WO2024032822A1 - 改进的β淀粉样蛋白寡聚体特异性结合抗体 - Google Patents

改进的β淀粉样蛋白寡聚体特异性结合抗体 Download PDF

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WO2024032822A1
WO2024032822A1 PCT/CN2023/121350 CN2023121350W WO2024032822A1 WO 2024032822 A1 WO2024032822 A1 WO 2024032822A1 CN 2023121350 W CN2023121350 W CN 2023121350W WO 2024032822 A1 WO2024032822 A1 WO 2024032822A1
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antibody
antigen
binding fragment
amino acid
specific antibody
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French (fr)
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刘瑞田
于晓琳
卢帅
杨金菊
朱杰
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深圳智源生物医药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P25/00—Drugs for disorders of the nervous system
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to antibodies that specifically bind to amyloid beta oligomers (A ⁇ Os), and in particular to improved antibodies that specifically bind to specific A ⁇ Os.
  • a ⁇ Os amyloid beta oligomers
  • AD Alzheimer's disease
  • the main clinical symptoms are memory decline, cognitive impairment and loss of self-care ability.
  • AD has been listed as the fourth leading cause of death in humans, after cancer, heart disease and stroke, and its incidence is significantly age-dependent.
  • AD has been listed as the fourth leading cause of death in humans, after cancer, heart disease and stroke, and its incidence is significantly age-dependent.
  • AD has been listed as the fourth leading cause of death in humans, after cancer, heart disease and stroke, and its incidence is significantly age-dependent.
  • AD patients worldwide As the global population ages, the number of AD patients is increasing year by year. According to statistics, there were approximately 50 million AD patients worldwide in 2018, and it is expected that the number of AD patients worldwide will reach 130 million in 2050. AD medical expenses are huge.
  • the global cost of treatment and care for AD patients was approximately US$818 billion in 2015, and is expected to increase to US$2 trillion in 2030. In the past ten years, approximately US$110 billion has been invested in the research and development of AD treatment drugs worldwide.
  • AD Alzheimer's disease
  • a ⁇ oligomers which are the most cytotoxic, rather than A ⁇ monomers and fibers.
  • the level of A ⁇ oligomers in the brains of AD patients is highly correlated with the occurrence, development and severity of AD.
  • a ⁇ oligomers have a high affinity for synapses.
  • a ⁇ oligomers binding to synapses can activate the complement system, prompting microglia to excessively prune and phagocytose synapses, destroy the structure of synapses, induce synaptic dysfunction, and cause synaptic damage and excessive loss. Lead to cognitive decline. Therefore, targeting A ⁇ oligomers and inhibiting A ⁇ oligomer levels and induced neurotoxicity is one of the ideal strategies to treat AD based on the cause.
  • Treatment options targeting A ⁇ monomers can cause adverse reactions such as inflammation and synaptic loss, leading to repeated failures in clinical trials. Eliminating A ⁇ and reducing the level of A ⁇ oligomers through immunotherapy strategies has always been the focus of research in the field of AD treatment. In recent years, dozens of antibodies and vaccines targeting A ⁇ have entered the market. Clinical trial stage.
  • Such as the first-generation vaccine AN1792 targeting A ⁇ fibers, and antibodies targeting A ⁇ monomers or fibers such as bapineuzumab (anti-A ⁇ 1-5), solanezumab (anti-A ⁇ 13-28), gantenerumab (anti-A ⁇ 1-11) and ponezumab (anti-A ⁇ C-terminus), etc.
  • amyloid proteins such as A ⁇ , tau, ⁇ -synuclein, mHTT, SOD1 and other proteins.
  • the oligomers formed by the aggregation of these protein monomers are responsible for AD and Parkinson's disease ( It is a key pathogenic factor in the development of PD, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), etc., and is also an effective target for the diagnosis and treatment of such 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.
  • amyloid proteins Although the primary sequences of these amyloid proteins are different, the oligomers formed by 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. In addition, studies have shown that various amyloid proteins have a synergistic promotion effect in amyloid diseases. The aggregation of a certain amyloid protein and the occurrence and development of the disease will promote or induce the aggregation of other amyloid proteins and participate in in the pathological process of the disease.
  • amyloid oligomer-specific antibodies Since 2005, the inventor has carried out research on amyloid oligomer-specific antibodies, and was the first in the world to screen and obtain a fully human single-chain antibody W20 that can recognize a variety of amyloid oligomers (Chinese patent CN101463082A ). This antibody can bind to oligomers such as A ⁇ , ⁇ -synuclein, mHTT, and SOD1, which cause various amyloid diseases such as AD, PD, HD, and ALS, but not to the monomers of each amyloid protein.
  • oligomers such as A ⁇ , ⁇ -synuclein, mHTT, and SOD1
  • the antibody can also inhibit the aggregation and cytotoxicity of various amyloid proteins; improve the cognitive function and behavioral coordination of disease model mice regulate ability and reduce neuropathological changes in mice (Biochimica et Biophysica Acta 2011, 1814, 1703-1712; AD: Current Alzheimer Research, 2014, 11, 69-78; PD&HD: Scientific Reports, 2016, 6, 36631; ALS: International Immunopharmacology 2018, 65, 413-421).
  • One aspect of the invention relates to an improved amyloid oligomer-specific antibody, or antigen-binding fragment thereof, at one amino acid residue position selected from the group consisting of positions 226, 227, and 228 relative to the W20 antibody. Or there are amino acid substitutions at multiple positions.
  • the amino acid sequence of the W20 antibody is shown in SEQ ID No. 1.
  • the amino acid position number of the antibody is determined using the Kabat numbering system.
  • the amino acid residue at position 226 of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is replaced with a non-polar, hydrophobic amino acid with properties similar to alanine.
  • a non-polar, hydrophobic amino acid with properties similar to alanine.
  • Acid is substituted by glycine, valine, leucine, isoleucine, phenylalanine, tryptophan or proline.
  • the 227th amino acid residue of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is replaced by a hydrophobic amino acid with properties similar to valine, preferably by The substitution is phenylalanine, tryptophan, tyrosine, alanine, leucine or isoleucine, more preferably, the substitution is phenylalanine, isoleucine or leucine.
  • the 228th amino acid residue of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is replaced by an aliphatic amino acid with a similar structure to arginine, preferably by Substituted for alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, glycine, serine, threonine, cysteine, asparagine Or glutamine, more preferably, is substituted with lysine, asparagine, glutamine.
  • amino acid residues 226-228 of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof are substituted with AVR, GSR, WVR, FER, NFR or VRR, respectively.
  • the 224th and 225th amino acid residues of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof are replaced with glutamine and threonine respectively.
  • the improved amyloid oligomer-specific antibodies or antigen-binding fragments thereof are capable of specifically binding A ⁇ o*3F oligomers.
  • the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is an antigen-binding fragment, preferably selected from the group consisting of scFv, F(ab') 2 , Fab', Fab, Fd , Fv, bispecific antibody, camel antibody, CDR and antibody minimal recognition unit (dAb) antigen-binding fragment of the group consisting of, more preferably, selected from the group consisting of scFv, F(ab') 2 , Fab', Fab Antigen-binding fragments.
  • the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is scFv or F(ab') 2 .
  • the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is a monoclonal antibody, a chimeric antibody, a humanized antibody or a fully human antibody.
  • the antibody is selected from the group consisting of From the group consisting of IgG, IgM, IgA, IgD and IgE and subtypes thereof, more preferably the antibody is selected from the group consisting of subtypes IgG1-4, more preferably the antibody is of the IgG4 subtype.
  • the heavy chain amino acid sequence of the antibody is set forth in any one of SEQ ID Nos. 18-21, and/or the light chain amino acid sequence of the antibody is set forth in SEQ ID No. 17.
  • the heavy chain constant region amino acid sequence of the antibody is as set forth in any one of SEQ ID No. 23-26, and/or the light chain constant region amino acid sequence of the antibody is as set forth in SEQ ID No. 22 Show.
  • the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is a scFv and its amino acid sequence is as shown in any one of SEQ ID Nos. 5, 4 and 6-9.
  • sequence of the CDR3 of the light chain of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is shown in SEQ ID No. 15.
  • the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof has an arginine substituted at position 98 relative to the 3F antibody.
  • the heavy chain amino acid sequence of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is as shown in any one of SEQ ID No. 33-36, and the light chain amino acid sequence is as shown above.
  • the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof is scFv and its amino acid sequence is shown in SEQ ID No. 27.
  • the invention relates to variants of improved amyloid oligomer specific antibodies, or antigen-binding fragments thereof, as described above, which are relative to the improved amyloid oligomer specificity as described above.
  • Antibodies or antigen-binding fragments thereof have substitutions, insertions, or deletions of one or more amino acid residues, such as 1, 2, 3, 4, 5, 10, 15, 20, 25, 30 or more amino acid residues substitutions, insertions, or deletions while still substantially retaining one, more, or all of the biological activities of the improved amyloid oligomer-specific antibodies or antigen-binding fragments thereof as described above.
  • the substitution of an amino acid residue is a substitution of a conservative amino acid residue.
  • the invention relates to improved amyloid oligomer-specific antibodies as described above, or variants of antigen-binding fragments thereof, which are combined with improved amyloid oligomer-specific antibodies as described above.
  • its antigen-binding fragment has at least 85% identity, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.3%, 99.6% or higher identity while still substantially retaining one, more or all of the biological activities of the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof as described above.
  • identity calculation is based on a single amino acid residue. Therefore, the above-mentioned numerical values are only approximate values. Those skilled in the art can accurately calculate the above-mentioned identity based on the actual number of substitutions, insertions or deletions of amino acid residues. Calculate the corresponding percent identity.
  • Another aspect of the invention relates to an isolated nucleic acid molecule selected from:
  • a further aspect of the invention relates to an expression vector comprising operably linked nucleic acid molecules as described above.
  • Yet another aspect of the invention relates to a host cell comprising a nucleic acid molecule or expression vector as described above.
  • compositions comprising an improved amyloid oligomer-specific 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 carrier, diluent or excipient.
  • Yet another aspect of the invention relates to a method of producing an improved amyloid oligomer-specific antibody or 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 improved amyloid oligomer-specific antibody or antigen-binding fragment thereof, and optionally, the resulting product is isolated and purified.
  • Yet another aspect of the invention relates to a composition
  • a composition comprising an improved amyloid oligomer-specific antibody or an antigen-binding fragment thereof, a nucleic acid molecule, an expression vector, a host cell as described above, or obtained by a method as described above.
  • the therapeutic agent is selected from acetylcholinesterase inhibitors such as donepezil, galantamine, carbastine, etc., and aspartate receptor antagonists such as memantine, etc.
  • Another aspect of the present invention relates to the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, host cell or composition prepared for inhibiting the aggregation of A ⁇ in a subject. and/or A ⁇ oligomer-induced cytotoxicity, or use in a medicament for treating and/or preventing neurodegenerative diseases in a subject, or for diagnosing whether a subject suffers from a neurodegenerative disease.
  • the improved amyloid oligomer-specific antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, host cell or composition is used to inhibit the aggregation of A ⁇ in a subject and/or A ⁇ oligomer induces induced cytotoxicity, or for treating and/or preventing neurodegenerative diseases in a subject, or for diagnosing whether a subject suffers from a neurodegenerative disease.
  • Another aspect of the invention relates to a method for inhibiting A ⁇ aggregation and/or A ⁇ oligomer-induced cytotoxicity in a subject, or treating and/or preventing neurodegenerative diseases in a subject, or for diagnosing a subject.
  • a method for treating a subject or a subject suffering from a neurodegenerative disease comprising administering to the subject or cells of the subject a therapeutically effective amount of an improved amyloid oligomer-specific antibody or antigen-binding fragment thereof as described above. , nucleic acid molecules, expression vectors, host cells or compositions.
  • the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia, or spinocerebellar ataxia, preferably Preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis.
  • Yet another aspect of the present invention relates to the use of the improved amyloid oligomer-specific antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, host cells or compositions for diagnosing amyloid in a subject sample.
  • the presence and/or levels of proteotoxic forms and uses of the reagent are particularly useful.
  • Yet another aspect of the invention relates to an improved amyloid oligomer-specific antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, host cell or composition as described above in preparation for specific binding to a subject
  • Use of the highly toxic amyloid oligomer A ⁇ o*3F in a drug that inhibits its neurotoxicity, wherein the highly toxic amyloid oligomer A ⁇ o*3F is obtained from an A ⁇ oligomer mixture by immunoprecipitation of an antibody against 3F Isolated from A ⁇ its typical characteristics are A ⁇ high molecular weight oligomers. Based on size exclusion chromatography (SEC) analysis, its molecular weight is approximately 588kDa and its diameter is approximately 10nm.
  • SEC size exclusion chromatography
  • the amyloid is selected from amyloid beta, alpha-synuclein, mHTT, and SOD1.
  • the present invention carries out mature mutation transformation on the W20 antibody.
  • the main mutation site is located in the CDR3 region of the light chain, for example, the QTHRP sequence is mutated into the NSAVR sequence.
  • Molecular simulation test results show that the improved antibodies of the present invention, such as antibody 3F, can recognize specific structures on oligomers formed by amyloid protein aggregation. Moreover, the affinity of improved antibodies such as antibody 3F for binding to antigen A ⁇ oligomers is significantly increased, and the half-life in the body is also significantly extended.
  • 3F can inhibit the aggregation and cytotoxicity of A ⁇ , ⁇ -synuclein, mHTT and other amyloid proteins in vitro; intranasal administration at a ⁇ g level for 20 days can significantly improve the memory of AD transgenic mice and reduce brain damage. Pathological changes and inflammatory reactions in the body. The results of acute toxicity tests using antibodies 1,000 times greater than the therapeutic dose showed that the animals tolerated it well and no pathological changes were found in organs and tissues. Therefore, 3F has obvious therapeutic effect, good safety, good stability, and good medicinal properties.
  • the A ⁇ oligomers specifically recognized by this antibody are super toxic oligomers and are a mixture of A ⁇ oligomers.
  • the main toxic component in the compound has a strong pathogenic effect and plays a key role in the occurrence and development of AD.
  • the highly toxic oligomers recognized by 3F are present in the CSF and blood of AD patients and AD-derived MCI patients. and/or brain tissue, and its levels show extremely significant differences in the CSF, blood and/or brain of AD patients, MCI patients and healthy elderly people, so AD patients, MCI patients and healthy elderly people can be accurately distinguished crowd. This toxic oligomer also exists in AD transgenic mice and is directly related to the pathogenesis of AD transgenic mice.
  • the highly toxic A ⁇ oligomer recognized by 3F is called A ⁇ O*3F, which can be separated from the A ⁇ oligomer mixture by immunoprecipitation (3F antibody).
  • a ⁇ O*3F immunoprecipitation
  • Its typical characteristics are A ⁇ high molecular weight oligomers, based on Size exclusion chromatography (SEC) analysis shows that its molecular weight is about 588kDa and its diameter is about 10nm. It has a strong toxic effect on neurons and is more than 200 times more toxic than the A ⁇ oligomer mixture.
  • a ⁇ o*3F can activate microglia and/or astrocytes and secrete a large number of inflammatory factors. More importantly, the present invention prepares A ⁇ o*3F in vitro.
  • the A ⁇ o*3F prepared in vitro is consistent in composition with the product immunoprecipitated from the cerebrospinal fluid or plasma of AD patients using 3F antibodies, and has physical and chemical properties and functions. Same as above. Although some prior art monoclonal antibodies such as lecanemab also target A ⁇ aggregates, the A ⁇ aggregates they target are different from those targeted by the improved antibodies of the present invention, such as antibody 3F. Differently, the 3F antibody specifically targets the potent neurotoxic oligomer A ⁇ o*3F as mentioned above.
  • the improved antibodies of the present invention such as antibody 3F, have the following advantageous features:
  • a ⁇ o*3F The highly toxic amyloid oligomer A ⁇ o*3F that specifically binds to 3F is present in the cerebrospinal fluid (CSF), blood and/or brain tissue of AD patients and AD-derived mild cognitive impairment (MCI) patients, and The levels show extremely significant differences in the CSF, blood and/or brain of AD patients, MCI patients and healthy elderly people. It is a super toxic oligomer and the main toxic component in the A ⁇ oligomer mixture. It has a strong pathogenic effect and plays a key role in the occurrence and development of AD. Therefore, A ⁇ o*3F can be used as a target for diagnosis, prevention and/or treatment of MCI and/or AD in subjects, targeting A ⁇ o* 3F of 3F has good application value in AD diagnosis and treatment.
  • CSF cerebrospinal fluid
  • MCI AD-derived mild cognitive impairment
  • 3F specifically binds to the key pathogenic factor, the highly toxic amyloid oligomer A ⁇ o*3F, does not recognize monomers and fibers, and does not cause autoimmune reactions. Since amyloid monomers, such as A ⁇ and ⁇ -synuclein, have normal physiological functions in the body, antibodies that bind to these monomers can easily cause autoimmune reactions and reduce the effective concentration of antibody targets in the body. This is one of the main reasons why many antibody and vaccine drugs have failed in clinical trials. In addition, most of the dozens of A ⁇ -targeting antibodies and vaccines that have entered clinical trials have poor therapeutic effects or serious adverse reactions.
  • 3F can bind to a variety of amyloid proteins with different primary sequences, reducing their toxicity and promoting their clearance.
  • Amyloid proteins with different primary structures can form similar structures when aggregated. For example, both are rich in ⁇ -sheet structures and can combine with thioflavin (ThT) and Congo red. This constitutes the formation of 3F and various amyloid protein oligomers.
  • the basis of body integration 3F can specifically bind to oligomers of A ⁇ , ⁇ -synuclein, mHTT and SOD1, and inhibit the aggregation and cytotoxicity of each amyloid protein. Compared with W20, 3F is effective in AD, PD, HD, ALS and other models. The therapeutic effect of animals is more significant and has greater potential for application and transformation.
  • a ⁇ o*3F is an oligomeric form aggregated from A ⁇ monomers
  • all antibodies targeting A ⁇ monomers may bind to A ⁇ o*3F.
  • the antibody specifically recognizes and binds to A ⁇ o*3F.
  • Antibodies targeting A ⁇ monomers in the prior art can also bind to various forms such as A ⁇ monomers, oligomers, and fibers. Therefore, they are not compatible with the strong neurotoxic oligomers of the present invention.
  • the binding opportunity of A ⁇ o*3F in vivo is greatly reduced, and accordingly, its efficacy is also dispersed, resulting in a decrease in the therapeutic effect of AD.
  • Figure 1 Polyclonal ELISA showing phage antibody library.
  • Figure 2 Phage monoclonal ELISA shown.
  • Figure 3 Molecular simulation showing the structure of single chain antibody W20 and its key amino acid residues.
  • Figure 4 Shows that 3F antibody specifically binds A ⁇ oligomers. Dot blot experiment detects the binding ability of 3F antibody to A ⁇ 42 monomers and aggregates.
  • Figure 5 Shows that 3F antibody can significantly inhibit A ⁇ aggregation and A ⁇ oligomer-induced cytotoxicity:
  • Thioflavin T (ThT) experiment detects the inhibitory effect of 3F and W20 antibodies on A ⁇ aggregation
  • B.MTT experiment detects the inhibitory effect of 3F and W20 antibodies on neuronal cytotoxicity induced by A ⁇ oligomers.
  • FIG. 6 Shows that 3F antibody can significantly improve the cognitive function of AD transgenic mice:
  • Figure 7 Shows that 3F antibody can significantly reduce A ⁇ plaque levels in the brains of AD transgenic mice:
  • Figure 8 Shows that 3F antibody can significantly reduce A ⁇ levels in the brains of AD transgenic mice.
  • the ELISA method was used to detect the content of A ⁇ 40/42 in the brain homogenate of APP/PS1 mice.
  • Figure 9 Shows that 3F antibody can significantly reduce glial cell activation in the brains of AD transgenic mice:
  • Figure 10 Shows that 3F antibody can significantly reduce the levels of inflammatory factors in the brains of AD transgenic mice.
  • the ELISA method was used to detect the contents of IL-1 ⁇ and IL-6 in the brain homogenate of APP/PS1 mice.
  • Figure 11 Shows that 3F antibody can significantly improve the behavioral coordination and cognitive functions of PD transgenic mice:
  • Figure 12 Shows that 3F antibody can significantly reduce ⁇ -synuclein levels in the brains of PD transgenic mice:
  • A. Use pSer129- ⁇ -syn antibody to immunostain the brain slices of A53T ⁇ -synuclein transgenic mice to detect pathological ⁇ -synuclein levels in the mouse brain;
  • Figure 13 Shows that 3F antibody can significantly increase the level of tyrosine hydroxylase in the brain of PD transgenic mice:
  • TH tyrosine hydroxylase
  • Figure 14 Shows that the 3F antibody can significantly improve the spontaneous activity and anxiety behavior of HD transgenic mice: the total distance traveled by each group of mice in the open field experiment (A), the number of standings in all areas (B) and the stay in the central area Statistical analysis of time (C).
  • Figure 15 Shows that 3F antibody can significantly reduce mHTT aggregate levels in the brains of HD transgenic mice:
  • A. Use EM48 antibody to immunostain the brain slices of R6/2 mice to detect the level of mHTT aggregates in the mouse brain;
  • Figure 16 Shows that 3F antibody can significantly improve the motor function of ALS transgenic mice:
  • mice in each group on the rotarod in the rotarod experiment The duration of mice in each group on the rotarod in the rotarod experiment.
  • Figure 17 Shows that 3F antibody can significantly reduce SOD1 aggregate levels in the brainstem of ALS transgenic mice:
  • Figure 18 Shows that 3F antibody can significantly reduce the activation of glial cells in the brain of ALS transgenic mice:
  • Figure 19 Shows that antibodies can improve cognitive function in AD transgenic mice. The residence time of each group of APP/PS1 mice in the new arm in the Y maze experiment.
  • Figure 20 shows the results of sequence alignment of positive clones and W20.
  • Figure 21 Shows that the 3FI4 antibody (IgG4 subtype of the A16 antibody) is able to specifically bind to A ⁇ oligomers.
  • Figure 22 Schematic showing immunoprecipitation preparation of A ⁇ o*3F, A ⁇ *6E10 and A ⁇ -ID.
  • Figure 23 Shows the molecular weight size, morphological characterization and cytotoxicity of A ⁇ o*3F:
  • SEC Size exclusion chromatography
  • E IC50 of hA ⁇ o*3F toxicity to primary neurons.
  • Figure 24 Shows that A ⁇ o*3F significantly reduces the cognitive function of mice and damages neurons in the mouse brain:
  • Figure 25 shows that after the K at position 98 of the single chain antibody heavy chain is mutated to R, the affinity is further improved, suggesting that the amino acid at position 98 plays an important role in the binding process of the single chain antibody to A ⁇ oligomers.
  • Amyloid proteins include ⁇ -amyloid, microtubule-associated protein tau, ⁇ -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, prefibrillar forms, etc.
  • Amyloid oligomers are nonfibrillar aggregates formed by the aggregation of two or more amyloid monomer molecules.
  • 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.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4 subtypes
  • 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.
  • an antibody of the invention that was originally an IgG can be class-switched to an IgM antibody of the invention.
  • class switching technology can be used to convert one IgG subclass to another, for example from IgG1 to IgG2.
  • 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.
  • 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.
  • 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).
  • Heavy and light chains are variable Regions are typically responsible for antigen recognition, whereas the heavy and light chain constant domains can mediate interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells), Fc receptors, and third elements of the classical complement system. Binding of one component (C1q).
  • 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 hypervariable regions called "Complementarity Determining Regions (CDRs)" (HVR), which are interspersed with more conserved regions called “backbone regions” (FR).
  • CDRs Complementarity Determining Regions
  • FR backbone regions
  • Each VH and VL consists of three CDR domains and four FR domains, arranged in the following order from the amino terminus to the carboxyl terminus : FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • 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.
  • HVR hypervariable region
  • 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.
  • monoclonal antibody 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.
  • 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).
  • 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.
  • bisAb refers to having two different antigen-binding sites, which can bind to two target antigens at the same time.
  • Antibodies are both targeting and mediating It can induce another special function.
  • the special function effector molecules mediated can also be toxins, enzymes, cytokines, radionuclides, etc.
  • the two arms of the bispecific antibody binding to the antigen can come from Fab, Fv, ScFv respectively. Or dSFv etc.
  • 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.
  • 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.
  • 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
  • the antibodies and antigen-binding fragments thereof of the invention are single chain antibodies.
  • 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.
  • scFv single chain Fv
  • Methods for generating 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.
  • 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.
  • 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 to the extent that they exhibit the desired biological learning activity.
  • the invention provides variable region antigen-binding sequences from human antibodies.
  • 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.
  • 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.
  • the antibodies and antigen-binding fragments thereof of the invention are humanized antibodies.
  • 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.
  • the antibodies and antigen-binding fragments thereof of the invention are human antibodies or fully human antibodies.
  • 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).
  • 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.
  • amino acid substitutions can be used to obtain antibodies with further improved affinity.
  • 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 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 , 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.
  • the antibodies of the invention, or antigen-binding fragments thereof are expressed in at least Binds human amyloid with an affinity of 10 -7 M, especially toxic forms thereof, such as 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or higher.
  • the antibody or antigen-binding fragment thereof binds under physiological conditions (eg, in vivo).
  • 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.
  • binding 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 is at least 10 times lower, such as at least 100 times lower, at least 1,000 times lower.
  • 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.
  • ka (M-1x sec-1 or 1/Msec) refers to the binding rate constant of a particular antibody-antigen interaction.
  • KD (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing kd by ka .
  • 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 .
  • the polynucleotide encoding the CDRs is mutagenized (eg via random mutagenesis or by site-directed methods) to generate CDRs with substituted amino acid residues.
  • 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 trusted alignment (Eddy, SR, (2004), Where Did The BLOSUM62Alignment Score Matrix Come From?, Nature Biotech., 22(8):1035- 1036; Henikoff, JG, (1992), Amino acid substitution matrices from protein blocks), Proc. Natl. Acad. Sci. (USA), 89: 10915-10919; Karlin, S.
  • 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.
  • conservative substitutions may be defined by substitutions within an amino acid class that are reflected in one or more of the following three tables:
  • More conservative substitution groupings include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine Aminoamide.
  • 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.
  • 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.
  • amino acids in the CDR region may be substituted with conservative substitutions, as defined in the above 3 tables.
  • 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.
  • 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.
  • the invention provides polypeptide fragments comprising contiguous stretches of various lengths of the amino acid sequences disclosed herein.
  • 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.
  • 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.
  • VH domain includes the amino-terminal variable domain of an immunoglobulin heavy chain
  • 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.
  • CH2 domain includes a portion of a heavy chain molecule that ranges, for example, from about residue 244 to residue 360 of an antibody, using conventional numbering schemes (residue 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 does not pair closely with another domain. Instead, two N-linked branched sugar chains are inserted between the two CH2 domains of an intact native IgG molecule.
  • the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.
  • 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 to 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 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.
  • 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
  • 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.
  • the antibodies of the invention are monovalent antibodies comprising:
  • 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 )
  • the domains of other domains in the CH region do not contain any amino acid residues. Any of these amino acid residues
  • the group is capable of forming disulfide bonds with the same CH region or, in the presence of polyclonal human IgG, other covalent or stable non-covalent inter-heavy chain bonds with the same CH region.
  • the heavy chain of the monovalent antibody is modified such that the entire hinge region is deleted.
  • sequence of the monovalent antibody is modified such that it does not contain any acceptor sites for N-linked glycosylation.
  • 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 (such as 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.
  • 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.
  • “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.
  • 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.
  • 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 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.
  • 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 polybasic Alcohols and polyethylene glycols (PEG) (eg, PEG having a molecular weight between about 1,000 and 40,000 D, such as between about 2,000 and 20,000 D, such as about 3,000 and 12,000 D).
  • 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.
  • 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.
  • 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).
  • suitable expression control elements 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.
  • nucleic acids encoding antibodies of the invention are contained in naked DNA or RNA vectors, including, for example, linear expression elements (as described, for example, in Sykes and Johnston, Nat Biotech, 12, 355-59 (1997)), compact type nucleic acid vectors (as described, for example, in US 6,077,835 and/or WO 00/70087), plasmid vectors (such as pBR322, pUC 19/18 or pUC 118/119), minimally sized nucleic acid vectors (as described, for example, in 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 Cell Genetic
  • 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., Biotechnol.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH
  • 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.
  • suitable promoter enhancer and other elements that facilitate expression.
  • 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 (such as CMV IE).
  • 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.
  • host cells include yeast, bacteria, and mammalian cells (such as CHO or HEK cells).
  • 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.
  • 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.
  • a non-integrating nucleic acid such as a plasmid, cosmid, phagemid or linear expression element
  • 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).
  • human 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • preparations should meet the sterility, pyrogenicity, general safety and purity standards required by regulatory agencies (such as the Office of the FDA or EMA).
  • the glycosylation of the antibodies of the invention is modified.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • nucleotide sequence defined above is DNA
  • nucleotide sequence may be RNA.
  • 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”.
  • 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 acid, many Nucleotides or nucleotide sequences may be wholly or partially synthetic or recombinant.
  • 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.
  • 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.
  • 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.
  • “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.
  • 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.
  • compositions of the present invention may be administered in a single dose or in multiple doses.
  • 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.
  • 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.
  • a suitable pharmaceutical excipient diluent or carrier selected based on the intended route of administration and standard pharmaceutical practice.
  • the antibodies, or antigen-binding fragments thereof, nucleic acid molecules, or compositions of the invention can be administered in a variety of dosage forms.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Subjects are adults (e.g., subjects over 18 years of age).
  • the subject is a child (eg, a subject under 18 years of age).
  • the subject is male.
  • 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.
  • 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, ⁇ -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.
  • paramagnetic labels may also be employed, and detection is preferably performed using positron emission tomography (PET) or single photon emission computed tomography (SPECT).
  • 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
  • the sample is a biological sample.
  • 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.
  • sample is intended to mean a sample taken from an individual prior to any analysis.
  • the phage antibody library (W20 random mutation library, which is constructed by introducing random mutations in the antibody CDR region using error-prone PCR, and constructing the phage antibody library by efficiently transforming E.
  • helper phage M13K07 purchased from the MRC Center in the UK
  • 10mL of culture incubate at 37°C for 30min, centrifuge the bacterial solution at 3000g at 4°C for 10min, resuspend the pellet in 50mL of 2xTY culture medium, and add the final Amp with a concentration of 100 ⁇ g/mL, kanamycin (Kana) 50 ⁇ g/mL and 0.1% glucose was cultured overnight at 30°C on a shaker.
  • the phage stock solution was serially diluted, infected with E. coli at 37°C for 30 minutes, then spread on a 2xTY plate, and cultured at 37°C overnight. on 2xTY tablet The growing colonies were counted, and the number of phage clones per unit volume of the phage stock solution was calculated based on the dilution ratio. Repeat the above enrichment screening process three times, that is, perform the second, third, and fourth rounds of screening.
  • the results are shown in Figure 1.
  • the M13 phage group is a negative control
  • the PBS group is a blank control. It can be seen from the figure that from the first round of phage antibody library to the fourth round of phage antibody library, its binding ability to A ⁇ oligomers shows a gradually increasing trend. , and higher than the negative control group.
  • the phages that have gone through the above four rounds of enrichment screening are infected with E. coli HB2151 (purchased from the MRC Center in the UK) and spread on a plate for incubation. Single clones are picked and placed on a 96-well cell culture plate. 200 ⁇ L of 2xTY medium is added to each well. , the medium contains 100 ⁇ g/mL Amp and 1% glucose. Incubate overnight at 37°C on a shaker (300r/min). Take 2 ⁇ L of bacterial liquid from each well and add it to another new 96-well bacterial culture plate. Add 200 ⁇ L of 2xTY culture medium to each well. The culture medium contains 100 ⁇ g/mL Amp and 1% glucose.
  • the above-mentioned positive clones were sequenced and identified for sequence analysis, using sequencing primers LMB3:5'CAGGAAACAGCTATGAC 3'(SEQ ID No.2), pHEN seq:5'CTATGCGGCCCCATTCA 3'(SEQ ID No.3), consistent with the antibodies in the antibody library
  • the basic structure of the clone is a complete single-chain genetically engineered antibody.
  • the measured sequences are shown in SEQ ID No. 4-9 respectively, and the heavy chain and light chain CDR sequences of antibody 3F are shown in SEQ ID No. 10-15 respectively.
  • the antibody amino acid numbering is the position of the single-chain antibody where the amino acid is located.
  • the correspondence between the light chain CDR3 region and the Kabat numbering system is: (223, L89), (224, L90), (225, L91), (226, L92), (227, L93), (228, L94), (229, L95), (230, L96), (231, L97).
  • P228 in W20 was mutated to R228 in all mutant sequences, indicating that amino acid position 228 is critical for the binding of single-chain antibodies to A ⁇ oligomers (the Kabat numbering system is used for amino acid numbering, the same below).
  • X226 is glycine, valine, Any amino acid among leucine, isoleucine, phenylalanine, tryptophan or proline
  • X227 is phenylalanine, tryptophan, tyrosine, alanine, leucine or Any amino acid in isoleucine
  • X228 is alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, glycine, serine, threonine
  • Any amino acid (amino acid, cysteine, asparagine or glutamine) fragment is the key site for the binding of single-chain antibodies to A ⁇ oligomers.
  • This example is based on the original pdb file created by the homology modeling method based on the single-chain antibody protein sequence; on the basis of the original pdb file, add water and Cl - and Na + to adjust the pH value, and run the molecular dynamics simulation program , obtain the pdb file of human at normal temperature (298K); based on the pdb file at normal temperature, the molecular dynamics software Gromacs is used to calculate the all-atom structure of the single-chain antibody protein. By mutating the 226th, 227th, and 228th amino acid residues of the single-chain antibody W20 to alanine, the structure of the single-chain antibody was calculated through molecular simulation. The results are shown in Figure 3.
  • Example 6 3F antibody specifically binds to A ⁇ oligomers
  • Dot blot experiments are used to evaluate antibody binding to A ⁇ 42 monomers and A ⁇ oligomers.
  • a ⁇ samples incubated for 0, 0.5 min, 1 min, 2 min and 120 min were spotted on the nitrocellulose membrane and blocked with 5% skim milk at room temperature for 1 h.
  • the membrane was incubated with detection antibody 3F or 4G8 antibody (Biolegend, Cat. No.: 800704) for 1-2 hours at room temperature, then washed three times with 0.1% TBST, 5 minutes each time, and HRP-labeled anti-c-myc secondary antibody (Santa Cruz, Santa Cruz, Cat. No. H2317) or goat anti-mouse secondary antibody (Abcam, Cat. No. ab6789) and incubated at room temperature for 1 hour.
  • Example 8 3F antibody can significantly inhibit A ⁇ aggregation and A ⁇ oligomer-induced cytotoxicity
  • the ThT fluorescence method was used to evaluate the effect of 3F antibody on A ⁇ aggregation
  • the MTT method was used to evaluate the effect of 3F antibody on cytotoxicity induced by A ⁇ oligomers.
  • ThT fluorescence results showed that both 3F and W20 antibodies could significantly reduce A ⁇ aggregation, while A ⁇ alone continued to accumulate ( Figure 5, panel A).
  • the results of cell MTT experiments showed that 3F can significantly inhibit SH-SY5Y cytotoxicity induced by A ⁇ oligomers, and its inhibitory activity is better than that of W20 antibody (Figure 5, Panel B).
  • Example 9 3F antibody can significantly improve the cognitive function of AD transgenic mice
  • mice Six-month-old male APP/PS1 mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were randomly divided into 3 groups, with 8 mice in each group. They were given 3F, W20 or PBS (AD CON.) intranasally, once a day. times for a total of 28 days; mice in the wild-type control group were intranasally administered an equal amount of PBS. According to the water maze and Y maze experimental methods described by Yu et al. (Br J Pharmacol. 2020; 177:2860–2871.), the spatial memory and cognitive ability of mice were detected. The results showed that compared with the AD CON.
  • Example 10 3F antibody can significantly reduce A ⁇ levels in the brains of AD transgenic mice
  • Example 11 3F antibody can significantly reduce glial cell activation and inflammatory factor levels in the brains of AD transgenic mice
  • the main characteristics of neuroinflammation include excessive activation of glial cells and the massive release of inflammatory factors.
  • the inventors detected the activation of microglia and astrocytes in the mouse brain through Iba-1 immunostaining and GFAP immunostaining respectively.
  • the results showed that compared with mice in the AD CON. group, the Iba-1 and GFAP positive staining areas in the brain cortex and hippocampus of APP/PS1 mice treated with 3F were significantly reduced (Figure 9), indicating that 3F antibody can significantly reduce AD.
  • ELISA was used to determine the contents of IL-1 ⁇ and IL-6 in mouse brain homogenate. Compared with mice in the AD CON.
  • Example 12 3F antibody can significantly improve the behavioral coordination and cognitive functions of PD transgenic mice
  • mice 12-month-old male A53T ⁇ -synuclein transgenic mice (purchased from JACKSON LAB) were randomly divided into 3 groups, 8 mice in each group, and were given 3F, W20 or PBS (PD CON.) intranasally, once a day, for a total of On day 28, mice in the wild-type control group were given an equal amount of PBS intranasally.
  • the pole-climbing test was used to detect the motor coordination ability of mice.
  • Pole climbing test The pole used is a wooden pole with a rough surface and a base, 1 cm in diameter and 50 cm in length. Place the wooden pole stably in the mouse cage, and place the mouse on the top of the wooden pole with its head facing up. Let it automatically turn its head and then crawl its head down along the rod into the cage. The time it took for the mice to turn their heads and climb into the cage was recorded. If the mouse fell, slipped, or was unable to complete the task, the time it took to turn its head was recorded as 30 seconds, and the time it climbed back into the cage was recorded as 60 seconds. Perform this exercise 5 times a day, with the first two days being the training period and the third day being the testing period. Statistical analysis was performed on the time it took for the mice in each group to turn their heads and climb into the cage during the test period.
  • mice in the 3F group spent less time than the mice in the W20 group ( Figure 11, A and B).
  • the new object recognition experiment 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.
  • the cognitive index is determined by the following calculation formula: (number of new objects – number of old objects)/(number of new objects + the number of old objects). To avoid the influence of mouse odor, the box was wiped with 75% alcohol after each mouse ended its exploration.
  • mice in the 3F group also had a stronger cognitive ability for new things (Figure 11, C).
  • Example 13 3F antibody can significantly reduce ⁇ -synuclein levels in the brain of PD transgenic mice
  • Phosphorylated Ser129 ⁇ -synuclein is a specific pathological form of ⁇ -synuclein in the brains of PD patients.
  • the inventors used pSer129- ⁇ -syn antibody (Abcam, Cat. No. ab59264) as the primary antibody and used immunohistochemistry to detect pathological ⁇ -synuclein levels in the brains of A53T ⁇ -synuclein transgenic mice. There was an obvious pSer129- ⁇ -syn positive area in the brainstem of mice in the PD CON.
  • Example 14 3F antibody can significantly increase the level of tyrosine hydroxylase in the brain of PD transgenic mice
  • Tyrosine hydroxylase is the rate-limiting enzyme for dopamine synthesis and plays an important role in dopamine signaling. There is a negative correlation between the expression of TH and the severity of PD.
  • the inventors used immunohistochemical methods to detect TH levels in the brains of A53T ⁇ -synuclein transgenic mice. The results of the study showed that compared with WT mice, the expression level of TH in the brainstem of A53T ⁇ -synuclein transgenic mice was significantly reduced, while the expression of TH in the brainstem of PD mice treated with 3F antibody was significantly increased ( Figure 13). Compared with W20, 3F can significantly increase TH levels in the brains of PD transgenic mice.
  • Example 15 3F antibody can significantly improve the spontaneous activity and anxiety behavior of HD transgenic mice
  • mice Five-week-old male R6/2 mice (purchased from JACKSON LAB) were randomly divided into 3 groups, 8 mice in each group, and were given 3F, W20 or PBS (PD CON.) intranasally, once a day for a total of 28 days; wild-type mice The mice in the control group were given an equal amount of PBS intranasally. The open field test was used to detect the motor coordination ability of mice.
  • Open field experiment is a method to evaluate the autonomous behavior, exploratory behavior and tension of experimental animals in novel environments.
  • the open field is a white open plastic box (27cm ⁇ 27cm ⁇ 20.3cm), which is evenly divided into 9 areas in the form of a nine-square grid.
  • a camera is installed above the box to capture the movement trajectory of the mice.
  • the mice were placed in the open field in turn, one at a time. After adapting for 1 minute, the mice's movement trajectories were recorded with video for 30 minutes. Each mouse was treated with 70% B before the experiment. Alcohol cleans the inside of the box.
  • mice in the HD CON. group showed a significant reduction in autonomous activity and obvious anxiety symptoms, including the total distance traveled in the open field, the number of hind limb stances, and the stay in the central area. Time decreases. 3F antibody treatment can significantly increase the total activity distance, the number of standing times and the time spent in the central area of R6/2 mice ( Figure 14). Moreover, compared with W20, 3F can more significantly improve the spontaneous activity ability of R6/2 mice and reduce their anxiety state.
  • Example 16 3F antibody can significantly reduce the level of mHTT aggregates in the brain of HD transgenic mice
  • the EM48 antibody (Merck Millipore, Cat. No. MAB5374) can specifically recognize mHTT aggregates in pathological conditions.
  • the EM48 antibody was used to perform immunofluorescence staining on the brain tissue of R6/2 mice. The results showed that there was obvious positive staining of EM48 in the striatum and cortical areas of the brain of mice in the HD CON. group. After treatment with 3F, R6/2 The EM48-positive area in the mouse brain was significantly reduced ( Figure 15), indicating that 3F treatment effectively reduced the mHTT level in the brain of HD transgenic mice, and its effect was better than that of W20.
  • Example 17 3F antibody can significantly improve the motor function of ALS transgenic mice
  • mice Eleven-week-old male SOD1-G93A transgenic mice (purchased from the Model Animal Institute of Nanjing University) were randomly divided into 3 groups, with 8 mice in each group. 3F, W20 or PBS (ALS CON.) were administered intranasally once a day. A total of 28 days; wild-type control mice were given an equal amount of PBS intranasally. The suspension test and rotarod test were used to detect the motor coordination ability of mice.
  • mice in the ALS CON. group The results showed that compared with mice in the ALS CON. group, the retention time of SOD1-G93A mice treated with 3F in the suspension test was significantly increased, indicating that 3F can effectively enhance the muscle strength of mice ( Figure 16, Panel A) . Moreover, 3F is better than W20.
  • Rotarod test Rotarod test is conducted continuously for 3 days, and each day consists of two phases: training phase and test phase.
  • Training phase The rotation speed of the rotary rod is 4 rpm. The mice are trained on the rotary rod for 5 minutes and then returned to the cage. After 1 hour, they enter the test phase.
  • Test phase The rotating rod speed is accelerated from rest to 40 rpm within 5 minutes. The duration of the mouse on the rotating rod is recorded. The time when the mouse does not fall is recorded as 300 seconds. Test 3 times a day with 30 minutes interval between each time. 3 days for each group of mice There were a total of 9 tests within 3 days, and statistical analysis was performed on each group of mice for a total of 9 tests within 3 days, and the duration of each time on the rotarod was analyzed.
  • mice in the ALS CON. group on the rotarod was significantly lower than that of WT mice, while the residence time of SOD1-G93A mice in the 3F treatment group on the rotarod was significantly increased, and compared with mice in the W20 group. Compared with mice in group 3F, the duration of mice in group 3F was also longer on the rotarod (panel B of Figure 16).
  • Example 18 3F antibody can significantly reduce the level of SOD1 aggregates in the brainstem of ALS transgenic mice
  • Example 19 3F antibody can significantly reduce the activation degree of glial cells in the brain of ALS transgenic mice
  • the inventors evaluated the activation status of microglia and astrocytes in the brains of SOD1-G93A mice by immunostaining Iba-1 and GFAP. The results showed that compared with WT mice, the number of activated glial cells in the brains of mice in the ALS CON. group was significantly increased, while treatment with 3F could significantly reduce the number of microglia and astrocytes in the brains of SOD1-G93A mice. activation level. Compared with W20, 3F has a better ability to reduce glial cell activation in the brains of ALS transgenic mice (Figure 18).
  • Example 20 3F and other improved antibodies can significantly improve the cognitive function of AD transgenic mice
  • mice Six-month-old male APP/PS1 mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) were randomly divided into 8 groups, with 8 mice in each group, and were administered intranasally with 3F, W20, 3A, 6A, 8H, 11G, and 13A. Or PBS (AD CON.), once a day for 28 days; mice in the wild-type control group were given the same amount of PBS intranasally. According to the Y maze experimental method described by Yu et al. (Br J Pharmacol. 2020; 177:2860–2871.), the cognitive ability of mice was detected. The results showed that mice in the AD CON. group had no obvious preference for the new arm. The residence time of APP/PS1 mice in each antibody group in the new arm was significantly increased (Figure 19). Among them, 3F antibody has the most significant ability to improve cognition in AD transgenic mice.
  • Example 21 3FI4 antibody (IgG4 subtype of 3F antibody) specifically binds to A ⁇ oligomers
  • the present invention uses conventional methods to transform 3F antibodies into IgG4 subtype antibodies, named 3FI4 (3FI4-1, 3FI4-2, 3FI4-3, 3FI4-4, whose light chain sequences are all as shown in SEQ ID No. 17, and the heavy chain
  • the chain sequences are shown in SEQ ID No. 18-21, the light chain constant region sequences are shown in SEQ ID No. 22, and the heavy chain constant region sequences are shown in SEQ ID No. 23-26. shown).
  • the ELISA method is used to determine the binding ability of antibodies to A ⁇ oligomers. Specifically, according to /wells were coated with A ⁇ oligomers and coated overnight at 4°C.
  • Example 22 A ⁇ oligomer A ⁇ o*3F that specifically binds to 3F has super toxicity
  • a ⁇ monomer was incubated statically at 25°C for 2 days, and then incubated with Protein A magnetic beads cross-linked with 3F antibody at 4°C overnight. The next day, the magnetic beads were washed three times with 0.1% PBST, and then eluted with 20-100mM glycine (pH 2.0) for 3-5 minutes, eluted twice, and the eluent was neutralized to pH 7 with 1M Tris to obtain sA ⁇ o* 3F (A ⁇ os prepared in vitro).
  • APP/PS1 mouse brain homogenates or AD patient CSF samples from the First affiliated Hospital of Zhengzhou University, signed informed consent and obtained from the First affiliated Hospital of Zhengzhou University Approved by the Hospital Ethical Review Committee
  • AD patient CSF samples from the First affiliated Hospital of Zhengzhou University, signed informed consent and obtained from the First affiliated Hospital of Zhengzhou University Approved by the Hospital Ethical Review Committee
  • Protein A magnetic beads cross-linked with 3F antibody The next day, after the magnetic beads were washed three times with 0.1% PBST, they were eluted with 20mM-100mM glycine (specifically, 20mM, pH 2.0) for 3 minutes, and eluted twice.
  • a ⁇ o*3F was neutralized with 1M Tris to pH 7 to obtain A ⁇ o*3F in the brains of APP/PS1 mice (mA ⁇ o*3F, A ⁇ o*3F isolated from the brains of AD mice), or A ⁇ o*3F extracted from the CSF of AD patients (hA ⁇ o*3F, A ⁇ o* isolated from the cerebrospinal fluid of AD patients 3F).
  • the A ⁇ aggregate mixture after 3F immune depletion is called A ⁇ -ID, which are sA ⁇ -ID (prepared in vitro), mA ⁇ -ID (isolated and prepared in the brain of APP/PS1 mice) and hA ⁇ -ID (isolated from the CSF of AD patients). Preparation), used as a control.
  • the A ⁇ aggregate mixture A ⁇ *6E10 is prepared by mixing APP/PS1 mouse brain homogenate or AD patient CSF with Protein G magnetic beads cross-linked with 6E10 antibody, reacting at 4°C overnight, and then adding 20mM-100mM glycine (with Use 20mM, pH 2.0) to elute for 3-5 min (specifically 3 min), and elute twice.
  • the eluate is neutralized to pH 7 with 1M Tris to obtain A ⁇ o*6E10 (mA ⁇ o*6E10) in the brain of APP/PS1 mice.
  • a ⁇ o*6E10 isolated from the brains of AD mice), or A ⁇ o*6E10 extracted from the CSF of AD patients (hA ⁇ o*6E10, A ⁇ o*6E10 isolated from the CSF of AD patients) ( Figure 22).
  • the concentration of A ⁇ obtained by immunoprecipitation was measured using an A ⁇ detection kit.
  • Negative staining was performed with 2% uranyl acetate for 30 s.
  • the filter paper was blotted and air-dried.
  • the samples were examined under a transmission electron microscope (TEM, Hitachi H7700, Japan) at an operating voltage of 120 kV at 100,000 ⁇ magnification.
  • TEM transmission electron microscope
  • the results showed that mA ⁇ o*3F was a particle with a diameter of approximately 10 nm (panel A of Figure 23).
  • the molecular weight size and distribution of sA ⁇ o*3F and A ⁇ o*3F (hA ⁇ o*3F) extracted from the CSF of AD patients are consistent with the results of mA ⁇ o*3F.
  • MTT results show that the half inhibitory concentration (IC50) of sA ⁇ o*3F on N2a cytotoxicity is about 0.186nM, while the IC50 of sA ⁇ os before immunoprecipitation is about 63.26nM, and the IC50 difference between the two is 340 times ( Figure 23, B and C) .
  • the MTT method detects that the IC50 of mA ⁇ o*3F against N2a cells is about 0.111nM; the IC50 against primary neuron cells is about 0.057nM (Figure 23, D).
  • MTT assay showed that the IC50 of hA ⁇ o*3F on primary neurons was approximately 0.076 nM ( Figure 23, Panel E).
  • mice Three-month-old C57BL/6 mice were randomly divided into 6 groups, 6-8 mice in each group, and injected into the lateral cerebral ventricle. Grouping: group injected with 2.5nM (45.5pg) mA ⁇ o*3F, injected with 2.5nM (45.5pg) mA ⁇ *6E10 group, the group injected with 2.5nM (45.5pg) mA ⁇ -ID, the group injected with 600nM (10.9ng) mA ⁇ *6E10, the group injected with 600nM (10.9ng) mA ⁇ -ID, and the control group injected with Tris-Gly solvent. The behavioral and cognitive abilities of the mice were tested 24 hours after injection, and then the mice were dissected for brain pathological analysis.
  • mice treated with 2.5nM mA ⁇ o*3F and 600nM mA ⁇ *6E10 in the new arm was significantly reduced compared with the control mice.
  • mA ⁇ o*3F, but not mA ⁇ *6E10 or mA ⁇ -ID causes severe memory impairment in mice at low concentrations.
  • mA ⁇ *6E10 only increasing the concentration to 600 nM can cause similar symptoms in mice. memory impairment ( Figure 24, Panel A).
  • K98R mutant Mutation of amino acid K at position 100 of the 3F antibody to R (excluding the MA residue introduced for expression as a single-chain antibody, it is position 98 of the antibody number).
  • This variant is named K98R mutant, and the sequence is as SEQ ID NO: 27), the effect of K98R mutation on the affinity of this single-chain antibody was studied. How to use it is as follows.
  • the 98th amino acid is also the residue of the heavy chain CDR3 of the 3F antibody (according to the IMGT numbering, the heavy chain/light chain CDR sequences of the 3F antibody are respectively : CDR-H1: GTFFSSYA (SEQ ID NO: 28); CDR-H2: ISNLGLTT (SEQ ID NO: 29); CDR-H3: AKTTSRFDY (SEQ ID NO: 30); CDR-L1: QSISSY (SEQ ID NO: 31); CDR-L2: KAS; CDR-L3: QNSAVRPVT (SEQ ID NO: 32)).
  • the inventors also studied the in vivo activity of the K98R variant and found that it was further improved relative to the 3F antibody (data not shown).
  • 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.
  • other elements may optionally be present, whether related or unrelated to those specifically identified elements, unless expressly stated otherwise.
  • 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.
  • 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.
  • “at least one of A and B” 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, there is no A (and optionally includes elements other than A); in yet another In embodiments, refers to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.
  • 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.

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Abstract

本发明涉及改进的β淀粉样蛋白寡聚体(AβOs)特异性结合抗体。具体而言，本发明涉及一种W20抗体的改进形式，其相对于W20抗体，与AβOs的亲和力显著提高，能够更显著地抑制Aβ的聚集和AβOs诱导的神经细胞毒性，更有效地改善阿尔茨海默病模型小鼠的认知和记忆功能，降低小鼠脑内的病理变化。改进形式的抗体能够与β淀粉样蛋白、α-突触核蛋白、mHTT和SOD1的寡聚体特异结合，并抑制各淀粉样蛋白的聚集和细胞毒性，比W20抗体具有更优的治疗帕金森病、亨廷顿舞蹈病、肌萎缩侧索硬化症等多种淀粉样蛋白类疾病的潜力，该改进型抗体能够特异性结合的强毒性淀粉样蛋白寡聚体Aβo*3F，具有更好的AD诊断价值，所述W20抗体的氨基酸序列如SEQ ID No.1所示。

Description

改进的β淀粉样蛋白寡聚体特异性结合抗体 技术领域
本发明涉及特异性结合β淀粉样蛋白寡聚体(AβOs)的抗体，尤其涉及改进的特异性结合特定AβOs的抗体。
背景技术
阿尔茨海默病(AD)是常发于老年人的一种慢性神经退行性疾病，临床症状主要表现为记忆力下降，认知障碍以及自理能力的丧失。AD已被列为导致人类死亡的第四大疾病，仅次于癌症、心脏病和中风，其发病率表现为明显的年龄依赖性。随着全球人口老龄化加剧，AD患病人数逐年增加。据统计，2018年全球大约有5000万AD患者，预计2050年全球AD患者可高达1.3亿人。AD医疗费用支出巨大，2015年全球用于AD病人的治疗和护理费用约8180亿美元，预计2030年将增至2万亿美元。近十余年来，全球用于AD治疗药物的研发已投入约1100亿美元，但巨大的人力物力投入仍未促使特异性治疗药物面世，上百种治疗药物在临床试验中相继失败。开发针对AD发病机理的药物已成为许多国际制药公司和研究单位竞相追逐的研究热点。
AD是由淀粉样蛋白Aβ和微管相关蛋白tau聚集形成具有毒性作用的寡聚物而引起的老年人主要以记忆力下降和脑部形成老年斑、神经元内形成神经缠绕为特征的神经退行性疾病。国际上普遍认为，引起AD发生发展的最主要因素为细胞毒性最大的Aβ寡聚体，而非Aβ单体和纤维。AD患者脑内Aβ寡聚体水平与AD的发生发展及严重程度高度相关。Aβ寡聚体对神经突触有高度的亲和性，它可以触发关键性突触蛋白的重新分布，诱导突触相关谷氨酸受体的过度活化，还可破坏神经细胞膜，扰乱突触Ca2+稳态水平，进而提高细胞内的氧化应激和线粒体的损伤。Aβ寡聚体结合至神经突触上可以激活补体系统，促使小胶质细胞对突触过度修剪和吞噬，破坏神经突触的结构，诱发突触功能紊乱，造成突触的损伤和过度丢失，导致认知功能下降。因此，靶向Aβ寡聚体，抑制Aβ寡聚体水平及其诱导的神经毒性是针对病因治疗AD的理想策略之一。而靶向Aβ单体的治疗方案，特别是靶向Aβ单体的免疫治疗则可带来炎症及突触丢失等不良反应，导致临床试验屡屡失败。通过免疫治疗策略清除Aβ，降低Aβ寡聚体的水平，一直为AD治疗领域研究的焦点。近年来，先后有数十种靶向Aβ的抗体和疫苗进入了 临床试验阶段。如靶向Aβ纤维的第一代疫苗AN1792，以及靶向Aβ单体或纤维的抗体如bapineuzumab(anti-Aβ1-5)、solanezumab(anti-Aβ13-28)、gantenerumab(anti-Aβ1-11)和ponezumab(anti-AβC端)等。虽然这些制剂在动物试验阶段表现出了良好的治疗效果，显著改善AD转基因动物的认知水平，降低动物脑内老年斑的量及其他病理变化，但在AD临床试验中，由于治疗效果欠佳或严重的副作用，迄今鲜有通过临床III期试验。2021年6月，美国FDA通过加速审批项目批准了Aduhelm(aducanumab)抗体上市。Aducanumab可特异性地识别Aβ寡聚体，在临床试验中显示出一定的疗效，但该抗体仍存在脑水肿、炎症等不良反应，抗体疗效也颇受争议。
许多神经退行性疾病以淀粉样蛋白异常聚集为特征，如Aβ、tau、α-突触核蛋白、mHTT和SOD1等蛋白，这些蛋白单体聚集形成的寡聚体是导致AD、帕金森病(PD)、亨廷顿舞蹈病(HD)、肌萎缩侧索硬化症(ALS)等发生发展的关键致病因素，也是诊断和治疗该类疾病的有效靶点。不同淀粉样蛋白的寡聚体和纤维沉积于大脑的不同部位，损伤相应区域的神经细胞，导致认知和行为障碍。这些淀粉样蛋白虽然一级序列不同，但其聚集而成的寡聚体能够形成相似的空间立体结构，并具有相似的毒性机制。因此，靶向淀粉样蛋白毒性寡聚体的治疗策略，对于多种神经退行性疾病的诊断和治疗具有重要意义。此外，研究表明，在淀粉样蛋白疾病中各种淀粉样蛋白之间具有协同促进的作用，某一种淀粉样蛋白的聚集和疾病的发生发展会促进或诱导其他淀粉样蛋白的聚集并参与到疾病的病理过程中。例如，临床研究发现，50％的AD患者脑内除了存在Aβ斑块的沉积和tau蛋白引起的神经纤维缠绕外，还存在着明显的由α-突触核蛋白聚集和沉积产生的路易小体(LB)。与没有LB沉积的AD患者相比，这类患者具有更严重的病理特征和认知功能的下降。转基因动物实验研究表明，Aβ、tau和α-突触核蛋白能够相互协同作用，促进彼此的聚集和沉积，并加速了转基因小鼠脑内的病理变化和认知能力的损伤。因此，仅针对一种淀粉样蛋白的治疗方式或许不足以治愈淀粉样蛋白疾病，特异性靶向多种淀粉样蛋白寡聚体所共有的结构特征而不依赖其一级氨基酸序列的制剂将成为治疗一种或多种淀粉样蛋白疾病的理想策略。
自2005年起，本发明人开展了淀粉样蛋白寡聚体特异抗体的研究，在国际上率先筛选获得了能够识别多种淀粉样蛋白寡聚体的全人源单链抗体W20(中国专利CN101463082A)。该抗体能够与引起AD、PD、HD和ALS等多种淀粉样蛋白类疾病的Aβ、α-突触核蛋白、mHTT、和SOD1等寡聚体结合，而不与各淀粉样蛋白的单体和纤维结合；该抗体还可抑制各淀粉样蛋白的聚集和细胞毒性；改善疾病模型小鼠的认知功能和行为协 调能力，降低小鼠的神经病理变化(Biochimica et Biophysica Acta 2011，1814，1703-1712；AD:Current Alzheimer Research,2014，11，69-78；PD&HD:Scientific Reports，2016，6，36631；ALS:International Immunopharmacology 2018，65，413-421)。
截至目前，本领域依然存在进一步增强淀粉样蛋白寡聚体特异抗体的成药性，以及对有效治疗和/或预防神经退行性疾病的抗体药物的迫切需求。
发明内容
本发明的一个方面涉及一种改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段，其相对于W20抗体在选自第226位、第227位和第228位氨基酸残基位置的一个或多个位置处具有氨基酸取代，所述W20抗体的氨基酸序列如SEQ ID No.1所示，所述抗体的氨基酸位置编号采用Kabat编号系统确定。
在一些实施方案中，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段的第226位的氨基酸残基被取代为与丙氨酸性质相近的非极性、疏水性氨基酸，优选地，被取代为甘氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、色氨酸或脯氨酸，更优选地，被取代为甘氨酸、苯丙氨酸、色氨酸。
在一些实施方案中，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段的第227位氨基酸残基被取代为与缬氨酸性质相近的疏水性氨基酸，优选地，被取代为苯丙氨酸、色氨酸、酪氨酸、丙氨酸、亮氨酸或异亮氨酸，更优选地，被取代为苯丙氨酸、异亮氨酸、亮氨酸。
在一些实施方案中，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段的第228位氨基酸残基被取代为与精氨酸结构相近的脂肪族氨基酸，优选地，被取代为丙氨酸、缬氨酸、亮氨酸、异亮氨酸、蛋氨酸、天冬氨酸、谷氨酸、赖氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺或谷氨酰胺，更优选地，被取代为赖氨酸、天冬酰胺、谷氨酰胺。
在一些实施方案中，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段的第226-228位氨基酸残基分别被取代为AVR、GSR、WVR、FER、NFR或VRR。
在一些实施方案中，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段的第224位、第225位氨基酸残基分别被取代为谷氨酰胺、苏氨酸。
在一些实施方案中，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段都能特异性结合Aβo*3F寡聚体。
在一些实施方案中，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段为抗原结合片段，优选地，选自由scFv、F(ab')2、Fab'、Fab、Fd、Fv、双特异抗体、骆驼抗体、CDR和抗体最小识别单位(dAb)组成的组的抗原结合片段，更优选地，选自由scFv、F(ab')2、Fab'、Fab组成的组的抗原结合片段。在一些实施方案中，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段为scFv或F(ab’)2。
在一些实施方案中，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段为单克隆抗体、嵌合抗体、人源化抗体或全人抗体，优选地，所述抗体选自由IgG、IgM、IgA、IgD和IgE及其亚型组成的组，更优选地，所述抗体选自亚型IgG1-4组成的组，更优选地，所述抗体是IgG4亚型。在一些实施方案中，所述抗体的重链氨基酸序列如SEQ ID No.18-21任一项所示，和/或所述抗体的轻链氨基酸序列如SEQ ID No.17所示。在一些实施方案中，所述抗体的重链恒定区氨基酸序列如SEQ ID No.23-26任一项所示，和/或所述抗体的轻链恒定区氨基酸序列如SEQ ID No.22所示。
在一些实施方案中，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段为scFv且其氨基酸序列如SEQ ID No.5、4和6-9任一项所示。
在一些实施方案中，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段的轻链的CDR3的序列如SEQ ID No.15所示。
在一些实施方案中，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段相对于3F抗体在第98位被取代为精氨酸。在一些实施方案中，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段的重链氨基酸序列如SEQ ID No.33-36任一项所示，轻链氨基酸序列如上所述，在一些实施方案中，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段为scFv且其氨基酸序列如SEQ ID No.27所示。
在一些实施方案中，本发明涉及如上所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段的变体，其相对于如上所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段具有1个或多个氨基酸残基的取代、插入、或缺失，如1、2、3、4、5、10、15、20、25、30或更多个氨基酸残基的取代、插入、或缺失，同时仍实质上保持如上所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段的一种、多种或全部生物活性。在一些实施方案中，所述氨基酸残基的取代是保守氨基酸残基的取代。在一些实施方案中，本发明涉及如上所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段的变体，其与如上所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段具有至少85％的同一性，如至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.1％、 99.3％、99.6％或更高的同一性，同时仍实质上保持如上所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段的一种、多种或全部生物活性。本领域技术人员知晓，基于单个氨基酸残基是计算上述同一性的基础，因此，上述数值仅是近似数值，本领域技术人员根据实际发生的氨基酸残基的取代、插入或缺失的数量可以精确地计算相应的同一性百分比。
本发明的另一个方面涉及一种分离的核酸分子，其选自：
(1)DNA或RNA，其编码如上所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段；
(2)与(1)中定义的DNA或RNA完全互补的核酸分子。
本发明的进一步的方面涉及一种表达载体，其包含有效连接的如上所述的核酸分子。
本发明的再一个方面涉及一种宿主细胞，其包含如上所述的核酸分子或表达载体。
本发明的另一个方面涉及一种组合物，其包含如上所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段、核酸分子、表达载体或宿主细胞，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明的又一个方面涉及一种生产如上所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段的方法，其包括步骤：
将如上所述的宿主细胞在适合所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段表达的培养条件下培养，任选地，分离、纯化所得的产物。
本发明的再一个方面涉及一种组合物，其包含如上所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段、核酸分子、表达载体、宿主细胞，或如上所述方法获得的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段、核酸分子、表达载体、宿主细胞，以及另一种神经退行性疾病治疗剂，优选地，所述另一种神经退行性疾病治疗剂选自乙酰胆碱酯酶抑制剂如多奈哌齐、加兰他敏、卡巴斯汀等，和天门冬氨酸受体拮抗剂如美金刚等。
本发明的另一个方面涉及所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段、核酸分子、表达载体、宿主细胞或组合物在制备用于抑制受试者中Aβ的聚集和/或Aβ寡聚体诱导的细胞毒性，或用于治疗和/或预防受试者中神经退行性疾病，或用于诊断受试者是否患有神经退行性疾病的药物中的用途。
在一些实施方案中，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段、核酸分子、表达载体、宿主细胞或组合物用于抑制受试者中Aβ的聚集和/或Aβ寡聚体诱 导的细胞毒性，或用于治疗和/或预防受试者中神经退行性疾病，或用于诊断受试者是否患有神经退行性疾病。
本发明的另一个方面涉及一种抑制受试者中Aβ的聚集和/或Aβ寡聚体诱导的细胞毒性，或治疗和/或预防受试者中神经退行性疾病，或用于诊断受试者是否患有神经退行性疾病的方法，其包括向所述受试者或受试者的细胞施用治疗有效量的如上所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段、核酸分子、表达载体、宿主细胞或组合物。
在一些实施方案中，所述神经退行性疾病选自由阿尔兹海默病、帕金森病、亨廷顿舞蹈症、肌萎缩性侧索硬化、额颞叶痴呆或脊髓小脑共济失调组成的组，优选地，所述神经退行性疾病选自由阿尔兹海默病、帕金森病、亨廷顿舞蹈症和肌萎缩性侧索硬化组成的组。
本发明的又一个方面涉及所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段、核酸分子、表达载体、宿主细胞或组合物在制备用于诊断受试者样品中淀粉样蛋白毒性形式的存在和/或水平的试剂中的用途。
本发明的又一个方面涉及如上所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段、核酸分子、表达载体、宿主细胞或组合物在制备用于特异性结合受试者中的强毒性淀粉样蛋白寡聚体Aβo*3F并抑制其神经毒性的药物中的用途，其中所述强毒性淀粉样蛋白寡聚体Aβo*3F采用针对3F抗体的免疫沉淀从Aβ寡聚体混合物中分离而来，其典型特征为Aβ高分子量寡聚体，基于分子排阻色谱(SEC)分析，其分子量大小约为588kDa，直径约为10nm。
在一些实施方案中，所述的淀粉样蛋白选自β淀粉样蛋白、α-突触核蛋白、mHTT和SOD1。
换言之，为了增强抗体的成药性，本发明对W20抗体进行了成熟突变改造，主要突变位点位于轻链CDR3区域，例如将QTHRP序列突变为NSAVR序列。分子模拟试验结果表明，本发明的改进的抗体如抗体3F能够识别淀粉样蛋白聚集形成的寡聚体上的特异结构。并且，改进的抗体如抗体3F与抗原Aβ寡聚体结合的亲和力显著提高，在体内的半衰期也明显延长。3F在体外可以抑制Aβ、α-突触核蛋白、mHTT等多种淀粉样蛋白的聚集和细胞毒性；以μg级别剂量经鼻给药20天，可以显著提高AD转基因小鼠的记忆力，降低脑部的病理变化和炎症反应。应用大于治疗量1000倍的抗体开展的急性毒性试验结果表明，动物的耐受性良好，脏器和组织未见病理变化。因此，3F治疗效果明显，安全性好，稳定性好，具有较好的成药性。
更有意义的是，该抗体特异识别的Aβ寡聚体为超强毒性寡聚体，是Aβ寡聚体混 合物中最主要的毒性成分，具有强烈的致病作用，在AD的发生和发展中起着关键作用，3F识别的强毒性寡聚体存在于AD患者和AD源性MCI患者的CSF、血液和/或脑组织中，且其水平在AD患者、MCI患者和健康老年人三类人群的CSF、血液和/或脑中呈现极显著差别，因此可精确区分出AD患者、MCI患者和健康老年人群。该种毒性寡聚体也存在于AD转基因小鼠中，并与AD转基因小鼠的发病直接相关。本发明中将3F识别的强毒性Aβ寡聚体称为AβO*3F，可采用免疫沉淀(3F抗体)从Aβ寡聚体混合物中分离而来，其典型特征为Aβ高分子量寡聚体，基于分子排阻色谱(SEC)分析，其分子量大小约为588kDa，直径约为10nm，对神经元有强毒性作用，其毒性比Aβ寡聚体混合物毒性强200倍以上。Aβo*3F可激活小胶质细胞和/或星形胶质细胞，分泌大量的炎症因子。更重要的是，本发明在体外制备出了Aβo*3F，体外制备的Aβo*3F与利用3F抗体从AD患者的脑脊液或血浆中免疫沉淀而来的产物在组成上一致，在理化性质和功能上相同。虽然某些现有技术的单抗如仑卡奈单抗(lecanemab)也靶向Aβ聚集体，但其靶向的Aβ聚集体与本发明的改进的抗体如抗体3F所靶向的Aβ聚集体不同，3F抗体特异靶向如上所述的强神经毒性寡聚体Aβo*3F。
具体地，本发明的改进的抗体如抗体3F具有如下优势特点：
1)与W20抗体相比，3F与强毒性淀粉样蛋白寡聚体Aβo*3F间的亲和力显著提高，能够更好地抑制淀粉样蛋白的聚集和细胞毒性，具有更好的治疗潜力。
2)3F特异性结合的强毒性淀粉样蛋白寡聚体Aβo*3F存在于AD患者和AD源性轻度认知障碍(MCI)患者的脑脊液(CSF)、血液和/或脑组织中，且水平在AD患者、MCI患者和健康老年人三类人群中的CSF、血液和/或脑中呈现极显著差别，为超强毒性寡聚体，是Aβ寡聚体混合物中最主要的毒性成分，具有强烈的致病作用，在AD的发生和发展中起着关键作用，因此，Aβo*3F可以作为诊断、预防和/或治疗受试者中MCI和/或AD的靶点，靶向Aβo*3F的3F具有较好的AD诊断和治疗的应用价值。
3)3F特异性结合致病关键因素强毒性淀粉样蛋白寡聚体Aβo*3F，不识别单体和纤维，不引起自身免疫性反应。由于淀粉样蛋白单体，如Aβ和α-突触核蛋白在体内具有正常生理功能，与这些单体结合的抗体易造成自身免疫性反应，同时降低了机体中抗体靶向靶标的有效浓度，这也是许多抗体和疫苗药物在临床试验中遭到失败的主要原因之一。此外，目前进入了临床试验阶段的数十种靶向Aβ的抗体和疫苗绝大部分都存在治疗效果欠佳或严重的不良反应的情况。研究表明，由于很多抗体靶向Aβ单体以及其抗原抗体复合物会引发炎症反应，并引起神经突触丢失，导致靶向Aβ免疫治疗AD失 败，而3F抗体能够克服这种副作用，达到较好的AD治疗效果。
4)3F可与多种具有不同一级序列的淀粉样蛋白结合，降低他们的毒性，促进其清除。不同一级结构的淀粉样蛋白聚集时可形成相似的结构，如都富β片层结构，都能够与硫黄素(ThT)和刚果红结合等，这构成了3F与多种淀粉样蛋白寡聚体结合的基础。3F可与Aβ、α-突触核蛋白、mHTT和SOD1的寡聚体特异结合，并抑制各淀粉样蛋白的聚集和细胞毒性，与W20相比，3F对AD、PD、HD、ALS等模型动物的治疗作用更显著，具有更大的应用转化潜力。
5)3F的治疗用量小，治疗效果明显，治疗机理清楚，安全性高，性质稳定。
6)由于Aβo*3F是一种由Aβ单体聚集而成的寡聚体形式，因此，所有靶向Aβ单体的抗体都可能与Aβo*3F结合，然而，相对于本发明的抗体如3F抗体特异性识别和结合Aβo*3F，现有技术的靶向Aβ单体的抗体也能够结合Aβ单体、寡聚体和纤维等多种形式，因此，其与本发明的强神经毒性寡聚体Aβo*3F的结合机会大大降低，相应地，其作用效力也被分散，导致AD治疗效果下降。
附图说明
图1：显示了噬菌体抗体库多克隆ELISA。
图2：显示了噬菌体单克隆ELISA。
图3：显示了单链抗体W20及其关键氨基酸残基的结构分子模拟。
图4：显示了3F抗体特异结合Aβ寡聚体。Dot blot实验检测3F抗体与Aβ42单体和聚集体的结合能力。
图5：显示了3F抗体能够显著抑制Aβ的聚集及Aβ寡聚体诱导的细胞毒性：
A.硫磺素T(ThT)实验检测3F和W20抗体对Aβ聚集的抑制作用；
B.MTT实验检测3F和W20抗体对Aβ寡聚体诱导的神经细胞毒性的抑制作用。
图6：显示了3F抗体能够显著改善AD转基因小鼠的认知功能：
A.水迷宫实验中的训练期各组小鼠找到平台的潜伏期；
B.水迷宫实验中撤除平台后的探索期，小鼠穿过平台所在位置的次数；
C.Y迷宫实验中各组小鼠在新臂的停留时间。
图7：显示了3F抗体能够显著降低AD转基因小鼠脑内Aβ斑块水平：
A.使用4G8抗体对APP/PS1小鼠脑片进行免疫染色以检测小鼠脑内老年斑水平；
B.对小鼠皮层和海马区域的老年斑面积的定量统计。
图8：显示了3F抗体能够显著降低AD转基因小鼠脑内Aβ水平。应用ELISA方法检测APP/PS1小鼠脑匀浆中Aβ40/42的含量。
图9：显示了3F抗体能够显著降低AD转基因小鼠脑内的胶质细胞活化程度：
A.通过Iba-1免疫染色和GFAP免疫染色检测APP/PS1小鼠脑内小胶质细胞和星形胶质细胞的活化情况；
B.小鼠脑内皮层和海马区域的Iba-1和GFAP阳性染色面积的定量统计。
图10：显示了3F抗体能够显著降低AD转基因小鼠脑内炎症因子水平。应用ELISA方法检测APP/PS1小鼠脑匀浆中IL-1β和IL-6的含量。
图11：显示了3F抗体能够显著改善PD转基因小鼠的行为协调能力及认知功能：
A.爬杆实验中各组小鼠转头所用时间；
B.爬杆实验中各组小鼠爬至笼内所用时间；
C.新事物识别实验中各组小鼠的认知指数。
图12：显示了3F抗体能够显著降低PD转基因小鼠脑内α-突触核蛋白水平：
A.使用pSer129-α-syn抗体对A53Tα-突触核蛋白转基因小鼠脑片进行免疫染色以检测小鼠脑内病理性α-突触核蛋白水平；
B.对小鼠脑干区pSer129-α-syn阳性染色面积的定量统计。
图13：显示了3F抗体能够显著提升PD转基因小鼠脑内酪氨酸羟化酶水平：
A.使用酪氨酸羟化酶(TH)抗体对A53Tα-突触核蛋白转基因小鼠脑片进行免疫染色以检测小鼠脑内TH水平；
B.对小鼠脑干区TH阳性染色面积的定量统计。
图14：显示了3F抗体能够显著改善HD转基因小鼠的自发活动能力和焦虑行为：旷场实验中各组小鼠总路程(A)、在全部区域的站立次数(B)和中央区域的停留时间(C)的统计分析。
图15：显示了3F抗体能够显著降低HD转基因小鼠脑内mHTT聚集体水平：
A.使用EM48抗体对R6/2小鼠脑片进行免疫染色以检测小鼠脑内mHTT聚集体水平；
B.对小鼠脑内EM48阳性染色面积的定量统计。
图16：显示了3F抗体能够显著改善ALS转基因小鼠的运动机能：
A.悬挂实验中各组小鼠的停留时间；
B.转棒实验中各组小鼠在转棒上的持续时间。
图17：显示了3F抗体能够显著降低ALS转基因小鼠脑干中SOD1聚集物水平：
A.使用SOD1抗体对SOD1-G93A小鼠脑片进行免疫染色以检测小鼠脑内SOD1聚集体水平；
B.对小鼠脑干中SOD1阳性染色面积的定量统计。
图18：显示了3F抗体能够显著降低ALS转基因小鼠脑内胶质细胞的活化程度：
A.通过Iba-1免疫染色和GFAP免疫染色检测SOD1-G93A小鼠脑内小胶质细胞和星形胶质细胞的活化情况；
B.小鼠脑内Iba-1和GFAP阳性染色面积的定量统计。
图19：显示了抗体能够改善AD转基因小鼠的认知功能。Y迷宫实验中各组APP/PS1小鼠在新臂的停留时间。
图20：显示了阳性克隆的序列与W20的序列比对的结果。
图21：显示了3FI4抗体(A16抗体的IgG4亚型)能够与Aβ寡聚体特异结合。
图22：显示了免疫沉淀制备Aβo*3F、Aβ*6E10和Aβ-ID的示意图。
图23：显示了Aβo*3F的分子量大小、形态表征和细胞毒性：
A：分子排阻色谱(SEC)分析mAβo*3F分子量大小；mAβo*3F和SEC标准品的SEC分析(Superdex 200 10/300)，SEC标准品：1.甲状腺球蛋白(669kDa)，2.铁蛋白(440kDa)，3.醛缩酶(158kDa)，4.伴清蛋白(75kDa)，5.卵清蛋白(44kDa)，6.碳酸酐酶(29kDa)；
B：sAβos对N2a细胞毒性的IC50；
C：sAβo*3F对N2a细胞毒性的IC50；
D：mAβo*3F对N2a细胞和原代神经元毒性的IC50；
E：hAβo*3F对原代神经元毒性的IC50。
图24：显示了Aβo*3F显著降低小鼠认知功能并损伤小鼠脑内神经元：
A：Y迷宫实验中各组小鼠在新臂的持续时间；
B：Golgi染色检测小鼠神经元树突棘的密度；
C：对B中树突棘密度的统计分析。
图25：显示了单链抗体重链98位K突变成R后，亲和力得到进一步提升，提示98位氨基酸在该单链抗体与Aβ寡聚体结合过程中起到重要作用。
具体实施方式
定义
淀粉样蛋白包括β-淀粉样蛋白、微管相关蛋白tau、α-突触核蛋白、亨廷顿蛋白、胰淀粉样蛋白、超氧化物歧化酶1(SOD1)和TDP-43蛋白等，包括其单体、寡聚体、前纤维或纤维等聚集形式。
淀粉样蛋白毒性形式是指在神经退行性疾病的发生发展中发挥负面作用的淀粉样蛋白的形式，例如寡聚体形式、前纤维形式等。
淀粉样蛋白寡聚体是指由两个或多个淀粉样蛋白单体分子聚集而成的非纤维样聚集体形式。
术语“抗体”是指免疫球蛋白分子或免疫球蛋白分子的具有结合到抗原的表位上的能力的片段。天然存在的抗体典型地包含四聚体，其通常由至少两条重(H)链和至少两条轻(L)链构成。免疫球蛋白包括如下同种型：IgG、IgA、IgM、IgD以及IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4亚型，IgA可分为IgA1和IgA2亚型。轻链根据恒定区的不同分为κ链和λ链。本发明的抗体可以具有任何同种型。同种型的选择通常由希望的效应物功能(如ADCC诱导)来决定。示例性同种型是IgG1、IgG2、IgG3和IgG4。可以使用人轻链恒定域κ或λ中任一者。如果需要，可以通过已知方法转换本发明抗体的类别。例如，最初是IgG的本发明抗体可以类别转换为本发明的IgM抗体。此外，类别转换技术可以用来将一个IgG亚类转化成另一亚类，例如从IgGl转换为IgG2。因此，本发明抗体的效应物功能可以通过同种型切换变为例如IgG1、IgG2、IgG3、IgG4、IgD、IgA、IgE或IgM抗体，以用于各种治疗用途，条件是所述抗体的补体如C1q和/或Fc受体结合活性降低或消除。在一些实施方案中，本发明的抗体是IgM或IgG1、2、3或4型抗体。如果抗体的氨基酸序列相对于其他同种型与该同种型大部分同源，则该抗体属于特定同种型。
在本文中，术语“抗体”以最广泛意义使用，指包含抗原结合位点的蛋白质，涵盖各种结构的天然抗体和人工抗体，包括但不限于完整抗体和抗体的抗原结合片段。
“可变区”或“可变结构域”是抗体的重链或轻链中参与抗体与其抗原的结合的结构域。抗体的每条重链由重链可变区(本文中简称为VH)和重链恒定区(本文中简称为CH)构成，重链恒定区通常由3个结构域(CH1、CH2和CH3)构成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区(本文中缩写为CL)组成。重链和轻链可变 区典型地负责抗原识别，而重链和轻链恒定域可以介导免疫球蛋白与宿主组织或因子(包括免疫系统的各种细胞(例如，效应细胞)、Fc受体和经典补体系统的第一组分(C1q)的结合。重链和轻链可变区含有与抗原相互作用的结合区。VH和VL区可以进一步细分成称作“互补性决定区(CDR)”的超变区(HVR)，它们中间穿插着更保守的称为“骨架区”(FR)的区域。每个VH和VL由三个CDR域和四个FR域构成，按以下顺序从氨基末端排到羧基末端：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
术语“互补决定区”或“CDR区”或“CDR”(在本文中与超变区“HVR”可以互换使用)即指抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。在本文中，重链的三个CDR称为HCDR1、HCDR2和HCDR3，轻链的三个CDR称为LCDR1、LCDR2和LCDR3。
应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
术语“单抗”、“单克隆抗体”或“单克隆抗体组合物”是指单分子组合物的抗体分子制剂，指从一群基本上同质的抗体获得的抗体，即包含个体抗体的群体除可能少量存在的天然发生的突变之外是相同的。常规的单克隆抗体组合物表现出对特定表位的单一结合特异性和亲和力。在某些实施方案中，单克隆抗体可以由多于一种Fab域构成，由此增加对多于一种靶标的特异性。术语“单克隆抗体”或“单克隆抗体组合物”并不受限于任何具体的产生方法(例如，重组的、转基因的、杂交瘤等)。
本发明还包括“双特异性抗体”，其中本发明的抗体是靶向一种以上表位的二价或多价双特异性框架的一部分(例如第二表位可以包含主动转运受体的表位，这样使得该双特异性抗体将展现出跨生物屏障(如血脑屏障)的改进的胞转作用)。因此，在另外的实施方案中，本发明抗体的单价Fab可以连接到另外的靶向不同蛋白的Fab或scfv，以产生双特异性抗体。双特异性抗体可以具有双重功能，例如由本发明赋予的治疗功能和可以结合到受体分子以增强跨生物屏障(如血脑屏障)转移的转运功能。术语“双抗”、“双功能抗体”“双特异性抗体”、“bispecific antibody”或“BsAb”是指拥有2个不同的抗原结合位点，从而可以同时与两个靶抗原结合，在发挥抗体靶向性的同时具有介 导另外一种特殊功能的作用，所介导的特殊功能效应分子还可以是毒素、酶、细胞因子、放射核素等，双特异性抗体结合抗原的两条臂可分别来自Fab、Fv、ScFv或dSFv等。
术语“抗体的抗原结合片段”指能够与表位结合的抗体的片段、部分、区域或结构域(例如可经由切割、重组、合成等获得)。抗原结合片段可以含有该类抗体的1、2、3、4、5或所有6个CDR域，并且尽管能够结合到所述表位，仍可以展现出不同的特异性、亲和力或选择性。优选地，抗原结合片段含有所述抗体的所有6个CDR域。抗体的抗原结合片段可以是单条多肽链(例如，scFv)的一部分或包含单条多肽链，或者可以是两条或更多条多肽链(各自具有氨基末端和羧基末端)(例如，双抗体、Fab片段、F(ab')2片段等)的一部分或包含两条或更多条多肽链。
本发明所包括的抗原结合片段的实例包括(a)Fab'或Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(b)F(ab')2片段，包含两个由二硫键在铰链结构域连接的Fab片段的二价片段；(c)由VH区和CHl域组成的Fd片段；(d)由抗体单臂的VL区和VH区组成的Fv片段；(e)单链抗体(single chain Fv，scFv)，用基因工程方法将抗体VH和VL通过一段链接肽连接而成的重组蛋白；(f)dAb片段(Ward等人，Nature，341，544-546(1989))，其基本上由VH区组成并且也称为结构域抗体(Holt等人，Trends Biotechnol.，2i(ll):484-90)；(g)骆驼或纳米抗体(Revets等人，Expert Opin Biol Ther.，5(l):111-24)以及(h)分离的互补决定区(CDR)。
在一些实施方案中，本发明的抗体及其抗原结合片段是单链抗体。在一些实施方案中，本发明提供了单链Fv(scFv)，其中本发明的抗体的Fv中的重链和轻链由柔性肽接头(典型地为约10、12、15或更多个氨基酸残基)连接成单条肽链。产生此类抗体的方法描述于例如US 4,946,778；Pluckthun,The Pharmacology of Monoclonal Antibodies,Vol.113，Rosenburg和Moore ed.，Springer-Verlag,New York,第269-315页(1994)；Bird等人，Science,242，423-426(1988)；Huston等人，PNAS USA 85，5879-5883(1988)和McCafferty等人，Nature,348，552-554(1990)。如果仅使用单个VH和VL，单链抗体是单价的；如果使用两个VH和VL，则是二价的；或如果使用两个以上VH和VL，则是多价的。
在一些实施方案中，本发明的抗体及其抗原结合片段是嵌合抗体。术语“嵌合抗体”，指重链和/或轻链的一部分与来自特定物种的或属于特定抗体类别或亚类的抗体的相应序列是相同或同源的，而链的其余部分与来自另一个物种或属于另一个抗体类别或亚类的抗体以及此类抗体的片段的相应序列是相同的或同源的，只要它们展现出期望的生物 学活性。本发明提供了来自人抗体的可变区抗原结合序列。因此，本文主要关注的嵌合抗体包括具有一个或多个人抗原结合序列(如CDR)并含有一个或多个来自非人抗体的序列如FR或C区序列的抗体。此外，本文所述的嵌合抗体是包含一种抗体类别或亚类的人可变区域抗原结合序列以及来自另一个抗体类别或亚类的另一个序列如FR或C区序列的抗体。
在一些实施方案中，本发明的抗体及其抗原结合片段是人源化抗体。术语“人源化抗体”指其中来源于另一种哺乳动物物种如小鼠的种系的CDR序列已被移植到人框架序列上的抗体。可以在人框架序列内进行额外的框架区修饰。
在一些实施方案中，本发明的抗体及其抗原结合片段是人抗体或全人抗体。术语“人抗体”或“全人抗体”(“humAb”或“HuMab”)指包括具有衍生自人种系免疫球蛋白序列的可变域和恒定域的抗体。本发明的人抗体可以包括不是由人种系免疫球蛋白序列编码的氨基酸残基(例如，在体外通过随机或位点特异性诱变或在基因重排期间或在体内通过体细胞突变引入的突变)。
变体抗体也包括在本发明范围之内。因此，申请中所列举的序列的变体也包括在本发明范围之内。可以通过使用本领域已知的方法获得具有改良的亲和性的抗体序列的其他变体并且这些变体也包括在本发明范围之内。例如，氨基酸替换可用于获得具有进一步改良的亲和性的抗体。或者，核苷酸序列的密码子优化可用于改善抗体生产中的表达系统的翻译效率。
这样的变体抗体序列与申请中列举的序列具有70％或更多(例如80％、85％、90％、95％、97％、98％、99％或更高)的序列同源性。这样的序列同源性是相对于参考序列(即，申请中列举的序列)的全长计算获得的。
本发明中的氨基酸残基的编号是根据 或Kabat,E.A.，Wu,T.T.，Perry,H.M.，Gottesmann,K.S.&Foeller,C.,(1991),Sequences of Proteins of Immunological Interest，第5版，NIH公开号91-3242，美国卫生与公众服务部；Chothia,C.&Lesk,A.M.,(1987),Canonical structures For The Hypervariable domains Of Immunoglobulins.,J.Mol.Biol.,196,901-917进行的。在没有明确指明的情况下，本发明中的氨基酸残基的编号是根据Kabat编号系统进行。
抗体或其抗原结合片段“特异性地”结合另一分子的区域(即，表位)是指，它相对于另外的表位与该表位更加频繁地、更加快速地、以更长的持续时间和/或以更大的亲和力或亲合力反应或结合。在一些实施方案中，本发明的抗体或其抗原结合片段以至少 10-7M的亲和力结合人淀粉样蛋白，尤其是其毒性形式，例如10-8M、10-9M、10-10M、10-11M或更高。优选地，抗体或其抗原结合片段在生理条件下(例如，在体内)结合。因此，特异性结合淀粉样蛋白尤其是其毒性形式是指该抗体或其抗原结合片段以上述特异性和/或在这样的条件下结合到淀粉样蛋白尤其是其毒性形式上的能力。适合于确定所述结合的方法是本领域已知的。
在抗体与指定抗原结合的背景下，术语“结合”通常是指以对应于约10-6M或更小的KD的亲和力结合，该KD比该抗体对与除指定抗原或紧密相关抗原之外的非特异性抗原(例如，BSA、酪蛋白)结合的亲和力低至少10倍，如低至少100倍、至少1,000倍。
如本文所用，术语“kd”(sec-1或1/s)是指特定抗体-抗原相互作用的解离速率常数。所述值又称为koff值。
如本文所用，术语“ka”(M-1x sec-1或1/Msec)是指特定抗体-抗原相互作用的结合速率常数。
如本文所用，术语“KD”(M)是指特定抗体-抗原相互作用的解离平衡常数并且通过kd除以ka获得。
如本文所用，术语“KA”(M-1或1/M)是指特定抗体-抗原相互作用的结合平衡常数并且通过ka除以kd获得。
利用CDR残基的单个氨基酸改变可以导致失去功能性结合的事实(Rudikoff,S.等人，(1982),Single Amino Acid Substitution Altering Antigen-binding Specificity,Proc.Natl.Acad.Sci.(USA))79(6):1979-1983)，可以系统性鉴定可替代的功能性CDR序列。在一种用于获得此类变体CDR的优选方法中，将编码CDR的多核苷酸诱变(例如经由随机诱变或通过位点定向方法)以产生具有取代的氨基酸残基的CDR。通过比较原始(功能性)CDR序列中的相关残基的身份与取代的(非功能性)变体CDR序列的身份，可以鉴定出该取代的BLOSUM62.iij取代得分。BLOSUM系统提供了通过分析序列数据库创建的氨基酸取代矩阵，用于可信比对(Eddy,S.R.,(2004),Where Did The BLOSUM62Alignment Score 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.等人，(1990),Methods For Assessing The Statistical Significance Of Molecular Sequence Features By Using General Scoring Schemes),PNAS,87:2264-2268；Altschul,S.F.,(1991),Amino Acid Substitution Matrices From An Information Theoretic Perspective,J.Mol.Biol.,219，555-565。目前，最先进的BLOSUM数据库是 BLOSUM62数据库(BLOSUM62.iij)。表1呈现了BLOSUM62.iij取代得分(得分越高取代越保守，并且因此更加可能地，该取代将不会影响功能)。例如，如果包含所得CDR的抗原结合片段不能结合到PD-L1，则BLOSUM62.iij取代得分被认为是不充分保守的，并且选择且产生新的具有更高取代得分的候选取代。因此，例如，如果原始残基是谷氨酸(E)并且非功能性取代残基是组氨酸(H)，则BLOSUM62.iij取代得分将为0，并且更保守的变化(如到天冬氨酸、天冬酰胺、谷氨酰胺或赖氨酸)是优选的。
本发明因此考虑了随机诱变用于鉴定改进的CDR的用途。在本发明的背景下，保守取代可以由反映在以下三个表中的一个或多个中的氨基酸类别内的取代定义：
保守取代的氨基酸残基类别：
替代性保守氨基酸残基取代类别：
氨基酸残基的物理和功能替代性分类：

更保守的取代分组包括：缬氨酸-亮氨酸-异亮氨酸、苯丙氨酸-酪氨酸、赖氨酸-精氨酸、丙氨酸-缬氨酸以及天冬酰胺-谷氨酰胺。
在一些实施方案中，亲水性氨基酸选自Arg、Asn、Asp、Gln、Glu、His、Tyr和Lys。
还可以使用描述于例如Creighton,(1984),Proteins:Structure and Molecular Properties,W.H.Freeman and Company)中的原理制定另外的氨基酸组群。
因此，所包含的抗体或其抗原结合片段的CDR变体的序列可以通过取代而不同于亲本抗体的CDR的序列；例如4、3、2或1个氨基酸残基的取代。根据本发明的实施方案，CDR区中的氨基酸可以用保守取代进行取代，如在以上3个表中所定义的。
“同源性”或“序列同一性”是指在序列比对和引入缺口后，多核苷酸或多肽序列变体的残基与非变体序列的相同的百分比。在具体实施方式中，多核苷酸和多肽变体与本文所述的多核苷酸或多肽具有至少约70％、至少约75％、至少约80％、至少约90％、至少约95％、至少约98％、或至少约99％的多核苷酸或多肽同源性。
这样的变体多肽序列与申请所列举的序列具有70％或以上(即80％、85％、90％、95％、97％、98％、99％或更多)的序列同一性。在其他实施方式中，本发明提供了多肽片段，其包括本文所公开的氨基酸序列的各种长度的连续延伸段。例如，适用的情况下，本发明提供的肽序列包含至少约5、10、15、20、30、40、50、75、100、150或更多的本发明公开的一个或多个序列的连续氨基酸的肽以及其之间所有的中间长度的肽。
本发明的抗体可以是通过重组DNA产生的单克隆抗体。
在一些实施方案中，本发明的抗体是全长抗体，优选IgG1-4抗体或IgM抗体。在另一些实施方案中，本发明的抗体是抗体抗原结合片段或单链抗体。
各种免疫球蛋白类的恒定区的亚基结构和三维结构是已知的。本文使用的，术语“VH结构域”包括免疫球蛋白重链的氨基末端可变结构域，而术语“CH1结构域”包括免疫球蛋白重链的第一(多数为氨基末端)恒定区。CH1结构域邻近VH结构域并且是免疫球蛋白重链分子的铰链区的氨基末端。本文使用的术语“CH2结构域”包括一部分的重链分子，该部分范围，例如，从抗体的约残基244到残基360，使用常规的编号方案(残基244 至360，Kabat编号系统；和残基231-340，EU编号系统；见Kabat等，美国卫生和公共服务部，“Sequences of Proteins of Immunological Interest”(1983)。CH2结构域是独特的，因为它与另一个结构域配对不紧密。相反，两个N-连接的支链的糖链插入至完整的天然IgG分子的两个CH2结构域之间。有文献记载，CH3结构域从CH2结构域延伸至IgG分子的C-末端，并包含约108个残基。本文使用的术语“铰链区”包括重链分子的将CH1结构域连接至CH2结构域的那一部分。该铰链区包含约25个残基并且是柔性的，从而使两个N-末端抗原结合区独立地移动。铰链区可分为三个不同的结构域：上部、中部、和下部铰链结构域(Roux等人，J.Immunol 161：4083(1998))。
在一些实施方案中，本发明的抗体是单价抗体，优选如在WO 2007059782(通过引用将其全文并入此处)中描述的具有铰链区缺失的单价抗体。因此，在一些实施方案中，抗体是单价抗体，其中所述抗体通过以下方法构建：i)提供编码单价抗体的轻链的核酸构建体，所述构建体包含编码所选抗原特异性抗体的VL区的核苷酸序列和编码Ig的恒定CL区的核苷酸序列，其中编码所选抗原特异性抗体的VL区的所述核苷酸序列和编码Ig的CL区的所述核苷酸序列被有效连接在一起，并且其中，在IgG1亚型的情况下，编码CL区的核苷酸序列已经被修饰，这样使得在多克隆人IgG的存在下或当给予动物或人时，该CL区不含有能够与包含该CL区的一致氨基酸序列的其他肽形成二硫键或共价键的任何氨基酸；ii)提供编码单价抗体的重链的核酸构建体，所述构建体包含编码所选抗原特异性抗体的VH区的核苷酸序列和编码人Ig的恒定CH区的核苷酸序列，其中编码CH区的核苷酸序列已经被修饰，这样使得在多克隆人IgG的存在下或当给予动物人时，对应于铰链区和(如由Ig亚型所要求的)CH区的其他区域(如CH3区)的区域不包含参与和包含人Ig的CH区的一致氨基酸序列的其他肽形成二硫键或共价或稳定的非共价重链间键的任何氨基酸残基，其中编码所选抗原特异性抗体的VH区的所述核苷酸序列和编码所述Ig的CH区的所述核苷酸序列被有效连接在一起；iii)提供用于产生单价抗体的细胞表达系统；iv)通过在(iii)的细胞表达系统的细胞中共表达(i)和(ii)的核酸构建体来产生所述单价抗体。
类似地，在一些实施方案中，本发明的抗体是单价抗体，其包含：
(i)如本文所述的本发明抗体的可变区或所述结构域的抗原结合部分，以及
(ii)免疫球蛋白的CH区或其包含CH2和CH3域的结构域，其中该CH区或其结构域已经被修饰，这样使得对应于铰链区和(如果该免疫球蛋白不是IgG4亚型的话)CH区的其他结构域(如CH3域)的结构域不包含任何氨基酸残基，这些任何氨基酸残 基能够与相同CH区形成二硫键或在多克隆人IgG的存在下与相同CH区形成其他共价或稳定的非共价重链间键。
在另外的一些实施方案中，单价抗体的重链被修饰，使得缺失整个铰链区。
在另外的实施方案中，单价抗体的序列被修饰，使得它不包含用于N-连接的糖基化的任何受体位点。
通过本领域已知的任何技术产生本发明的抗体，如但不限于任何化学、生物学、遗传学或酶学技术，可单独使用或组合使用。通常，知道期望序列的氨基酸序列，本领域技术人员可通过用于产生多肽的标准技术容易地产生所述抗体。例如，可以使用公知的固相方法合成这些抗体，优选使用市售的肽合成装置(如Applied Biosystems,Foster City,California制造的装置)并遵循制造商的说明书合成这些抗体。或者，可以通过本领域熟知的重组DNA技术合成本发明的抗体。例如，在将编码抗体的DNA序列并入表达载体并将这些载体导入合适的表达所需抗体的真核或原核宿主中后，可以获得作为DNA表达产物的抗体，之后可以使用已知技术从宿主分离抗体。
可以通过包含任何“适合”数目的经修饰的氨基酸和/或与偶联取代基结合来修饰本发明的抗体及其抗原结合片段。在这种情况下，“适合”通常由至少基本上保留与非衍生化亲本抗体相关的淀粉样蛋白尤其是其毒性形式选择性和/或淀粉样蛋白尤其是其毒性形式特异性的能力决定。包含一个或多个经修饰的氨基酸在例如增加多肽血清半衰期、降低多肽抗原性或增加多肽储存稳定性中可以是有利的。对一种或多种氨基酸进行修饰，例如，在重组生产的过程中与翻译同时进行或在翻译之后进行(例如，在哺乳动物细胞表达过程中在N-X-S/T基序上的N-连接的糖基化作用)，或通过合成手段进行修饰。经修饰的氨基酸的非限制性实例包括糖基化的氨基酸、硫酸化的氨基酸、异戊二烯化(例如，法尼基化、香叶基-香叶基化)的氨基酸、乙酰化的氨基酸、酰化的氨基酸、聚乙二醇化的氨基酸、生物素酰化的氨基酸、羧基化的氨基酸、磷酸化的氨基酸等。进行氨基酸修饰的参考文献在本领域中司空见惯，参见例如Walker,(1998),Protein Protocols On CD-Rom,Humana Press,Totowa,New Jersey。经修饰的氨基酸可以例如选自糖基化的氨基酸、聚乙二醇化的氨基酸、法尼基化的氨基酸、乙酰化的氨基酸、生物素酰化的氨基酸、偶联到脂质部分的氨基酸或偶联到有机衍化剂的氨基酸。
本发明的抗体及其抗原结合片段还可以通过共价偶联到聚合物而化学修饰，以增加其循环半衰期。示例性聚合物以及将它们连接到肽的方法参见例如US 4,766,106；US 4,179,337；US 4,495,285和US 4,609,546。另外的示例性聚合物包括聚氧乙基化的多元 醇和聚乙二醇(PEG)(例如，分子量在约1,000～40,000D之间，如在约2,000～20,000D之间，例如约3,000～12,000D的PEG)。
本发明的抗体及其抗原结合片段还包括上述淀粉样蛋白抗体的一个或数个氨基酸替换、缺失或添加所形成的突变体。
在另外的方面，本发明涉及编码本发明的抗体或其抗原结合片段的一条或多条多肽链的表达载体。此类表达载体可以用于重组产生本发明的抗体和其抗原结合片段。
在本发明中，表达载体可以是任何适合的DNA或RNA载体，包括染色体载体、非染色体载体和合成核酸载体(包含一组适合的表达控制元件的核酸序列)。此类载体的实例包括SV40的衍生物、细菌质粒、噬菌体DNA、杆状病毒、酵母质粒、衍生自质粒与噬菌体DNA的组合的载体以及病毒核酸(RNA或DNA)载体。在一些实施方案中，编码本发明抗体的核酸被包含在裸DNA或RNA载体中，包括例如线性表达元件(如描述于例如Sykes and Johnston,Nat Biotech,12，355-59(1997))、紧凑型核酸载体(如描述于例如US 6,077,835和/或WO 00/70087)、质粒载体(如pBR322、pUC 19/18或pUC 118/119)、最小尺寸的核酸载体(如描述于例如Schakowski等人，MoI Ther,3，793-800(2001))，或作为沉淀型核酸载体构建体，如CaPO4沉淀型构建体(如描述于例如WO 00/46147；Benvenisty和Reshef，PNAS USA 83，9551-55(1986)；Wigler等人，Cell,14，725(1978)以及Coraro和Pearson，Somatic Cell Genetics,2，603(1981))。此类核酸载体及其使用在本领域是熟知的(参见例如US 5,589,466和US 5,973,972)。
在一些实施方案中，载体适用于在细菌细胞中表达本发明抗体或其抗原结合片段。此类载体的实例包括例如BlueScript(Stratagene)、pIN载体(Van Heeke&Schuster,J Biol Chem,264，5503-5509(1989))、pET载体(Novagen,Madison,Wisconsin)等。
表达载体还可以是适用于在酵母系统中进行表达的载体。可以采用任何适用于在酵母系统中进行表达的载体。适合的载体包括例如包含组成型或诱导型启动子(如α因子、醇氧化酶和PGH)的载体(综述于：F.Ausubel等人，ed.,Current Protocols in Molecular Biology,Greene Publishing and Wiley InterScience,New York(1987)；Grant等人，Methods in Enzymol,153，516-544(1987)；Mattanovich,D.等人，Methods Mol.Biol.,824，329-358(2012)；Celik,E.等人，Biotechnol.Adv.,30(5)，1108-1118(2012)；Li,P.等人，Appl.Biochem.Biotechnol.,142(2)，105-124(2007)；E.等人，Appl.Microbiol.Biotechnol.,77(3)，513-523(2007)；van der Vaart,J.M.,Methods Mol.Biol.,178，359-366(2002)和Holliger,P.,Methods Mol.Biol.,178，349-357(2002))。
在本发明的表达载体中，编码本发明抗体的核酸可以包含任何适合的启动子、增强子和其他有助于表达的元件或者与其结合。此类元件的实例包括强表达型启动子(例如，人CMV IE启动子/增强子以及RSV、SV40、SL3-3、MMTV和HIV LTR启动子)、有效的聚(A)终止序列、用于在大肠杆菌中产生质粒的复制起点、作为选择性标记的抗生素抗性基因和/或便利的克隆位点(例如，多聚接头)。核酸还可以包含与组成型启动子(如CMV IE)相对的诱导型启动子。
在另外的方面，本发明涉及重组真核或原核宿主细胞(如转染瘤)，其产生本发明的抗体或其抗原结合片段或本发明的双特异性分子。宿主细胞的实例包括酵母、细菌和哺乳动物细胞(如CHO或HEK细胞)。例如，在一些实施方案中，本发明提供了包含稳定整合进细胞基因组中的核酸的细胞，该基因组包含编码本发明抗体或其抗原结合片段的核酸序列。在另一些实施方案中，本发明提供了包含非整合型核酸(如质粒、粘粒、噬菌粒或线性表达元件)的细胞，该核酸包含编码本发明抗体或其抗原结合片段的序列。
本发明的抗体及其抗原结合片段可以在不同细胞系中产生，如人细胞系、非人哺乳动物细胞系和昆虫细胞系，例如CHO细胞系、HEK细胞系、BHK-21细胞系、鼠类细胞系(如骨髓瘤细胞系)、纤维肉瘤细胞系、PER.C6细胞系、HKB-11细胞系、CAP细胞系和HuH-7人细胞系(Dumont等人，2015,Crit Rev Biotechnol.,Sep.18,1-13.，其内容通过引用并入本文)。
通过常规免疫球蛋白纯化方法适当地将本发明的抗体与培养基分离，所述方法如蛋白A-Sepharose、羟磷灰石色谱法、凝胶电泳、透析或亲和色谱法。
本发明进一步涉及组合物，其包含本发明的抗体、由其组成或基本上由其组成。
如本文所用，关于组合物，“基本上由......组成”是指至少一种如上所述的本发明抗体是所述组合物中具有生物学活性的唯一一种治疗剂或试剂。
在一个实施方案中，本发明的组合物是药物组合物，并且还包含药学上可接受的赋形剂、稀释剂或载体。
本发明进一步涉及药物，其包含本发明的抗体、由其组成或基本上由其组成，并且还包含药学上可接受的赋形剂、稀释剂或载体。
术语“药学上可接受的载体”是指当施用于动物，优选人时，不产生不利的、过敏或其他不良反应的赋形剂。其包括任何和所有溶剂、分散介质、涂层、抗细菌和抗真菌剂、等渗和吸收延迟剂等。对于人类施用，制剂应满足监管机构(例如FDA办公室或EMA)所要求的无菌、致热原性、一般安全性和纯度标准。
在一些实施方案中，修饰本发明抗体的糖基化。例如，可以制备无糖基化的抗体(即，抗体缺少糖基化)。可以改变糖基化以例如增加抗体对抗原的亲和力或改变抗体的ADCC活性。这种碳水化合物修饰可以通过例如改变抗体序列内的一个或多个糖基化位点来实现。例如，可以进行一个或多个氨基酸取代，其导致消除一个或多个可变区框架糖基化位点，从而消除该位点的糖基化。这种无糖基化可以增加抗体对抗原的亲和力。在Co等人的美国专利No.5,714,350和No.6,350,861(通过引用并入本文)中进一步详细描述了这种方法。另外或可替代地，可以制备具有改变的糖基化类型的抗体。这种碳水化合物修饰可以通过例如在具有改变的糖基化机制的宿主细胞中表达抗体来实现。具有改变的糖基化机制的细胞已在本领域中描述，并且可用作宿主细胞，在该宿主细胞中表达本发明的重组抗体，从而产生具有改变的糖基化的抗体。
可以将重组表达载体引入宿主细胞中以产生转化的宿主细胞。术语“用...转化”、“用...转染”、“转化”、“转导”和“转染”旨在包括通过本领域已知的许多可能技术之一将核酸(例如载体)引入细胞中。如本文所用的，术语“转化的宿主细胞”或“转导的宿主细胞”还意图包括已经用本发明的重组表达载体转化的细胞。可以通过例如电穿孔或氯化钙介导的转化用核酸转化原核细胞。例如，可以通过常规技术，例如磷酸钙或氯化钙共沉淀、DEAE-葡聚糖介导的转染、脂质体转染、电穿孔或显微注射将核酸引入哺乳动物细胞。用于转化和转染宿主细胞的合适方法可以在Sambrook,J.,Fritsch,E.F.and Maniatis,T.(1989)Molecular Cloning:A Laboratory Manual.Cold Spring Harbor Press,New York和其他实验室教科书中找到。
尽管上面定义的核苷酸序列是DNA，但在本发明的备选实施方案中，核苷酸序列可以是RNA。因此涵盖与本文所述DNA序列的相应RNA序列。技术人员将理解如何将编码相同蛋白质/多肽产物的RNA序列衍生为上文所示的DNA序列。“T”应当替换为“U”。
如本文所用，术语“核酸序列”或“核酸分子”或“多核苷酸”、“多核苷酸序列”或“核苷酸序列”是指由天然存在的碱基、糖和糖间(骨架)连接组成的核苷或核苷酸单体的序列。该术语还包括含有非天然存在的单体或其部分的修饰或取代的序列。本发明的核酸、多核苷酸或核苷酸序列可以是脱氧核糖核酸序列(DNA)或核糖核酸序列(RNA)，并且可以包括天然存在的碱基，包括腺嘌呤、鸟嘌呤、胞嘧啶、胸苷和尿嘧啶。序列还可含有修饰的碱基。此类修饰碱基的实例包括氮杂和去氮腺嘌呤、鸟嘌呤、胞嘧啶、胸苷和尿嘧啶；和黄嘌呤和次黄嘌呤。核酸、多核苷酸或核苷酸序列可以是双链或单链的。核酸、多 核苷酸或核苷酸序列可以是完全或部分合成的或重组的。
尽管本发明的抗体、核酸、载体或细胞在单独使用时可以有效对抗疾病，但是可以将另外的治疗剂与本发明的抗体、核酸、载体或细胞组合使用以对抗疾病。因此，在本发明的另一个实施方案中，可以向受试者施用至少一种另外的或额外的治疗剂(例如其他神经退行性疾病治疗药物)。因此，可以向受试者施用本发明的抗体或其抗原结合片段、核酸、载体或细胞和其他治疗剂(例如其他神经退行性疾病治疗药物)。因此，本发明的组合物或药物组合物可以包含其他活性剂或治疗剂，以及本发明的抗体或其抗原结合片段、核酸、载体和/或细胞。然而，应当理解，本发明的抗体或其抗原结合片段、核酸、载体或细胞和其他治疗剂(例如其他神经退行性疾病治疗药物)可以分开施用，例如通过分开施用途径。另外，本发明的抗体或其抗原结合片段、核酸、载体或细胞和至少一种其他治疗剂(例如其他神经退行性疾病这类药物)可以序贯或(基本上)同时施用。它们可以在相同的药物制剂或药物中施用，或者它们可以分别配制和给药。对于序贯施用，可以在施用抗体或其抗原结合片段/核酸/载体/细胞之前或之后至少1分钟、10分钟、1小时、6小时、12小时、1天、5天、10天、2周、4周或6周施用另外的治疗剂。
“药学上可接受的”包括制剂是无菌和无热原的。合适的药物载体、稀释剂和赋形剂在药学领域是公知的。在与药物相容并且对其接受者无害的意义上，载体必须是“可接受的”。通常，载体将是盐水或输注介质(或者称为输注溶液)，其将是无菌和无热原的；但是，可以使用其他可接受的载体。
本发明的药物组合物可以适合于待治疗(或预防)的疾病的方式施用。施用的数目和频率将由诸如患者的状况以及患者的疾病的类型和严重性等因素确定，尽管适当的剂量可以通过临床试验确定。
本发明的组合物可以以单剂量或多剂量施用。特别地，该组合物可以单次一次性施用。
本发明的抗体或其抗原结合片段、核酸、载体或组合物可以通过任何胃肠外途径以包含活性成分的药物制剂的形式施用。取决于病症和待治疗的患者以及施用途径，组合物可以以不同剂量施用。在任何情况下，内科医生将确定最适合于任何个体患者的实际剂量，并且它将随着特定患者的年龄、体重和反应而变化。
在人疗法中，本发明的抗体或其抗原结合片段、核酸或组合物通常与根据预期施用途径和标准药学实践选择的合适的药物赋形剂、稀释剂或载体混合施用。对于每个所述实施方案，本发明的抗体或其抗原结合片段、核酸分子或组合物可以通过多种剂型施用。 此类剂型的实例包括但不限于可重构的粉末、酏剂、液体、溶液、悬浮液、乳液、粉末、颗粒、粒子、微粒、可分散颗粒、扁囊剂、吸入剂、气雾剂吸入剂、贴剂、粒子吸入剂、植入物、长效植入物、注射剂(包括皮下、肌内、静脉内和皮内，优选静脉内)，输注及其组合。通常，本发明的细胞可以在注射或输注缓冲液中施用。示例性制剂可以在例如Remington’s Pharmaceutical Sciences,第19版.,Grennaro,A.,编辑,1995中找到，其通过引用并入本文。
本发明的抗体或其抗原结合片段、核酸或组合物还可以以胃肠外施用，例如静脉内、动脉内、腹膜内、鞘内、颅内、局部、肌内、经颊、皮下、经表皮、硬膜外、吸入、心内、脑室内、眼内、脊柱内、经鼻、舌下、透皮或透粘膜，或者它们可以通过输注技术施用。它们最好以无菌水溶液的形式使用，其可以含有其他物质，例如足够的盐或葡萄糖以使溶液与血液等张。若必要的话，水溶液应当适当缓冲(优选pH为3至9)。在无菌条件下制备合适的胃肠外制剂可通过本领域技术人员公知的标准制药技术容易地完成。
制剂可以在单位剂量或多剂量容器，例如密封的安瓿、袋和小瓶中呈现。
在一些实施方案中，本发明的抗体或其抗原结合片段、核酸或组合物用于治疗和/或预防受试者中的神经退行性疾病，其包括向有需要的受试者施用治疗或预防有效量的所述抗体或其抗原结合片段、核酸或组合物。在一些实施方案中，所述神经退行性疾病选自由阿尔兹海默病、帕金森病、亨廷顿舞蹈症、额颞叶痴呆、肌萎缩性侧索硬化或脊髓小脑共济失调组成的组。
术语“治疗”指改善、减缓、减弱或逆转疾病或病状的进展或严重性，或者改善、减缓、减弱或逆转这种疾病或病状的一种或多种症状或副作用。在本发明中，“治疗”还指用于获得有益的或希望的临床结果的方法，其中“有益的或希望的临床结果”包括但不限于症状的缓解、病状或疾病程度的减小、稳定化的(即没有恶化的)疾病或病状状态、疾病或病状状态进展的延缓或减缓、疾病或病状状态的改善或减轻以及疾病或病状的缓解，不论是部分地或全部地、可检出的或不可检出的。
术语“预防”指施用本发明的抗体及其功能片段，从而阻止或阻碍疾病或病况的至少一种症状的发展。该术语还包括治疗缓解中的受试者以预防或阻碍复发。
术语“受试者”是指温血动物，优选哺乳动物(包括人、家畜和农场动物、动物园动物、运动动物或宠物动物，如狗、猫、牛、马、绵羊、猪、山羊、兔等)，更优选人。在一个实施方案中，受试者可以是“患者”，即，温血动物，更优选是人，其正在等待接收或正在接受医疗护理或将是医疗程序、或疾病发展监测的对象。在一个实施方案中， 受试者是成年人(例如18岁以上的受试者)。在另一个实施方案中，受试者是儿童(例如，18岁以下的受试者)。在一个实施方案中，受试者是男性。在另一个实施方案中，受试者是女性。
本发明的抗体及其抗原结合片段可以进一步用在诊断方法中或用作诊断成像配体。在一些实施方案中，本发明的抗体及其抗原结合片段可以用放射性标记、荧光标记、荧光素型标记、罗丹明型标记、藻红蛋白、伞形酮、丽丝胺、花菁、德克萨斯红、BODIPY(英杰公司)或其类似物、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、乙酰胆碱酯酶、链霉亲和素/生物素和亲和素/生物素进行标记或修饰。这样的标记或修饰的抗体或其抗原结合片段可分别用于检测样品中淀粉样蛋白的存在和/或浓度，用于临床诊断、试验检测等。利用的实验技术包括但不限于ELISA、Dot-blot、Western-blot、化学发光、电化学发光、Simoa技术、放射性技术等。在一些实施方案中，还可以采用顺磁性标记，并且优选地使用正电子发射断层术(PET)或单光子发射计算机断层术(SPECT)进行检测。这样的顺磁性标记包括但不限于含有铝(Al)、钡(Ba)、钙(Ca)、铈(Ce)、镝(Dy)、铒(Er)、铕(Eu)、钆(Gd)、钬(Ho)、铱(Ir)、锂(Li)、镁(Mg)、锰(Mn)、钼(M)、钕(Nd)、锇(Os)、氧(O)、钯(Pd)、铂(Pt)、铑(Rh)、钌(Ru)、钐(Sm)、钠(Na)、锶(Sr)、铽(Tb)、铥(Tm)、锡(Sn)、钛(Ti)、钨(W)和锆(Zi)并且特别是Co+2、CR+2、Cr+3、Cu+2、Fe+2、Fe+3、Ga+3、Mn+3、Ni+2、Ti+3、V+3和V+4的顺磁离子的化合物，使用各种正电子发射断层术的正电子发射金属以及非放射性顺磁金属离子。
在本发明的一个实施方案中，样品是生物样品。生物样品的实例包括但不限于从患病组织，体液，优选血液，更优选血清、血浆、滑液、支气管肺泡灌洗液、痰、淋巴液、腹水、尿液、羊水、腹膜液、脑脊液、胸膜液、心包液和肺泡巨噬细胞制备的组织裂解物和提取物。
在本发明的一个实施方案中，术语“样品”旨在表示在任何分析之前取自个体的样品。
下文将通过实施例来具体描述本发明的技术方案，这些实施例是描述性、例证性的，而不意味着限制。下述实施例中所用的试剂，如无特殊标注，均可从试剂公司如Sigma Aldrich、Merck容易地商购，所述试验方法，如无特殊备注，均可以从教科书如Sambrook,J.,Fritsch,E.F.和Maniatis,T.(1989)Molecular Cloning:A Laboratory Manual.Cold Spring Harbor Press,New York中找到。
实施例
实施例1 W20抗体亲和力成熟
以包被缓冲液(PBS，pH＝7.4)稀释Aβ寡聚体(制备方法参见CN101463082A)至10～100μg/mL，取4mL加入到免疫管中，4℃包被过夜。弃上清，以PBS迅速洗管3次。用3％牛血清白蛋白(BSA)注满免疫管，室温垂直封闭2h。弃上清，以PBS迅速洗管3次。将噬菌体抗体库(W20随机突变库，该库的构建是通过使用易错PCR的方法在抗体CDR区引入随机突变，并通过高效转化大肠杆菌构建的噬菌体抗体库)悬浮于4mL 3％BSA中并加入到免疫管，室温颠倒孵育1h后，垂直孵育1h。用0.1％Tween-20的PBS洗涤10次(第二轮筛选及其以后洗涤20次)。将PBS吸干后，加入500μL甘氨酸溶液(0.1M，pH＝7.4)洗脱噬菌体，室温颠倒孵育10min。于1.75mL OD600＝0.4的大肠杆菌TG1(购自英国MRC中心)中加入250μL洗脱下来的噬菌体，37℃静止孵育30min。剩余洗脱下来的噬菌体4℃保存。分别取TG1培养液的104、106、108倍稀释液涂于2xTY平板上，37℃培养过夜。将剩余的TG1培养液4℃ 11600g离心5min，用100μL培养基重悬沉淀，涂于2xTY平板上，37℃培养过夜。对2xTY平板上生长的克隆计数计算投入产出比(投入产出比＝富集后噬菌体抗体滴度/富集前噬菌体抗体滴度)。
向长满细菌克隆的平板中加入2mL 2xTY培养基，并用玻璃棒刮取细菌，收集菌体悬液，取50μL加入到50mL 2xTY培养基中，培养基中加入终浓度为100μg/mL的氨苄青霉素(Sigma，货号A9518)，1％的葡萄糖，37℃摇至OD600＝0.4。剩余菌液加入15％甘油，-70℃保存。在10mL培养物中加入1x1011辅助噬菌体M13K07(购自英国的MRC中心)，37℃孵育静置30min，并将菌液于3000g 4℃离心10min，重悬沉淀于50mL 2xTY培养基中，加入终浓度为100μg/mL的Amp、50μg/mL卡那霉素(Kana)和0.1％葡萄糖，于30℃摇床培养过夜。将过夜培养物于3300g，4℃离心15min，收集上清，加入1/4体积的PEG(聚乙二醇，PEG-6000)(20％)和NaCl(2.5M)的混合溶液，充分混匀后4℃放置1小时以上。3300g 4℃离心30min并充分丢弃上清后重悬沉淀于2mL PBS中。4℃ 11600g离心10min，得到的上清即为富集第一轮后的噬菌体抗体展示文库。每次筛选完成后，取1μL噬菌体上清测定滴度，具体地，将噬菌体原液进行梯度稀释后，于37℃感染大肠杆菌30min后，涂于2xTY平板上，37℃培养过夜。对2xTY平板上 生长的克隆进行计数，通过稀释比例计算噬菌体原液单位体积的噬菌体克隆数。将以上富集筛选过程重复3次，即进行第二、三、四轮的筛选。
表5针对Aβ寡聚体AβO*W20噬菌体抗体库筛选
结果如表5显示，经过四轮筛选，噬菌体抗体投入产出比逐步提高。第四轮投入产出比是第一轮的200多倍，说明展示针对Aβ寡聚体的高亲和力抗体的噬菌体得到了有效富集。同时，对筛选过程中的四轮抗体库进行了多克隆ELISA分析，具体地，通过ELISA的方法测定每轮所得噬菌体库与Aβ寡聚体的结合能力，其实验原理是将Aβ寡聚体包被到ELISA板上，随后加入噬菌体库(1E10cfu)与其进行结合，然后通过辣根过氧化物酶(HRP)偶联的抗M13噬菌体抗体以间接ELISA的方式进行检测。结果如图1所示。其中M13噬菌体组是阴性对照，PBS组是空白对照，从图中可以看出，从第一轮噬菌体抗体库到第四轮噬菌体抗体库，其与Aβ寡聚体的结合能力呈逐步上升的趋势，且高于阴性对照组。
实施例2单链抗体单克隆的ELISA鉴定
将经过上述4轮富集筛选的噬菌体感染大肠杆菌HB2151(购自英国的MRC中心)并涂到平板上进行孵育，挑取单克隆到96孔细胞培养板上，每孔中加入2xTY培养基200μL，培养基中含有100μg/mL Amp和1％的葡萄糖。37℃摇床(300r/min)培养过夜。每孔吸取2μL菌液，加入到另外一块新的96孔细菌培养板中，每孔添加2xTY培养基200μL，培养基中含有100μg/mL Amp和1％的葡萄糖，37℃摇床培养，OD600达到1.0后再向第一块板中加入甘油，使甘油终浓度为15％，-70℃保存。向第二块96孔板中每孔加入IPTG至终浓度1mmol/L，30℃摇床培养过夜。通过300多个单克隆的ELISA分 析，从中挑选了6个优势突变体(阳性克隆)进行梯度ELISA，具体地，通过ELISA的方法测定单克隆噬菌体(1E10cfu)与Aβ寡聚体的结合能力，其实验原理是将Aβ寡聚体包被到ELISA板上，随后加入单克隆噬菌体与其进行结合，然后通过HRP偶联的抗M13噬菌体抗体以间接ELISA的方式进行检测分析。具体而言，对所述6个优势突变体选择4个梯度稀释，依次为1：10、1：20、1：40和1：80，W20(1：10)为对照，实验重复三次，取平均值，结果如图2所示。可见，6个优势突变体的亲和力均得到很大的提升，尤其是3F，在1：80的稀释条件下仍然比1：10稀释的W20高，亲和力显著提升。
实施例3单链抗体序列测定
将上述阳性克隆进行测序鉴定进行序列分析，使用测序引物LMB3:5'CAGGAAACAGCTATGAC 3'(SEQ ID No.2)，pHEN seq:5'CTATGCGGCCCCATTCA 3'(SEQ ID No.3)，符合抗体库中抗体基本结构的克隆则为完整的单链基因工程抗体。所测得的序列分别如SEQ ID No.4-9所示，其中抗体3F的重链和轻链CDR序列分别如SEQ ID No.10-15所示。
实施例4单链抗体序列比对
使用ClustalW软件将上述阳性克隆的序列与W20进行序列比对，结果如图20所示。抗体氨基酸编号为该氨基酸所在单链抗体的位置，其中轻链CDR3区域与Kabat编号系统对应关系为：(223，L89)，(224，L90)，(225，L91)，(226，L92)，(227，L93)，(228，L94)，(229，L95)，(230，L96)，(231，L97)。W20中的P228在所有的突变体序列中均突变为R228，说明氨基酸位置228对于单链抗体与Aβ寡聚体结合至关重要(氨基酸编号采用Kabat编号系统，下同)。通过比对13A与3F的序列，发现将G226S227替换为A226V227后，单链抗体的亲和力得到进一步提升。由于第226位的甘氨酸与丙氨酸性质相近，提示位于轻链CDR3的第227位的氨基酸是该单链抗体与Aβ寡聚体结合的重要位点。此外，通过比对6A、8H、3A的序列可以发现，将V226突变为W226或F226等更为疏水的氨基酸残基时，其与Aβ寡聚体亲和力进一步增强。以上结果提示，单链抗体序列中位于轻链CDR3的第226位的氨基酸在该单链抗体与Aβ寡聚体结合过程中起到重要作用。以上结果说明，抗体的轻链CDR3序列中(N/Q)224-(S/T)225-X226-X227-X228(SEQ ID No.16)(其中，X226为甘氨酸、缬氨酸、 亮氨酸、异亮氨酸、苯丙氨酸、色氨酸或脯氨酸中任意一种氨基酸；X227为苯丙氨酸、色氨酸、酪氨酸、丙氨酸、亮氨酸或异亮氨酸中任意一种氨基酸；X228为丙氨酸、缬氨酸、亮氨酸、异亮氨酸、蛋氨酸、天冬氨酸、谷氨酸、赖氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺或谷氨酰胺中任意一种氨基酸)片段是单链抗体与Aβ寡聚体结合的关键位点。
实施例5单链抗体结构分子模拟
本实施例基于单链抗体蛋白序列，通过同源建模的方法建立的原始pdb文件；在原始pdb文件的基础上，加入水和调节pH值的Cl-和Na+，运行分子动力学模拟程序，得到人常温(298K)状态下的pdb文件；基于常温状态下的pdb文件，采用分子动力学软件Gromacs计算单链抗体蛋白质的全原子结构。通过将单链抗体W20的226位、227位、228位氨基酸残基分别突变为丙氨酸，再通过分子模拟对单链抗体结构进行计算，结果如图3所示。通过对CDR3区域进行分析，发现在226位进行丙氨酸突变后，单链抗体227位精氨酸残基侧链残基朝向发生改变，而227位氨基酸进行丙氨酸突变后，其结构无明显变化。由实施例4结论可知，226和227位氨基酸残基是与Aβ寡聚体结合的关键残基。在本实施例中，由抗体结构分析可知，单链抗体通过227位氨基酸残基与Aβ寡聚体进行结合，而226位氨基酸残基能够影响227位氨基酸残基朝向，因而对于该结合具有重要影响。而在228位进行丙氨酸突变后，单链抗体结构发生较大变化，说明228位氨基酸残基对单链抗体结构维持具有重要作用。
实施例6 3F抗体与Aβ寡聚体特异结合
Dot blot实验用于评估抗体与Aβ42单体和Aβ寡聚体的结合情况。将分别孵育了0、0.5min、1min、2min和120min的Aβ样品点在硝酸纤维素膜上，用5％脱脂牛奶室温封闭1h。将膜与检测抗体3F或4G8抗体(Biolegend，货号：800704)室温孵育1-2h，然后用0.1％TBST洗涤三次，每次5min，并加入HRP标记的抗c-myc的二抗(Santa Cruz,货号H2317)或山羊抗小鼠二抗(Abcam,货号ab6789)室温孵育1h。用0.1％TBST洗涤三次，每次5min，膜上加入ECL化学发光液，使用Amersham Imager 680成像系统进行分析，结果显示，3F抗体不与Aβ单体结合，而主要与Aβ寡聚体结合(图4)。
实施例7抗体亲和常数KD的测定
利用表面等离子共振法(SPR)对抗体KD进行测定。在检测芯片(CM5)(购自瑞典Biacore公司)上固定3F，并用HBS-EP缓冲液平衡过夜，将Aβ寡聚体用HBS-EP缓冲液稀释成不同浓度梯度。检测时上样量为35μL，流速为5μL/min，上样后数据滞后时间为120秒，并检测蛋白结合信号，最后用BIAcore公司分析软件读取数据并计算各抗体的KD值。测得3F与Aβ寡聚体之间的亲和力常数为KD＝8.47×l0-10M。
实施例8 3F抗体能够显著抑制Aβ的聚集及Aβ寡聚体诱导的细胞毒性
依据Liu等(J Nanobiotechnol(2020)18:160.)所载方法，以ThT荧光方法评价3F抗体对Aβ的聚集的作用，以MTT方法评价3F抗体对Aβ寡聚体诱导的细胞毒性的作用。ThT荧光结果表明，3F和W20抗体均能显著降低Aβ聚集，而单独Aβ则持续聚集(图5的A图)。细胞MTT实验结果表明，3F能够显著抑制Aβ寡聚体诱导的SH-SY5Y细胞毒性，其抑制活性优于W20抗体(图5的B图)。
实施例9 3F抗体能够显著改善AD转基因小鼠的认知功能
将6月龄雄性APP/PS1小鼠(购自北京华阜康生物科技股份有限公司)随机分为3组，每组8只，经鼻给予3F、W20或PBS(AD CON.)，每天1次，共28天；野生型对照组小鼠经鼻给予等量PBS。依据Yu等(Br J Pharmacol.2020；177:2860–2871.)所载水迷宫及Y迷宫实验方法，检测小鼠的空间记忆和认知能力。结果表明，与AD CON.组相比，3F治疗的APP/PS1小鼠在训练期到达平台的时间显著缩短(图6的A图)，在撤去平台的探索期，3F组APP/PS1小鼠表现出明显的空间定向游泳行为，穿台次数明显增多(图6的B图)。同样地，在Y迷宫实验中，AD CON.组小鼠对新臂并没有明显偏好。而3F组APP/PS1小鼠在新臂停留时间均显著增加(图6的C图)。说明3F抗体能够显著改善AD转基因小鼠的认知功能。并且，与W20相比，3F具有更显著的改善AD转基因小鼠认知的能力。
实施例10 3F抗体能够显著降低AD转基因小鼠脑内的Aβ水平
应用4G8抗体作为一抗，采用免疫组化检测APP/PS1小鼠脑内Aβ斑块水平。与AD CON.组小鼠相比，3F治疗的APP/PS1小鼠脑内皮层和海马区的斑块面积显著降低(图7)。进一步应用ELISA检测小鼠脑匀浆中Aβ40/42的含量。结果表明，3F能够显著降低APP/PS1小鼠脑内Aβ40和Aβ42的水平(图8)。并且，与W20相比，3F能 够更显著地降低APP/PS1小鼠脑内斑块和Aβ40/Aβ42水平。
实施例11 3F抗体能够显著降低AD转基因小鼠脑内的胶质细胞活化程度和炎症因子水平
神经炎症的主要特征包括胶质细胞的过度激活和炎症因子的大量释放。本发明人分别通过Iba-1免疫染色和GFAP免疫染色检测小鼠脑内小胶质细胞和星形胶质细胞的活化情况。结果显示，与AD CON.组小鼠相比，3F治疗的APP/PS1小鼠脑内皮层和海马区的Iba-1以及GFAP阳性染色面积显著减少(图9)，表明3F抗体能够显著降低AD转基因小鼠脑内的胶质细胞活化。进一步地，应用ELISA测定了小鼠脑匀浆中IL-1β和IL-6的含量。与AD CON.组小鼠相比，3F治疗的APP/PS1小鼠脑内炎症细胞因子水平显著降低(图10)。并且，与W20相比，3F具有更显著的降低AD转基因小鼠脑内神经炎症水平的能力。
实施例12 3F抗体能够显著改善PD转基因小鼠的行为协调能力及认知功能
将12月龄雄性A53Tα-突触核蛋白转基因小鼠(购自JACKSON LAB)随机分为3组，每组8只，经鼻给予3F、W20或PBS(PD CON.)，每天1次，共28天；野生型对照组小鼠经鼻给予等量PBS。应用爬杆实验检测小鼠的运动协调能力。
爬杆试验：所用杆是一个表面粗糙、带有基座的木质杆，直径1厘米，长度50厘米。将木杆平稳放在小鼠笼内，小鼠头部朝上放在木杆顶部，使其自动转头然后头部向下顺杆爬至笼内。分别记录小鼠转头以及爬至笼内所用时间。如果小鼠跌落、滑落，或不能完成任务，则转头时间记为30秒，爬回笼内记为60秒。每天连续进行5次，前两天为训练期，第三天为测试期。统计分析各组小鼠测试期的转头以及爬至笼内所用时间。
结果表明，3F治疗的A53Tα-突触核蛋白转基因小鼠转头以及爬至笼内所用时间明显低于PD CON.组小鼠。并且，3F组比W20组小鼠所用时间更少(图11的A、B图)。
新事物识别实验基于小鼠乐于探索新事物的天性而设计，其装置是一个大小为40cm×40cm×40cm的白色盒子。实验分为三个阶段：适应阶段、训练阶段和检测阶段；在适应阶段，小鼠被放置于空盒子中自发适应5min；24h后进入训练阶段，盒子里放入两个相同的物体，让小鼠在盒子里熟悉5min；间隔6h后进入检测阶段，将盒中的一个旧物体换成新物体，让小鼠在盒子里自由探索5min，记录小鼠探索新旧物体的次数。认知指数通过以下计算公式确定：(新物体的次数–旧物体的次数)/(新物体的次数 +旧物体的次数)。为了避免小鼠气味的影响，每只小鼠探索结束后，用75％酒精擦拭盒子。
结果表明，3F治疗的A53Tα-突触核蛋白转基因小鼠对于新事物的认知能力明显高于PD CON.组小鼠。并且，与W20组小鼠相比，3F组小鼠对于新事物的认知能力也更强(图11的C图)。
实施例13 3F抗体能够显著降低PD转基因小鼠脑内α-突触核蛋白水平
磷酸化Ser129α-突触核蛋白(pSer129-α-syn)是PD患者脑内α-突触核蛋白的特异性病理形式。本发明人应用pSer129-α-syn抗体(Abcam,货号ab59264)作为一抗，采用免疫组化检测A53Tα-突触核蛋白转基因小鼠脑内病理性α-突触核蛋白水平。PD CON.组小鼠脑干中存在明显的pSer129-α-syn阳性区域，而3F治疗的A53Tα-突触核蛋白转基因小鼠脑干区pSer129-α-syn阳性面积显著降低(图12)。并且，与W20相比，3F能够更显著地降低A53Tα-突触核蛋白转基因小鼠脑干区域pSer129-α-syn水平。
实施例14 3F抗体能够显著提升PD转基因小鼠脑内酪氨酸羟化酶水平
酪氨酸羟化酶(TH)是多巴胺合成的限速酶，在多巴胺信号传递中发挥着重要作用。TH的表达量与PD的严重程度存在负相关关系。本发明人应用免疫组化方法检测了A53Tα-突触核蛋白转基因小鼠脑内TH水平。研究结果显示，与WT小鼠相比，A53Tα-突触核蛋白转基因小鼠脑干中TH的表达水平明显降低，而经3F抗体治疗后的PD小鼠脑干中TH的表达明显升高(图13)。与W20相比，3F能够更显著提升PD转基因小鼠脑内TH水平。
实施例15 3F抗体能够显著改善HD转基因小鼠的自发活动能力和焦虑行为
将5周龄雄性R6/2小鼠(购自JACKSON LAB)随机分为3组，每组8只，经鼻给予3F，W20或PBS(PD CON.)，每天1次，共28天；野生型对照组小鼠经鼻给予等量PBS。应用旷场实验检测小鼠的运动协调能力。
旷场实验是评价实验动物在新异环境中自主行为、探究行为与紧张度的一种方法。旷场为一白色敞口的塑料盒子(27cm×27cm×20.3cm)，以九宫格的形式平均分为9个区域，盒子上方装有摄像头，负责摄取小鼠的运动轨迹。依次将小鼠放置在旷场中，每次一只，适应1min后，摄像记录小鼠的运动轨迹30min。每只小鼠实验前用70％乙 醇清洁盒子内壁。对以下3个指标进行定量分析：总路程、在全部区域的站立次数、在中心区域的停留时间。
实验结果表明，与WT小鼠相比，HD CON.组小鼠表现出明显的自主活动能力的减少以及明显的焦虑症状，包括在旷场中的运动总距离、后肢站立次数以及中心区域的停留时间减少。3F抗体治疗能够明显提高R6/2小鼠的活动总路程、站立次数以及中心区域停留时间(图14)。并且，与W20相比，3F可以更显著地改善R6/2小鼠的自发活动能力，减轻其焦虑状态。
实施例16 3F抗体能够显著降低HD转基因小鼠脑内mHTT聚集体水平
在HD患者或HD转基因动物脑中，mHTT蛋白在神经元核和细胞质中的大量聚集和沉积是重要的病理特征。EM48抗体(Merck Millipore,货号MAB5374)可以特异性识别病理状态的mHTT聚集体。应用EM48抗体对R6/2小鼠脑组织进行免疫荧光染色，结果表明，HD CON.组小鼠的大脑纹状体和皮层区均存在明显的EM48阳性染色，经过3F的治疗后，R6/2小鼠大脑内EM48阳性面积显著减少(图15)，说明3F治疗有效降低了HD转基因小鼠脑内mHTT水平，其作用优于W20。
实施例17 3F抗体能够显著改善ALS转基因小鼠的运动机能
将11周龄雄性SOD1-G93A转基因小鼠(购自南京大学模式动物研究所)随机分为3组，每组8只，经鼻给予3F、W20或PBS(ALS CON.)，每天1次，共28天；野生型对照组小鼠经鼻给予等量PBS。应用悬挂实验和转棒实验检测小鼠的运动协调能力。
悬挂实验：小鼠放置于笼盖上，将笼盖翻转并放置于离鼠笼50cm处，观察60秒，记录小鼠在笼盖上悬挂停留的时间。小鼠停留时间超过60秒统一记为60秒。
结果表明，与ALS CON.组小鼠相比，经过3F治疗后的SOD1-G93A小鼠在悬挂实验中保持的时间明显提高，表明3F能够有效增强小鼠的肌肉强度(图16的A图)。并且，3F的作用优于W20。
转棒试验：转棒试验连续进行3天，每天由两个阶段组成：训练阶段和测试阶段。训练阶段：转棒转速为4转/分，小鼠在转棒上训练5分钟后放回笼内，1小时之后进入测试阶段。测试阶段：转棒转速为5分钟内由静止匀加速至40转/分，记录小鼠在转棒上的持续时间，不跌落记为300秒。每天测试3次，每次间隔30分钟。每组小鼠3天 内总计9次测试，统计分析每组小鼠3天内总计9次测试，每次在转棒仪上的持续时间。
结果表明，ALS CON.组小鼠比WT小鼠在转棒上的持续时间明显降低，而3F治疗组SOD1-G93A小鼠在转棒上的停留时间显著提高，并且，与W20组小鼠相比，3F组小鼠在转棒上的持续时间也更长(图16的B图)。
实施例18 3F抗体能够显著降低ALS转基因小鼠脑干中SOD1聚集物水平
ALS患者及转基因小鼠的脑干中存在着大量的突变SOD1聚集体的沉积。本发明人对SOD1-G93A小鼠脑切片进行了SOD1免疫染色。结果表明，与ALS CON.组小鼠相比，3F治疗组SOD1-G93A小鼠脑干中的SOD1聚集物显著减少(图17)。并且，与W20相比，3F能够更有效地降低ALS转基因小鼠脑干中SOD1聚集物水平。
实施例19 3F抗体能够显著降低ALS转基因小鼠脑内胶质细胞的活化程度
本发明人通过对Iba-1和GFAP进行免疫染色评定SOD1-G93A小鼠脑中小胶质细胞和星形胶质细胞的激活状况。结果表明，与WT小鼠相比，ALS CON.组小鼠脑中激活的胶质细胞均明显增加，而3F的治疗能显著降低SOD1-G93A小鼠脑中小胶质细胞与星形胶质细胞的活化水平。与W20相比，3F具有更优的降低ALS转基因小鼠脑内胶质细胞活化的能力(图18)。
实施例20 3F等改进型抗体能够显著改善AD转基因小鼠的认知功能
将6月龄雄性APP/PS1小鼠(购自北京华阜康生物科技股份有限公司)随机分为8组，每组8只，经鼻给予3F、W20、3A、6A、8H、11G、13A或PBS(AD CON.)，每天1次，共28天；野生型对照组小鼠经鼻给予等量PBS。依据Yu等(Br J Pharmacol.2020；177:2860–2871.)所载Y迷宫实验方法，检测小鼠的认知能力。结果表明，AD CON.组小鼠对新臂并没有明显偏好。而各抗体组APP/PS1小鼠在新臂停留时间均显著增加(图19)。其中，3F抗体具有最显著的改善AD转基因小鼠认知的能力。
实施例21 3FI4抗体(3F抗体的IgG4亚型)与Aβ寡聚体特异结合
本发明利用常规方法将3F抗体改造为IgG4亚型抗体，命名为3FI4(3FI4-1，3FI4-2，3FI4-3，3FI4-4，其轻链序列均如SEQ ID No.17所示，重链序列分别如SEQ ID No.18-21所示，轻链恒定区序列如SEQ ID No.22所示，重链恒定区序列分别如SEQ ID No.23-26 所示)。应用ELISA的方法测定抗体与Aβ寡聚体的结合能力，具体地，按/孔包被Aβ寡聚体，4℃包被过夜。次日，将稀释后的各种3FI4抗体按100μL/孔加入至包被的ELISA板，37℃孵育1h；PBST洗3次，拍干残留PBST；加入1：10000稀释的山羊抗人HRP二抗，100μL/孔，37℃孵育1h，PBST洗3次，去除残留PBST。加入TMB显色液，100μL/孔，37℃或室温显色，用2M盐酸终止反应，酶标仪检测OD450nm的吸光值(图21)。结果表明，3FI4抗体能够与Aβ寡聚体特异结合。
实施例22与3F特异性结合的Aβ寡聚体Aβo*3F具有超强毒性
22.1与3F特异性结合的Aβ寡聚体的获取
22.1.1 Aβo*3F的体外制备
将1mg Aβ42、Aβ40或其他形式的Aβ(中肽生化有限公司)溶于1mL 100％六氟异丙醇(HFIP)，涡旋5min，水浴超声10min后，分装到EP管中，过夜挥发溶剂，并放于-20℃保存。在使用之前，将HFIP处理分装的Aβ溶解在50mM NaOH中，浓度为1mg/mL，涡旋3-5min，超声1min，然后用预冷的PBS稀释至10μM。4℃，21000g离心30-40min，丢弃沉淀部分(约为起始体积的5％)，得到Aβ单体。将Aβ单体在25℃静止孵育2天，将其与交联了3F抗体的Protein A磁珠4℃孵育过夜。第二天，磁珠用0.1％PBST洗三遍后，用20-100mM甘氨酸(pH 2.0)洗脱3-5min，洗脱两次，洗脱液用1M Tris中和到pH 7，得到sAβo*3F(体外制备的Aβos)。
22.1.2 APP/PS1小鼠脑匀浆以及AD患者CSF中Aβo*3F的分离制备
为了分离制备3F特异性识别的Aβos(Aβo*3F)，将APP/PS1小鼠脑匀浆或AD患者CSF样品(来自郑州大学第一附属医院，签署知情同意书并获得了郑州大学第一附属医院伦理审查委员会批准)与交联了3F抗体的Protein A磁珠4℃孵育过夜。第二天，磁珠用0.1％PBST洗三遍后，用20mM-100mM甘氨酸(具体使用20mM，pH 2.0)洗脱3min，洗脱两次，洗脱液用1M Tris中和到pH 7，得到APP/PS1小鼠脑内的Aβo*3F(mAβo*3F，AD小鼠脑内分离的Aβo*3F)，或AD患者CSF中提取的Aβo*3F(hAβo*3F，AD患者脑脊液分离的Aβo*3F)。3F免疫耗竭后的Aβ聚集体混合物被称为Aβ-ID，分别是sAβ-ID(体外制备)、mAβ-ID(APP/PS1小鼠脑内分离制备)和hAβ-ID(AD患者CSF中分离制备)，用作对照。
Aβ聚集体混合物Aβ*6E10是通过将APP/PS1小鼠脑匀浆或AD患者CSF与交联了6E10抗体的Protein G磁珠混合，4℃反应过夜，然后用20mM-100mM甘氨酸(具 体使用20mM，pH 2.0)洗脱3-5min(具体为3min)，洗脱两次，洗脱液用1M Tris中和到pH 7得到APP/PS1小鼠脑内的Aβo*6E10(mAβo*6E10，AD小鼠脑内分离的Aβo*6E10)，或AD患者CSF中提取的Aβo*6E10(hAβo*6E10，AD患者CSF内分离的Aβo*6E10)(图22)。免疫沉淀得到的Aβ浓度通过Aβ检测试剂盒进行测定。
22.2 Aβo*3F分子量大小和形态表征
将500μL APP/PS1小鼠脑内的Aβo*3F(mAβo*3F)或100μL SEC Marker上样到与AKTA pure系统相连的Superdex 200 10/300GL分子筛色谱柱中。色谱柱预先用Tris-Gly缓冲液平衡，以0.5mL/min的流速洗脱。通过与Marker比对发现mAβo*3F的分子量大小为588kDa(图23的A图)。此外，将10μL mAβo*3F滴在200目铜网上吸附20min，滤纸吸干，2％醋酸双氧铀负染30s，滤纸吸干后风干。在透射电子显微镜(TEM，日立H7700，日本)下以120kV的工作电压在100,000×倍数下检查样品。结果显示，mAβo*3F是直径约为10nm的颗粒(图23的A图)。sAβo*3F与AD患者CSF中提取的Aβo*3F(hAβo*3F)分子量大小和分布与mAβo*3F结果一致。
22.3 Aβo*3F的强细胞毒性
MTT结果显示sAβo*3F对N2a细胞毒性的半抑制浓度(IC50)约为0.186nM，而免疫沉淀前的sAβos的IC50约为63.26nM，两者IC50相差340倍(图23的B、C图)。MTT法检测mAβo*3F对N2a细胞的IC50约为0.111nM；对原代神经元细胞的IC50约为0.057nM(图23的D图)。MTT检测hAβo*3F对原代神经元的IC50约为0.076nM(图23的E图)。这些结果表明，Aβo*3F是一种具有强神经毒性的寡聚体。
22.3 Aβo*3F导致小鼠的认知功能显著下降，并严重损伤小鼠脑内神经元功能
将3月龄C57BL/6小鼠随机分为6组，每组6-8只，侧脑室注射，分组：注射2.5nM(45.5pg)mAβo*3F组，注射2.5nM(45.5pg)mAβ*6E10组，注射2.5nM(45.5pg)mAβ-ID组，注射600nM(10.9ng)mAβ*6E10组，注射600nM(10.9ng)mAβ-ID组，以及注射Tris-Gly溶剂的对照组。注射24h后检测小鼠的行为和认知能力，然后解剖小鼠进行脑部病理分析。
Y迷宫结果显示，2.5nM mAβo*3F和600nM mAβ*6E10处理的小鼠在新臂的持续时间与对照组小鼠相比明显减少。而注射2.5nM mAβ*6E10、2.5nM mAβ*ID和600nM mAβ*ID的小鼠与对照组相比无明显差异。这表明mAβo*3F，而不是mAβ*6E10或mAβ-ID，在低浓度时就会引起小鼠严重的记忆障碍，对于mAβ*6E10，只有当浓度增加到600nM时，才能使小鼠出现类似程度的记忆损伤(图24的A图)。
使用FD快速Golgi染色试剂盒对小鼠的大脑样本进行染色。通过计算树突棘密度发现，与对照组相比，2.5nM mAβo*3F和600nM mAβ*6E10处理的小鼠脑CA1区神经元的树突棘密度明显下降，但2.5nM mAβ*6E10、2.5nM mAβ-ID或600nM mAβ-ID则对小鼠脑神经元树突棘密度无明显作用(图24的B、C图)。
实施例23 3F抗体的进一步优化与活性测定
将3F抗体的第100位氨基酸K突变为R(不算入为了表达为单链抗体而引入的MA残基时，为抗体编号的第98位，该变体命名为K98R突变体，序列如SEQ ID NO：27所示)，研究了K98R突变对该单链抗体亲和力的影响。使用方法如下。
将上述Aβ寡聚体按100ng/孔包被至96孔板，4℃包被过夜。次日用3％的BSA室温封闭2h，PBST洗涤2遍后拍干备用。稀释K98R突变体和3F，从1μg/ml开始，行倍比稀释，共14个梯度。将稀释好的上述抗体溶液加入至包被上述Aβ寡聚体的ELISA板中，做平行复孔，37℃孵育1h，PBST洗涤3次，拍干。加入1:10000稀释的羊抗人IgG-HRP，37℃孵育45min，PBST洗涤3次，拍干。加入TMB显色液显色15分钟左右，终止液终止后，OD450nm下读数，数值用graphpad进行分析比较并作图处理。
结果如图25所示，实验表明，将单链抗体重链98位K突变成R后，亲和力得到进一步提升，提示98位氨基酸在该单链抗体与Aβ寡聚体结合过程中起到重要作用。本发明人考虑了导致该结果的原因，发现按照IMGT编号进行编号时，第98位氨基酸也是3F抗体的重链CDR3的残基(按照IMGT编号，3F抗体的重链/轻链CDR序列分别为：CDR-H1：GFTFSSYA(SEQ ID NO：28)；CDR-H2：ISNLGLTT(SEQ ID NO：29)；CDR-H3：AKTTSRFDY(SEQ ID NO：30)；CDR-L1：QSISSY(SEQ ID NO：31)；CDR-L2：KAS；CDR-L3：QNSAVRPVT(SEQ ID NO：32))。本发明人还研究了K98R变体的体内活性，发现其相对于3F抗体得以进一步提高(数据未显示)。
等同方案
虽然本文已经描述和示出了本发明的多个实施方案，但本领域普通技术人员将容易预想到用于实现本文所述的功能和/或获得本文所述的结果和/或一个或多个优点的各种其他手段和/或结构，并且认为每一个这样的变化和/或修改均在本发明的范围内。更广泛地，本领域技术人员将容易理解，本文所述的所有参数、材料和设定意为示例性的，并且实际的参数、材料和/或设定将取决于使用本发明的教导的具体应用。本领域技术人 员仅使用常规实验将认识到或能够确定本文所述的本发明的具体实施方案的许多等同方案。因此，应当理解，前述实施方案和实施例仅通过示例的方式呈现，并且在所附权利要求及其等同方案的范围内，本发明可以以不同于具体描述和要求保护的方式实施。如果这样的特征、系统、物品、材料和/或方法不是相互冲突的话，则两个或更多个这样的特征、系统、物品、材料和/或方法的任意组合包括在本发明的范围内。
本发明说明书和权利要求书中使用的短语“和/或”应理解为意指如此结合的元素的“任一或两者”，即在一些情况下结合存在而在其他情况下不结合存在的元素。除了由“和/或”从句具体标识的元素之外，可以任选地存在其他元素，无论与那些具体标识的元素相关或不相关，除非另外明确指出。因此，作为非限制性示例，当与开放式语言例如“包含”结合使用时，对“A和/或B”的引用在一个实施方案中可以指有A没B(任选地包括除B之外的其他元素)；在另一个实施方案中，指有B没A(任选地包括除A之外的元素)；在又一个实施方案中，指有A和B两者(任选地包括其他元素)；等等。
如本文在说明书和权利要求中所用，“或”应理解为与如上定义的“和/或”具有相同的含义。例如，当分隔列表中的项时，“或”或“和/或”应理解为包含性的，即包括多个元素或元素列表的至少一个，但也包括多于一个，以及任选地，其他未列出的项。只有明确指出相反的术语，例如“仅其一”或“正好其一”，或当用于权力要求中时，“由……组成”将指包含多个元素或元素列表的正好一个元素。通常，当前面有排他性术语，例如“任一”、“其一”、“仅其一”或“正好其一”时，本文所用的术语“或”应该仅理解为表示排他性的替代方案(即，“一个或另一个但不是两者”)。“基本上由……组成”用于权利要求中时，应具有其在专利法领域中的普通含义。
如本文在说明书和权利要求中所用，指代一个或多个元素的列表时，短语“至少一个”应理解为意指选自所述元素列表的任意一个或多个元素中的至少一个元素，但不一定包括所述元素列表中具体列出的每个元素的至少一个，并且不排除所述元素列表中元素的任意组合。这个定义还允许除短语“至少一个”指代的元素列表中具体标识的元素之外的元素可以任选地存在，无论与那些具体标识的元素相关或不相关。因此，作为非限制性示例，在一个实施方案中，“A和B的至少一个”(或等价地，“A或B的至少一个”，或等价地，“A和/或B的至少一个”)可以指至少一个，任选地包括多于一个A，不存在B(并且任选地包括除B之外的元素)；在另一个实施方案中，指至少一个，任选地包括多于一个B，不存在A(并且任选地包括除A之外的元素)；在又一个 实施方案中，指至少一个，任选地包括多于一个A，和至少一个，任选地包括多于一个B(并且任选地包括其他元素)；等等。
在权利要求以及上述说明书中，所有连接词，例如“包含”、“包括”、“带有”、“具有”、“含有”、“涉及”、“拥有”等理解为是开放式的，即意味着包括但不限于。仅连接词“由……组成”和“基本上由……组成”应分别是封闭式或半封闭式的连接词。
在权利要求中使用顺序术语，例如“第一”、“第二”、“第三”等来修改权利要求元素本身并不意味着一个权利要求元素相对于另一个权利要求元素的任何优先级、优先性或顺序，或一个方法中动作进行的时间顺序，而仅仅用作将具有某一名称的一个权利要求元素与具有相同名称的另一个元素区分开(但用于序数术语)的标签，以区分权利要求元素。

Claims (22)

  1. 一种改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段，其相对于W20抗体在选自第226位、第227位和第228位氨基酸残基位置的一个或多个位置处具有氨基酸取代，所述W20抗体的氨基酸序列如SEQ ID No.1所示，所述抗体的氨基酸位置编号采用Kabat编号系统确定。
  2. 根据权利要求1所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段，其中第226位的氨基酸残基被取代为与丙氨酸性质相近的非极性、疏水性氨基酸，优选地，被取代为甘氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、色氨酸或脯氨酸，更优选地，被取代为甘氨酸、苯丙氨酸、色氨酸。
  3. 根据权利要求1或2所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段，其中第227位氨基酸残基被取代为与缬氨酸性质相近的疏水性氨基酸，优选地，被取代为苯丙氨酸、色氨酸、酪氨酸、丙氨酸、亮氨酸或异亮氨酸，更优选地，被取代为苯丙氨酸、异亮氨酸、亮氨酸。
  4. 根据权利要求1-3任一项所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段，其中第228位氨基酸残基被取代为与精氨酸结构相近的脂肪族氨基酸，优选地，被取代为丙氨酸、缬氨酸、亮氨酸、异亮氨酸、蛋氨酸、天冬氨酸、谷氨酸、赖氨酸、甘氨酸、丝氨酸、苏氨酸、半胱氨酸、天冬酰胺或谷氨酰胺，更优选地，被取代为赖氨酸、天冬酰胺、谷氨酰胺。
  5. 根据权利要求1-4任一项所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段，其中第226-228位氨基酸残基分别被取代为AVR、GSR、WVR、FER、NFR或VRR。
  6. 根据权利要求1-5任一项所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段，其中第224位、第225位氨基酸残基分别被取代为谷氨酰胺、苏氨酸。
  7. 根据权利要求1-6任一项所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段，其为抗原结合片段，优选地，选自由scFv、F(ab')2、Fab'、Fab、Fd、Fv、双特异抗体、骆驼抗体、CDR和抗体最小识别单位(dAb)组成的组的抗原结合片段，更优选地，选自由scFv、F(ab')2、Fab'、Fab组成的组的抗原结合片段。
  8. 根据权利要求1-6任一项所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结 合片段，其为单克隆抗体、嵌合抗体、人源化抗体或全人抗体，优选地，所述抗体选自由IgG、IgM、IgA、IgD和IgE及其亚型组成的组，更优选地，所述抗体选自IgG1-4亚型组成的组，更优选地，所述抗体是IgG4亚型。
  9. 根据权利要求1-8任一项所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段，其为scFv且其氨基酸序列如SEQ ID No.5、4和6-9任一项所示。
  10. 根据权利要求1-9任一项所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段，其轻链的CDR3的序列如SEQ ID No.15所示。
  11. 根据权利要求1-9任一项所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段，其相对于3F抗体在第98位被取代为精氨酸，优选地，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段的重链氨基酸序列如SEQ ID No.33-36任一项所示，优选地，所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段为scFv且其氨基酸序列如SEQ ID No.27所示。
  12. 一种分离的核酸分子，其选自：
    (1)DNA或RNA，其编码权利要求1-11任一项所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段；
    (2)与(1)中定义的DNA或RNA完全互补的核酸分子。
  13. 一种表达载体，其包含有效连接的权利要求12所述的核酸分子。
  14. 一种宿主细胞，其包含权利要求12所述的核酸分子或权利要求13所述的表达载体。
  15. 一种组合物，其包含权利要求1-11任一项所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段、权利要求12所述的核酸分子、权利要求13所述的表达载体或权利要求14所述的宿主细胞，以及一种或多种药学上可接受的载体、稀释剂或赋形剂，任选地，所述组合物还包含另一种神经退行性疾病治疗剂，优选地，所述另一种神经退行性疾病治疗剂选自乙酰胆碱酯酶抑制剂如多奈哌齐、加兰他敏、卡巴斯汀，和天门冬氨酸受体拮抗剂如美金刚。
  16. 一种生产权利要求1-11任一项所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段的方法，其包括步骤：
    将权利要求14所述的宿主细胞在适合所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段表达的培养条件下培养，任选地，分离、纯化所得的产物。
  17. 权利要求1-11任一项所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合 片段、权利要求12所述的核酸分子、权利要求13所述的表达载体、权利要求14所述的宿主细胞或权利要求15所述的组合物在制备用于抑制受试者中Aβ的聚集和/或Aβ寡聚体诱导的细胞毒性，或用于治疗和/或预防受试者中神经退行性疾病，或用于诊断受试者是否患有神经退行性疾病的药物中的用途。
  18. 根据权利要求1-11任一项所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段、权利要求12所述的核酸分子、权利要求13所述的表达载体、权利要求14所述的宿主细胞或权利要求15所述的组合物，其用于抑制受试者中Aβ的聚集和/或Aβ寡聚体诱导的细胞毒性，或用于治疗和/或预防受试者中神经退行性疾病，或用于诊断受试者是否患有神经退行性疾病。
  19. 一种抑制受试者中Aβ的聚集和/或Aβ寡聚体诱导的细胞毒性，或治疗和/或预防受试者中神经退行性疾病，或用于诊断受试者是否患有神经退行性疾病的方法，其包括向所述受试者或受试者的细胞施用治疗有效量的权利要求1-11任一项所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段、权利要求12所述的核酸分子、权利要求13所述的表达载体、权利要求14所述的宿主细胞或权利要求15所述的组合物。
  20. 根据权利要求17所述的用途，权利要求18所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段、核酸分子、表达载体、宿主细胞或组合物，权利要求19所述的方法，其中所述神经退行性疾病选自由阿尔兹海默病、帕金森病、亨廷顿舞蹈症、肌萎缩性侧索硬化、额颞叶痴呆或脊髓小脑共济失调组成的组，优选地，所述神经退行性疾病选自由阿尔兹海默病、帕金森病、亨廷顿舞蹈症和肌萎缩性侧索硬化组成的组。
  21. 权利要求1-11任一项所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段、权利要求12所述的核酸分子、权利要求13所述的表达载体、权利要求14所述的宿主细胞或权利要求15所述的组合物在制备用于诊断受试者样品中淀粉样蛋白毒性形式的存在和/或水平的试剂中的用途。
  22. 权利要求1-11任一项所述的改进的淀粉样蛋白寡聚体特异性抗体或其抗原结合片段、权利要求12所述的核酸分子、权利要求13所述的表达载体、权利要求14所述的宿主细胞或权利要求15所述的组合物在制备用于特异性结合受试者中的强毒性淀粉样蛋白寡聚体Aβo*3F并抑制其神经毒性的药物中的用途，其中所述强毒性淀粉样蛋白寡聚体Aβo*3F采用针对3F抗体的免疫沉淀从Aβ寡聚体混合物中分离而来，其典型特征为Aβ高分子量寡聚体，基于分子排阻色谱(SEC)分析，其分子量大小约为588kDa，直径约为10nm。
PCT/CN2023/121350 2022-08-09 2023-09-26 改进的β淀粉样蛋白寡聚体特异性结合抗体 WO2024032822A1 (zh)

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CN101463082A (zh) * 2009-01-16 2009-06-24 清华大学 与A-beta寡聚体特异性结合的基因工程单克隆抗体
US20100202968A1 (en) * 2007-01-05 2010-08-12 University Of Zurich Method of Providing Disease-Specific Binding Molecules and Targets
WO2011130377A2 (en) * 2010-04-15 2011-10-20 Abbott Laboratories Amyloid-beta binding proteins
CN102597234A (zh) * 2009-08-07 2012-07-18 协和发酵麒麟株式会社 人源化抗淀粉样-b寡聚体抗体
CN103179981A (zh) * 2010-07-30 2013-06-26 Ac免疫有限公司 安全和功能性的人源化抗β-淀粉样蛋白抗体
WO2013167681A1 (en) * 2012-05-10 2013-11-14 Georg-August-Universität Göttingen CONFORMATIONAL-SPECIFIC ANTIBODIES AGAINST Αβ OLIGOMERS

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Publication number Priority date Publication date Assignee Title
US20100202968A1 (en) * 2007-01-05 2010-08-12 University Of Zurich Method of Providing Disease-Specific Binding Molecules and Targets
CN101463082A (zh) * 2009-01-16 2009-06-24 清华大学 与A-beta寡聚体特异性结合的基因工程单克隆抗体
CN102597234A (zh) * 2009-08-07 2012-07-18 协和发酵麒麟株式会社 人源化抗淀粉样-b寡聚体抗体
WO2011130377A2 (en) * 2010-04-15 2011-10-20 Abbott Laboratories Amyloid-beta binding proteins
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