Multispecific antibodies and uses thereof

Abstract

Provided is a multispecific antibody or antigen-binding fragment thereof, comprising at least one first antigen-binding region capable of specifically binding to pyroglutamate amyloid-β and a second antigen-binding region capable of specifically binding to transferrin receptor (TfR). Also provided are methods of treating or detecting neurological disorders and / or delivering therapeutic or diagnostic agents across the blood-brain barrier. Also described are nucleic acids encoding the antibodies, vectors comprising the nucleic acids, recombinant host cells comprising the nucleic acids and / or vectors, and methods of making the multispecific antibody or antigen-binding fragment thereof.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 269,204, filed March 11, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] (Reference to electronically submitted sequence listing) This application contains a sequence listing that has been submitted electronically. The contents of the electronic sequence listing (065768-121WO1_Sequence Listing.xml; size: 43,618 bytes; and creation date: March 2, 2023) are incorporated herein by reference in their entirety.

[0003] FIELD OF THEINVENTION The present application relates to anti-pyroglutamate amyloid-β / anti-transferrin receptor (TfR) antibodies, antibody conjugates, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the expression vectors, and compositions comprising the antibodies. Methods of making the antibodies, methods of using the antibodies to treat conditions, including neurological disorders (e.g., amyloid-related disorders), and methods of using the antibodies to diagnose neurological disorders are also provided. [Background technology]

[0004] Alzheimer's disease (AD) is a degenerative brain disorder clinically characterized by progressive loss of memory, cognition, reasoning, judgment, and emotional stability, which leads to gradually and severely impaired mental function and ultimately to death. Alzheimer's disease is a common cause of progressive mental disability (dementia) in the elderly. Alzheimer's disease is identified worldwide and represents a major public health problem. The disease is currently estimated to affect more than 5 million individuals in the United States alone. Currently, it is incurable, and no treatment effectively suppresses AD or reverses its symptoms or course.

[0005] The brains of individuals with AD exhibit characteristic lesions called amyloid plaques, amyloid angiopathy (amyloid deposits in blood vessels) and neurofibrillary tangles. The majority of these lesions, particularly amyloid plaques and neurofibrillary tangles, are commonly found in several areas of the brain important for memory and cognitive function. Amyloid plaques and amyloid angiopathy also characterize the brains of individuals with Trisomy 21 (Down's Syndrome), Diffuse Lewy Body Disease and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch Type (HCHWA-D).

[0006] The major components of amyloid plaques are various amyloid-beta (Aβ) peptides produced by cleavage of the β-amyloid precursor protein (APP). The deposition of Aβ peptides in the brain is hypothesized to be an early and necessary step in the disease cascade that leads to AD. The identification of mutations in the amyloid precursor protein and presenilin genes that lead to altered Aβ production and cause familial early-onset AD provides strong evidence that alterations in amyloid metabolism are central events in the pathogenic process underlying the disease.

[0007] Amyloid-β peptide with pyroglutamate at the third residue (3pE Aβ) is the major species accumulated in the brain of AD patients. 3pE Aβ is present in almost all diffuse and mature plaques in AD, is metabolically stable, and may play a role in both plaque seeding and stabilization (Cynis et al., Molecular Neurodegeneration, 2016; 11: 48). No detectable amount of 3pE Aβ has been reported in CSF or plasma, suggesting that the target peptide is pathology specific (DeMattos et al., Neuron, 2012; 76: 1-13). Antibodies that selectively bind to 3pE Aβ may be useful for immunotherapy.

[0008] The blood-brain barrier (BBB) ​​prevents harmful substances from entering the brain and is essential for brain homeostasis, but it also represents a formidable obstacle to the efficient delivery of drugs to the brain. Large molecules such as monoclonal antibodies and other biological therapeutics have great potential as therapeutic / diagnostic agents for treating / detecting pathologies in the central nervous system (CNS). However, their path into the brain is hindered by the BBB. Previous studies have shown that only a small percentage (approximately 0.1%) of IgG injected into the bloodstream is able to cross the BBB and enter the CNS compartment (Felgenhauer, Klin. Wschr. 52:1158-1164 (1974)). This limits any pharmacological effects due to the low concentration of antibodies in the CNS.

[0009] Numerous approaches have been investigated to improve brain delivery of therapeutic monoclonal antibodies (mAbs), including the use of receptor-mediated transcytosis (RMT). RMT exploits receptors abundantly expressed on the luminal side of the BBB for transport through brain endothelial cells. Previous efforts to create clinically feasible platforms for delivering therapeutic mAbs into the brain have focused on antibody engineering to enhance the efficiency of transcytosis, with progress being made through observations of valency, pH dependency, and affinity (reviewed in Goulatis et al., 2017, Curr Opin Struct Biol 45:109-115). However, translation to NHPs and the clinic has been limited by rapid peripheral clearance from target-mediated pharmacokinetics (TMDD), and safety from acute reticulocyte depletion (Gadkar, 2016, Eur J Pharm Biopharm. 2016 Apr;101:53-61). Transferrin receptors (TfR), particularly TfR1, mediate the transport of iron-loaded transferrin (Tf) from the blood to the brain and the return of iron-depleted 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, safety issues and poor pharmacokinetics (PK) of anti-TfR1 monoclonal antibodies have hindered their clinical development as BBB carriers.

[0010] Therefore, there is a need for anti-pyroglutamate amyloid-β / anti-TfR antibodies or antigen-binding fragments thereof that can cross the BBB and be used to target 3pEAβ for immunotherapy. Summary of the Invention

[0011] As embodied and fully described, the present invention relates to multispecific antibodies and antigen-binding fragments thereof that bind to amyloid-β having a pyroglutamate third residue (3pE Aβ) and that bind to the transferrin receptor (TfR), methods of making multispecific antibodies or antigen-binding fragments thereof that bind to 3pE Aβ and TfR, assays using such multispecific antibodies or antigen-binding fragments thereof, and the use of a multispecific antibody or antigen-binding fragment thereof of the invention in the manufacture of a medicament for treating, delaying the onset of, or reversing at least one disease state or symptom, such as a neurological disorder, e.g., Alzheimer's disease and other β-amyloid related diseases.

[0012] In one general aspect, the present application relates to a multispecific antibody or antigen-binding fragment thereof, comprising at least one first antigen-binding region capable of specifically binding to pyroglutamate amyloid-β and a second antigen-binding region capable of specifically binding to transferrin receptor (TfR). In a particular embodiment, (a) the first antigen-binding region comprises (i) a first heavy chain variable region (VH1) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO:8 or SEQ ID NO:16, SEQ ID NO:9 or SEQ ID NO:17, and SEQ ID NO:10, respectively, and (ii) a first light chain variable region (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively. and (b) a second antigen-binding region comprising (i) a second heavy chain variable region (VH2) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and (ii) a second light chain variable region (VL2) comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0013] In certain embodiments, VH1 comprises an amino acid sequence at least 90% identical to SEQ ID NO: 14, and VL1 comprises an amino acid sequence 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.

[0014] In certain embodiments, the second antigen-binding region comprises a single chain variable fragment (scFv) antibody or antigen-binding fragment thereof comprising a VH2 and a VL2. The scFv can comprise, for example, an amino acid sequence that is at least 90% identical to SEQ ID NO: 7. In certain embodiments, the scFV comprises the amino acid sequence of SEQ ID NO: 7.

[0015] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises two first antigen-binding regions. The multispecific antibody or antigen-binding fragment thereof may, for example, comprise: (i) a first heavy chain (HC1) comprising a VH1, a first heavy chain constant region comprising a first Fc region (Fc1), and an scFv; (ii) a second heavy chain (HC2) comprising a VH1 and a second heavy chain constant region comprising a second Fc region (Fc2); and (iii) a first and a second light chain (LC), each comprising a VL1 and a light chain constant region.

[0016] In a particular embodiment, the scFv is linked to the carboxy terminus of the first heavy chain constant region via a linker, more specifically, a linker comprising the amino acid sequence of SEQ ID NO:27.

[0017] In certain embodiments, Fc1 and Fc2 each comprise one or more heterodimeric mutations, such as a first modified heterodimeric CH3 domain and a second modified heterodimeric CH3 domain, respectively, compared to a wild-type Fc region; specifically, Fc1 comprises amino acid modifications at positions T350, L351, F405, and Y407; 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; and the amino acid modification at position F405 is F405A, F405V, F405T, or is 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 the numbering of amino acid residues is according to the EU index as set forth in Kabat, more specifically, Fc1 comprises the amino acid modifications T350V, L351Y, F405A, and Y407V, and Fc2 comprises the amino acid modifications T350V, T366L, K392L, and T394W.

[0018] In certain embodiments, Fc1 and Fc2 each comprise one or more heterodimeric mutations, such as a first modified heterodimeric CH3 domain and a second modified heterodimeric CH3 domain, respectively, compared to a wild-type Fc region, Fc1 comprises amino acid modifications at positions T364, L366, and Y406, and Fc2 comprises an amino acid modification at position T365, wherein the amino acid modification at position T364 is T364S, the amino acid modification at position L366 is L366A, the amino acid modification at position Y406 is Y406V, and the amino acid modification at position T365 is T365W, and the numbering of the amino acid residues is according to the EU index as set forth in Kabat, and more specifically, Fc1 comprises the amino acid modifications T364S, L366A, and Y406V, and Fc2 comprises the amino acid modification T365W.

[0019] 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 at least one of Fc1 and Fc2 has M252Y / S254T / T256E (YTE) mutations, where the numbering of amino acid residues is according to the EU index as described in Kabat.

[0020] 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, e.g., one, two, or three of the following mutations: L234A, L235A, and P331S, where the numbering of amino acid residues is according to the EU index as described in Kabat.

[0021] 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, a second heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 26, and a second light chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 25. In a particular embodiment, 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, the second heavy chain comprises the amino acid sequence of SEQ ID NO: 26, and the second light chain comprises the amino acid sequence of SEQ ID NO: 25.

[0022] Another general aspect of the present application relates to an isolated nucleic acid encoding a multispecific antibody or antigen-binding fragment thereof of the present application. Also provided are vectors comprising the isolated nucleic acid of the present application, and host cells comprising the nucleic acid or vector of the present application.

[0023] Another general aspect of the present application relates to a method of making the multispecific antibodies or antigen-binding fragments thereof of the present application, the method comprising culturing a host cell of the present application under conditions to 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.

[0024] Further provided is a pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof of the present application and a pharma- ceutically acceptable carrier.

[0025] Another general aspect of the present application relates to a method of treating or detecting a neurological disorder in a subject in need thereof, comprising administering to the subject an effective amount of a multispecific antibody or antigen-binding fragment thereof or a pharmaceutical composition of the present application. Preferably, the neurological disorder is a neurodegenerative disease (e.g., Lewy body disease, post-poliomyelitis syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, spinocerebellar degeneration, spinal muscular atrophy), tauopathy (e.g., Alzheimer's disease and supranuclear palsy), prion disease (e.g., bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob disease, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and nervous system heterodegenerative disorder. and / or cerebrovascular disease (e.g., Canavan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Hallervorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (e.g., Pick's disease, and spinocerebellar degeneration), and cancer of the CNS and / or brain (e.g., brain metastases resulting from cancer elsewhere in the body).

[0026] Also provided is a method of treating a condition associated with the formation of plaques containing β-amyloid protein in a subject in need thereof. The method comprises administering to a subject in need thereof a multispecific antibody or antigen-binding fragment thereof, 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 the group consisting of dementia associated with trisomy 21 (Down's syndrome), diffuse Lewy body disease, inclusion body myositis, cerebral amyloid angiopathy, and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch Type (HCHWA-D).

[0027] Also provided is a method of reducing plaques associated with Alzheimer's disease in a subject in need thereof, comprising 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.

[0028] Also provided is a method of inhibiting the seeding activity of 3pE Aβ in a subject in need thereof, comprising administering to a subject in need thereof a multispecific antibody or antigen-binding fragment thereof, or a pharmaceutical composition of the present application.

[0029] Other aspects, features, and advantages of the present invention will become apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments, and the appended claims. [Brief description of the drawings]

[0030] The foregoing Summary of the Invention and the following Detailed Description of the Invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise embodiments shown in the drawings. [Figure 1] FIG. 1 is a diagram of a multispecific antibody or antigen-binding fragment thereof of the present application. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] In the "Background" section and throughout the specification, various publications, articles and patents are cited or described, and each of these references is incorporated herein by reference in its entirety. The discussion of documents, operations, materials, devices, articles and the like which is included in the specification is for the purpose of providing a background for the invention. Such discussion is not an admission that any or all of these items constitute part of the prior art to any invention disclosed or claimed.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Unless otherwise specified, certain terms used herein have the meaning as described herein. All patents, published patent applications and publications cited herein are incorporated by reference as if set forth herein in their entirety.

[0033] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0034] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0035] Unless otherwise stated, any numerical value, such as a concentration or concentration range, described herein should be understood in all cases as being modified by the term "about". Thus, numerical values ​​typically include ±10% of the described value. For example, a dosage of 10 mg includes 9 mg to 11 mg. As used herein, the use of numerical ranges expressly includes all possible subranges, all individual numerical values ​​within the range, including integers and fractions of values ​​within the range, unless otherwise expressly specified in the context.

[0036] As used herein, it will be understood that the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variation thereof, are intended to include the stated element or elements, but not to exclude other elements or elements other than those, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or device that includes a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or that are inherent to such composition, mixture, process, method, article, or device. Further, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not an exclusive "or." For example, condition A or B is satisfied by one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0037] As used herein, the connective term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are connected by "and / or," the first option refers to the first element being applicable without the second element. The second option refers to the second element being applicable without the first element. The third option refers to the first and second elements being applicable together. Any one of these options is understood to be within the meaning and thus meets the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the options is also understood to be within the meaning and thus meets the requirements of the term "and / or."

[0038] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As 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 an aspect or embodiment of the invention, any of the above terms "comprise", "contain", "include", and "have" can be replaced with the term "comprise" or "consist essentially of" in order to vary the scope of the disclosure.

[0039] As used herein, the term "antibody" is used broadly and specifically includes full-length monoclonal antibodies, polyclonal antibodies, and, unless otherwise indicated or contrary to the context, antigen-binding fragments, antibody variants, and multispecific molecules thereof, so long as they exhibit the desired biological activity. In general, a full-length antibody is a glycoprotein that includes at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, inter-connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further divided into hypervariable regions called complementarily determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. General principles of antibody molecular structure and various techniques for generating antibodies are provided, for example, in Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988).

[0040] Depending on the amino acid sequence of the constant domain of their heavy chains, full-length antibodies can be assigned to different "classes". There are five major classes of antibodies, namely IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to these different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0041] An "antibody" may also be a single variable domain on the heavy chain (VHH) antibody, also called a heavy chain antibody (HcAb), which is devoid of light chains and may be naturally produced by camelids or sharks. The antigen-binding portion of an HcAb consists of a VHH fragment.

[0042] The term "recombinant antibody," as used herein, refers to an antibody (e.g., a chimeric, humanized or human antibody, or an antigen-binding fragment thereof) expressed by a recombinant host cell containing nucleic acid encoding the antibody. Examples of "host cells" for producing recombinant antibodies include: (1) mammalian cells, such as Chinese hamster ovary (CHO), COS, myeloma cells (including YO cells and NSO cells), baby hamster kidney (BHK), 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.

[0043] An "antigen-binding fragment" of an antibody is a molecule that contains a portion of a full-length antibody that is capable of detectably binding to an antigen, typically comprising at least one or more portions of the VH region. An antigen-binding fragment is a multivalent molecule that contains one, two, three, or more antigen-binding portions of an antibody and a single-chain construct, in which the VL and VH regions, or selected portions thereof, are joined by a synthetic linker or by recombinant techniques to form a functional antigen-binding molecule. An antigen-binding fragment can also be a single-domain antibody (sdAb), also known as a nanobody, which is an antibody fragment consisting of a single monomeric variable antibody domain (VHH). Some antigen-binding fragments of antibodies can be obtained by actual cleavage (e.g., enzymatic cleavage) of a larger antibody molecule, but are often typically produced by recombinant techniques. The antibodies of the present invention can be prepared as full-length antibodies or antigen-binding fragments thereof. Examples of antigen-binding fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)3, Fv (typically the VL and VH domains 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 (typically the VH and CH1 domains), and dAb (typically 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 kappabodies (see, e.g., Ill et al., Protein Eng 1997;10:949-57); camelid IgG; IgNAR; and one or more isolated CDRs or functional paratopes, which isolated CDRs, or antigen-binding residues or antigen-binding polypeptides can be associated or linked together to form a functional antibody fragment.Various types of antibody fragments are described or reviewed in, for example, Holliger and Hudson, Nat Biotechnol 2005;23:1126-1136, WO 2005040219, and published U.S. Patent Applications Nos. 20050238646 and 20020161201. Antibody fragments can be obtained using conventional recombinant or protein engineering techniques, and the fragments can be screened for antigen binding or other functions in the same manner as intact antibodies.

[0044] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments have been obtained by proteolytic digestion of full-length antibodies (see, for example, 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 E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). By another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. In another embodiment, 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. An antibody fragment may also be a "linear antibody" as described, for example, in U.S. Patent No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

[0045] The term "antibody derivative" as used herein refers to a molecule comprising a full-length antibody or an antigen-binding fragment thereof, in which one or more amino acids have been chemically modified or substituted. Chemical modifications that can be used in antibody derivatives include, for example, alkylation, PEGylation, acylation, ester formation, or amide formation, for example, to link the antibody to a second molecule. Exemplary modifications include PEGylation (e.g., cysteine ​​PEGylation), biotinylation, radiolabeling, and conjugation with a second reagent (e.g., a cytotoxic agent).

[0046] Antibodies herein include "amino acid sequence variants" that have altered antigen-binding or biological activity. Examples of such amino acid modifications include antibodies with enhanced affinity for the antibody (e.g., "affinity matured" antibodies), and antibodies with altered Fc regions (if present), such as antibodies with altered (enhanced or decreased) antibody dependent cellular cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC) (see, e.g., WO 00 / 42072 (Presta, L.) and WO 99 / 51642 (Iduosogie et al.)), and / or antibodies with increased or decreased serum half-life (see, e.g., WO 00 / 42072, Presta, L.).

[0047] A "multispecific molecule" includes an antibody or antigen-binding fragment thereof that associates or binds to at least one other functional molecule (e.g., another antibody or another peptide or protein, such as a ligand for a receptor), thereby forming at least two different binding sites or molecules that bind to target molecules. Exemplary multispecific molecules include bispecific antibodies and antibodies that bind to soluble receptor fragments or ligands.

[0048] The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from (i.e., identical or essentially identical to) a human germline immunoglobulin. Furthermore, if the antibody contains a constant region, the constant region is also "derived from" a human germline immunoglobulin. The human antibodies of the invention may include amino acid residues not encoded in human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0049] A "humanized" antibody is a human / non-human chimeric antibody that contains minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate) having the desired specificity, affinity, and capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to the CDR regions of a non-human immunoglobulin and all or substantially all of the FR residues are FR residues of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see 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), International Publication No. 92 / 02190, U.S. Patent Application No. 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, and 6,180,370. See Nos. 5,895,205, 5,886,152, 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.

[0050] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. Hypervariable regions generally consist of amino acid residues from the "complementarity determining regions" or "CDRs" (residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242) and / or residues from the "hypervariable loops" (residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain); Chothia and Lesk, J. Mol. Biol. 1987;196:901-917). Typically, the numbering of amino acid residues in this region is performed according to the method described in Kabat et al., supra. Expressions such as "Kabat position", "variable domain residue numbering in Kabat" and "according to Kabat" herein refer to this numbering system for 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 of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of CDR H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (such as residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by aligning the sequence of that antibody with a "standard" Kabat numbered sequence at the regions of homology.

[0051] "Framework Region" or "FR" residues are those VH or VL residues outside the CDRs as herein defined.

[0052] An "epitope" or "binding site" is an area or region on an antigen to which an antigen-binding peptide (such as an antibody) specifically binds. A protein epitope can include amino acid residues that are directly involved in binding (also called the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, e.g., amino acid residues that are effectively blocked by the peptide that specifically binds to the antigen (i.e., amino acid residues that are within the "solvent-excluded surface" and / or "footprint" of the peptide that specifically binds to the antigen).

[0053] A "paratope" is an area or region of the antigen-binding portion of an antibody that specifically binds to an antigen. Unless otherwise stated or contrary to context, the paratope can include amino acid residues that are directly involved in epitope binding (some of which are typically in the CDRs) and other amino acid residues that are not directly involved in binding, e.g., amino acid residues that are effectively blocked by the specifically bound antigen (i.e., amino acid residues that are in the "solvent-excluded surface" and / or "footprint" of the specifically bound antigen).

[0054] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay.

[0055] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B, 848:79-87 (2007).

[0056] The term "administration" with respect to the methods of the invention refers to a method of therapeutically or prophylactically suppressing, treating, or ameliorating a syndrome, disorder, or disease described herein by using the complexes, or formulations, compositions, or medicaments thereof of the invention. Such methods include administering an effective amount of said antibodies, antigen-binding fragments thereof, or conjugates thereof, or formulations, compositions, or medicaments at different times during the course of treatment or simultaneously in a combined form. The methods of the invention are to be understood as embracing all known therapeutic treatment regimes.

[0057] The ability of a targeting antibody to "block" binding of a target molecule to a natural target ligand means that the antibody detectably reduces binding of the target molecule to the ligand in a dose-dependent manner in an assay using soluble or cell surface bound target and ligand molecules, and the target molecule detectably binds to the ligand in the absence of the antibody.

[0058] "Blood-brain barrier" or "BBB" refers to the physiological barrier between the peripheral circulation and the brain and spinal cord, formed by tight junctions in the brain capillary endothelial cell membranes, forming a tight barrier that restricts the transport of molecules into the brain. The BBB can restrict the transport of even very small molecules, such as urea (60 daltons), into the brain. Examples of the BBB include the BBB in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retina barrier in the retina, which are all continuous capillary barriers in the CNS. The BBB also encompasses the blood-CSF barrier (choroid plexus), which is composed of ependymal cells rather than capillary endothelial cells.

[0059] "Blood-brain barrier receptors" (abbreviated herein as "R / BBB") are extracellular membrane-bound receptor proteins that are expressed on brain endothelial cells and can transport molecules across the BBB or are utilized to transport exogenously administered molecules. Examples of R / BBB include, but are not limited to, transferrin receptor (TfR), insulin receptor, insulin-like growth factor receptor (IGF-R), low-density lipoprotein receptors, 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 includes the transferrin receptor (TfR).

[0060] "Central nervous system" or "CNS" refers to the complex of nervous tissue that controls bodily functions and includes the brain and spinal cord.

[0061] As used herein, "conjugate" refers to a protein covalently attached to one or more heterologous molecules, including but not limited to a therapeutic peptide or protein, an antibody, a label, or a neuropathic drug.

[0062] As used herein, the term "conjugated / linked" refers to joining or connecting two or more entities together. When referring to a chemical or biological compound, conjugated / linked may refer to a covalent connection between two or more chemical or biological compounds. As a non-limiting example, an antibody of the present invention may be linked to a peptide of interest to form an antibody-linked peptide. The antibody-linked peptide may be formed by a specific chemical reaction designed to conjugate the antibody to the peptide. In certain embodiments, an antibody of the present invention may be covalently linked to a peptide of the present invention by a linker. The linker may, for example, first be covalently linked to the antibody or peptide, and then covalently linked to the peptide or antibody.

[0063] An "effective amount" or "therapeutically effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0064] As used herein, "linker" refers to a chemical linker or a single-chain peptide linker that covalently links two different entities. A linker can be used to link any two of the antibodies or fragments thereof, blood-brain barrier shuttles, fusion proteins, and conjugates of the present invention. The linker can link, for example, the VH and VL in an scFv, or an antibody or antigen-binding fragment thereof to a therapeutic molecule, such as a second antibody. In some embodiments, when the monovalent binding entity comprises an scFv against TfR, preferably huTfR1, and the therapeutic molecule comprises a CNS target, such as an antibody against pyroglutamate amyloid-β, the linker can link the scFv to an antibody against pyroglutamate amyloid-β. A single peptide linker consisting of 1-25 amino acids linked by peptide bonds, for example 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, can be used. In certain embodiments, the amino acids are selected from the 20 naturally occurring amino acids. In certain other embodiments, the one or more 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, hybrid linkers consisting of PEG and embedded heterocycles, and hydrocarbon chains can also be used.

[0065] As used herein, "neurological disorder" refers to a disease or disorder that affects the CNS and / or has a CNS etiology. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, ocular disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, stroke, behavioral disorder, and lysosomal storage disease. For the purposes of this application, the CNS is understood to include the eye, which is normally separated 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-poliomyelitis syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, spinocerebellar degeneration, and spinal muscular atrophy), tauopathy (including, but not limited to, Alzheimer's disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob disease, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and nervous system heterodegenerative disorders. disorders (including but not limited to Canavan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Hallervorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (including but not limited to Pick's disease, and spinocerebellar degeneration), and cancer (e.g., cancer of the CNS and / or brain, including brain metastases resulting from cancer elsewhere in the body).

[0066] A "neuropathy drug" is a drug or therapeutic agent useful for treating or ameliorating the effects of one or more neuropathies. Neuropathic 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 natural ligands of one or more CNS targets, aptamers, inhibitory nucleic acids (i.e., small inhibitory RNAs (siRNAs) and small hairpin RNAs (shRNAs)), ribozymes, or active fragments of any of the above. Exemplary neuropathic agents of the invention are described herein and include, but are not limited to, antibodies, aptamers, proteins, peptides, inhibitory nucleic acids and small molecules, as well as active fragments of any of the above, which themselves or which specifically recognize and / or act on (i.e., inhibit, activate, or detect) a CNS antigen or target molecule (such as, but not limited to, amyloid precursor protein or a portion thereof, amyloid beta, beta-secretase, gamma-secretase, tau, alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer markers, and neurotrophins). Non-limiting examples of neuropathic drugs and corresponding disorders that may be treated using them include brain-derived neurotrophic factor (BDNF), chronic brain injury (neurogenesis), fibroblast growth factor 2 (FGF-2), anti-epidermal growth factor receptor brain cancer, (EGFR) antibodies, glial cell line-derived neurofactor Parkinson's disease, (GDNF), brain-derived neurotrophic factor (BDNF) amyotrophic lateral sclerosis, depression, brain lysosomal enzyme lysosomal storage disease, ciliary neurotrophic factor (CNTF) amyotrophic lateral sclerosis, neuregulin-1 schizophrenia, anti-HER2 antibodies (e.g., trastuzumab) brain metastases from HER2-positive cancers.

[0067] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient it contains to be effective and that does not contain additional ingredients that exhibit unacceptable toxicity to a subject to which the formulation may be administered.

[0068] As used herein, "a pharma- ceutically acceptable carrier or diluent" refers to any substance suitable for use in administration to an individual. For example, a pharma- ceutically acceptable carrier may be a sterile aqueous solution such as phosphate buffered saline (PBS) or water for injection.

[0069] As used herein, "pharmaceutically acceptable salts" refers to physiologically and pharma- ceutically acceptable salts of compounds, such as oligomeric compounds or oligonucleotides, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects.

[0070] Pharmaceutically acceptable acid / anionic salts for use in the present invention include acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estrus, esylate, fumarate, grisepate, gluconate, glutamate, glycolylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxyl, hydroxypropyl ... The acid salts include, but are not limited to, naphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucoate, napsylate, nitrate, pamoate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theoclate, tosylate, and triethiodide.The organic or inorganic acid includes, but is not limited to, hydroiodic acid, perchloric acid, sulfuric acid, phosphoric acid, propionic acid, glycolic acid, methanesulfonic acid, hydroxyethanesulfonic acid, oxalic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, cyclohexanesulfamic acid, saccharic acid, or trifluoroacetic acid. Pharmaceutically acceptable basic / cationic salts include, but are not limited to, aluminum, 2-amino-2-hydroxymethyl-propane-1,3-diol (also known as tris(hydroxymethyl)aminomethane, tromethane, or "TRIS"), ammonia, benzathine, t-butylamine, calcium, chloroprocaine, choline, cyclohexylamine, diethanolamine, ethylenediamine, lithium, L-lysine, magnesium, meglumine, N-methyl-D-glucamine, piperidine, potassium, procaine, quinine, sodium, triethanolamine, or zinc.

[0071] "Polypeptide" or "protein" refers to a molecule containing 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."

[0072] When used in reference to amino acid sequences, the phrase "sequence identity" or "percent (%) sequence identity" or "% identity" describes the number of identical amino acid matches ("hits") of two or more aligned amino acid sequences compared to the number of amino acid residues that make up the full length of the amino acid sequence. In other words, the alignment can be used to determine the percentage of amino acid residues that are the same (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identity over the full length of the amino acid sequence) when comparing and aligning the sequences for maximum correspondence as determined using sequence comparison algorithms known in the art, or when manually aligning the sequences and visually inspecting them. Thus, the sequences compared to determine sequence identity may differ due to amino acid substitutions, additions, or deletions. Suitable programs for aligning protein sequences are known to those skilled in the art. The percentage of sequence identity of protein sequences can be determined, for example, using programs such as CLUSTALW, Clustal Omega, FASTA, or BLAST, for example, using the NCBI BLAST algorithm (Altschul SF, et al (1997), Nucleic Acids Res. 25:3389-3402).

[0073] The term "substantially identical" in the context of two amino acid sequences means that the sequences, when optimally aligned (e.g., with programs such as GAP or BESTFIT using default gap weighting), share at least about 50 percent sequence identity. Typically, substantially identical sequences exhibit at least about 60 percent, at least about 70 percent, at least about 80 percent, at least about 90 percent, at least about 95 percent, at least about 98 percent, or at least about 99 percent sequence identity.

[0074] "Specific binding" or "specifically binds" or "binds" refers to an antibody binding to an antigen or an epitope within that antigen with higher affinity than to other antigens. Typically, antibodies bind to an antigen with a high affinity dissociation constant (K D ) is the K for binding to non-specific antigens (e.g., BSA, casein) D At least 100 times smaller than about 1 × 10 -8 M or less, for example, about 1×10 -9 M or less, approximately 1×10 -10 M or less, approximately 1×10 -11 M or less, or about 1 x 10 -12 K below M D It binds to an antigen or an epitope within an antigen with K D is the equilibrium dissociation constant, k between an antibody and its antigen off / k on It is the ratio of K D The on-rate (k on ) is a constant used to characterize how quickly an antibody binds to its target. off ) is a constant used to characterize how quickly an antibody dissociates from its target. The dissociation constant, K D can be measured using standard procedures. For example, the K Dcan be determined by using surface plasmon resonance techniques, for example by using a biosensor system, such as the Biacore® system, or by using biolayer interferometry techniques, for example the Octet RED96 system. D The smaller the value of , the higher the affinity that the antibody binds to the target antigen. However, an antibody that specifically binds to an antigen or an epitope within that antigen may have cross-reactivity to other related antigens, or to the same antigen (homologs) from other species, such as humans or monkeys, such as cynomolgus (Macaca fascicularis), chimpanzees (Pan troglodytes), or common marmosets (Callithrix jacchus). A monospecific antibody specifically binds to one antigen or one epitope, whereas a bispecific antibody specifically binds to two different antigens or two different epitopes.

[0075] The term "subject" as used herein refers to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, 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."

[0076] The term "transferrin receptor" or "TfR" as used herein refers to a cell surface receptor required for cellular iron uptake by the process of receptor-mediated endocytosis. It is the carrier protein for transferrin. TfR is involved in iron uptake in vertebrates and is regulated in response to intracellular iron concentration. TfR takes up iron by internalizing the transferrin-iron complex by receptor-mediated endocytosis. Two transferrin receptors have been characterized in humans: transferrin receptor 1 and transferrin receptor 2. Both receptors are transmembrane glycoproteins. TfR1 is a ubiquitously expressed receptor with high affinity. TfR2 binds transferrin with 25-30 times lower affinity than TfR1. Expression of TfR2 is restricted to certain cell types and is not affected by intracellular iron concentration. In one embodiment, the TfR is a human TfR, including the amino acid sequence described, for example, in Schneider et al. Nature 311:675-678 (1984). The TfR may have a molecular weight of about 180,000 daltons and two subunits each having an apparent molecular weight of about 90,000 daltons. Preferably, the TfR is human TfR1.

[0077] "Target antigen" or "brain target" as used herein refers to an antigen and / or molecule that is expressed in the CNS, including the brain, and can be targeted with an antibody or small molecule. Examples of such antigens and / or molecules include, but are not limited to, beta-secretase 1 (BACE1), amyloid beta (Aβ), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. In some embodiments, the target antigen is BACE1. In some embodiments, the target antigen is amyloid-beta, in particular pyroglutamate amyloid-beta.

[0078] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical disease. Desirable effects of treatment include, but are not limited to, inhibiting the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, inhibiting metastasis, reducing the rate of disease progression, improving or ameliorating the disease state, and achieving remission or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or to slow the progression of the disease.

[0079] Antibodies or immunoglobulins can be assigned to five main classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy chain constant domain. IgG is the most stable of the five immunoglobulins, with a serum half-life of about 23 days in humans. IgA and IgG are further subclassified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Each of the four IgG subclasses has a different biological function, known as the effector function. These effector functions are generally mediated by interaction with Fc receptors (FcγR) and / or by binding with C1q and fixation of complement. Binding with FcγR can cause antibody-dependent cell-mediated cytolysis or antibody-dependent cellular cytotoxicity (ADCC), whereas binding with complement factors can cause complement-mediated cytolysis or complement-dependent cytotoxicity (CDC). The multispecific antibodies or antigen-binding fragments thereof of the invention, or therapeutic or diagnostic antibodies conjugated or fused to the multispecific antibodies or antigen-binding fragments thereof, may have no or minimal effector functions, but retain the ability to bind to FcRn, which binding may be the primary means by which the antibodies have a long in vivo half-life.

[0080] Binding of FcγR or complement (e.g., C1q) to antibodies is caused by specific protein-protein interactions at so-called Fc-part binding sites. Such Fc-part binding sites are known in the art. Such Fc-part binding sites include, for example, those characterized by amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat). In some embodiments, the multispecific antibodies or antigen-binding fragments thereof of the invention, or therapeutic or diagnostic antibodies conjugated or fused to the multispecific antibodies or antigen-binding fragments thereof, comprise one or more substitutions in one or more Fc-part binding sites to eliminate effector functions. For example, a multispecific antibody or antigen-binding fragment thereof of the invention, or a therapeutic or diagnostic antibody conjugated or fused to the multispecific antibody or antigen-binding fragment thereof, may comprise an Fc region comprising one or more of the following substitutions: glutamic acid to proline at residue 233, phenylalanine to alanine or valine at residue 234, and leucine to alanine or glutamic acid at residue 235 (EU numbering, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. US Pet. of Health and Human Services, Bethesda, Md., NIH Publication no. 91-3242). Preferably, the antibody of interest comprises one, two or three of the following mutations: L234A, L235A, and P331S (EU numbering, Kabat).

[0081] Antibodies of subclasses IgG1, IgG2 and IgG3 usually exhibit complement activation, including binding of C1q and C3, whereas IgG4 does not activate the complement system and does not bind C1q and / or C3. Human IgG4 Fc region has a reduced ability to bind FcγR and complement factors compared to other IgG subtypes. Preferably, the multispecific antibody or antigen-binding fragment thereof of the invention, or therapeutic or diagnostic antibody conjugated or fused to the multispecific antibody or antigen-binding fragment thereof, comprises an Fc region derived from a human IgG4 Fc region. More preferably, the Fc region comprises a human IgG4 Fc region with substitutions that eliminate effector function. Removal of N-linked glycosylation sites in the IgG4 Fc region, for example by using Ala instead of Asn at residue 297 (EU numbering), is another way to ensure that residual effector activity is eliminated.

[0082] Multispecific anti-pyroglutamate amyloid-β / anti-transferrin receptor (TfR) antibodies and their antigen-binding fragments Anti-pyroglutamate amyloid-β binding domain / anti-pyroglutamate amyloid-β antibody, and antigen-binding fragment thereof Anti-pyroglutamate amyloid-β antibodies and antigen-binding fragments thereof have been previously disclosed in International Publication No. WO 2020 / 193644, the entire contents of which are incorporated herein by reference. In one general aspect, the present application relates to a multispecific antibody or antigen-binding fragment thereof comprising an antigen-binding region based on the aforementioned anti-pyroglutamate amyloid-β antibodies and antigen-binding fragments thereof.

[0083] As used herein, "pyroglutamate amyloid-β", "AβpE3", or "3pE Aβ" refers to modified Aβ peptides that oligomerize with Aβ42 and deposit in the brain in Alzheimer's disease (AD). Pyroglutamate amyloid-β may act as a seed for misfolding Abs as an early stage of AD. Pyroglutamate amyloid-β is known in the art, see, for example, 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).

[0084] Anti-TfR antigen-binding region / anti-TfR antibody and antigen-binding fragment thereof Anti-TfR antibodies and antigen-binding fragments thereof have been previously disclosed in WO 2021 / 0205358, the entire contents of which are incorporated herein by reference. In one general aspect, the present application relates to a multispecific antibody or antigen-binding fragment thereof, comprising an antigen-binding region based on the aforementioned anti-TfR antibodies and antigen-binding fragments thereof. The anti-TfR antigen-binding region can bind to a primate TfR, such as a human TfR or a monkey TfR, and the antigen-binding region can be optimized to deliver an agent to the brain of a subject in need thereof. The relationship between binding affinity of anti-TfR antibodies to TfR and transcytosis efficiency has been previously described as improved transcytosis with reduced affinity to TfR (Yu, Zhang et al. 2011, Sci Transl Med 3(84):84ra44), and WO 2021 / 0205358 (incorporated herein by reference in its entirety) surprisingly found a more nuanced relationship between affinity and transcytosis efficiency than previously described, with effects from both on-rates and off-rates affecting brain concentrations. In particular, an unbiased off-rate that is neither too fast nor too slow is required for optimal brain PK and PD of an agent (such as a mAb) to be efficiently delivered by an anti-TfR antibody or antigen-binding fragment thereof.

[0085] Preferably, the anti-TfR antigen-binding region of the present application is pH sensitive, e.g., its binding affinity to TfR varies with pH. For example, the anti-TfR antigen-binding region of the present application can bind with high affinity to cell surface TfR at neutral pH, such as physiological pH (e.g., pH 7.4), but dissociates from TfR at relatively low acidic pH, such as pH 5.0-6.0, upon internalization to endosomal compartments. Affinity is a measure of the binding strength between two moieties, e.g., an antibody and an antigen. Affinity can be expressed in several ways. One method is to measure the dissociation constant (K D ) perspective. D can be measured by standard methods including equilibrium dialysis, or by the dissociation and association rates of the antigen-antibody bond (koff (kd or k dis ) velocity and k on (or k a ) rate) directly (see, for example, Nature, 1993 361:186-87). off / k on The ratio of D (See generally, Davies et al., Annual Rev Biochem, 1990 59:439-473). Thus, K D The smaller the affinity, the higher the affinity. Another expression for affinity is K a This means that K D or k on / k off Therefore, K a For example, an anti-TfR antigen-binding domain for use in the compositions and / or methods of the present application may have a specific binding affinity of 1 nanomolar (nM, 10 -9 M) or more K D and binds to TfR at 10 at acidic pH such as pH 5.0 (e.g., pH 4.5 to 6.0). -4 sec -1 More than k dis The antigen-binding region may be an anti-TfR antigen-binding region that dissociates from TfR at

[0086] Thus, a general aspect of the present application is an anti-TfR antigen binding region for delivering an agent to the brain of a subject in need thereof, the anti-TfR antigen binding region having a dissociation constant K of at least 1 nM at neutral pH, preferably between 1 nM and 500 nM. D At an acidic pH, preferably pH 5, the -4 sec -1 , preferably 10 -4 ~10 -1 sec -1 Dissociation rate constant k d and an anti-TfR antigen-binding region that binds to the transferrin receptor (TfR), preferably human TfR1.

[0087] In one embodiment, the anti-TfR antigen binding region of the present application has a cytotoxicity of 2×10 -2 ~2×10 -4 sec -1 , for example 2×10 -2 , 1×10 -2 , 9×10 -3 , 8×10 -3 , 7×10 -3 , 6×10 -3 , 5×10 -3 , 4×10 -3 , 3×10 -3 , 2×10 -3 , 1×10 -3 , 9×10 -4 , 8×10 -4 , 7×10 -4 , 6×10 -4 , 5×10 -4 , 4×10 -4 , 3×10 -4 , 2×10 -4 sec -1 , or any value between the off-rate constant k d has.

[0088] Multispecific antibodies and antigen-binding fragments thereof In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises at least one first antigen-binding region capable of specifically binding to pyroglutamate amyloid-β and a second antigen-binding region capable of specifically binding to transferrin receptor (TfR). In certain embodiments, (a) the first antigen-binding region comprises (i) a first heavy chain variable region (VH1) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO:8 or SEQ ID NO:16, SEQ ID NO:9 or SEQ ID NO:17, and SEQ ID NO:10, respectively, and (ii) a first light chain variable region (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively. and (b) a second antigen-binding region comprising (i) a second heavy chain variable region (VH2) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and (ii) a second light chain variable region (VL2) comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0089] In certain embodiments, VH1 comprises an amino acid sequence at least 90% identical to SEQ ID NO: 14, and VL1 comprises an amino acid sequence 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.

[0090] In certain embodiments, the second antigen-binding region comprises a single chain variable fragment (scFv) antibody or antigen-binding fragment thereof comprising a VH2 and a VL2. The scFv can comprise, for example, an amino acid sequence that is at least 90% identical to SEQ ID NO: 7. In certain embodiments, the scFV comprises the amino acid sequence of SEQ ID NO: 7.

[0091] In certain embodiments, the multispecific antibody or antigen-binding fragment thereof comprises two first antigen-binding regions. The multispecific antibody or antigen-binding fragment thereof may, for example, comprise: (i) a first heavy chain (HC1) comprising a VH1, a first heavy chain constant region comprising a first Fc region (Fc1), and an scFv; (ii) a second heavy chain (HC2) comprising a VH1 and a second heavy chain constant region comprising a second Fc region (Fc2); and (iii) a first and a second light chain (LC), each comprising a VL1 and a light chain constant region.

[0092] Preferably, the anti-TfR antigen-binding region is linked to a light chain variable region (L V ) covalently linked to the heavy chain variable region (H V ) is a single chain variable fragment (scFv). scFv can retain the specificity of the original immunoglobulin even if the constant region is removed and a linker is introduced. In scFv, the order of the domains is H V -Linker L V , or L V -linkerH VThe linker may be either de novo designed or derived from a known, publicly known protein structure, such that the linker is of a length and conformation compatible with crosslinking the variable domains of the scFv without significant steric hindrance. The linker may be 10 to about 25 amino acids in length. Preferably, the linker is a peptide linker that spans about 3.5 nm (35 Å) between the carboxy terminus of the variable domain and the amino terminus of the other domain without affecting the ability of the domains to fold and form an intact antigen-binding site (Huston et al., Methods in Enzymology, vol. 203, pp. 46-88, 1991, incorporated herein by reference in its entirety). The linker preferably contains a hydrophilic sequence to avoid peptide insertion within or between the variable domains throughout protein folding (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 with charged residues such as Glu, Thr, and Lys interspersed to enhance solubility. In one embodiment, the linker has the amino acid sequence of SEQ ID NO: 27 (GGGSGGSGGCPPCGGSGG). Any other suitable linker may also be used, taking into account the present disclosure. In a particular embodiment, the scFv is linked to the carboxy terminus of the first heavy chain constant region via a linker, more specifically, a linker comprising the amino acid sequence of SEQ ID NO: 27.

[0093] 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, a second heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 26, and a second light chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 25. In a particular embodiment, 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, the second heavy chain comprises the amino acid sequence of SEQ ID NO: 26, and the second light chain comprises the amino acid sequence of SEQ ID NO: 25.

[0094] In another particular 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.

[0095] In another particular 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.

[0096] In another general aspect, the present invention relates to an isolated nucleic acid encoding a multispecific antibody or antigen-binding fragment thereof of the present invention. It will be understood by those skilled in the art that the coding sequence of a protein can be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, it will be understood by those skilled in the art that the nucleic acid sequence encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention can be altered without changing the amino acid sequence of the protein.

[0097] In another general aspect, the present invention relates to a vector comprising an isolated nucleic acid encoding the multispecific antibody or antigen-binding fragment thereof of the present invention. In view of the present disclosure, any vector known to the skilled artisan can be used, such as a plasmid, cosmid, phage vector, or viral vector. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector can include any element for establishing the conventional functions of 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 can be a constitutive, inducible, or repressible promoter. Numerous expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein to produce antibodies or antigen-binding fragments thereof in cells. Conventional cloning techniques, or artificial gene synthesis methods, can be used to generate recombinant expression vectors according to embodiments of the present invention.

[0098] 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 the skilled artisan can be used for recombinant expression of the antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the host cell is an E. coli TG1 or BL21 cell (e.g., for expression of scFv or Fab antibodies), a CHO-DG44 or CHO-K1 cell, or a HEK293 cell (e.g., for expression of full-length IgG antibodies). According to certain embodiments, the recombinant expression vector is transformed into the host cell by conventional methods such as chemical transfection, heat shock, or electroporation, in which the recombinant nucleic acid is stably integrated into the host cell genome for efficient expression.

[0099] Brain shuttle construct An optimized RMT brain delivery platform is developed using the transferrin receptor (TfR) to enhance endogenous transcytosis efficiency, extend peripheral pharmacokinetics, and obtain an acceptable safety profile while maintaining efficacy of therapeutic mAbs. The interplay between transcytosis receptor affinity and brain concentration is studied in human TfR knock-in mice. Extensive binding kinetics studies reveal that optimal brain PK and PD of mAbs require unbiased off-rates that are neither too fast nor too slow. The enhanced brain delivery observed in mice is confirmed in cynomolgus monkeys.

[0100] It has also been discovered that engineered antibody constant regions with increased binding to the neonatal Fc receptor (FcRn) resulted in decreased peripheral clearance and increased brain concentrations.

[0101] Further Fc mutations are introduced to abolish binding to Fcγ receptors (FcγR) to avoid effector function-mediated toxicity. When combined with a high-affinity anti-pyroglutamate amyloid-β-binding mAb, these mutations prevent peripheral effector function-mediated toxicity while preserving antibody-dependent phagocytosis (ADP) through a novel non-FcγR mechanism for microglial uptake and target degradation. This mechanism relies on TfR receptor-mediated uptake and is more efficient at promoting target degradation than conventional FcγR-mediated ADP without stimulating secretion of pro-inflammatory cytokines.

[0102] In certain embodiments, Fc1 and Fc2 each comprise one or more heterodimeric mutations, such as a first modified heterodimeric CH3 domain and a second modified heterodimeric CH3 domain, respectively, compared to a wild-type Fc region; specifically, Fc1 comprises amino acid modifications at positions T350, L351, F405, and Y407; 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; and the amino acid modification at position F405 is F405A, F405V, F405T, or is 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 the numbering of amino acid residues is according to the EU index as set forth in Kabat, more specifically, Fc1 comprises the amino acid modifications T350V, L351Y, F405A, and Y407V, and Fc2 comprises the amino acid modifications T350V, T366L, K392L, and T394W.

[0103] In certain embodiments, Fc1 and Fc2 each comprise one or more heterodimeric mutations, such as a first modified heterodimeric CH3 domain and a second modified heterodimeric CH3 domain, respectively, compared to a wild-type Fc region, Fc1 comprises amino acid modifications at positions T364, L366, and Y406, and Fc2 comprises an amino acid modification at position T365, wherein the amino acid modification at position T364 is T364S, the amino acid modification at position L366 is L366A, the amino acid modification at position Y406 is Y406V, and the amino acid modification at position T365 is T365W, and the numbering of the amino acid residues is according to the EU index as set forth in Kabat, and more specifically, Fc1 comprises the amino acid modifications T364S, L366A, and Y406V, and Fc2 comprises the amino acid modification T365W.

[0104] 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 at least one of Fc1 and Fc2 has M252Y / S254T / T256E (YTE) mutations, where the numbering of amino acid residues is according to the EU index as described in Kabat.

[0105] 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, e.g., one, two, or three of the following mutations: L234A, L235A, and P331S, where the numbering of amino acid residues is according to the EU index as described in Kabat.

[0106] Thus, in one general aspect, the present application relates to an antibody-targeted brain delivery system comprising the anti-TfR antigen binding region of the present application. The anti-TfR antigen binding region can be used to deliver therapeutic or diagnostic agents intracellularly (e.g., into cancer cells) or into the BBB system. The agents that can be delivered include any neuropathic drug or any agent that can be used to detect or analyze a neuropathic drug. For example, such agents may 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), gamma-amino-butyric acid (GABA), dopamine, cholecystokinin (CCK), endorphins, enkephalins, and thyrotropin releasing hormone (TRH); cytokines; anxiolytics; anticonvulsants, polynucleotides and transgenes, including small interfering RNA and / or antisense oligos; or antibodies or antigen-binding fragments thereof that bind to brain targets. The anti-hTfR antigen-binding regions of the present application provide an effective means for enhancing delivery of agents of interest from the blood to the brain and can function in the brain.

[0107] In particular, the agent of interest can be delivered parenterally, e.g., intravenously, in a combined form or linked to the anti-TfR antigen binding region of the present application. For example, the agent can be non-covalently linked to the anti-TfR antigen binding region. The agent can also be covalently linked to the anti-TfR antigen binding region to form a conjugate. In certain embodiments, the conjugation is by constructing a protein fusion (i.e., by fusing two genes encoding the anti-TfR antigen binding region and the neuropathic drug and expressing them as a single protein). In view of the present disclosure, the agent can be linked to the antibody or antigen-binding fragment thereof 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.

[0108] In some embodiments, the therapeutic or diagnostic agent to be delivered to the brain and the anti-TfR antigen binding region can be covalently linked (or conjugated) to each other via a non-peptide or 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 ether, biodegradable polymers, polymerized lipids, chitin and hyaluronic acid, or derivatives thereof, or combinations thereof. The peptide linker can be a peptide chain or derivatives thereof consisting of 1 to 50 amino acids linked by peptide bonds, the N-terminus and C-terminus of which can be covalently linked to the anti-TfR antigen binding region.

[0109] In certain embodiments, the conjugate of the present application is a multispecific antibody or antigen-binding fragment thereof, comprising a first antigen-binding region that binds pyroglutamate amyloid-β and a second antigen-binding region that binds TfR. Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-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 "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Also, by manipulating electrostatic steering effects (WO 2009 / 089004(A1)), by 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), by using leucine zippers (see, e.g., Kostelny et al, J. Immunol., 148(5):1547-1553, 1992)), by using "diabody" technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448, 1993), by using single-chain Fv (sFv) dimers (see, e.g., Gruber et al. J. Immunol, 152:5368 (1994)), and by using other methods, such as those described, for example, in Tutt et al. by preparing trispecific antibodies as described in, e.g., et al. J. Immunol. 147:60-1991. Multispecific antibodies can also be made. The multispecific antibodies of the present application also include antibodies with three or more functional antigen binding sites, including "Octopus antibodies" or "Dual Variable Region Immunoglobulins" (DVDs) (see, e.g., US Patent Application Publication No. 2006 / 0025576(A1) and Wu et al. Nature Biotechnology, 25(11):1290-7, 2007). The multispecific antibodies of the present application also include "Dual Acting Fabs" or "DAFs" that contain antigen binding regions that bind to TfR and pyroglutamate amyloid-β (see, e.g., US Patent Application Publication No. 2008 / 0069820). In one embodiment, the antibody is an antibody fragment, and a variety of such fragments are disclosed herein.

[0110] 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 antibody or antigen-binding fragment thereof. Preferably, the second antibody or antigen-binding fragment thereof binds to a brain target such as pyroglutamate amyloid-β as described herein. The anti-TfR antigen-binding region can be fused directly or via a linker to the carboxy and / or amino terminus of the light and / or heavy chain of the second antibody or antigen-binding fragment thereof.

[0111] In one embodiment, the anti-TfR antigen-binding region is fused directly or via a linker to the carboxy terminus of the light chain of a second antibody or antigen-binding fragment thereof.

[0112] 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.

[0113] In another embodiment, the anti-TfR antigen-binding region is fused directly or via a linker to the carboxy terminus of the heavy chain of a second antibody or antigen-binding fragment thereof.

[0114] 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.

[0115] In a preferred embodiment, the fusion construct of the present application comprises an anti-TfR antigen-binding region of the present application, preferably an anti-huTfR1 VHH or scFv fragment, covalently linked via a linker to the carboxy terminus of only one of the two heavy chains of a second antibody or antigen-binding fragment thereof that binds to pyroglutamate amyloid-β. Preferably, the linker has the amino acid sequence of SEQ ID NO:27.

[0116] Heterodimer mutations introduced into the Fc of two heavy chains to promote the formation of heterodimers between two heavy chains, e.g., a heavy chain with and without a fusion of an anti-TfR antigen binding region, or a heavy chain with an Fc of an anti-TfR arm and a heavy chain with an Fc of an anti-pyroglutamate amyloid-β arm. Examples of such Fc mutations include, but are not limited to, Zymework mutations (see, e.g., U.S. Pat. No. 10,457,742) and "knob-in-hole" mutations (see, e.g., Ridgway et al., Protein Eng., 9(7):617-621, 1996). Other heterodimer mutations can also be used in the present invention. In some embodiments, the modified CH3 described herein is used to promote the formation of heterodimers between two heavy chains.

[0117] In addition to the heterodimer mutations, other mutations can 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 alter (increase or decrease), preferably abolish, ADCC / CDC, and / or one or more mutations (such as YTE mutations described herein) that alter (increase or decrease), preferably increase, 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 substituted with another amino acid, such as serine.

[0118] Conjugates such as the multispecific antibodies or fusion constructs of the present application can be produced by any of a number of techniques known in the art, taking into account the present disclosure. For example, the conjugates can be expressed from recombinant host cells, in which one or more expression vectors encoding the heavy and light chains of the fusion construct or multispecific antibody are transfected into the host cell by standard techniques. The host cell can be a prokaryotic or eukaryotic host cell.

[0119] In an exemplary system, one or more recombinant expression vectors encoding the two heavy and light chains of the heterodimer 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, which is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vectors, transfect the host cells, select for transformants, culture the host cells, and recover the protein construct from the culture medium.

[0120] Pharmaceutical Compositions and Related Methods The present invention further relates to pharmaceutical compositions, methods for their preparation, and methods of their use.

[0121] 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 pharma- ceutically acceptable carrier. The multispecific antibody or antigen-binding fragment thereof of the present invention is also useful for the manufacture of a medicament for the therapeutic applications mentioned herein. A pharma- ceutically acceptable carrier can be any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation, or other material known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application.

[0122] Thus, in one embodiment, the present application relates to a method of transporting a therapeutic or diagnostic agent across the blood-brain barrier (BBB), comprising exposing a multispecific antibody or antigen-binding fragment thereof of the present invention coupled to a therapeutic or diagnostic agent to the blood-brain barrier, such that the multispecific antibody or antigen-binding fragment thereof transports the agent coupled thereto across the blood-brain barrier. In one embodiment, the agent is a neurological disorder drug. In another embodiment, the agent is an imaging agent, or an agent for detecting a neurological disorder. Preferably, the multispecific antibody or antigen-binding fragment thereof does not impair 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 present in a mammal, preferably a primate, such as a human, more preferably a human with a neurological disorder. In one embodiment, the neurological disorder is selected from the group consisting of 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.

[0123] In one embodiment, the multispecific antibodies or antigen-binding fragments thereof of the present application are used to detect neurological damage before symptoms appear and / or to assess the severity or duration of a disease or disorder. The multispecific antibodies or antigen-binding fragments thereof allow for detection and / or imaging of neurological damage, including imaging by radiography, tomography, or magnetic resonance imaging (MRI).

[0124] 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 to a subject in need of treatment an effective amount of the multispecific antibody or antigen-binding fragment thereof, in some embodiments, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent.

[0125] In another embodiment, the present invention relates to a method for inhibiting, ameliorating, treating, and / or reducing amyloid-β deposition in an amyloid-β associated condition, comprising administering to a subject in need thereof a therapeutically effective amount of a multispecific antibody or antigen-binding fragment thereof disclosed herein. A further aspect of the present invention includes a pharmaceutical composition for inhibiting, ameliorating, treating, and / or reducing amyloid deposition in an amyloid-β associated condition, comprising an antibody or antigen-binding fragment thereof disclosed herein. The method of the present invention comprises administering to a subject in need thereof an effective amount of one or more of the antibodies or antigen-binding fragments thereof described herein.

[0126] In one aspect, the invention relates to a method for inhibiting, ameliorating, treating and / or reducing amyloid-β deposition in a condition characterized by the formation of plaques containing β-amyloid protein in a human, comprising administering, preferably peripherally, to a human in need of such treatment, a therapeutically or prophylactically effective amount of a multispecific antibody according to the invention or an immunologically reactive fragment thereof, which multispecific antibody specifically binds to human Aβ3pE. In another aspect, the invention relates to a method for inhibiting amyloid plaque formation and / or removing amyloid plaques in a human, comprising administering to a human subject in need of such inhibition or removal an effective amount of a multispecific antibody according to the invention, wherein the multispecific antibody sequesters Aβ3pE peptide in the brain and causes altered Aβ3pE clearance in the brain.

[0127] A subject in need thereof is a human suffering from or predisposed to a condition characterized by the formation of plaques containing β-amyloid protein. In one embodiment, the condition is Alzheimer's disease. In another embodiment, the condition is dementia associated with trisomy 21 (Down's syndrome), diffuse Lewy body disease, inclusion body myositis, cerebral amyloid angiopathy, or Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch Type (HCHWA-D).

[0128] In one embodiment of the present invention, the multispecific antibody or antigen-binding fragment thereof binds to 3pE Aβ in plaque deposits. By binding to 3pE Aβ in plaque deposits, the multispecific antibody or antigen-binding fragment thereof can cause plaque removal. Induction of plaque removal can be by activation of microglia surrounding the plaque and destabilization of the plaque by removing stable Aβ forms. Furthermore, the antibody or antigen-binding fragment thereof can suppress the plaque seeding activity of 3pE Aβ. The possible enrichment of 3pE Aβ in plaques compared to vascular amyloid can increase the therapeutic safety margin for immunotherapy.

[0129] In another embodiment, the present application relates to the use of a 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 for the treatment of a neurological disease or disorder. In a further embodiment, the medicament is for use in a method of treating a neurological disease or disorder comprising administering an effective amount of the medicament to an individual having a neurological disease or disorder.

[0130] Another general aspect of the present application relates to a method of inducing antibody-dependent phagocytosis (ADP) in a subject in need thereof without stimulating the secretion of proinflammatory cytokines, comprising administering to the subject a conjugate comprising a therapeutic antibody or antigen-binding fragment thereof coupled, preferably covalently, to a multispecific antibody or antigen-binding fragment thereof of the present application, wherein the therapeutic antibody or antigen-binding fragment thereof has no effector function. For example, the therapeutic antibody or antigen-binding fragment thereof may comprise one or more amino acid modifications that reduce or eliminate an effector function, such as ADCC or CDC, such as a mutation that reduces or eliminates binding to an Fcγ receptor. Such mutations may be one, two or three mutations at positions L234, L235, D270, N297, E318, K320, K322, P331 and P329, e.g., L234A, L235A and P331S, where the numbering of the amino acid residues is according to the EU index as set forth in Kabat. In one embodiment, the therapeutic antibody or antigen-binding fragment thereof specifically binds to tau aggregates.

[0131] In some embodiments, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent, hi certain embodiments, the additional therapeutic agent is a therapeutic agent effective to treat the same or a different neurological disorder that the multispecific antibody or antigen-binding fragment thereof is being used to treat. Exemplary additional therapeutic agents include, but are not limited to, the various neurological drugs described above, cholinesterase inhibitors (e.g., donepezil, galantamine, rovastigmine, and tacrine), NMDA receptor antagonists (e.g., memantine), amyloid beta peptide aggregation inhibitors, antioxidants, gamma-secretase modulators, nerve growth factor (NGF) mimetics or NGF gene therapy, PPAR gamma agonists, HMS-CoA reductase inhibitors (statins), ampakines, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulin, muscarinic receptor agonists, nicrotinic 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 for its ability to reduce one or more side effects of the neurological agent. The additional therapeutic agent can be administered in the same or separate formulation and can be administered together with or separately from the multispecific antibody or antigen-binding fragment thereof. The multispecific antibody or antigen-binding fragment thereof of the present application can be administered prior to, simultaneously with, and / or after administration of the additional therapeutic agent and / or adjuvant. The multispecific antibody or antigen-binding fragment thereof of the present application can also be used in combination with other interventional therapies, such as, but not limited to, radiation therapy, behavioral therapy, or other therapies known in the art and appropriate for the neurological disorder to be treated or suppressed.

[0132] 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 desired for localized treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration, depending in part on whether administration is brief or chronic. Various dosing regimens are contemplated herein, including, but not limited to, single or multiple doses over various time periods, bolus administration, and pulse infusion.

[0133] The appropriate dosage of the multispecific antibody or antigen-binding fragment thereof of the present application for the prevention or treatment of a disease (when used alone or in combination with one or more other additional therapeutic agents) depends on various factors, such as the type of disease being treated, the type of antibody or conjugate, the severity and course of the disease, whether the multispecific antibody or antigen-binding fragment thereof is being administered for prevention or treatment, previous therapy, the patient's medical history and response to the antibody, the physiological condition of the subject (including, for example, age, weight, health condition), and the discretion of the attending physician. Treatment dosages are optimally titrated to optimize safety and efficacy. The multispecific antibody or antigen-binding fragment thereof is suitably administered to the patient at one time or over a series of treatments.

[0134] According to certain embodiments, a therapeutically effective amount refers to an amount of treatment sufficient to achieve one, two, three, four or more of the following effects: (i) reducing or ameliorating the severity of the disease, disorder or condition being treated or symptoms associated therewith; (ii) shortening the duration of the disease, disorder or condition being treated or symptoms associated therewith; (iii) inhibiting the progression of the disease, disorder or condition being treated or symptoms associated therewith; (iv) causing regression of the disease, disorder or condition being treated or symptoms associated therewith; (v) inhibiting the progression or onset of the disease, disorder or condition being treated or symptoms associated therewith. (vi) inhibiting the recurrence of the disease, disorder or condition being treated, or symptoms associated therewith; (vii) reducing hospitalization of a subject having the disease, disorder or condition being treated, or symptoms associated therewith; (viii) shortening the length of hospitalization of a subject having the disease, disorder or condition being treated, or symptoms associated therewith; (ix) increasing the survival rate of a subject having the disease, disorder or condition being treated, or symptoms associated therewith; (xi) inhibiting or alleviating the disease, disorder or condition being treated, or symptoms associated therewith in a subject; and / or (xii) enhancing or improving the prophylactic or therapeutic efficacy of another therapy.

[0135] In another aspect, the present application relates to an article of manufacture (such as a kit) that includes materials useful for treating, inhibiting, and / or diagnosing the above disorders. The article of manufacture may include a container and a label or package insert attached or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV fluid bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds the composition alone or in combination with another composition effective for treating, inhibiting, and / or diagnosing a condition and may have a sterile access port (e.g., the container may be an intravenous fluid bag or a vial with a stopper pierceable 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 the selected condition. Additionally, the article of manufacture may include (a) a first container containing a composition, the composition comprising the multispecific antibody or antigen-binding fragment thereof of the present application, and (b) a second container containing a composition, the composition comprising an additional cytotoxic or other therapeutic agent. The article of manufacture of this embodiment of the invention may further include a package insert indicating that the composition may be used to treat a particular condition. Optionally, the article of manufacture may further include a second (or third) container containing a pharma- ceutical acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0136] In vitro methods It should be understood that any format of immunoassay using a multispecific antibody or antigen-binding fragment thereof is contemplated for use in accordance with the presently preferred embodiments, including assays in which the multispecific antibody or antigen-binding fragment thereof is bound to a solid phase and assays in which the antibody is in a liquid medium. Immunoassay methods that may be used to detect an analyte using a multispecific antibody embodying features of the present invention include, but are not limited to, competitive (reagent-limited) assays in which a labeled analyte (analog of the analyte) competes with the analyte in the sample for the antibody, and single-site immunometric assays in which the multispecific antibody is labeled.

[0137] The multispecific antibodies or antigen-binding fragments thereof according to the invention can be used in conventional immunological techniques for the detection of Aβ3pE or TfR wherever peptides may be present, including biological samples and conditioned media of cell cultures. 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., all of which are well known to rely on the formation of antigen-antibody immune complexes, but where for the purposes of the assay the antibody or antigen-binding fragment thereof can be detectably labeled, for example with a radioactive, enzymatic, luminescent or fluorescent label, or the antibody or antigen-binding fragment thereof can be immobilized on an insoluble carrier. It is therefore an object of the present invention to provide an immunoassay for the measurement or detection of Aβ3pE and / or TfR, or fragments thereof, in a sample, which method comprises contacting a sample having an antibody or antigen-binding fragment thereof according to the present invention with Aβ3pE or a fragment thereof and determining whether an immune complex is formed between the antibody or antigen-binding fragment thereof and Aβ3pE and / or TfR, or fragments thereof. These methods can be performed on either tissue or body fluid samples, and generally include obtaining a sample from the subject's body, contacting the sample with an effective amount of an antibody or antigen-binding fragment thereof according to the present invention that is detectably labeled, and detecting the label to confirm the presence of Aβ3pE and / or TfR, or fragments thereof, in the sample. The measurement method using the antibody or antigen-binding fragment thereof of the present invention is not particularly limited. Any measurement method can be used as long as the amount of antibody, antigen, or antigen-antibody complex corresponding to the amount of antigen, particularly the amount of Aβ3pE and / or TfR, or fragments thereof, in the solution to be measured, is detected by chemical or physical means and calculated from a standard curve generated by using a standard solution containing a known amount of antigen. For example, turbidimetric methods, competitive methods, immunoassays, and sandwich methods are preferably used. In terms of sensitivity and specificity, it is particularly preferred to use a sandwich method.

[0138] In the sandwich method, a test solution is reacted with an insolubilized polyspecific antibody or an insolubilized antibody of the present invention or the like (first reaction), and then a labeled secondary antibody is reacted (second reaction), and the activity of the labeling agent on the insolubilized carrier is then assayed, thereby determining the amount of Aβ3pE and / or TfR, or a fragment thereof, in the test solution. The first reaction and the second reaction may be performed simultaneously or successively.

[0139] In the measurement 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 14 C. Enzymes are usually made detectable by binding to a suitable substrate which then catalyzes a detectable reaction. Examples thereof include, for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase and malate dehydrogenase, preferably horseradish peroxidase. Luminescent substances include, for example, luminol, luminol derivatives, luciferin, aequorin and luciferase. Furthermore, the avidin-biotin system can also be used to label the antibodies and immunogens of the present invention. When the immunogen or antibody is insolubilized, either physical adsorption or chemical binding, which is usually used for insolubilization or immobilization 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 silicon polymers, and glass.

[0140] In a further embodiment for detecting or diagnosing β-amyloid-related diseases and / or other neurological diseases or conditions, a biological sample, including tissue, bodily fluids, such as cerebrospinal fluid (CSF), blood, plasma, serum, urine, etc., is placed in contact with an appropriate amount of a primary antibody to generate immune complexes. Contacting typically involves adding the sample to a solid matrix coated with the primary antibody. The complexes generated from the contact of the sample with the primary antibody are separated from the sample by elution. However, other recovery methods may be used. The recovered complexes are contacted with at least one secondary antibody, directed against an antigenic determinant on the antigen and capable of binding to the antigen in the complex. The antigenic determinant against which the secondary antibody is directed may be the same as the antigenic determinant against which the primary antibody is directed due to the multi-epitopic nature of the antigen entity. Either the primary or secondary antibody may be made detectable using any of the labels described above. In a preferred embodiment, the secondary antibody is made detectable. The presence of a detectable antibody bound to a complex consisting of the primary antibody and the antigen bound to the secondary antibody may be readily detected using techniques known in the art. By comparing the results obtained with the biological sample with those obtained with a control sample, the presence or concentration of altered Aβ3pE and / or TfR, or fragments thereof, can be determined.

[0141] Embodiment The present invention also provides the following non-limiting embodiments.

[0142] Embodiment 1 is a multispecific antibody or antigen-binding fragment thereof comprising at least one first antigen-binding region capable of specifically binding to pyroglutamate amyloid-β and a second antigen-binding region capable of specifically binding to transferrin receptor (TfR), wherein: a. the first antigen-binding region comprises: i. a first heavy chain variable region (VH1) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO:8 or SEQ ID NO:16, SEQ ID NO:9 or SEQ ID NO:17, and SEQ ID NO:10, respectively; ii. a first light chain variable region (VL1) comprising a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively; Including, b. The second antigen-binding region is i. a second heavy chain variable region (VH2) comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; ii. a second light chain variable region (VL2) comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0143] Embodiment 2 is the multispecific antibody or antigen-binding fragment thereof of 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.

[0144] Embodiment 3 is the multispecific antibody or antigen-binding fragment thereof of 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.

[0145] Embodiment 4 is the multispecific antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 3, wherein the second antigen-binding region comprises a single-chain variable fragment (scFv) antibody comprising VH2 and VL2, or an antigen-binding fragment thereof.

[0146] Embodiment 5 is the multispecific antibody or antigen-binding fragment thereof of embodiment 4, wherein the scFv comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:7.

[0147] Embodiment 6 is the multispecific antibody or antigen-binding fragment thereof of embodiment 5, wherein the scFV comprises the amino acid sequence of SEQ ID NO:7.

[0148] Embodiment 7 is the multispecific antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 6, which comprises two first antigen-binding regions.

[0149] Embodiment 8 is the multispecific antibody or antigen-binding fragment thereof of embodiment 7, (i) a first heavy chain (HC1) comprising VH1, 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); (iii) a first and a second light chain (LC), each of which comprises a VL1 and a light chain constant region, or an antigen-binding fragment thereof.

[0150] Embodiment 9 is the multispecific antibody or antigen-binding fragment thereof of embodiment 8, wherein the scFv is linked to the carboxy terminus of the first heavy chain constant region via a linker, more specifically a linker comprising the amino acid sequence of SEQ ID NO: 27.

[0151] Embodiment 10 is the multispecific antibody or antigen-binding fragment thereof of embodiment 8 or 9, wherein Fc1 and Fc2 each comprise one or more heterodimeric mutations, such as a first modified heterodimeric CH3 domain and a second modified heterodimeric CH3 domain, respectively, compared to a wild-type Fc region, 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, and the amino acid modification at position F405 is 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 the numbering of amino acid residues is according to the EU index as described in Kabat. More specifically, Fc1 comprises the amino acid modifications T350V, L351Y, F405A, and Y407V, and Fc2 comprises the amino acid modifications T350V, T366L, K392L, and T394W.

[0152] Embodiment 11 is the multispecific antibody or antigen-binding fragment thereof of any one of embodiments 8 to 10, wherein Fc1 and Fc2 each comprise one or more heterodimeric mutations, such as a first modified heterodimeric CH3 domain and a second modified heterodimeric CH3 domain, respectively, compared to the wild-type Fc region, and Fc1 comprises amino acid modifications at positions T364, L366, and Y406, and Fc2 comprises an amino acid modification at position T365, wherein the amino acid modification at position T364 is T364S, the amino acid modification at position L366 is L366A, the amino acid modification at position Y406 is Y406V, and the amino acid modification at position T365 is T365W, and the numbering of the amino acid residues is according to the EU index as described in Kabat, more specifically, Fc1 comprises the amino acid modifications T364S, L366A, and Y406V, and Fc2 comprises the amino acid modification T365W.

[0153] Embodiment 12 is the multispecific antibody or antigen-binding fragment thereof of any one of embodiments 8 to 11, wherein 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 at least one of Fc1 and Fc2 has M252Y / S254T / T256E (YTE) mutations, and the numbering of the amino acid residues is according to the EU index as described in Kabat.

[0154] Embodiment 13 is the multispecific antibody or antigen-binding fragment thereof of any one of embodiments 8 to 12, 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, and wherein the numbering of the amino acid residues is according to the EU index as described in Kabat.

[0155] Embodiment 14 is a multispecific antibody, or an antigen-binding fragment thereof, comprising a first heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:24, a first light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:25, a second heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:26, and a second light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:25.

[0156] Embodiment 15 is the multispecific antibody or antigen-binding fragment thereof of embodiment 14, 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, the second heavy chain comprises the amino acid sequence of SEQ ID NO: 26, and the second light chain comprises the amino acid sequence of SEQ ID NO: 25.

[0157] Embodiment 16 is an isolated nucleic acid sequence encoding the multispecific antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 16.

[0158] Embodiment 17 is a vector comprising the isolated nucleic acid of embodiment 16.

[0159] Embodiment 18 is a host cell comprising the isolated nucleic acid of embodiment 16 or the vector of embodiment 17.

[0160] Embodiment 19 is a method of making a multispecific antibody or antigen-binding fragment thereof, comprising culturing a host cell of embodiment 18 under conditions to produce the multispecific antibody or antigen-binding fragment thereof, and recovering the multispecific antibody or antigen-binding fragment thereof.

[0161] Embodiment 20 is a pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 15 and a pharma- ceutically acceptable carrier.

[0162] Embodiment 21 is a method of treating or detecting a disorder, preferably a neurological disorder, in a subject in need thereof, comprising administering to the subject the multispecific antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 15, or the pharmaceutical composition of Embodiment 20, preferably wherein the neurological disorder is selected from the group consisting of neurodegenerative diseases (e.g., Lewy body disease, post-poliomyelitis syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, spinocerebellar degeneration, spinal muscular atrophy), tauopathy (e.g., Alzheimer's disease and supranuclear palsy), prion diseases (e.g., bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob disease, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and nervous system heterodegenerative disorders. amyloidosis, cerebrovascular disease, myelopathy, encephalopathy, pulmonary edema ...

[0163] Embodiment 22 is a method for treating a condition associated with the formation of plaques containing β-amyloid protein in a subject in need thereof, comprising administering to a subject in need thereof a multispecific antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 15, or a pharmaceutical composition of Embodiment 20.

[0164] Embodiment 23 is the method of embodiment 22, wherein the condition is Alzheimer's disease.

[0165] Embodiment 24 is the method of embodiment 22, wherein the condition is selected from the group consisting of dementia associated with trisomy 21 (Down's syndrome), diffuse Lewy body disease, inclusion body myositis, cerebral amyloid angiopathy, and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch Type (HCHWA-D).

[0166] Embodiment 25 is a method for reducing plaques associated with Alzheimer's disease in a subject in need thereof, comprising administering to a subject in need thereof a multispecific antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 15, or a pharmaceutical composition of Embodiment 20.

[0167] Embodiment 26 is a method for suppressing the seeding activity of 3pE Aβ in a subject in need thereof, comprising administering to a subject in need thereof a multispecific antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 15, or a pharmaceutical composition of Embodiment 20.

[0168] Embodiment 27 is a method for producing a pharmaceutical composition comprising the multispecific antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 15, comprising combining the multispecific antibody or antigen-binding fragment thereof with a pharma- ceutically acceptable carrier to obtain the pharmaceutical composition.

[0169] The following examples of the present invention are presented to further illustrate the nature of the present invention, but it should be understood that the following examples are not intended to limit the present invention, the scope of which is defined by the appended claims. EXAMPLES

[0170] Example 1: Generation of anti-Aβ / anti-transferrin receptor (TfR) expression constructs The cell line development (CLD) process incorporated the use of ATUM's Leap-In Transposase® technology and Horizon's Chinese Hamster Ovary (CHO) host cell line. ATUM's Leap-In Transposase® system was engineered from the frog Xenopus tropicalis. In this system, a synthetic transposon is cloned onto a single plasmid that contains a selectable marker and the biotherapeutic gene of interest between Leap-In inverted terminal repeats (ITRs). Each gene was placed under the control of its own regulatory elements, including a promoter and polyadenylation sequence.

[0171] DNA encoding the anti-pyroglutamate amyloid-β / anti-TfR antibodies was synthesized by Golden Gate Assembly using type IIS restriction enzymes BsaI and SapI at ATUM (California, USA) and subcloned into the Leap-In Transposase® glutamine synthetase expression vector backbone pD2546ht+_n. The plasmids were designated Plasmid 1 [light chain, heavy chain 1] and Plasmid 2 [light chain, heavy chain 2].

[0172] The vector elements of Plasmid 1 and Plasmid 2 are described in Tables 1 and 2, respectively. All plasmids were bi-directionally sequence verified at NeoGenomics Laboratories (California, USA).

[0173] 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.

[0174] Nucleotide sequence of heavy chain 1 of plasmid 1 with signal peptide underlined (SEQ ID NO:18):

[0175] [ka]

[0176] [ka]

[0177] Nucleotide sequence of the light chain of Plasmid 1 with the signal peptide underlined (SEQ ID NO:19):

[0178] [ka]

[0179] Nucleotide sequence of heavy chain 2 of plasmid 2 (SEQ ID NO:20) with signal peptide underlined:

[0180] [ka]

[0181] Amino acid sequence of heavy chain 1 of plasmid 1 (SEQ ID NO: 21) with the signal peptide underlined. The Gln (Q) residue at position 1 of heavy chain 1 constitutes the N-terminus of the mature chain.

[0182] [ka]

[0183] Amino acid sequence of the light chain of plasmid 1 (SEQ ID NO: 22) with the signal peptide underlined. The Asp (D) residue at position 1 of the light chain constitutes the N-terminus of the mature chain.

[0184] [ka]

[0185] Amino acid sequence of heavy chain 2 of plasmid 2 (SEQ ID NO: 23) with the signal peptide underlined. The Gln (Q) residue at position 1 of the heavy chain constitutes the N-terminus of the mature chain.

[0186] [ka]

[0187] Mature amino acid sequence of heavy chain 1 of plasmid 1 (SEQ ID NO:24):

[0188] [ka]

[0189] Mature amino acid sequence of light chain 1 of plasmid 1 (SEQ ID NO:25):

[0190] [ka]

[0191] Mature amino acid sequence of heavy chain 2 of plasmid 2 (SEQ ID NO:26):

[0192] [ka]

[0193] A cell line expressing the anti-pyroglutamate amyloid-β / anti-TfR construct was generated.

[0194] Example 2: Characterization of Binding by Surface Plasmon Resonance The binding interaction for recombinant β-amyloid peptide (3pE-28) and human transferrin receptor (TfR; TFRW2) is tested by SPR at 25° C. using a Biacore 8k instrument with buffers of pH 7.4 or pH 5.0 supplemented with 3 mM EDTA and 0.05% Tween 20. Briefly, the biosensor surface is prepared by coupling anti-human IgG Fcγ-fragment specific antibodies to the surface of a C1 sensor chip using the supplier's recommended protocol for amine coupling chemistry (>400 response units (RU)). The coupling buffer is 10 mM sodium acetate (pH 4.5). The test substance is diluted in running buffer and injected over the anti-human IgG and allowed to be captured sufficiently to allow detection of antigen binding. After capture of the test substances, three recombinant antigens are injected in a single cycle kinetic mode at different concentrations (TfR in a 3-fold dilution series from 300 nM to 11.1 nM, 3pE-28 in a 2-fold dilution series from 12 nM to 0.8 nM). Association and dissociation are monitored for 60 min (for 3pE-28 antigen) and for 2 and 5 min for TfR antigen at a flow rate of 50 μL / min. Dissociation profiles of TfR are measured at both pH 7.4 and pH 5.0. Regeneration of the sensor surface is performed with 0.85% H3PO4. Binding sensorgrams are fitted using a 1:1 Langmuir binding model to obtain on-rates, off-rates, and affinities. Parent bivalent antibody against β-amyloid was included as a control.

[0195] Those skilled in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept. It is understood, therefore, that the invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined by the appended claims.

Claims

1. A multispecific antibody comprising (i) a first antigen-binding region and a second antigen-binding region, each capable of specifically binding to pyroglutamate amyloid-β, and (ii) an antigen-binding region capable of specifically binding to a transferrin receptor (TfR), (a) Fab comprising a first antigen-binding region and a second antigen-binding region that can specifically bind to pyroglutamate amyloid-β, each comprising a first heavy chain variable region (VH1) containing the amino acid sequence of SEQ ID NO: 14 and a first light chain variable region (VL1) containing the amino acid sequence of SEQ ID NO: 15, (b) A single-chain variable fragment (scFv) comprising: a second heavy-chain variable region (VH2) having an antigen-binding region that can specifically bind to TfR, comprising heavy-chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3, each containing the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; and a second light-chain variable region (VL2) comprising light-chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, wherein the scFv contains an amino acid sequence that is at least 95% identical to SEQ ID NO:

7. (c) The first heavy chain (HC1) comprises a first antigen-binding region VH1 that can specifically bind to pyroglutamate amyloid-β, a first heavy chain constant region including a first Fc region (Fc1), and the scFv, wherein the scFv is linked to the carboxyl terminus of the first heavy chain constant region via a linker. (d) The second heavy chain (HC2) includes a second constant heavy chain region comprising a second antigen-binding region VH1 and a second Fc region (Fc2) that can specifically bind to pyroglutamate amyloid-β, and (e) A multispecific antibody in which each Fab of a first antigen-binding region and a second antigen-binding region that can specifically bind to pyroglutamate amyloid-β contains the VL1 and a light chain (LC) including the light chain constant region.

2. The multispecific antibody according to claim 1, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

7.

3. The multispecific antibody according to claim 1, wherein the linker comprises the amino acid sequence of SEQ ID NO:

27.

4. The multispecific antibody according to claim 1, wherein each of Fc1 and Fc2 contains the amino acid mutations M252Y, S254T, and T256E, and the numbering of the amino acid residues follows the EU index described in Kabat.

5. The multispecific antibody according to claim 1, wherein each of Fc1 and Fc2 contains the amino acid mutations L234A and L235A, and the numbering of the amino acid residues follows the EU index described in Kabat.

6. The multispecific antibody according to claim 1, wherein HC1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 24, HC2 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 26, and each LC comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

25.

7. The multispecific antibody according to claim 1, wherein Fc1 and Fc2 comprise a first modified heterodimer CH3 domain and a second modified heterodimer CH3 domain compared to the wild-type Fc region, Fc1 comprises amino acid modifications T364S, L366A, and Y406V, and Fc2 comprises amino acid modification T365W, and the numbering of amino acid residues follows the EU index described in Kabat.

8. The multispecific antibody according to claim 1, wherein Fc1 includes an R residue at amino acid position 334 and an F residue at amino acid position 335, and the numbering of the amino acid residues follows the EU index described in Kabat.

9. The multispecific antibody according to claim 1, wherein the HC1 comprises a C-terminal histidine cap.

10. A multispecific antibody that can specifically bind to pyroglutamate amyloid-β and transferrin receptor (TfR), comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 24, a first light chain containing the amino acid sequence of SEQ ID NO: 25, a second heavy chain containing the amino acid sequence of SEQ ID NO: 26, and a second light chain containing the amino acid sequence of SEQ ID NO:

25.

11. An isolated nucleic acid sequence encoding a multispecific antibody according to any one of claims 1 to 10.

12. A vector comprising the isolated nucleic acid described in claim 11.

13. A host cell comprising the isolated nucleic acid described in claim 11.

14. A method for producing a multispecific antibody, comprising: culturing the host cells described in claim 13 under conditions that produce the multispecific antibody; and recovering the multispecific antibody.

15. A pharmaceutical composition comprising a multispecific antibody according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

16. A drug for treating or detecting neurological disorders in a target area, comprising a multispecific antibody as described in any one of claims 1 to 10.

17. A drug for treating a condition associated with the formation of plaques containing β-amyloid protein in a target subject, comprising a multispecific antibody according to any one of claims 1 to 10.

18. A drug for inhibiting the seeding activity of 3pE Aβ in a target area, comprising a multispecific antibody according to any one of claims 1 to 10.

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