 0 1.0 (2 × 0.5) 0 0 females 2 24 ACI-24-Empty 0 1.0 (2 × 0.5) 0 76 females 3 28 ACI-24 0.4 0.2 80 15 females 4 28 ACI-24 0.4 0.4 160 30 females 5 28 ACI-24 0.4 1.0 (2 × 0.5) 400 76 females [0276] Dose administered seven times at following intervals: Day 1, Week 3, 5, 7, 9, 11 and 13.
Results
[0277] ACI-24 immunization raised a dose-dependent humoral anti-Aβ immune response, characterized by mainly anti-Aβ IgGs and less anti-Aβ IgMs, but did not cause: [0278] Treatment-related death [0279] Enhanced incidence of mortality [0280] Significant changes in clinical signs [0281] Change in body weight or in relative or absolute organ weight [0282] Dose-dependent changes in haematology and blood biochemistry. Some non-dose-dependent changes were considered of limited toxicological significance.
[0283] ACI-24 treatment led to minimal-to-moderate subcutis fibroplasia in the injections sites of all treated groups, with a minimal increase in incidence and severity in liposome treated groups (ACI-24 or empty liposomes), when compared to the PBS control group.
T-Cell Response:
[0284] Splenocytes isolated from mice immunized with the high dose of ACI-24 (400 μg) and re-stimulated in vitro with Aβ1-15 peptide significantly increased the number of IL-4 secreting cells, suggesting that ACI-24 preferentially induces a Th2 response. No T-cell proliferation could be observed.
Local Brain Inflammation:
[0285] Immunization with ACI-24 did not induce pro-inflammatory cytokine release (IFN-γ, TNF-α, IL-6) in brains of immunized mice but was associated with slightly decreasing levels of IFN-γ, TNF-α and IL-6. [0286] Immunization with high doses of ACI-24 (400 μg) did not enhance the presence of T-cells (CD3, CD4 and CD8), macrophages (F4/80) nor B-cells (B220 or CD45R) in the brains of immunized mice as evaluated by immunohistochemistry. [0287] Immunization with ACI-24 did not increase the incidence of micro-hemorrhages (Perl's hemosiderin) nor the severity of perivascular brown pigment-laden macrophages in the brain at any dose level, when compared to the PBS control group. [0288] Immunization with ACI-24 did not change the density of vessels (collagen type-IV) nor enhance Thioflavin-S positive amyloid plaques in vessels, indicating that there is only a low risk of cerebral amyloid angiopathy (CAA).
Conclusion
[0289] These data demonstrate that immunization with ACI-24 does not induce micro-haemorrhage, local brain inflammation, penetration of peripheral inflammatory cells (T-, B-cell or macrophages) nor perivascular accumulation of AP, indicating that ACI-24 as a vaccine does present a promising safety potential. As supported by the results of this study, the NOAEL (No Observed Adverse Effect Level) was set at 400 μg/injection of ACI-24 for systemic toxicity.
4.2.4 12-Week Subcutaneous Immunogenicity and Toxicity Study in the Cynomolgus Monkey
Objective
[0290] The purpose of this GLP study was to assess the toxicity and immunogenicity of different batches of ACI-24 when administered once every two weeks for a total of five occasions, subcutaneously to cynomolgus monkeys.
Design
[0291] The study design is explained in Table 6.
TABLE-US-00009 TABLE 6 Study design Number of Dosage Peptide MPLA animals Route of volume Dose- dose Group Male Female administration (mL/injection) level (ug)* (ug) Group 3 3 s.c. 3 0 (PBS) 0 1 Group 3 3 3 1323 μg/inject. 204 2 (Old, comparator)* Group 3 3 2 880 μg/inject. 214 3 (New, 6.9 batch)* Group 3 3 1320 μg/inject. 321 4 (New, 6.9 batch)* Group 3 3 3 1278 μg/inject. 207 5 (New, 7.4 batch)* *The details given refer to changes in the manufacturing techniques utilized to produce the various batches. Group 2 were administered with the batch previously assessed in toxicological studies and was therefore used as a comparator group. Groups 3, 4 and 5 were administered the additional batches produced under revised manufacturing conditions which lead to a limited the hydrolysis of the MPLA during the first steps of the manufacturing process (as described in WO2012/055933, incorporated herein by reference). In addition the pH of the final solution was decreased from 7.4 to 6.5 to improve the stability of MPLA during storage. [0292] Dose administered seven times at following intervals: Day 1, 15, 29, 43 and 57. [0293] Blood sample withdrawn at following intervals: Pre-dose, Day 15, 29, 43, 57 and 71.
[0294] Throughout the study, all animals were observed at least twice daily for viability/mortality and clinical signs. Injection areas were observed daily during treatment and recovery periods.
[0295] Food consumption was estimated for each cage (qualitatively) twice daily during the study.
[0296] All animals were weighed twice a week during pretest and then weekly during treatment and recovery periods.
[0297] Blood samples were collected for clinical laboratory investigations during pretest and at the end of the treatment period (Week 12).
[0298] Blood samples were taken for IgG anti-Abeta determination once during pretest and 14 days after each administration.
[0299] At termination blood samples were collected to obtain serum, plasma and PBMCs which were either stored or analysed as part of a separate study.
[0300] Following completion of the scheduled treatment period, animals from Groups 1, 3 and 4 were necropsied and various organs were weighed. Macroscopic alterations were recorded. A full set of tissues and organs were collected, processed and examined histologically. Animals from Groups 2 and 5 were retained for future investigation work and therefore subsequently removed from the study.
Results
[0301] No death occurred during the study protocol. There were no relevant clinical signs or local effects at the injection sites. Neither effects on food consumption nor on body weight occurred throughout the treatment period.
[0302] Subcutaneous administration of three formulations of ACI-24 induced a comparable profile in anti-Aβ IgG antibody across all groups, therefore indicating suitable correlation between the batches. One animal vaccinated with ACI-24 “New 6.9 batch” (Group 3 Female 24), showed a sustained anti-Aβ IgG titers from Day 43 onwards that were three-fold higher than those typically seen. Monkeys dosed with PBS did not show any detectable anti-Aβ IgG antibodies, as expected.
[0303] There were no relevant changes in the hematology or blood chemistry parameters.
[0304] No relevant macroscopic findings or noteworthy changes in the organ weights were recorded at necropsy.
[0305] Histological findings at the injection sites consisted of mononuclear cell focus/foci in subcutaneous tissue, with an increased incidence in Group 3 and increased incidence and severity in Group 4. These findings were present in monkeys of all groups examined (1, 3 and 4), including one control male. These changes were of minimal-slight intensity and their distribution was strictly local.
Conclusion
[0306] Five subcutaneous administration, once every two weeks, using different batches of ACI-24, to cynomolgus monkeys (up to approximately 1320 μg/injection), was well tolerated with no effects on body weight, food consumption or clinical pathology parameters.
[0307] Based on the results obtained and under these study conditions, all batches of ACI-24 assessed were considered comparable in terms of toxicity and immunogenicity with an approximate dose of 1320 μg/injection currently considered as a NOAEL (No Observed Adverse Effects Level).
Example 5: A Phase 2 Double-Blind, Randomized, Placebo-Controlled Study to Assess the Safety, Tolerability and Target Engagement of ACI-24 in Adults with Down Syndrome
Primary Outcome Measures:
[0308] Number of participants with Adverse Events (AEs) assessed by intensity (mild, moderate or severe) and causal relationship (unrelated, unlikely, possibly or probably related) [0309] [Time Frame: from screening up to week 100] [0310] Mean change from baseline in vital signs [0311] systolic and diastolic blood pressure (mmHg), hear rate (bpm), body temperature (degree Celsius) [0312] [Time Frame: from baseline up to week 100] [0313] Mean change from baseline in suicidal ideation/behavior using Columbia-Suicide Severity Rating Scale (C-SSRS) [0314] [Time Frame: from baseline up to week 100] [0315] Number of participants reporting suicidal ideation or behavior using Columbia-Suicide Severity Rating Scale (C-SSRS) [Time Frame: from baseline up to week 100] [0316] Number of participants with abnormal MRI results [0317] Occurrence of Amyloid-related imaging abnormalities (ARIA) [0318] [Time Frame: from baseline up to week 100]
Secondary Outcome Measures:
[0319] Change from baseline of composite standardized uptake value ratio (SUVR) assessed by amyloid PET imaging using florbetaben [0320] [Time Frame: from baseline up to week 76] [0321] Change from baseline in anti-Aβ antibody titers in blood [0322] [Time Frame: from baseline up to week 100] [0323] Change from baseline of amyloid-related biomarkers (Aβ1-40, Aβ1-42), total tau, phosphorylated tau and NfL in blood/CSF (pg/mL) (CSF is optional). [0324] [Time Frame: from baseline up to week 100] [0325] Change from baseline of brain tau load assessed by tau PET imaging [0326] [Time Frame: from screening up to week 74] [0327] Change from baseline of cognitive performance using Cambridge Neuropsychological Test Automated Battery—Paired Associates Learning [CANTAB-PAL] [0328] Score is a z-score ranging from −7.5 to 0. A higher score (eg., 0) indicates a better outcome. [0329] [Time Frame: from baseline up to week 100] [0330] Change from baseline of cognitive performance using Cambridge Cognitive Examination—Down Syndrome [CAMCOG-DS] [0331] [Time Frame: from baseline up to week 100] [0332] The total score ranges from 0 to 107. A higher score indicates a better outcome. [0333] Change from baseline in adaptive behavior (Vineland Adaptive Behavior Scale) [0334] [Time Frame: from baseline up to week 100] [0335] The composite score ranges from 20 to 140. A higher score indicates a better outcome. [0336] Change from baseline in Clinical Global Impression of Change (CGIC) [0337] [Time Frame: from baseline up to week 100] [0338] The score ranges from 1 to 7. A higher score indicates a worse outcome.
Method:
[0339] This study is a prospective multicenter, placebo-controlled, double-blind, randomized study to assess the effect of one dose of the ACI-24 vaccine, versus placebo over a 74-week treatment period and 26-week safety follow-up period.
[0340] After the screening period, eligible subjects are randomized in a 1:1 ratio to receive either ACI-24 or corresponding placebo, both given by the intramuscular route. Approximately 72 subjects (36 subjects receiving ACI-24 1000 μg and 36 subjects receiving placebo) are randomized in the study.
[0341] Subjects are treated with repeated administrations of ACI-24 (1000 μg dose) or corresponding placebo using the intramuscular route. ACI-24 (1000 μg dose) or placebo is administered 8 times (each time, the dose of study treatment is administered in 2 separate concomitant intramuscular injections): the first 4 administrations are at 4-week intervals (W0, W4, W8, and W12); the next 3 administrations are at 12-week intervals (W24, W36, and W48); and the last administration is at W74 (26-week interval from previous administration). The 74-week treatment period is followed by a 26-week safety follow-up period.
Inclusion Criteria:
[0342] Male or female subjects with DS with a cytogenetic diagnosis being either trisomy 21 or complete unbalanced translocation of the chromosome 21. [0343] Age ≥40 and ≤50 years at screening. [0344] Elevated brain Aβ as evidenced by composite SUVR ≥1.25 on florbetaben PET scan assessed by central reading. [0345] Subjects, their legal representatives (if applicable) and/or their study partners in the opinion of the investigator, are able to understand and to provide written informed consent before starting any study-related activities. [0346] In the opinion of the investigator, subjects, their legal representatives (if applicable) and/or their study partners are able to fully participate in the study, be sufficiently proficient in the official languages(s) of the country they are living in, and be capable of reliably completing study assessments. [0347] Mild to moderate intellectual disability as per Diagnostic and Statistical Manual of Mental Disorders (DSM-5) classification. [0348] Subjects must have a study partner who has direct and regular contact with the subject and who is able to provide reliable answers to questions related to the subject, according to the study investigator. [0349] Subjects in preclinical stage of AD or with mild cognitive impairment due to AD.
REFERENCE UST
[0350] Belichenko P V, Madani R, Rey-Bellet L, et al. An Anti-β-Amyloid Vaccine for Treating Cognitive Deficits in a Mouse Model of Down Syndrome. PLOS ONE. 2016; 11(3):e0152471. [0351] Folstein M F, Folstein S E, McHugh P R (1975) “Mini-Mental State”: a practical method for grading the cognitive state of patients for the clinician J Psychiatr Res 12: 189-198 [0352] Gilman S., Koller M., Black R. S., Jenkins L., Griffith S. G., Fox N. C., Eisner L., Kirby L., Boada Rovira M., Forette F., Orgogozo J. M., Clinical effect of Aβ immunization (AN1792) in patients with AD in an interrupted trial. Neurology 64, 1553-1562 (2005). [0353] Hartley S L, Handen B L, Devenny D, et al. Cognitive decline and brain amyloid-β accumulation across 3 years in adults with Down syndrome. Neurobiology of aging. 2017; 58:68-76. [0354] Head E, Powell D, Gold B T, Schmitt F A. Alzheimer's Disease in Down Syndrome. European journal of neurodegenerative disease. 2012; 1(3):353-364. [0355] Hughes C P, Berg L, Danzinger W L et al (1982) A new clinical scale for the staging of dementia. Am J Psychiatry; 140: 566-572 [0356] Monsonego A., Weiner H. L., Immunotherapeutic approaches to Alzheimer's disease. Science. 31; 302(5646):834-8 (2003). [0357] Muhs A., Hickman D. T., Pihlgren M., Chuard N., Giriens V., Meerschman C., van der Auwera I., van Leuven F., Sugawara M., Weingertner M.-C., Bechinger B., Greferath R., Kolonko N., Nagel-Steger L., Riesner D., Brady R. O., Pfeifer A., Nicolau C., Liposomal vaccines with conformation-specific amyloid peptide antigens define immune response and efficacy in APP transgenic mice. PNAS, 104 23:9810-9815 (2007). [0358] Nasreddine Z S, Phillips N A, et al. The Montreal Cognitive Assessment, MoCA: A brief screening tool for mild cognitive impairment. J Am Geriatr Soc. 2005; 53:695-699. [0359] Orgogozo J. M., Gilman S., Dartigues J. F., Laurent B., Puel M., Kirby L. C., Jouanny P., Dubois B., Eisner L., Flitman S., Michel B. F., Boada M., Frank A., Hock C., Subacute meningoencephalitis in a subset of patients with AD after Abet42 immunization. Neurology 61: 46-54 (2003). [0360] Prasher V P, Huxley A, Haque M S (2002) A 24-week, doubleblind, placebo-controlled trial of donepezil in patients with Down syndrome and Alzheimer's disease—pilot study. Int J Geriatr Psychiatry 17(3):270-278 (PMID: 11921156) [0361] Pihlgren M., Silva A. B., Madani R., Giriens V., Waeckerle-Men Y., Fettelschoss A., Hickman D. T., López-Deber M. P., Ndao D. M., Vukicevic M., Buccarello A. L., Gafner V., Chuard N., Reis P., Piorkowska K., Pfeifer A., Kundig T. M., Muhs A., Johansen P., TLR4- and TRIF-dependent stimulation of B lymphocytes by peptide liposomes enables T cell-independent isotype switch in mice. Blood. January 3; 121(1):85-94 (2013). [0362] Soto C., Plaque busters: strategies to inhibit amyloid formation in Alzheimer's disease. Molecular Medicine Today (vol 5), August 1999. [0363] Winblad B., Graf A., Riviere M. E., Andreasen N., Ryan J. M., Active immunotherapy options for Alzheimer's disease. Alzheimers Res Ther. 2014 Jan. 30; 6(1):7.
[0364] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications and patents specifically mentioned herein are incorporated by reference in their entirety for all purposes in connection with the invention.
[0365] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Moreover, all aspects and embodiments of the invention described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, including those taken from other aspects of the invention (including in isolation) as appropriate.