Multispecific antibodies and uses thereof

Abstract

A multispecific antibody or antigen binding fragment thereof is provided, the multispecific antibody or antigen-binding fragment thereof comprises at least one first antigen-binding region capable of specifically binding to pyroglutamate amyloid-beta, a second antigen-binding region capable of specifically binding to transferrin receptor (TfR), and a third antigen-binding region capable of specifically binding to paired spiral filaments (PHF)-tau. Methods of treating or detecting nervous system disorders and / or delivering therapeutic or diagnostic agents across the blood-brain barrier are also provided. Nucleic acids encoding the antibodies, vectors comprising the nucleic acids, recombinant host cells comprising the nucleic acids and / or vectors, and methods of producing the multispecific antibodies or antigen-binding fragments thereof are also described.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 269,206, filed on March 11, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Reference sequence listing submitted electronically

[0004] This application contains a sequence listing submitted electronically. The contents of the electronic sequence listing (065768-122WO1_Sequence Listing.xml; size: 55,091 bytes; creation date: March 2, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present application relates to anti-paired helical filament tau / anti-pyroglutamate amyloid-β / anti-transferrin receptor (TfR) multispecific antibodies, antibody conjugates, nucleic acids encoding antibodies and expression vectors, recombinant cells containing expression vectors, and compositions containing antibodies. Also provided are methods for preparing antibodies, methods for using antibodies to treat conditions including nervous system disorders (e.g., amyloid-related disorders), and methods for diagnosing nervous system disorders using antibodies. Background Art

[0006] Alzheimer's disease (AD) is a degenerative brain disorder characterized by progressive loss of memory, cognition, reasoning, judgment and emotional stability, which gradually leads to extreme mental decline and ultimate death. Alzheimer's disease is a common cause of progressive mental decline (dementia) in the elderly. Alzheimer's disease has been found worldwide and represents a major public health problem. It is estimated that the disease currently affects more than about five million people in the United States alone. It is currently incurable, and any treatment does not effectively prevent AD or reverse its symptoms or processes.

[0007] The brains of individuals with AD show characteristic lesions called amyloid plaques, amyloid angiopathy (deposition of amyloid in blood vessels), and neurofibrillary tangles. These lesions, particularly amyloid plaques and neurofibrillary tangles, are generally found in large numbers in several areas of the brain that are important for memory and cognitive function. Amyloid plaques and amyloid angiopathy also characterize the brains of individuals with trisomy 21 (Down syndrome), diffuse Lewy body disease, and hereditary cerebral hemorrhage with Dutch amyloidosis (HCHWA-D).

[0008] The major components of amyloid plaques are various amyloid-β (Aβ) peptides, which are generated by the cleavage of β-amyloid precursor protein (APP). It is assumed that the deposition of Aβ peptides in the brain is an early and necessary step in the disease cascade leading to AD. The identification of mutations in the amyloid precursor protein and presenilin genes that lead to altered Aβ production and cause early-onset AD in families provides strong evidence that altered amyloid metabolism is a central event in the pathogenic process that underlies the disease.

[0009] Amyloid-β peptide (3pE Aβ) with pyroglutamic acid on the third residue is the main substance deposited in the brain of AD patients. 3pE Aβ is present in almost all diffuse and mature plaques in AD, is metabolically stable and can play a role in both plaque seeding and stabilization (Cynis et al., Molecular Neurodegeneration, 2016; 11: 48). The detectable amount of 3pE Aβ in CSF or plasma has not been reported, thus indicating that the target peptide is pathologically specific (DeMattos et al., Neuron, 2012; 76: 1-13). Antibodies that selectively bind to 3pE Aβ can be used for immunotherapy.

[0010] Current AD treatment options include only approved therapies for treating cognitive symptoms in patients with dementia. There are no approved therapies that alter or slow the progression of AD. Potential disease modifiers are anti-amyloid antibodies, including Eli Lilly's Donetuzumab, a humanized IgG1 monoclonal antibody that recognizes Aβ (p3-42), the pyroglutamate form of Aβ, and Aducanumab, a human IgG1 monoclonal antibody directed against a conformational epitope found on Aβ. These therapies, as well as most other potential disease modifiers that may be on the market in the next decade, target Aβ, the main component of amyloid plaques that are one of the two "hallmark" pathological signs of AD.

[0011] Neurofibrillary tangles, the second hallmark pathological sign of AD, are mainly composed of aggregates of hyperphosphorylated tau protein. The main physiological function of tau is microtubule polymerization and stabilization. The binding of tau to microtubules occurs through ionic interactions between the positive charge of the microtubule binding region of tau and the negative charge on the microtubule network (Butner and Kirschner, J Cell Biol. 115 (3): 717-30, 1991). Tau protein contains 85 possible phosphorylation sites, and phosphorylation at many of these sites interferes with the main function of tau. Tau bound to the axonal microtubule network is in a hypophosphorylated state, while the aggregated tau in AD is hyperphosphorylated, thereby providing a unique epitope different from the physiologically active library of tau.

[0012] The tauopathy propagation and spread hypothesis has been described and is based on the Braak stage of tauopathy development in the human brain and the spread of tauopathy after tau aggregate injection in preclinical tau models (Frost et al., J Biol Chem. 284:12845-52, 2009; Clavaguera et al., Nat Cell Biol. 11:909-13, 2009).

[0013] The development of therapeutic agents to prevent or eliminate tau aggregation has been of interest for many years, and candidate drugs (including anti-aggregation compounds and kinase inhibitors) have entered clinical testing (Brunden et al., Nat Rev Drug Discov. 8:783-93, 2009). A number of studies have been published showing the beneficial therapeutic effects of both active and passive tau immunity in transgenic mouse models (Chai et al., J Biol Chem. 286:34457-67, 2011; Boutajangout et al., J Neurochem. 118:658-67, 2011; Boutajangout et al., J Neurosci. 30:16559-66, 2010; Asuni et al., J Neurosci. 27:9115-29, 2007). The activity of both phospho-directed antibodies and non-phospho-directed antibodies has been reported (Schroeder et al., J Neuroimmune Pharmacol. 11(1):9-25, 2016). Therefore, antibodies that selectively prevent tau aggregation and tauopathy progression could be used to treat tauopathies such as AD and other neurodegenerative diseases.

[0014] Although the blood-brain barrier (BBB) ​​prevents harmful substances from entering the brain and is necessary for brain homeostasis, it constitutes an insurmountable obstacle for effectively delivering drugs to the brain. Macromolecules such as monoclonal antibodies and other biotherapeutics have huge therapeutic / diagnostic potential for treating / detecting pathologies in the central nervous system (CNS). However, their access to the brain is blocked by the BBB. Previous studies have shown that only a very small percentage (about 0.1%) of IgG injected in the bloodstream can penetrate the BBB and enter the CNS compartment (Felgenhauer, Klin. Wschr. 52: 1158-1164, 1974). Due to the low concentration of the antibody in the CNS, this will limit any pharmacological action.

[0015] Many methods have been studied to improve the brain delivery of therapeutic monoclonal antibodies (mAbs), including the use of receptor-mediated transcytosis (RMT). RMT utilizes receptors expressed in large quantities on the luminal side of the BBB to transport through brain endothelial cells. The attempt to establish a clinically feasible platform for delivering therapeutic mAbs to the brain has been focused on antibody engineering to improve the efficiency of transcytosis, and progress has been made by observing binding titer, pH dependence and affinity (reviewed in Goulatis et al., 2017, Curr Opin Struct Biol 45: 109-115). However, transfer to NHP and clinical is subject to the rapid peripheral clearance of target-mediated drug disposal (TMDD) and the safety restrictions from acute reticulocyte depletion (Gadkar, 2016, Eur J Pharm Biopharm. April 2016; 101: 53-61). Transferrin receptor (TfR), especially TfR1 mediates the transport of iron-carrying transferrin (Tf) from the blood to the brain and the return of iron-deficient Tf to the blood (Kawabata, Free Radical Biology & Medicine, 133, 46-54, 2019). Anti-TfR1 monoclonal antibodies have been used to deliver drugs to the brain (Burkhart et al., Progress in neurobiology, 181, 101665, 2019). However, the safety tendency and poor pharmacokinetics (PK) of anti-TfR1 monoclonal antibodies have hindered their clinical development as BBB carriers.

[0016] Therefore, there is a need for a PHF-tau / anti-pyroglutamate amyloid-β / anti-TfR antibody or antigen-binding fragment thereof that can be used to cross the BBB and target 3pE Aβ and / or anti-paired helical filament tau for immunotherapy. Summary of the invention

[0017] As embodied and fully described, the present invention relates to multispecific antibodies and antigen-binding fragments thereof that bind to amyloid-β with pyroglutamate at the third residue (3pE Aβ), to transferrin receptor (TfR), and to paired helical filament (PHF)-tau, methods for producing multispecific antibodies or antigen-binding fragments thereof that bind to 3pE Aβ, TfR and PHF-tau, assays using such multispecific antibodies or antigen-binding fragments thereof, and the use of the multispecific antibodies or antigen-binding fragments thereof of the present invention in the manufacture of medicaments for treating neurological disorders such as, for example, Alzheimer's disease and other β-amyloid protein-related diseases, delaying their onset or reversing at least one pathology or symptom thereof.

[0018] In a general aspect, the present application relates to a multispecific antibody or an antigen-binding fragment thereof, which comprises at least a first antigen-binding region capable of specifically binding to pyroglutamate amyloid-β, a second antigen-binding region capable of specifically binding to transferrin receptor (TfR), and a third antigen-binding region capable of specifically binding to paired helical filaments (PHF)-tau. In certain embodiments, (a) the first antigen binding region comprises (i) a first heavy chain variable region (VH1), which VH1 comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2 and HCDR3, which comprise the amino acid sequences of SEQ ID NOs: 8 or 16, 9 or 17 and 10, respectively; and (ii) a first light chain variable region (VL1), which VL1 comprises a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, which comprise the amino acid sequences of SEQ ID NOs: 11, 12 and 13, respectively; (b) the second antigen binding region comprises (i) a second heavy chain variable region (VH2), which VH2 comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2 and HCDR3, which comprise the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively; and (ii) a second light chain variable region (VL2), which VL2 comprises a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, which comprise the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively. IDNO:4, 5 and 6 amino acid sequences; and (c) the third antigen binding region comprises (i) a third heavy chain variable region (VH3), which VH3 comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2 and HCDR3, which respectively comprise the amino acid sequences of SEQ ID NO:28, 29 and 30; and (ii) a third light chain variable region (VL3), which VL3 comprises a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, which respectively comprise the amino acid sequences of SEQ ID NO:31, 32 and 33.

[0019] In certain embodiments, VH1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 14; and VL1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 15. In certain embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 14; and VL1 comprises the amino acid sequence of SEQ ID NO: 15.

[0020] In certain embodiments, the second antigen binding region comprises a single chain fragment variable (scFv) antibody or antigen binding fragment thereof comprising VH2 and VL2. The scFv may, for example, comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 7. In certain embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 7.

[0021] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof may, for example, comprise (i) a first heavy chain (HC1) comprising VH3 and VL3, a first heavy chain constant region comprising a first Fc region (Fc1), and an scFv; (ii) a second heavy chain (HC2) comprising VH1 and a second heavy chain constant region comprising a second Fc region (Fc2); and (iii) a first light chain (LC), each comprising VL1 and a light chain constant region.

[0022] In certain embodiments, the scFv is linked to the carboxyl terminus of the first heavy chain constant region via a linker, more specifically via a linker comprising the amino acid sequence of SEQ ID NO:27.

[0023] In certain embodiments, Fc1 and Fc2 each comprise one or more heterodimer mutations compared to a wild-type Fc region, such as a first modified heterodimer CH3 domain and a second modified heterodimer CH3 domain, respectively; specifically, Fc1 comprises amino acid modifications at positions T350, L351, F405, and Y407, and Fc2 comprises amino acid modifications at positions T350, T366, K392, and T394, wherein the amino acid modification at position T350 is T350V, T350I, T350L, or T350M; the amino acid modification at position L351 is L351Y; the amino acid modification at position F405 is F405A, F405V, F405I, T350L, or T350M; F405T or F405S; the amino acid modification at position Y407 is Y407V, Y407A or Y407I; the amino acid modification at position T366 is T366L, T366I, T366V or T366M, the amino acid modification at position K392 is K392F, K392L or K392M, and the amino acid modification at position T394 is T394W, and wherein the numbering of amino acid residues is performed according to the EU index as described in Kabat, more specifically, Fc1 comprises amino acid modifications T350V, L351Y, F405A and Y407V, and Fc2 comprises amino acid modifications T350V, T366L, K392L and T394W.

[0024] In certain embodiments, at least one of Fc1 and Fc2 comprises one or more mutations that enhance binding of the multispecific antibody or antigen-binding fragment thereof to the neonatal Fc receptor (FcRn), preferably, the one or more mutations enhance binding at acidic pH, more preferably, the at least one of Fc1 and Fc2 has M252Y / S254T / T256E (YTE) mutations, wherein the numbering of amino acid residues is performed according to the EU index as set forth in Kabat.

[0025] In certain embodiments, at least one of Fc1 and Fc2 comprises one or more mutations that reduce or eliminate effector function, preferably at least one of Fc1 and Fc2 has one or more amino acid modifications at positions L234, L235, D270, N297, E318, K320, K322, P331 and P329, such as one, two or three mutations of L234A, L235A and P331S, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.

[0026] Also provided is a multispecific antibody or antigen-binding fragment thereof, comprising a first heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 24, a first light chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 25, and a second heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 26. In certain embodiments, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 24, the first light chain comprises the amino acid sequence of SEQ ID NO: 25, and the second heavy chain comprises the amino acid sequence of SEQ ID NO: 26.

[0027] Another general aspect of the present application relates to an isolated nucleic acid encoding the multispecific antibody or antigen-binding fragment thereof of the present application. A vector comprising the isolated nucleic acid of the present application, and a host cell comprising the nucleic acid or vector of the present application are also provided.

[0028] Another general aspect of the present application relates to a method for producing a multispecific antibody or antigen-binding fragment thereof of the present application. The method comprises culturing the host cell of the present application under conditions that produce the multispecific antibody or antigen-binding fragment thereof, and recovering the multispecific antibody or antigen-binding fragment thereof from the cell or cell culture.

[0029] Also provided is a pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof of the present application and a pharmaceutically acceptable carrier.

[0030] Another general aspect of the present application relates to a method for treating or detecting a neurological disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a multispecific antibody or antigen-binding fragment thereof or a pharmaceutical composition of the present application. Preferably, the neurological disorder is selected from a neurodegenerative disease (such as Lewy body disease, post-polio syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy), tauopathies (such as Alzheimer's disease and supranuclear palsy), prion diseases (such as bovine spongiform encephalopathy, scrapie, Creutz-Feldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease and fatal familial Insomnia), bulbar palsy, motor neurone disease and heterogeneous degenerative disorders of the nervous system (such as Canavan disease, Huntington disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette syndrome, Menkes curly hair syndrome, Cockne syndrome, Halervorden-Spatz syndrome, Raffaello disease, Rett syndrome, Wilson's disease, Lesch-Nyhan syndrome and Unverricht-Lundborg syndrome), dementia (such as Pick's disease and spinocerebellar ataxia), and CNS cancer and / or brain cancer (such as brain metastases caused by cancer elsewhere in the body).

[0031] Also provided are methods for treating a condition associated with the formation of plaques containing beta-amyloid in a subject in need thereof. These methods comprise administering to a subject in need thereof a multispecific antibody or antigen-binding fragment thereof of the present application or a pharmaceutical composition of the present application. The condition may be, for example, Alzheimer's disease. The condition may be, for example, selected from dementia associated with trisomy 21 (Down syndrome), diffuse Lewy body disease, inclusion body myositis, cerebral amyloid angiopathy, and hereditary cerebral hemorrhage with Dutch amyloidosis (HCHWA-D).

[0032] Also provided are methods of reducing plaques associated with Alzheimer's disease in a subject in need thereof. These methods include administering the multispecific antibody or antigen-binding fragment thereof of the present application or the pharmaceutical composition of the present application to a subject in need thereof.

[0033] Also provided are methods for preventing the vaccination activity of 3pE Aβ in a subject in need thereof. These methods comprise administering the multispecific antibody or antigen-binding fragment thereof of the present application or the pharmaceutical composition of the present application to a subject in need thereof.

[0034] Also provided are methods for blocking tau seeding in a subject in need thereof. These methods include administering the multispecific antibody or antigen-binding fragment thereof of the present application or the pharmaceutical composition of the present application to a subject in need thereof.

[0035] Also provided are methods for treating tauopathy in a subject in need thereof. These methods include administering the multispecific antibody or antigen-binding fragment thereof of the present application or the pharmaceutical composition of the present application to a subject in need thereof.

[0036] Also provided are methods for reducing pathological tau aggregation or the spread of tauopathy in a subject in need thereof. These methods include administering the multispecific antibody or antigen-binding fragment thereof of the present application or the pharmaceutical composition of the present application to a subject in need thereof. In certain embodiments, the tauopathy is selected from the group consisting of familial Alzheimer's disease, sporadic Alzheimer's disease, frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle-only dementia, diffuse neurofibrillary tangles with calcifications, argyrophilic grain dementia, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Strauss-Schaef disease, Hallevorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, chronic traumatic encephalopathy, and dementia pugilista (boxing disease).

[0037] Other aspects, features, and advantages of the invention will be apparent from the following disclosure, including the detailed description of the invention and preferred embodiments thereof, and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] When read in conjunction with the accompanying drawings, the above summary of the invention and the detailed description of the invention below will be better understood. For the purpose of illustrating the present invention, the drawings show the currently preferred embodiments. However, it should be understood that the present invention is not limited to the precise embodiments shown in the drawings.

[0039] Figure 1 is a diagram of a multispecific antibody or antigen-binding fragment thereof of the present application. DETAILED DESCRIPTION

[0040] Various publications, articles, and patents are cited or described in the background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, etc. included in this specification is intended to provide a context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Otherwise, certain terms used herein have the meanings described in this specification. All patents, published patent applications, and publications cited herein are incorporated herein by reference as if fully set forth herein.

[0042] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0043] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or will be able to ascertain using only routine experimentation, a variety of equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0044] Unless otherwise indicated, any numerical value, such as a concentration or concentration range described herein is understood to be modified in all cases by the term "about". Therefore, numerical values ​​generally include ± 10% of the value described. For example, a dose of 10 mg includes 9 mg to 11 mg. Unless the context clearly indicates otherwise, as used herein, the numerical range used explicitly includes all possible subranges, all individual values ​​within the range, including integers within such ranges and fractions of these values.

[0045] As used herein, the terms "comprises," "including," "having," or "containing," or any other variation thereof, should be understood to mean including the specified integer or group of integers but not excluding any other integer or group of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to these compositions, mixtures, processes, methods, articles, or apparatuses. In addition, unless expressly indicated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0046] As used herein, the connection term "and / or" between multiple listed elements is understood to include both single options and combined options. For example, where two elements are connected by "and / or", the first option refers to the application of the first element without the second element. The second option refers to the application of the second element without the first element. The third option refers to the application of the first element and the second element together. Any of these options is understood to fall within the meaning and therefore meets the requirements of the term "and / or" as used herein. The parallel applicability of more than one option is also understood to fall within the meaning and therefore meets the requirements of the term "and / or".

[0047] When used herein, "consisting of excludes any element, step, or ingredient not specified in the claim elements. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Whenever used herein in the context of one aspect or embodiment of the present invention, any of the aforementioned terms "comprising," "containing," "including," and "having" may be replaced with the terms "consisting of" or "consisting essentially of" to alter the scope of the present disclosure.

[0048] The term "antibody" is used in the broadest sense herein, and specifically includes full-length monoclonal antibodies, polyclonal antibodies, and unless otherwise specified or contradictory to the context, includes antigen-binding fragments thereof, antibody variants and multispecific molecules, as long as they exhibit the desired biological activity. Typically, a full-length antibody is a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises a domain CL. The VH region and the VL region can be further subdivided into hypervariable regions, which are called complementary determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The general principles of antibody molecular structure and various techniques related to antibody production are provided in, for example, Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988).

[0049] Full-length antibodies can be assigned to different "classes" based on the amino acid sequence of the constant domain of their heavy chains. There are five major classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these five classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0050] An "antibody" may also be a single variable domain (VHH) antibody located on the heavy chain, also known as a heavy chain-only antibody (HcAb), which lacks a light chain and may be naturally produced by camelids or sharks. The antigen-binding portion of an HcAb is composed of a VHH fragment.

[0051] As used herein, the term "recombinant antibody" refers to an antibody (eg, a chimeric, humanized, or human antibody or antigen-binding fragments thereof) expressed by a recombinant host cell comprising a nucleic acid encoding the antibody. Examples of "host cells" for producing recombinant antibodies include: (1) mammalian cells, such as Chinese hamster ovary (CHO) cells, COS cells, myeloma cells (including YO and NSO cells), baby hamster kidney (BHK) cells, Hela cells and Vero cells; (2) insect cells, such as sf9, sf21 and Tn5; (3) plant cells, such as plants belonging to the genus Nicotiana (e.g., Nicotiana tabacum); (4) yeast cells, such as yeast cells belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or yeast cells belonging to the genus Aspergillus (e.g., Aspergillus niger); (5) bacterial cells, such as Escherichia coli cells or Bacillus subtilis cells, etc.

[0052] The "antigen binding fragment" of an antibody is a molecule that can detectably bind to a part of an antigen comprising a full-length antibody, typically comprising one or more parts of at least a VH region. Antigen binding fragments include multivalent molecules and single-chain constructs, which include one, two, three or more antigen binding moieties of an antibody, wherein the VL region and the VH region or their selected parts are connected by synthetic joints or by a recombinant method to form a functional antigen binding molecule. Antigen binding fragments can also be single-domain antibodies (sdAbs), also referred to as nanobodies, which are antibody fragments consisting of a single monomer variable antibody domain (VHH). Although some antigen binding fragments of antibodies can be obtained by actual fragmentation (e.g., enzymatic cleavage) of larger antibody molecules, most antibodies are typically produced by recombinant techniques. The antibodies of the present invention can be prepared as full-length antibodies or their antigen binding fragments. Examples of antigen-binding fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)3, Fv (usually the VL domain and VH domain of a single arm of an antibody), single-chain Fv (scFv, see, e.g., Bird et al., Science 1988; 242:423-426; and Huston et al. PNAS 1988; 85:5879-5883), dsFv, Fd (usually the VH domain and CH1 domain), and dAb (usually the VH domain) fragments; VH, VL, VHH, and V-NAR domains; monovalent molecules comprising a single VH chain and a single VL chain; minibodies, diabodies, triabodies, tetrabodies, and kappa-body antibodies (see, e.g., Ill et al., Protein Eng 1997; 10:949-57); camel IgG; IgNAR; and one or more isolated CDRs or functional paratopes, wherein the isolated CDRs or antigen binding residues or polypeptides can be associated or linked together to form a functional antibody fragment. Various types of antibody fragments have been described or reviewed in, for example, Holliger and Hudson, Nat Biotechnol 2005; 23:1126-1136; WO2005040219 and published U.S. patent applications 20050238646 and 20020161201. Antibody fragments can be obtained using conventional recombinant or protein engineering techniques, and these fragments can be screened for antigen binding or other functions in the same manner as intact antibodies.

[0053] Various techniques have been developed to produce antibody fragments. Traditionally, these fragments are obtained via proteolysis of full-length antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods, 24: 107-117 (1992); and Brennan et al., Science, 229: 81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Alternatively, Fab'-SH fragments can be directly recovered from Escherichia coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). According to another method, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See WO 1993 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. The antibody fragment may also be a "linear antibody," e.g., as described in U.S. Pat. No. 5,641, 870. Such linear antibody fragments may be monospecific or bispecific.

[0054] As used herein, the term "antibody derivative" refers to a molecule comprising a full-length antibody or an antigen-binding fragment thereof, wherein one or more amino acids are chemically modified or substituted. Chemical modifications that can be used for antibody derivatives include, for example, alkylation, PEGylation, acylation, ester formation, or amide formation, for example, for connecting the antibody to a second molecule. Exemplary modifications include PEGylation (e.g., cysteine ​​PEGylation), biotinylation, radiolabeling, and conjugation to a second agent (such as a cytotoxic agent).

[0055] Antibodies herein include "amino acid sequence variants" with altered antigen binding or biological activity. Examples of such amino acid changes include antibodies with enhanced affinity for antigen (e.g., "affinity matured" antibodies), and antibodies with altered Fc regions (if present), such as antibodies with altered (increased or decreased) antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) (see, e.g., WO 00 / 42072, Presta, L. and WO99 / 51642, Iduosogie et al.); and / or antibodies with increased or decreased serum half-life (see, e.g., WO 00 / 42072, Presta, L.).

[0056] A "multispecific molecule" comprises an antibody or antigen-binding fragment thereof associated or linked to at least one other functional molecule (e.g., another peptide or protein, such as another antibody or ligand for a receptor) to form a molecule that binds to at least two different binding sites or target molecules. Exemplary multispecific molecules include bispecific antibodies and antibodies linked to soluble receptor fragments or ligands.

[0057] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which the framework and CDR regions are derived from (i.e., identical or substantially identical to) human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also "derived from" human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted to human framework sequences.

[0058] "Humanized" antibodies are human / non-human chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. To a large extent, humanized antibodies are human immunoglobulins (receptor antibodies), wherein the residues in the hypervariable regions of the receptor are replaced by residues in the hypervariable regions of non-human species (donor antibodies) such as mice, rats, rabbits or non-human primates with desired specificity, affinity and ability. In some cases, the FR residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues that are not present in receptor antibodies or donor antibodies. These modifications are made to further improve antibody performance. Generally speaking, humanized antibodies will include substantially all at least one, and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins, and all or substantially all of the FR residues are those of human immunoglobulin sequences. Humanized antibodies may optionally also include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a constant region of a human immunoglobulin. For more details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992); WO 92 / 02190; U.S. Patent Application 20060073137; and U.S. Patent Nos. 6,750,325, 6,632,927, 6,639,055, 6,548,640, 6,407,213, 6,180,370, 6,054,297, 5,929,212, 5,895,205, 5,886,15 2, 5,877,293, 5,869,619, 5,821,337, 5,821,123, 5,770,196, 5,777,085, 5,766,886, 5,714,350, 5,693,762, 5,693,761, 5,530,101, 5,585,089 and 5,225,539.

[0059] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from the "complementarity determining region" or "CDR" (residues 24 to 34 (L1), 50 to 56 (L2), and 89 to 97 (L3) in the light chain variable domain, and residues 31 to 35 (H1), 50 to 65 (H2), and 95 to 102 (H3) in the heavy chain variable domain; (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) and / or those residues from the "hypervariable loop" (residues 26 to 32 (L1), 50 to 52 (L2), and 91 to 96 (L3) in the light chain variable domain, and residues 26 to 32 (H1), 53 to 55 (H2), and 96 to 101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol., 1987; 196: 901-917). In general, the numbering of amino acid residues in this region is performed by the method described by Kabat et al., supra. Phrases such as "Kabat position", "variable domain residue numbering as in Kabat", and "according to Kabat" refer herein to this numbering system for either the heavy chain variable domain or the light chain variable domain. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening or insertion of a FR or CDR of the variable domain. For example, the heavy chain variable domain may include a CDR A single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat residue numbering for a given antibody can be determined by aligning the homologous regions of the antibody sequence with the "standard" Kabat numbering sequence.

[0060] "Framework Region" or "FR" residues are those VH residues or VL residues other than the CDRs as herein defined.

[0061] An "epitope" or "binding site" is a region or area on an antigen to which an antigen-binding peptide (such as an antibody) specifically binds. A protein epitope may include amino acid residues that are directly involved in binding (also referred to as the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by the specific antigen-binding peptide (in other words, the amino acid residues are within the "solvent-excluded surface" and / or "footprint" of the specific antigen-binding peptide).

[0062] A "paratope" is a region or area of ​​the antigen-binding portion of an antibody that specifically binds an antigen. Unless otherwise specified or clearly contradicted by context, a paratope may include amino acid residues that are directly involved in epitope binding (several of which are typically in a CDR) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by the specifically bound antigen (in other words, the amino acid residues are within the "solvent exclusion surface" and / or "footprint" of the specifically bound antigen).

[0063] An "antibody that binds to the same epitope as a reference antibody" is an antibody that blocks binding of the reference antibody to its antigen by 50% or more in a competition assay, whereas the reference antibody blocks binding of the antibody to its antigen by 50% or more in a competition assay.

[0064] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0065] In the context of the methods of the invention, the term "administering" means a method of therapeutically or prophylactically preventing, treating or ameliorating the syndromes, disorders or diseases described herein by using the conjugates of the invention or forms, compositions or medicaments thereof. Such methods include administering an effective amount of the antibody, antigen-binding fragment thereof or conjugate, forms, compositions or medicaments thereof at different times during the course of treatment, or administering them simultaneously in a combined form. The methods of the invention should be understood to encompass all known therapeutic treatment regimens.

[0066] The ability of a target antibody to "block" the binding of a target molecule to a natural target ligand means that the antibody can detectably reduce the binding of the target molecule to the ligand in an assay using soluble or cell surface-associated target and ligand molecules in a dose-dependent manner, wherein the target molecule detectably binds to the ligand in the absence of the antibody.

[0067] "Blood-brain barrier" or "BBB" refers to the physiological barrier between peripheral circulation and the brain and spinal cord, which is formed by tight junctions in the cortical membrane of brain capillaries, thereby producing a tight barrier that limits the transport of molecules to the brain. The BBB can limit even very small molecules such as urea (60 daltons) to be transported to the brain. Examples of BBB include the BBB in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retinal barrier in the retina, all of which are continuous capillary barriers in the CNS. The BBB also encompasses the blood-CSF barrier (choroid plexus), in which the barrier is composed of ependymal cells rather than capillary endothelial cells.

[0068] "Blood-brain barrier receptor" (abbreviated herein as "R / BBB") is a receptor protein connected to the extracellular membrane expressed on brain endothelial cells, which can transport molecules across the BBB or for transporting molecules of exogenous administration. The example of R / BBB includes but is not limited to transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), low-density lipoprotein receptor (including but not limited to low-density lipoprotein receptor-related protein 1 (LRP1) and low-density lipoprotein receptor-related protein 8 (LRP8)) and heparin-binding epidermal growth factor-like growth factor (HB-EGF). Exemplary R / BBB herein is transferrin receptor (TfR).

[0069] The "central nervous system" or "CNS" refers to the complex of nervous tissue that controls body functions, including the brain and spinal cord.

[0070] As used herein, "conjugate" refers to a protein covalently linked to one or more heterologous molecules, including but not limited to therapeutic peptides or proteins, antibodies, markers, or neurological disorder drugs.

[0071] As used herein, the term "coupling" refers to two or more objects that are combined or connected together. When referring to chemical or biological compounds, coupling can refer to the covalent connection between two or more chemical or biological compounds. By way of non-limiting example, an antibody of the present invention can be coupled to a peptide of interest to form an antibody-coupled peptide. The antibody-coupled peptide can be formed by a specific chemical reaction designed to conjugate the antibody to the peptide. In certain embodiments, the antibody of the present invention can be covalently coupled to the peptide of the present invention through a linker. The linker can, for example, be first covalently connected to the antibody or peptide, and then covalently connected to the peptide or antibody.

[0072] An "effective amount" or "therapeutically effective amount" of an agent, such as a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0073] As used herein, "linker" refers to a chemical linker or a single-chain peptide linker that covalently links two different entities. The linker can be used to connect any two of the antibodies or fragments thereof, blood-brain barrier shuttles, fusion proteins, and conjugates of the present invention. The linker can connect, for example, VH and VL in a scFv, or an antibody or its antigen-binding fragment with a therapeutic molecule, such as a second antibody. In some embodiments, if the monovalent binding entity comprises an scFv for TfR (preferably huTfR1), and the therapeutic molecule comprises an antibody for at least one CNS target (such as pyroglutamate amyloid-β and / or PHF-tau), the linker can connect the scFv to the antibody for pyroglutamate amyloid-β and / or PHF-tau. A single-chain peptide linker consisting of 1 to 25 amino acids (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids) connected by peptide bonds can be used. In certain embodiments, these amino acids are selected from 20 naturally occurring amino acids. In certain other embodiments, one or more of these amino acids are selected from glycine, alanine, proline, asparagine, glutamine and lysine. Chemical linkers such as hydrocarbon linkers, polyethylene glycol (PEG) linkers, polypropylene glycol (PPG) linkers, polysaccharide linkers, polyester linkers, mixed linkers consisting of PEG and embedded heterocycles, and hydrocarbon chains can also be used.

[0074] As used herein, "nervous system disorder" refers to a disease or disorder that affects the CNS and / or has a pathogenic source in the CNS. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, eye disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, epileptic seizure, behavioral disorder, and lysosomal storage disease. For purposes of this application, the CNS will be understood to include the eye, which is usually isolated from the rest of the body by the blood-retinal barrier. Specific examples of neurological disorders include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy), tauopathies (including, but not limited to, Alzheimer's disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Schenck disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neurone disease and heterogeneous degenerative disorders of the nervous system (including but not limited to Canavan disease, Huntington disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette syndrome, Menkes curly hair syndrome, Cockne syndrome, Hallerwarden-Spatz syndrome, Raffaello disease, Rett syndrome, Wilson's disease, Lesch-Nyhan syndrome and Ong-Lung syndrome), dementia (including but not limited to Pick's disease and spinocerebellar ataxia), cancer (e.g., CNS cancer and / or brain cancer, including brain metastases caused by cancer elsewhere in the body).

[0075] "Nervous system disorder drugs" are drugs or therapeutic agents that can be used to treat or improve the consequences of one or more nervous system disorders. Nervous system disorder drugs of the present invention include, but are not limited to, small molecule compounds, antibodies, peptides, proteins, natural ligands of one or more CNS targets, modified forms of natural ligands of one or more CNS targets, oligonucleotide aptamers, inhibitory nucleic acids (i.e., small inhibitory RNA (siRNA) and short hairpin RNA (shRNA)), nucleases, or active fragments of any of the foregoing substances. Exemplary nervous system disorder drugs of the present invention are described herein, including, but not limited to, antibodies, oligonucleotide aptamers, proteins, peptides, inhibitory nucleic acids and small molecules, and active fragments of any of the foregoing, which themselves or specifically recognize and / or act on (i.e., inhibit, activate or detect) CNS antigens or target molecules (such as, but not limited to, amyloid precursor protein or a portion thereof, amyloid β, β-secretase, γ-secretase, tau, α-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer markers, and neurotrophic factors). Non-limiting examples of drugs for nervous system disorders and the corresponding disorders they can be used to treat: Brain-derived neurotrophic factor (BDNF), chronic brain damage (neurogenesis); Fibroblast growth factor 2 (FGF-2), anti-epidermal growth factor receptor brain cancer; (EGFR)-antibodies, glial-derived neurotrophic factor (GDNF) Parkinson's disease; Brain-derived neurotrophic factor (BDNF), amyotrophic lateral sclerosis, depression, lysosomal enzymes of the brain lysosomal storage diseases; Ciliary neurotrophic factor (CNTF), amyotrophic lateral sclerosis; Neuregulin-1, schizophrenia; anti-HER2 antibodies (e.g., trastuzumab), brain metastases caused by HER2-positive cancers.

[0076] The term "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredients contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

[0077] As used herein, "pharmaceutically acceptable carrier or diluent" means any substance suitable for administration to an individual. For example, a pharmaceutically acceptable carrier can be a sterile aqueous solution, such as phosphate buffered saline (PBS) or water for injection.

[0078] As used herein, "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of compounds (such as oligomeric compounds or oligonucleotides), ie, salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

[0079] Pharmaceutically acceptable acid salts / anionic salts used in the present invention include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camphorsulfonate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, etopate, esylate, fumarate, glucoheptonate, gluconate, glutamate, p-hydroxyacetamidophenylarsonic acid salt, hexylresorcinol salt, hydrazine, hydrobromide, Hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, methanesulfonate, methyl bromide, methyl nitrate, methyl sulfate, mucate, naphthenate, nitrate, pamoate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theochlorate, toluenesulfonate and triethiodide. Organic or inorganic acids also include, but are not limited to, hydroiodic acid, perchloric acid, sulfuric acid, phosphoric acid, propionic acid, glycolic acid, methanesulfonic acid, isethionic acid, oxalic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, cyclohexanesulfamic acid, saccharinic acid or trifluoroacetic acid. Pharmaceutically acceptable base / cationic salts include, but are not limited to, aluminum, 2-amino-2-hydroxymethyl-propane-1,3-diol (also known as tris(hydroxymethyl)aminomethane, tromethamine, or "TRIS"), ammonia, benzathine, tert-butylamine, chloroprocaine, choline, cyclohexylamine, diethanolamine, ethylenediamine, lithium, L-lysine, magnesium, meglumine, N-methyl-D-glucamine, piperidine, potassium, procaine, quinine, sodium, triethanolamine, or zinc.

[0080] "Polypeptide" or "protein" means a molecule comprising at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than 50 amino acids may be referred to as "peptides."

[0081] The phrases "sequence identity" or "percent (%) sequence identity" or "identity" or "% identity" when used with reference to amino acid sequences describe the number of matches ("hits") of identical amino acids of two or more aligned amino acid sequences compared to the number of amino acid residues that make up the total length of the amino acid sequences. In other words, using alignment, the percentage of identical amino acid residues (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity over the entire length of the amino acid sequence) can be determined for two or more sequences when the sequences are compared and aligned for maximum correspondence (as measured using a sequence comparison algorithm known in the art), or when aligned manually and visually inspected. Thus, sequences compared to determine sequence identity may differ by substitutions, additions or deletions of amino acids. Suitable programs for aligning protein sequences are known to the skilled artisan. The percent sequence identity of protein sequences can be determined, for example, using programs such as CLUSTALW, ClustalOmega, FASTA or BLAST, for example using the NCBI BLAST algorithm (Altschul SF et al., (1997), Nucleic Acids Res. 25:3389-3402).

[0082] In the context of two amino acid sequences, the term "substantially identical" means that when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, the sequences share at least about 50% sequence identity. Typically, substantially identical sequences will exhibit at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity.

[0083] "Specific binding", "specifically binds" or "binding" means that the antibody binds to the antigen or an epitope within the antigen with a higher affinity than for other antigens. -8 M or less, for example, about 1×10 -9 M or smaller, about 1×10 -10 M or smaller, about 1×10 -11 M or smaller, or about 1×10 -12 M or less dissociation constant (K D ), usually expressed as a ratio of its K binding to nonspecific antigens (e.g., BSA, casein). D K is at least a hundred times smaller D Binds to an antigen or an epitope within an antigen. D is the equilibrium dissociation constant between the antibody and its antigen, i.e., k off / k on The ratio of K Dand affinity are negatively correlated. on ) is a constant used to characterize how quickly an antibody binds to its target. off ) is a constant used to characterize the rate at which an antibody dissociates from its target. The dissociation constant, K, can be measured using standard procedures. D For example, the K D This can be accomplished by using surface plasmon resonance, such as by using a biosensor system (e.g. The K of the antibody can be determined by using biolayer interferometry techniques such as the Octet RED96 system. D The smaller the value, the higher the affinity of the antibody to the target antigen. However, antibodies that specifically bind to an antigen or an epitope within an antigen may have cross-reactivity with other related antigens, such as with the same antigen from other species (homologs) such as humans or monkeys, such as macaques (Macacafascicularis) (cynomolgus, cyno), chimpanzees (Pan troglodytes, chimpanzee, chimp) or common marmosets (Callithrix jacchus) (common marmosets, marmosets)). Although monospecific antibodies specifically bind to one antigen or one epitope, bispecific antibodies specifically bind to two different antigens or two different epitopes.

[0084] The term "subject" as used herein refers to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human. When the subject is a human, it may also be referred to as a "patient."

[0085] As used herein, the term "transferrin receptor" or "TfR" refers to a cell surface receptor necessary for the uptake of cellular iron by a receptor-mediated endocytosis process. A carrier protein of transferrin. TfR participates in iron uptake in vertebrates and is regulated in response to intracellular iron concentrations. It imports iron by internalizing transferrin-iron complexes through receptor-mediated endocytosis. Two transferrin receptors in humans have been characterized: transferrin receptor 1 and transferrin receptor 2. Both receptors are transmembrane glycoproteins. TfR1 is a high-affinity ubiquitously expressed receptor. TfR2 binds to transferrin with an affinity 25 to 30 times lower than that of TfR1. The expression of TfR2 is limited to certain cell types and is not affected by intracellular iron concentrations. In one embodiment, TfR is a human TfR comprising an amino acid sequence as described in Schneider et al., Nature 311: 675-678 (1984). It may have a molecular weight of about 180,000 Daltons, with two subunits each having an apparent molecular weight of about 90,000 Daltons. Preferably, the TfR is human TfR1.

[0086] As used herein, "target antigen" or "brain target" refers to antigens and / or molecules expressed in the CNS (including the brain) that can be targeted with antibodies or small molecules. Examples of such antigens and / or molecules include, but are not limited to, β-secretase 1 (BACE1), amyloid β (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), Parkin, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), and caspase 6. In some embodiments, the target antigen is BACE1. In some embodiments, the target antigen is β-amyloid, particularly pyroglutamate amyloid-β.

[0087] As used herein, the term "tau" or "tau protein" refers to the abundant central and peripheral nervous system proteins with multiple isotypes. In the human central nervous system (CNS), due to alternative splicing, there are six major tau isotypes (Hanger et al., Trends Mol Med.15:112-9,2009) with a size range of 352 to 441 amino acid lengths. Isotypes are different from each other by the regulatory inclusions of 0 to 2 N-terminal insertion sequences and 3 or 4 microtubules arranged in series in combination with repeat sequences, and are referred to as 0N3R, 1N3R, 2N3R, 0N4R, 1N4R and 2N4R. As used herein, the term "control tau" refers to the 2N4R tau isotype without phosphorylation and other post-translational modifications. As used herein, the term "tau" includes mutations comprising full-length wild-type tau, such as point mutations, fragments, insertions, deletions and splice variants. The term "tau" also encompasses post-translational modifications of the tau amino acid sequence. Post-translational modifications include, but are not limited to, phosphorylation. As used herein, the phrase "phosphorylation S433 of tau protein" and similar phrases refer to the phosphorylated amino acid at a certain position of the full-length wild-type tau protein, for example, the serine at position 433.

[0088] Tau binds to microtubules and regulates the transport of cargo through cells, which is a process that can be regulated by tau phosphorylation. In AD and related disorders, abnormal phosphorylation of tau is common and is thought to precede and / or trigger tau aggregation into fibrils called paired helical filaments (PHFs). The main component of PHF is highly phosphorylated tau. As used herein, the term "paired helical filaments-tau" or "PHF-tau" refers to tau aggregates in paired helical filaments. The two main areas in the PHF structure are more obvious in electron microscopy, namely the villous shell and the core filament; the villous shell is sensitive to proteolysis and is located outside the filament, and the protease-resistant core of the filament forms the main chain of the PHF (Wischik et al., Proc Natl Acad Sci USA. 85: 4884-8, 1988).

[0089] As used herein, "tauopathy" encompasses any neurodegenerative disease involving pathological aggregation of tau within the brain. In addition to familial and sporadic AD, other exemplary tauopathies are frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, dementia with tangles only, diffuse neurofibrillary tangles with calcifications, argyrophilic grain dementia, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Strauss-Schaef disease, Hallevorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, and chronic traumatic encephalopathy such as dementia pugilistica (boxing disease) (Morris et al., Neuron, 70:410-26, 2011).

[0090] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or in the course of clinical pathology. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

[0091] Based on the heavy chain constant domain amino acid sequence, antibodies or immunoglobulins can be assigned to five major categories, namely IgA, IgD, IgE, IgG and IgM. IgG is the most stable of the five classes of immunoglobulins, with a serum half-life of about 23 days in humans. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Each of the four IgG subclasses has different biological functions, which are called effector functions. These effector functions are usually mediated by interaction with Fc receptors (FcγR) and / or by binding to C1q and fixing complement. Binding to FcγR can lead to antibody-dependent cell-mediated cytolysis or antibody-dependent cellular cytotoxicity (ADCC), while binding to complement factors can lead to complement-mediated cell lysis or complement-dependent cytotoxicity (CDC). The multispecific antibodies or antigen-binding fragments thereof of the present invention, or therapeutic or diagnostic antibodies to be conjugated or fused to the multispecific antibodies or antigen-binding fragments thereof, may have no or minimal effector function, but retain their ability to bind to FcRn, which binding may be the primary means by which the antibody has a prolonged in vivo half-life.

[0092] The binding of FcγR or complement (e.g., C1q) to the antibody at the so-called Fc portion binding site is caused by a defined protein-protein interaction. Such Fc portion binding sites are known in the art. Such Fc portion binding sites include, for example, amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbered according to the EU index of Kabat). In some embodiments, the multispecific antibody or antigen-binding fragment thereof of the present invention, or a therapeutic or diagnostic antibody to be conjugated or fused with the multispecific antibody or antigen-binding fragment thereof, contains one or more substitutions in one or more Fc portion binding sites to eliminate effector functions. For example, the multispecific antibody or antigen-binding fragment thereof of the invention, or a therapeutic or diagnostic antibody to be conjugated or fused to the multispecific antibody or antigen-binding fragment thereof, may contain an Fc region comprising one or more of the following substitutions: substitution of proline for glutamic acid at residue 233, substitution of alanine or valine for phenylalanine at residue 234, and substitution of alanine or glutamic acid for leucine at residue 235 (EU numbering, Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDept. of Health and Human Services, Bethesda, Md., NIH Publication No. 91-3242). Preferably, the antibody of interest contains one, two or three mutations among L234A, L235A and P331S (EU numbering, Kabat).

[0093] Antibodies of subclasses IgG1, IgG2, and IgG3 typically show complement activation (including C1q and C3 binding), while IgG4 does not activate the complement system and does not bind C1q and / or C3. Compared to other IgG subtypes, the human IgG4 Fc region has a reduced ability to bind FcγRs and complement factors. Preferably, the multispecific antibody or antigen-binding fragment thereof of the present invention, or a therapeutic or diagnostic antibody to be conjugated or fused with the multispecific antibody or antigen-binding fragment thereof comprises an Fc region derived from a human IgG4 Fc region. More preferably, the Fc region contains a human IgG4 Fc region with a substitution that eliminates effector function. For example, removing the N-linked glycosylation site in the IgG4 Fc region by replacing Asn with Ala at residue 297 (EU numbering) is another way to ensure elimination of residual effector activity.

[0094] Multispecific anti-pyroglutamate amyloid-β / anti-transferrin receptor (TfR) / anti-paired helical filaments (PHF)- Tau antibodies and antigen-binding fragments thereof

[0095] Anti-pyroglutamate amyloid protein-β binding region / anti-pyroglutamate amyloid protein-β antibody and antigen binding fragment thereof part

[0096] Anti-pyroglutamate amyloid-β antibodies and antigen-binding fragments thereof were previously disclosed in WO2020 / 193644, the entire text of which is incorporated herein by reference. In a general aspect, the present application relates to a multispecific antibody or antigen-binding fragment thereof, which comprises an antigen-binding region based on the previously described anti-pyroglutamate amyloid-β antibodies and antigen-binding fragments thereof.

[0097] As used herein, "pyroglutamate amyloid-β," "AβpE3" or "3pE Aβ" refers to a modified Aβ peptide that oligomerizes with Aβ42 and deposits in the Alzheimer's disease (AD) brain. Pyroglutamate amyloid-β can serve as a seed for misfolded Ab, which is a major step in AD. Pyroglutamate amyloid-β is known in the art, see, e.g., Perez-Garmendia et al., Curr. Neuropharma. 11(5):491-8 (2013); Wittnam et al., JBC 287(11):8154-62 (2012); and Wang et al., Alzheimer's Dement 12:e12029 (2020).

[0098] Anti-TfR antigen binding region / anti-TfR antibody and antigen binding fragment thereof

[0099] Anti-TfR antibodies and antigen-binding fragments thereof were previously disclosed in WO2021 / 0205358, the entire text of which is incorporated herein by reference. In a general aspect, the present application relates to a multispecific antibody or antigen-binding fragment thereof, which comprises an antigen-binding region based on the previously described anti-TfR antibody and antigen-binding fragment thereof. The anti-TfR antigen-binding region is capable of binding to primate TfR, such as human TfR or monkey TfR, and the antigen-binding region can be optimized to deliver the agent to the brain of a subject in need thereof. The relationship between the binding affinity of anti-TfR antibodies to TfR and the efficiency of transcytosis has previously been described as transcytosis improving as the affinity for TfR decreases (Yu, Zhang et al., 2011, Sci Transl Med 3(84):84ra44). In WO2021 / 0205358 (the entire text of which is incorporated herein by reference), it was surprisingly found that the relationship between affinity and transcytosis efficiency is more subtle and complex than previously described, with brain concentration being affected by both association rate and dissociation rate. Specifically, in order to achieve optimal brain PK and PD of an agent (such as a mAb) effectively delivered by an anti-TfR antibody or antigen-binding fragment thereof, a moderate dissociation rate that is neither too fast nor too slow is required.

[0100] Preferably, the anti-TfR antigen binding region of the present application is pH sensitive, for example, it has different binding affinities to TfR at different pH. For example, the anti-TfR antigen binding region of the present application can bind to the cell surface TfR with high affinity at a neutral pH such as physiological pH (e.g., pH 7.4), but when internalized into the endosomal compartment, it dissociates from TfR at an acidic pH such as a relatively low pH (pH 5.0-6.0). Affinity is a measure of the strength of binding between two parts (e.g., an antibody and an antigen). Affinity can be expressed in several ways. One way is to use the dissociation constant (K) of the interaction. D ) is used to represent. D It can be measured by conventional methods, including equilibrium dialysis or by directly measuring the rates of antigen-antibody dissociation and association, i.e., k off (kd or k dis ) rate and k on (or ka) rate (see, e.g., Nature, 1993 361:186-87). off / k on The ratio of 2 eliminates all parameters not related to affinity and is equal to the dissociation constant K. D (See generally Davies et al., Annual Rev Biochem, 1990 59:439-473). D The smaller the value, the higher the affinity. Another way to express affinity is K a , that is, K D The reciprocal of, or k on / k off Therefore, K a The larger the affinity, the higher the affinity. For example, the anti-TfR antigen binding region used in the composition and / or method of the present application can be an anti-TfR antigen binding region that has a binding affinity of 1 nanomolar (nM, 10 -9 M) or larger K D Binds to TfR and is activated at 10 ng / mL at an acidic pH (e.g., pH 4.5-6.0) such as pH 5.0. -4 sec -1 K dis Dissociation from TfR.

[0101] Therefore, a general aspect of the present application relates to an anti-TfR antigen binding region for delivering an agent to the brain of a subject in need thereof, wherein the anti-TfR antigen binding region binds to the TfR antigen at a neutral pH with a dissociation constant K of at least 1 nM, preferably 1 nM to 500 nM. DBinds to transferrin receptor (TfR), preferably human TfR1, and is activated by at least 10 ng / mL at acidic pH, preferably pH 5. - 4 sec -1 , preferably 10 -4 sec -1 Up to 10 -1 sec -1 The dissociation rate constant k d Binds to TfR, preferably human TfR1.

[0102] In one embodiment, the anti-TfR antigen binding region of the present application has a pH of 2×10 -2 sec -1 Up to 2×10 -4 sec -1 , such as 2×10 -2 sec -1 , 1×10 -2 sec -1 ,9×10 -3 sec -1 , 8×10 -3 sec -1 ,7×10 -3 sec -1 , 6×10 -3 sec -1 , 5×10 -3 sec -1 , 4×10 -3 sec -1 , 3×10 -3 sec -1 , 2×10 -3 sec -1 , 1×10 -3 sec -1 ,9×10 -4 sec -1 , 8×10 -4 sec -1 ,7×10 -4 sec -1 , 6×10 -4 sec -1 , 5×10 -4 sec -1 , 4×10 -4 sec -1 , 3×10 -4 sec -1 , 2×10 -4 sec -1 or any value in between of the dissociation rate constant k d .

[0103] Anti-PHF-tau antigen binding region / anti-PHF-tau antibody and antigen binding fragment thereof

[0104] Anti-PHF-tau antibodies and antigen-binding fragments thereof were previously disclosed in U.S. Provisional Application No. 63 / 166,254 filed on March 26, 2021, which is incorporated herein by reference in its entirety. In one general aspect, the present application relates to a multispecific antibody or antigen-binding fragment thereof, which comprises an antigen-binding region based on the previously described anti-paired helical filament (PHF)-tau antibodies and antigen-binding fragments thereof.

[0105] In certain embodiments, the anti-PHF-tau antigen binding region or antigen binding fragment thereof binds to PHF-tau. Such anti-PHF-tau antigen binding region may have the property of binding to a phosphorylated epitope on PHF-tau or to a non-phosphorylated epitope on PHF-tau. The anti-PHF-tau antigen binding region may be used as a therapeutic agent, as well as a research or diagnostic reagent to detect PHF-tau in a biological sample such as a tissue or a cell.

[0106] According to a specific aspect, the anti-PHF-tau antigen binding region or its antigen binding fragment binds to tau protein at an epitope in the C-terminal domain of tau protein. In some embodiments, the anti-PHF-tau antigen binding region or its antigen binding fragment binds to tau protein at an epitope of tau protein having an amino acid sequence of SEQ ID NO: 36 or within the amino acid sequence, wherein the antigen binding region or its antigen binding fragment binds to PHF-tau, preferably human PHF-tau. Preferably, the anti-PHF-tau antigen binding region or its antigen binding fragment binds to tau protein at an epitope of tau protein consisting of or within the amino acid sequence of SEQ ID NO: 36, wherein the antigen binding region or its antigen binding fragment binds to PHF-tau, preferably human PHF-tau.

[0107] In some embodiments, the epitope of tau protein comprises one or more of phosphorylated T427, phosphorylated S433, and phosphorylated S435 of tau protein, but does not comprise all of phosphorylated T427, phosphorylated S433, and phosphorylated S435;

[0108] Multispecific antibodies and antigen-binding fragments thereof

[0109] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding region capable of specifically binding to pyroglutamate amyloid-β, a second antigen-binding region capable of specifically binding to transferrin receptor (TfR), and a third antigen-binding region capable of specifically binding to paired helical filament (PHF)-tau. In certain embodiments, (a) the first antigen binding region comprises (i) a first heavy chain variable region (VH1), which VH1 comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2 and HCDR3, which comprise the amino acid sequences of SEQ ID NOs: 8 or 16, 9 or 17 and 10, respectively; and (ii) a first light chain variable region (VL1), which VL1 comprises a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, which comprise the amino acid sequences of SEQ ID NOs: 11, 12 and 13, respectively; (b) the second antigen binding region comprises (i) a second heavy chain variable region (VH2), which VH2 comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2 and HCDR3, which comprise the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively; and (ii) a second light chain variable region (VL2), which VL2 comprises a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, which comprise the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively. NO:4, 5 and 6; and (c) the third antigen binding region comprises (i) a third heavy chain variable region (VH3), which VH3 comprises a heavy chain complementary determining region 1 (HCDR1), HCDR2 and HCDR3, which respectively comprise the amino acid sequences of SEQ ID NO:28, 29 and 30; and (ii) a third light chain variable region (VL3), which VL3 comprises a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, which respectively comprise the amino acid sequences of SEQ ID NO:31, 32 and 33.

[0110] In certain embodiments, VH1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 14; and VL1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 15. In certain embodiments, VH1 comprises the amino acid sequence of SEQ ID NO: 14; and VL1 comprises the amino acid sequence of SEQ ID NO: 15.

[0111] In certain embodiments, the second antigen binding region comprises a single chain fragment variable (scFv) antibody or antigen binding fragment thereof comprising VH2 and VL2. The scFv may, for example, comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 7. In certain embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 7.

[0112] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof may, for example, comprise (i) a first heavy chain (HC1) comprising VH3 and VL3, a first heavy chain constant region comprising a first Fc region (Fc1), and an scFv; (ii) a second heavy chain (HC2) comprising VH1 and a second heavy chain constant region comprising a second Fc region (Fc2); and (iii) a first light chain (LC) comprising VL1 and a light chain constant region.

[0113] Preferably, the anti-TfR antigen binding region is a single chain variable fragment (scFv), which comprises a light chain variable region (L V ) covalently linked heavy chain variable region (H V ). Despite the removal of the constant region and the introduction of a linker, scFvs can retain the specificity of the original immunoglobulin. In scFvs, the order of the domains can be H V -Connector-L V or L V -Connector-H V The linker can be designed de novo or derived from a known protein structure to provide a compatible length and conformation when bridging the variable domains of the scFv without severe steric interference. The linker can have a length of 10 to about 25 amino acids. Preferably, the linker is a length that spans about 3.5 nm between the carboxyl terminus of the variable domain and the amino terminus of the other domain. Without affecting the ability of domain folding and forming a complete antigen binding site, the peptide linker (Huston et al., Methods in Enzymology, Vol. 203, pp. 46-88, 1991, the entire text of which is incorporated herein by reference). The linker preferably comprises a hydrophilic sequence to avoid the embedding of the peptide within or between the variable domains during the entire protein folding process (Argos, Journal of Molecular Biology, Vol. 211, No. 4, pp. 943-958, 1990). For example, the linker may comprise Gly and Ser residues and / or interspersed with charged residues such as Glu, Thr and Lys to improve solubility. In one embodiment, the linker has the amino acid sequence (GGAGGA) of SEQ ID NO: 27. According to the present disclosure, any other suitable linker may also be used. In certain embodiments, the scFv is connected to the carboxyl terminus of the first heavy chain constant region via a linker, more specifically via a linker comprising the amino acid sequence of SEQ ID NO: 27.

[0114] Also provided is a multispecific antibody or antigen-binding fragment thereof, comprising a first heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 24, a first light chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 25, and a second heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 26. In certain embodiments, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 24, the first light chain comprises the amino acid sequence of SEQ ID NO: 25, and the second heavy chain comprises the amino acid sequence of SEQ ID NO: 26.

[0115] According to another specific aspect, the present invention relates to an isolated multispecific antibody or antigen-binding fragment thereof of the present invention, wherein the multispecific antibody or antigen-binding fragment thereof is chimeric.

[0116] According to another specific aspect, the present invention relates to an isolated multispecific antibody or antigen-binding fragment thereof of the present invention, wherein the multispecific antibody or antigen-binding fragment thereof is human or humanized.

[0117] In another general aspect, the invention relates to an isolated nucleic acid encoding a multispecific antibody or antigen-binding fragment thereof of the invention. One skilled in the art will appreciate that the coding sequence of a protein may be altered (e.g., replaced, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, one skilled in the art will appreciate that the nucleic acid sequence encoding a monoclonal antibody or antigen-binding fragment thereof of the invention may be altered without changing the amino acid sequence of the protein.

[0118] In another general aspect, the present invention relates to a vector comprising an isolated nucleic acid encoding a multispecific antibody or antigen-binding fragment thereof of the present invention. According to the present disclosure, any vector known to those skilled in the art, such as a plasmid, a cosmid, a phage vector or a viral vector, may be used. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any element of the conventional function of establishing an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selection marker and an origin of replication. The promoter may be a constitutive, inducible or repressible promoter. A variety of expression vectors capable of delivering nucleic acids to cells are known in the art and may be used herein to produce antibodies or antigen-binding fragments thereof in cells. Conventional cloning techniques or artificial gene synthesis may be used to generate recombinant expression vectors according to embodiments of the present invention.

[0119] In another general aspect, the present invention relates to a host cell comprising an isolated nucleic acid encoding a multispecific antibody or antigen-binding fragment thereof of the present invention. In view of the present disclosure, any host cell known to those skilled in the art can be used to recombinantly express the antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the host cell is an Escherichia coli TG1 or BL21 cell (for expressing, for example, scFv or Fab antibodies), a CHO-DG44 or CHO-K1 cell or a HEK293 cell (for expressing, for example, a full-length IgG antibody). According to a specific embodiment, the recombinant expression vector is transformed into a host cell by conventional methods such as chemical transfection, heat shock or electroporation, wherein the recombinant expression vector is stably integrated into the host cell genome so that the recombinant nucleic acid is effectively expressed.

[0120] Brain spindle construct

[0121] An optimized RMT brain delivery platform was developed using transferrin receptor (TfR) by improving intrinsic transcytosis efficiency, extending peripheral pharmacokinetics, and designing an acceptable safety profile while maintaining the efficacy of therapeutic mAbs. The interaction between transcytosis receptor affinity and brain concentration in human TfR knock-in mice was studied. In-depth studies of binding kinetics showed that a moderate dissociation rate that was neither too fast nor too slow was required to achieve optimal brain PK and PD of mAbs. This enhanced brain delivery observed in mice was confirmed in cynomolgus monkeys.

[0122] It was also found that enhanced binding of the engineered antibody constant regions to the neonatal Fc receptor (FcRn) resulted in reduced peripheral clearance and increased brain concentrations.

[0123] Additional Fc mutations were introduced to abolish binding to Fcγ receptors (FcγRs) and avoid effector function-mediated toxicity. When coupled with a high-affinity anti-pyroglutamate amyloid-β binding mAb and a high-affinity anti-PHF-tau binding mAb, these mutations prevent effector function-mediated toxicity in the periphery while maintaining antibody-dependent phagocytosis (ADP) through a novel FcγR-independent mechanism of microglial uptake and target degradation. This mechanism relies on internalization through the TfR receptor and is more effective than conventional FcγR-mediated ADP in promoting target degradation without stimulating the secretion of proinflammatory cytokines.

[0124] In certain embodiments, Fc1 and Fc2 each comprise one or more heterodimer mutations compared to a wild-type Fc region, such as a first modified heterodimer CH3 domain and a second modified heterodimer CH3 domain, respectively; specifically, Fc1 comprises amino acid modifications at positions T350, L351, F405, and Y407, and Fc2 comprises amino acid modifications at positions T350, T366, K392, and T394, wherein the amino acid modification at position T350 is T350V, T350I, T350L, or T350M; the amino acid modification at position L351 is L351Y; the amino acid modification at position F405 is F405A, F405V, F405I, T350L, or T350M; F405T or F405S; the amino acid modification at position Y407 is Y407V, Y407A or Y407I; the amino acid modification at position T366 is T366L, T366I, T366V or T366M, the amino acid modification at position K392 is K392F, K392L or K392M, and the amino acid modification at position T394 is T394W, and wherein the numbering of amino acid residues is performed according to the EU index as described in Kabat, more specifically, Fc1 comprises amino acid modifications T350V, L351Y, F405A and Y407V, and Fc2 comprises amino acid modifications T350V, T366L, K392L and T394W.

[0125] In certain embodiments, at least one of Fc1 and Fc2 comprises one or more mutations that enhance binding of the multispecific antibody or antigen-binding fragment thereof to the neonatal Fc receptor (FcRn), preferably, the one or more mutations enhance binding at acidic pH, more preferably, the at least one of Fc1 and Fc2 has M252Y / S254T / T256E (YTE) mutations, wherein the numbering of amino acid residues is performed according to the EU index as set forth in Kabat.

[0126] In certain embodiments, at least one of Fc1 and Fc2 comprises one or more mutations that reduce or eliminate effector function, preferably at least one of Fc1 and Fc2 has one or more amino acid modifications at positions L234, L235, D270, N297, E318, K320, K322, P331 and P329, such as one, two or three mutations of L234A, L235A and P331S, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.

[0127] Therefore, in a general aspect, the present application relates to a brain delivery system targeted by an antibody comprising an anti-TfR antigen binding region of the present application. The anti-TfR antigen binding region can be used to deliver therapeutic agents or diagnostic agents to cells (e.g., cancer cells) or BBB systems. The agents that can be delivered include any nervous system disorder drugs or agents that can be used to detect or analyze nervous system disorder drugs. For example, such agents can be neurotrophic factors, including but not limited to nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell line neurotrophic factor (GDNF) and insulin-like growth factor (IGF); neuropeptides, including but not limited to substance P, neuropeptide Y, vasoactive intestinal peptide (VIP), γ-aminobutyric acid (GABA), dopamine, cholecystokinin (CCK), endorphins, enkephalins and thyrotropin-releasing hormone (TRH); cytokines; antianxiety agents; anticonvulsants; polynucleotides and transgenics, including, for example, small interfering RNA and / or antisense oligonucleotides; or antibodies or antigen-binding fragments thereof that bind to brain targets. The anti-hTfR antigen-binding region of the present application may be an effective means to enhance the delivery of an agent of interest from the blood to the brain and its action in the brain.

[0128] Specifically, the agent of interest can be delivered parenterally, for example, intravenously, in a combined form or in a form connected to the anti-TfR antigen binding region of the present application. For example, the agent can be non-covalently attached to the anti-TfR antigen binding region. The agent can also be covalently attached to the anti-TfR antigen binding region to form a conjugate. In certain embodiments, conjugation is achieved by constructing a protein fusion (i.e., by fusing two genes encoding the anti-TfR antigen binding region with the gene of a drug for a nervous system disorder and expressing it as a single protein). According to the present disclosure, the agent can be connected to an antibody or its antigen-binding fragment using known methods. See, e.g., Wu et al., Nat Biotechnol., 23(9): 1137-46, 2005; Trail et al., Cancer Immunol Immunother., 52(5): 328-37, 2003; Saito et al., Adv Drug Deliv Rev., 55(2): 199-215, 2003; Jones et al., Pharmaceutical Research, 24(9): 1759-1771, 2007.

[0129] In some embodiments, the therapeutic agent or diagnostic agent to be delivered to the brain and the anti-TfR antigen binding region can be covalently linked (or conjugated) together via a non-peptide linker or a peptide linker. Examples of non-peptide linkers include, but are not limited to, polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethers, biodegradable polymers, polymerized lipids, chitin and hyaluronic acid, or their derivatives, or combinations thereof. The peptide linker can be a peptide chain consisting of 1 to 50 amino acids connected by a peptide bond, or a derivative thereof, and its N-terminus and C-terminus can be covalently linked to the anti-TfR antigen binding region.

[0130] In certain embodiments, the conjugate of the present application is a multispecific antibody or an antigen-binding fragment thereof, which comprises a first antigen-binding region that binds to pyroglutamate amyloid protein-β, a second antigen-binding region that binds to TfR, and a third antigen-binding region that binds to PHF-tau. Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537, 1983; WO 93 / 08829 and Traunecker et al., EMBO J. 10:3655, 1991) and "knobs into holes" engineering (see, for example, U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by: engineering electrostatic steering effects (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229:81, 1985); using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553, 1992); using "diabody" technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448, 1993); using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol, 152:5368 (1994)); and preparing trispecific antibodies as described in Tutt et al., J. Immunol. 147:60, 1991. The multispecific antibodies of the present application also encompass antibodies with three or more functional antigen binding sites, including "octopus antibodies" or "dual variable domain immunoglobulins" (DVD) (see, e.g., US2006 / 0025576A1 and Wu et al., Nature Biotechnology, 25(11):1290-7, 2007). The multispecific antibodies of the present application also encompass "dual-acting Fab" or "DAF", which comprises an antigen binding region that binds to TfR and PHF-tau (see, e.g., US2008 / 0069820). In one embodiment, the antibody is an antibody fragment, and various such fragments are disclosed herein.

[0131] In one embodiment, the multispecific antibody of the present application is a fusion construct comprising an anti-TfR antigen binding region of the present application covalently linked (or fused) to a second antigen binding region or an antigen binding fragment thereof. Preferably, the second antigen binding region or an antigen binding fragment thereof binds to a brain target, such as PHF-tau as described herein. The anti-TfR antigen binding region can be fused directly or via a linker to the carboxyl and / or amino terminus of the light chain and / or heavy chain of the second antibody or its antigen binding fragment.

[0132] In one embodiment, the anti-TfR antigen binding region is fused directly or via a linker to the carboxyl terminus of the light chain of a second antibody or antigen binding fragment thereof.

[0133] In another embodiment, the anti-TfR antigen binding region is fused directly or via a linker to the amino terminus of the light chain of a second antibody or antigen binding fragment thereof.

[0134] In another embodiment, the anti-TfR antigen binding region is fused directly or via a linker to the carboxyl terminus of the heavy chain of a second antibody or antigen binding fragment thereof.

[0135] In another embodiment, the anti-TfR antigen binding region is fused directly or via a linker to the amino terminus of the heavy chain of a second antibody or antigen-binding fragment thereof.

[0136] In a preferred embodiment, the fusion construct of the present application comprises the anti-TfR antigen binding region of the present application, preferably anti-huTfR1 VHH or scFv fragment, which is covalently linked to the carboxyl terminus of the heavy chain of the second antigen binding region or antigen binding fragment thereof that binds to PHF-tau via a linker. Preferably, the linker has the amino acid sequence of SEQ ID NO: 27.

[0137] In order to promote the formation of heterodimers between two heavy chains, for example, a fusion with an anti-TfR antigen binding region and one without; or an Fc containing an anti-TfR arm and an Fc containing an anti-PHF-tau arm, heterodimer mutations are introduced into the Fc of the two heavy chains. Examples of such Fc mutations include, but are not limited to, Zymework mutations (see, e.g., US10,457,742) and "knobs into holes" mutations (see, e.g., Ridgway et al., Protein Eng., 9(7): 617-621, 1996). Other heterodimer mutations may also be used in the present invention. In some embodiments, a modified CH3 as described herein is used to promote the formation of heterodimers between two heavy chains.

[0138] In addition to heterodimer mutations, other mutations may also be introduced. In some embodiments, the Fc region of the fusion construct or bispecific antibody further comprises one or more mutations (such as AAS mutations described herein) that change (increase or decrease), preferably eliminate ADCC / CDC and / or one or more mutations (such as YTE mutations described herein) that change (increase or decrease), preferably increase the binding of the fusion construct or bispecific antibody to FcRn. In some embodiments, one or more cysteine ​​residues in the fusion construct or bispecific antibody are replaced by other amino acids (such as serine).

[0139] The conjugates of the present application, such as multispecific antibodies or fusion constructs, can be produced according to the present disclosure by any of a variety of techniques known in the art. For example, they can be expressed from recombinant host cells, wherein an expression vector encoding the heavy and light chains of the fusion construct or multispecific antibody is transfected into the host cell by standard techniques. The host cell can be a prokaryotic host cell or a eukaryotic host cell.

[0140] In an exemplary system, one or more recombinant expression vectors encoding the two heavy chains and light chains of the heterodimerization of the fusion construct of the present application are introduced into a host cell by transfection or electroporation. The selected transformed host cells are cultured to allow expression of the heavy and light chains under conditions sufficient to produce the fusion construct, and the fusion construct is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select the transformants, culture the host cells and recover the protein construct from the culture medium.

[0141] Pharmaceutical compositions and related methods

[0142] The present invention further relates to pharmaceutical compositions, methods of preparation, and methods of use thereof.

[0143] In another general aspect, the present invention relates to a pharmaceutical composition comprising a multispecific antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier. The multispecific antibodies or antigen-binding fragments thereof of the present invention can also be used to manufacture medicaments for therapeutic applications mentioned herein. The pharmaceutically acceptable carrier can be any suitable excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation or other materials well known in the art for use in pharmaceutical formulations. It should be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for the specific application.

[0144] Therefore, in one embodiment, the present application relates to a method for transporting a therapeutic agent or a diagnostic agent across the blood-brain barrier (BBB), the method comprising exposing a multispecific antibody or an antigen-binding fragment thereof of the present invention coupled to a therapeutic agent or a diagnostic agent to the blood-brain barrier, so that the multispecific antibody or its antigen-binding fragment transports the agent coupled thereto across the blood-brain barrier. In one embodiment, the agent is a drug for a nervous system disorder. In another embodiment, the agent is an imaging agent or an agent for detecting a nervous system disorder. Preferably, the multispecific antibody or its antigen-binding fragment does not weaken the binding of TfR to its natural ligand transferrin. The antibody specifically binds to TfR in a manner that does not inhibit the binding of TfR to transferrin. In some embodiments, the BBB is in a mammal, preferably a primate, such as a human, more preferably in a human suffering from a nervous system disorder. In one embodiment, the neurological disorder is selected from Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer and traumatic brain injury.

[0145] In one embodiment, the multispecific antibodies or antigen-binding fragments thereof of the present application are used to detect a neurological disorder and / or assess the severity or duration of a disease or disorder prior to the onset of symptoms. The multispecific antibodies or antigen-binding fragments thereof allow for detection and / or imaging of a neurological disorder, including by radiography, tomography, or magnetic resonance imaging (MRI).

[0146] In another embodiment, the multispecific antibody or antigen-binding fragment thereof is used to treat a neurological disorder (e.g., Alzheimer's disease), comprising administering an effective amount of the multispecific antibody or antigen-binding fragment thereof to a subject in need of treatment. In some embodiments, the method further comprises administering an effective amount of at least one additional therapeutic agent to the subject.

[0147] In another embodiment, the present invention relates to a method for preventing, ameliorating, treating and / or reducing amyloid-β deposition in an amyloid-β-related disorder, the method comprising administering a multispecific antibody or antigen-binding fragment thereof as disclosed herein to a subject in need thereof in a therapeutically effective amount. Additional aspects of the present invention include pharmaceutical compositions for preventing, ameliorating, treating and / or reducing amyloid deposition in an amyloid-β-related disorder, comprising an antibody or antigen-binding fragment thereof as disclosed herein. The method of the present invention comprises administering an effective amount of one or more antibodies or antigen-binding fragments thereof as described herein to a subject in need thereof.

[0148] In one aspect, the present invention relates to a method for preventing, ameliorating, treating and / or reducing amyloid-β deposition in a disorder characterized by the formation of plaques containing β-amyloid in a human, the method comprising administering to a person in need of such treatment, preferably peripherally, a therapeutically effective amount or a prophylactically effective amount of a multispecific antibody according to the present invention or an immunologically reactive fragment thereof, the multispecific antibody specifically binding to human Aβ3pE. In another aspect, the present invention relates to a method for inhibiting the formation of amyloid plaques and / or a method for clearing amyloid plaques in humans, the method comprising administering to a human subject in need of such inhibition or clearance an effective amount of a multispecific antibody according to the present invention, wherein the multispecific antibody sequesters Aβ3pE peptides in the brain and induces altered Aβ3pE clearance in the brain.

[0149] A subject in need thereof is a person suffering from or susceptible to a disorder characterized by the formation of plaques comprising beta amyloid. In one embodiment, the disorder is Alzheimer's disease. In other embodiments, the disorder is dementia associated with trisomy 21 (Down syndrome), diffuse Lewy body disease, inclusion body myositis, cerebral amyloid angiopathy, or hereditary cerebral hemorrhage with Dutch amyloidosis (HCHWA-D).

[0150] In one embodiment of the invention, the multispecific antibodies or antigen-binding fragments thereof of the invention bind to 3pE Aβ in plaque deposits. By binding to 3pE Aβ in plaque deposits, the multispecific antibodies or antigen-binding fragments thereof can induce plaque removal. Induction of plaque removal can be performed by activating microglia surrounding the plaque and destabilizing the plaque by removing stable Aβ forms. In addition, the antibodies or antigen-binding fragments thereof of the invention can inhibit the plaque seeding activity of 3pE Aβ. The possible enrichment of 3pE Aβ in plaques compared to vascular amyloid can increase the therapeutic safety window of immunotherapy.

[0151] In another general aspect, the present application relates to a method for treating or alleviating the symptoms of a disease, disorder or condition (such as tauopathy) in a subject in need thereof, the method comprising administering to the subject the multispecific antibody or antigen-binding fragment thereof of the present application or the pharmaceutical composition of the present application.

[0152] In another general aspect, the present application relates to a method for reducing pathological tau aggregation or the spread of tauopathy in a subject in need thereof, the method comprising administering to the subject the multispecific antibody or antigen-binding fragment thereof of the present application or the pharmaceutical composition of the present application.

[0153] According to specific embodiments, the disease, disorder or condition to be treated is a tauopathy. According to more specific embodiments, the diseases, disorders or conditions to be treated include, but are not limited to, familial Alzheimer's disease, sporadic Alzheimer's disease, frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle-only dementia, diffuse neurofibrillary tangles with calcifications, argyrophilic grain dementia, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Strauss-Schaefer disease, Hallevorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, chronic traumatic encephalopathy, or dementia pugilista (boxing disease).

[0154] Tauopathies-associated behavioral phenotypes include, but are not limited to, cognitive impairment, early personality changes and loss of control, apathy, avolition, mutism, apraxia, perseverative speech, stereotyped movements / behaviors, hypersexuality, disorganization, inability to plan or organize sequential tasks, selfishness / callousness, antisocial traits, lack of perspective-taking, hesitancy, agrammatic speech with frequent semantic errors but relatively well-preserved comprehension, impaired comprehension and word selection, slowly progressive gait instability, paragastases, rigidity, frequent falls, axial rigidity that is not levodopa-responsive, supranuclear gaze palsy, square-wave jerks, slow vertical saccades, pseudobulbar palsy, limb apraxia, dystonia, cortical sensory loss, and tremor.

[0155] Patients who comply with treatment include, but are not limited to, asymptomatic individuals who are at risk of AD or other tauopathy, and patients who currently show symptoms. Patients who comply with treatment include individuals with known AD genetic risks such as AD family history or genetic risk factors in the genome. Exemplary risk factors are mutations in amyloid precursor protein (APP), especially mutations at position 717 and position 670 and 671 (Hardy mutation and Swedish mutation, respectively). Other risk factors are mutations in presenilin genes, PS1 and PS2, and ApoE4, family history of hypercholesterolemia or atherosclerosis. Individuals who currently suffer from AD can be identified by the characteristic dementia of the presence of the above-mentioned risk factors. In addition, multiple diagnostic tests can be used to identify individuals who suffer from AD. These tests include measuring cerebrospinal fluid tau and Aβ42 levels. The Aβ42 levels of elevated tau and reduced represent the presence of AD. Individuals who suffer from AD can also be diagnosed by AD and related disease association (AD and RelatedDisorders Association) standards.

[0156] The multispecific antibodies or antigen-binding fragments thereof comprising the anti-PHF-tau antigen-binding region or antigen-binding fragments of the present application are suitable as both therapeutic agents and preventive agents for treating or preventing neurodegenerative diseases, which involve pathological aggregation of tau, such as AD or other tauopathies. In asymptomatic patients, treatment can be started at any age (e.g., at about 10, 15, 20, 25, 30 years old). However, generally, treatment does not need to be started until the patient reaches about 40, 50, 60 or 70 years old. Treatment generally requires multiple doses over a period of time. Treatment can be monitored by measuring antibodies to the therapeutic agent over time, or activated T cells or B cell responses. If the response decreases, a booster dose may be indicated.

[0157] In preventive applications, pharmaceutical compositions or agents are administered to patients susceptible to AD or otherwise at risk for AD in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes present during the course of disease progression. In therapeutic applications, compositions or agents are administered to patients suspected of having or already having such a disease in an amount sufficient to reduce, prevent, or delay any of the symptoms of the disease (biochemical, histological, and / or behavioral). Administration of a therapeutic agent can reduce or eliminate mild cognitive impairment in a patient who has not yet developed characteristic Alzheimer's pathology.

[0158] According to a specific embodiment, the composition used in the treatment of tau disease can be used in combination with other agents that effectively treat related neurodegenerative diseases. In the case of AD, the multispecific antibodies of the present application can be co-administered with agents that reduce or prevent the deposition of amyloid beta (Aβ). Synergistic effects of PHF-tau and Aβ pathologies are possible. Therefore, a combined treatment that simultaneously targets the removal of both PHF-tau and Aβ and Aβ-related pathologies may be more effective than individual targeting of each. In the case of Parkinson's disease and related neurodegenerative diseases, immunomodulation that removes aggregated forms of alpha-synuclein is also an emerging treatment. A combined treatment that simultaneously targets the removal of both tau and alpha-synuclein may be more effective than individual targeting of either protein.

[0159] In another embodiment, the present application relates to the use of the multispecific antibody or antigen-binding fragment thereof of the present application in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat a nervous system disease or disorder. In another embodiment, the medicament is used in a method for treating a nervous system disease or disorder, the method comprising administering an effective amount of the medicament to an individual suffering from a nervous system disease or disorder.

[0160] Another general aspect of the present application relates to a method for inducing antibody-dependent phagocytosis (ADP) in a subject in need thereof without stimulating the secretion of proinflammatory cytokines, the method comprising administering to the subject a compound comprising a therapeutic antibody or its antigen-binding fragment coupled to the multispecific antibody or its antigen-binding fragment of the present application, preferably covalently conjugated, wherein the therapeutic antibody or its antigen-binding fragment does not have effector function. For example, the therapeutic antibody or its antigen-binding fragment may include one or more amino acid modifications that reduce or eliminate effector function (such as ADCC or CDC), such as mutations that reduce or abolish binding to Fcγ receptors. Such mutations may be at positions L234, L235, D270, N297, E318, K320, K322, P331 and P329, such as one, two or three mutations in L234A, L235A and P331S, wherein the numbering of amino acid residues is performed according to the EU index as described in Kabat. In one embodiment, the therapeutic antibody or its antigen-binding fragment specifically binds to tau aggregates.

[0161] In some embodiments, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is a therapeutic agent that is effective for treating a nervous system disorder that is the same or different from the nervous system disorder that the multispecific antibody or its antigen-binding fragment is used to treat. Exemplary additional therapeutic agents include, but are not limited to, the various nervous system drugs described above, cholinesterase inhibitors (such as donepezil, galantamine, rivastigmine and tacrine), NMDA receptor antagonists (such as memantine), amyloid beta peptide aggregation inhibitors, antioxidants, γ-secretase modulators, nerve growth factor (NGF) mimetics or NGF gene therapy, PPARy agonists, HMS-CoA reductase inhibitors (inhibitors), ampakins, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulins, muscarinic receptor agonists, nicotinic receptor modulators, active or passive amyloid beta peptide immunization, phosphodiesterase inhibitors, serotonin receptor antagonists, and anti-amyloid beta peptide antibodies. In certain embodiments, the at least one additional therapeutic agent is selected according to its ability to alleviate one or more side effects of nervous system drugs. The additional therapeutic agent can be applied in the same preparation or a separate preparation, and is applied together or separately with the multispecific antibody or its antigen-binding fragment. The multispecific antibody or its antigen-binding fragment of the present application can be applied before, after and / or simultaneously with the additional therapeutic agent and / or adjuvant. The multispecific antibody or its antigen-binding fragment of the present application can also be used in combination with other interventional therapies, such as but not limited to radiotherapy, behavioral therapy or other therapies known in the art and suitable for the nervous system disorder to be treated or prevented.

[0162] The multispecific antibodies or antigen-binding fragments thereof (and any additional therapeutic agents) of the present application can be administered by any suitable means, including parenteral, intrapulmonary and intranasal administration, and if necessary for local treatment, the means include intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration, depending in part on whether the administration is short-lived or long-term. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations at multiple time points, bolus administration and pulse infusions.

[0163] For disease prevention or treatment, the appropriate dosage of the multispecific antibodies or antigen-binding fragments thereof of the present application (when used alone or in combination with one or more other additional therapeutic agents) will depend on a variety of factors, such as the type of disease to be treated, the type of antibody or conjugate, the severity and course of the disease, whether the multispecific antibody or antigen-binding fragment thereof is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, the subject's physiological state (including, for example, age, weight, health status), and the judgment of the attending physician. The therapeutic dose is optimally titrated to optimize safety and efficacy. The multispecific antibody or antigen-binding fragment thereof is appropriately administered to the patient at one time or over a series of treatments.

[0164] According to certain embodiments, a therapeutically effective amount refers to a therapeutic amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder, or condition being treated, or symptoms associated therewith; (ii) reduce the duration of the disease, disorder, or condition being treated, or symptoms associated therewith; (iii) prevent the development of the disease, disorder, or condition being treated, or symptoms associated therewith; (iv) cause regression of the disease, disorder, or condition being treated, or symptoms associated therewith; (v) prevent the development or onset of the disease, disorder, or condition being treated, or symptoms associated therewith; vi) prevent recurrence of the disease, disorder or condition to be treated, or symptoms associated therewith; (vii) reduce hospitalization of a subject suffering from the disease, disorder or condition to be treated, or symptoms associated therewith; (viii) reduce the length of hospital stay of a subject suffering from the disease, disorder or condition to be treated, or symptoms associated therewith; (ix) improve survival of a subject suffering from the disease, disorder or condition to be treated, or symptoms associated therewith; (xi) inhibit or reduce the disease, disorder or condition to be treated, or symptoms associated therewith, in a subject; and / or (xii) enhance or improve the prophylactic or therapeutic effect of another therapy.

[0165] Diagnostic methods and kits

[0166] In another aspect, the present application relates to an article (such as a kit) containing materials that can be used to treat, prevent and / or diagnose the above-mentioned disorders. The article includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed of a variety of materials, such as glass or plastic. The container holds a composition that is effective for treating, preventing and / or diagnosing a condition when it is itself or in combination with another composition, and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a multispecific antibody or antigen-binding fragment thereof of the present application. The label or package insert indicates that the composition is used to treat a selected condition. In addition, the article may include (a) a first container containing a composition, wherein the composition comprises a multispecific antibody or antigen-binding fragment thereof of the present application; and (b) a second container containing a composition, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent. The article in this embodiment of the present invention may also include a package insert indicating that the composition can be used to treat a specific condition. Optionally, the article of manufacture may further include a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may also include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0167] In vitro methods

[0168] It should be understood that all forms of immunoassays employing multispecific antibodies or antigen-binding fragments thereof are contemplated for use according to the presently preferred embodiments, including assays in which the multispecific antibodies or antigen-binding fragments thereof are bound to a solid phase and assays in which the antibodies are in a liquid medium. Immunoassay methods that can be used to detect analytes using multispecific antibodies embodying features of the invention include, but are not limited to, competitive (reagent-limited) assays in which a labeled analyte (analyte analog) in a sample and the analyte competes for the antibody, as well as single-site immunoassays in which the multispecific antibody is labeled; etc.

[0169] The multispecific antibodies or antigen-binding fragments thereof according to the present invention can be used in conventional immunological techniques for detecting Aβ3pE, TfR or PHF-tau, wherever the peptide may be present, including biological samples and conditioned medium from cell culture. Suitable immunological techniques are well known to those skilled in the art and include, for example, ELISA, Western blot analysis, competitive or sandwich immunoassays, etc., and as is also well known, they all rely on the formation of antigen-antibody immune complexes, wherein for the purpose of the assay, the antibody or antigen-binding fragment thereof can be detectably labeled with, for example, a radioactive, enzymatic, luminescent or fluorescent label, or the antibody or antigen-binding fragment thereof can be immobilized on an insoluble carrier. Therefore, it is an object of the present invention to provide an immunoassay for determining or detecting Aβ3pE, TfR and / or PHF-tau or fragments thereof in a sample, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof against Aβ3pE, TfR and / or PHF-tau or fragments thereof according to the present invention, and determining whether an immune complex is formed between the antibody or antigen-binding fragment thereof and Aβ3pE, TfR and / or PHF-tau or fragments thereof. These methods can be performed on tissue samples or body fluid samples, and generally include obtaining a sample from the subject's body; contacting the sample with an imaging effective amount of a detectably labeled antibody or antigen-binding fragment thereof according to the present invention; and detecting the label to determine the presence of Aβ3pE and / or TfR or a fragment thereof in the sample. There is no particular limitation on the measurement method using the antibody or antigen-binding fragment thereof of the present invention. Any measurement method can be used, as long as the amount of antibody, antigen, or antigen-antibody complex corresponding to the amount of antigen in the solution to be tested (particularly the amount of Aβ3pE and / or TfR or a fragment thereof) is detected by chemical or physical means and calculated by using a standard curve obtained by using a standard solution containing a known amount of antigen. For example, turbidimetry, competition, immunoassay, and sandwich methods are suitable. In terms of sensitivity and specificity, the sandwich method is particularly preferred.

[0170] In the sandwich method, the test solution is reacted with an insoluble antibody (such as an insoluble multispecific antibody or the present invention) (first reaction), and in addition, a labeled second antibody is reacted (second reaction); the activity of the labeling agent on the insoluble carrier is then measured, thereby determining the amount of Aβ3pE, TfR and / or PHF-tau or a fragment thereof in the test solution. The first reaction and the second reaction can be performed simultaneously or sequentially.

[0171] In the measuring method, a labeling substance, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, etc. are used as a labeling agent. Examples of radioisotopes include 125 1. 131 I. 3 H and 14C. Enzymes are usually made detectable by conjugation to an appropriate substrate that then catalyzes a detectable reaction. Examples include, for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, and malate dehydrogenase, preferably horseradish peroxidase. The luminescent material includes, for example, luminol, luminol derivatives, luciferin, aequorin, and luciferase. In addition, avidin-biotin systems can also be used to label antibodies and immunogens of the present invention. When the immunogen or antibody is insoluble, physical adsorption or chemical binding commonly used for insolubilization or fixation of proteins or enzymes can be used. Examples of carriers include insoluble polysaccharides such as agarose, dextran and cellulose, synthetic resins such as polystyrene, polyacrylamide and silicone polymers, and glass.

[0172] In another embodiment for detecting or diagnosing β-amyloid protein related diseases, tau related diseases and / or other nervous system diseases or conditions, a biological sample including tissues, body fluids such as cerebrospinal fluid (CSF), blood, plasma, serum, urine, etc. is included and contacted with an appropriate amount of a first antibody to produce an immune complex. The contact generally involves adding the sample to a solid matrix coated with the first antibody. The fluoride produced by contacting the sample with the first antibody is separated from the sample by elution. However, other recovery methods can be used. The recovered complex is contacted with at least one second antibody, which is directed to an antigenic determinant on the antigen and is capable of binding to the antigen on the complex. The antigenic determinant targeted by the second antibody can be the same antigenic determinant targeted by the first antibody, due to the multi-epitope characteristics of the antigenic entity. Any of the above-mentioned labels can be used to make the first antibody or the second antibody detectable. In a preferred embodiment, the second antibody is detectable. The presence of a detectable antibody bound to a complex can be easily detected using techniques known in the art, and the complex consists of antigens bound to the first antibody and the second antibody. By comparing the results obtained in the biological sample with those obtained on a control sample, the presence or level of altered A[beta]3pE, TfR and / or PHF-tau or fragments thereof can be determined.

[0173] Implementation

[0174] The present invention also provides the following non-limiting embodiments.

[0175] Embodiment 1 is a multispecific antibody or an antigen-binding fragment thereof, comprising at least a first antigen-binding region capable of specifically binding to pyroglutamate amyloid-β, a second antigen-binding region capable of specifically binding to transferrin receptor (TfR), and a third antigen-binding region capable of specifically binding to paired helical filaments (PHF)-tau, wherein:

[0176] a. The first antigen binding region comprises:

[0177] i. a first heavy chain variable region (VH1), comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, which comprise the amino acid sequences of SEQ ID NOs: 8 or 16, 9 or 17 and 10, respectively; and

[0178] ii. a first light chain variable region (VL1), the VL1 comprising a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, which comprise the amino acid sequences of SEQ ID NOs: 11, 12 and 13, respectively; and

[0179] b. The second antigen binding region comprises:

[0180] i. a second heavy chain variable region (VH2), the VH2 comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, which comprise the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively; and

[0181] ii. a second light chain variable region (VL2), the VL2 comprising a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, which comprise the amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively; and

[0182] c. The third antigen binding region comprises:

[0183] i. a third heavy chain variable region (VH3), comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, which comprise the amino acid sequences of SEQ ID NOs: 28, 29 and 30, respectively; and

[0184] ii. a third light chain variable region (VL3), which VL3 comprises light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, which comprise the amino acid sequences of SEQ ID NOs: 31, 32 and 33, respectively.

[0185] Embodiment 2 is a multispecific antibody or antigen-binding fragment thereof according to embodiment 1, wherein VH1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:14; and VL1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:15.

[0186] Embodiment 3 is a multispecific antibody or antigen-binding fragment thereof according to embodiment 1, wherein VH1 comprises the amino acid sequence of SEQ ID NO:14; and VL1 comprises the amino acid sequence of SEQ ID NO:15.

[0187] Embodiment 4 is a multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, wherein the second antigen-binding region comprises a single-chain fragment variable (scFv) antibody or antigen-binding fragment thereof comprising VH2 and VL2.

[0188] Embodiment 5 is a multispecific antibody or antigen-binding fragment thereof according to embodiment 4, wherein the scFv comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:7.

[0189] Embodiment 6 is a multispecific antibody or antigen-binding fragment thereof according to embodiment 5, wherein the scFv comprises the amino acid sequence of SEQ ID NO:7.

[0190] Embodiment 7 is a multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, comprising:

[0191] (i) a first heavy chain (HC1) comprising VL3 and VL3, a first heavy chain constant region comprising a first Fc region (Fc1), and a scFv;

[0192] (ii) a second heavy chain (HC2), comprising VH1 and a second heavy chain constant region comprising a second Fc region (Fc2);

[0193] (iii) the first light chain (LC), which comprises VL1 and a light chain constant region.

[0194] Embodiment 8 is a multispecific antibody or antigen-binding fragment thereof according to embodiment 7, wherein the scFv is connected to the carboxyl terminus of the first heavy chain constant region via a linker, more specifically via a linker comprising the amino acid sequence of SEQ ID NO:27.

[0195] Embodiment 9 is a multispecific antibody or antigen-binding fragment thereof according to embodiment 7 or 8, wherein compared to the wild-type Fc region, Fc1 and Fc2 each comprise one or more heterodimer mutations, such as a first modified heterodimer CH3 domain and a second modified heterodimer CH3 domain, respectively; specifically, the Fc1 comprises amino acid modifications at positions T350, L351, F405 and Y407, and the Fc2 comprises amino acid modifications at positions T350, T366, K392 and T394, wherein the amino acid modification at position T350 is T350V, T350I, T350L or T350M; the amino acid modification at position L351 is L351Y; the amino acid modification at position F405 is The amino acid modification is F405A, F405V, F405T or F405S; the amino acid modification at position Y407 is Y407V, Y407A or Y407I; the amino acid modification at position T366 is T366L, T366I, T366V or T366M, the amino acid modification at position K392 is K392F, K392L or K392M, and the amino acid modification at position T394 is T394W, and wherein the numbering of amino acid residues is performed according to the EU index as described in Kabat, more specifically, Fc1 comprises amino acid modifications T350V, L351Y, F405A and Y407V, and Fc2 comprises amino acid modifications T350V, T366L, K392L and T394W.

[0196] Embodiment 10 is a multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 7 to 9, wherein at least one of Fc1 and Fc2 comprises one or more mutations that enhance the binding of the multispecific antibody or antigen-binding fragment thereof to the neonatal Fc receptor (FcRn), preferably, the one or more mutations enhance binding at acidic pH, more preferably, the at least one of Fc1 and Fc2 has M252Y / S254T / T256E (YTE) mutations, wherein the numbering of amino acid residues is performed according to the EU index as described in Kabat.

[0197] Embodiment 11 is a multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 7 to 10, wherein at least one of Fc1 and Fc2 comprises one or more mutations that reduce or eliminate effector function, preferably at least one of Fc1 and Fc2 has one or more amino acid modifications at positions L234, L235, D270, N297, E318, K320, K322, P331 and P329, such as one, two or three mutations of L234A, L235A and P331S, wherein the numbering of amino acid residues is according to the EU index as described in Kabat.

[0198] Embodiment 12 is a multispecific antibody or an antigen-binding fragment thereof, which comprises a first heavy chain, a first light chain, and a second heavy chain, wherein the first heavy chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:24, the first light chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:25, and the second heavy chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:26.

[0199] Embodiment 13 is a multispecific antibody or antigen-binding fragment thereof according to embodiment 12, wherein the first heavy chain comprises the amino acid sequence of SEQ ID NO:24, the first light chain comprises the amino acid sequence of SEQ ID NO:25, and the second heavy chain comprises the amino acid sequence of SEQ ID NO:26.

[0200] Embodiment 14 is an isolated nucleic acid sequence encoding the multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 13.

[0201] Embodiment 15 is a vector comprising the isolated nucleic acid according to embodiment 14.

[0202] Embodiment 16 is a host cell comprising the isolated nucleic acid according to embodiment 14 or the vector according to claim 15.

[0203] Embodiment 17 is a method for producing a multispecific antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell according to embodiment 16 under conditions where the multispecific antibody or the antigen-binding fragment thereof is produced, and recovering the multispecific antibody or the antigen-binding fragment thereof.

[0204] Embodiment 18 is a pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 13 and a pharmaceutically acceptable carrier.

[0205] Embodiment 19 is a method of treating or detecting a disorder, preferably a nervous system disorder, in a subject in need thereof, the method comprising administering to the subject a multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 13 or a pharmaceutical composition according to embodiment 18, preferably a nervous system disorder selected from a neurodegenerative disease (such as Lewy body disease, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy), tauopathies (such as Alzheimer's disease and supranuclear palsy), prion diseases (such as bovine spongiform encephalopathy), The present invention relates to a variety of diseases including encephalopathy, pruritus, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Schenck disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and heterogeneous degenerative disorders of the nervous system (such as Canavan disease, Huntington disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette syndrome, Menkes kinky hair syndrome, Cockne syndrome, Hallervorden-Spatz syndrome, Raffaello disease, Rett syndrome, Wilson's disease, Lesch-Nyhan syndrome, and Ong-Long syndrome), dementias (such as Pick's disease and spinocerebellar ataxia), and CNS cancers and / or brain cancers (such as brain metastases caused by cancer elsewhere in the body).

[0206] Embodiment 20 is a method of treating a condition associated with the formation of plaques containing β-amyloid in a subject in need thereof, the method comprising administering to a subject in need thereof the multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 13 or the pharmaceutical composition according to Embodiment 18.

[0207] Embodiment 21 is a method according to embodiment 20, wherein the disorder is Alzheimer's disease.

[0208] Embodiment 22 is a method according to embodiment 20, wherein the condition is selected from: dementia associated with trisomy 21 (Down syndrome), diffuse Lewy body disease, inclusion body myositis, cerebral amyloid angiopathy, and hereditary cerebral hemorrhage with Dutch amyloidosis (HCHWA-D).

[0209] Embodiment 23 is a method of reducing plaques associated with Alzheimer's disease in a subject in need thereof, the method comprising administering to a subject in need thereof the multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 13 or the pharmaceutical composition according to Embodiment 18.

[0210] Embodiment 24 is a method of preventing vaccination activity of 3pE Aβ in a subject in need thereof, the method comprising administering to a subject in need thereof the multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 13 or the pharmaceutical composition according to embodiment 18.

[0211] Embodiment 25 is a method of blocking tau seeding in a subject in need thereof, the method comprising administering to the subject the multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 13 or administering to the subject in need thereof the pharmaceutical composition according to embodiment 18.

[0212] Embodiment 26 is a method of treating tauopathy in a subject in need thereof, the method comprising administering to the subject the multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 13 or administering to the subject in need thereof the pharmaceutical composition according to Embodiment 18.

[0213] Embodiment 27 is a method of reducing pathological tau aggregation or spread of tauopathy in a subject in need thereof, the method comprising administering to the subject the multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 13 or administering to the subject the pharmaceutical composition according to embodiment 18.

[0214] Embodiment 28 is a method according to embodiment 26 or 27, wherein the tauopathy is selected from the group consisting of familial Alzheimer's disease, sporadic Alzheimer's disease, frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle-only dementia, diffuse neurofibrillary tangles with calcifications, argyrophilic grain dementia, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Strauss-Schaefer disease, Hallevorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, chronic traumatic encephalopathy, and dementia pugilista (boxing disease).

[0215] Embodiment 29 is a method for producing a pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 13, the method comprising combining the multispecific antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0216] The following examples of the present invention are intended to further illustrate the nature of the present invention. It should be understood that the following examples do not limit the present invention, and the scope of the present invention is determined by the appended claims.

[0217] Example

[0218] Example 1: Generation of anti-Aβ / anti-transferrin receptor (TfR) expression constructs

[0219] The cell line development (CLD) process incorporates ATUM’s Leap-In Technology and use of the Horizon Chinese Hamster Ovary (CHO) host cell line. ATUM Leap-In The system was engineered from Xenopus tropicalis. In this system, a synthetic transposon is cloned into a single plasmid containing a selectable marker and the desired biotherapeutic gene between Leap-In inverted terminal repeats (ITRs). Each gene is placed under the control of its own regulatory elements, including a promoter and polyadenylation sequence.

[0220] DNA encoding anti-PHF tau / anti-pyroglutamate amyloid-β / anti-TfR antibodies was synthesized and subcloned into Leap-In at ATUM (CA, USA) by Golden Gate Assembly technology using type IIS restriction enzymes BsaI and SapI. The glutamine synthetase expression vector backbone pD2546ht+_n was used. The plasmids were named plasmid 1 [light chain, heavy chain 1] and plasmid 2 [light chain, heavy chain 2].

[0221] The entire plasmid was bidirectionally sequenced at NeoGenomics Laboratories (CA, USA).

[0222] The primary transcript nucleotide sequences of the heavy chain 1, light chain, and heavy chain 2 genes are listed below. The predicted amino acid sequences of the heavy chain 1, light chain, and heavy chain 2 genes are listed below. Note that the light chain genes are identical in plasmid 1 and plasmid 2.

[0223] The nucleotide sequence of heavy chain 1 of plasmid 1 with the signal peptide underlined (SEQ ID NO: 18): ATGGCCAGG AAGTCCGCTCTGCTCGCTCTGGCACTTCTGCTTCTGGGATTTGGACCTGCTTGGGCT

[0224] The nucleotide sequence of the light chain of plasmid 1 with the signal peptide underlined (SEQ ID NO: 19):

[0225] ATGGCCAGGAAGTCCGCTCTGCTCGCTCTGGCACTTCTGCTTCTGGGATTTGGACCTGCTTGGGCT GACGTGGTCATGACACAGAGCCCTCTGAGCCTGCCTGTGACATTGGGACAGCCTGCCTCCATCTCCTGCAAGTCCTCTCAGTCCCTGCTGGACTCCAGAGCCAAGACCTATCTGACCTGGCTGCAGCAGAGGCCTGGCCAGTCTCCTAGAAGGCTGATCTACCT GGTGTCCAAGCTGGACTCTGGCGTGCCCGATAGATTCTCCGGCTCTGGCTCTGGCACCGACTTCACCCTGAAGATCTCCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTTGGCAGGGCACCCACTTTCCATACACCTTCGGCCAGGGCACAAAGCTGG AAATCAAGCGTACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0226] The nucleotide sequence of heavy chain 2 of plasmid 2 with the signal peptide underlined (SEQ ID NO: 20): ATGGCCAGG AAGTCCGCTCTGCTCGCTCTGGCACTTCTGCTTCTGGGATTTGGACCTGCTTGGGCT

[0227] Amino acid sequence of heavy chain 1 of plasmid 1 with the signal peptide underlined (SEQ ID NO: 21). The Asp (D) residue at position 1 of heavy chain 1 constitutes the N-terminus of the mature chain. MARKSALLALALLLLGFGPAWA DIVMTQTPLSSPVTLGQPASISCRSSKSLLYKDGKTYLNWLQQRPGCPPRLLIYLMSTRASGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCQQLVDYPLTFGQGTKLEIKGGGSGGSGGCPPCGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWITWVRQRPGQGLEWMGDIH PGRGSTKYAQKLQGRVTMTTDTSTAYMELRSLRSDDTAVYYCARRWGFDYWGCGTLVTVSSEPKSSDKTHTCPPCPAPEAAGGPSSVFLFPPKDTLYITREPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAEVQLLESGGGLVQPGGSLRLSCADSGFTFSSYAMNWVRQAPGCGLEWVSGISGSGGHTYYADSV KGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCAREGYDSSGYNPFDYWGQGTQVTVSSGGGSGGSGGCPPCGGSGGSYELTQPPSVSVSPGQTASITCSGDKLGDKYASWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTVVFGCGTKLTVLH

[0228] Amino acid sequence of the light chain of plasmid 1 where the signal peptide is underlined (SEQ ID NO: 22). The Asp (D) residue at position 1 of the light chain constitutes the N-terminus of the mature chain. MARKSALLALALLLLGFGPAWADVVMTQSPLSLPVTLGQPASISCKSSQSLLDSRAKTYLTWLQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0229] Amino acid sequence of heavy chain 2 of plasmid 2 with the signal peptide underlined (SEQ ID NO: 23). The Gln (Q) residue at position 1 of the heavy chain constitutes the N-terminus of the mature chain. MARKSALLALALLLLGFGPAWA QVQLVQSGAEVKKPGASVKVSCKASGHVFTSYDMYWVRQAPGQGLEWIGYIDSDSGDTSYNQKFKGRVTLTVDTSTSTVYMELSSLRSEDTAVYYCAYYRYAMDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0230] Mature amino acid sequence of the heavy chain 1 of plasmid 1 (SEQ ID NO: 24): DIVMTQTPLSSPVTLGQPASISCRSSKSLLYKDGKTYLNWLQQRPGCPPRLLIYLMSTRASGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCQQLVDYPLTFGQGTKLEIKGGGSGGSGGCPPCGGSGGQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWITWVRQRPGQGLEWMGDIHPGRGSTKYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRWGFDYWGCGTLVTVSSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAEVQLLESGGGLVQPGGSLRLSCADSGFTFSSYAMNWVRQAPGCGLEWVSGISGSGGHTYYADSVKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCAREGYDSSGYNPFDYWGQGTQVTVSSGGGSGGSGGCPPCGGSGGSYELTQPPSVSVSPGQTASITCSGDKLGDKYASWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTVVFGCGTKLTVLH

[0231] The mature amino acid sequence of light chain 1 of plasmid 1 (SEQ ID NO: 25) is: DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSRAKTYLTWLQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0232] Mature amino acid sequence of heavy chain 2 of plasmid 2 (SEQ ID NO:26):QVQLVQSGAEVKKPGASVKVSCKASGHVFTSYDMYWVRQAPGQGLEWIGYIDSDSGDTSYNQKFKGRVTLTVDTSTSTVYMELSSLRSEDTAVYYCAYYRYAMDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0233] Generation of cell lines expressing anti-PHF tau / anti-pyroglutamate amyloid-β / anti-TfR constructs.

[0234] Example 2: Binding Characterization by Surface Plasmon Resonance

[0235] The binding interactions of the test articles with recombinant human full-length recombinant tau (2N4R; PT1W1.ECO1), β-amyloid peptide (3pE-28) and human transferrin receptor (TfR; TFRW2) were studied by SPR using a Biacore 8k instrument at 25°C, pH 7.4 or pH 5.0 buffer, supplemented with 3mM EDTA and 0.05% Tween 20. In brief, the biosensor surface was prepared by coupling anti-human IgG Fcg-fragment specific antibodies to the surface of the C1 sensor chip using the protocol recommended by the supplier for amine coupling chemistry (>400 response units (RU)). The coupling buffer was 10mM sodium acetate (pH 4.5). The test articles were diluted in running buffer and injected onto anti-human IgG to obtain sufficient capture to enable detection of antigen binding. After capturing the test article, three recombinant antigens of different concentration series were injected in a single cycle kinetic mode (30nM to 1.1nM series of 3-fold dilution for 2N4R, 300nM to 11.1nM series of 3-fold dilution for TfR, and 12nM to 0.8nM series of 2-fold dilution for 3pE-28). The binding and dissociation of 2N4R and 3pE-28 antigens were monitored for 3 minutes and 60 minutes, respectively, at a flow rate of 50 μL / min, and the binding and dissociation of TfR antigen were monitored for 2 minutes and 5 minutes. The dissociation characteristics of TfR were measured at both pH 7.4 and pH 5.0. The sensor surface was regenerated with 0.85% H3PO4. The binding sensor diagram was fitted using a 1:1 Langmuir binding model to obtain the binding rate, dissociation rate and affinity. Parental bivalent antibodies against Tau and β-amyloid protein were included as controls.

[0236] It will be appreciated by those skilled in the art that modifications may be made to the above embodiments without departing from the broad inventive concept of the present invention. Therefore, it will be appreciated that the present invention is not limited to the specific embodiments disclosed, but the present invention is intended to cover modifications consistent with the spirit and scope of the present invention, as defined in the appended claims.

Claims

1. A multispecific antibody or antigen-binding fragment thereof, comprising a first antigen-binding region capable of specifically binding to pyroglutamate amyloid-β, a second antigen-binding region capable of specifically binding to transferrin receptor (TfR), and a third antigen-binding region capable of specifically binding to paired helical filaments (PHF)-tau, wherein: a. The first antigen binding region comprises: i. a first heavy chain variable region (VH1), said VH1 comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, each comprising SEQ ID NO: 8 or 16, 9 or 17 and 10; and ii. a first light chain variable region (VL1), said VL1 comprising a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, each comprising SEQ ID NO: The amino acid sequences of 11, 12 and 13; b. The second antigen binding region comprises: i. a second heavy chain variable region (VH2), said VH2 comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, each comprising SEQ ID NO: 1, 2 and 3; and ii. a second light chain variable region (VL2), said VL2 comprising a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, each comprising SEQ ID NO: 4, 5 and 6; and c. the third antigen binding region comprises: i. a third heavy chain variable region (VH3), said VH3 comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3, each comprising SEQ ID NO: The amino acid sequences of 28, 29 and 30; and ii. a third light chain variable region (VL3), the VL3 comprising a light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3, each comprising SEQ ID NO: The amino acid sequences of 31, 32 and 33.

2. The multispecific antibody or antigen-binding fragment thereof according to claim 1, wherein the VH1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 14; and The VL1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

15. 3 . The multispecific antibody or antigen-binding fragment thereof according to claim 1 , wherein the VH1 comprises the amino acid sequence of SEQ ID NO: 14; and the VL1 comprises the amino acid sequence of SEQ ID NO:

15.

4. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the second antigen-binding region comprises a single-chain fragment variable (scFv) antibody or antigen-binding fragment thereof comprising the VH2 and the VL2. 5 . The multispecific antibody or antigen-binding fragment thereof according to claim 4 , wherein the scFv comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

7. 6 . The multispecific antibody or antigen-binding fragment thereof according to claim 5 , wherein the scFv comprises the amino acid sequence of SEQ ID NO:

7.

7. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising: (j) a first heavy chain (HC1), said HC1 comprising said VL3 and said VL3, a first heavy chain constant region comprising a first Fc region (Fc1), and said scFv; (iv) a second heavy chain (HC2), said HC2 comprising said VH1 and a second heavy chain constant region comprising a second Fc region (Fc2); (v) a first light chain (LC), said LC comprising said VL1 and a light chain constant region.

8. The multispecific antibody or antigen-binding fragment thereof according to claim 7, wherein the scFv is connected to the carboxyl terminus of the first heavy chain constant region via a linker, more specifically via a linker comprising the amino acid sequence of SEQ ID NO:

27.

9. The multispecific antibody or antigen-binding fragment thereof according to claim 7 or 8, wherein the Fc1 and the Fc2 each comprise one or more heterodimer mutations compared to a wild-type Fc region, such as a first modified heterodimer CH3 domain and a second modified heterodimer CH3 domain, respectively; specifically, the Fc1 comprises amino acid modifications at positions T350, L351, F405 and Y407, and the Fc2 comprises amino acid modifications at positions T350, T366, K392 and T394, wherein the amino acid modification at position T350 is T350V, T350I, T350L or T350M; The amino acid modification at position L351 is L351Y; the amino acid modification at position F405 is F405A, F405V, F405T or F405S; The amino acid modification at position Y407 is Y407V, Y407A or Y407I; the amino acid modification at position T366 is T366L, T366I, T366V or T366M, the amino acid modification at position K392 is K392F, K392L or K392M, and the amino acid modification at position T394 is T394W, and wherein the numbering of amino acid residues is performed according to the EU index as described in Kabat, more specifically, Fc1 comprises amino acid modifications T350V, L351Y, F405A and Y407V, and Fc2 comprises amino acid modifications T350V, T366L, K392L and T394W.

10. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 7 to 9, wherein at least one of the Fc1 and the Fc2 comprises one or more mutations that enhance the binding of the multispecific antibody or the antigen-binding fragment thereof to the neonatal Fc receptor (FcRn), preferably, the one or more mutations enhance the binding at acidic pH, more preferably, the at least one of the Fc1 and the Fc2 has M252Y / S254T / T256E (YTE) mutations, wherein the numbering of amino acid residues is performed according to the EU index as described in Kabat.

11. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 7 to 10, wherein at least one of the Fc1 and the Fc2 comprises one or more mutations that reduce or eliminate effector function, preferably, the at least one of the Fc1 and the Fc2 has one or more amino acid modifications at positions L234, L235, D270, N297, E318, K320, K322, P331 and P329, such as one, two or three mutations of L234A, L235A and P331S, wherein the numbering of amino acid residues is performed according to the EU index as described in Kabat.

12. A multispecific antibody or an antigen-binding fragment thereof, comprising a first heavy chain, a first light chain, and a second heavy chain, wherein the first heavy chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:24, the first light chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:25, and the second heavy chain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

26.

13. The multispecific antibody or antigen-binding fragment thereof according to claim 12, wherein the first heavy chain comprises the amino acid sequence of SEQ ID NO: 24, the first light chain comprises the amino acid sequence of SEQ ID NO: 25, and the second heavy chain comprises the amino acid sequence of SEQ ID NO:

26.

14. An isolated nucleic acid sequence encoding the multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

15. A vector comprising the isolated nucleic acid according to claim 14.

16. A host cell comprising the isolated nucleic acid according to claim 14 or the vector according to claim 15.

17. A method for producing a multispecific antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell according to claim 16 under conditions in which the multispecific antibody or the antigen-binding fragment thereof is produced, and recovering the multispecific antibody or the antigen-binding fragment thereof. 18 . A pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

19. A method for treating or detecting a disorder, preferably a nervous system disorder, in a subject in need thereof, the method comprising administering to the subject a multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or a pharmaceutical composition according to claim 18, preferably, the nervous system disorder is selected from neurodegenerative diseases (such as Lewy body disease, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy), tauopathies (such as Alzheimer's disease and supranuclear palsy), prion diseases (such as bovine spongiform encephalopathy), The present invention relates to a variety of diseases including encephalopathy, pruritus, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Schenck disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and heterogeneous degenerative disorders of the nervous system (such as Canavan disease, Huntington disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette syndrome, Menkes kinky hair syndrome, Cockne syndrome, Hallervorden-Spatz syndrome, Raffaello disease, Rett syndrome, Wilson's disease, Lesch-Nyhan syndrome, and Ong-Long syndrome), dementias (such as Pick's disease and spinocerebellar ataxia), and CNS cancers and / or brain cancers (such as brain metastases caused by cancer elsewhere in the body).

20. A method of treating a condition associated with the formation of plaques containing β-amyloid in a subject in need thereof, the method comprising administering to the subject in need thereof the multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or the pharmaceutical composition according to claim 18.

21. The method of claim 20, wherein the disorder is Alzheimer's disease.

22. The method of claim 20, wherein the condition is selected from the group consisting of dementia associated with trisomy 21 (Down syndrome), diffuse Lewy body disease, inclusion body myositis, cerebral amyloid angiopathy, and hereditary cerebral hemorrhage with Dutch amyloidosis (HCHWA-D).

23. A method of reducing plaques associated with Alzheimer's disease in a subject in need thereof, the method comprising administering to the subject in need thereof the multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or the pharmaceutical composition according to claim 18.

24. A method of preventing vaccination activity of 3pE Aβ in a subject in need thereof, the method comprising administering to the subject in need thereof the multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or the pharmaceutical composition according to claim 18.

25. A method for blocking tau seeding in a subject in need thereof, the method comprising administering to the subject a multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or administering to the subject in need thereof a pharmaceutical composition according to claim 18.

26. A method of treating tauopathy in a subject in need thereof, the method comprising administering to the subject a multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or administering to the subject in need thereof a pharmaceutical composition according to claim 18.

27. A method for reducing pathological tau aggregation or spread of tauopathy in a subject in need thereof, the method comprising administering to the subject a multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or administering to the subject in need thereof a pharmaceutical composition according to claim 18.

28. The method of claim 26 or 27, wherein the tauopathy is selected from the group consisting of familial Alzheimer's disease, sporadic Alzheimer's disease, frontotemporal dementia and parkinsonism associated with chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, tangle-only dementia, diffuse neurofibrillary tangles with calcifications, argyrophilic grain dementia, amyotrophic lateral sclerosis-parkinsonism-dementia complex, Down syndrome, Gerstmann-Strauss-Schaefer disease, Hallevorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, chronic traumatic encephalopathy, and dementia pugilista (boxing disease).

29. A method for producing a pharmaceutical composition comprising a multispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, the method comprising combining the multispecific antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

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