CN116854815A - Modified antibodies targeting amyloid and uses thereof - Google Patents

Abstract

The present invention relates to modified antibodies targeting amyloid and uses thereof. Specifically, the invention relates to a modified antibody for reducing or eliminating the complement and/or Fc receptor binding activity of an antibody targeting amyloid protein and application thereof, and solves the problem that a full-length antibody induces excessive phagocytosis of synapses by glial cells due to formation of an amyloid protein-antibody-complement complex by reducing or eliminating the complement and/or Fc receptor binding activity of the antibody, and discloses the reason of failure of clinical test of immune treatment of neurodegenerative diseases, thereby providing a new theoretical basis for the treatment of the neurodegenerative diseases and obtaining an immune preparation of the neurodegenerative diseases with better application prospect.

Description

Modified antibodies targeting amyloid and their uses

Technical field

The present invention relates to modified antibodies targeting amyloid and their uses. In particular, the present invention relates to modified antibodies that reduce or eliminate the complement and/or Fc receptor binding activity of amyloid-targeting antibodies and their uses.

Background technique

Neurodegenerative diseases are a general term for diseases caused by chronic progressive degeneration and loss of nerve cells, including Alzheimer's Disease (AD), Parkinson's Disease (PD), and Huntington's disease (Huntingdon's Disease, HD) and amyotrophic lateral sclerosis (ALS), etc., are characterized by neuronal structure and dysfunction, neuron loss, and cognitive and behavioral abnormalities. AD, commonly known as Alzheimer's disease, is a neurodegenerative disease characterized by progressive memory impairment, impaired cognitive function, personality changes, and language impairment. The pathogenesis of AD is mainly due to the abnormal aggregation of amyloid-β (Aβ) and highly phosphorylated tau protein to form toxic oligomers, which lead to the formation of senile plaques and neurofibrillary tangles in the brain, causing neuronal death. PD is a movement disorder disease with an incidence rate of approximately 0.3%. The main pathological characteristics of PD are the death of dopamine (DA) neurons and the occurrence of Lewy bodies (Lewy bodies) formed by abnormal aggregation of α-synuclein in substantia nigra neurons. Abnormally aggregated α-synuclein can destroy the integrity of synaptic vesicles, interfere with dopamine metabolism, damage mitochondrial function, interfere with intracellular transport, and ultimately trigger the death of dopaminergic neurons. HD is a neurodegenerative disease characterized by chorea-like symptoms, manifesting as PD-like movement disorders and ataxia, and AD-like dementia. HD is caused by the accumulation of a large amount of mutated huntingtin protein (mHTT) containing polyglutamine in nerve cells, leading to dysfunction and apoptosis of nerve cells in the cortex, striatum and other parts of the body. ALS, also known as Lou Gehrig’s disease, is a fatal neurodegenerative disease characterized by the progressive degeneration of motor neurons in the brain and spinal cord. Clinical manifestations include muscle weakness, atrophy, difficulty swallowing, and respiratory failure. Cu/Zn superoxide dismutase 1 (SOD1) is the earliest discovered dominant mutation causative gene of ALS. The accumulation and deposition of SOD1 monomers in motor neurons is one of the main pathogenesis of ALS. The aggregation of SOD1 induces oxidation. Stress causes excessive activation of glial cells and neuroinflammation, interferes with RNA metabolism, and causes many pathological changes such as glutamate toxicity, mitochondrial dysfunction, axonal conduction disorders, and even motor nerve cell apoptosis.

The causes of neurodegenerative diseases are complex. Many neurodegenerative diseases are characterized by abnormal aggregation of amyloid proteins, such as Aβ, tau, α-synuclein, mHTT, SOD1 and other proteins. The oligomers formed by the aggregation of these protein monomers are responsible for AD, It is a key pathogenic factor in the development of PD, HD, ALS, etc., and is also an effective target for diagnosis and treatment of such diseases. Among all aggregate forms, oligomers are the most cytotoxic and are directly related to the occurrence and development of diseases. Oligomers and fibers of different amyloid proteins are deposited in different parts of the brain, damaging nerve cells in corresponding areas, leading to cognitive and behavioral disorders. Although these amyloid proteins have different primary sequences, their aggregation can form similar three-dimensional structures and have similar toxicity mechanisms. Therefore, therapeutic strategies targeting amyloid toxic oligomers are of great significance for the diagnosis and treatment of various neurodegenerative diseases.

Among them, Aβ oligomers have a high affinity for neural synapses. They can trigger the redistribution of key synaptic proteins, induce excessive activation of synapse-related glutamate receptors, damage nerve cell membranes, and disrupt synapses. Touch the steady-state level of Ca ²⁺ , thereby increasing intracellular oxidative stress, inflammatory factor levels and mitochondrial damage. It can be seen that drugs that accelerate Aβ clearance, inhibit Aβ aggregation and cytotoxicity, especially immunotherapy drugs with high affinity for Aβ that people have high hopes for, should have therapeutic effects on AD.

Glial cells are another major type of cells in the nervous system besides neurons, mainly including: microglia, astrocytes, oligodendrocytes and oligodendrocyte precursor cells (OPC), etc. . In the central nervous system, glial cells play a variety of important physiological functions, such as regulating the normal physiological functions of synapses and synaptic regeneration, removing foreign matter, participating in the formation of myelin around axons, and maintaining the blood-brain barrier ( BBB) integrity and the homeostasis of ions and neurotransmitters in the brain.

Microglia are widely distributed in the brain and spinal cord. They are the most important innate immune cells in the central nervous system. They play an important role in immune surveillance and endogenous immune defense in the brain and play an important role in the occurrence and development of neurodegenerative diseases.

In AD, microglia play a key role in the clearance of Aβ from the brain. Aβ oligomers can interact with certain microglia surface receptors. On the one hand, they induce microglia to phagocytose and clear Aβ, protecting neurons from Aβ. On the other hand, large amounts of Aβ oligomers can overactivate microglia. Plasma cells promote the release of inflammatory factors, leading to neuroinflammation. At the same time, the phagocytosis of Aβ by overactivated microglia is inhibited, further causing the deposition of Aβ. Aβ oligomers can also activate complement-related clearance pathways, leading to abnormal phagocytosis of synapses by microglia, resulting in excessive synapse loss and cognitive decline. Synapses are the basis for the interconnection between neurons and are key components that constitute neural circuits, produce memory and neural activity. Studies have shown that in healthy brains, one of the normal physiological functions of microglia is to participate in the regulation of synaptic development and guide synaptic plasticity related to cognition, and its mechanism of action relies on the C3 receptor on microglia. Identification of unique C1q-C3 complexes at hypoactive synapses. During the developmental stage, microglia build mature central nervous networks by pruning redundant synapses. In the growth and maturity stage, learning and memory functions are highly dependent on nerve regeneration, and microglia are important factors in regulating synaptic plasticity and nerve regeneration, as well as cognitive function. Loss of synapses is closely associated with decline in cognitive function. Many recent studies believe that excessive pruning of synapses by microglia and erroneous up-regulation of the complement system mediate the disorder and loss of synaptic function and are one of the important pathogenic mechanisms of AD. Aβ oligomers can upregulate the expression of C1q in microglia. Excessive C1q binds to synapses, activates the classical complement pathway, forms a C1q-C3 complex, and further activates C3 receptors on microglia, leading to microglia. Glial cells excessively engulf and clear synapses in a manner similar to normal pruning of synapses, resulting in synapse loss and cognitive decline. In addition, Aβ oligomers can cause autophagy dysfunction in microglia and reduce their ability to degrade Aβ. Therefore, microglia play a "double-edged sword" role in the pathological process of AD. Maintaining the normal activation state of microglia and accelerating the phagocytosis and clearance of Aβ toxic oligomers are the key to AD treatment.

There are Fc receptors for immunoglobulin G antibodies on the surface of microglia, called Fcγ receptors. Fcγ receptors can mediate phagocytosis through immune complexes and can also mediate the release of pro-inflammatory factors from microglia. In the process of immunotherapy for neurodegenerative diseases such as AD, antigen-antibody complexes can activate Fcγ receptors on the surface of microglia, promote the occurrence of inflammatory reactions in the brain, lead to damage to the patient's blood-brain barrier, and cause cerebral edema or cerebral infarction. Bleeding and other serious adverse reactions. (Salloway et al., 2009).

However, taking AD as an example, in the past decade or so, approximately US$110 billion has been invested globally in the research and development of AD therapeutic drugs, but the huge investment in manpower and material resources has not yet led to the emergence of specific therapeutic drugs. Although the development of drugs targeting the pathogenesis of AD is still a research hotspot for many large international pharmaceutical companies and research units, only by clarifying the reasons for the failure of drug clinical trials can we guide the direction of future drug research and development.

Immunotherapy has always been the focus of research in the field of AD treatment. In recent years, dozens of antibodies and vaccines targeting Aβ have entered clinical trials. Although these preparations have shown good therapeutic effects in the animal testing phase, significantly improving the cognitive level of AD transgenic animals and reducing the amount of senile plaques and other pathological changes in the animals' brains, in AD clinical trials, due to poor therapeutic effects or Serious side effects, so far no antibody or vaccine has passed Phase III clinical trials. Although the U.S. Food and Drug Administration (FDA) has approved the monoclonal antibody drug aducanumab for the treatment of AD, its efficacy It’s not clear, and it initially failed in two randomized, double-blind, placebo-controlled Phase 3 clinical trials in 2019.

Aβ antibodies and vaccines have gone through the development of three generations of products targeting the overall molecule of Aβ, the N-terminus of Aβ, and the spatial structure of Aβ aggregates. The first and second generation antibodies targeting Aβ such as bapineuzumab (anti-Aβ1-5), solanezumab (anti-Aβ13-28), gantenerumab (anti-Aβ1-11) and ponezumab (anti-Aβ C-terminus), as well as third-generation antibodies targeting Aβ aggregates such as aducanumab, crenezumab, BAN2401, etc., although they can reduce Aβ in the brains of AD patients level and number of age spots, but did not reach the expected endpoints in terms of improving cognitive function and slowing down memory loss. Similarly, the results of the Phase II clinical trial using IVIG to treat AD showed that the agent can reduce Aβ levels in the cerebrospinal fluid of patients with mild to moderate AD and stabilize or improve cognitive levels. However, in the Phase III clinical trial, although APOE-e4 Gene-positive AD patients experienced significant cognitive improvements, but overall trial results fell short of expectations. The continuous failure of clinical trials of immunotherapy for AD has caused some scholars to question the theory of Aβ pathogenicity, which has seriously affected the development of new drugs to treat AD.

Therefore, there is an urgent need in the field for immunotherapeutic agents that can overcome the shortcomings of existing amyloid-targeting proteins and achieve effective treatment of human neurodegenerative diseases.

Contents of the invention

One aspect of the invention relates to an antibody specifically targeting amyloid or an antigen-binding fragment thereof, characterized in that it has reduced complement and/or Fc receptor binding activity or no complement and/or Fc receptor binding active.

In some embodiments, the amyloid protein is in its monomeric, oligomeric, profibrillar or fibrillar form.

In some embodiments, the amyloid protein is selected from the group consisting of beta amyloid, microtubule associated protein tau, alpha-synuclein, huntingtin, pancreatic amyloid, SOD1 and TDP-43, preferably , the amyloid protein is β-amyloid protein or microtubule-associated protein tau.

In some embodiments, the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody or a fully human antibody, preferably the antibody is selected from the group consisting of IgG, IgM, IgA, IgD and IgE and subtypes thereof Group.

In some embodiments, the antibody is IgG, IgM, or a subtype thereof, wherein the complement and/or Fc receptor binding activity of the antibody is modified by deletion of the entire complement and/or Fc receptor binding segment of the Fc fragment of the antibody. or partially, reduced or eliminated by mutation that reduces or eliminates the complement and/or Fc receptor binding segment of the antibody Fc fragment, preferably the mutation is a complement of the antibody Fc fragment and/or substitution or insertion of one or more amino acid residues within the Fc receptor binding segment.

In some embodiments, the antigen-binding fragment is selected from the group consisting of F(ab') ₂ , Fab', Fab, Fd, Fv, scFv, diabodies, camel antibodies, CDRs, and minimal recognition units of antibodies (dAbs) , preferably, the antigen-binding fragment is Fab, F(ab') ₂ or scFv.

In some embodiments, the antibody is selected from the group consisting of A16, adunumumab, BAN2401, bapineuzumab, solanezumab, gantenerumab, ponezumab, crizumab, Donanemab, Gosuranemab, Tilavonemab, Semorinemab, Zagotenemab, and PRX002, wherein the A16 antibody The CDR sequences are shown in SEQ ID NO: 1-6.

In a further embodiment, the antigen-binding fragment is F(ab') ₂ of the monoclonal antibody A16 targeting amyloid-beta, and the CDR sequence of the A16 antibody is as shown in SEQ ID NO: 1-6.

Another aspect of the invention relates to an isolated nucleic acid molecule selected from:

(1) DNA or RNA encoding the antibody or antigen-binding fragment thereof as described above;

(2) A nucleic acid that is completely complementary to the nucleic acid defined in (1).

Another aspect of the invention relates to an expression vector comprising a nucleic acid molecule operably linked as described above.

Another aspect of the invention relates to a host cell comprising a nucleic acid molecule as described above or an expression vector as described above.

Another aspect of the invention relates to a composition comprising an antibody or antigen-binding fragment thereof, a nucleic acid molecule, an expression vector or a host cell as described above, and one or more pharmaceutically acceptable carriers, diluents or excipient.

Another aspect of the present invention relates to a method of producing an antibody or an antigen-binding fragment thereof as described above, comprising the steps of:

The host cells as described above are cultured under culture conditions suitable for expression of the antibody or antigen-binding fragment thereof, and optionally, the resulting product is isolated and purified.

Another aspect of the invention relates to the use of antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, host cells or compositions as described above in preparations for promoting the clearance of toxic forms of amyloid in cells of a subject, reducing the risk of Age spots, use in drugs to treat and/or prevent neurodegenerative diseases, inhibit gliosis, and improve synaptic levels in the brain.

In some embodiments, the antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, host cells or compositions as described above are used to promote the clearance of toxic forms of amyloid in cells of a subject, reduce senile plaques in the brain, and treat And/or prevent neurodegenerative diseases, inhibit gliosis, and improve brain synapse levels.

Another aspect of the invention relates to a method that promotes the clearance of toxic forms of amyloid in cells of a subject, reduces senile plaques in the brain, treats and/or prevents neurodegenerative diseases, inhibits gliosis, and improves synaptic levels in the brain. A method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, host cell or composition as described above.

In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, frontotemporal dementia, Huntington's disease, amyotrophic lateral sclerosis, or spinocerebellar ataxia, preferably Preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.

Another aspect of the invention relates to the use of an antibody, or antigen-binding fragment thereof, nucleic acid molecule or expression vector as described above, for the preparation of a reagent for diagnosing the presence and/or levels of toxic forms of amyloid in a subject sample.

In other words, amyloid proteins such as Aβ oligomers, which cause neurodegenerative diseases such as AD, have a high affinity to synapses and can easily bind to synapses and neurons, leading to the destruction of synapses and neurons. damage. Synapses are a large number of key components formed by neurons that connect neurons. They are an important basis for memory and other neural activities. Under physiological conditions, microglia in the brain have a pruning function on less active synapses. The implementation of this function relies on complement C1q binding to low-activity synapses and then activating the classical pathway to form a C1-C3 complex at the synapse. Microglia carry characteristic C3 receptors, which bind to the complement complex on synapses and pruning and engulfing the corresponding synapses.

In the treatment of neurodegenerative diseases such as AD, whether it is antibodies used for passive immunotherapy or antibodies produced by the body induced by active immunity, they can directly target amyloid proteins such as Aβ oligomers that bind to synapses. Antigen-antibody immune complexes are formed at synapses. At the same time, the Fc segment of the antibody activates the classical complement pathway, allowing C1q to bind to it, and then the activated complement C3 binds to C1q to form an antigen-antibody-C1q-C3 complex. Furthermore, C3 binds to the unique C3 receptor on microglia, activating microglia and causing them to excessively prune synapses, causing further loss of synapses and damage to cognitive functions. Therefore, although the levels of amyloid proteins such as Aβ in the brain are significantly reduced at this time, the patient's cognitive function cannot be restored. In addition, while microglia engulf antibody-antigen complexes on synapses, they can also activate Fcγ receptors, which mediate microglia to phagocytose antigen-antibody complexes and phagocytose synapses together, and, It will also stimulate microglia to release a large amount of inflammatory factors, further causing neuronal dysfunction and apoptosis, leading to the failure of AD immunotherapy.

In this regard, the inventor believes that immunotherapy preparations for neurodegenerative diseases such as AD that enter clinical trials should have good therapeutic effects on experimental animals. Otherwise, it is impossible to enter clinical trials. For example, in the preclinical stage of AD immunotherapy preparations, AD transgenic mice are used for pharmacological efficacy testing. Mice have undergone several immunotherapy treatments, and their cognitive abilities and brain pathological changes are usually measured 1-2 weeks after the last treatment. At this time, effective immune preparations can significantly reduce brain pathological changes and Aβ levels in mice, and improve synaptic levels and cognitive abilities. Why can’t immunotherapy effects seen in mice be replicated in AD patients? The present inventors believe that adverse effects caused by immunotherapy of neurodegenerative diseases such as AD exist in both the brains of patients with neurodegenerative diseases such as AD and in the brains of mice. Within days of immunotherapy, synapses in the brains of both mice and patients were subject to excessive pruning and phagocytosis. While Aβ levels decreased, cognitive abilities also declined significantly. But unlike mice, primates such as patients with neurodegenerative diseases such as AD have very slow growth and regeneration of synapses. Within 1 to a few weeks after treatment, the number of synapses in mice can be rapidly regenerated and restored, especially in an environment where Aβ levels in the brain are significantly reduced. The cognitive abilities of mice are significantly enhanced, and neurodegeneration is The number of synapses in the brains of patients with diseases such as AD rarely exceeds that of untreated patients within weeks. If the recovery time is too long, the levels of amyloid proteins such as Aβ that have been reduced by antibodies will increase again, causing the patient's synapses and neuronal functions to be damaged again, making it difficult for the patient to show good therapeutic effects. In short, within a limited time window, the synaptic level of mice treated with full antibody can exceed that of untreated control mice, while the synaptic level of patients with neurodegenerative diseases such as AD who receive full antibody treatment cannot exceed that of untreated control mice. of patients.

In order to confirm the above speculation, the present invention studied the mechanism of action of antibodies in AD immunotherapy. By constructing a primary hippocampal neuron-microglia co-culture system, the present invention explores the effects of full-effect antibodies targeting Aβ and antibody F(ab') ₂ fragments on microglia phagocytosis of synapses, and further To analyze the mechanism by which the Fc fragment of the antibody promotes phagocytosis of synapses by microglia, the whole antibody (A16) targeting Aβ and its F(ab') ₂ fragment (Fab16) were administered to AD transgenic mice via intracerebroventricular injection, respectively. The changes in cognitive ability, number of synapses in the brain, inflammation, complement and Aβ levels of mice at different time points were detected. The results showed that the memory and synaptic levels of mice treated with whole antibodies were significantly lower than those of untreated and mice treated with F(ab') ₂ fragments. However, due to the rapid regeneration ability of mouse synapses, both whole antibodies and F(ab') ₂ antibodies can improve the memory and synaptic levels of mice and reduce pathological changes in the brain 14 days after the last treatment. Similarly, in the primary neuron-microglia co-culture system, whole antibodies but not F(ab') ₂ antibodies can significantly promote the phagocytosis of synapses by microglia. These results preliminarily confirm the hypothesis of the present invention from one aspect.

Since neurodegenerative diseases due to amyloid have a considerable degree of commonality, the conclusions of the present invention are also applicable to neurodegenerative diseases due to other amyloid forms, such as PD, HD, ALS, etc.

Description of the drawings

Figure 1: Preparation and functional characterization of A16 whole antibody and its F(ab') ₂ fragment Fab16.

A. Reducing or non-reducing gel electrophoresis to detect the purity of A16 antibody and Fab16 antibody;

B. Use microthermal molecular interaction electrophoresis instrument to detect the affinity between A16 and Fab16 and Aβ;

C. Thioflavin T (ThT) experiment detects the inhibitory effect of A16 and Fab16 on Aβ aggregation;

D. MTT experiment detects the inhibitory effect of A16 and Fab16 on neuronal cytotoxicity induced by Aβ oligomers;

E. Use A16, Fab16 or isotype antibody control (IgG control) as the primary antibody for immunohistochemical staining to mark senile plaques in the brains of AD mice. The scale bar is 100 μm.

Figure 2: Antibody A16 but not Fab16 mediates microglial phagocytosis of synapses.

A. Add Aβ oligomers to the co-culture system of neurons and microglia, and then add A16, Fab16, or solvent as a control. Laser confocal microscopy was used to detect the phagocytosis of synapses by microglia. PSD95: red; Iba-1: green; Hoechst: blue. Scale bar: white (lower), 25 μm; cyan (upper), 5 μm.

B. Statistics on the number of phagocytosed PSD95 in microglia.

C. Count of PSD95 near microglia.

D. Western blot analysis of PSD95 levels in cell lysates of the co-culture system 36 hours after treatment with different antibodies, with β-actin as the internal control.

E. Quantitative analysis of PSD95 levels in each group in D.

Figure 3: Antibody A16 but not Fab16 mediates microglial phagocytosis of synapse/antigen-antibody/complement complexes.

A. Confocal microscopy detects phagocytosed PSD95 and Aβ in microglia. PSD95: red; Aβ: magenta; Iba-1: green; Hoechst: blue. Scale bar is 2.5 μm.

B. Quantity statistics of co-localized PSD95/Aβ in microglia.

C. PSD95 and C1q phagocytosed in microglia. PSD95: red; C1q: cyan; Iba-1: green; Hoechst: blue. Scale bar is 2.5 μm.

D. Quantitative statistics of co-localized PSD95/C1q in microglia.

E. PSD95, C3 and C1q phagocytosed in microglia. PSD95: red; C3: green; C1q: cyan; Hoechst: blue. Scale bar is 2.5 μm.

F. Quantitative statistics of co-localized PSD95/C3/C1q in microglia.

G. PSD95, Aβ and synaptophysin phagocytosed in microglia. PSD95: red; synaptophysin: green; Aβ: magenta; Hoechst: blue. Scale bar is 2.5 μm.

H. Quantitative statistics of co-localized PSD95/Aβ, synaptophysin/Aβ, and PSD95/synaptophysin in microglia.

I. Quantity statistics of PSD95, synaptophysin, Aβ, C1q and C3 phagocytosed in microglia.

Figure 4: Antibody A16 mediates synaptic phagocytosis through microglia surface CR3 and Fcγ receptors.

A. First add functional antibody CD11b antibody or CD32 antibody to block CR3 and FcγRIIb receptors on the surface of microglia respectively, use isotype antibodies as controls, or add scavenger receptor blocker fucose to block microglia. scavenger receptors on the surface, then microglia and neurons were co-cultured, Aβ oligomers and A16 were added to the system, and laser confocal microscopy was used to detect the phagocytosis of synapses and Aβ by microglia. PSD95: red; Aβ: magenta; Iba-1: green; Hoechst: blue. Scale bar is 5 μm.

B. Statistics on the number of PSD95 and Aβ phagocytized in microglia.

C. Use lentivirus to construct an shRNA system, knock down the gene expression of C1qa, C3, CD11b and FcγRIIb in microglia, and then detect the expression levels of each gene by qPCR.

D. Co-culture microglia with knockdown of relevant genes and neurons, add Aβ oligomers and A16 to the system, and use laser confocal microscopy to detect the phagocytosis of synapses by microglia. PSD95: red;

GFP: lentiviral vector fluorescence; Hoechst: blue. Scale bar is 5 μm.

Statistics on the number of phagocytosed PSD95 in microglia in E.D.

Figure 5: 24 hours after injection of A16 but not Fab16 antibody, the cognitive ability and synaptic level of APP/PS1 mice were significantly reduced.

A. The residence time of mice in the new arm in the Y maze experiment;

B. Cognitive index of each group of mice in the new object recognition experiment;

C. Glogi staining detects the number of dendritic spines of cortical neurons in mouse brain;

D. Number statistics of neuron dendritic spines;

E. Use immunohistofluorescence staining to detect PSD95 and synaptophysin in the mouse cerebral cortex and hippocampus. PSD95: red; synaptophysin: green, scale bar is 2.5 μm.

F. Number statistics of PSD95, synaptophysin and intact synapses in the cortex and hippocampus.

Figure 6: A large number of microglia engulfed synapses in the brain of APP/PS1 transgenic mice after injection of A16 but not Fab16 antibody for 48 hours.

A. Confocal microscopy detects the phagocytosis of PSD95 and Aβ in microglia in the mouse brain. Green is Iba-1, red is PSD95, magenta is Aβ, and blue is Hoechst. Scale bar = 3 μm;

B. Use confocal microscopy to detect intracellular phagocytosis of PSD95 and C1q in microglia. Green is Iba-1, red is PSD95, cyan is C1q, and blue is Hoechst. Scale bar = 3 μm;

C. Quantitative statistics of co-localized PSD95/Aβ and PSD95/C1q in microglia;

D. Statistics on the number of PSD95, Aβ, and C1q phagocytosed in microglia.

E.Iba-1 antibody staining detects the morphology of microglia in mouse cerebral cortex;

F. Statistics of the proportion of branched microglia to total microglia.

Figure 7: Increased inflammation levels in the brains of APP/PS1 transgenic mice 48 hours after injection of A16 but not Fab16 antibodies

A. Immunostaining of Iba-1 and GFAP in brain sections of APP/PS1 mice, scale bar = 200 μm;

B. Statistics of Iba-1 positive area in picture A;

C. Statistics of GFAP positive area in picture A;

D. ELISA technology detects the levels of inflammatory factors IL-1β, IL-6, and TNF-α in brain homogenates of APP/PS1 mice.

E: q-PCR technology was used to detect the mRNA levels of inflammatory and complement-related genes in the cerebral cortex microglia of APP/PS1 mice.

Figure 8: Change trend in cognitive level of APP/PS1 transgenic mice treated with A16

A. Experimental flow chart of the change trend of cognitive ability in APP/PS1 mice;

B. The residence time of APP/PS1 mice in the new arm in the Y-maze experiment at different time points;

Figure 9: A large number of microglia engulf synapses in the brain of APP/PS1 transgenic mice 48 hours after injection of A16I4 antibody

A. Confocal microscopy detects the phagocytosis of PSD95 and Aβ in microglia in the mouse brain. Green is Iba-1, red is PSD95, magenta is Aβ, and blue is Hoechst. Scale bar = 3 μm;

B. Use confocal microscopy to detect intracellular phagocytosis of PSD95 and C1q in microglia. Green is Iba-1, red is PSD95, cyan is C1q, and blue is Hoechst. Scale bar = 3 μm;

C. Use confocal microscopy to detect intracellular phagocytosis of PSD95 and C3 in microglia. Green is Iba-1, red is PSD95, yellow is C3, and blue is Hoechst. Scale bar = 3 μm;

D. Quantitative statistics of co-localized PSD95/Aβ, PSD95/C1q and PSD95/C3 in microglia;

E. Statistics on the number of PSD95, Aβ, C1q, and C3 phagocytosed in microglia.

Figure 10: Human neurons regenerate synapses much slower than mice

A. After treating the neurons derived from mouse neural stem cells with 1 μM AβOs for 24 hours, replace the culture medium with new ones, and take Western blot at different time points to detect the PSD95 levels of the cell lysate; PSD95: postsynaptic protein, NeuN: the nucleus of mature neurons. Protein, Tuj1: Neuronal Class IIIβ-Tubulin;

B. After treating neurons derived from human neural stem cells with 1 μM AβOs for 24 hours, replace the medium with new ones, and take Western blot at different time points to detect the PSD95 levels of the cell lysate; PSD95: postsynaptic protein, NeuN: nuclear protein of mature neurons. , Tuj1: Neuronal Class IIIβ-Tubulin;

C. Statistical analysis of A-B plots.

Detailed ways

definition

The term "glia" refers to another major type of cells in the nervous system other than neurons, mainly including: microglia, astrocytes, oligodendrocytes and oligodendrocyte precursor cells (OPC) etc.

Amyloid proteins include β-amyloid, microtubule-associated protein tau, α-synuclein, huntingtin, pancreatic amyloid, superoxide dismutase 1 (SOD1) and TDP-43 proteins, including their individual Aggregated forms such as bodies, oligomers, prefibers or fibers.

Toxic forms of amyloid refer to forms of amyloid that play a negative role in the development of neurodegenerative diseases, such as oligomer forms, profibrillar forms, fibrillar forms, etc.

Amyloid oligomers are nonfibrillar aggregates formed by the aggregation of two or more amyloid monomer molecules.

Complement refers to a serum protein that exists in the serum and tissue fluid of humans and vertebrates. It is not heat-resistant. After activation, it has enzymatic activity and can mediate immune responses and inflammatory reactions. It can be activated by antigen-antibody complexes or microorganisms, leading to lysis or phagocytosis of pathogenic microorganisms. It can be activated through three independent and intersecting pathways, namely the classical pathway, the alternative pathway and the lectin pathway. The complement system is composed of more than 30 glycoproteins such as plasma complement components, soluble and membrane-type complement regulatory proteins, and complement receptors. It is a protein response system or multi-molecular system with a precise regulatory mechanism, including soluble proteins and membrane-bound proteins. and complement receptors. According to the biological functions of each component of the complement system, it can be divided into intrinsic components of complement, regulatory components of complement and complement receptors (CR). Components involved in the classical activation pathway of complement include C1-C9. According to their role in the activation process, they are artificially divided into three groups, namely recognition units (C1q, C1r, C1s), activation units (C4, C2, C3) and membrane attack units (C5-C9). It plays a role in the recognition stage, activation stage and membrane attack stage. During the activation process of complement, C2, C3, C4, and C5 are each cleaved into 2 or more fragments, which are marked with symbols such as a and b, such as C3a, C3b, C3c, etc. Among them, C2b, C3b, C4b, and C5b are directly or indirectly bound to the target cells and participate in the cell lysis process in the form of solid phase, while C3a and C5a are free in the liquid phase. During the activation process of complement, C5, C6, and C7 can also polymerize into C567 after activation, and together with C3a and C5a, they can exert special biological functions. The difference between the bypass activation pathway and the classic activation pathway is that the activation bypasses the three components of C1, C4, and C2, directly activating C3 and then completing the chain reaction of each component from C5 to C9.

C1q: The C1q molecule has 6 sites that can bind to the complement binding sites on immunoglobulin molecules. When more than two binding sites bind to immunoglobulin molecules, that is, after Clq bridges the immunoglobulin, subsequent complement components can be activated. IgG is a monomer. Only when it binds to an antigen can two or more IgG molecules be brought closer to each other and provide more than two adjacent complement binding points to contact C1q. Only when IgM binds to the antigen, undergoes a conformational change, and exposes the complement binding site, can it bind to C1q. The ability of a molecule of IgM to activate complement is greater than that of IgG. After C1q bridges with the complement binding site, its configuration changes, resulting in the sequential activation of Clr and Cls.

Fc receptor: The Fc receptor of immunoglobulin G antibodies is called the Fcγ receptor. Fc receptors are specialized cell surface receptors on macrophages or microglia, which can mediate phagocytosis through immune complexes and the release of pro-inflammatory factors.

The Fcγ receptor (FcγR) plays a crucial role in the elimination or enhancement of immune recruitment. Currently, three types of Fcγ receptors are identified on the cells of the immune system: the high-affinity receptor Fcγ receptor I (CD64), which is capable of binding haplotype immunoglobulin G; the low-affinity receptor Fcγ receptor I (CD64); Receptors Fcγ receptor II (CD32), and Fcγ receptor III (CD16), which preferentially interact with complex immunoglobulin G. In addition, the Fcγ receptor II and Fcγ receptor III types include “A” form and “B” form (see Gessner-JE et al. 1998 Ann Heamatol 76:231-248 The article "The IgG Fc receptor family" published in ("The Fc receptor family of immunoglobulin G")).

The term "antibody" refers to an immunoglobulin molecule or a fragment of an immunoglobulin molecule that has the ability to bind to an epitope on an antigen. Naturally occurring antibodies typically comprise tetramers, usually composed of at least two heavy (H) chains and at least two light (L) chains. Immunoglobulins include the following isotypes: IgG, IgA, IgM, IgD, and IgE, and their corresponding heavy chains are mu chain, delta chain, gamma chain, alpha chain, and epsilon chain, respectively. The same type of Ig can be divided into different subtypes based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4 subtypes, and IgA can be divided into IgA ₁ and IgA ₂ subtypes. Light chains are divided into kappa and lambda chains based on different constant regions. Antibodies of the invention can be of any isotype. The choice of isoform is usually determined by the desired effector function (eg ADCC induction). Exemplary isotypes are IgGl, IgG2, IgG3 and IgG4. Either human light chain constant domain kappa or lambda can be used. If necessary, the class of the antibody of the present invention can be converted by known methods. For example, an antibody of the invention that was originally an IgG can be class-switched to an IgM antibody of the invention. In addition, class switching technology can be used to convert one IgG subclass to another, for example from IgG1 to IgG2. Thus, the effector function of the antibodies of the invention can be isotype-switched to, for example, an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibody for various therapeutic uses, provided that the complement of said antibody Such as C1q and/or Fc receptor binding activity is reduced or eliminated. In some embodiments, the antibodies of the invention are IgM or IgG type 1, 2, 3 or 4 antibodies. An antibody belongs to a particular isotype if its amino acid sequence is mostly homologous to that isotype relative to other isotypes.

As used herein, the term "antibody" is used in the broadest sense to refer to proteins containing an antigen-binding site, encompassing natural and artificial antibodies of various structures, including but not limited to intact antibodies and antigen-binding fragments of antibodies.

A "variable region" or "variable domain" is the domain of the heavy or light chain of an antibody that is involved in the binding of the antibody to its antigen. Each heavy chain of an antibody is composed of a heavy chain variable region (herein referred to as VH) and a heavy chain constant region (herein referred to as CH). The heavy chain constant region usually consists of 3 domains (CH1, CH2 and CH3) constitute. Each light chain consists of a light chain variable region (herein abbreviated as VL) and a light chain constant region (herein abbreviated as CL). The heavy and light chain variable regions are typically responsible for antigen recognition, while the heavy and light chain constant domains can mediate the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells), Fc receptors, The binding of the body to the first component (C1q) of the classical complement system. The heavy and light chain variable regions contain the binding regions that interact with the antigen. The VH and VL regions can be further subdivided into what are called "complementarity determining regions" ( CDR)" hypervariable regions (HVR), which are interspersed with more conservative regions called "backbone regions" (FR). Each VH and VL consists of three CDR domains and four FR domains, in the following order Arranged from amino terminus to carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The term "complementarity determining region" or "CDR region" or "CDR" (used interchangeably herein with hypervariable region "HVR") refers to an antibody variable domain that is hypervariable in sequence and forms a structurally defined loops ("hypervariable loops") and/or regions containing antigen contact residues ("antigen contact points"). CDRs are mainly responsible for binding to antigenic epitopes. In this article, the three CDRs of the heavy chain are called HCDR1, HCDR2 and HCDR3, and the three CDRs of the light chain are called LCDR1, LCDR2 and LCDR3.

It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the same antibody variable region defined under different assignment systems are different. Therefore, when referring to an antibody defined by a specific CDR sequence as defined in the invention, the scope of said antibody also encompasses antibodies whose variable region sequences comprise said specific CDR sequence but which differ due to the application of different protocols (e.g. Different assignment system rules or combinations) cause the claimed CDR boundaries to be different from the specific CDR boundaries defined in the present invention.

The term "monoclonal antibody", "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules as a single molecule composition and refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., a population that contains individual antibodies except Identical except for naturally occurring mutations that may be present in small amounts. Conventional monoclonal antibody compositions exhibit a single binding specificity and affinity for a specific epitope. In certain embodiments, a monoclonal antibody can be composed of more than one Fab domain, thereby increasing specificity for more than one target. The term "monoclonal antibody" or "monoclonal antibody composition" is not limited to any specific method of production (eg, recombinant, transgenic, hybridoma, etc.).

The invention also includes "bispecific antibodies", wherein the antibody of the invention is part of a bivalent or multivalent bispecific framework targeting more than one epitope (e.g. the second epitope may comprise an epitope of an active transport receptor) position such that the bispecific antibody will exhibit improved transcytosis across biological barriers (such as the blood-brain barrier). Thus, in additional embodiments, a monovalent Fab of an antibody of the invention can be linked to an additional Fab or scfv targeting a different protein to generate a bispecific antibody. Bispecific antibodies can have dual functions, such as therapeutic functions conferred by the present invention and transport functions that can bind to receptor molecules to enhance transfer across biological barriers, such as the blood-brain barrier. The terms "bisAb", "bifunctional antibody", "bispecific antibody", "bispecific antibody" or "BsAb" refer to having two different antigen-binding sites, which can bind to two target antigens at the same time. Antibodies are not only targeted but also mediate another special function. The special function effector molecules mediated can also be toxins, enzymes, cytokines, radionuclides, etc. Bispecific antibodies bind the two arms of the antigen. Can be from Fab, Fv, ScFv or dSFv, etc.

The term "antigen-binding fragment of an antibody" refers to a fragment, part, region or domain of an antibody capable of binding to an epitope (e.g., obtainable via cleavage, recombination, synthesis, etc.). Antigen-binding fragments may contain 1, 2, 3, 4, 5 or all 6 CDR domains of such antibodies and, despite being able to bind to the epitope, may exhibit different specificities, affinities or selectivities. Preferably, the antigen-binding fragment contains all 6 CDR domains of the antibody. Antigen-binding fragments of an antibody can be part of or comprise a single polypeptide chain (e.g., scFv), or can be two or more polypeptide chains (each having an amino terminus and a carboxyl terminus) (e.g., diabodies, Fab fragment, F(ab') ₂ fragment, etc.) or contain two or more polypeptide chains.

Examples of antigen-binding fragments encompassed by the invention include (a) Fab' or Fab fragments, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (b) F(ab') ₂ fragments, consisting of two Bivalent fragments of Fab fragments connected by disulfide bonds in the hinge domain; (c) Fd fragments composed of VH region and CH1 domain; (d) Fv fragments composed of VL region and VH region of one arm of the antibody; (e) ) Single chain antibody (single chain Fv, scFv), a recombinant protein formed by connecting the antibodies VH and VL through a linking peptide using genetic engineering methods; (f) dAb fragment (Ward et al., Nature, 341, 544-546 ( 1989)), which essentially consist of VH regions and are also called domain antibodies (Holt et al., Trends Biotechnol., 2i(ll):484-90); (g) Camel or Nanobodies (Revets et al., Expert Opin Biol Ther., 5(l):111-24) and (h) isolated complementarity determining regions (CDRs).

In some embodiments, the antibodies and antigen-binding fragments thereof of the invention are single chain antibodies. In some embodiments, the invention provides a single chain Fv (scFv), wherein the heavy and light chains in the Fv of an antibody of the invention are linked by a flexible peptide (typically about 10, 12, 15 or more amino acids residues) are linked into a single peptide chain. Methods of producing such antibodies are described, for example, in US 4,946,778; Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore ed., Springer-Verlag, New York, pp. 269-315 (1994); Bird et al., Science, 242, 423-426 (1988); Huston et al., PNAS USA 85, 5879-5883 (1988) and McCafferty et al., Nature, 348, 552-554 (1990). A single chain antibody is monovalent if only a single VH and VL is used, bivalent if two VH and VL are used, or multivalent if more than two VH and VL are used.

In some embodiments, the antibodies and antigen-binding fragments thereof of the invention are chimeric antibodies. The term "chimeric antibody" means that a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence of an antibody from a specific species or belonging to a specific antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody from another species or belonging to a specific antibody class or subclass. Corresponding sequences of antibodies from one species or belonging to another antibody class or subclass and fragments of such antibodies are identical or homologous as long as they exhibit the desired biological activity. The invention provides variable region antigen-binding sequences from human antibodies. Therefore, chimeric antibodies of primary concern herein include antibodies that have one or more human antigen-binding sequences (eg, CDRs) and contain one or more sequences from a non-human antibody, such as FR or C region sequences. Furthermore, chimeric antibodies as described herein are antibodies that comprise a human variable region antigen-binding sequence from one antibody class or subclass and another sequence from another antibody class or subclass, such as an FR or C region sequence.

In some embodiments, the antibodies and antigen-binding fragments thereof of the invention are humanized antibodies. The term "humanized antibody" refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequence.

In some embodiments, the antibodies and antigen-binding fragments thereof of the invention are human antibodies or fully human antibodies. The term "human antibody" or "fully human antibody" ("humAb" or "HuMab") refers to antibodies having variable and constant domains derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., introduced by random or site-specific mutagenesis in vitro or during gene rearrangement or by somatic mutation in vivo mutation).

Variant antibodies are also included within the scope of the invention. Therefore, variants of the sequences listed in the application are also included within the scope of the present invention. Other variants of antibody sequences with improved affinity can be obtained using methods known in the art and are included within the scope of the present invention. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of nucleotide sequences can be used to improve the translation efficiency of expression systems in antibody production.

Such variant antibody sequences have 70% or more (eg, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence homology to sequences listed in the application. Such sequence homology is calculated relative to the full length of a reference sequence (ie, the sequence listed in the application).

The numbering of amino acid residues in the present invention is based on (the internationalImMunoGeneTics information system)/> Or Kabat, EA, Wu, TT, Perry, HM, Gottesmann, KS & Foeller, C., (1991), Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242, U.S. Department of Health and Human Services; Chothia, Conducted by C. & Lesk, AM, (1987), Canonical structures For The Hypervariable domains Of Immunoglobulins., J. Mol. Biol., 196, 901-917. Unless otherwise specified, the numbering of amino acid residues in the present invention is based on the Kabat numbering system.

An antibody or antigen-binding fragment thereof "specifically" binds a region (i.e., an epitope) of another molecule when it binds to that epitope more frequently, more rapidly, and for a longer duration than another epitope. time and/or react or bind with greater affinity or avidity. In some embodiments, the antibodies of the invention, or antigen-binding fragments thereof, bind human amyloid, particularly toxic forms thereof, with an affinity of at least 10 ^-7 M, e.g., 10 ^-8 M, 10 ^-9 M, 10 ^-10 M , 10 ^-11 M or higher. Preferably, the antibody or antigen-binding fragment thereof binds under physiological conditions (eg, in vivo). Thus, specifically binding to amyloid, in particular its toxic form, refers to the ability of the antibody or antigen-binding fragment thereof to bind to amyloid, in particular its toxic form, with the above specificity and/or under such conditions. Methods suitable for determining such binding are known in the art.

In the context of an antibody binding to a specified antigen, the term "binds" generally refers to binding with an affinity corresponding to a K of about 10 ^-6 M or less than that of the _antibody pair with other than the _specified antigen or a closely related antigen. The binding affinity of non-specific antigens (eg, BSA, casein) is at least 10 times lower, such as at least 100 times lower, at least 1,000 times lower.

As used herein, the term "k _d " (sec-1 or 1/s) refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is also called the k _off value.

As used herein, the term " _ka " (M-1x sec-1 or 1/Msec) refers to the binding rate constant of a particular antibody-antigen interaction.

As used herein, the term " _KD " (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing _kd by _ka .

As used herein, the term " _KA " (M-1 or 1/M) refers to the binding equilibrium constant for a particular antibody-antigen interaction and is obtained by dividing _ka by _kd .

Taking advantage of the fact that single amino acid changes in CDR residues can result in loss of functional binding (Rudikoff, S. et al., (1982), Single Amino Acid Substitution Altering Antigen-binding Specificity, Proc. Natl. Acad. Sci. (USA)) 79 (6):1979-1983), alternative functional CDR sequences can be systematically identified. In a preferred method for obtaining such variant CDRs, the polynucleotide encoding the CDRs is mutagenized (eg via random mutagenesis or by site-directed methods) to generate CDRs with substituted amino acid residues. By comparing the identity of the relevant residues in the original (functional) CDR sequence with the identity of the substituted (non-functional) variant CDR sequence, the BLOSUM62.iij substitution score for the substitution can be identified. The BLOSUM system provides an amino acid substitution matrix created by analyzing sequence databases for confident alignment (Eddy, S.R., (2004), Where Did The BLOSUM62 AlignmentScore Matrix Come From?, Nature Biotech., 22(8):1035- 1036; Henikoff, J.G., (1992), Amino acid substitution matrices from protein blocks), Proc. Natl. Acad. Sci. (USA), 89: 10915-10919; Karlin, S. et al., (1990), Methods For Assessing TheStatistical Significance Of Molecular Sequence Features By Using GeneralScoring Schemes),PNAS,87:2264-2268;Altschul,S.F.,(1991),Amino AcidSubstitution Matrices From An Information Theoretic Perspective,J.Mol.Biol.,219,555-565 . Currently, the most advanced BLOSUM database is the BLOSUM62 database (BLOSUM62.iij). Table 1 presents the BLOSUM62.iij substitution scores (the higher the score the more conservative the substitution is, and therefore the more likely it is that the substitution will not affect function). For example, if the antigen-binding fragment containing the resulting CDR is unable to bind to PD-L1, the BLOSUM62.iij substitution score is considered insufficiently conserved, and new candidate substitutions with higher substitution scores are selected and generated. So, for example, if the original residue was glutamic acid (E) and the non-functional substituted residue was histidine (H), the BLOSUM62.iij substitution score would be 0, and a more conservative change (e.g. to Aspartame acid, asparagine, glutamine or lysine) are preferred.

The present invention therefore contemplates the use of random mutagenesis for identifying improved CDRs. In the context of the present invention, conservative substitutions may be defined by substitutions within an amino acid class that are reflected in one or more of the following three tables:

Conservatively substituted amino acid residue categories:

Alternative Conservative Amino Acid Residue Substitution Categories:

Classification of physical and functional alternatives to amino acid residues:

More conservative substitution groupings include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine Aminoamide.

In some embodiments, the hydrophilic amino acid is selected from the group consisting of Arg, Asn, Asp, Gln, Glu, His, Tyr, and Lys.

Additional amino acid groups can also be formulated using principles described, for example, in Creighton, (1984), Proteins: Structure and Molecular Properties, W.H. Freeman and Company).

Thus, the sequence of the CDR variants of the included antibody or antigen-binding fragment thereof may differ from the sequence of the CDR of the parent antibody by substitution; for example, substitution of 4, 3, 2 or 1 amino acid residues. According to embodiments of the present invention, amino acids in the CDR region may be substituted with conservative substitutions, as defined in the above 3 tables.

"Homology" or "sequence identity" refers to the percentage of residues of a polynucleotide or polypeptide sequence variant that are identical to a non-variant sequence after sequence alignment and introduction of gaps. In specific embodiments, polynucleotide and polypeptide variants are at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least About 98%, or at least about 99% polynucleotide or polypeptide homology.

Such variant polypeptide sequences have 70% or more (ie, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity with the sequences listed in the application. In other embodiments, the invention provides polypeptide fragments comprising contiguous stretches of various lengths of the amino acid sequences disclosed herein. For example, where applicable, the peptide sequences provided by the invention comprise at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150 or more consecutive sequences of one or more sequences disclosed in the invention. Amino acid peptides and all intermediate length peptides in between.

The antibodies of the invention may be monoclonal antibodies produced by recombinant DNA.

In some embodiments, the antibodies of the invention are full-length antibodies, preferably IgG1-4 antibodies or IgM antibodies. In other embodiments, the antibodies of the invention are antibody antigen-binding fragments or single chain antibodies.

The subunit structure and three-dimensional structure of the constant regions of various immunoglobulin classes are known. As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (mostly amino-terminal) constant region of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is the amino terminus of the hinge region of the immunoglobulin heavy chain molecule. As used herein, the term "CH2 domain" includes a portion of a heavy chain molecule that ranges, for example, from approximately residue 244 to residue 360 of an antibody, using the conventional numbering scheme (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983). The CH2 domain is unique in that it is not closely paired with another domain .In contrast, two N-linked branched sugar chains are inserted between the two CH2 domains of the intact native IgG molecule. It has been documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule. and contains approximately 108 residues. As used herein, the term "hinge region" includes that portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region contains approximately 25 residues and is flexible, allowing The two N-terminal antigen-binding regions move independently. The hinge region can be divided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

The complement binding site of an antibody refers to the position in the antibody structure that binds to the complement component. For IgM, the complement binding site of the antibody is located in CH3. For IgG1-3, the complement binding site of the antibody is located in CH2. At the same time, the hinge region of the antibody also has an important impact on complement binding. IgG4 cannot activate the classical complement pathway, but can activate complement via the alternative pathway or the lectin pathway. In some embodiments, when the antibody is IgG1, such as antibody A16 of the present invention, the CH1 region of the antibody refers to positions 120-216 of SEQ ID NO: 10, the CH2 region refers to positions 231-336, and CH3 The region refers to positions 337-441, and its complement binding site is located in CH2, that is, positions 231-336. Based on the complement binding site information of the A16 antibody of the IgG1 class, those skilled in the art can easily determine the complement binding site information of other classes of antibodies, such as IgG2-3 and IgM.

Fc receptors include IgG-FcR (FcγR), IgA-FcR (FcαR (CD89)), medium-affinity receptors, distributed on phagocytes and certain T and B cell subpopulations, mediating phagocytosis of phagocytes and superoxide production. , release inflammatory mediators and ADCC effect) and IgE-FcR (FcεR (CD23)), among which Fcγ receptor (sometimes referred to as Fcγ receptor, FcγR or FcgR in this specification) refers to a receptor that can be combined with IgG1, IgG2, IgG3, and IgG4 The receptor to which the Fc region of a cloned antibody binds essentially also refers to any member of the protein family encoded by the Fcγ receptor gene. For humans, this family includes FcγRI (CD64, high-affinity receptor, expressed on monocytes and macrophages), FcγRII (CD32, medium-affinity receptor, widely distributed) and FcγRIII (CD16, low-affinity receptor, distributed on NK cells, macrophages).

The Fc receptor binding site refers to the site in the Fc fragment of the antibody that binds to the Fc receptor. For human IgG antibodies, the Fc receptor binding site is located within the CH2 and/or CH3 domains. In some embodiments, when the antibody is IgG1, such as antibody A16 of the present invention, the CH1 region of the antibody refers to positions 120-216 of SEQ ID NO: 10, the CH2 region refers to positions 231-336, and CH3 The region refers to positions 337-441, and its Fc binding site is located in CH2 and/or CH3, that is, positions 231-441. Based on the Fc receptor binding site information of the IgG class, those skilled in the art can easily determine the Fc receptor binding site information of other classes of antibodies, such as IgA and IgE.

In some embodiments, the complement, such as Clq and/or Fc receptor binding activity of the antibodies of the invention is reduced or completely eliminated. In some embodiments, the complement, such as Clq and/or Fc receptor binding activity of an antibody of the invention is reduced by at least 5%, such as by at least 10-100%, such as at least 10%, 15%, 20%, 25%, 30 %, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

There is no limitation on the method of reducing or eliminating the complement, such as C1q and/or Fc receptor binding activity of the antibody of the present invention. For example, proteases can be used to remove the complete Fc fragment of the antibody, such as ficin, papain, pepsin and other proteases for digestion.

For example, the complement of the antibody such as C1q and/or the binding site of the Fc receptor can be deleted through recombinant DNA technology, for example, the CH3 region of the IgM antibody or the CH2 region and/or CH3 region of the IgG1-3 antibody can be deleted, and the complement such as C1q can be deleted. The entire region where the binding site of Fc receptor and/or Fc receptor is located, or only part of the region can be deleted, provided that the binding activity of complement such as C1q and/or Fc receptor of the antibody of the present invention is reduced or eliminated, for example, it can be deleted At least 5%, such as 10-100%, of the CH3 region of an IgM antibody or the CH2 region and/or CH3 region of an IgG1-3 antibody, such as at least 10%, 15%, 20%, 25%, 30%, 35%, 40 %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, such as deletion of at least 1 amino acid residue, such as deletion of 2-106 amino acid residues, for example, at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 or more amino acid residues, until the entire CH3 region of the IgM antibody or the CH2 region and/or CH3 region of the IgG1-3 antibody is deleted. The deletion can be carried out continuously along the N-terminal or C-terminal of the region where the binding site of complement such as C1q and/or Fc receptor is located or from the middle region, or it can be carried out at intervals in the region, for example, 2 of the region where the complement is located can be deleted at the same time. , 3, 4, 5 or more segments, each segment has the same or different length, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20 or more amino acid residues. In some embodiments, the deletion preferably comprises one or more key amino acid residues, such as 1, 2, 3, 4, 5 or More amino acid residues. For human antibody complement binding activity, the key amino acid residues for human IgG1 are L234, L235, D270, E318, K320, K322, K326, P329, P331, E333 (see, e.g., Michaelsen et al., Structural Difference in the ComplementActivation Site of Human IgG1 and IgG3, Scandinavian Journal of Immunology 70,553–564). For human antibody Fc receptor binding activity, the key amino acid residues refer to N297, L234, L235, P331, and G237 for human IgG1, and refer to V234, G237, P238, H268, V309, A330, and P331 for human IgG2. . Since human IgG subclasses display more than 95% amino acid sequence homology in the CH2 and CH3 regions and share extensive structural similarities, those skilled in the art can easily determine the key amino acids of IgG2 and IgG3 from the key amino acid residue information of IgG1 Residue information. Similarly, based on the above key amino acid residue information of IgG1-3, those skilled in the art can also easily determine the corresponding key amino acid residue information of IgM and other antibody types or antibody subtypes, as well as the relevant key amino acid residues of antibodies derived from other species. Residue information.

In some embodiments, the deletion is to delete from the C-terminus of the intact antibody a segment that contains part or all of the binding sites for the antibody's complement, such as C1q and/or Fc receptors, in which case the complement is e.g. All the antibody segments after the binding site of C1q and/or Fc receptors (from N-terminus to C-terminus) are deleted. For example, for IgM antibodies, the CH4 domain will be deleted at the same time. For IgG1-3 antibodies, CH2 and/or CH3 Domains will be deleted at the same time, and the binding site sequence of the antibody's complement, such as C1q and/or Fc receptor, will be deleted at least 5%, such as 10-100%, such as at least 10%, 15%, 20%, 25 %, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, e.g. simultaneously missing at least 1 amino acid residue, for example, 2-106 amino acid residues are deleted, for example, at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50 are deleted , 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 or more amino acid residues, until the CH3 region of the IgM antibody or the CH2 region and/or CH3 region of the IgG1-3 antibody is deleted All, provided that the complement, such as C1q and/or Fc receptor binding activity of the antibody of the invention is reduced or eliminated. In a further embodiment, the deletion is to delete from the C-terminus of the intact antibody a segment comprising all binding sites for the antibody's complement, such as C1q and/or Fc receptors, and part or all of the other Fc preceding this binding site. segment (from N-terminus to C-terminus), for example, for IgM antibodies, part or all of the CH2 and CH1 domains will also be deleted simultaneously, and for IgG1-3 antibodies, part or all of the CH1 domain will also be deleted simultaneously, e.g. At least 5%, such as 10-100%, of the CH2 and/or CH1 domains may be deleted simultaneously, such as at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% ,55%,60%,65%,70%,75%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, for example, at least 1 amino acid residue is deleted at the same time, for example, 2-217 amino acid residues are deleted, For example, at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 are missing , 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210 or more amino acid residues, until the entire Fc fragment of the IgM or IgG1-3 antibody is deleted.

In some embodiments, the complement of the antibody of the invention, such as C1q and/or Fc receptor, can be reduced or eliminated by replacing part or all of the amino acid residues in the binding site of the antibody's complement, such as C1q and/or Fc receptor. body binding activity. For example, at least 5%, e.g., 10-100%, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, of the binding sites of the antibody's complement, such as Clq and/or Fc receptors, may be replaced. 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid residues, for example, replacing at least 1 amino acid residue, For example, replace 2-106 amino acid residues, for example, replace at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 , 70, 75, 80, 85, 90, 95, 100, 105 or more amino acid residues, provided that the complement, such as C1q and/or Fc receptor binding activity of the antibody of the present invention is reduced or eliminated. In a further embodiment, the substitution is located at one or more key amino acid residues, such as 1, 2, 3, 4, 5, in the binding site of the antibody's complement, such as Clq and/or Fc receptors. or more key amino acid residues, the key amino acid residues being as defined above. In some embodiments, the substitution of amino acid residues involves substitution with a different type of amino acid residue, for example, replacing the key amino acid residue with a non-conservative amino acid residue as described above to reduce or eliminate the activity of the antibody of the invention. Binding activity of complement such as C1q and/or Fc receptors. In some embodiments, the substitution is located outside the binding site of the antibody complement, such as C1q, and/or the Fc receptor, preferably within the Fc fragment of the antibody, provided that the complement of the antibody of the invention, such as C1q and/or the Fc receptor binding activity is reduced or eliminated.

In some embodiments, one or more amino acid residues can be inserted into the Fc fragment of the antibody of the invention by recombinant DNA technology to reduce or eliminate the complement, such as C1q and/or Fc receptor binding activity of the antibody of the invention, for example at least 2 amino acid residues, for example 2-150 amino acid residues, for example at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 , 60, 65, 70, 75, 80 or more amino acid residues. In some embodiments, the insertion is within the binding site of the antibody's complement, such as Clq and/or Fc receptors. In some embodiments, the insertion is outside the binding site of the antibody's complement, such as Clq and/or Fc receptors. In some embodiments, the insertion is located within and outside the binding site of the antibody's complement, such as Clq and/or Fc receptors. In some embodiments, the plurality of amino acid residues is a contiguous stretch of amino acids. In some embodiments, the plurality of amino acid residues are located in a stretch of contiguous amino acids. In some embodiments, the plurality of amino acid residues are separated by a single amino acid residue, i.e., a combination of a plurality of separated single amino acid substitutions separated by at least one amino acid residue. groups, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more amino acid residues. In some embodiments, some of the plurality of amino acid residues are located in one or more stretches of contiguous amino acids, while other residues are single amino acid residues that are separated. In some embodiments, the insertion is located at one or more critical amino acid residues, such as 1, 2, 3, 4, 5 or At more key amino acid residues, the key amino acid residues are as defined above. Regardless of the site of insertion, it is provided that the complement, eg, Clq and/or Fc receptor binding activity of the antibody of the invention is reduced or eliminated.

In some embodiments, one or more combinations of deletions, substitutions and/or insertions of amino acid residues of the above-mentioned antibodies can be used to reduce or eliminate the complement, such as C1q and/or Fc receptor-binding activity of the antibodies of the invention. .

In some embodiments, reducing or eliminating the complement, e.g., C1q and/or Fc receptor binding activity of an antibody of the invention can be achieved by providing inhibition of complement, e.g., C1q and/or Fc receptor binding activity concurrently with the anti-amyloid antibody. Examples of such inhibitors are substances that compete with complement such as C1q and/or Fc receptors for binding to antibodies, such as small molecule compounds, specific anti-C1q antibodies, specific anti-Fc receptor antibodies or antigen-binding fragments thereof, Such as ANX005, TNT009, Cinryze, OMS721, AMY101, APL2, Mirococept, AMY201, CLG561, Lampalizumab, ACH-4471, Bikaciomab, H17, S77, CB2782, BI-1206, AFM24, AFM13, anti-CD64 antibody, anti-CD32 antibody, anti- CD16 antibodies and the like and combinations of one or more thereof, such as combinations of 2, 3, 4, 5 or more, preferably anti-FcγR antibodies are disclosed in WO94/10332, US4,954,617 and CN200710085297.6 (each of which The full text of is incorporated herein by reference in its entirety).

In some embodiments, the antibodies of the invention are monovalent antibodies, preferably monovalent antibodies with a hinge region deletion as described in WO 2007059782, which is incorporated herein by reference in its entirety. Accordingly, in some embodiments, the antibody is a monovalent antibody, wherein the antibody is constructed by: i) providing a nucleic acid construct encoding the light chain of the monovalent antibody, the construct comprising a VL encoding the selected antigen-specific antibody The nucleotide sequence of the region and the nucleotide sequence encoding the constant CL region of an Ig, wherein said nucleotide sequence encoding the VL region of the selected antigen-specific antibody and said nucleotide sequence encoding the CL region of the Ig are operably linked together and wherein, in the case of the IgG1 subtype, the nucleotide sequence encoding the CL region has been modified such that in the presence of polyclonal human IgG or when administered to an animal or human, the CL region Not containing any amino acid capable of forming disulfide bonds or covalent bonds with other peptides comprising the consensus amino acid sequence of the CL region; ii) providing a nucleic acid construct encoding the heavy chain of a monovalent antibody, said construct comprising a nucleic acid construct encoding a selected antigen The nucleotide sequence of the VH region of the specific antibody and the nucleotide sequence encoding the constant CH region of human Ig, wherein the nucleotide sequence encoding the CH region has been modified such that in the presence or when polyclonal human IgG When administered to animals, the regions corresponding to the hinge region and (as required by Ig subtype) other regions of the CH region (such as the CH3 region) do not contain other peptides involved in the formation of the consensus amino acid sequence of the CH region of a human Ig. Any amino acid residue of a disulfide bond or a covalent or stable non-covalent inter-heavy chain linkage in which the nucleotide sequence encoding the VH region of the selected antigen-specific antibody and the CH region encoding the Ig The nucleotide sequences described above are effectively linked together; iii) providing a cell expression system for producing monovalent antibodies; iv) by co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii) to produce the monovalent antibodies.

Similarly, in some embodiments, the antibodies of the invention are monovalent antibodies comprising:

(i) a variable region of an antibody of the invention as described herein or an antigen-binding portion of said domain, and

(ii) The CH region of an immunoglobulin or a domain thereof comprising a CH2 and CH3 domain, wherein the CH region or a domain thereof has been modified such that it corresponds to the hinge region and (if the immunoglobulin is not of the IgG4 subtype ) domains of other domains of the CH region (e.g. CH3 domain) do not contain any amino acid residues capable of forming disulfide bonds with the same CH region or with the same CH region in the presence of polyclonal human IgG Other covalent or stable non-covalent inter-heavy chain bonds.

In some additional embodiments, the heavy chain of the monovalent antibody is modified such that the entire hinge region is deleted.

In additional embodiments, the sequence of the monovalent antibody is modified such that it does not contain any acceptor sites for N-linked glycosylation.

In some embodiments, the antibody of the invention is A16 as described below or any antibody known in the art directed against amyloid, e.g., directed against the monomeric form, the oligomeric form, the profibrillar form, and/or the fibrillar form Known antibodies to amyloid, such as Aducanumab, Crenezumab, BAN2401, bapineuzumab (anti-Aβ1-5), solanezumab (anti-Aβ13-28), gantenerumab (anti-Aβ1- 11), ponezumab (anti-Aβ C-terminal), Donanemab (anti-pyroglutamate Aβ), and anti-tau antibodies Gosuranemab, Tilavonemab, Semorinemab, Zagotenemab, PRX002 (anti-α-synuclein antibody), etc. In some embodiments, these antibodies are active in binding complement, such as Clq and/or Fc receptors. In some embodiments, the antibodies of the invention are not antigen-binding fragments of these known antibodies. In some embodiments, after the present invention modifies the prior art antibody as described above, it no longer has the activity of binding complement such as C1q and/or Fc receptor or binding complement such as C1q and/or Fc receptor. Body activity is reduced.

The antibody of the present invention acts on human amyloid, especially its toxic form, while having reduced or missing complement such as C1q and/or Fc receptor binding activity, and therefore does not activate the complement and/or Fc receptor system or activate complement. and/or the Fc receptor system is to a low degree and cannot lead to the formation of amyloid-antibody-complement complexes, preventing excessive phagocytosis of synapses by microglia in a mechanism similar to physiological synaptic pruning. Due to the reduced or missing Fc receptor binding activity, it does not promote the release of inflammatory factors by microglia and avoids the incorrect phagocytosis of synapses by microglia. Based on this, the present invention reveals the reasons for the failure of immunotherapy for various amyloid diseases, especially neurodegenerative diseases, lays a theoretical foundation for the design and development of immune preparations for such diseases, and provides a method for the treatment of such diseases. A promising preparation. As far as the inventor is aware, as of the filing of this invention, the antibodies against amyloid used in clinical trials for the treatment of neurodegenerative diseases such as AD in this field are all intact antibodies, although their corresponding patent applications are often The use of antigen-binding fragments will also be mentioned. As demonstrated for the first time by the present invention, such antibodies all have complement binding activity such as C1q and Fc receptor binding activity. When applied to the human body, they lead to the activation of the human complement system and the formation of amyloid-antibody-complement complexes. Microglia use a mechanism similar to physiological synapse pruning to excessively phagocytose synapses, and at the same time mediate microglia to release inflammatory factors and mistakenly phagocytose synapses, leading to the failure of immunotherapy for neurodegenerative diseases. The present invention overcomes the shortcomings of the prior art by reducing or eliminating the complement, such as C1q binding activity, and Fc receptor binding activity of such antibodies.

Antibodies of the invention are produced by any technique known in the art, such as, but not limited to, any chemical, biological, genetic or enzymatic technique, alone or in combination. In general, knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce such antibodies by standard techniques for producing polypeptides. For example, these antibodies can be synthesized using well-known solid phase methods, preferably using commercially available peptide synthesis equipment (eg, equipment manufactured by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, the antibodies of the invention can be synthesized by recombinant DNA techniques well known in the art. For example, antibodies can be obtained as DNA expression products after incorporating DNA sequences encoding the antibodies into expression vectors and introducing these vectors into suitable eukaryotic or prokaryotic hosts for expressing the desired antibodies, which can then be obtained from the host using known techniques. Isolate antibodies.

Antibodies and antigen-binding fragments thereof of the invention may be modified by including any "suitable" number of modified amino acids and/or in combination with coupling substituents. In this context, "suitable" is generally determined by the ability to at least substantially retain amyloid selectivity, especially toxic forms thereof, and/or amyloid specificity, especially toxic forms thereof, relative to the non-derivatized parent antibody. The inclusion of one or more modified amino acids may be advantageous, for example, to increase the serum half-life of the polypeptide, reduce the antigenicity of the polypeptide, or increase the storage stability of the polypeptide. Modification of one or more amino acids, e.g., concurrently with or after translation during recombinant production (e.g., N-linked sugars on the N-X-S/T motif during expression in mammalian cells tylation), or modification by synthetic means. Non-limiting examples of modified amino acids include glycosylated amino acids, sulfated amino acids, prenylated (e.g., farnesylated, geranyl-geranylated) amino acids, acetylated amino acids , acylated amino acids, PEGylated amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, etc. References to amino acid modifications are common in the art, see, for example, Walker, (1998), Protein Protocols On CD-Rom, Humana Press, Totowa, New Jersey. The modified amino acid may, for example, be selected from glycosylated amino acids, pegylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids coupled to lipid moieties, or amino acids coupled to lipid moieties. Amino acids linked to organic derivatizing agents.

The antibodies and antigen-binding fragments thereof of the invention may also be chemically modified by covalent coupling to polymers to increase their circulating half-life. Exemplary polymers and methods of linking them to peptides are provided in, for example, US 4,766,106; US 4,179,337; US 4,495,285 and US 4,609,546. Additional exemplary polymers include polyoxyethylated polyols and polyethylene glycols (PEG) (e.g., having a molecular weight between about 1,000 and 40,000D, such as between about 2,000 and 20,000D, such as about 3,000 and 12,000D D's PEG).

The antibodies and antigen-binding fragments thereof of the present invention also include mutants formed by one or several amino acid substitutions, deletions, or additions of the above-mentioned amyloid antibody.

In a further aspect, the invention relates to an expression vector encoding one or more polypeptide chains of an antibody of the invention or an antigen-binding fragment thereof. Such expression vectors can be used to recombinantly produce the antibodies and antigen-binding fragments thereof of the invention.

In the present invention, the expression vector can be any suitable DNA or RNA vector, including chromosomal vectors, non-chromosomal vectors and synthetic nucleic acid vectors (nucleic acid sequences containing a set of suitable expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In some embodiments, nucleic acids encoding antibodies of the invention are contained in naked DNA or RNA vectors, including, for example, linear expression elements (as described in, for example, Sykes and Johnston, Nat Biotech, 12, 355-59 (1997)), compact Nucleic acid vectors (as described in eg US 6,077,835 and/or WO 00/70087), plasmid vectors (eg pBR322, pUC 19/18 or pUC 118/119), minimal size nucleic acid vectors (as described in eg Schakowski et al., MoI Ther, 3, 793-800 (2001)), or as a precipitating nucleic acid vector construct, such as a CaPO ₄ precipitating construct (as described, for example, in WO 00/46147; Benvenisty and Reshef, PNAS USA 83, 9551-55 ( 1986); Wigler et al., Cell, 14, 725 (1978) and Coraro and Pearson, Somatic CellGenetics, 2, 603 (1981)). Such nucleic acid vectors and their use are well known in the art (see, eg, US 5,589,466 and US 5,973,972).

In some embodiments, the vector is suitable for expression of the antibodies of the invention or antigen-binding fragments thereof in bacterial cells. Examples of such vectors include, for example, BlueScript (Stratagene), pIN vector (Van Heeke & Schuster, J Biol Chem, 264, 5503-5509 (1989)), pET vector (Novagen, Madison, Wisconsin), and the like.

The expression vector may also be a vector suitable for expression in yeast systems. Any vector suitable for expression in yeast systems may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience, New York (1987); Grant et al., Methods in Enzymol, 153, 516-544 (1987); Mattanovich, D. et al., Methods Mol. Biol., 824, 329-358 (2012); Celik, E. et al. Human, Biotechnol. Adv., 30(5), 1108-1118(2012); Li, P. et al., Appl. Biochem. Biotechnol., 142(2), 105-124(2007); E. et al., Appl. Microbiol. Biotechnol., 77(3), 513-523 (2007); van der Vaart, JM, Methods Mol. Biol., 178, 359-366 (2002) and Holliger, P., Methods Mol. Biol., 178, 349-357 (2002)).

In the expression vector of the present invention, the nucleic acid encoding the antibody of the present invention may contain or be combined with any suitable promoter, enhancer and other elements that facilitate expression. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter/enhancer and RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), efficient poly(A) termination sequences, Generate the origin of replication of the plasmid, an antibiotic resistance gene as a selectable marker, and/or a convenient cloning site (eg, a polylinker) in E. coli. The nucleic acid may also contain an inducible promoter as opposed to a constitutive promoter (eg, CMV IE).

In a further aspect, the invention relates to recombinant eukaryotic or prokaryotic host cells (eg, transfectomas) that produce an antibody of the invention or an antigen-binding fragment thereof or a bispecific molecule of the invention. Examples of host cells include yeast, bacteria, and mammalian cells (such as CHO or HEK cells). For example, in some embodiments, the invention provides a cell comprising a nucleic acid stably integrated into the cell's genome comprising a nucleic acid sequence encoding an antibody of the invention or an antigen-binding fragment thereof. In other embodiments, the invention provides cells comprising a non-integrating nucleic acid (such as a plasmid, cosmid, phagemid or linear expression element) comprising a sequence encoding an antibody of the invention or an antigen-binding fragment thereof.

The antibodies and antigen-binding fragments thereof of the invention can be produced in different cell lines, such as human cell lines, non-human mammalian cell lines and insect cell lines, such as CHO cell lines, HEK cell lines, BHK-21 cell lines, murine Cell lines (such as myeloma cell lines), fibrosarcoma cell lines, PER.C6 cell lines, HKB-11 cell lines, CAP cell lines, and HuH-7 human cell lines (Dumont et al., 2015, Crit Rev Biotechnol., Sep .18,1-13., the contents of which are incorporated herein by reference).

The antibodies of the invention are suitably separated from the culture medium by conventional immunoglobulin purification methods, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

The invention further relates to compositions comprising, consisting or consisting essentially of an antibody of the invention.

As used herein, with respect to a composition, "consisting essentially of" means that at least one antibody of the invention, as described above, is the only biologically active therapeutic agent in the composition or Reagents.

In one embodiment, the composition of the invention is a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient, diluent or carrier.

The invention further relates to a medicament comprising, consisting or consisting essentially of an antibody of the invention and further comprising a pharmaceutically acceptable excipient, diluent or carrier.

The term "pharmaceutically acceptable carrier" refers to an excipient that does not produce adverse, allergic or other adverse reactions when administered to animals, preferably humans. This includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. For human administration, preparations should meet the sterility, pyrogenicity, general safety and purity standards required by regulatory agencies (such as the Office of the FDA or EMA).

In some embodiments, the glycosylation of the antibodies of the invention is modified. For example, aglycosylated antibodies can be prepared (i.e., antibodies lack glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen or alter the ADCC activity of the antibody. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. This aglycosylation increases the affinity of the antibody for the antigen. This approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al. (incorporated herein by reference). Additionally or alternatively, antibodies with altered glycosylation patterns can be produced. Such carbohydrate modification can be accomplished, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which recombinant antibodies of the invention are expressed, thereby producing antibodies with altered glycosylation.

Recombinant expression vectors can be introduced into host cells to produce transformed host cells. The terms "transformed with," "transfected with," "transformed," "transduced" and "transfection" are intended to include the transfer of a nucleic acid, such as a vector, by one of the many possible techniques known in the art. ) introduced into cells. As used herein, the term "transformed host cell" or "transduced host cell" is also intended to include cells that have been transformed with the recombinant expression vector of the invention. Prokaryotic cells can be transformed with nucleic acids, for example, by electroporation or calcium chloride-mediated transformation. For example, nucleic acids can be introduced into mammalian cells by conventional techniques such as calcium phosphate or calcium chloride coprecipitation, DEAE-dextran mediated transfection, lipofection, electroporation, or microinjection. Suitable methods for transforming and transfecting host cells can be found in Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York and other laboratory textbooks .

Although the nucleotide sequence defined above is DNA, in alternative embodiments of the invention the nucleotide sequence may be RNA. The corresponding RNA sequences to the DNA sequences described herein are therefore encompassed. The skilled person will understand how to derive an RNA sequence encoding the same protein/polypeptide product into the DNA sequence shown above. "T" should be replaced with "U".

As used herein, the term "nucleic acid sequence" or "nucleic acid molecule" or "polynucleotide", "polynucleotide sequence" or "nucleotide sequence" refers to a naturally occurring sequence of bases, sugars, and sugars (backbone). A sequence of linked nucleoside or nucleotide monomers. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid, polynucleotide or nucleotide sequence of the invention may be a deoxyribonucleic acid sequence (DNA) or a ribonucleic acid sequence (RNA), and may include naturally occurring bases, including adenine, guanine, cytosine, thoracic acid, Glycosides and uracil. The sequence may also contain modified bases. Examples of such modified bases include aza and deazaadenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. A nucleic acid, polynucleotide or nucleotide sequence may be double-stranded or single-stranded. Nucleic acids, polynucleotides or nucleotide sequences may be wholly or partially synthetic or recombinant.

Although the antibodies, nucleic acids, vectors or cells of the invention are effective against disease when used alone, additional therapeutic agents can be used in combination with the antibodies, nucleic acids, vectors or cells of the invention to combat disease. Accordingly, in another embodiment of the invention, at least one additional or additional therapeutic agent (eg, other neurodegenerative disease treatment agents) may be administered to the subject. Accordingly, the antibodies or antigen-binding fragments thereof, nucleic acids, vectors, or cells of the invention and other therapeutic agents (eg, other neurodegenerative disease treatments) of the invention can be administered to a subject. Accordingly, compositions or pharmaceutical compositions of the invention may comprise other active or therapeutic agents, as well as antibodies or antigen-binding fragments thereof, nucleic acids, vectors and/or cells of the invention. However, it is to be understood that the antibodies, or antigen-binding fragments thereof, nucleic acids, vectors or cells thereof, and other therapeutic agents (eg, other neurodegenerative disease treatments) of the invention may be administered separately, for example, by separate routes of administration. Additionally, the antibodies or antigen-binding fragments thereof, nucleic acids, vectors or cells of the invention and at least one other therapeutic agent (eg, other neurodegenerative diseases) may be administered sequentially or (substantially) simultaneously. They may be administered in the same pharmaceutical preparation or drug, or they may be formulated and administered separately. For sequential administration, at least 1 minute, 10 minutes, 1 hour, 6 hours, 12 hours, 1 day, 5 days, 10 days, 2 weeks before or after administration of the antibody or antigen-binding fragment/nucleic acid/vector/cell thereof , 4 weeks or 6 weeks to administer additional therapeutic agents.

"Pharmaceutically acceptable" includes preparations that are sterile and pyrogen-free. Suitable pharmaceutical carriers, diluents and excipients are well known in the pharmaceutical arts. The carrier must be "acceptable" in the sense of being compatible with the drug and not deleterious to the recipient thereof. Typically, the carrier will be saline or an infusion medium (alternatively referred to as an infusion solution), which will be sterile and pyrogen-free; however, other acceptable carriers may be used.

The pharmaceutical compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although appropriate dosages can be determined through clinical trials.

The compositions of the present invention may be administered in a single dose or in multiple doses. In particular, the composition can be administered in a single, disposable application.

The antibodies or antigen-binding fragments thereof, nucleic acids, vectors or compositions of the invention may be administered by any parenteral route in the form of a pharmaceutical preparation containing the active ingredient. The compositions may be administered in varying dosages depending on the condition and patient to be treated, as well as the route of administration. In any case, the physician will determine the actual dosage that is most appropriate for any individual patient, and it will vary with the age, weight, and response of the particular patient.

In human therapy, the antibodies or antigen-binding fragments thereof, nucleic acids or compositions of the invention are typically administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected based on the intended route of administration and standard pharmaceutical practice. For each of the described embodiments, the antibodies, or antigen-binding fragments thereof, nucleic acid molecules, or compositions of the invention can be administered in a variety of dosage forms. Examples of such dosage forms include, but are not limited to, reconstitutable powders, elixirs, liquids, solutions, suspensions, emulsions, powders, granules, granules, microgranules, dispersible granules, cachets, inhalants, aerosols for inhalation agents, patches, particle inhalants, implants, long-acting implants, injections (including subcutaneous, intramuscular, intravenous and intradermal, preferably intravenous), infusions and combinations thereof. Typically, the cells of the invention can be administered in injection or infusion buffer. Exemplary formulations can be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition., Grennaro, A., Editor, 1995, which is incorporated herein by reference.

The antibodies or antigen-binding fragments thereof, nucleic acids or compositions of the invention may also be administered parenterally, such as intravenously, intraarterially, intraperitoneally, intrathecally, intracranially, topically, intramuscularly, bucally, subcutaneously, transdermally. , epidural, inhalation, intracardiac, intracerebroventricular, intraocular, intraspinal, nasal, sublingual, transdermal or transmucosal, or they may be administered by infusion techniques. They are best used in the form of sterile aqueous solutions, which may contain other substances, such as sufficient salt or glucose to make the solution isotonic with the blood. If necessary, the aqueous solution should be appropriately buffered (preferably pH 3 to 9). The preparation of suitable parenteral formulations under sterile conditions can be readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

Formulations may be presented in unit-dose or multi-dose containers, such as sealed ampoules, bags, and vials.

In some embodiments, the antibodies, or antigen-binding fragments, nucleic acids, or compositions thereof of the invention are used to treat and/or prevent neurodegenerative diseases in a subject, comprising administering to a subject in need thereof the treatment or prevention. An effective amount of the antibody or antigen-binding fragment thereof, nucleic acid or composition. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, amyotrophic lateral sclerosis, or spinocerebellar ataxia.

The term "treatment" means ameliorating, slowing, attenuating or reversing the progression or severity of a disease or condition, or ameliorating, slowing, attenuating or reversing one or more symptoms or side effects of such disease or condition. In the present invention, "treatment" also refers to methods for obtaining beneficial or desired clinical results, where "beneficial or desired clinical results" include but are not limited to relief of symptoms, reduction, and stabilization of symptoms or disease severity. Progression (i.e., no worsening) of a disease or condition, delay or slowing of the progression of a disease or condition, improvement or alleviation of a disease or condition, and alleviation of a disease or condition, whether partial or total, detectable or undetectable.

The term "prevention" refers to the administration of the antibodies and functional fragments thereof of the invention to prevent or hinder the development of at least one symptom of a disease or condition. The term also includes treating a subject in remission to prevent or hinder relapse.

The term "subject" refers to a warm-blooded animal, preferably a mammal (including humans, domestic and farm animals, zoo animals, sport animals or pet animals such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. ), more preferably humans. In one embodiment, the subject may be a "patient," ie, a warm-blooded animal, more preferably a human, who is waiting to receive or is receiving medical care or will be the subject of a medical procedure, or monitoring for the development of a disease. In one embodiment, the subject is an adult (eg, a subject over 18 years of age). In another embodiment, the subject is a child (eg, a subject under 18 years of age). In one embodiment, the subject is male. In another embodiment, the subject is female.

The antibodies and antigen-binding fragments thereof of the invention may further be used in diagnostic methods or as diagnostic imaging ligands. In some embodiments, the antibodies and antigen-binding fragments thereof of the invention can be labeled with radioactive labels, fluorescent labels, fluorescein-type labels, rhodamine-type labels, phycoerythrin, umbelliferone, lissamine, cyanine, Dekker Saas red, BODIPY (Invitrogen) or its analogs, horseradish peroxidase, alkaline phosphatase, β-galactosidase, acetylcholinesterase, streptavidin/biotin and avidin/biotin for labeling or modification . Such labeled or modified antibodies or antigen-binding fragments thereof can be used to detect the presence and/or concentration of amyloid in samples, respectively, for clinical diagnosis, experimental detection, etc. The experimental techniques used include but are not limited to ELISA, Dot-blot, Western-blot, chemiluminescence, electrochemiluminescence, Simoa technology, radioactive technology, etc. In some embodiments, paramagnetic labels may also be employed, and detection is preferably performed using positron emission tomography (PET) or single photon emission computed tomography (SPECT). Such paramagnetic labels include, but are not limited to, aluminum (Al), barium (Ba), calcium (Ca), cerium (Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), Holmium (Ho), iridium (Ir), lithium (Li), magnesium (Mg), manganese (Mn), molybdenum (M), neodymium (Nd), osmium (Os), oxygen (O), palladium (Pd), Platinum (Pt), rhodium (Rh), ruthenium (Ru), samarium (Sm), sodium (Na), strontium (Sr), terbium (Tb), thulium (Tm), tin (Sn), titanium (Ti), Tungsten (W) and zirconium (Zi) and in particular Co ⁺² , CR ⁺² , Cr ⁺³ , Cu ⁺² , Fe ⁺² , Fe ⁺³ , Ga ⁺³ , Mn ⁺³ , Ni ⁺² , Ti ^{+ 3.} Compounds of paramagnetic ions of V ⁺³ and V ⁺⁴ , using various positron emission tomography positron emitting metals and non-radioactive paramagnetic metal ions.

In one embodiment of the invention, the sample is a biological sample. Examples of biological samples include, but are not limited to, samples from diseased tissues, body fluids, preferably blood, more preferably serum, plasma, synovial fluid, bronchoalveolar lavage fluid, sputum, lymph fluid, ascites, urine, amniotic fluid, peritoneal fluid, cerebrospinal fluid, Tissue lysates and extracts prepared from pleural fluid, pericardial fluid, and alveolar macrophages.

In one embodiment of the invention, the term "sample" is intended to mean a sample taken from an individual prior to any analysis.

In some embodiments, the invention relates to the following:

Item 1. An antibody specifically targeting amyloid or an antigen-binding fragment thereof, characterized in that it has reduced complement and/or Fc receptor binding activity or no complement and/or Fc receptor binding activity.

Item 2. The antibody or antigen-binding fragment thereof according to item 1, wherein the amyloid protein is in its monomer, oligomer, profibrillar or fibrillar form.

Item 3. The antibody or antigen-binding fragment thereof according to Item 1 or 2, wherein the amyloid protein is selected from the group consisting of β-amyloid protein, microtubule-associated protein tau, α-synuclein, huntingtin, and pancreatic amyloid. Preferably, the amyloid protein is β-amyloid protein or microtubule-associated protein tau.

Item 4. The antibody or antigen-binding fragment thereof according to any one of items 1 to 3, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody or a fully human antibody. Preferably, the antibody is selected from The group consisting of IgG, IgM, IgA, IgD and IgE and their subtypes.

Item 5. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 4, wherein the antibody is IgG, IgM or a subtype thereof, wherein the complement and/or Fc receptor binding activity of the antibody is determined by deletion All or part of the complement and/or Fc receptor-binding segment of the antibody Fc fragment is mutated to reduce or eliminate the complement and/or Fc receptor-binding activity of the complement and/or Fc receptor-binding segment of the antibody Fc fragment. To reduce or eliminate, preferably, the mutation is a substitution or insertion of one or more amino acid residues within the complement and/or Fc receptor binding segment of the antibody Fc fragment.

Item 6. The antibody or antigen-binding fragment thereof according to any one of items 1 to 5, wherein the antigen-binding fragment is selected from the group consisting of F(ab') ₂ , Fab', Fab, Fd, Fv, scFv, bispecific antibodies , a group consisting of camel antibodies, CDRs and antibody minimal recognition units (dAb). Preferably, the antigen-binding fragment is Fab, F(ab') ₂ or scFv.

Item 7. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 6, wherein the antibody is selected from the group consisting of A16, aducanumab, BAN2401, bapineuzumab, solanezumab, gantenerumab, ponezumab, and crizumab , Donanemab, Gosuranemab, Tilavonemab, Semorinemab, Zagotenemab and PRX002, in which the CDR sequence of the A16 antibody is shown in SEQ ID NO: 1-6.

Item 8. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 7, wherein the antigen-binding fragment is F(ab') 2 of the monoclonal antibody A16 targeting amyloid-beta, or F(ab') ₂ of the A16 antibody. The CDR sequences are shown in SEQ ID NO: 1-6.

Item 9. An isolated nucleic acid molecule selected from:

(1) DNA or RNA encoding the antibody or antigen-binding fragment thereof described in any one of items 1 to 8;

(2) A nucleic acid that is completely complementary to the nucleic acid defined in (1).

Item 10. An expression vector comprising the nucleic acid molecule described in Item 9 operably linked.

Item 11. A host cell comprising the nucleic acid molecule described in Item 9 or the expression vector described in Item 10.

Item 12. A composition comprising the antibody or antigen-binding fragment thereof according to any one of Items 1 to 8, the nucleic acid molecule according to Item 9, the expression vector according to Item 10, or the host cell according to Item 11 , and one or more pharmaceutically acceptable carriers, diluents or excipients.

Item 13. A method for producing the antibody or antigen-binding fragment thereof according to any one of Items 1 to 8, which includes the steps of:

The host cell described in Item 11 is cultured under culture conditions suitable for expression of the antibody or antigen-binding fragment thereof, and optionally, the resulting product is isolated and purified.

Item 14. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 8, the nucleic acid molecule according to Item 9, the expression vector according to Item 10, the host cell according to Item 11, or the combination according to Item 12 The use of substances in the preparation of drugs for promoting the clearance of toxic forms of amyloid in cells of subjects, reducing senile plaques in the brain, treating and/or preventing neurodegenerative diseases, inhibiting gliosis, and improving the level of brain synapses .

Item 15. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 8, the nucleic acid molecule according to Item 9, the expression vector according to Item 10, the host cell according to Item 11, or the host cell according to Item 12 The composition is used to promote the clearance of toxic forms of amyloid in cells of a subject, reduce senile plaques in the brain, treat and/or prevent neurodegenerative diseases, inhibit gliosis, and improve the level of brain synapses.

Item 16. A method for promoting the clearance of toxic forms of amyloid in cells of a subject, reducing senile plaques in the brain, treating and/or preventing neurodegenerative diseases, inhibiting gliosis, and improving the level of brain synapses, which includes Administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof described in any one of Items 1-8, the nucleic acid molecule described in Item 9, the expression vector described in Item 10, and the host described in Item 11 cells or the composition described in item 12.

Item 17. The use according to Item 14, the antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, host cell or composition according to Item 15, or the method according to Item 16, wherein the neurodegenerative disease is selected The group consisting of Alzheimer's disease, Parkinson's disease, frontotemporal dementia, Huntington's disease, amyotrophic lateral sclerosis or spinocerebellar ataxia, preferably said neurodegenerative disease is selected from the group consisting of Alzheimer's disease Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis.

Item 18. The antibody or antigen-binding fragment thereof described in any one of Items 1 to 8, the nucleic acid molecule described in Item 9, or the expression vector described in Item 10 is used in the preparation of samples for diagnosing amyloid toxic forms in subjects. The presence and/or level of the reagent in use.

The technical solutions of the present invention will be specifically described below through examples. These examples are descriptive and illustrative, and are not meant to be limiting. The reagents used in the following examples, unless otherwise noted, can be easily purchased from reagent companies such as SigmaAldrich and Merck. The test methods, unless otherwise noted, can be obtained from textbooks such as Sambrook, J., Fritsch, Found in E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York.

Example

Example 1. Preparation and functional characterization of A16 whole antibody F(ab') ₂ fragment Fab16.

1.1 Experimental materials and methods

1.1.1 Experimental materials

Aβ42 peptide and FAM-Aβ42 peptide were purchased from Zhongtide Biochemical Co., Ltd.; DMEM cell culture medium, PS, and FBS were purchased from ThermoFisher Scientific; Pierce ^TM Mouse IgG1 Fab and F(ab') ₂ Preparation Kit (Thermo, Cat. No.: 44980), Non-reducing protein loading buffer (5X) (P0016N) was purchased from Beyotime Biotechnology Co., Ltd.

The antibodies used were as follows: 4G8 antibody (Biolegend, product number: 800704) and HRP-labeled goat anti-mouse secondary antibody were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

1.1.2 Experimental instruments

The equipment used in the experiment mainly includes: high-speed cryogenic centrifuge (Hitachi, Japan), IX73 fluorescence microscope (Olympus), protein electrophoresis instrument (Biorad, United States), MD-M5 microplate reader (Molecular Devices, United States) and Odyssey two-color infrared laser Imaging system (LICOR, United States), microthermophore (MST, NanoTemperTechnologies, Germany), ultra-clean workbench (Shanghai Boxun), Tecan Safire2 microplate reader (Tecan, Switzerland).

1.1.3 Main solution preparation

(1) Phosphate buffer solution (PBS): 0.62g sodium hydrogen phosphate dihydrate, 5.73g disodium hydrogen phosphate dodecahydrate, and 9g sodium chloride were dissolved in deionized water, diluted to 1L, and filtered with a 0.22 μm filter. Sterilize.

(2) 0.1% PBST: Dissolve 1mL Tween 20 in 1L PBS.

(3) 10× electrophoresis buffer: Dissolve 144g glycine, 30g Tris, and 10g sodium dodecyl sulfate in deionized water and adjust the volume to 1L.

(4) 5× reducing electrophoresis loading buffer: 2.5mL 1M Tris-HCl (pH 6.8), 1g sodium dodecyl sulfate, 5mL glycerol, 0.5mL β-mercaptoethanol, 50mg bromophenol blue dissolved in deionized In water, adjust the volume to 10mL.

(5) ThT solution: Use 50mM PB (pH=6.5) solution to prepare 100X ThT stock solution (500μM), and dilute it to 1X with 50mMPB (pH=6.5) before use.

(6)MTT: 5mg/mL MTT is dissolved in deionized water, filtered and stored in the dark at -20°C. It is valid within three months.

Note: The relevant reagents, equipment and solutions mentioned in Examples 2, 3 and 4 but whose sources are not given are the same as those in Example 1.

1.2 Preparation of A16 antibody F(ab') ₂ fragment

The present invention applies hybridoma screening and monoclonal antibody preparation technology to obtain a monoclonal antibody (antibody A16) targeting Aβ1-16. The amino acid sequences of its light and heavy chain CDRs are shown in SEQ ID NO: 1-6 respectively. The light and heavy chains can The amino acid sequences of the variable region are shown in SEQ ID NO: 7-8 respectively, and the amino acid sequences of the light and heavy chains are shown in SEQ ID NO: 9-10 respectively). This antibody has a high affinity for Aβ1-16 and is suitable for imitation. Antibody therapy for AD in animals and humans.

Use Pierce ^TM Mouse IgG1 Fab and F(ab')2Preparation Kit (Thermo, 44980) to digest A16 according to the instructions. The detailed steps are as follows:

Fig protease is activated by digestive juice containing 4mM cysteine before use. The A16 antibody was desalted through a desalting column (Thermo Scientific Zeba Spin Desalting Column) to replace its solution with digestion solution, and then incubated with spare ficin at 37°C for 24-30 hours, and then passed through NAb Protein A Spin Columns (Thermo Scientific, Cat. No.: 89956) was purified to obtain Fab16. A16 and Fab16 were treated with reducing electrophoresis loading buffer and non-reducing electrophoresis loading buffer, and subjected to SDS-PAGE electrophoresis (first electrophoresis at 80V for 15 minutes, then adjusting the voltage to 100V, and electrophoresis on ice for 100 minutes). As shown in Panel A of Figure 1, the present invention purifies an F(ab') ₂ fragment with a molecular weight of approximately 110 kD, which is named Fab16.

1.3 Functional characterization of Fab16 and A16

1.3.1 Use microthermal molecular interaction electrophoresis instrument to detect the affinity between Fab16 and A16 and Aβ. Both MST power and LED excitation power were set to 40%. Measurements were performed in PBS buffer (containing 0.05% Tween 20) and standard capillary tubes. The concentration of FAM-Aβ was held constant at 40 nM, and Fab16 and A16 were serially diluted twofold (14 points per curve). Curve fits and binding affinities were calculated using the Kd model in MST analysis software MO. The results showed that A16 and Fab16 had similar affinities, with A16 being 5.35±0.84nM and Fab16 being 6.55±3.86nM (Figure 1, Panel B).

1.3.2 Thioflavin T (ThT) experiment detects the inhibitory effect of Fab16 and A16 on Aβ aggregation.

Mix 20 μM Aβ with 2 μM antibody (Fab16 or A16) and incubate at 37°C. Incubation samples at different time points were taken, 10 μL aliquots were mixed with 190 μL ThT solution (5 μM), and the fluorescence values (excitation wavelength 450 nm, emission wavelength 482 nm) were measured using a Tecan Safire2 microplate reader (Tecan, Switzerland), and the readings were subtracted The background fluorescence of ThT is the actual fluorescence value. Each group was tested in parallel more than 3 times. The results showed that both A16 and Fab16 could significantly inhibit the aggregation of Aβ (Figure 1, Panel C).

1.3.3 MTT assay to detect the inhibitory effect of Fab16 and A16 on neuronal cytotoxicity induced by Aβ oligomers

N2a cells (purchased from the Cell Line Resource Center of the Chinese Academy of Medical Sciences and Peking Union Medical College) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C, 5% Culture in a CO ₂ incubator. The cells were then seeded in a 96-well plate at a density of approximately 5,000 cells in 100 μL of culture medium per well. The 96-well plate was incubated at 37 °C for 12 h to allow cell attachment. Add 0.5 μM antibody (Fab16 or A16) and 1 μM Aβ oligomer, and incubate at 37°C for 72 hours. Then add 25 μL of 5 mg/mL MTT to each well. After continuing to incubate at 37°C for 3 hours, discard the cell culture medium. Add 150 μL DMSO to each well and shake for 10 min in the dark. The absorbance at 570/630nm was measured using MD SpectraMax M5 microplate reader. The results demonstrate that both Fab16 and A16 can effectively inhibit Aβ-mediated neuronal cell toxicity and have similar biological activities. (Panel D in Figure 1).

1.3.4 Fab16 and A16 recognize and bind to senile plaques in the brain of AD mice

Using Fab16 and A16 as primary antibodies, immunohistochemistry was used to detect the levels of Aβ plaques in the brains of APP/PS1 mice (Jackson Lab, 004462). Specifically, APP/PS1 mice were deeply anesthetized with sodium pentobarbital (50 mg/kg), cardiac perfusion was performed with pre-cooled PBS (containing heparin 10 U/mL), and then the mice were sacrificed. Peel off the brain, cut it into two parts along the sagittal plane, fix the left cerebral hemisphere in 4% paraformaldehyde at 4°C overnight, and dehydrate the next day (30% ethanol for 1 hour, 50% ethanol for 1 hour, 75% ethanol 1h, 100% ethanol 1h, 50% xylene 1h, 100% xylene 1h, 100% xylene 1h), paraffin embedding for 3h. 5 μm continuous paraffin-embedded sections were cut on a microtome. Place the slices at 65°C for 45 minutes for dewaxing; then soak the slices in xylene, 50% xylene alcohol, 100% alcohol, 90% alcohol, 75% alcohol, 50% alcohol, 30% alcohol and deionized water. 5 minutes; then place the slices in boiling citric acid repair solution and boil continuously for 15 minutes for antigen repair. Then the Fab16 or A16 antibody of the same concentration was incubated with the brain tissue sections overnight at 4°C, washed three times with PBS, 5 minutes each time; HRP-labeled goat anti-mouse was added as a secondary antibody, washed three times with PBS after 1 hour at room temperature, and developed with DAB. . Immunohistochemistry experiments showed that both Fab16 and A16 can effectively recognize and bind to senile plaques in brain slices of APP/PS1 mice (Figure 1, Panel E).

Example 2 Antibody A16 but not Fab16 mediates microglia phagocytosis of synapses

2.1 Experimental materials and equipment

SD rats at 17-18 days of gestation were purchased from Beijing Huafukang Biotechnology Co., Ltd.; Neurobasal medium, DMEM cell culture medium, PS, and FBS were purchased from Thermo Fisher Scientific, B27, L-GlutaMAX, and confocal microscope (Leica TCS SP8, Germany).

The antibodies used were as follows: C1q antibody (Invitrogen, product number: MA1-40311), C3 antibody (Abcam, product number: ab200999, Hycult, product number: HM1065), Iba-1 antibody (Abcam, product number: ab178847, GeneTex, product number: GTX101495), PSD95 antibody (Abcam, Catalog No.: ab12093, Catalog No.: ab18258), Synaptophysin antibody (Abcam, Catalog No.: ab32127), 4G8 antibody (Biolegend, Catalog No.: 800704), β-actin antibody (MBL, Catalog No.: M177-3), mouse anti-rat CD11b (BD Biosciences, Cat. No. 554980), mouse IgA, kappa isotype control (BD Biosciences, Cat. No. 553476), mouse anti-rat CD32 (BD Biosciences, Cat. No. 550273 ), mouse IgG1κ isotype control (BD Biosciences, Cat. No. 553447).

2.2 Culture of primary neurons

Take SD rat embryos at 17-18 days of pregnancy, soak and disinfect them in 75% alcohol, take out the brain and soak it in HBSS buffer, remove the blood meninges, peel off the hippocampal tissue and cut it into pieces with tissue scissors, and place it in a 15mL centrifuge tube. Add 5 mL of tissue digestion solution (DMEM medium containing 0.25% trypsin and 1 mg/mL DNase I), digest at 37°C for 20 minutes, pass the digested cell suspension through a 70 μm cell sieve to remove undigested tissue blocks, and centrifuge at 500g Collect cells after 10 minutes, spread the cells in a 12-well plate containing cell slides (PDL coated at room temperature overnight), and culture using Neurobasal medium (containing B27, L-GlutaMAX).

2.3 Culture of primary microglia and co-culture of primary neurons and microglia

Take 1-2 days old SD newborn rats, soak and disinfect them in 75% alcohol, take out the brain and soak it in HBSS buffer, peel off the blood meninges, cut the brain into pieces with tissue scissors, place it in a 50mL centrifuge tube, and add 20mL of tissue Digestion solution (DMEM medium containing 0.25% trypsin and 1 mg/mL DNase I), digest at 37°C for 20 minutes, pass the digested cell suspension through a 70 μm cell sieve to remove incompletely digested tissue blocks, and centrifuge at 500g for 10 minutes to collect cells. , spread the cells in T-75 culture flasks, and culture them in DMEM medium at 37°C for 5-7 days. Change the culture medium every 2-3 days. After 7 days of culture, cells were taken out and placed in a cell shaker. Shake at 220 rpm for 30 min. Put the supernatant into a centrifuge tube and centrifuge at 500g for 10 min to collect microglia.

For the co-culture system, centrifuge the collected microglia and resuspend them in neuron culture medium. Count the cells and add microglia at a ratio of neurons: microglia = 3:1. Culture neurons in culture plates.

2.4 Antibody A16 but not Fab16 mediates microglia phagocytosis of synapses

1 μM Aβ oligomer was added to the neuron-microglia co-culture system, and 0.5 μM antibody (Fab16 or A16) was added after 30 min to incubate overnight. The cells were washed three times with PBS for immunofluorescence staining. Specifically: fixed with 4% paraformaldehyde; then soaked in 0.3% Triton :100 dilution, incubate overnight at 4°C; wash 3 times with PBS, 5 min each time; add fluorescently labeled secondary antibody, use Hoechst 33258 (Molecular Probes, USA) for nuclear staining, and use a laser confocal scanning microscope (Leica TCS SP8, Germany) ) to detect the phagocytosis of synapses by microglia (Panel A in Figure 2). The results showed that the amount of postsynaptic protein PSD95 in the microglia of the A16 group was significantly higher than that of the Aβ control group (Figure 2, B), and the level of PSD95 near the microglia decreased significantly (Figure 2, C ). However, Fab16 did not promote phagocytosis of PSD95 by microglia.

In addition, western blot (WB) analysis of cell lysates cultured for 36 hours after antibody stimulation showed that the level of PSD95 in the A16 group was significantly lower than that of the Fab16 group and the Aβ control group (Figure 2, D and E). These results are consistent with the above-mentioned cellular immunofluorescence results.

The present invention further explored the intracellular phagocytic components of microglia. The results showed that compared with the Fab16 group and the solvent control group, there were a large number of co-localized PSD95, synaptophysin, Aβ, C1q and C3 (Fig. 3). These results confirm that A16 targets Aβ oligomers that bind to synapses and form antigen-antibody immune complexes at the synapses. At the same time, the Fc segment of A16 activates the classical complement pathway, allowing C1q to bind to it, and then the activated complement C3 binds to C1q to form an antigen-antibody-C1q-C3 complex. Further activation of microglia causes microglia to phagocytose antigen-antibody-complement complexes as well as synapses, resulting in further loss of synapses.

2.5 Antibody A16 mediates synaptic phagocytosis through microglia surface CR3 and Fcγ receptors

The present invention blocks the CR3 and FcγRIIb receptors on the surface of microglia by adding functional antibodies CD11b antibody or CD32 antibody respectively, or adding the scavenger receptor blocker fucose to block the scavenger receptors on the surface of microglia. , to investigate through which receptor the antibody A16 mediates the phagocytosis of synapses by microglia. Specifically: for CR3 blockade, neurons were pretreated with Aβ oligomers (AβOs) (1 μM) for 30 min, and then treated with A16 (0.5 μM) and then treated with 15 μg/ml anti-CD11b functional antibody or 15 μg/ml isotype control antibody for 30 min. Microglia were then added to the neurons, and the co-cultured cell system was placed in an incubator at 37°C and 5% _CO2 for 10 h. Cells were immunofluorescently stained using anti-PSD95, anti-Iba-1, and 4G8 antibodies, respectively, and images were captured using a Leica TCS SP8 confocal microscope. For FcγRIIb blocking, use 10 μg/ml anti-CD32 functional antibody or 10 μg/ml isotype control and perform the experiment in the same manner. The results showed that after the two receptors were blocked, the number of microglia phagocytosis of PSD95 and Aβ was significantly reduced (Figure AB in Figure 4). Fucoidan is a scavenger receptor blocker, and blocking scavenger receptors does not affect the phagocytosis of PSD95 and Aβ by microglia, indicating that the antibody does not mediate synaptic phagocytosis through the scavenger receptor pathway. Subsequently, the present invention uses lentivirus to construct shRNA systems of different viruses, knocking down the gene expression of C1qa, C3, CD11b and FcγRIIb in microglia (Figure 4, C), and comparing the gene knockdown of microglia and nerve cells for 48 hours. After co-culture with Aβ and full antibody A16, confocal microscopy was used to observe the phagocytosis of PSD95 by microglia. The results showed that after knocking down C1qa, C3, CD11b and FcγRIIb for 48 hours, the ability of microglia to phagocytose PSD95 was significantly improved. decreased, indicating that A16 mediates the phagocytosis of Aβ and PSD95 through the classical complement pathway (C1q-C3-CR3) and the Fcγ receptor pathway (DE diagram in Figure 4).

Example 3 After 24 hours of injection of A16 but not Fab16 antibody, the cognitive ability and synaptic level of APP/PS1 mice were significantly reduced.

In order to explore whether the antibody A16 can activate microglia in the brain of APP/PS1 transgenic mice to phagocytose synapses, thereby leading to a decline in cognitive function, the present invention injected equal concentrations of A16 and isotype into the brains of APP/PS1 transgenic mice. Antibody control, Fab16 and PBS solvent control, after 24 hours, the cognitive ability of mice was detected through Y maze test and novel object recognition test. Immunohistochemistry was used to detect synaptic levels in the brains of APP/PS1 transgenic mice 48 hours later.

3.1 Main experimental materials and equipment

In addition to the experimental materials and equipment used in Examples 1 and 2, this example also uses the following main experimental materials and equipment: KD-BM paraffin embedding machine (KEDEE), RM2245 paraffin microtome (Leica), new object identification Equipment (Chinese Academy of Medical Sciences), Y-maze equipment (Chinese Academy of Medical Sciences). 50% xylene: Equal volumes of ethanol and xylene are mixed thoroughly. Different percentages of ethanol: Different volumes of absolute ethanol and deionized water are thoroughly mixed. Citric acid antigen retrieval solution: Dissolve 3g trisodium citrate and 0.4g citric acid in deionized water and adjust the volume to 1L.

3.2 Experimental animals

Six-month-old APP/PS1 transgenic mice and 6-month-old male wild-type mice were purchased from Jackson Laboratory004462. Mice were raised in a clean room with a room temperature of 22±2°C and a humidity of 45%±10%. The mice had free access to food and water. All animal experiments were conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals of the Chinese Public Health Service" conduct. Experiments involving mice were approved by the Animal Care and Use Committee of Tsinghua University.

3.3 Animal experiment design

APP/PS1 transgenic mice were divided into 5 groups, with 8 mice in each group. The dosing strategy was as follows: A16, Fab16, isotype control antibody and solvent carrier (AD-A16, AD-Fab16, AD-Iso, AD- Veh) were injected into the lateral ventricles of AD mice via a stereotaxic instrument (0.5 mm posterior, 1 mm right, 2.5 mm deep). The WT mice in the same litter were also given solvent carriers into the lateral ventricles (WT-Veh). Each Mice were given 24 μM, each dose was 5 μL, and behavioral tests were performed 24 hours after injection.

3.4 The cognitive ability of APP/PS1 transgenic mice was significantly reduced 24 hours after injection of A16 but not Fab16 antibodies.

After 24 hours of administration to the mice in each group, the cognitive ability of the mice was examined through the Y maze test and the new object recognition test.

The specific experimental methods are as follows:

3.4.1 Y maze

The Y-maze is composed of three arms A, B, and C, and includes two test stages with an interval of 1 hour. In the first stage, mice were allowed to explore two arms (A, B) of the maze, and the third arm (new arm C) was blocked for 10 min. The second stage is to place the mouse on the same starting arm as in the first stage, open the new arm C, and allow it to freely enter and exit all three arms for 5 min. Use a camera to record the exploration trajectory, record the number of times the new arm is explored, and the time the new arm stays.

3.4.2 Recognition of new things

The new object recognition device is designed based on mice's willingness to explore new things. The device is a white box with a size of 40cm × 40cm × 40cm. The experiment is divided into three stages: adaptation stage, training stage and detection stage; in the adaptation stage, mice were placed in an empty box to adapt spontaneously for 5 minutes; after 24 hours, the training stage entered, and two identical objects were placed in the box, allowing the mice to The mice were familiarized with the box for 5 minutes; after an interval of 6 hours, the detection phase was entered, an old object in the box was replaced with a new object, and the mice were allowed to explore freely in the box for 5 minutes, and the number of times the mice explored the old and new objects was recorded. To avoid the influence of mouse odor, the box was wiped with 75% alcohol after each mouse ended its exploration.

The results showed that compared with Fab16 and PBS solvent control group APP/PS1 mice, APP/PS1 mice in the A16 group showed shorter new arm exploration time, indicating a decline in their cognitive ability (Figure 5, Panel A) . The results of the new object recognition experiment showed that the cognitive ability of mice in the A16 group was lower than that of the APP/PS1 mice in the Fab16 group (Figure 5, B), while the APP/PS1 mice in the Fab16 group did not show cognitive decline. The phenomenon.

3.5 Synaptic levels in APP/PS1 transgenic mice were significantly reduced 48 hours after injection of A16 but not Fab16 antibodies.

Golgi staining and immunohistochemistry were used to measure synaptophysin and PSD95 protein levels in the mouse brain to evaluate synaptic levels in the mouse brain.

For Golgi staining, fresh cerebral hemispheres were processed using the FD Rapid GolgiStainTM kit (Cat#PK401, FDNeuroTechnologies, Columbia, MD) according to the kit instructions. 100 μm thick coronal serial sections were cut on a Lecia CM1850 microtome (Leica Biosystems, Buffalo Grove, IL). Brightfield images of Golgi-impregnated dendrites from cortical neurons were captured using an Olympus IX73 microscope and an oil immersion lens (100×, Olympus, Japan). Dendritic spine density was quantified by manual counting in ImageJ. Analyze 10-12 fragments per mouse. The results showed that the number of neuronal dendritic spines in the brains of APP/PS1 mice injected with A16 antibody was significantly lower than that of Fab16, isotype antibody control and PBS solvent control APP/PS1 mice (Figure 5, C-D). Immunohistochemical staining of synaptophysin and PSD95 in the brain (Figure 5, E-F) also revealed the same conclusion, that is, the injection of A16 antibody for 48 hours reduced the level of synapses in the mouse brain. However, Fab16 did not cause a decrease in synaptic levels in APP/PS1 mice.

3.6 A large number of microglia phagocytosed synapses in the brains of APP/PS1 transgenic mice 48 hours after injection of A16 but not Fab16 antibodies

The present invention uses immunohistofluorescence technology to detect intracellular PSD95, Aβ and C1q contents in microglia. Specifically, paraffin sections of brain tissue were placed in a 60°C oven for 1 h, and then dewaxed in 100% xylene, 50% xylene, 100% alcohol, 70% alcohol, 50% alcohol and 30% alcohol. Soak the slices in deionized water for 10 minutes, add antigen retrieval solution and boil for 15 minutes, then cool to room temperature for later use. Wash three times with PBS and block for 1 h at room temperature in PBST containing 10% sheep serum and 0.3% TritonX-100. Subsequently, they were incubated with Iba-1, PSD95, Aβ, and C1q (1:100) antibodies respectively, washed three times with PBS, 5 min each time, and then incubated with the corresponding fluorescently labeled secondary antibodies, washed three times with PBS, 5 min each time, and washed with a total of Focused microscope observation. The results showed that in the brains of A16-treated mice, the level of PSD95 in microglia was about 4 times higher than that in the Fab16 group, and it was highly co-localized with Aβ and C1q (Figure 6, A-D). And compared with the Fab16 group, the number of non-activated branched microglia in the brains of mice in the A16 group was significantly reduced (Figure 6 E-F). These results indicate that the A16 antibody can mediate massive phagocytosis of synapses by microglia in the brains of APP/PS1 transgenic mice, while Fab16 does not cause this adverse reaction.

Example 4 After 48 hours of injection of A16 but not Fab16 antibody, the level of neuroinflammation in the brain of APP/PS1 mice increased significantly.

4.1 The degree of glial cell activation in the brains of APP/PS1 transgenic mice increased 48 hours after injection of A16 but not Fab16 antibodies.

Neuroinflammation plays a key role in the occurrence and development of AD, and is characterized by excessive activation of microglia and astrocytes, and the massive release of inflammatory factors. The present invention detects the activation of astrocytes and microglia in the mouse brain through GFAP and Iba-1 immunostaining respectively. Compared with the Fab16 group and the solvent control group, the degree of activation of microglia and astrocytes in the brains of mice in the A16 antibody group was significantly increased, and the degree of clustering was intensified (Figure 7, A-C). In addition, the ELISA results of mouse brain homogenates showed that the levels of IL-1β, IL-6 and TNF-α inflammatory factors in the brains of mice in the A16 group were also significantly increased (Figure 7, D). Inflammation and complement-related The expression of the gene was also significantly increased (panel E of Figure 7). This shows that injection of A16 antibody can promote inflammatory response in the brain of APP/PS1 transgenic mice 48 hours later, while Fab16 does not cause inflammation.

Example 5 Change trends in cognitive level of APP/PS1 transgenic mice treated with A16

In order to further explore the impact of A16 antibody treatment on the cognitive level of APP/PS1 transgenic mice, the present invention used a Y-maze experiment to observe the changing trend of the cognitive ability of mice (Figure 8, A-B). First, the A16 antibody was injected into the right cerebral ventricle of APP/PS1 transgenic mice through stereotaxic injection technology, and Y maze experiments were performed at 24 hours, 3 days, 5 days, 7 days, and 9 days after injection. The results showed that compared with the solvent control Compared with the APP/PS1 transgenic mice group, the cognitive ability of the mice decreased significantly 24 hours after injection of A16 antibody. As time went by, their cognitive level gradually recovered and improved, and surpassed the solvent control group. On the 9th day, the cognitive ability of the mice in the A16 group was significantly higher than that of the mice in the control group. Subsequently, in order to further clarify whether the A16 antibody caused synaptic damage in the brains of APP/PS1 transgenic mice in the short term and led to cognitive function impairment, the present invention conducted a second test of the A16 antibody on APP/PS1 mice on the 10th day. injection, and a Y maze experiment was conducted 24 hours after the injection. The results showed that the cognitive ability of the mice declined again, but due to the basis of the previous treatment effect, the level of cognitive decline this time was not significantly lower than that of the solvent control. group of mice. The present invention then conducted a Y maze experiment on the APP/PS1 transgenic mice on the 10th day after the second injection. The results found that after two injections of A16 antibody, the cognitive ability of APP/PS1 transgenic mice was significantly improved. These results suggest that A16 antibody treatment of APP/PS1 transgenic mice will cause synapses and Aβ to be engulfed by microglia, resulting in excessive synapse loss and cognitive impairment. Over time, within one to several weeks after treatment, the number of synapses in mice can be rapidly regenerated and restored. Especially in an environment where Aβ levels in the brain are significantly reduced, the cognitive abilities of mice are significantly enhanced. , showing immunotherapeutic effects. However, unlike mice, the growth and regeneration rate of synapses in primates such as AD patients is very slow. The number of synapses in the brains of AD patients cannot exceed that of untreated AD patients within a few weeks. If the recovery time is too long, the Aβ level that has been reduced by the antibody will increase again, causing the patient's synapses and neuronal functions to be damaged again, making it difficult for the patient to show good therapeutic effects. In short, within a limited time window, the synaptic levels of mice treated with full antibodies can exceed those of untreated control mice, while the synaptic levels of AD patients treated with full antibodies cannot exceed those of untreated patients.

Example 6 The antibody aducanumab mediates microglial phagocytosis of synapses, and its F(ab') ₂ antibody that removes Fc does not cause this adverse reaction.

The present invention applied the method of Example 1 to prepare the F(ab') ₂ fragment (Fab-adu) of adenumab (Adu), and characterized its function and activity. The results show that Adu and Fab-adu have similar Aβ affinity, and both can effectively recognize and bind to senile plaques in the brains of APP/PS1 transgenic mice (data not shown).

The method of Example 2 was used to detect the phagocytosis of synapses by microglia in the presence of Aβ oligomers in a primary neuron-microglia co-culture system. The results showed that Adu promoted the phagocytosis of synapses by microglia. Phagocytosis of synaptic proteins by cytoplasmic cells, whose Fc-removing F(ab') ₂ antibody Fab-adu did not cause this adverse reaction (data not shown).

According to the method of Example 3, APP/PS1 transgenic mice were injected with equal concentrations of Adu, Fab-adu and PBS solvent control respectively. After 24 hours, the cognitive ability of the mice was detected. After 48 hours, the synaptic levels and brain levels of the mice were detected. Pathological changes. The experimental results showed that, similar to A16, the memory and synaptic levels of APP/PS1 transgenic mice treated with Adu were significantly lower than those of mice treated with Fab-adu or the solvent control group. Moreover, Adu treatment mediated excessive phagocytosis of synapses by microglia in the brains of APP/PS1 transgenic mice, leading to increased activation levels of glial cells and a large release of inflammatory factors (data not shown). However, Fab-adu treatment did not result in these adverse effects.

Example 7 The number of microglia engulfing synapses in the brain of APP/PS1 mice increased 48 hours after injection of A16I4 antibody (IgG4 subtype of A16 antibody)

The present invention uses conventional methods to transform the A16 antibody into an IgG4 subtype antibody, named A16I4, and gives it to APP/PS1 mice through tail vein injection. The injection dose is 20 mg/kg. At the same time, equal concentrations of A16, control group, and PBS solvent control and WT control group. After 48 hours, the phagocytosis of synapses by microglia in the mouse brain was detected according to the method 2.4 of Example 2. Experimental results show that, similar to A16, A16I4 can significantly induce excessive phagocytosis of synapses by microglia in APP/PS1 mice. The level of PSD95 in microglia of mice in the A16I4 group was significantly higher than that in the solvent control group (Figure 9 Figures A-E), and highly co-localized with Aβ and C3, but not with C1q. It is suggested that although the IgG4 subtype cannot activate the classical complement pathway, it can still activate the alternative pathway or the lectin complement pathway, that is, activate C3, leading to excessive synaptic phagocytosis.

Example 8 The growth and regeneration rate of human synapses is much lower than that of mice

8.1 Human neural stem cell differentiation:

Human neuronal stem cells (NSC, purchased from Wuhan Shangen Biotechnology Co., Ltd.) (50,000 cells per well) were cultured on Matrigel-coated 12-well plates for 3 days, and then the cells were cultured in DMEM/F12 medium to induce neurons. Differentiation contained 1% N2 supplement, 1% B27 supplement, 200 μM ascorbic acid (MCE), 400 μM dbcAMP (MCE), 10 ng/ml GDNF (Peprotech) and 10 ng/ml BDNF (Peprotech). Laminin (Gibco) was added to the culture after 2 days to promote differentiation. Cells were maintained in the above-mentioned medium for 14 days, and the medium was changed daily.

8.2 Differentiation of mouse neuronal stem cells:

Neural stem cells were obtained from 13-16 day old fetal rat brains (Pollard SM, Conti L, Sun Y, GoffredoD, Smith A (2006) Adherent Neural Stem (NS) Cells from Fetal and Adult Forebrain. Cerebral Cortex 16:i112-i120 ) and cultured in growth medium containing 2% N2 supplement, 2% B27 supplement, bFGF 20ng/ml (Peprotech), EGF 20ng/ml (Peprotech). Cells grow to form neurospheres. Adjust the cell density to 50,000/mL and inoculate the PDL-coated 12-well plate. After the cells were basically cultured for 8 days, the medium was replaced with differentiation medium containing 1% N2 supplement, 1% B27 supplement and 0.5% FBS.

8.3 Human synapses grow and regenerate much slower than mice

In order to examine the difference in synaptic regeneration between humans and mice, the present invention added 1 μM AβOs to human and mouse neuron cell culture systems and incubated them for 24 hours to clear synapses. Then, the AβOs were removed, and fresh medium was replaced to continue cell culture. Western blot analysis of PSD95 was performed on cell lysates at different time points.

The results showed that incubation with AβOs for 24 hours resulted in a 53% reduction in mouse synapse levels (PSD95) and a 42% reduction in human synapse levels (PSD95). Then, 96 hours after replacement of fresh medium, mouse synaptic levels returned to 91%, while human synaptic levels only reached 65% at 192 hours and 81% at 528 hours (Figure 10, A-C). These results indicate that human synapses grow and regenerate much slower than mice.

Equivalent solution

Although various embodiments of the invention have been described and illustrated herein, one of ordinary skill in the art will readily envision one or more of the inventions being used to perform the functions described herein and/or to obtain the results described herein. Various other means and/or structures may be used to advantage, and each such change and/or modification is deemed to be within the scope of the invention. More broadly, those skilled in the art will readily appreciate that all parameters, materials, and settings described herein are intended to be exemplary and that the actual parameters, materials, and/or settings will depend on the specific circumstances in which the teachings of the present invention are utilized. application. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments and examples are presented by way of illustration only and that within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and claimed. Any combination of two or more such features, systems, articles, materials and/or methods is included within the scope of the invention provided that such features, systems, articles, materials and/or methods are not in conflict with each other.

As used in the description and claims of the present invention, the phrase "and/or" should be understood to mean "either or both" of the elements so combined, that is, present in combination in some cases and not in other cases. element. In addition to the elements specifically identified by the "and/or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified elements, unless expressly stated otherwise. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprises," a reference to "A and/or B" in one embodiment may refer to both A and B (optionally including anything other than B). elements other than A); in another embodiment, it refers to B and A (optionally including elements other than A); in yet another embodiment, it refers to both A and B (optionally including other elements); etc.

As used herein in the specification and claims, "or" is to be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" should be understood to be inclusive, i.e. including a plurality of elements or at least one of the list of elements, but also more than one, and optionally, Other items not listed. Only terms that expressly indicate the contrary, such as "only one of" or "exactly one of", or when used in claims, "consisting of" will refer to exactly one element of a plurality of elements or a list of elements. Generally, when preceded by an exclusive term such as "any", "an", "only one" or "exactly one", the term "or" as used herein should only be understood to mean exclusive alternatives (i.e. , "one or the other but not both"). “Consisting essentially of” when used in a claim shall have its ordinary meaning in the field of patent law.

As used herein in the specification and claims, when referring to a list of one or more elements, the phrase "at least one" shall be understood to mean at least one element selected from any one or more elements of the list of elements, However, it does not necessarily include at least one of each element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows that elements other than those specifically identified in the list of elements referred to by the phrase "at least one" may optionally be present, whether or not related to those specifically identified elements. Thus, by way of non-limiting example, in one embodiment, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently, "at least one of A and/or B" "A") may refer to at least one, optionally including more than one A, without B (and optionally including elements other than B); in another embodiment, refers to at least one, optionally including more than In a B, A is absent (and optionally includes elements other than A); in yet another embodiment, refers to at least one, optionally including more than one A, and at least one, optionally including more than one A to a B (and optionally other elements); etc.

In the claims and the above description, all connectives such as "includes", "includes", "with", "has", "contains", "involves", "has", etc. are to be understood as open-ended, i.e. Means including but not limited to. Only the connectives "consisting of" and "consisting essentially of" shall be closed or semi-closed connectives respectively.

The use of sequential terms in the claims, such as "first", "second", "third", etc., to modify a claim element does not itself imply any priority of one claim element over another claim element. precedence or order, or the temporal order in which actions are performed in a method, and merely used as a label to distinguish one claim element with a certain name from another element with the same name (but in ordinal terms), to Distinguish claim elements.

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Claims (10)

1. An antibody specifically targeting amyloid or an antigen-binding fragment thereof, characterized in that it has reduced complement and/or Fc receptor binding activity or no complement and/or Fc receptor binding activity. 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amyloid is in its monomeric, oligomeric, profibrillar or fibrillar form. 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the amyloid protein is selected from the group consisting of beta amyloid, microtubule-associated protein tau, alpha-synuclein, huntingtin, pancreatic amyloid Preferably, the amyloid protein is β-amyloid protein or microtubule-associated protein tau. 4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody or a fully human antibody. Preferably, the antibody is selected from The group consisting of IgG, IgM, IgA, IgD and IgE and their subtypes. 5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody is IgG, IgM or a subtype thereof, wherein the complement and/or Fc receptor binding activity of the antibody is determined by deletion All or part of the complement and/or Fc receptor-binding segment of the antibody Fc fragment is mutated to reduce or eliminate the complement and/or Fc receptor-binding activity of the complement and/or Fc receptor-binding segment of the antibody Fc fragment. To reduce or eliminate, preferably, the mutation is a substitution or insertion of one or more amino acid residues within the complement and/or Fc receptor binding segment of the antibody Fc fragment. 6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antigen-binding fragment is selected from the group consisting of F(ab') ₂ , Fab', Fab, Fd, Fv, scFv, bispecific antibodies , a group consisting of camel antibodies, CDRs and antibody minimal recognition units (dAb). Preferably, the antigen-binding fragment is Fab, F(ab') ₂ or scFv. 7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody is selected from the group consisting of A16, adunumumab, BAN2401, bapineuzumab, solanezumab, gantenerumab, ponezumab, and crizumab. , Donanemab, Gosuranemab, Tilavonemab, Semorinemab, Zagotenemab and PRX002, in which the CDR sequence of the A16 antibody is shown in SEQ ID NO: 1-6. 8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antigen-binding fragment is F(ab') 2 of the monoclonal antibody A16 targeting amyloid beta protein, F(ab') ₂ of the A16 antibody. The CDR sequences are shown in SEQ ID NO: 1-6. 9. An isolated nucleic acid molecule selected from: (1) DNA or RNA encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8; (2) A nucleic acid that is completely complementary to the nucleic acid defined in (1). 10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 or the nucleic acid molecule according to claim 9 is used in preparation to promote the clearance of toxic forms of amyloid in cells of a subject and reduce senile plaques in the brain. , use in drugs to treat and/or prevent neurodegenerative diseases, inhibit gliosis, and improve the level of brain synapses.