Protofibril-binding antibodies and their use for the treatment and diagnosis of parkinson's disease, dementia with lewy bodies and other alpha-synucleinopathies
阅读说明：本技术 原细纤维结合抗体及其治疗和诊断帕金森氏症、路易体痴呆和其他α-共核蛋白病的应用 (Protofibril-binding antibodies and their use for the treatment and diagnosis of parkinson's disease, dementia with lewy bodies and other alpha-synucleinopathies ) 是由 埃娃·努德斯特伦 亚里克斯·卡斯拉亚恩 莫妮卡·埃克贝里 松德奎斯特 瓦伦丁娜·斯克雷潘蒂 于 2011-02-25 设计创作，主要内容包括：本发明涉及原细纤维结合抗体及其治疗和诊断帕金森氏症、路易体痴呆和其他α-共核蛋白病的应用。抗体及其片段具有对人类α-突触核蛋白原细纤维高的亲和力和α-突触核蛋白单体的低的结合。组合物包含这样的抗体或片段。利用这样的抗体或片段来检测α-突触核蛋白原细纤维；预防、推迟具有α-突触核蛋白病理的神经退化性疾病的发作或治疗所述疾病；制备治疗具有α-突触核蛋白病理的神经退化性疾病的的药物组合物；诊断或监测具有α-突触核蛋白病理的神经退化性疾病进展,以及减少或抑制α-突触核蛋白聚集。(The present invention relates to protofibril-binding antibodies and their use in the treatment and diagnosis of Parkinson's disease, dementia with Lewy bodies and other alpha-synucleinopathies. Antibodies and fragments thereof have high affinity for human α -synuclein protofibrils and low binding of α -synuclein monomers. Compositions comprise such antibodies or fragments. Detecting α -synuclein protofibrils using such antibodies or fragments; preventing, delaying the onset of or treating a neurodegenerative disease having an alpha-synuclein pathology; preparing a pharmaceutical composition for the treatment of neurodegenerative diseases having an alpha-synuclein pathology; diagnosing or monitoring the progression of a neurodegenerative disease with α -synuclein pathology, and reducing or inhibiting α -synuclein aggregation.)
1. An antibody having high affinity for human a-synuclein protofibrils and low binding of a-synuclein monomers and β -synuclein monomers, wherein the antibody binds human a-synuclein protofibrils more than 100-fold greater than binding of a-synuclein monomers and more than 100-fold greater than binding of β -synuclein monomers, the antibody having a combination of three variable heavy chain (VH) CDR sequences and three variable light chain (VL) CDR sequences selected from the following combinations:
(a) VH-CDR-1: 22, VH-CDR-2: 28, VH-CDR-3: 35, VL-CDR-1: 41, VL-CDR-2: 47 and VL-CDR-3: the amino acid sequence shown in SEQ ID NO. 50,
(b) VH-CDR-1: 24, VH-CDR-2: 30, VH-CDR-3: 37, VL-CDR-1: 43, VL-CDR-2: 48 and VL-CDR-3: the amino acid sequence shown in SEQ ID NO:51,
(c) VH-CDR-1: 25, VH-CDR-2: 31, VH-CDR-3: 38, VL-CDR-1: 44, VL-CDR-2: 47 and VL-CDR-3: the amino acid sequence shown in SEQ ID NO. 52,
(d) VH-CDR-1: 23, VH-CDR-2: 33, VH-CDR-3: 37, VL-CDR-1: 43, VL-CDR-2: 48 and VL-CDR-3: 54 of SEQ ID NO, and
(e) VH-CDR-1: 27, VH-CDR-2: 34, VH-CDR-3: 40, VL-CDR-1: 46, VL-CDR-2: 49 and VL-CDR-3: SEQ ID NO: 55.
2. The antibody of claim 1, wherein the antibody has the CDR sequence combination of (a).
3. The antibody of claim 1, wherein the antibody has the CDR sequence combination of (b).
4. The antibody of claim 1, wherein the antibody has the CDR sequence combination of (c).
5. The antibody of claim 1, wherein the antibody has the CDR sequence combination of (d).
6. The antibody of claim 1, wherein the antibody has the CDR sequence combination of (e).
7. The antibody of any one of claims 1-6, wherein the antibody is a monoclonal antibody.
8. The antibody of any one of claims 1-7, wherein the antibody binds to alpha-synuclein fibrils more than 100-fold greater than to alpha-synuclein fibrils.
9. A pharmaceutical composition comprising the antibody of any one of claims 1-8, and a pharmaceutically acceptable carrier.
10. Use of an antibody according to any one of claims 1 to 8 or a composition according to claim 9 in the preparation of a medicament for detecting the presence of alpha-synuclein protofibrils in a biological sample comprising or suspected of comprising alpha-synuclein protofibrils.
11. Use of an antibody according to any one of claims 1-8 or a composition according to claim 9 for the preparation of a medicament for preventing, delaying the onset of or treating a neurodegenerative disease having an alpha-synuclein pathology in an individual.
12. The use according to claim 11, wherein the neurodegenerative disease is Parkinson's Disease (PD), lewy body Dementia (DLB), lewy body variants of alzheimer's disease, down's syndrome, multiple system atrophy, schizophrenia or creutzfeldt-jakob disease.
13. The use according to claim 11, wherein the neurodegenerative disease is Parkinson's Disease (PD), lewy body Dementia (DLB), lewy body variants of alzheimer's disease, alzheimer's disease or multiple system atrophy.
14. The use according to claim 13, wherein the medicament is for the treatment of the neurodegenerative disease.
15. The use according to claim 11, wherein the medicament is for the treatment of the neurodegenerative disease.
16. Use of an antibody according to any one of claims 1-8 or a composition according to claim 9 for the preparation of a medicament for diagnosing or monitoring the progression of a neurodegenerative disease having an alpha-synuclein pathology.
17. The use according to claim 16, wherein the neurodegenerative disease is Parkinson's Disease (PD), lewy body Dementia (DLB), lewy body variants of alzheimer's disease, down's syndrome, multiple system atrophy, schizophrenia or creutzfeldt-jakob disease.
18. The use of claim 16, wherein the neurodegenerative disease is Parkinson's Disease (PD), lewy body Dementia (DLB), lewy body variants of alzheimer's disease, alzheimer's disease or multiple system atrophy.
19. Use of an antibody according to any one of claims 1-8 or a composition according to claim 9 in the preparation of a medicament for reducing or inhibiting alpha-synuclein aggregation in a sample comprising soluble alpha-synuclein species.
20. Use of an antibody according to any one of claims 1-8 or a composition according to claim 9 in the manufacture of a medicament for reducing the amount of alpha-synuclein protofibrils in a subject.
Technical Field
The present invention relates to antibodies or fragments thereof having high affinity for human alpha-synuclein protofibrils (protofibrils) and low binding of alpha-synuclein monomers, wherein the antibodies or fragments have specific Complementarity Determining Region (CDR) sequences. The invention also relates to compositions comprising such antibodies or fragments, and to methods of using such antibodies or fragments to detect alpha-synuclein fibrils. In further embodiments, the present invention relates to methods for preventing, delaying the onset of or treating neurodegenerative diseases having an alpha-synuclein pathology by administering such antibodies or fragments, and to the use of such antibodies or fragments in the preparation of pharmaceutical compositions for the treatment of neurodegenerative diseases having an alpha-synuclein pathology. The invention also relates to the use of such antibodies or fragments in the diagnosis or monitoring of the development of neurodegenerative diseases having alpha-synuclein pathology, and to methods of reducing or inhibiting alpha-synuclein aggregation by administration of such antibodies or fragments.
Background
Parkinson's Disease (PD) and dementia with Lewy bodies (DLB) are the two most common examples of neurodegenerative diseases with alpha-synuclein brain pathology. PD is the most common movement disorder, and PD is characterized by rigidity, hypokinesia, tremors, and postural instability (postural instability). Approximately four to six million people worldwide are believed to suffer from PD. DLB represents 5-15% of all dementias. In addition to amnesia and other mad symptoms that fluctuate frequently, DLB patients typically suffer recurrent falls and visual hallucinations.
Intracellular (intracellular) accumulation of alpha-synuclein leads to the formation of lewy bodies, inclusions of spherical eosinophilic vitreous 10-20 μm in size, or lewy neurites, elongated, threadlike, misshapen axons and dendrites. In PD brain, deposits of lewy bodies and lewy neurites are usually confined to nerve cells connecting the striatum and substantia nigra. These cells are essential for the completion of motor and postural functions, explaining the nature of the PD symptoms. In DLB brain, widely distributed deposits of lewy bodies and lewy neurites are found in both midbrain and cortical areas.
Alpha-synuclein is a protein found primarily within nerves. Within nerve cells, α -synuclein is predominantly presynaptic (presynaptic), and thus it is presumed to play a role in the regulation of synaptic activity. Three major subtypes of alpha-synuclein are identified, the longest and most common form of which comprises 140 amino acids. Such a subtype has been applied and the α -synuclein (α -synuclein) -related feature of the antibody according to the invention relates to this subtype of α -synuclein.
In addition to α -synuclein, the lewy body is composed of a variety of molecules, one of which is 4-hydroxy-2-nonenal (HNE), α, β -unsaturated hydroxyolefin (hydroxylkenal) (Qin et al, 2007). HNE has been shown in vitro to modify α -synuclein and thus promote oligomerization of α -synuclein. In particular, HNE has been shown to increase and immobilize fibril formation, e.g., the soluble, more oligomeric form of alpha-synuclein (Qin et al, 2007; WO 2009/133521, incorporated herein by reference).
Oxidative stress has been implicated in a number of neurodegenerative diseases characterized by the pathological accumulation of misfolded α -synuclein. Various reactive oxygen species induce peroxidation of lipids, such as cell membranes or lipoproteins, and also lead to the production of highly reactive aldehydes from polyunsaturated fatty acids (yoritaka et al, 1996).
Brain pathological indications of Alzheimer's Disease (AD), such as amyloid plaques (amyloid plaques) and neurofibrillary tangles (neurofibrillary tangles), are seen in approximately 50% of cases with DLB. It is unclear whether the presence of parallel pathologies implies two different diseases or merely represents a variant of each respective disorder. Cases of co-pathology are sometimes described as lewy body variants with AD (Hansen et al, 1990).
Recent studies have suggested a role for α -synuclein in AD and Down's syndrome, as accumulation of α -synuclein protein in the limbic region in these disorders has been demonstrated (cress et al, 2009).
HNE reacts with and modifies the side chains of cysteine, histidine and lysine, substantially altering the structure and physical properties of these side chains. Thus, HNE can react with C-3 carbons or with aldehyde groups or combinations thereof. Thus, HNE can modify proteins covalently, either intermolecularly or intramolecularly.
Genetics of Parkinson's disease and dementia with Lewy bodies
The rare predominantly inherited forms of PD and DLB can be formed by point mutations or repeats of the alpha-synuclein gene. Pathogenic mutations A30P and A53T (Kruger et al, 1998) and gene duplication (chartier-Harlin et al, 2004) have been described which cause family PD, whereas one other alpha-synuclein mutation, E46K (Zarranz et al, 2004) and triploid of the alpha-synuclein gene (Singleton et al, 2003) have been reported which cause PD or DLB.
The consequences of the pathogenesis of alpha-synuclein mutants are only partially understood. However, in vitro data have shown that the a30P and a53T mutations increase the aggregation rate (Conway et al, 2000). A variety of different compositions of a-synuclein species (monomers, dimers, oligomers, including fibrils) are involved in the aggregation process, all of which can have different toxicities. It is unclear which molecular species have toxic effects in the brain. However, recent studies have shown that oligomeric forms of α -synuclein are significantly neurotoxic. The observation that certain alpha-synuclein mutants (A30P and A53T) cause inherited Parkinson's disease, resulting in increased rates of oligomerization, gives additional evidence for the effect of oligomers.
How the alpha-synuclein aggregation cascade begins is not fully understood. It is likely that the altered conformation of monomeric alpha-synuclein begins the formation of dimers and trimers, which continue to form larger soluble oligomers, including fibrils, before these intermediate size species are deposited as insoluble fibrils in the lewy body. It will also be appreciated that oligomers of alpha-synuclein, once they are formed, are able to bind new monomers and/or smaller multimers of alpha-synuclein, thus accelerating the fibril (fibrils) formation process. Such seeding effects may also occur in the extracellular space, as recent evidence suggests that α -synuclein pathology may spread from nerve cell to nerve cell in diseased brain.
In addition to the changes in neuropathology in α -synucleinopathies (synucleinopathies), the levels of α -synaptoproteins are often increased in affected brain regions (Klucken et al, 2006).
The major pathology of α -synucleinopathies is intracellular, which poses a challenge in immunotherapeutic approaches. However, it is possible that actively induced fragments or passively administered antibodies could also bind their target antigen within neurons. Furthermore, the recognition of α -synuclein in both plasma and cerebrospinal fluid (El-Agnaf et al, 2006) suggests that the protein is not found solely within nerve cells. A reduction of such extracellular α -synuclein may alter the balance between intracellular and extracellular protein aggregation and also result in a reduction of intracellular α -synuclein. Evidence suggests that α -synuclein in solution is able to penetrate the lipid bilayer in the cell membrane and thus internalize or export the cell. Recent findings demonstrate that α -synuclein has toxic effects in the extracellular space, thus providing a plausible explanation as to how α -synuclein pathology propagates throughout the brain as disease progression. Studies have shown that the road pathology spreads to transplanted nerve cells in transplanted PD patients (Li et al, 2008). Furthermore, α -synuclein is transmitted to neighboring neural cells by endocytosis (endocytosis), and cell-to-cell transmission of α -synuclein aggregates has been linked to neuronal cell death and pathogenesis in PD and other α -synucleinopathies (dessplats et al, 2009).
Diagnosis of PD and DLB
There is a need for improved diagnostic tools and methods to identify the risk of neurodegenerative diseases with α -synuclein pathology. Today, no biochemical approach can help clinicians diagnose clinical symptoms in patients in the early stages of the disease before actual damage to the brain has occurred.
Accurate diagnostic analysis becomes more important than the possibility of new treatments emerging. To date, only symptomatic treatment (by replacing the loss of active dopamine in the brain) is available for PD patients. For DLB, even fewer treatment options are available. However, clinicians often evaluate the possible beneficial effects of standard treatments for AD, e.g., cholinesterase inhibitors, on DLB patients. In either approach, none of the existing treatment strategies for α -synucleinopathies can be directed against the underlying disease process. In addition, there is also a need to monitor disease progression and therapeutic efficacy. For an evaluation of different methods of altering the development of parkinson's disease, see George et al, 2009.
In view of the above mentioned α -synuclein involvement in several neurodegenerative diseases, there is a need for new treatments that eliminate or reduce the impact of toxic α -synuclein species and for good biomarkers to monitor new interventions and provide good predictive specificity.
Disclosure of Invention
The present invention relates to improved antibodies and fragments thereof having high affinity for human alpha-synuclein protofibrils and low binding of alpha-synuclein monomers. The invention also relates to compositions comprising such antibodies or fragments and to methods of using such antibodies or fragments to detect alpha-synuclein fibrils. In further embodiments, the present invention relates to methods of preventing, delaying the onset of, or treating a neurodegenerative disease having an alpha-synuclein pathology by administering such an antibody or fragment, and to the preparation of such an antibody or fragment for use in a pharmaceutical composition for the treatment of a neurodegenerative disease having an alpha-synuclein pathology. The invention also relates to the use of such antibodies or fragments in the diagnosis or monitoring of the development of neurodegenerative diseases having alpha-synuclein pathology, and to methods of reducing or inhibiting alpha-synuclein aggregation by administration of such antibodies or fragments.
In one embodiment, the antibody or fragment thereof has high affinity for human α -synuclein protofibrils and low binding of α -synuclein monomers, and has three variable heavy chain (VH) CDR sequences (VH-CDR-1, VH-CDR-2, and VH-CDR-3 and three variable light chain (VL) CDR sequences (VL-CDR-1, VL-CDR-2, and VL-CDR-3), wherein the six CDR sequences of the antibody or fragment thereof are selected from the following respective groups:
VH-CDR-1 SEQ ID NOS 22, 23, 24, 25, 26 or 27
VH-CDR-2 SEQ ID NOS 28, 29, 30, 31, 32, 33 or 34
VH-CDR-3
VL-CDR-1 SEQ ID NOS 41, 42, 43, 44, 45 or 46
VL-CDR-2 SEQ ID NOS:47, 48 or 49
VL-CDR-3 SEQ ID NOS 50, 51, 52, 53, 54 or 55
In another embodiment, the antibody or fragment thereof has high affinity for human α -synuclein protofibrils and low binding of α -synuclein monomers, and has three variable heavy chain (VH) CDR sequences (VH-CDR-1, VH-CDR-2, and VH-CDR-3 and three variable light chain (VL) CDR sequences (VL-CDR-1, VL-CDR-2, and VL-CDR-3), wherein the six CDR sequences of the antibody or fragment thereof are selected from the following respective groups, and sequences having greater than 70, 80, 90, 95, or 98% similarity to the sequences of any one of the respective groups:
VH-CDR-1 SEQ ID NOS 22, 23, 24, 25, 26 or 27
VH-CDR-2 SEQ ID NOS 28, 29, 30, 31, 32, 33 or 34
VH-CDR-3
VL-CDR-1 SEQ ID NOS 41, 42, 43, 44, 45 or 46
VL-CDR-2 SEQ ID NOS:47, 48 or 49
VL-CDR-3 SEQ ID NOS 50, 51, 52, 53, 54 or 55
Wherein the antibody or fragment thereof binds to an epitope within amino acid region 113-.
The antibodies, fragments, compositions and methods according to the invention provide improvements in the diagnosis, monitoring, prevention, delaying the onset and/or treatment of neurodegenerative diseases having alpha-synuclein pathology in individuals having and/or at risk of developing such diseases.
Further aspects, embodiments and advantages of various embodiments of the present invention will become apparent from the detailed description.
Drawings
The embodiments will be more fully understood from the accompanying drawings, in which:
figure 1 shows the performance of protofibril-specific monoclonal antibodies as determined by competitive ELISA. The assay was performed as described in example 4 using HNE-immobilized (stabilized) alpha-synuclein protofibrils.
Fig. 2A and 2B show the performance of the fibril-specific antibody mAb49/G analyzed by competitive ELISA as described in example 4. FIG. 2A shows that the protofibril-specific monoclonal antibody mAb49/G binds with high affinity to human α -synuclein protofibrils immobilized by HNE or ONE. FIG. 2B shows that monoclonal antibodies also bind with high affinity to human mutated forms of alpha-synuclein HNE-immobilized protofibrils, A30P and A53T.
Fig. 3A-3C show the performance of fibril-specific antibodies analyzed by competitive ELISA as described in example 4. The protofibril-specific monoclonal antibody binds with high affinity to wild-type human alpha-synuclein protofibrils immobilized by HNE (PF-HNE) or ONE (PF-ONE). Monoclonal antibodies also bind with high affinity to human mutated forms of alpha-synuclein HNE-immobilized fibril, a30P (a30P-HNE) and a53T (a 30P-HNE).
Fig. 4A and 4B relate to quantification of α -synuclein protofibrils by sandwich ELISA (sandwich ELISA) as described in example 5. FIG. 4A shows a schematic representation of the protofibril-specific antibody mAb49/G used for both the capture and detection antibody effects. FIG. 4B shows a plot of a standard generated using HNE-immobilized α -synuclein protofibrils. The experimental performance reached the limit of quantification of LOQ ═ 9 pM.
Fig. 5A and 5B show the results of analysis of Diseased (DLB) and control human brain extracts using an α -synuclein protofibril specific sandwich ELISA as described in example 6.
Figure 6 shows analysis of brain extracts from control mice (ntg, non-transgenic) and 5 month old mice from the Khale transgenic (tg) mouse PD model as described in example 7. Brain tissue was extracted using Tris Buffered Saline (TBS) and TBS containing Triton. The analysis was performed using an α -synuclein protofibril sandwich ELISA as described in example 5. The fibril-specific antibody mAb49/G was used as a capture antibody and a detection antibody. In the graph, the y-axis represents the absorbance at OD 450.
Fig. 7A-7F show Immunohistochemical (IHC) analysis of tissues as described in example 8. FIG. 7A shows 38E2/7 binding of Lewy bodies, and neurites in PD substantia nigra and positive α - α -synuclein control. FIG. 7B shows Lewy body 38E2/7 binding and neurites in DLB cortex and substantia nigra and a positive α - α -synuclein control. Figure 7C shows various antibody binding lewy bodies and neurites in DLB cortex and substantia nigra and negative controls. Figure 7D shows that various antibodies bind to lewy bodies and neurites in PD substantia nigra and negative controls. FIG. 7E shows 38E2/7 is not bound and a positive α - α -synuclein control in non-disease-associated substantia nigra. FIG. 7F shows a comparison of 38E2/7 binding and a positive α -A β control in the cortex of Alzheimer's disease patients.
FIGS. 8A and 8B show immunoprecipitation of human brain extracts with crude fiber-selective monoclonal antibody 38E2/7 extracted using the brain extraction protocol as described in example 9.
Fig. 9A and 9B show fluorescence data measured using an Axiovert 200 microscope equipped with a FITC epifluorescence (epifluorescence) filter as described in example 10. Fig. 9A shows treated cells, while fig. 9B shows data calculated as a relative% reduction in fluorescence intensity, set to 100%, compared to antibody untreated α -synuclein overexpressed cells.
The various figures will be more fully understood in view of the examples set forth below.
Detailed Description
In a first embodiment, the present invention relates to improved antibodies and fragments thereof having high affinity for human alpha-synuclein protofibrils and low binding of alpha-synuclein monomers. In particular embodiments, the antibody is a class of IgG antibody or a variant thereof. Within the present disclosure, a high affinity for human a-synuclein protofibrils means that the antibody or fragment exhibits less than 10 for human a-synuclein protofibrils-7Separation constant K of Md. As known in the art, fibril is a soluble oligomer of alpha-synuclein. Typical protofibrils have molecular weights ranging from about 1000 to about 5000kDa, as measured appropriately using size exclusion chromatography with reference to globular proteins, but the present invention is not limited to such typical protofibrils. Further, within the present disclosure, low binding of alpha-synuclein monomers means that the binding of an antibody or fragment according to the invention to alpha-synuclein monomers is at least 100-fold less than to alpha-synuclein protofibrils. In specific examples, these binding affinities are measured according to a competitive ELISA, e.g., as described in example 4.
The invention further relates to methods and uses of such antibodies and fragments for improvement in the prevention, delay of onset of, treatment, monitoring and/or diagnosis of neurodegenerative diseases having alpha-synuclein pathology, including, but not limited to, Parkinson's Disease (PD), Lewy body Dementia (DLB), Lewy body variant of Alzheimer's disease, multiple system atrophy, psychosis, schizophrenia and Creutzfeldt-Jakob disease. In α -synucleinopathies, α -synuclein, which is an aggregation of lewy bodies and lewy neurites, accumulates in the brain, in certain indications, as well as in other organs.
Examples of antibodies according to the invention have been disclosed by classical hybridoma cell technology. The antibody may be a polyclonal antibody or a monoclonal antibody. In particular embodiments, the antibody is a monoclonal antibody. In many instances where the disclosure relates to antibodies and fragments thereof, for convenience purposes, the term "antibody" in the disclosure includes fragments thereof, meaning that the active fragments thereof, e.g., fragments, have the same characteristics as defined for the antibodies of the invention, i.e., high affinity for α -synuclein oligomers/fibrils and low binding of α -synuclein monomers. Antibodies and fragments thereof exhibit high efficiency in the clearance of pathological forms of alpha-synuclein.
The antibodies of the invention bind aggregated forms, in particular protofibrils, including unmodified or bound alpha-synuclein, for example, to 4-hydroxy-2-nonenal (HNE) or 4-oxo-nonenal (4-oxo-2-nonenal) (ONE), or other alpha, beta-unsaturated hydroxyalkenes, or poly-unsaturated fatty acids, which binds to immobilized pathogenic protofibril/oligomeric alpha-synuclein epitopes. The epitopes are present on conformationally altered or modified alpha-synuclein, e.g., alpha-synuclein protofibrils and oligomers present in the brain of patients with alpha-synucleinopathies, such as, but not limited to, Parkinson's disease, DLB, and the like. The antibodies of the invention also bind to pathogenic protofibril/oligomeric structures formed by alpha-synuclein mutants, e.g., A30P and A53T (Kruger et al, 1998) (Polymeropoulos et al, 1997) which have been described to cause family PD. Another example of such a target object of the antibodies of the invention is a protofibril formed by the mutant alpha-synuclein E46K causing PD or DLB.
In a specific embodiment of the invention, monoclonal antibodies are provided for differentiating, diagnosing, identifying the risk of developing, and/or treating diseases associated with α -synuclein pathology, including, but not limited to, for example, parkinson's disease, dementia with lewy bodies, lewy body variants of alzheimer's disease, down's syndrome, multiple system atrophy, confusion, schizophrenia, Creutzfeldt-Jakob disease (Creutzfeldt-Jakob disease), and other neurodegenerative diseases.
The antibodies or fragments of the invention comprise amino acid sequences defined by the CDR1-3 regions on the Variable Light (VL) and Variable Heavy (VH) chains of an antibody, wherein the antibody has high affinity for soluble α -synuclein fibrils that contain "epitopes of PD and/or DLB disease". In particular embodiments, the CDR regions bind to variants of the Fc region in order to modulate effector functions such as, but not limited to, Fc receptor binding, complement factor C1q binding, effective half-life (effective half-life), complement activation and inflammatory processes. The constant region of an antibody has many important functions, notably binding to Fc-receptors and complement factor C1 q. Can prevent later activation of functions in order to avoid inflammatory reactions.
As compared to other known immunotherapeutic therapeutics, the antibodies and fragments of the invention, which have high affinity for α -synuclein protofibrils and low binding to α -synuclein monomers, have the following unique advantages:
1) the antibodies and fragments of the invention target and inactivate or at least reduce disease-induced alpha-synuclein fibrils, e.g., by inhibiting oligomerization (see example 10) or by other mechanisms.
2) The high affinity for α -synuclein fibrils exhibited by the antibodies and fragments of the invention reduces the effective clinical dose required for treatment.
3) The antibodies and fragments of the invention provide a precise dosage pattern in elderly patients compared to active immunization methods such as vaccines.
4) Low binding to alpha-synuclein monomers in the marginal zone/whole body, thus allowing more antibodies/fragments available for binding and clearance of oligomeric forms of alpha-synuclein in the brain.
5) Antibodies and fragments reduce the risk of inflammatory side effects, e.g., meningoencephalitis (meninogenecephalitis), by low or no binding to complement factor C1 q.
One aspect of the invention is the discovery of antibody amino acid sequences for CDR regions that play an important role in the binding of human wild-type and mutant α -synuclein protofibrils. Antibodies with binding sites (CDR regions) according to the invention are characterized by a high affinity for wild-type human alpha-synuclein oligomers/fibrils for use as therapeutics or diagnostics.
The basic structure of an immunoglobulin (IgG) molecule comprises two identical light chains and two identical heavy chains linked together by disulfide bridges. The light chain, which is either lambda or kappa, has a variable region (VL) and a constant region (CL) of about 110 amino acid residues each. The heavy chain has a variable region (VH) of about 110 amino acid residues, but has a larger constant region (CH) of 300-400 amino acid residues, including
The constant region (Fc) activates the complement system and binds to Fc receptors on macrophages, microglia and neutrophils, which take up and destroy infected microorganisms or foreign/non-self antigens. This function is important because it is part of the therapeutic principle of antibodies, e.g., Fc receptors mediate phagocytosis of microglia and clearance of α -synuclein fibrils. Regulated clearance of alpha-synuclein oligomers by lysosomal degradation pathways has been demonstrated (Lee et al, 2004). This process involves endocytosis of receptor-dependent or non-receptor-dependent antibody/fibril complexes following fusion of lysosomes in which alpha-synuclein fibrils are degraded (Masliah et al, 2005). Receptors that have been shown to control this process include the thy1.1 receptor and the lipoprotein receptor associated protein (LPR).
Other anti-alpha-synuclein clearance mechanisms may also be manipulated. The clearance of soluble α -synuclein fibrils is an important mechanism of treatment according to the present invention, and α -synuclein fibrils are believed to be highly neurotoxic, initiating and accelerating disease processes. Clearance of α -synuclein protofibrils in the brain is an important mechanism for clinical evaluation. In addition to clearance of α -synuclein fibrils, clearance by precursor forms of α -synuclein fibrils, e.g., α -synuclein fibrils, dimers, trimers, tetramers, and higher oligomeric forms, will indirectly simplify other forms of α -synuclein aggregation including α -synuclein fibrils. The different forms of alpha-synuclein, including fibrils and fibrils, remain in equilibrium. Treatment with high affinity protofibril-binding antibodies and clearance of α -synuclein protofibrils by the antibodies also has the advantage of indirectly simplifying other α -synuclein aggregated or oligomerized forms. Yet another mechanism of action of antibodies is to block or inhibit the toxicity of alpha-synuclein by binding to toxic alpha-synuclein species and preventing their interaction with nerve cells.
The variable regions of each of the heavy and light chains contain three hypervariable regions (hypervariable regions), known as complementarity determining regions or CDRs. CDR regions are short stretches of about 7-23, e.g., 13-23 amino acids, located in the VL and VH regions. The six CDR regions on one "arm" of the antibody form a "pocket" that binds antigen. Several definitions of CDR-sequences are used in the literature. 1-21 define the inventive CDR-sequences using a first recognition system, thus the recognized CDR sequences are shown in Table 1 (see example 2) by the underlined regions in VL and VH in human wild-type and mutant α -synuclein protofibril-specific monoclonal antibodies. 22-55 utilize the known Kabat system to identify the invention of CDR-sequences, thus in table 2 (see example 2) through the underlined region in human wild type and mutant alpha-synuclein fibril specific monoclonal antibody VL and VH show the identification of Kabat CDR sequences. Identification of the CDR-sequences invented according to Kabat (SEQ ID NOS:22-55) is used in the present disclosure.
Thus, in one embodiment, an antibody according to the invention is characterized by having six CDR sequences (VH-CDR-1, VH-CDR-2, VH-CDR-3, VL-CDR-1, VL-CDR-2, and VL-CDR-3), wherein the CDR sequences are selected from each of the following respective sets of CDR sequences in any combination.
VH CDR-1
GFTFNTYAM SEQ ID NO:1 GFTFNTYAMN SEQ ID NO:22GFTFSNYAM SEQ ID NO:2 GFTFSNYAMS SEQ ID NO:23GFTFSSYAM SEQ ID NO:3 GFTFSSYAMS SEQ ID NO:24GDSFTSGYWSEQ ID NO:4 GDSFTSGYWN SEQ ID NO:25
GFSLTSYGVH SEQ ID NO:26
GFTFTDYYMS SEQ ID NO:27
VH CDR-2
RIRTKSNDYATYYADSVKG SEQ ID NO:5
RIRTKSNDYATYYADSV SEQ ID NO:28
TVTSGGSYTYYPDSVRG SEQ ID NO:6
TVTSGGSYTYYPDSV SEQ ID NO:29
TISNGGS YT Y YPD S VKG SEQ ID NO:7
TISNGGS YT Y YPD S V SEQ ID NO:30
YIRYSGNTYYNPSLKS SEQ ID NO:8
YIRYSGNTYYNPSL SEQ ID NO:31
VIWRGGSTDYSAAF SEQ ID NO:32
TISTGGSYTYYPDSV SEQ ID NO:33
FIRNKANGYTTEYSASV SEQ ID NO:34
VH CDR-3
VGYRPYAMDY SEQ ID NO:9(SEQ ID NO:35)
QNFGSRGWYFDV SEQ ID NO:10(SEQ ID NO:36)
HSDYSGAWFAY SEQ ID NO:11(SEQ ID NO:37)
SYYDYDRAWFAY SEQ ID NO:12(SEQ ID NO:38)
LLRSVGGFAD SEQ ID NO:39
DYGNYAMDY SEQ ID NO:40
VL CDR-1
RSSQNIVHSNGNTYLE SEQ ID NO:13(SEQ ID NO:41)
RSSQSIVNSNGNTYLE SEQ ID NO:14(SEQ ID NO:42)
SASSSVSYMY SEQ ID NO:15(SEQ ID NO:43)
RSSQSLVHSNGNTYLH SEQ ID NO:16(SEQ ID NO:44)
RSSQTIVHNNGNTYLE SEQ ID NO:45
KSSQSLLYSSNQKNYLA SEQ ID NO:46
VL CDR-2
KVSNRFS SEQ ID NO:17(SEQ ID NO:47)
RTSNLAS SEQ ID NO:18(SEQ ID NO:48)
WASTRES SEQ ID NO:49
VL CDR-3
FQGSHVPLT SEQ ID NO:19(SEQ ID NO:50)
QQYHSYPYT SEQ ID NO:20(SEQ ID NO:51)
SQSTHVPWT SEQ ID NO:21(SEQ ID NO:52)
FQGSHVPFT SEQ ID NO:53
QQFHSYPYT SEQ ID NO:54
QQYYSYPYT SEQ ID NO:55
An antibody originally selected for certain features of interest is excluded because it does not meet the defined antibody according to the criteria of the invention. An important parameter for this exclusion is the rather short VH CDR-3 sequence with five amino acids exposed by this antibody. Therefore, it was concluded that the VH CDR-3 sequence required more than 5 amino acids. In particular embodiments, the VHCDR-3 sequence is 9, 10, 11, or 12 amino acids.
In particular embodiments, antibodies and fragments according to the invention have six CDR sequences selected from the following combinations:
22, 28, 35, 41, 47 and 50 in SEQ ID NOS,
23, 29, 36, 42, 47 and 50,
24, 30, 37, 43, 48 and 51 of SEQ ID NOS,
25, 31, 38, 44, 47 and 52 of SEQ ID NOS,
26, 32, 39, 45, 47 and 53 in SEQ ID NOS,
23, 33, 37, 43, 48 and 54 of SEQ ID NOS, and
In further specific embodiments, the antibody that provides the desired specificity for α -synuclein protofibrils, while meeting other important features defined herein, has six CDR sequences of an antibody or fragment selected from the following respective groups:
VH CDR-1 SEQ ID NOS 23, 24, 25 or 26
VH CDR-2 SEQ ID NOS 29, 30, 31 or 32
VH CDR-3 SEQ ID NO:36
VL CDR-1 SEQ ID NOS 42, 43, 44 or 45
VL CDR-2 SEQ ID NOS:47 or 48
VL CDR-3 SEQ ID NOS 50, 51, 52 or 53
Or selected from the following groups:
VH CDR-1 SEQ ID NOS 23, 24, 25 or 26
VH CDR-2 SEQ ID NOS 29, 30, 31 or 32
VH CDR-3 SEQ ID NO:37
VL CDR-1 SEQ ID NOS 42, 43, 44 or 45
VL CDR-2 SEQ ID NOS:47 or 48
VL CDR-3 SEQ ID NOS 50, 51, 52 or 53
Or selected from the following groups:
VH CDR-1 SEQ ID NOS 23, 24, 25 or 26
VH CDR-2 SEQ ID NOS 29, 30, 31 or 32
VH CDR-3 SEQ ID NO:38
VL CDR-1 SEQ ID NOS 42, 43, 44 or 45
VL CDR-2 SEQ ID NOS:47 or 48
VL CDR-3 SEQ ID NOS 50, 51, 52 or 53
Or selected from the following groups:
VH CDR-1 SEQ ID NOS 23, 24, 25 or 26
VH CDR-2 SEQ ID NOS 29, 30, 31 or 32
VH CDR-3 SEQ ID NO:39
VL CDR-1 SEQ ID NOS 42, 43, 44 or 45
VL CDR-2 SEQ ID NOS:47 or 48
VL CDR-3 SEQ ID NOS:50, 51, 52 or 53.
As previously mentioned, the antibodies and fragments according to the invention bound by α -synuclein protofibrils are characterized by high affinity to the target. High affinity, expressed as the separation constant Kd, of less than 10-7And M. In further embodiments, the separation constant Kd for human α -synuclein protofibrils is less than 10-8M, less than 10-9M, less than 10-10M, or even less than 10-11And M. And has a diameter of 10-6These antibodies and fragments have the advantage that they can be administered at lower doses compared to antibodies of around M or higher affinity. High affinity antibodies, such as these, which can be administered by injection, can be administered subcutaneously, which has important clinical advantages because only low amounts of antibody are required for efficacy. The mode of administration is not limited to subcutaneous or intravenous injection. Furthermore, the lower dosage required for efficacy would reduce the cost of materials used for antibody production.
In addition to the high affinity of antibodies for α -synuclein fibrils, antibodies and fragments exhibit low binding to α -synuclein monomers, and optionally to α -synuclein fibrils. As mentioned above, a low binding to alpha-synuclein monomers means that the binding of an antibody or fragment according to the invention to alpha-synuclein monomers is at least 100 times less than to alpha-synuclein fibrils. In a more specific embodiment, the binding of the antibody or fragment according to the invention to α -synuclein fibrils is more than 500-fold or even more than 1000-fold higher than to α -synuclein monomers.
In another embodiment, the antibodies and fragments exhibit low binding to α -synuclein fibrils. In more specific embodiments, the binding of an antibody or fragment according to the invention to α -synuclein fibrils is more than 100-fold, more than 500-fold, or even more than 1000-fold greater than to α -synuclein fibrils.
In yet another embodiment of the invention, the antibodies and fragments exhibit low binding (e.g., Kd) to amyloid-beta (A β) fibrils>10-5M) and exhibits low binding to beta-amyloid monomers (e.g., Kd>10-5M)。
In yet another embodiment of the invention, the antibodies and fragments exhibit low binding to β -synuclein monomers, γ -synuclein monomers, IAPP (islet amyloid polypeptide), and/or Medin polypeptides, e.g., binding of the antibodies and fragments to one or more of these peptides/proteins is at least 100-fold less than binding to human α -synuclein fibrils.
According to another embodiment of the present invention, the binding of a linear epitope in α -synuclein within the amino acid (aa) region 113-. In this model system, 15-mer α -synuclein peptides with 11 amino acid sequence overlaps were used (see example 3 below).
According to a further embodiment of the invention, there is provided an antibody or fragment having high affinity for human α -synuclein protofibrils and low binding of α -synuclein monomers and comprising a combination of one CDR-sequence of each of the six CDR sequence sets selected from SEQ ID NOs 22-27, 28-34, 35-40, 41-46, 47-49 and 50-52, and having a sequence similarity of greater than 70, 80, 90, 95 or 98% to any one of the sequences in each respective set. The antibody or fragment binds to an epitope within amino acid (aa) region 113-.
According to another specific embodiment of the invention, high affinity α -synuclein protofibril-bound antibodies can reduce or inhibit α -synuclein aggregation, thereby reducing the level of soluble oligomeric α -synuclein forms in the brain.
According to another specific embodiment of the invention, the high affinity α -synuclein protofibrils-bound antibody is also able to bind α -synuclein oligomers/protofibrils outside the CNS, thus altering the equilibrium of said α -synuclein form on the blood-brain barrier in such a way as to reduce the CNS levels (exclusion) of said α -synuclein form.
According to another particular embodiment of the invention, the antibody is of the IgG type, suitable for therapeutic applications capable of crossing the blood-brain barrier. High affinity α -synuclein protofibril-bound IgG antibodies can be designed to reduce complement factor C1q binding to the CH2 domain of IgG1 and reduce the risk of complement activation and inflammation. This modification can be accomplished in several different ways. One way is to make a chimeric antibody in which the
Yet another way to reduce the risk of inflammation is to alter the oligosaccharide structure of antibodies, which will reduce complement factor C1q binding and complement activation. Thirty different structures of synthetic hybrid oligosaccharides at Asn-297 in human IgG1 have been described. It is believed that the deletion of the CH 2-associated carbohydrate causes a conformational change in the "hinge" region of the antibody, reducing the efficacy of the interaction with the effector molecule and the loss of complement activation function and C1q binding.
Modification of high affinity human α -synuclein protofibril-binding antibodies by site-directed mutagenesis at position Asn-297 to any other amino acid will result in antibodies retaining Fc-receptor binding with less C1q binding, thus reducing the risk of inflammation, in particular on the blood brain barrier. Alternatively, the glycosylation is modified on the antibody by expressing the antibody in a cell type in which N-acetylglucosamine transferase has been inhibited. This will result in an antibody with an altered carbohydrate structure at Asn-297. A structure of Man5GlcNAc2 is formed, but not limited to this structure. This carbohydrate modification will reduce complement factor C1q binding and inhibit inflammation (Wright et al, 1998). Alternatively, aglycosylated (aglycosylated) fibril-binding antibody can be obtained by culturing antibody-expressing cells in the presence of tunicamycin, wherein the tunicamycin inhibits glycosylation. These antibodies have altered complement activation activity and altered Fc-receptor function (Leatherbarrow et al, 1985). Screening for clonal expression of expressed antibodies with low complement activation and high Fc-receptor binding will result in protofibril-binding antibodies that exhibit high Fc-mediated clearance of alpha-synuclein protofibrils and low C1q binding.
In another embodiment, the high affinity human α -synuclein fibril-binding antibody is of the IgG subtype, e.g., IgG1 or IgG4, wherein the complement factor C1q binding site has been modified in such a way, e.g., Pro331> Ser331(Xu et al, 1994), so as to reduce or inhibit the binding of complement factor C1 q. Such antibodies are particularly suitable, for example, for use in treating, preventing or delaying the onset of a neurodegenerative disease having an alpha-synuclein pathology, for administration to an individual having or at risk of developing such a disease, such as, but not limited to, an individual having or at risk of developing PD. The proline residue at position 331 in human IgG1 can also be changed to threonine or glycine or any other polar amino acid. Such modifications can be accomplished by standard molecular biology techniques such as site-directed mutagenesis or DNA deletion.
Yet another aspect of the invention relates to the use of high affinity human α -synuclein protofibril binding antibodies, in particular to determine the level of protofibrils in human or animal tissue, e.g. in cerebrospinal fluid (CSF), blood, urine, saliva, brain tissue, as diagnostic tools or biomarkers for, or for monitoring, neurodegenerative diseases with α -synuclein pathology. Parkinson's Disease (PD), Lewy body dementia (DLB0, Lewy body variants of Alzheimer's disease, multiple system atrophy, confusion, schizophrenia, and Creutzfeldt-Jakob disease are merely illustrative of such neurodegenerative diseases with alpha-synuclein pathology for example, a PD patient may have a different level of human alpha-synuclein protofibrils in CSF or blood as compared to a matched aged control group without Parkinson's disease or any other alpha-synucleinopathies It is possible that the fiber-bound antibody completes the assay, and, in particular embodiments, the assay can be completed in conjunction with a sandwich ELISA method (see example 5), in which alpha-synuclein protofibrils reduced to a level of 9pM have been assayed. Interference of other forms of alpha-synuclein, specifically alpha-synuclein monomers, and optionally alpha-synuclein fibrils and alpha-synuclein fragments in the assay, may be ignored.
Examples of suitable methods for analyzing α -synuclein protofibrils in these tissues and cell culture media using anti- α -synuclein protofibril antibodies include immunoassays such as ELSA, RIA, Western blotting, or dot blotting (dotblotting). These methods are suitable for the following therapeutic efficacy as measured by fibril reduction in clinical trials and/or as diagnostic tests. Since the alpha-synuclein protofibrils levels in CSF and blood are very low, the high affinity alpha-synuclein protofibril binding antibodies of the invention are advantageous for use in diagnostic assays, e.g., diagnostic assays based on ELISA methods, to allow measurement of low levels of alpha-synuclein protofibrils.
According to such a method, an antibody or fragment according to the invention is added to a biological sample comprising or suspected of comprising alpha-synuclein fibrils, and the presence of a complex (complex) formed between the alpha-synuclein fibrils and the antibody or fragment is detected. The complex may be detected qualitatively, e.g., the presence of the complex is detected, or qualitatively, e.g., the concentration of the complex or a threshold concentration of the complex may be detected, as desired.
In further embodiments, the invention includes the use of high affinity protofibril-specific antibodies and fragments in imaging for the detection, localization and quantification of alpha-synuclein protofibrils in human and animal tissues. The antibody or fragment may be labelled with a detectable label, e.g. a radioactive ligand such as I131、C14、H3Or gallium68But are not limited to, these radioisotopes, and are administered in contact with a sample or for detection purposes. Such methods are suitable as diagnostic tools for neurodegenerative diseases with α -synuclein pathology, including, but not limited to, parkinson's disease, dementia with lewy bodies, and other α -synuclein-associated neurodegenerative diseases. In particular embodiments, such methods may guide monitoring the progression of an α -synuclein-associated disease in a subject without or under drug treatment or other possible treatment.
Thus, in one aspect of the invention, an antibody is added to a biological sample comprising or suspected of comprising alpha-synuclein protofibrils, and the concentration of the complex formed between the protofibrils and the antibody is measured for detection and/or quantification of the protofibrils in the sample. In specific embodiments, the detection methods include immunoassays and proximity ligation technologies (proximity ligation assay). The biological sample may be an in vitro sample and an in vivo liquid obtained from a subject.
Yet another aspect of the invention is the manufacture of specific antibody classes for veterinary use. The listed diagnostic methods are also suitable for veterinary applications.
Another aspect of the invention is the humanization of the antibodies in order to avoid side effects, e.g., to avoid an immune response against the antibodies in humans when used as a therapeutic or diagnostic agent. Such humanization techniques are within the ability of one of ordinary skill in the art.
The pharmaceutical composition according to the present invention comprises the antibody or fragment described herein, and a pharmaceutically acceptable carrier. In a specific embodiment of the therapeutic application, the composition is a physiologically acceptable formulation comprising a therapeutically active amount of the antibody or fragment according to the invention in a physiological buffer suitable for human and/or animal administration, such as, but not limited to, PBS. For better immobilization, the antibody or fragment can be lyophilized. The lyophilized formulation may contain any suitable conventional excipient including fixatives, lyoprotectants, buffers, and the like, such as, but not limited to, mannitol, for protecting and/or immobilizing the product during and/or after lyophilization and/or subsequent storage.
Alternatively, the antibody formulation may contain an antibacterial agent or other preservative or additive that does not interfere with the function or efficacy of the fibril-binding antibody or fragment.