Cell permeabilizing agents and their use

Abstract

This disclosure provides cell permeabilizers comprising an intracellular integration module and an antibody or antigen-binding antibody fragment that specifically binds to human beta-amyloid, and methods for treating patients with beta-amyloid-related diseases, including inclusion body myositis (IBM), using these cell permeabilizers. This disclosure relates to cell permeabilizers ("CPA") comprising an intracellular integration module ("CIM") and an antibody (and antigen-binding antibody fragment) that specifically binds to Aβ. This disclosure is based at least in part on the identification and characterization of antibodies that specifically bind to Aβ peptides, reduce deposition load, and are effective in neutralizing soluble Aβ species associated with IBM.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 538,600, filed September 15, 2023, the contents of which said application are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing submitted electronically as an XML file named "50887-0048WO1_ST26_SL.XML". The XML file, created on September 5, 2024, is 373,575 bytes in size. The entire contents of the XML file are incorporated herein by reference.

[0003] This disclosure relates to the technology fields of immunology and medicine. [Background technology]

[0004] Inclusion body myositis (IBM), sometimes called sporadic inclusion body myositis (sIBM), is a group of muscle disorders known as inflammatory myopathy. These myopathy disorders are generally characterized by chronic, progressive inflammation of the muscles, usually accompanied by muscle weakness.

[0005] The onset of muscle weakness in IBM typically develops gradually over several months and years, affecting both sides of the body, although in some cases, the weakness may affect only one side of the body. Symptoms may begin with falls, stumbling, and difficulty with motor tasks such as picking up objects, buttoning, and grasping. In many cases, diagnosis is not made until after age 50, and while IBM is primarily diagnosed in men, women can also be affected.

[0006] While average life expectancy generally does not decrease in individuals with IBM, the disease progresses over time, which may result in the need for lifestyle adaptations and mobility aids such as canes, walkers, or wheelchairs.

[0007] The cause(s) of IBM are largely unknown, but one theory suggests that IBM is an autoimmune disorder that causes inflammation of muscle cells. Persistent inflammation leads to degeneration of muscle fibers and proteins, and as a result, symptoms become observed in patients as the disease progresses. Additionally and / or alternatively, IBM may result from abnormal beta-amyloid protein deposition in muscle cells. The main component of Aβ (A-beta) or β-amyloid deposits is a peptide. Aβ peptide is a 4 kDa internal fragment of 39 to 43 amino acids of a larger transmembrane glycoprotein called amyloid precursor protein (APP). As a result of proteolytic processing of APP by different secretase enzymes, Aβ exists primarily in both a short form of 40 amino acids in length and a long form ranging from 42 to 43 amino acids in length. A portion of the hydrophobic transmembrane domain of APP is found at the carboxy terminus of Aβ, which may account for the ability of Aβ to aggregate into plaques, particularly in the case of the long form. Accumulation of amyloid deposits in muscle is thought to play a role in the phenotype associated with IBM (see, for example, Benveniste, O., et al., Amyloid deposits and inflammatory infiltrates in sporadic inclusion body myositis: the inflammatory egg comes before the degenerative chicken, Acta Neuropahol., 139(5):611-624 (2015)). Although the specific cause(s) of IBM remain unknown, beta-amyloid vaccines have shown efficacy against IBM in mouse models (Kitazawa, M., et al., Immunization with amyloid-β attenuates inclusion body myositis-like myopathology and motor impairment in a transgenic mouse model, The Journal of Neuroscience, 29(19):6132-41 (2009)). However, treatment for IBM remains in need in human patients.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

[0009] This disclosure relates to a cell permeabilizer ("CPA") comprising an intracellular module ("CIM") and an antibody (and antigen-binding antibody fragment) that specifically binds to Aβ. This disclosure also features methods for producing such CPAs and related nucleic acids, for example, pharmaceutical formulations and compositions of CPAs comprising an antibody exhibiting high affinity binding to Aβ for prophylactic and / or therapeutic use to treat amyloidogenic diseases, reduce their risk or delay their onset, prevent amyloidogenic diseases, mitigate them or inhibit their markers. In some examples, amyloidogenic diseases are inclusion body myositis (IBM), also known as sporadic IBM (sIBM).

[0010] This disclosure is based, at least in part, on the identification and characterization of antibodies that specifically bind to Aβ peptides, reduce deposition load, and are effective in neutralizing soluble Aβ species associated with IBM.

[0011] Accordingly, provided herein is an anti-amyloid beta peptide (Aβ) cell-penetrating agent (CPA) comprising (i) a cellular internalization module, and (ii) an antibody that specifically binds to amyloid beta (Aβ) peptide.

[0012] In some embodiments, the CIM comprises a cell membrane internalization peptide (CMIP).

[0013] In another aspect, the present disclosure relates to Formula (IA) to Formula (ID): I-X 2 -X 3 -T-A-L-X 7 -F-X 9 -G-X 11 -A-A-X 14 -K-X 16 -E-A-X 19 -Q-F-L-X 23 -X 24 -L(I-A) (SEQ ID NO: 395), I-W-X 3 -X 4 -A-L-X 7 -F-X 9 -G-X 11 -X 12 -X 13 -A-X 15 -A-E-A-X 19 -X 20 -F-X 22 -S-X 24 -L(I-B), X 1 -X 2 -L-T-X 5 -L-K-X 8 -S-X 10 -K-A-A-A-X 15 -A-E-A-K-Q-X 21 -L-S-X 24 -L(I-C), I-W-L-T-X 5 -X 6 -K-F-S-X 10 -K-X 12 -A-A-K-A-X 17 -X 18 -K-Q-F-L-X 23 -X 24-X 25 A polypeptide of an expression selected from (ID), During the ceremony, X 1 However, it is a hydrophobic amino acid residue or a positively charged amino acid residue, X 2 However, it is an aromatic amino acid residue, a positively charged amino acid residue, or a negatively charged amino acid residue, X 3 However, it is an aromatic amino acid residue, a hydrophobic amino acid residue, a positively charged amino acid residue, or a negatively charged amino acid residue, X 4 However, it is a neutral hydrophilic amino acid residue or an aromatic amino acid residue, X 5 However, it is a hydrophobic amino acid residue or an aromatic amino acid residue, X 6 However, it is a hydrophobic amino acid residue, X 7 However, it is a hydrophobic amino acid residue or a positively charged amino acid, X 8 However, it is an aromatic amino acid residue, X 9 However, X is a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively charged amino acid residue. 10 However, X is a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively charged amino acid residue. 11 However, X is a neutral hydrophilic amino acid residue, a hydrophobic amino acid residue, or a positively charged amino acid residue. 12 However, X is a neutral hydrophilic amino acid residue, a hydrophobic amino acid residue, or a positively charged amino acid residue. 13 However, it is a neutral hydrophilic amino acid residue or a hydrophobic amino acid residue, X 14 However, it is a hydrophobic amino acid residue or a positively charged amino acid, X 15 However, it is an aromatic amino acid residue, a positively charged amino acid residue, or a negatively charged amino acid residue, X 16 However, X is a hydrophobic amino acid residue, an aromatic amino acid residue, a positively charged amino acid residue, or a negatively charged amino acid residue. 17 However, it is either a positively charged amino acid residue or a negatively charged amino acid residue, X 18 However, X is a hydrophobic amino acid residue, an aromatic amino acid residue, or a positively charged amino acid residue. 19However, it is a hydrophobic amino acid residue or a positively charged amino acid residue, X 20 However, it is a neutral hydrophilic amino acid residue or a negatively charged amino acid residue, X 21 However, it is a hydrophobic amino acid residue or an aromatic amino acid residue, X 22 However, it is absent, a hydrophobic amino acid residue, an aromatic amino acid residue, or a positively charged amino acid residue, X 23 However, it is absent, a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively charged amino acid residue, X 24 However, it is absent, an aromatic amino acid residue, or a positively charged amino acid residue, X 25 The present invention provides polypeptides, or pharmaceutically acceptable salts thereof, that are absent or hydrophobic amino acids.

[0014] In some embodiments, CMIP is given by formula (IA): IX 2 -X 3 -TALX 7 -FX 9 -GX 11 -AAX 14 -KX 16 -EAX 19 -QFLX 23 -X 24 -L(IA) or a pharmaceutically acceptable salt thereof.

[0015] In some embodiments, CMIP is given by formula (IB): IWX 3 -X 4 -ALX 7 -FX 9 -GX 11 -X 12 -X 13 -AX 15 -AEAX 19 -X 20 -FX 22 -SX 24 -L(IB) or a pharmaceutically acceptable salt thereof.

[0016] In some embodiments, CMIP is of Formula (IC): X 1 -X 2 -L-T-X 5 -L-K-X 8 -S-X 10 -K-A-A-A-X 15 -A-E-A-K-Q-X 21 -L-S-X 24 -L(IC) or a pharmaceutically acceptable salt thereof.

[0017] In some embodiments, CMIP is of Formula (ID): I-W-L-T-X 5 -X 6 -K-F-S-X 10 -K-X 12 -A-A-K-A-X 17 -X 18 -K-Q-F-L-X 23 -X 24 -X 25 (ID) or a pharmaceutically acceptable salt thereof.

[0018] In some embodiments, CMIP consists essentially of an amino acid sequence selected from Formulas (IA) to (ID). In some embodiments, CMIP consists essentially of the amino acid sequence of Formula (IA). In some embodiments, CMIP consists essentially of the amino acid sequence of Formula (IB). In some embodiments, CMIP consists essentially of the amino acid sequence of Formula (IC). In some embodiments, CMIP consists essentially of the amino acid sequence of Formula (ID).

[0019] In another aspect, the present disclosure provides Formulas (IIA) to (IIC): I-X 2 -X 3 -T-A-L-X 7 -F-X 9 -G-X 11 -A-A-X 14 -K-X 16 -E-A-X 19 -Q-F-L-X 23 -X24 -L(IIA)(Sequence ID 399), IWX 3 -X 4 -ALX 7 -FX 9 -GX 11 -X 12 -X 13 -AX 15 -AEAX 19 -QFX 22 -SX 24 -L(IIB)(Sequence ID 400), X 1 -X 2 -LTALKFSX 10 -KAAAX 15 -AEAKQX 21 -LSX 24 CMIP containing an amino acid sequence having a formula selected from -L(IIC)(SEQ ID NO: 401), or a pharmaceutically acceptable salt thereof, wherein X 1 However, it is isoleucine or lysine, X 2 However, it is either arginine or tryptophan, X 3 However, it is arginine, glutamic acid, leucine, or tryptophan, X 4 However, it is threonine or tryptophan, X 7 However, it is arginine, isoleucine, or lysine, X 9 However, it is arginine, histidine, or serine, X 10 However, it is glycine or tyrosine, X 11 However, X is arginine, isoleucine, lysine, or threonine. 12 However, it is alanine or glutamine, X 13 However, it is alanine or serine, X 14 However, it is alanine, histidine, leucine, or valine, X 15 However, it is arginine, glutamic acid, or lysine, X 16 However, it is alanine or lysine, X 18 However, it is alanine, methionine, or tryptophan, X 19is arginine or lysine, X 21 is arginine or phenylalanine, X 22 is arginine, leucine, or tryptophan, X 23 is arginine or serine, X 24 is arginine, lysine, or tryptophan, there is provided CMIP, or a pharmaceutically acceptable salt thereof.

[0020] In some embodiments, CMIP comprises the amino acid sequence of Formula (IIA). In some embodiments, CMIP comprises the amino acid sequence of Formula (IIB). In some embodiments, CMIP comprises the amino acid sequence of Formula (IIC).

[0021] In some embodiments, CIM comprises a wild-type M-lycotoxin peptide. In some embodiments, CIM comprises an M-lycotoxin derivative. In some embodiments, CIM comprises a polyarginine amino acid sequence. In some embodiments, CIM comprises a plurality of polyarginine amino acid sequences. In some embodiments, CIM comprises a TAT amino acid sequence. In some embodiments, CIM comprises a plurality of TAT amino acid sequences.

[0022] In some embodiments, CIM comprises a macrocycle. In some embodiments, the macrocycle is formed by a covalent bond between two amino acid residues of CIM. In some embodiments, the macrocycle is formed by a disulfide bond between two cysteine residues of CIM.

[0023] In some embodiments, CIM comprises a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 101 to 107, 110, and 116 to 338.

[0024] In some embodiments, the CIM includes one or more spacer regions. In some embodiments, at least one of the one or more spacer regions includes one or more amino acid residues. In some embodiments, at least one of the one or more spacer regions includes one or more glycine residues.

[0025] In some embodiments, CIM is a polypeptide having an amino acid sequence selected from SEQ ID NOs: 101-338.

[0026] In some embodiments, CIM is covalently bound to the antibody. In some embodiments, CIM is noncovalently bound to the antibody.

[0027] In some embodiments, the CPA includes a linker that connects the CIM to the antibody. In some embodiments, the linker is covalently bound to both the CIM and the antibody. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker includes a polypeptide. In some embodiments, the linker includes one or more glycine residues. In some embodiments, the linker includes a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs. 343-346.

[0028] In some embodiments, the antibody is ligated to the C-terminus of the CIM. In some embodiments, the antibody is ligated to the N-terminus of the CIM.

[0029] In some embodiments, the antibody includes a heavy chain or a portion thereof. In some embodiments, the CIM is covalently bonded to the C-terminus of the heavy chain. In some embodiments, the CIM is covalently bonded to the N-terminus of the heavy chain.

[0030] In some embodiments, the antibody includes a light chain or a portion thereof. In some embodiments, the CIM is covalently bonded to the C-terminus of the light chain. In some embodiments, the CIM is covalently bonded to the N-terminus of the light chain.

[0031] In some embodiments, the antibody competes for binding to a reference antibody that includes heavy chain variable domains comprising heavy chains CDR1, CDR2, and CDR3, and light chain variable domains comprising light chains CDR1, CDR2, and CDR3, where heavy chains CDR1, CDR2, and CDR3, and light chains CDR1, CDR2, and CDR3 are as shown for one of the antibodies in Tables 1A and 1B.

[0032] In some embodiments, the antibody comprises a heavy chain variable domain including heavy chains CDR1, CDR2, and CDR3, and a light chain variable domain including light chains CDR1, CDR2, and CDR3, where the heavy chains CDR1, CDR2, and CDR3, and the light chains CDR1, CDR2, and CDR3 are as shown for one of the antibodies in Tables 1A and 1B.

[0033] In some embodiments, the antibody includes a heavy chain variable domain, as shown for one of the antibodies in Tables 1A and 1B. In some embodiments, the antibody includes a light chain variable domain, as shown for one of the antibodies in Tables 1A and 1B.

[0034] In some embodiments, the antibody comprises a heavy chain variable domain including heavy chain CDR1, CDR2, and CDR3, and a light chain variable domain including light chain CDR1, CDR2, and CDR3, where heavy chain CDR1 comprises one of SEQ ID NOs: 16, 19, or 20; heavy chain CDR2 comprises one of SEQ ID NOs: 20, 21, 22, or 23; heavy chain CDR3 comprises one of SEQ ID NOs: 18, 24, or 25; light chain CDR1 comprises one of SEQ ID NOs: 26, 29, 31, or 32; light chain CDR2 comprises one of SEQ ID NOs: 33, 34, 35, or 36; and light chain CDR3 comprises one of SEQ ID NOs: 28, 38, or 39.

[0035] In some embodiments, the heavy chain variable domain is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, 6, and 7, excluding the CDR, and the light chain variable domain is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15, excluding the CDR.

[0036] In some embodiments, the heavy chain variable domain is at least 98% identical to an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, 6, and 7, excluding the CDR, and the light chain variable domain is at least 98% identical to an amino acid sequence selected from SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15, excluding the CDR.

[0037] In some embodiments, the heavy chain variable domain is selected from SEQ ID NOs: 3, 4, 5, 6, and 7, and the light chain variable domain is selected from SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15.

[0038] In some embodiments, the antibody comprises a heavy chain variable domain including heavy chains CDR1, CDR2, and CDR3, and a light chain variable domain including light chains CDR1, CDR2, and CDR3, where heavy chain CDR1 is the amino acid sequence GFTFSNX A GMS (in the formula, X A (It contains Y or F) (SEQ ID NO: 88), and the heavy chain CDR2 has the amino acid sequence SX A RSGSGRTYYSDNVKG (where X A It contains (SEQ ID NO: 89) which is I or V, and the heavy chain CDR3 has the amino acid sequence YDHYX A GX B SDY (in the formula, X A is S or T, X B (is S or T) (SEQ ID NO: 90), light chain CDR1 contains amino acid sequence KSSQSLLDYDGKTYLN (SEQ ID NO: 91), light chain CDR2 contains amino acid sequence X A VX B NRDX C (In the formula, X A is K or R, X Bis S or T, X C (SEQ ID NO: 92) contains S or T, and the light chain CDR3 has the amino acid sequence WQGTHFPRX A (In the formula, X A (is S or T) (Sequence ID 93). In some embodiments, the light chain CDR3 is WQGTHFPRX A FX B (In the formula, X A is S or T, X B (is F or Y) (Sequence ID 94) is included.

[0039] In some embodiments, the antibody comprises a heavy chain variable domain including heavy chains CDR1, CDR2, and CDR3, and a light chain variable domain including light chains CDR1, CDR2, and CDR3, where heavy chain CDR1 is the amino acid sequence GFTFX A NX B GMS (in the formula, X A is S or A, X B (It is Y or F) (SEQ ID NO: 95), and the heavy chain CDR2 has the amino acid sequence SX A RSG X B X C RTYYSDNVKG(where X A is I or V, X B is S or G, X C (SEQ ID NO: 96) contains S or G, and the heavy chain CDR3 has the amino acid sequence YDHYX A GX B SDY (in the formula, X A is S or T, X B (SEQ ID NO: 90) contains S or T, and the light chain CDR1 has amino acid sequence X A SSQSLX B DX C DGKTYLN(where X A is K or R, X B is V, M, or L, X C (is Y, T, or S) (Sequence ID 97), and the light chain CDR2 contains amino acid sequence X A VX B NRX C X D(In the formula, X A is K or R, X B is S or T, X C is E or D, X D (SEQ ID NO: 98) contains S or T, and the light chain CDR3 has the amino acid sequence WQGX A HFPRX B (In the formula, X A is S or T, X B (is S or T) (Sequence ID 99) is included. In some embodiments, the light chain CDR3 is WQGTHFPRX A FX B X C (In the formula, X A is S or T, X B is S or T, X C (is F or Y) (Sequence ID 100) is included.

[0040] In some embodiments, the antibody is humanized. In some embodiments, the antibody is a human IgG1 isotype. In some embodiments, the antibody is a complete antibody, a chimeric antibody, a CDR-transplanted antibody, or a recombinant antibody. In some embodiments, the fragment is Fab, Fab', F(ab')2, Fabc, or Fv.

[0041] In some embodiments, the antibody includes a heavy chain constant domain having an amino acid sequence at least 95% identical to SEQ ID NO: 40. In some embodiments, the antibody includes a light chain constant domain having an amino acid sequence at least 95% identical to SEQ ID NO: 41.

[0042] In some embodiments, the heavy chain variable domain is fused to the heavy chain constant domain, and the light chain variable domain is fused to the light chain constant domain. In some embodiments, the heavy chain constant domain is a variant of the native human heavy chain constant domain, with reduced binding to the Fcγ receptor compared to the native heavy chain constant domain.

[0043] In some embodiments, the heavy chain constant domain is of the IgG1 isotype.

[0044] In some embodiments, the antibody has at least one mutation in its constant domain. In some embodiments, at least one mutation reduces complement binding or activation by the constant domain. In some embodiments, at least one mutation is located at one or more of the following positions in EU numbering: 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331. In some embodiments, the antibody has alanine at positions 318, 320, and 322 in EU numbering.

[0045] In some embodiments, the antibody specifically binds to an epitope having an amino acid sequence that includes three or more amino acid positions of amino acids 1-7 of Aβ.

[0046] In some embodiments, the antibody is selected from aducanumab, lecanumab, bapineuzumab, solanezumab, ganterelumab, crenezumab, and ponezumab.

[0047] In some embodiments, the anti-Aβ CPA comprises a polypeptide that is at least 95% identical to a sequence selected from any one of SEQ ID NOs: 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, and 394. In some embodiments, the anti-Aβ CPA comprises a polypeptide that is at least 98% identical to a sequence selected from any one of SEQ ID NOs: 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, and 394. In some embodiments, the anti-Aβ CPA comprises a polypeptide sequence selected from any one of SEQ ID NOs: 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, and 394.

[0048] In some embodiments, the anti-Aβ CPA comprises a polypeptide that is at least 95% identical to a sequence selected from any one of SEQ ID NOs: 350, 352, 354, 356, 358, 360, 362, 364, 366, and 368-393. In some embodiments, the anti-Aβ CPA comprises a polypeptide that is at least 98% identical to a sequence selected from any one of SEQ ID NOs: 350, 352, 354, 356, 358, 360, 362, 364, 366, and 368-393. In some embodiments, the anti-Aβ CPA comprises a polypeptide sequence selected from any one of SEQ ID NOs: 350, 352, 354, 356, 358, 360, 362, 364, 366, and 368-393.

[0049] In some embodiments, the anti-Aβ CPA comprises a first polypeptide and a second polypeptide, further comprising, where the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 349 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 350; or the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 351 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 352; or the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 353 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 354; or the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 355 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 356; or the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 357 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 358; or the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 359 The first polypeptide contains an identical sequence, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 360; the first polypeptide contains a sequence that is at least 95% identical to sequence number 361, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 362; the first polypeptide contains a sequence that is at least 95% identical to sequence number 363, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 364; the first polypeptide contains a sequence that is at least 95% identical to sequence number 365, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 366; the first polypeptide contains a sequence that is at least 95% identical to sequence number 367, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 368; or the first polypeptide contains a sequence that is at least 95% identical to sequence number 394, and the second polypeptide contains a sequence that is at least 95% identical to any one of sequence numbers 369 to 393.

[0050] In some embodiments, anti-Aβ CPA comprises a first polypeptide and a second polypeptide, further comprising, where the first polypeptide comprises SEQ ID NO: 349 and the second polypeptide comprises SEQ ID NO: 350, or the first polypeptide comprises SEQ ID NO: 351 and the second polypeptide comprises SEQ ID NO: 352, or the first polypeptide comprises SEQ ID NO: 353 and the second polypeptide comprises SEQ ID NO: 354, or the first polypeptide comprises SEQ ID NO: 355 and the second polypeptide comprises SEQ ID NO: 356, or the first polypeptide comprises SEQ ID NO: 357 and the second polypeptide comprises SEQ ID NO: 358, or the first polypeptide comprises SEQ ID NO: 359 and the second polypeptide comprises SEQ ID NO: 360, or the first polypeptide comprises SEQ ID NO: 361 and the second polypeptide comprises SEQ ID NO: 362, or the first polypeptide comprises SEQ ID NO: 363 and the second polypeptide comprises SEQ ID NO: 364, or the first polypeptide comprises SEQ ID NO: 365 and the second polypeptide comprises SEQ ID NO: 366. The first polypeptide contains SEQ ID NO: 367 and the second polypeptide contains SEQ ID NO: 368, or the first polypeptide contains SEQ ID NO: 394 and the second polypeptide contains any one of SEQ ID NOs: 369-393.

[0051] In some embodiments, the antibody is conjugated with a therapeutic agent, cytotoxic agent, cell proliferation inhibitor, immunomodulator, neurotrophic agent, or neuroprotective agent.

[0052] In some embodiments, the heavy chain does not contain a C-terminal lysine residue.

[0053] Accordingly, this specification provides pharmaceutical compositions comprising any of the anti-Aβ CPAs described herein and a pharmaceutically acceptable carrier or diluent.

[0054] This specification also provides nucleic acids encoding at least a portion of any of the anti-Aβ CPAs described herein. In some embodiments, the nucleic acid encodes CIM or a portion thereof. In some embodiments, the nucleic acid encodes CIM or a portion thereof, as well as at least one of the heavy chain variable domain and the light chain variable domain of the antibody. In some embodiments, the nucleic acid encodes CIM or a portion thereof, and the heavy chain variable domain or a portion thereof of the antibody. In some embodiments, the nucleic acid encodes CIM or a portion thereof, and the light chain variable domain or a portion thereof of the antibody.

[0055] This specification also provides vectors comprising one of the nucleic acids described herein operably ligated to one or more regulatory sequences to bring about the expression of any of the cell permeabilizers described herein in mammalian cells. In some embodiments, the one or more regulatory sequences comprise one or more of enhancers, ribosome binding sites, transcription termination signals, and promoters, and optionally, the promoter is a eukaryotic promoter. In some embodiments, the nucleic acids are codon-optimized for expression in host cells.

[0056] This specification also provides host cells transformed with any of the vectors described herein.

[0057] This specification also provides host cells containing any of the nucleic acids described herein.

[0058] This specification also provides a method for delivering an antibody that specifically binds to Aβ into a cell, comprising contacting the cell with one of the anti-Aβ CPAs described herein, thereby resulting in the internalization of at least an antigen-binding fragment of the antibody into the cell. In some embodiments, the method comprises at least the transfer of the antigen-binding fragment of the antibody into the cell's cytosol.

[0059] This specification also provides a method for binding to an intracellular Aβ protein in a cell, the method comprising contacting the cell with one of the anti-Aβ CPAs described herein, thereby resulting in at least internalization and transfer of the antigen-binding fragment of the antibody into the cytosol. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are in vitro. In some embodiments, the cells are within a subject.

[0060] This specification also provides a method for inhibiting or reducing the aggregation of Aβ in a subject having or at risk of developing an Aβ-related disease, comprising administering an effective amount of one of the anti-Aβ CPAs described herein to the subject, thereby inhibiting or reducing the aggregation of Aβ in the subject.

[0061] This specification also provides a method for treating or preventing an Aβ-related disease in a subject, comprising administering a therapeutically effective amount of one of the cell permeabilizing agents described herein, thereby treating or preventing an Aβ-related disease. In some embodiments, the Aβ-related disease is inclusion body myositis.

[0062] This specification also provides a method for detecting Aβ deposition in subjects who have or are at risk of developing an Aβ-related disease, comprising administering one of the cell permeabilizing agents described herein to the subject and detecting antibodies bound to Aβ in the subject.

[0063] This specification also provides a method for detecting Aβ in a sample obtained from a patient who has or is at risk of developing an Aβ-related disease, comprising contacting the sample with one of the cell permeabilizing agents described herein and detecting the binding of an antibody to Aβ in the sample.

[0064] In some embodiments, the antibody is labeled. In some embodiments, the antibody is labeled with a fluorescent label, a paramagnetic label, or a radioactive label. In some embodiments, the radioactive label is detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

[0065] All publications, patents, and patent applications described herein are incorporated by reference to the same extent that each individual publication, patent, patent application, or information is explicitly and individually indicated as being incorporated by reference. If any publication, patent, patent application, or information incorporated by reference conflicts with any disclosure contained herein, this specification is intended to supersede and / or take precedence over all such conflicts.

[0066] When values ​​are described in terms of ranges, please understand that this description includes disclosure of all possible subranges within such ranges, and any specific numerical values ​​that fall within such ranges, regardless of whether a particular numerical value or subrange is explicitly stated.

[0067] The following drawings illustrate specific embodiments that illustrate the features and advantages of this disclosure. These embodiments are not intended to limit in any way the scope of the appended claims. Similar reference numerals in each drawing refer to similar elements. [Brief explanation of the drawing]

[0068] [Figure 1] Graphs A-C show the results for Aβ model cells treated with anti-Aβ cell permeabilizing agents, specifically for focus area (A), number of foci (B), and average focus area (C). [Figure 2] Graphs A-C show the results for Aβ model cells treated with anti-Aβ cell permeabilisants compared to isotype controls for focus area (A), number of foci (B), and average focus area (C). [Figure 3A]This graph shows the results for Aβ model cells treated with an anti-Aβ cell permeabilizer compared to isotype controls in terms of focus area. [Figure 3B] This graph shows the results for Aβ model cells treated with an anti-Aβ cell permeabilizing agent compared to isotype controls in terms of the number of foci. [Figure 3C] This graph shows the results for Aβ model cells treated with an anti-Aβ cell permeabilizer compared to isotype controls in terms of average focus area. [Figure 4A] This graph shows the results for Aβ model cells treated with anti-Aβ cell permeabilizing agents containing FcK or FcRn mutations, regarding the total Aβ focus area per cell. [Figure 4B] This graph shows the results for Aβ model cells treated with anti-Aβ cell permeabilizers containing FcK or FcRn mutations, regarding the number of Aβ focus areas per cell. [Figure 4C] This graph shows the results for cell-to-cell permeabilization of Aβ model cells treated with anti-Aβ cell permeabilizers containing FcK or FcRn mutations. [Figure 5] Graphs A-C show the results for Aβ model cells treated with the anti-Aβ cell permeabilizer (mzd-2931-CMIP4) in terms of focus area (A), number of foci per cell (B), and internalization of antibody or cell permeabilizer per cell. [Figure 6A] This graph shows the results for total Aβ focus area per cell in Aβ model cells treated with the CMIP-4-derived intracellular integration module. [Figure 6B] This graph shows the results for the average number of Aβ foci per cell in Aβ model cells treated with the CMIP-4-derived intracellular integration module. [Figure 6C] This graph shows the results for antibody or cell permeabilizer internalization of Aβ model cells treated with the CMIP-4-derived intracellular internalization module. [Figure 7A] This image shows the results regarding Aβ aggregates in untreated model Aβ cells. [Figure 7B]This image shows the results regarding Aβ aggregates in model Aβ cells treated with untagged antibodies. [Figure 7C] This image shows the results regarding Aβ aggregates in model Aβ cells treated with an anti-Aβ cell permeabilizing agent. [Figure 8] Graphs A-C show the results for Aβ model cells treated with anti-Aβ cell permeabilizing agents regarding total Aβ focus area per cell (A), number of foci (B), and internalization (C). [Figure 9] A and B show staining of Aβ aggregates with two different antibodies (A), and a graph (B) showing the percentage of stable cells containing Aβ aggregates compared to a control sample. [Figure 10] Graphs A-C show the results for Aβ model cells treated with mouse-type cell permeabilis for total Aβ focus area per cell (A), number of Aβ foci per cell (B), and internalization of cell permeabilis (C). [Modes for carrying out the invention]

[0069] This disclosure provides systems and methods for delivering antibodies into cells. More specifically, this disclosure provides systems and methods for delivering anti-amyloid beta (Aβ) cell permeabilizers into cells. Also provided are pharmaceutical compositions comprising these cell permeabilizers and pharmaceutically acceptable carriers, nucleic acids and / or vectors encoding these cell permeabilizers, and host cells expressing the aforementioned nucleic acids and / or vectors. The specific examples and embodiments disclosed herein are not limiting and merely illustrate the breadth of the approaches.

[0070] In some embodiments, the present disclosure provides a cell permeabilizer ("CPA") comprising (i) an internalization module ("CIM") and (ii) an antibody that specifically binds to an Aβ peptide. The CIM facilitates the internalization of the cell permeabilizer into cells. In some embodiments, the CIM further facilitates the movement of the cell permeabilizer from internalization vesicles (e.g., endosomes) to the cytosol. In some embodiments, the CIM disrupts the membrane of the internalization vesicle, resulting in vesicle rupture and facilitating the movement of the cell permeabilizer from the vesicle to the cytosol.

[0071] Monoclonal antibodies (mAbs) targeting the N-terminus of amyloid beta (Aβ) have been clinically demonstrated to reduce amyloid plaque load, with aducanumab, one such antibody, showing a significant reduction in plaque load. Preclinical studies have also shown that monoclonal antibodies (mAbs) targeting the N-terminal epitope of Aβ induce antibody-dependent microglia-mediated Aβ plaque clearance and neutralization of soluble toxic Aβ oligomers both in vitro and in vivo. It is hypothesized that administration of cell permeabilizers containing Aβ-targeted mAbs may slow disease progression in IBM patients through intracellular Aβ plaque clearance.

[0072] Accordingly, this disclosure provides antibodies (and antigen-binding antibody fragments), encoding nucleic acids and / or vectors, methods for producing such antibodies and antibody fragments, pharmaceutical compositions, and methods for preventing or treating amyloidogenic diseases such as inclusion body myositis.

[0073] This disclosure is based at least in part on the characterization of a genus of monoclonal antibodies that are effective in binding to beta-amyloid protein (Aβ) (e.g., to soluble and / or aggregated Aβ), mediating phagocytosis (e.g., of aggregated Aβ), reducing plaque load and / or mitigating neurite dystrophy (e.g., in patients), and neutralizing soluble toxic Aβ species. The antibodies and fragments of this disclosure exhibit higher binding strength (affinity and / or binding activity) against pathological fibrous Aβ than reported current experimental therapies and higher affinity for soluble toxic Aβ forms. These antibodies may enable simpler administration strategies and enhanced patient access.

[0074] I. Definition The term "antibody" includes complete antibodies and their conjugated fragments. Typically, fragments compete with the intact antibody from which they originate for specific binding to a target. Fragments include separate heavy chains, light chains Fab, Fab', F(ab')2, F(ab)c, Fv, and single-domain antibodies. Single (variable) domain antibodies include VH regions isolated from the VL partner in conventional antibodies (or vice versa) (Ward et al., 1989, Nature 341:544-546), as well as VH regions from species such as camelid or cartilaginous fish (e.g., nurse shark) where the VH region is not associated with the VL region (sometimes known as VHH) (see, e.g., WO9404678). Single-domain antibodies in which one chain is isolated from its natural partner are sometimes known as Dab, and single-domain antibodies from camelid or cartilaginous fish are sometimes known as nanobodies. The constant region or a portion of the constant region may or may not be present in a single-domain antibody. For example, naturally occurring single-domain antibodies from camelids contain a VHH variable region, as well as CH2 and CH3 constant regions. Single-domain antibodies can be humanized using approaches similar to those for conventional antibodies. Dab-type antibodies are typically obtained from human-derived antibodies. Nanobody-type antibodies, originating from camelids or sharks, can be humanized. Fragments can be produced by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins. The term "antibody" also includes bispecific antibodies. Bispecific or bifunctional antibodies are artificial hybrid antibodies with two different heavy / light chain pairs and two different binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990), Kostelny et al., J. Immunol., 148:1547-53 (1992)).

[0075] The term “amyloidogenic disease” refers to inclusion body myositis (IBM) or sporadic inclusion body myositis (sIBM). Therefore, IBM or sIBM is, for example, an example of a “disease characterized by Aβ deposition” or “disease associated with Aβ deposition” in the muscles of a subject or patient. The terms “β-amyloid protein,” “β-amyloid peptide,” “β-amyloid,” “Aβ,” and “Aβ peptide” are used interchangeably herein.

[0076] As used herein, “Cell Permeation Agent” (also referred to herein as “CPA”) refers to a drug (e.g., a molecule and / or molecular complex) that can enter cells (e.g., mammalian cells in vitro and / or in vivo). In some embodiments, the CPA enters the cell and, after internalization by the cell, moves into the cytosol. In some embodiments, the CPA includes an intracellular internalization module (CIM) that facilitates the internalization of the CPA by the cell. In some cases, the CPA includes a CIM that facilitates the internalization of the CPA and the movement of the CPA into the cytosol. In some embodiments, the CPA further includes an anti-Aβ antibody linked to the CIM (e.g., covalently or non-covalently). In some embodiments, the CPA includes a CIM covalently linked to the anti-Aβ antibody (e.g., via a linker between the CIM and the anti-Aβ antibody). In other embodiments, the CPA includes a CIM non-covalently bound to an anti-Aβ antibody (e.g., via a streptavidin-biotin interaction) such that the CIM remains bound to the anti-Aβ antibody under relevant conditions (e.g., plasma). In some embodiments, the CPA further includes a linker that connects the CIM to the anti-Aβ antibody. In various cases, the linker may be cleavable or incleavable and / or can covalently or non-covalently connect the CIM to the anti-Aβ antibody. In some embodiments, a CPA containing an anti-Aβ antibody exhibits enhanced cell penetration compared to a reference anti-Aβ antibody that is not part of the CPA. In some embodiments, the CPA includes two or more anti-Aβ antibodies and / or two or more CIMs (e.g., the CPA includes multiple CIMs and / or dendrimers bound to anti-Aβ antibodies). Non-limiting features and examples of cell permeabilizers are described herein.

[0077] As used herein, the “internalization module” (also referred to herein as “CIM”) refers to a portion of CPA that facilitates the internalization of CPA (and, by extension, anti-Aβ antibodies) by cells. In various embodiments, the CIM may achieve internalization by utilizing one or more internalization processes, including both active and passive internalization. Exemplary processes include, but are not limited to, endocytosis (e.g., receptor-mediated endocytosis (RME), phagocytosis, phagocytosis) and membrane translocation (e.g., direct permeation and / or energy-independent internalization). In various non-limiting embodiments, the CIM includes, for example, a cell membrane internalization peptide (CMIP), a small molecule ligand (e.g., a vitamin, fatty acid, integrin-binding ligand, etc.), a portion of an antibody (e.g., an scFv portion that binds to an internalized cell surface target), or a decoy receptor ligand (e.g., a cytokine or its derivative). In some embodiments, the CIM includes a cell membrane internalization peptide (CMIP). Non-limiting features and examples of the internalization module are described herein.

[0078] As used herein, “Cell membrane internalization peptide” (also referred to herein as “CMIP”) is a sequence of three or more naturally occurring or non-naturally occurring amino acids that, when covalently or non-covalently bound to an anti-Aβ antibody, result in the internalization of at least an anti-Aβ antibody or an active fragment of an anti-Aβ antibody into a mammalian cell. In various embodiments, CMIP may achieve internalization by utilizing one or more internalization processes, including both active and passive internalization. Exemplary processes include, but are not limited to, endocytosis and membrane translocation. In some embodiments, CMIP interacts with the cell membrane and / or cell surface antigens, resulting in the internalization of CPA or a portion thereof (e.g., the anti-Aβ antibody or a portion thereof containing its functional part) via endocytic vesicles. In some embodiments, CMIP further facilitates the translocation of CPA or a portion thereof from endocytic vesicles into the cytoplasm. In some cases, CMIP further interacts with the membrane of the endocytic vesicle, resulting in vesicle rupture and endosomal exodus of CPA or a portion thereof into the cytosol. Therefore, in some embodiments, CMIP facilitates both the internalization of anti-Aβ antibodies or their functional portions into cells and the transfer of anti-Aβ antibodies or their functional portions into the cytosol. Non-limiting examples of CMIP include, for example, cationic peptides (e.g., M-lycotoxin, TAT peptide, penetratin, and polyarginine peptides, and their derivatives), amphiphilic peptides (e.g., MPG peptide, Pep-1 peptide, transportan peptide, and their derivatives), and proline-rich peptides (e.g., Bac7 peptide and its derivatives). In some embodiments, CMIP includes cell-permeable peptides and their derivatives, such as those described in I. Rueska and A. Zimmer, Internalization mechanisms of cell penetrating peptides, BEILSTEIN J. NANOTECHOL. 202;11:101-123. Non-limiting features and examples of CMIP are described herein.

[0079] The term "epitope" refers to a specific site on an antigen molecule to which an antibody binds. Epitopes can be formed from juxtaposed or discontinuous amino acids by the tertiary folding of one or more proteins. Epitopes formed from juxtaposed amino acids (also known as linear epitopes) are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding (also known as structural epitopes) are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly at least five or eight to ten, amino acids in their unique spatial structure. Methods for determining the spatial structure of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0080] Antibodies that recognize the same or overlapping epitopes can be identified by a simple immunoassay that demonstrates the ability of one antibody to compete for the binding of another antibody to a target antigen. Antibody epitopes can also be defined by identifying contact residues through X-ray crystallography of the antibody bound to the antigen. Alternatively, two antibodies can be said to have the same epitope if an amino acid mutation in the antigen reduces or eliminates the binding of one antibody. Two antibodies can be said to have overlapping epitopes if an amino acid mutation that reduces or eliminates the binding of one antibody also reduces or eliminates the binding of the other antibody.

[0081] The terms “humanized immunoglobulin” or “humanized antibody” refer to an immunoglobulin or antibody comprising at least one humanized immunoglobulin or antibody chain (i.e., at least one humanized light chain or heavy chain). The terms “humanized immunoglobulin chain” or “humanized antibody chain” (i.e., “humanized immunoglobulin light chain” or “humanized immunoglobulin heavy chain”) refer to an immunoglobulin or antibody chain (i.e., a light chain or heavy chain, respectively) having a variable domain comprising a variable framework region substantially derived from a human immunoglobulin or antibody and a complementarity-determining region (CDR) substantially derived from a non-human immunoglobulin or antibody (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs), and further comprising a constant region (e.g., at least one constant region or a portion thereof in the case of a light chain, or preferably three constant regions in the case of a heavy chain). The term “humanized variable region” (e.g., “humanized light chain variable region” or “humanized heavy chain variable region”) refers to a variable framework region substantially derived from human immunoglobulins or antibodies, as well as a variable region containing complementarity-determining regions (CDRs) substantially derived from non-human immunoglobulins or antibodies.

[0082] Antibody competition is determined by an assay in which the antibody under test inhibits the specific binding of a reference antibody (e.g., 3D6, aducanumab, bapineuzumab) to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess of the test antibody (e.g., at least 2x, 5x, 10x, 20x, or 100x) inhibits the binding of the reference antibody by at least 50%, preferably 75%, 90%, or 99%, as measured by a competitive binding assay. Antibodies identified by the competitive assay (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody, and antibodies that bind to an adjacent epitope that is sufficiently proximal to the epitope bound by the reference antibody to cause steric hindrance.

[0083] The light chain or heavy chain variable region of an immunoglobulin (sometimes referred to herein as the “light chain variable domain” (“VL domain”) or the “heavy chain variable domain” (“VH domain”), respectively) consists of a “framework” region and three “complementarity-determining regions” (“CDR”). The framework region plays a role in aligning the CDRs to specifically bind to the epitope of the antigen. The CDRs contain amino acid residues of the antibody that are primarily involved in antigen binding. From the amino terminus to the carboxyl terminus, both the VL domain and the VH domain contain the following framework (FR) region and CDR region: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, 2, and 3 of the VL domain are sometimes also called CDR-L1, CDR-L2, and CDR-L3, respectively, and the CDR1, 2, and 3 of the VH domain are sometimes called CDR-H1, CDR-H2, and CDR-H3, respectively. In this application, if a VL sequence having R as the C-terminal residue is disclosed, R can also be considered as the N-terminal residue of the light chain constant domain. Therefore, this application should be understood to disclose a VL sequence that does not contain the C-terminal R.

[0084] The amino acid assignments to each VL and VH domain conform to the conventional definition of CDR. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia & Lesk, J.Mol.Biol.196:901-917, 1987, Chothia et al., Nature 342:878-883, 1989), the CDR-H1 complex of Chothia and Kabat's CDRs, the AbM definition used in Oxford Molecular's antibody modeling software, and Martin et al. This includes the contact definition (bioinfo.org.uk / abs) (see Table 1). Kabat provides a widely used numbering rule (Kabat numbering) that assigns the same number to corresponding residues between different heavy chains or between different light chains. When an antibody is said to contain a CDR by a particular CDR definition (e.g., Kabat), that definition specifies the minimum number of CDR residues present in the antibody (i.e., Kabat CDRs). This does not rule out the possibility of other residues being present that are included in other conventional CDR definitions but not in the given definition. For example, an antibody containing a CDR as defined by Kabat includes, among other possibilities, an antibody in which the CDR contains Kabat CDR residues and does not contain other CDR residues, and an antibody in which CDR H1 is a compound Chothia-Kabat CDR H1 and other CDRs contain Kabat CDR residues and do not contain additional CDR residues based on other definitions. [Table 8]

[0085] In some embodiments, the CDR of the humanized antibody of this disclosure is defined as being selected from the group consisting of Kabat, Chothia, Kabat / Chothia complex, AbM, and Contact.

[0086] One or more amino acids at the amino or carboxyl termini of the light and / or heavy chains, for example, the C-terminal lysine of the heavy chain, may be omitted in part or all of the molecule, or may be derivatized. Substitutions can be made in the constant domain to reduce or increase effector functions such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to extend the half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary substitutions include Gln at position 250 and / or Leu at position 428 (EU numbering is used in this paragraph for constant domains) to increase the half-life of an antibody. Substitutions at any or all of the 234, 235, 236, and / or 237 positions reduce affinity for Fcγ receptors, particularly FcγRI receptors (see, e.g., US6,624,821). Alanine substitutions at the 234, 235, and 237 positions of human IgG1 can be used to reduce effector function. Some antibodies have alanine substitutions at the 234, 235, and 237 positions of human IgG1 to reduce effector function. Optionally, the 234, 236, and / or 237 positions of human IgG2 are substituted with alanine, and the 235 position is substituted with glutamine (see, e.g., US5,624,821). Some antibodies use mutations at one or more of the following positions in the EU numbering of human IgG1: 241, 264, 265, 270, 296, 297, 322, 329, and 331. Some antibodies use mutations at one or more of the following positions in the EU numbering of human IgG1: 318, 320, and 322. Some antibodies have alanine substitution at positions 234 and / or 235, and / or glycine substitution at position 329. Some antibodies have alanine substitution at positions 234 and 235.For some antibodies, the isotype is either human IgG2 or human IgG4.

[0087] As used herein, “linker” refers to a chemical moiety that does not exhibit catalytic or therapeutic activity in mammalian cells and is used to covalently link two different functional molecules (e.g., an intracellularization module and an Aβ antibody). For example, a linker may be a peptide of about 3 to 25 amino acids (e.g., about 3 to 20 amino acids, or about 3 to 12 amino acids). In other examples, a linker may be a bond (e.g., an amide bond, an ester bond, an ether bond, and a disulfide bond). In some examples, a linker may comprise a pair of affinity domains (e.g., the first domain of a pair of affinity domains may be interleukin-15, and the second domain of a pair of affinity domains may be the sushi domain of the interleukin-15 receptor alpha). In some examples, a linker is a glycine residue followed by a serine residue (GS). In some examples, a linker is three consecutive glycine residues (e.g., GGG). In some examples, a linker is one of sequence numbers 343–346.

[0088] The term "pharmaceutically acceptable" means that a carrier, diluent, excipient, or adjuvant is compatible with the other components of the formulation and is not substantially harmful to its recipient.

[0089] As used herein, “M-lycotoxin derivative” is a peptide comprising three or more amino acids (either naturally occurring or not) designed based on the starting M-lycotoxin peptide. In some embodiments, the “M-lycotoxin derivative” is a polypeptide having, for example, 60% to 99% sequence homology to wild-type M-lycotoxin. Additional non-limiting embodiments and examples of M-lycotoxin derivatives are described herein.

[0090] As used herein, the term "polymer" refers to a molecule having either a molecular weight of at least 5 kDa and / or a hydrodynamic radius of at least 1.0 nm. As used herein, the term "polymer" includes biomolecules, organic polymers, and organometallic complexes.

[0091] As used herein, “spacer” or “spacer region” refers to an amino acid that does not exhibit catalytic or therapeutic activity in mammalian cells. For example, a spacer may be a peptide of 1 to about 10 amino acids (e.g., 1 to about 8 amino acids, 1 to about 6 amino acids, or 1 to about 4 amino acids). In some examples, one or more spacer regions are contained within an intracellular integration module. For example, a spacer region can isolate amino acids within an intracellular integration module; for example, a spacer may be located after the first amino acid of the CIM. In some examples, a spacer may be located before the last amino acid of the CIM. In some examples, the CIM may have two or more spacer regions (e.g., two spacer regions, three space sequences, four spacer regions, five spacer regions, or more). In some examples, a spacer region is a single glycine residue. In some examples, a spacer region is a pair of glycine residues. In some examples, a spacer region is three glycine residues. In some examples, a spacer region is four glycine residues. In some examples, the spacer region consists of four glycine residues followed by a serine residue. In other examples, the spacer region is one of sequence numbers 339-342.

[0092] The terms “patient” or “subject” include human and other mammalian subjects (e.g., humans) receiving either prophylactic or therapeutic treatment.

[0093] If a subject has at least one known risk factor (e.g., genetic, biochemical, family history, and situational exposure), that individual is considered to have an increased risk of disease, and is statistically significantly more likely to develop the disease than an individual without the risk factor.

[0094] The term "biological sample" refers to a sample of biological material from a biological source, such as a human or mammalian subject, or a biological material obtainable from them. Such a sample may be an organ, organelle, tissue, tissue section, body fluid, peripheral blood, plasma, serum, cell, protein and peptide molecules, and any part or combination thereof. The term "biological sample" may also encompass any material obtained by processing the sample. The derived material may include cells or their offspring. Processing of a biological sample may include one or more of the following: filtration, distillation, extraction, concentration, fixation, inactivation of interfering components, etc.

[0095] A “control sample” refers to a biological sample that is not known or suspected to contain areas affected by Aβ, or that is not known or suspected to contain at least a specific type of diseased tissue. A control sample may be obtained from an individual that does not have Aβ-related disease. Alternatively, a control sample may be obtained from a patient with Aβ-related disease. Such a sample may be collected at the same time as the biological sample thought to contain Aβ-related disease, or at a separate occasion. Both the biological sample and the control sample may be collected from the same tissue. Preferably, the control sample consists of substantially or completely normal and healthy tissue and can be used for comparison with the biological sample thought to contain areas affected by Aβ-related disease. Preferably, the tissue in the control sample is of the same type as the tissue in the biological sample. Preferably, Aβ-related disease-affected cells thought to be present in the biological sample may originate from the same cell type (e.g., neurons or glial cells) as the cells in the control sample.

[0096] For the purpose of classifying amino acid substitutions as conserved or non-conserved, amino acids are classified as follows: Group I (hydrophobic side chains): Met, Ala, Val, Leu, Ile; Group II (neutral hydrophilic side chains): Cys, Ser, Thr; Group III (acidic side chains): Asp, Glu; Group IV (basic side chains): Asn, Gln, His, Lys, Arg; Group V (residues affecting chain orientation): Gly, Pro; and Group VI (aromatic side chains): Trp, Tyr, Phe. Conserved substitutions include substitutions between amino acids of the same class. Non-conservative substitutions include the exchange of one member of one of these classes with a member of another class.

[0097] The sequence identity percentage is determined by the antibody sequence that is most alignable according to the Kabat numbering rules. After alignment, if the target antibody region (e.g., the entire maturation variable region of the heavy or light chain) is compared to the same region of the reference antibody, the sequence identity percentage between the target antibody region and the reference antibody region is calculated by dividing the number of positions occupied by the same amino acids in both the target antibody region and the reference antibody region by the total number of aligned positions in the two regions (without counting gaps), and multiplying by 100 to convert it to a percentage.

[0098] Unless otherwise evident from the context, the term "approximately" includes small variations such as values ​​within the standard error of measurement (e.g., SEM) of the stated value.

[0099] The phrase "substantially derived from human immunoglobulin or antibody" means that, when aligned to a human immunoglobulin or antibody amino sequence for comparative purposes, the region has at least 80-90%, preferably 90-95%, more preferably 95-99% identity (i.e., local sequence identity) with the human framework or constant region sequence, allowing for, for example, conservative substitutions, consensus sequence substitutions, germline substitutions, reverse mutations, etc. The introduction of conservative substitutions, consensus sequence substitutions, germline substitutions, reverse mutations, etc., is often referred to as "optimization" of the humanized antibody or chain. The phrase "substantially derived from non-human immunoglobulin or antibody" or "substantially non-human" means that the immunoglobulin or antibody sequence is at least 80-95%, preferably 90-95%, more preferably 96%, 97%, 98%, or 99% identical to that of a non-human organism, e.g., a non-human mammal.

[0100] Therefore, all regions or residues of a humanized immunoglobulin or antibody, or a humanized immunoglobulin or antibody chain, except perhaps the CDR, are substantially identical to the corresponding regions or residues of one or more native human immunoglobulin sequences. The terms “corresponding region” or “corresponding residue” refer to the region or residue on the second amino acid or nucleotide sequence that occupies the same (i.e., equivalent) position as the region or residue on the first amino acid or nucleotide sequence when the first and second sequences are optimally aligned for comparison purposes.

[0101] "Specific binding" of an antibody means that the antibody exhibits clear affinity for the antigen or preferred epitope, preferably without significant cross-reactivity. Clear or preferred binding means at least 10 6 , 10 7 , 10 8 , 10 9 M -1 , or 10 10 M -1 This includes affinity binding. 10 7 M -1 More preferably 0 8 M-1 A higher affinity is more preferable. The midpoint of the values ​​described herein is also intended to be within the scope of this disclosure, and the preferred binding affinity is within the range of affinity, for example, 10 6 ~10 10 M -1 Preferably 10 7 ~10 10 M -1 , more 10 8 ~10 10 M -1 This can be expressed as follows: An antibody that "does not exhibit significant cross-reactivity" is an antibody that does not clearly bind to undesirable elements (e.g., undesirable protein entities). For example, an antibody that specifically binds to Aβ clearly binds to Aβ but does not significantly react with non-Aβ proteins or peptides (e.g., non-Aβ proteins or peptides contained in plaques). An antibody specific to a preferred epitope does not significantly cross-react with, for example, remote epitopes on the same protein or peptide. Specific binding can be measured according to any means recognized in the art for measuring such binding. Preferably, specific binding is determined by scatchard analysis and / or competitive binding assays.

[0102] II. Beta-amyloid Aβ (also referred herein as beta-amyloid peptide and A-beta) peptides are approximately 4 kDa internal fragments of APP consisting of 39–43 amino acids (referred to as Aβ39, Aβ40, Aβ41, Aβ42, and Aβ43, respectively). For example, Aβ40 consists of residues 672–711 of APP, and Aβ42 consists of residues 673–713 of APP. As a result of proteolytic processing of APP by different secretase enzymes in vivo or in situ, Aβ exists in both a short form of 40 amino acids and a long form ranging from 42–43 amino acids. Preferred epitopes or antigenic determinants described herein are located within the N-terminus of the Aβ peptide and include residues within amino acids 1–10 of Aβ, preferably residues 1–3, 1–4, 1–5, 1–6, 1–7, or 3–7 of Aβ42. Additional epitopes or antigenic determinants may include residues 2–4, 5, 6, 7, or 8 of Aβ, residues 3–5, 6, 7, 8, or 9 of Aβ, or residues 4–7, 8, 9, or 10 of Aβ42.

[0103] Unless otherwise evident from the context, references to Aβ or its fragments include native human amino acid sequences, including its isoforms, variants, and allele variants. The ability of an antibody or antigen-binding antibody fragment to bind to Aβ can be determined, for example, using surface plasmon resonance.

[0104] III. Anti-Aβ cell permeable agents This disclosure provides cell permeabilizers (as described herein) comprising an intracellular localization moiety that specifically binds to Aβ (e.g., human Aβ) and an antibody or its antigen-binding fragment. Furthermore, this disclosure relates to cell permeabilizers comprising an intracellular localization moiety and an antibody that specifically binds to human Aβ, compositions comprising these cell permeabilizers, and methods for treating Aβ-related diseases, including inclusion body myositis, using these cell permeabilizers.

[0105] Throughout this disclosure, references to anti-Aβ CPA and / or anti-Aβ antibodies are made in the context of intracellular internalization and / or intracellular function, but those skilled in the art will understand that such references include intact anti-Aβ CPA and / or anti-Aβ antibodies, chemically modified (e.g., oxidized, reduced) anti-Aβ CPA and / or anti-Aβ antibodies, and partially degraded functional fragments thereof (e.g., intact anti-Aβ CPA, CPA fragments, intact anti-Aβ antibodies, and / or anti-Aβ antibody fragments). Accordingly, references to the internalization, cytosolic transport, and target association by either anti-Aβ CPA or anti-Aβ antibody include references to intact anti-Aβ CPA and / or anti-Aβ antibodies, chemically modified anti-Aβ CPA and / or anti-Aβ antibodies, and partially degraded functional fragments thereof. For example, the methods of the present disclosure may refer to the internalization and / or transfer of anti-Aβ antibodies into the cytosol, but such references will be understood to include intact anti-Aβ CPAs and / or functional fragments of anti-Aβ CPAs containing anti-Aβ antibodies or portions thereof, as well as their chemically modified derivatives.

[0106] The cell permeabilizers of this disclosure may utilize one or more of several biochemical processes to achieve internalization of anti-Aβ antibodies. In various embodiments, the anti-Aβ CPAs of this disclosure may utilize passive internalization, active internalization, or a combination thereof. In some embodiments, the anti-Aβ CPAs utilize active internalization (e.g., endocytosis) to achieve intracellular delivery of anti-Aβ antibodies.

[0107] In various embodiments, anti-Aβ CPAs are internalized into cells by endocytosis. After internalization, anti-Aβ CPAs achieve endosomal escape. In such embodiments, anti-Aβ CPAs move into the cytosol, and anti-Aβ antibodies may bind to Aβ (e.g., human Aβ). In some embodiments, endosomal escape of anti-Aβ CPAs is achieved by interaction between a CIM and the endosomal membrane, thereby resulting in the disruption of the endosomal membrane. The CIM may utilize one or more methods to achieve endosomal escape. For example, the CIM may comprise a cationic moiety (e.g., positively charged amino acids, cationic polymers and / or oligomers, cationic lipids). In some such embodiments, the cationic moiety interacts with negatively charged phospholipids comprising the endosomal membrane, thereby disrupting the endosomal membrane and achieving endosomal escape of the anti-Aβ CPA. The anti-Aβ CPAs of this disclosure also comprise a CIM having an amphiphilic moiety (e.g., an amphiphilic peptide). In some such embodiments, the amphiphilic moiety interacts with the endosomal membrane via hydrophobic interactions with membrane lipids, thereby disrupting the endosomal membrane and achieving endosomal exodus of the anti-Aβ CPA. Following endosomal exodus of the anti-Aβ antibody, the anti-Aβ antibody can move into the cytosol, where it can bind to Aβ (e.g., human Aβ). Further examples of internalization mechanisms that can be utilized by the CPA are described in detail herein.

[0108] Accordingly, this specification provides a cell permeabilizer comprising (i) an intracellular integration module (CIM) and (ii) an antibody that specifically binds to Aβ (e.g., human Aβ). Various embodiments of the cell permeabilizers of this disclosure, including examples of the intracellular integration portion, anti-Aβ antibody, and / or optional linker, are described below. Those skilled in the art will understand how to combine the various CIMs, anti-Aβ antibodies, and optional linkers disclosed herein. Such examples are merely illustrative and not limiting to the scope of this disclosure.

[0109] Anti-Aβ antibody The cell permeabilizers and methods of this disclosure facilitate the intracellular integration and / or cytosolic release of a wide range of anti-Aβ antibodies, including cell permeabilizers comprising antibodies or fragments thereof that specifically bind to Aβ peptides. The antibodies or fragments comprise heavy-chain and light-chain CDRs from one of the constructs identified herein as h2726, h2731, h2831, h2931, h2926, h4921, h2828, h2929, h3818G, h2927, h49k3G, h4917G, h2727, and h4918G. Certain monoclonal antibodies of this disclosure may bind to epitopes within residues 1–6 of Aβ (the first N-terminal residue of natural Aβ is indicated as 1). Some monoclonal antibodies bind to epitopes within amino acids 1–6, some bind to epitopes within 1–5, and some bind to epitopes within 1–4. Some antibodies bind to epitopes within amino acids 1-3, 2-5, 3-5, 2-4, 2-5, 2-6, 3-5, or 3-6. When an antibody is said to bind to a specific residue, for example, an epitope within Aβ1-6, it means that the antibody specifically binds to a polypeptide containing that specific residue (i.e., Aβ1-6 in this example), but such an antibody does not necessarily come into contact with all residues within Aβ1-6.

[0110] In another embodiment, the antibody or fragment comprises a heavy chain variable region having heavy chains CDR1, CDR2, and CDR3 derived from the construct shown in Table 1A, and a light chain variable region containing light chains CDR1, CDR2, and CDR3. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0111] In another embodiment, the antibody or fragment of the present disclosure includes a heavy chain variable region (VH), as shown for one of the constructs in Table 1A. The antibody or fragment may also include a light chain variable region (VL), as shown for one of the constructs in Table 1A.

[0112] In one embodiment, the present disclosure relates to an antibody or fragment thereof comprising heavy chain CDR1, CDR2, and CDR3, wherein CDR1 may be selected from any one of SEQ ID NOs: 16, 19, and 20; CDR2 may be selected from any one of SEQ ID NOs: 17, 20, 21, 22, and 23; and CDR3 may be selected from any one of SEQ ID NOs: 18, 24, and 25. Furthermore, the antibody or fragment thereof comprises light chain CDR1, CDR2, and CDR3, wherein CDR1 may be selected from any one of SEQ ID NOs: 26, 29, 31, and 32; CDR2 may be selected from any one of SEQ ID NOs: 27, 33, 34, and 35; and CDR3 may be selected from any one of SEQ ID NOs: 28, 38, and 39. In each of these embodiments, the heavy chain CDR and light chain CDR are not simultaneously SEQ ID NOs: 16, 17, 18, 26, 27, and 28 in combination.

[0113] Analysis of the protein modeling information of the antibodies described above identified two changes in the CDR that were responsible for the increased binding activity / affinity properties of the antibodies disclosed herein: CDR-L1:S32Y (Ser to Tyr at position 32) and CDR-H2:G55S (Gly to Ser at position 54). Anti-Aβ antibodies having Tyr at position 32 of CDR-L1 and Ser at position 54 of CDR-H2 that bind to the same epitope as the antibodies listed herein are expected to have the same properties as the antibodies listed and identified (see Table 1A). The disclosed antibodies that do not have Tyr at position 32 of CDR-L1 and Ser at position 55 of CDR-H2 can be modified to have Tyr at position 32 of CDR-L1 and Ser at position 55 of CDR-H2, and it is expected that this will confer similar binding properties to such antibodies identified herein.

[0114] Examples of CDR-L1 with Tyr at position 32 include sequence numbers 29 and 31. Examples of CDR-H2 with Ser at position 55 include sequence numbers 20 and 21.

[0115] For example, antibodies containing CDR-L1 with Tyr at position 32 and CDR-H2 with Ser at position 55 include antibodies with CDRs h2726, h2731, h2727, h2826, h2831, h2926, h2927, h2931, and h2929 (see Table 1A). Additional such antibodies include antibodies containing LC CDR1, 2, and 3 and HC CDR1, 2, and 3 as listed in Table 1B below. [Table 1-10]

[0116] Considering the binding properties identified for the antibodies identified herein, consensus sequences that are expected to provide similar binding properties can be identified. For example, in embodiments of this disclosure, an antibody or binding fragment that specifically binds to an Aβ peptide may include a heavy chain variable region having heavy chains CDR1, CDR2 and CDR3, and a light chain variable region having light chains CDR1, CDR2 and CDR3, as follows: heavy chain CDR1 is the amino acid sequence GFTFSNX A GMS (in the formula, X A (It contains Y or F) (SEQ ID NO: 88), and the heavy chain CDR2 has the amino acid sequence SX A RSGSGRTYYSDNVKG (where X A It contains (SEQ ID NO: 89) which is I or V, and the heavy chain CDR3 has the amino acid sequence YDHYX A GX B SDY (in the formula, X A is S or T, X B (is S or T) (SEQ ID NO: 90), light chain CDR1 contains amino acid sequence KSSQSLLDYDGKTYLN (SEQ ID NO: 91), light chain CDR2 contains amino acid sequence X A VX B NRDX C (In the formula, X A is K or R, X B is S or T, X C (SEQ ID NO: 92) contains S or T, and the light chain CDR3 has the amino acid sequence WQGTHFPRX A (In the formula, X A (is S or T) (Sequence ID 93)

[0117] In some embodiments, the light chain CDR3 is WQGTHFPRX A FX B (In the formula, X A is S or T, X B (is F or Y) (Sequence ID 94) is included.

[0118] Similar consensus sequences that are expected to provide binding properties similar to those of the antibodies described herein may include a heavy chain variable region having heavy chains CDR1, CDR2 and CDR3, and a light chain variable region having light chains CDR1, CDR2 and CDR3, as follows: heavy chain CDR1 is the amino acid sequence GFTFX A NX B GMS (in the formula, X A is S or A, X B (It is Y or F) (SEQ ID NO: 95), and the heavy chain CDR2 has the amino acid sequence SX A RSG X B X C RTYYSDNVKG(where X A is I or V, X B is S or G, X C (SEQ ID NO: 96) contains S or G, and the heavy chain CDR3 has the amino acid sequence YDHYX A GX B SDY (in the formula, X A is S or T, X B (SEQ ID NO: 90) contains S or T, and the light chain CDR1 has amino acid sequence X A SSQSLX B DX C DGKTYLN(where X A is K or R, X B is V, M, or L, X C (is Y, T, or S) (Sequence ID 97), and the light chain CDR2 contains amino acid sequence X A VX B NRX C X D (In the formula, X A is K or R, X B is S or T, X C is E or D, X D (SEQ ID NO: 98) contains S or T, and the light chain CDR3 has the amino acid sequence WQGX A HFPRX B (In the formula, X A is S or T, X B (is S or T) (Sequence ID 99)

[0119] In some embodiments, the light chain CDR3 is WQGTHFPRX A FX B X C (In the formula, X A is S or T, X B is S or T, X C (is F or Y) (Sequence ID 100) is included.

[0120] Furthermore, the light chain and heavy chain variable regions may be at least 75% identical to the light chain and heavy chain variable regions shown in Table 1A. For example, the light chain and heavy chain variable regions may be 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, or 100% identical to the VH and / or VL sequences identified in Table 1A. In various embodiments, any sequence mutation in the VH and VL may be located outside the CDR, thereby the VH and VL sequences of this disclosure may include the CDR identified in Table 1A, but the regions of the VH and VL sequences outside the CDR may be at least 75% identical to the regions of the VH and VL sequences outside the CDR in Table 1A.

[0121] For example, the antibodies or fragments of the present disclosure may include a heavy chain variable region that is at least 95% identical to one of SEQ ID NOs: 3, 4, 5, 6, and 7, excluding the CDR, and a light chain variable region that is at least 95% identical to one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15, excluding the CDR.

[0122] The antibodies and fragments of this disclosure may also include a heavy chain constant region that is at least 75% identical to SEQ ID NO: 40. For example, the heavy chain constant region may be 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, 99% identical, or 100% identical to SEQ ID NO: 40.

[0123] The antibodies and fragments of this disclosure may also include a light chain constant region that is at least 75% identical to SEQ ID NO: 41. For example, the heavy chain constant region may be 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41.

[0124] Variant antibodies or fragments that are less than 100% identical to the sequences (+ any constant region) listed in Table 1A may differ from the anti-Aβ antibodies in Table 1A by as few as 1-15 amino acid residues, 1-10 amino acid residues, e.g., 6-10, just 5, just 4, 3, 2, or even just 1 amino acid residue. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, for example, mutations may be randomly introduced along all or part of the coding sequence by saturation mutagenesis, and the resulting mutants may be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind to Aβ polypeptides).

[0125] For example, it is possible to introduce mutations only in the framework region of an antibody molecule. The introduced mutations may be silent or neutral missense mutations, i.e., they have little to no effect on the antibody's ability to bind to an antigen. These types of mutations may be useful for optimizing codon use or improving antibody production in hybridomas. Alternatively, non-neutral missense mutations may alter the antibody's ability to bind to an antigen. Those skilled in the art will be able to design and test mutant molecules with desired properties, such as no change in antigen-binding activity or a change in binding activity (e.g., improved antigen-binding activity or a change in antibody specificity). After mutagenesis, the encoded protein can be conventionally expressed, and the functional and / or biological activity of the encoded protein (e.g., the ability to immune-specifically bind to at least one epitope of an Aβ polypeptide) can be determined using the techniques described herein or by conventionally modifying techniques known in the art.

[0126] In each of the embodiments described above, the antibody or fragment of the Disclosure may be a humanized antibody as described herein. For example, the antibody may be a human IgG1 antibody. Furthermore, the antibody may be a complete antibody, a chimeric antibody, a CDR-transplanted antibody, or a recombinant antibody. The antibody fragment may be Fab, Fab', F(ab')2, Fabc, or Fv. The fragment may be produced by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins.

[0127] Other exemplary Aβ antibodies can be used in the cell permeabilizing agents described herein. For example, anti-Aβ antibodies such as aducanumab, lecanumab, bapineuzumab, solanezumab, ganterelumab, crenezumab, donanemab, and ponezumab can be used in the cell permeabilizing agents described herein. Additional Aβ antibodies are available from Novus Biologicals (catalog no. NBP2-13075), Cell Signalling Technology (catalog no. 2454), Santa Cruz Biotechnology (catalog no. sc-28365), LS Bio (catalog no. LS-C186426), abcam (catalog no. ab11132), and Invitrogen (catalog no. MAI-24966).

[0128] Other Aβ antibodies that can be used as cell permeabilisants as described herein include U.S. Patent Nos. 8,491,903, 8,323,654, 8,614,298, 11,655,289, U.S. Patent Publication No. 2022 / 02181963, U.S. Patent Publication No. 2022 / 0372123, and the following references: Karran, E. and Strooper B., The amyloid hypothesis in Alzheimer disease: new insights from new therapeutics. Nat Rev Drug Discov. 21(4):306-318 (2022); Imbimbo, BP, et al., Role of monomeric amyloid-β in cognitive performance in Alzheimer's disease: Insights from clinical trials with secretase inhibitors and monoclonal antibodies. Pharmacol Res. Jan. (2023); Vitek, GE, et al., An Examples include those described in "Anti-beta-amyloid monoclonal antibody for the treatment of Alzheimer's disease," Expert Opin Investig Drugs, 32(2):89-94 (2023), and "Anti-β-amyloid monoclonal antibodies for the treatment of Alzheimer's disease," Panza, F. et al., Immunotherapy, 2(6):767-82 (2010), each of which is incorporated herein by reference in its entirety.

[0129] The antibodies or conjugated fragments, variants, or derivatives disclosed herein are 5 × 10 -2 sec -1 , 10 -2 sec -1 , 5×10 -3 sec -1 or 10-3 sec -1 The following off-rate (k(off)) may be said to bind to Aβ) or its fragment or variant. In certain embodiments, the antibody disclosed herein is 5 × 10 4 sec -1 , 10 -4 sec -1 , 5×10 -5 sec -1 , or 10 -5 sec -1 , 5×10 -6 sec -1 , 10 -6 sec -1 , 5×10 -7 sec -1 or 10 -7 sec -1 It can be said that Aβ or its fragments or variants bind at the following off-rate (k(off)).

[0130] The antibodies or antigen-binding fragments, variants, or derivatives disclosed herein are 10 3 M-1 sec-1, 5×10 3 M-1 sec-1, 10 4 M-1 sec-1 or 5×10 4 It can be said that the antibody of this disclosure binds to the target polypeptide (e.g., Aβ) or a fragment or variant thereof at an on-rate (k(on)) of M-1 sec-1 or higher. In certain embodiments, the antibody of this disclosure is 10 5 M-1 sec-1, 5×10 5 M-1 sec-1, 10 6 M-1 sec-1, or 5×10 6 M-1 sec-1 or 10 7 It can be said that the target polypeptide (e.g., Aβ) or a fragment or variant thereof disclosed herein binds at an on-rate (k(on)) of M-1 sec-1 or higher.

[0131] The anti-Aβ antibodies or their antigen-binding fragments, variants, or derivatives described herein may also be described or specified in terms of their binding affinity to Aβ. Binding affinity is 5 × 10⁻⁶. -2 M, 10 -2 M, 5×10 -3 M, 10 -3 M, 5×10 -4 M, 10 -4 M, 5×10 -5 M, 10 -5 M, 5×10 -6 M, 10 -6 M, 5×10 -7 M, 10 -7 M, 5×10 -8 M, 10 -8 M, 5×10 -9 M, 10 -9 M, 5×10 -10 M, 10 -10 M, 5×10 -11 M, 10 -11 M, 5×10 -12 M, 10 -12 M, 5×10 -13 M, 10 -13 M, 5×10 -14 M, 10 -14 M, 5×10 -15 M, or 10 -15 This may include those with a dissociation constant or Kd of less than M.

[0132] The humanized mature heavy chains of antibodies described herein may contain a lysine residue at their C-terminus. However, in some embodiments, the humanized mature heavy chains (e.g., any of the mature heavy chains described herein) do not contain a C-terminal lysine residue.

[0133] The humanized mature light chain variable domains described herein may contain a lysine residue at their C-terminus. However, in some embodiments, the humanized mature heavy chains (e.g., any of the mature heavy chains described herein) do not contain a C-terminal lysine residue. For example, any one of the mature heavy chains of SEQ ID NO: 349, SEQ ID NO: 351, SEQ ID NO: 353, SEQ ID NO: 355, SEQ ID NO: 357, SEQ ID NO: 359, SEQ ID NO: 361, SEQ ID NO: 363, SEQ ID NO: 365, SEQ ID NO: 367, or SEQ ID NO: 394 may lack a C-terminal lysine residue.

[0134] In some embodiments, the humanized antibody has a melting temperature of about 55°C or higher. In some embodiments, the humanized antibody has a melting temperature of about 56°C or higher, about 57°C or higher, about 58°C or higher, about 59°C or higher, about 60°C or higher, about 61°C or higher, about 62°C or higher, about 63°C or higher, about 64°C or higher, about 65°C or higher, about 66°C or higher, about 67°C or higher, about 68°C or higher, about 69°C or higher, about 70°C or higher, about 71°C or higher, about 72°C or higher, about 73°C or higher, about 74°C or higher, about 75°C or higher, about 76°C or higher, about 77°C or higher, about 78°C or higher, about 79°C or higher, about 80°C or higher, about 81°C or higher, about 82°C or higher, about 83°C or higher, about 84°C or higher, or about 85°C or higher. In some embodiments, the humanized antibody has a melting temperature of at least about 55°C, at least about 56°C, at least about 57°C, at least about 58°C, at least about 59°C, at least about 60°C, at least about 61°C, at least about 62°C, at least about 63°C, at least about 64°C, at least about 65°C, at least about 66°C, at least about 67°C, at least about 68°C, at least about 69°C, at least about 70°C, at least about 71°C, at least about 72°C, at least about 73°C, at least about 74°C, at least about 75°C, at least about 76°C, at least about 77°C, at least about 78°C, at least about 79°C, at least about 80°C, at least about 81°C, at least about 82°C, at least about 83°C, at least about 84°C, or at least about 85°C.In some embodiments, the humanized antibody is suitable for temperatures of approximately 55°C to 85°C, 55°C to 80°C, 55°C to 75°C, 55°C to 70°C, 55°C to 65°C, 55°C to 63°C, 55°C to 61°C, 55°C to 59°C, 55°C to 57°C, 57°C to 85°C, 57°C to 80°C, 57°C to 75°C, 57°C to 70°C, 57°C to 65°C, 57°C to 63°C, 57°C to 61°C, 57°C to 59°C, 59°C to 85°C, 59°C to 80°C, 59°C to 75°C, 59°C to 70°C, 59°C to 65°C, and approximately It has a melting temperature of 59°C to approximately 63°C, approximately 59°C to approximately 61°C, approximately 61°C to approximately 85°C, approximately 61°C to approximately 80°C, approximately 61°C to approximately 75°C, approximately 61°C to approximately 70°C, approximately 61°C to approximately 65°C, approximately 61°C to approximately 63°C, approximately 63°C to approximately 85°C, approximately 63°C to approximately 80°C, approximately 63°C to approximately 75°C, approximately 63°C to approximately 70°C, approximately 63°C to approximately 65°C, approximately 65°C to approximately 85°C, approximately 65°C to approximately 80°C, approximately 65°C to approximately 75°C, approximately 65°C to approximately 70°C, approximately 70°C to approximately 85°C, approximately 70°C to approximately 75°C, approximately 75°C to approximately 85°C, or approximately 80°C to approximately 85°C.

[0135] In some embodiments, the cell permeabilizing antibody is an intact antibody. In some embodiments, the antibody has a human IgG1 isotype.

[0136] In some embodiments, the heavy chain variable domains are fused to the heavy chain steady region (e.g., any of the heavy chain steady regions described herein), and the light chain variable domains are fused to the light chain steady region (e.g., any of the light chain steady regions described herein).

[0137] In some embodiments, the heavy chain constant region is a variant of the native human heavy chain constant region, exhibiting reduced binding to the Fcγ receptor compared to the native heavy chain constant region. In some embodiments, the heavy chain constant region is of the IgG1 isotype. In some embodiments, the antibody has at least one mutation in the constant region. In some embodiments, at least one mutation reduces complement binding or activation by the constant region. In some embodiments, at least one mutation is located at one or more of EU numbering positions 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11). In some embodiments, the antibody has alanine at EU numbering positions 318, 320, and 322.

[0138] In some embodiments, the cell permeability agent antibody or antigen-binding antibody fragment is higher than non-phosphorylated Aβ (e.g., human Aβ) (e.g., at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, at least 300 times, at least 350 times, at least 400 times, at least 450 times, at least 500 times, at least 550 times, at least 600 times, at least 650 times, at least 700 times, at least 750 times, less All are 800 times, at least 850 times, at least 900 times, at least 950 times, at least 1,000 times, at least 1,050 times, at least 1,100 times, at least 1,150 times, at least 1,200 times, at least 1,250 times, at least 1,300 times, at least 1,350 times, at least 1,400 times, at least 1,450 times, or at least 1,500 times higher, or about 10 to about 1,500 times, about 10 to about 1,400 times higher, about 10 to about 1,300 times, about 10 to about 1,200 times, about 10 to about 1,100 times, about 10 to Approx. 1,000x, approx. 10x to approx. 900x, approx. 10x to approx. 800x, approx. 10x to approx. 700x, approx. 10x to approx. 600x, approx. 10x to approx. 500x , about 10 times to about 400 times, about 10 times to about 300 times, about 10 times to about 200 times, about 10 times to about 100 times, about 10 times to about 50 times, about 50 times to about 1 500 times, approximately 50 to 1,400 times higher, approximately 50 to 1,300 times, approximately 50 to 1,200 times, approximately 50 to 1,100 times, approximately 50 to 1,000 times, approximately 50 to 900 times, approximately 50 to 800 times, approximately 50 to 700 times, approximately 50 to 600 times, approximately 50 to 500 Times, approximately 50 to 400 times, approximately 50 to 300 times, approximately 50 to 200 times, approximately 50 to 100 times, approximately 100 to 1,500 times, approximately 100 to 1,400 times higher, approximately 100 to 1,300 times, approximately 100 to 1,200 times, approximately 100 to 1,100 times, approximately 100 to 1 1,000 times, approximately 100 times to approximately 900 times, approximately 100 times to approximately 800 times, approximately 100 times to approximately 700 times, approximately 100 times to approximately 600 times, approximately 100 times to approximately 500 times, about 100 times to about 400 times, about 100 times to about 300 times, about 100 times to about 200 times, about 200 times to about 1,500 times, about 200 times to 1,400 times higher, approximately 200 to 1,300 times, approximately 200 to 1,200 times, approximately 200 to 1,100 times, approximately 200 to 1,000 times, approximately 200 to 900 times, approximately 200 to 800 times, approximately 200 to 700 times, approximately 200 to 600 times, approximately 200 to 500 times, approximately 200 to 400 times, approximately 200 to 300 times, approximately 500 to 1,500 times, approximately 500 to 1,400 times higher, approximately 500 to 1,300 times, approximately 500 to 1,200 times, approximately 500 to 1,100 times, approximately 500 to 1,000 times, approximately 500 to 900 times, It binds to phosphorylated Aβ (e.g., human Aβ) with affinity of approximately 500 to 800 times, 500 to 700 times, 500 to 600 times, 800 to 1,500 times, 800 to 1,400 times higher, 800 to 1,300 times, 800 to 1,200 times, 800 to 1,100 times, 800 to 1,000 times, 800 to 900 times, 1,000 to 1,500 times, 1,000 to 1,400 times higher, 1,000 to 1,300 times, 1,000 to 1,200 times, or 1,000 to 1,100 times higher.

[0139] In some embodiments, the cell permeabilizing antibody or a fragment thereof selectively binds to cytoplasmic aggregates of Aβ (e.g., cytoplasmic aggregates of human Aβ). In some embodiments, the cell permeabilizing antibody or a fragment thereof selectively binds to cytoplasmic aggregates of Aβ (e.g., cytoplasmic aggregates of human Aβ) compared to nuclear Aβ (e.g., nuclear human Aβ).

[0140] The heavy and light chain variable regions present in cell permeabilizers can be linked to at least a portion of the human constant region. The selection of the constant region depends in part on whether antibody conjugate-dependent cell-mediated cytotoxicity, antibody conjugate-dependent phagocytosis, and / or complement-dependent cytotoxicity are desired. For example, human isotypes IgG1 and IgG3 possess complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not. Human IgG1 and IgG3 also induce more potent cell-mediated effector function than human IgG2 and IgG4. The light chain constant region may be lambda or kappa. Examples of constant region numbering rules include EU numbering (Edelman, GM et al., Proc. Natl. Acad. Sci. USA 63:78-85 (1969)), Kabat numbering (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1991), IMGT unique numbering (Lefranc M.-P. et al., IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains, Dev. Comp. Immunol. 29:185-203 (2005)), and IMGT exon numbering (Lefranc, cited above).

[0141] One or more amino acids at the amino or carboxyl termini of the light and / or heavy chains, for example, the C-terminal lysine of the heavy chain, may be omitted in part or all of the molecule, or may be derivatized. Substitutions can be made in the constant region to reduce or increase effector functions such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to extend the half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary substitutions include Gln at position 250 and / or Leu at position 428 (EU numbering is used in this paragraph for the constant region) to increase the half-life of an antibody. Substitutions at any or all of the 234, 235, 236, and / or 237 positions reduce affinity for Fey receptors, particularly FcyRI receptors (see, e.g., U.S. Patent No. 6,624,821). Alanine substitutions at the 234, 235, and 237 positions of human IgG1 can be used to reduce effector function. Some antibodies have alanine substitutions at the 234, 235, and 237 positions of human IgG1 to reduce effector function. Optionally, the 234, 236, and / or 237 positions of human IgG2 are substituted with alanine, and the 235 position is substituted with glutamine (see, e.g., U.S. Patent No. 5,624,821). Some antibodies utilize mutations at one or more positions (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) of the EU numbering of human IgG1, specifically at positions 241, 264, 265, 270, 296, 297, 322, 329, and 331. Some antibodies utilize mutations at one or more positions (e.g., 2 or 3) of the EU numbering of human IgG1, specifically at positions 318, 320, and 322. Some antibodies have positions 234 and / or 235 substituted with alanine, and / or 329 substituted with glycine.In some antibodies, positions 234 and 235 are substituted with alanine. In some antibodies, the isotype is either human IgG2 or human IgG4.

[0142] Antibodies can be expressed as a tetramer containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab', F(ab')2, and Fv, or as a single-chain antibody in which the heavy and light chain maturation variable domains are linked via spacers.

[0143] The human constant region exhibits allotypic and isoallotypic variations between different individuals; that is, the constant region can differ from individual to individual at one or more polymorphic sites. Isoallotypes differ from allotypes in that the serum recognizing the isoallotype binds to the non-polymorphic region of one or more other isotypes. For example, another heavy chain constant region is that of IgG1 Glm3 with or without C-terminal lysine. References to the human constant region include any native allotype or any constant region with any substitution of residues occupying a position in the native allotype.

[0144] Intracellular relocation module As used herein, “internalization module” or “CIM” refers to a composition that, when covalently or noncovalently bound to an anti-Aβ antibody molecule, results in the internalization of at least an anti-Aβ antibody or its active fragment into a cell. Non-limiting features and examples of intracellularization modules are described herein.

[0145] In some embodiments, CIM is a peptide (e.g., an amino acid sequence). In such embodiments, CIM may also be referred to as a cell membrane internalized peptide or "CMIP," as further defined herein. Exemplary cell membrane internalized peptides include naturally occurring peptides and their derivatives, as well as synthetic peptides. Non-limiting examples of CMIPs include M-lycotoxin and its derivatives, TAT and its derivatives, PEPTH, polyarginine sequences, penetratin, DPT-C9h, DPT-C9, transportan, Xentry, Pep-1, Pep-7, Aurein 1.2, MTS, GFWFG, DPV1047, MPG, pVEC, ARF(1_22), BPrPr, MAP, p28, VT5, Bac7, C105Y, PFVYLI, and BR2. In some embodiments, CMIP includes any one of SEQ ID NOs: 101-338.

[0146] In some embodiments, CIM is a non-peptide portion (e.g., a ligand) that is internalized by a cell (e.g., a mammalian cell). In such examples, the ligand can induce receptor-mediated internalization of an anti-Aβ antibody. Generally, ligand internalization is a receptor-mediated endocytosis process in which a cell takes in an extracellular molecule (including a therapeutic agent) when the ligand binds to its homologous receptor protein on the cell surface. Receptor-mediated internalization also includes transcytosis.

[0147] CIM can result in the internalization of anti-Aβ antibodies into mammalian cells. Generally, the process of intracellular internalization is broadly classified as endocytosis. Typically, endocytic pathways can be divided into two larger categories: phagocytosis and phagocytosis. During phagocytosis, the cell membrane absorbs solutes, while during phagocytosis, the cell internalizes much larger vesicles. Phagocytosis is generally subdivided into macropinocytosis, clathrin-dependent endocytosis (e.g., receptor-mediated endocytosis), caveolin-dependent endocytosis, and clathrin / caveolin-independent endocytosis. (See, for example, Marsh, M. Endocytosis, Oxford University Press (2001), Doherty, GJ, and McMahon, HT, Mechanisms of Endocytosis, Annu. Rev. Biochem., 78:31.1-31.46 (2009), and Xu, Y., et al., Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom, Front Biosci, 22:1439-1457 (2017), each of which is incorporated herein by reference in its entirety.)

[0148] Ligand-mediated endocytosis is a mechanism by which cells internalize specific macromolecules. In some cases, the cell membrane (e.g., the plasma membrane) contains clathrin pits that protrude from the cell membrane and form small vesicles called clathrin-coated vesicles. These clathrin-coated vesicles contain receptors and bound macromolecules, i.e., ligands. The clathrin-coated vesicles then fuse with early endosomes (vesicles consisting of tubular extensions located around the cell). Endosomes have an acidic environment (pH 6.0-6.2) that facilitates the dissociation of receptors from ligands. The ingested contents are then sorted to either be recycled back into the plasma membrane or transported to lysosomes for degradation.

[0149] Peptide-based CIMs (e.g., CMIPs) internalize anti-Aβ antibodies through various mechanisms. Generally, CMIPs have been shown to utilize either endocytosis (e.g., energy-dependent internalization) or direct permeation (e.g., migration) (energy-independent internalization) as two main internalization mechanisms. For direct permeation, various mechanisms have been described, including the carpet-like model (membrane destabilization) and the pore formation model (barrel stave). Positively charged CMIPs can interact with negatively charged membrane components such as the phospholipid bilayer, leading to membrane destabilization and subsequent passage of CMIPs and anti-Aβ antibodies through the lipid bilayer. Studies have shown that some CMIPs can induce and shift different uptake mechanisms depending on their concentration, cargo, and / or the cell line used (e.g., Rueska, I. and Zimmer, A., Internalization mechanisms of cell penetting peptides, Beilstein J Nanotechnol, 11:101-123, (2020)).

[0150] Once internalized into the cell, anti-Aβ antibodies typically need to escape the endosomal pathway. Generally, the endocytosis pathway in mammalian cells consists of distinct membrane compartments that internalize molecules (i.e., cell permeability agents) from the plasma membrane and recycle membrane-bound receptors to the surface, or sort the internalized molecules into various degradation pathways. The main components of the endocytosis pathway include the early endosome, the first compartment of the endocytosis pathway. Early endosomes are usually located around the periphery of the cell and receive most types of vesicles arriving from the cell surface. They have a characteristic vesicular tubular structure and a weakly acidic pH. Early endosomes primarily sort organelles, where many endocytized ligands dissociate from their receptors in the acidic pH of the compartment and are recycled to the cell surface. Early endosomes are also sorted into transcytosis pathways to later compartments (e.g., late endosomes or lysosomes) via the transvesicular compartment.

[0151] Late endosomes generally receive endocytized substances via pathways leading to lysosomes, typically from early endosomes in the endocytosis pathway, from the trans-Golgi network (TGN) in the biosynthetic pathway, and from phagosomes in the phagocytic pathway. They are acidic (approximately pH 5.5) and are generally thought to mediate final sorting before delivery of substances to lysosomes. Lysosomes are the final compartment of the endocytosis pathway. Lysosomes break down cellular waste, fats, carbohydrates, proteins, and other macromolecules into simpler compounds, which are then returned to the cytoplasm as new cellular building blocks. Lysosomes contain many different types of hydrolytic enzymes that function in an acidic environment (e.g., pH approximately 4.8).

[0152] In some embodiments, CIM (as defined herein) comprises a cell membrane internalized peptide (e.g., CMIP4, SEQ ID NO: 143). In some embodiments, CIM comprises a wild-type M-lycotoxin peptide. In some embodiments, CIM comprises an M-lycotoxin derivative (e.g., SEQ ID NO: 104). In some embodiments, CIM comprises a penetratin amino acid sequence or a derivative thereof (e.g., SEQ ID NO: 110). In some embodiments, CIM comprises a Pepth amino acid sequence or a derivative thereof (e.g., SEQ ID NO: 107). In some embodiments, CIM comprises a polyarginine amino acid sequence (e.g., SEQ ID NO: 113, SEQ ID NO: 114, and / or SEQ ID NO: 115). In some embodiments, CIM comprises multiple polyarginine amino acid sequences (e.g., two, three, four, five or more polyarginine amino acid sequences). In some embodiments, CIM comprises three polyarginine amino acid sequences. In some embodiments, CIM comprises a TAT amino acid sequence (e.g., SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 109, and / or SEQ ID NO: 117). In some embodiments, CIM comprises a plurality of TAT amino acid sequences or derivatives thereof (e.g., two, three, four, five or more TAT amino acid sequences or derivatives thereof). In some embodiments, CIM comprises three TAT amino acid sequences.

[0153] In some embodiments, CIM includes macrocyclic molecules. Generally, macrocyclic molecules are molecules and ions containing a ring of 12 or more atoms. Classical examples include crown ethers, calixalene, porphyrins, and cyclodextrins. In some embodiments, the macrocyclic molecule is formed by a covalent bond between two amino acid residues of CIM. In some embodiments, the macrocyclic molecule is formed by a disulfide bond between two cysteine ​​residues of CIM.

[0154] In some embodiments, CIM comprises one or more histidine residues.

[0155] In some embodiments, CIM comprises a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 101-338. In some embodiments, CIM is a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 1101-338.

[0156] In some embodiments, the CIM is covalently bound to an antibody or its antigen-binding antibody fragment. In some embodiments, the CIM is non-covalently bound to an antibody or its antigen-binding antibody fragment. In some embodiments, the cell permeabilizer includes a linker that connects the CIM to the antibody. In some embodiments, the linker is covalently bound to both the CIM and the antibody. In some embodiments, the linker is a cleavable linker (e.g., a photocleavable linker, a chemical linker, an enzymatically cleavable linker, etc.). In some embodiments, the linker is an incleavable linker. In some embodiments, the linker comprises a polypeptide. In some embodiments, the linker comprises one or more glycine residues (e.g., two, three, four, five or more glycine residues). In some examples, the linker is a glycine residue followed by a serine residue (GS). In some embodiments, the linker comprises a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs. 343-346. In some embodiments, the linker is a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs. 343-346.

[0157] In some embodiments, the cell permeability agent antibody or antigen-binding antibody fragment is ligated to the C-terminus of the CIM. In some embodiments, the cell permeability agent antibody or antigen-binding antibody fragment is ligated to the N-terminus of the CIM. In some embodiments, the cell permeability agent antibody or antigen-binding antibody fragment is ligated to the C-terminus of the heavy chain of the CIM. In some embodiments, the cell permeability agent antibody or antigen-binding antibody fragment is ligated to the N-terminus of the heavy chain of the CIM. In some embodiments, the cell permeability agent antibody or antigen-binding antibody fragment is ligated to the C-terminus of the light chain of the CIM. In some embodiments, the cell permeability agent antibody or antigen-binding antibody fragment is ligated to the N-terminus of the light chain of the CIM.

[0158] Table 2 below shows a non-exclusive and exemplary list of elements that may be included in any of the CIMs, including M-lycotoxins and their derivatives, TATs and their derivatives, PEPTHs, polyarginine sequences, Pep1, Pep-7, etc. [Table 2-1] [Table 2-2]

[0159] This disclosure further provides an improved CMIP for use, in particular, in connection with the development of novel cell permeability agents.

[0160] In some embodiments, the CIM includes one or more spacer regions. For example, a CIM containing any one of sequence numbers 108, 109, and 111-115 includes one or more spacer regions. In some embodiments, the CIM does not include spacer regions. For example, a CIM containing polypeptide amino acid sequences selected from 101-107, 110, and 116-338 does not include spacer regions.

[0161] As used herein, a spacer refers to an amino acid that does not possess catalytic or therapeutic activity in mammalian cells. For example, a spacer may be a peptide of 1 to about 10 amino acids (e.g., 1 to about 8 amino acids, 1 to about 6 amino acids, or 1 to about 4 amino acids). In some examples, the spacer region is contained within an intracellular module sequence. For example, a spacer region can isolate amino acids within an intracellular module (CIM), and for example, the spacer may be located after the first amino acid of the CIM. In some examples, the spacer may be located before the last amino acid of the CIM. In some examples, a CIM may have one or more spacer regions (e.g., two spacer regions, three space sequences, four spacer regions, five spacer regions, or more). In some examples, the spacer region is a single glycine residue. In some examples, the spacer region is a pair of glycine residues. In some examples, the spacer region is three glycine residues. In some examples, the spacer region is four glycine residues. In some examples, the spacer region consists of four glycine residues followed by a serine residue. In some embodiments, at least one of the one or more spacer regions contains an amino acid sequence selected from any one of SEQ ID NOs. 339-342. In some embodiments, each of the one or more spacer regions contains an amino acid sequence selected from any one of SEQ ID NOs. 339-3342.

[0162] In some embodiments, the CIM includes spacers. In some embodiments, the spacers are the spacers shown in Table 3. [Table 3]

[0163] This disclosure further provides CMIP for use, in particular, in connection with the development of novel cell permeability agents. In some embodiments, the intracellular integration effect of CMIP can be improved by designing CMIP having an α-helix structure.

[0164] In another aspect, this disclosure relates to formulas (IA) to (ID): IX 2 -X 3 -TALX 7 -FX 9 -GX 11 -AAX 14 -KX 16 -EAX 19 -QFLX 23 -X 24 -L(IA)(Sequence ID 395), IWX 3 -X 4 -ALX 7 -FX 9 -GX 11 -X 12 -X 13 -AX 15 -AEAX 19 -X 20 -FX 22 -SX 24 -L(IB)(Sequence ID 396), X 1 -X 2 -LTX 5 -LKX 8 -SX 10 -KAAAX 15 -AEAKQX 21 -LSX 24 -L(IC)(Sequence ID 397), IWLTX 5 -X 6 -KFSX 10 -KX 12 -AAKAX 17 -X 18 -KQFLX 23 -X 24 -X 25 A polypeptide of the expression selected from (ID)(Sequence ID 395), During the ceremony, X 1 However, these are hydrophobic amino acid residues or positively charged amino acid residues. X 2However, these are aromatic amino acid residues, positively charged amino acid residues, or negatively charged amino acid residues. X 3 However, these are aromatic amino acid residues, hydrophobic amino acid residues, positively charged amino acid residues, or negatively charged amino acid residues. X 4 However, it is a neutral hydrophilic amino acid residue or an aromatic amino acid residue. X 5 However, it is a hydrophobic amino acid residue or an aromatic amino acid residue, X 6 However, it is a hydrophobic amino acid residue, X 7 However, these are hydrophobic amino acid residues or positively charged amino acids. X 8 However, it is an aromatic amino acid residue, X 9 However, these are neutral hydrophilic amino acid residues, aromatic amino acid residues, or positively charged amino acid residues. X 10 However, these are neutral hydrophilic amino acid residues, aromatic amino acid residues, or positively charged amino acid residues. X 11 However, these are neutral hydrophilic amino acid residues, hydrophobic amino acid residues, or positively charged amino acid residues. X 12 However, these are neutral hydrophilic amino acid residues, hydrophobic amino acid residues, or positively charged amino acid residues. X 13 However, it is a neutral hydrophilic amino acid residue or a hydrophobic amino acid residue. X 14 However, these are hydrophobic amino acid residues or positively charged amino acids. X 15 However, these are aromatic amino acid residues, positively charged amino acid residues, or negatively charged amino acid residues. X 16 However, these are hydrophobic amino acid residues, aromatic amino acid residues, positively charged amino acid residues, or negatively charged amino acid residues. X 17 However, these are positively charged amino acid residues or negatively charged amino acid residues. X18 However, these are hydrophobic amino acid residues, aromatic amino acid residues, or positively charged amino acid residues. X 19 However, these are hydrophobic amino acid residues or positively charged amino acid residues. X 20 However, these are neutral hydrophilic amino acid residues or negatively charged amino acid residues. X 21 However, it is a hydrophobic amino acid residue or an aromatic amino acid residue, X 22 However, it is absent, hydrophobic amino acid residue, aromatic amino acid residue, or positively charged amino acid residue. X 23 However, it is absent, a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively charged amino acid residue. X 24 However, it is absent, an aromatic amino acid residue, or a positively charged amino acid residue. X 25 The present invention provides polypeptides, or pharmaceutically acceptable salts thereof, that are absent or hydrophobic amino acids.

[0165] In some embodiments, CMIP is given by formula (IA): IX 2 -X 3 -TALX 7 -FX 9 -GX 11 -AAX 14 -KX 16 -EAX 19 -QFLX 23 -X 24 -L(IA)(Sequence ID 395) or a pharmaceutically acceptable salt thereof.

[0166] In some embodiments, CMIP is given by formula (IB): IWX 3 -X 4 -ALX 7 -FX 9 -GX 11 -X 12 -X 13 -AX15 -AEAX 19 -X 20 -FX 22 -SX 24 -L(IB)(Sequence ID 396) or a pharmaceutically acceptable salt thereof.

[0167] In some embodiments, CMIP is given by formula (IC): X 1 -X 2 -LTX 5 -LKX 8 -SX 10 -KAAAX 15 -AEAKQX 21 -LSX 24 -L(IC)(Sequence ID 397) or a pharmaceutically acceptable salt thereof.

[0168] In some embodiments, CMIP is expressed by formula (ID): IWLTX 5 -X 6 -KFSX 10 -KX 12 -AAKAX 17 -X 18 -KQFLX 23 -X 24 -X 25 (ID)(Sequence ID 398) or a pharmaceutically acceptable salt thereof.

[0169] In some embodiments, CMIP essentially consists of an amino acid sequence selected from formulas (IA) to (ID). In some embodiments, CMIP essentially consists of the amino acid sequence of formula (IA). In some embodiments, CMIP essentially consists of the amino acid sequence of formula (IB). In some embodiments, CMIP essentially consists of the amino acid sequence of formula (IC). In some embodiments, CMIP essentially consists of the amino acid sequence of formula (ID).

[0170] In some embodiments, CMIP consists of an amino acid sequence selected from formulas (IA) to (ID). In some embodiments, CMIP consists of the amino acid sequence of formula (IA). In some embodiments, CMIP consists of the amino acid sequence of formula (IB). In some embodiments, CMIP consists of the amino acid sequence of formula (IC). In some embodiments, CMIP consists of the amino acid sequence of formula (ID).

[0171] In some embodiments, for CMIPs of formulas (IA), (IB), (IC), and (ID), the polypeptide comprises at least three positively charged amino acid residues, each of which is X 7 , X 11 , X 15 , X 19 , or X 24 It occupies the position corresponding to X. In some embodiments, the polypeptide comprises at least four positively charged amino acid residues, each of which is X 7 , X 11 , X 15 , X 19 , or X 24 It occupies the position corresponding to X. In some embodiments, the polypeptide comprises five positively charged amino acid residues, each of which is X 7 , X 11 , X 15 , X 19 , or X 24 It occupies the corresponding position.

[0172] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), the polypeptide comprises at least four hydrophobic amino acid residues, each of which is X 3 , X 5 , X 6 , X 8 , X 13 , X 14 , X 16 , X 18 , or X 22 It occupies the corresponding position. In some embodiments, the polypeptide comprises at least five hydrophobic amino acid residues, each of which is X 3 , X5 , X 6 , X 8 , X 13 , X 14 , X 16 , X 18 , or X 22 It occupies the position corresponding to X. In some embodiments, the polypeptide comprises at least six hydrophobic amino acid residues, each of which is X 3 , X 5 , X 6 , X 8 , X 13 , X 14 , X 16 , X 18 , or X 22 It occupies the corresponding position. In some embodiments, the polypeptide comprises at least seven hydrophobic amino acid residues, each of which is X 3 , X 5 , X 6 , X 8 , X 13 , X 14 , X 16 , X 18 , or X 22 It occupies the position corresponding to X. In some embodiments, the polypeptide comprises at least eight hydrophobic amino acid residues, each of which is X 3 , X 5 , X 6 , X 8 , X 13 , X 14 , X 16 , X 18 , or X 22 It occupies the position corresponding to X. In some embodiments, the polypeptide comprises nine hydrophobic amino acid residues, each of which is X 3 , X 5 , X 6 , X 8 , X 13 , X 14 , X 16 , X 18 , or X 22 It occupies the corresponding position.

[0173] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X1 X is a hydrophobic amino acid residue or a positively charged amino acid residue. In some embodiments, X 1 X is a hydrophobic amino acid residue. In some embodiments, X 1 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 1 X is an amino acid residue selected from alanine, arginine, aspartic acid, isoleucine, and lysine. In some embodiments, X 1 isoleucine or lysine. In some embodiments, X 1 It is isoleucine.

[0174] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 2 X is an aromatic amino acid residue, a positively charged amino acid residue, or a negatively charged amino acid residue. In some embodiments, X 2 X is an aromatic amino acid residue. In some embodiments, X 2 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 2 X is an amino acid residue selected from alanine, arginine, aspartic acid, phenylalanine, tryptophan, and tyrosine. In some embodiments, X 2 X is arginine, aspartic acid, phenylalanine, tryptophan, or tyrosine. In some embodiments, X 2 It is tryptophan.

[0175] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 3 X is an aromatic amino acid residue, a hydrophobic amino acid residue, a positively charged amino acid residue, or a negatively charged amino acid residue. In some embodiments, X 3 X is a hydrophobic amino acid residue. In some embodiments, X 3 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 3 X is an amino acid residue selected from alanine, arginine, glutamic acid, leucine, lysine, phenylalanine, and tryptophan. In some embodiments, arginine, glutamic acid, leucine, phenylalanine, or tryptophan. In some embodiments, X 3 It is leucine.

[0176] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 4 X is a neutral hydrophilic amino acid residue or an aromatic amino acid residue. In some embodiments, X 4 X is a neutral hydrophilic amino acid residue. In some embodiments, X 4 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 4 X is an amino acid residue selected from alanine, histidine, phenylalanine, threonine, and tryptophan. In some embodiments, X 4 X is threonine or tryptophan. In some embodiments, X 4 It is threonine.

[0177] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 5 X is a hydrophobic amino acid residue or an aromatic amino acid residue. In some embodiments, X 5 X is a hydrophobic amino acid residue. In some embodiments, X 5 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 5 X is an amino acid residue selected from alanine, arginine, histidine, phenylalanine, and tryptophan. In some embodiments, X 5 X is alanine or phenylalanine. In some embodiments, X 5 It is alanine.

[0178] In some embodiments, X 5 is histidine. In some embodiments, X 5 is histidine, and X 3 X is an amino acid residue selected from alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 5 is histidine, and X 3 This is an amino acid residue selected from alanine, glutamic acid, leucine, lysine, phenylalanine, and tryptophan.

[0179] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 6 X is a hydrophobic amino acid residue. In some embodiments, X 6X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 6 X is an amino acid residue selected from alanine, arginine, glutamic acid, isoleucine, and leucine. In some embodiments, X 6 It is leucine.

[0180] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 7 X is a hydrophobic amino acid residue or a positively charged amino acid. In some embodiments, X 7 X is a positively charged amino acid. In some embodiments, X 7 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 7 X is an amino acid residue selected from alanine, arginine, aspartic acid, isoleucine, leucine, lysine, and phenylalanine. In some embodiments, X 7 X is arginine, isoleucine, leucine, or lysine. In some embodiments, X 7 It is lysine.

[0181] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 8 X is an aromatic amino acid residue. In some embodiments, X 8X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 8 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, glutamine, histidine, lysine, phenylalanine, serine, and tryptophan. In some embodiments, X 8 It is phenylalanine.

[0182] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 9 X is a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively charged amino acid residue. In some embodiments, X 9 X is a neutral hydrophilic amino acid residue. In some embodiments, X 9 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 9 X is an amino acid residue selected from alanine, arginine, histidine, leucine, phenylalanine, serine, and tryptophan. In some embodiments, X 9 X is arginine, histidine, phenylalanine, or serine. In some embodiments, X 9 It is serine.

[0183] In some embodiments, X 9 It is serine, and X 12 is not histidine. In some embodiments, X 9 It is serine, and X 12X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 9 It is serine, and X 12 This is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, phenylalanine, and tryptophan.

[0184] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 10 X is a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively charged amino acid residue. In some embodiments, X 10 X is a neutral hydrophilic amino acid residue. In some embodiments, X 10 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 10 X is an amino acid residue selected from alanine, arginine, glycine, tryptophan, and tyrosine. In some embodiments, X 10 X is arginine, glycine, or tyrosine. In some embodiments, X 10 It is glycine.

[0185] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 11 X is a neutral hydrophilic amino acid residue, a hydrophobic amino acid residue, or a positively charged amino acid residue. In some embodiments, X 11 X is a positively charged amino acid residue. In some embodiments, X 11X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 11 X is an amino acid residue selected from alanine, arginine, asparagine, glutamic acid, isoleucine, lysine, threonine, and tryptophan. In some embodiments, X 11 X is arginine, isoleucine, lysine, or threonine. In some embodiments, X 11 It is lysine.

[0186] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 12 X is a neutral hydrophilic amino acid residue, a hydrophobic amino acid residue, or a positively charged amino acid residue. In some embodiments, X 12 X is a hydrophobic amino acid residue. In some embodiments, X 12 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 12 X is an amino acid residue selected from alanine, glutamic acid, glutamine, histidine, phenylalanine, and tryptophan. In some embodiments, X 12 X is alanine or glutamine. In some embodiments, X 12 It is alanine.

[0187] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 13 X is a neutral hydrophilic amino acid residue or a hydrophobic amino acid residue. In some embodiments, X 13X is a hydrophobic amino acid residue. In some embodiments, X 13 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 13 X is an amino acid residue selected from alanine, arginine, aspartic acid, glutamic acid, glycine, leucine, phenylalanine, serine, and tryptophan. In some embodiments, X 13 X is alanine or serine. In some embodiments, X 13 It is alanine.

[0188] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 14 X is a hydrophobic amino acid residue or a positively charged amino acid. In some embodiments, X 14 X is a hydrophobic amino acid residue. In some embodiments, X 14 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 14 X is an amino acid residue selected from alanine, arginine, glutamic acid, histidine, isoleucine, leucine, phenylalanine, tryptophan, tyrosine, and valine. In some embodiments, X 14 X is alanine, histidine, leucine, or valine. In some embodiments, X 14 It is alanine.

[0189] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 15X is an aromatic amino acid residue, a positively charged amino acid residue, or a negatively charged amino acid residue. In some embodiments, X 15 X is a positively charged amino acid residue. In some embodiments, X 15 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 15 X is an amino acid residue selected from alanine, arginine, glutamic acid, lysine, threonine, and tryptophan. In some embodiments, X 15 X is arginine, glutamic acid, or lysine. In some embodiments, X 15 It is lysine.

[0190] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 16 X is a hydrophobic amino acid residue, an aromatic amino acid residue, a positively charged amino acid residue, or a negatively charged amino acid residue. In some embodiments, X 16 X is a hydrophobic amino acid residue. In some embodiments, X 16 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 16 X is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, histidine, lysine, serine, and tryptophan. In some embodiments, X 16 is not histidine. In some embodiments, X 16X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 16 X is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, lysine, serine, and tryptophan. In some embodiments, X 16 X is alanine, glutamic acid, or lysine. In some embodiments, X 16 It is alanine.

[0191] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 17 X is a positively charged amino acid residue or a negatively charged amino acid residue. In some embodiments, X 17 X is a negatively charged amino acid residue. In some embodiments, X 17 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 17 X is an amino acid residue selected from alanine, arginine, aspartic acid, glutamic acid, leucine, phenylalanine, and tryptophan. In some embodiments, X 17 X is arginine or glutamic acid. In some embodiments, X 17 It is glutamic acid.

[0192] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 18 X is a hydrophobic amino acid residue, an aromatic amino acid residue, or a positively charged amino acid residue. In some embodiments, X 18X is a hydrophobic amino acid residue. In some embodiments, X 18 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 18 X is an amino acid residue selected from alanine, arginine, glutamic acid, leucine, methionine, and tryptophan. In some embodiments, X 18 X is alanine, glutamic acid, methionine, or tryptophan. In some embodiments, X 18 It is alanine.

[0193] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 19 X is a hydrophobic amino acid residue or a positively charged amino acid residue. In some embodiments, X 19 X is a positively charged amino acid residue. In some embodiments, X 19 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 19 X is an amino acid residue selected from alanine, arginine, leucine, lysine, threonine, and tryptophan. In some embodiments, X 19 X is arginine, leucine, or lysine. In some embodiments, X 19 It is lysine.

[0194] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 20 X is a neutral hydrophilic amino acid residue or a negatively charged amino acid residue. In some embodiments, X20 X is a neutral hydrophilic amino acid residue. In some embodiments, X 20 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 20 X is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, methionine, serine, and tryptophan. In some embodiments, X 20 is glutamic acid or glutamine. In some embodiments, X 20 It is glutamine.

[0195] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 21 X is a hydrophobic amino acid residue or an aromatic amino acid residue. In some embodiments, X 21 X is an aromatic amino acid residue. In some embodiments, X 21 X is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 21 X is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, lysine, phenylalanine, and tryptophan. In some embodiments, X 21 X is an amino acid residue selected from alanine, arginine, lysine, phenylalanine, and tryptophan. In some embodiments, X 21 X is arginine, phenylalanine, or tryptophan. In some embodiments, X 21 It is phenylalanine.

[0196] In some embodiments, CMIP of formulas (IA), (IB), (IC), and (ID) is absent, hydrophobic amino acid residue, aromatic amino acid residue, or positively charged amino acid residue. In some embodiments, X 22 X is a hydrophobic amino acid residue. In some embodiments, X 22 X is either absent or one of the amino acid residues selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 22 X is either absent or one of the amino acid residues selected from alanine, arginine, aspartic acid, leucine, and lysine. In some embodiments, X 22 X is either absent or leucine. In some embodiments, X 22 X is absent. In some embodiments, X 22 X is arginine, leucine, or tryptophan. In some embodiments, X 22 It is leucine.

[0197] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 23 X is absent, a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively charged amino acid residue. In some embodiments, X 23 X is a neutral hydrophilic amino acid residue. In some embodiments, X 23 X is either absent or one of the amino acid residues selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 23X is either absent or one of the amino acid residues selected from alanine, arginine, aspartic acid, glutamic acid, leucine, lysine, threonine, tryptophan, and valine. In some embodiments, X 23 X is either absent or serine. In some embodiments, X 23 X is absent. In some embodiments, X 23 X is either absent or is arginine, methionine, serine, or tryptophan. In some embodiments, X 23 It is serine.

[0198] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 24 X is absent, an aromatic amino acid residue, or a positively charged amino acid residue. In some embodiments, X 24 X is a positively charged amino acid residue. In some embodiments, X 24 X is either absent or one of the amino acid residues selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 24 X is either absent or one of the amino acid residues selected from alanine, arginine, and lysine. In some embodiments, X 24 X is either absent or lysine. In some embodiments, X 24 X is absent. In some embodiments, X 24 X is either absent or is arginine, lysine, or tryptophan. In some embodiments, X 24 It is lysine.

[0199] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 25is an absent or hydrophobic amino acid. In some embodiments, X 25 X is a hydrophobic amino acid. In some embodiments, X 25 X is either absent or an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X 25 X is either absent or one of the amino acid residues selected from alanine, arginine, leucine, and lysine. In some embodiments, X 25 X is either absent or leucine. In some embodiments, X 25 X is absent. In some embodiments, X 25 It is leucine.

[0200] In some embodiments, for CMIP of formulas (IA), (IB), (IC), and (ID), X 22 , X 23 , X 24 , and X 25 They are all absent.

[0201] In another aspect, this disclosure relates to formulas (IIA) to (IIC) (Sequence ID 395: IX 2 -X 3 -TALX 7 -FX 9 -GX 11 -AAX 14 -KX 16 -EAX 19 -QFLX 23 -X 24 -L(IIA), IWX 3 -X 4 -ALX 7 -FX 9 -GX 11 -X 12 -X 13-AX 15 -AEAX 19 -QFX 22 -SX 24 -L(IIB), X 1 -X 2 -LTALKFSX 10 -KAAAX 15 -AEAKQX 21 -LSX 24 CMIP containing an amino acid sequence having a formula selected from -L(IIC), or a pharmaceutically acceptable salt thereof, wherein X 1 However, it is isoleucine or lysine, X 2 However, it is either arginine or tryptophan, X 3 However, it is arginine, glutamic acid, leucine, or tryptophan, X 4 However, it is threonine or tryptophan, X 7 However, it is arginine, isoleucine, or lysine, X 9 However, it is arginine, histidine, or serine, X 10 However, it is glycine or tyrosine, X 11 However, X is arginine, isoleucine, lysine, or threonine. 12 However, it is alanine or glutamine, X 13 However, it is alanine or serine, X 14 However, it is alanine, histidine, leucine, or valine, X 15 However, it is arginine, glutamic acid, or lysine, X 16 However, it is alanine or lysine, X 18 However, it is alanine, methionine, or tryptophan, X 19 However, it is arginine or lysine, X 21 However, it is either arginine or phenylalanine, X 22 However, it is arginine, leucine, or tryptophan, X 23 However, it is arginine or serine, X 24The present invention provides CMIP, or a pharmaceutically acceptable salt thereof, which is arginine, lysine, or tryptophan.

[0202] In some embodiments, CMIP comprises the amino acid sequence of formula (IIA). In some embodiments, CMIP comprises the amino acid sequence of formula (IIB). In some embodiments, CMIP comprises the amino acid sequence of formula (IIC).

[0203] In some embodiments, CMIP consists of an amino acid sequence selected from formulas (IIA) to (IIC). In some embodiments, CMIP consists of the amino acid sequence of formula (IIA). In some embodiments, CMIP consists of the amino acid sequence of formula (IIB). In some embodiments, CMIP consists of the amino acid sequence of formula (IIC).

[0204] In some embodiments, CMIP consists of an amino acid sequence selected from formulas (IIA) to (IIC). In some embodiments, CMIP consists of the amino acid sequence of formula (IIA). In some embodiments, CMIP consists of the amino acid sequence of formula (IIB). In some embodiments, CMIP consists of the amino acid sequence of formula (IIC).

[0205] In some embodiments, for CMIP of formulas (IIA), (IIB), and (IIC), X 1 isoleucine. In some embodiments, X 2 X is tryptophan. In some embodiments, X 3 is leucine. In some embodiments, X 4 X is threonine. In some embodiments, X 7 is lysine. In some embodiments, X 9 is serine. In some embodiments, X 10 is glycine. In some embodiments, X 11 is lysine. In some embodiments, X 12 is alanine. In some embodiments, X13 is alanine. In some embodiments, X 14 is alanine. In some embodiments, X 15 is lysine. In some embodiments, X 16 is alanine. In some embodiments, X 18 is alanine. In some embodiments, X 19 is lysine. In some embodiments, X 21 X is phenylalanine. In some embodiments, X 22 is leucine. In some embodiments, X 23 is serine. In some embodiments, X 24 It is lysine.

[0206] Table 4 below provides a non-limiting and exemplary list of CMIPs of this disclosure. In some embodiments, the CMIP is selected from the CMIPs in Table 4. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0207] In some embodiments, the CMIPs of this disclosure offer the additional benefit of reduced predicted immunogenicity compared to other cell-permeable peptides. One method for calculating the immunogenicity of a CMIP sequence is to assign an immunogenicity unit (IU) to each sequence, as described in Example 2 below. In some embodiments, CMIP has an immunogenicity value of 2.0 IU or less (e.g., 1.8 IU or less, 1.6 IU or less, 1.4 IU or less, 1.2 IU or less, or 1.0 IU or less). In some embodiments, CMIP has an immunogenicity value of 1.6 IU or less. In some embodiments, CMIP has an immunogenicity value of 0 to 2.0 IU (e.g., 0 to 1.8 IU, 0 to 1.6 IU, 0 to 1.4 IU, 0 to 1.2 IU, 0 to 1.0 IU, 0 to 0.8 IU, 0 to 0.6 IU, 0 to 0.4 IU, 0 to 0.2 IU). In some embodiments, CMIP has an immunogenicity value of 0 to 1.6 IU.

[0208] Linker In various embodiments of this disclosure, the anti-Aβ cell permeabilizer further comprises a linker that connects the CIM to an anti-Aβ antibody. The linker of this disclosure may connect the CIM to the anti-Aβ antibody via either covalent or non-covalent bonding. In some embodiments, the CIM is covalently bound to the anti-Aβ antibody via a linker (i.e., a covalent linker). In some embodiments, the CIM is non-covalently bound to the anti-Aβ antibody via a linker (i.e., a non-covalent linker). In some embodiments, the CIM is covalently bound to the linker. In some embodiments, the anti-Aβ antibody is covalently bound to the linker. In some embodiments, the linker is covalently bound to both the CPP and the anti-Aβ antibody.

[0209] In some embodiments, the linker is a non-covalent linker. Non-covalent bonding can be achieved using affinity pairs that strongly interact in a non-covalent manner (e.g., by hydrogen bonds, ionic bonds, van der Waals interactions, or any combination thereof). Numerous examples of non-covalent bonding are known in the art. For example, biotin and a biotin binder (e.g., streptavidin) are an example of an affinity pair. For example, non-covalent bonding can be achieved between CIM and an anti-Aβ antibody by attaching biotin to one side of CPA (e.g., CIM) and the biotin binder to the other side of CPA (e.g., an anti-Aβ antibody). In some embodiments, the linker includes a pair of affinity domains (e.g., the first domain of the pair of affinity domains may be interleukin 15, and the second domain of the pair of affinity domains may be the sushi domain of the interleukin 15 receptor alpha).

[0210] In some embodiments, the linker is a covalent linker. Many covalent linkers are known in the art. For example, in some embodiments, the covalent linker comprises an organic linker (e.g., an alkylene chain, a polyethylene glycol chain, a polyacrylamide, a polyacrylic acid, a polyvinyl alcohol, or a polyethyleneimine chain). In some cases, the covalent linker comprises an unsubstituted or substituted alkylene chain (e.g., a polyvinyl alcohol chain, a polyacrylamide chain, or a polyacrylic acid chain). In some cases, the covalent linker comprises an unsubstituted or substituted heteroalkylene chain (e.g., a polyethylene glycol chain or a polyethyleneimine chain). In various embodiments, the linker is a linear linker or a branched linker. In some such embodiments, the branched linker allows for the incorporation of two or more CIMs and / or anti-Aβ antibodies into a CPA (e.g., a dendrimer linker structure).

[0211] In some embodiments, the linker comprises amino acid residues. In some embodiments, the linker comprises polypeptides. In some exemplary embodiments, the linker may be a peptide of about 1 to about 50 amino acids (e.g., about 1 to about 40 amino acids, or about 1 to about 30 amino acids). In some exemplary embodiments, the linker may be a peptide of about 1 to about 25 amino acids (e.g., about 1 to about 20 amino acids, or about 1 to about 12 amino acids). In some exemplary embodiments, the linker may be a peptide of about 1 to about 10 amino acids (e.g., about 1 to about 6 amino acids, or about 1 to about 7 amino acids). In some exemplary embodiments, the linker may be a peptide of about 1 to about 5 amino acids (e.g., about 1 to about 4 amino acids, or about 1 to about 3 amino acids). In some exemplary embodiments, the linker may be a peptide of about 3 to about 20 amino acids (e.g., about 3 to about 15 amino acids, or about 3 to about 12 amino acids). In some exemplary embodiments, the linker may be a peptide of about 3 to about 10 amino acids (e.g., about 3 to about 8 amino acids, or about 3 to about 6 amino acids).

[0212] In some embodiments, the linker includes a glycine residue. In some embodiments, the linker includes two or more glycine residues. In some embodiments, the linker includes two or more consecutive glycine residues (e.g., 2-3 consecutive glycine residues, 2-4 consecutive glycine residues, 2-5 consecutive glycine residues, or 2-6 consecutive glycine residues). In some embodiments, the linker includes 2, 3, 4, 5, or 6 consecutive glycine residues. In some embodiments, the linker includes a serine residue. In some embodiments, the linker includes an amino acid sequence selected from GS, GGG, GGGGS, and GGGGSGGGGS. In some embodiments, the linker includes the amino acid sequence GGGGS.

[0213] In some embodiments, the CIM includes a linker. In some embodiments, the linker is one of the linkers shown in Table 5. [Table 5]

[0214] Exemplary anti-Aβ cell permeable agents Various components of cell permeabilizers, including anti-Aβ antibodies, linkers, and intracellular localization portions (e.g., CMIP), are disclosed herein. Those skilled in the art will understand how these features are combined to yield functional anti-Aβ cell permeabilizers. Cell permeabilizers having CMIP expressed as a single polypeptide (i.e., a fusion protein), an optional linker, and the light and / or heavy chains of an anti-Aβ antibody are also described herein.

[0215] In some embodiments, the anti-Aβ CPA comprises a polypeptide that is at least 95% identical to a sequence selected from any one of SEQ ID NOs: 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, and 394. In some embodiments, the anti-Aβ CPA comprises a polypeptide that is at least 98% identical to a sequence selected from any one of SEQ ID NOs: 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, and 394. In some embodiments, the anti-Aβ CPA comprises a polypeptide sequence selected from any one of SEQ ID NOs: 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, and 394.

[0216] In some embodiments, the anti-Aβ CPA comprises a polypeptide that is at least 95% identical to a sequence selected from any one of SEQ ID NOs: 350, 352, 354, 356, 358, 360, 362, 364, 366, and 368-393. In some embodiments, the anti-Aβ CPA comprises a polypeptide that is at least 98% identical to a sequence selected from any one of SEQ ID NOs: 350, 352, 354, 356, 358, 360, 362, 364, 366, and 368-393. In some embodiments, the anti-Aβ CPA comprises a polypeptide sequence selected from any one of SEQ ID NOs: 350, 352, 354, 356, 358, 360, 362, 364, 366, and 368-393.

[0217] In some embodiments, the anti-Aβ CPA comprises a first polypeptide and a second polypeptide, further comprising, where the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 349 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 350; or the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 351 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 352; or the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 353 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 354; or the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 355 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 356; or the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 357 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 358; or the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 359 The first polypeptide contains an identical sequence, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 360; the first polypeptide contains a sequence that is at least 95% identical to sequence number 361, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 362; the first polypeptide contains a sequence that is at least 95% identical to sequence number 363, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 364; the first polypeptide contains a sequence that is at least 95% identical to sequence number 365, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 366; the first polypeptide contains a sequence that is at least 95% identical to sequence number 367, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 368; or the first polypeptide contains a sequence that is at least 95% identical to sequence number 394, and the second polypeptide contains a sequence that is at least 95% identical to any one of sequence numbers 369 to 393.

[0218] In some embodiments, anti-Aβ CPA comprises a first polypeptide and a second polypeptide, further comprising, where the first polypeptide comprises SEQ ID NO: 349 and the second polypeptide comprises SEQ ID NO: 350, or the first polypeptide comprises SEQ ID NO: 351 and the second polypeptide comprises SEQ ID NO: 352, or the first polypeptide comprises SEQ ID NO: 353 and the second polypeptide comprises SEQ ID NO: 354, or the first polypeptide comprises SEQ ID NO: 355 and the second polypeptide comprises SEQ ID NO: 356, or the first polypeptide comprises SEQ ID NO: 357 and the second polypeptide comprises SEQ ID NO: 358, or the first polypeptide comprises SEQ ID NO: 359 and the second polypeptide comprises SEQ ID NO: 360, or the first polypeptide comprises SEQ ID NO: 361 and the second polypeptide comprises SEQ ID NO: 362, or the first polypeptide comprises SEQ ID NO: 363 and the second polypeptide comprises SEQ ID NO: 364, or the first polypeptide comprises SEQ ID NO: 365 and the second polypeptide comprises SEQ ID NO: 366. The first polypeptide contains SEQ ID NO: 367 and the second polypeptide contains SEQ ID NO: 368, or the first polypeptide contains SEQ ID NO: 394 and the second polypeptide contains any one of SEQ ID NOs: 369-393.

[0219] Cell permeabilizers can also be administered in the form of nucleic acids encoding antibodies present within the cell permeabilizer or cell permeabilizer itself. If both heavy and light chains are present, they are preferably linked as single-chain antibodies.

[0220] IV. Nucleic acids, vectors, and host cells This disclosure provides nucleic acids encoding at least some of the cell permeabilizers described herein. For example, the nucleic acid may encode any of the CIM, linker, and / or spacer described herein, in addition to any of the heavy chain and / or light chain described herein. In some embodiments, the nucleic acid encodes a first polypeptide comprising a heavy chain of an antibody that specifically binds to Aβ, the first polypeptide comprising any one of SEQ ID NOs. 349, SEQ ID NOs. 351, SEQ ID NOs. 353, SEQ ID NOs. 355, SEQ ID NOs. 357, SEQ ID NOs. 359, SEQ ID NOs. 361, SEQ ID NOs. 363, SEQ ID NOs. 365, SEQ ID NOs. 367, and SEQ ID NOs. 394. In some embodiments, the nucleic acid encodes a second polypeptide comprising a light chain of an antibody that specifically binds to Aβ, the second polypeptide comprising any one of SEQ ID NOs. 350, SEQ ID NOs. 352, SEQ ID NOs. 354, SEQ ID NOs. 356, SEQ ID NOs. 358, SEQ ID NOs. 360, SEQ ID NOs. 362, SEQ ID NOs. 364, SEQ ID NOs. 366, and SEQ ID NOs. 368-393.

[0221] In some embodiments, the nucleic acid encodes a first polypeptide comprising a heavy chain of an antibody that specifically binds to Aβ, wherein the first polypeptide comprises one of SEQ ID NOs: 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, and 394, and a second polypeptide comprising a light chain of an antibody that specifically binds to Aβ, wherein the second polypeptide comprises one of SEQ ID NOs: 350, 352, 354, 356, 358, 360, 362, 364, 366, and 368-393.

[0222] In some embodiments, the nucleic acid encodes a cell permeabilizer comprising a first polypeptide containing SEQ ID NO: 349 and a second polypeptide containing SEQ ID NO: 350. In some embodiments, the nucleic acid encodes a cell permeabilizer comprising a first polypeptide containing SEQ ID NO: 351 and a second polypeptide containing SEQ ID NO: 352. In some embodiments, the nucleic acid encodes a cell permeabilizer comprising a first polypeptide containing SEQ ID NO: 353 and a second polypeptide containing SEQ ID NO: 354. In some embodiments, the nucleic acid encodes a cell permeabilizer comprising a first polypeptide containing SEQ ID NO: 355 and a second polypeptide containing SEQ ID NO: 356. In some embodiments, the nucleic acid encodes a cell permeabilizer comprising a first polypeptide containing SEQ ID NO: 357 and a second polypeptide containing SEQ ID NO: 358. In some embodiments, the nucleic acid encodes a cell permeabilizer comprising a first polypeptide containing SEQ ID NO: 359 and a second polypeptide containing SEQ ID NO: 360. In some embodiments, the nucleic acid encodes a cell permeabilizer comprising a first polypeptide containing SEQ ID NO: 361 and a second polypeptide containing SEQ ID NO: 362. In some embodiments, the nucleic acid encodes a cell permeabilizer comprising a first polypeptide containing SEQ ID NO: 363 and a second polypeptide containing SEQ ID NO: 364. In some embodiments, the nucleic acid encodes a cell permeabilizer comprising a first polypeptide containing SEQ ID NO: 365 and a second polypeptide containing SEQ ID NO: 366. In some embodiments, the nucleic acid encodes a cell permeabilizer comprising a first polypeptide containing SEQ ID NO: 367 and a second polypeptide containing SEQ ID NO: 368. In some embodiments, the nucleic acid encodes a cell permeabilizer comprising a first polypeptide containing SEQ ID NO: 394 and a second polypeptide containing any one of SEQ ID NOs. 369 to 393.

[0223] Optionally, the nucleic acid may further encode a signal peptide, and the signal peptide may be expressed with the signal peptide ligated to the constant region (with or without CMIP). The coding sequence of the nucleic acid may be operably ligated with regulatory sequences to ensure the expression of coding sequences such as promoters, enhancers, ribosome binding sites, and transcription termination signals. The nucleic acids encoding the heavy and light chains may exist in isolated forms or be cloned into one or more vectors. The nucleic acids can be synthesized, for example, by solid-phase synthesis or PCR of overlapping oligonucleotides. The nucleic acids encoding the heavy and light chains may be linked together as a single continuous nucleic acid within an expression vector, or they may exist separately, for example, each cloned into its own expression vector. In some embodiments, the nucleic acids are codon-optimized for expression in host cells.

[0224] Several methods are known for producing chimeric and humanized antibodies using antibody-expressing cell lines (e.g., hybridomas) and can be used for the production of the anti-Aβ CPA of this disclosure. For example, the immunoglobulin variable region of the antibody can be cloned and sequenced using well-known methods. In one method, the heavy chain variable VH region is cloned by RT-PCR using mRNA prepared from hybridoma cells. Consensus primers are used on the VH region leader peptide containing the translation start codon as a 5' primer and a g2b constant region-specific 3' primer. Exemplary primers are described in U.S. Patent Publication US2005 / 0009150 by Schenk et al. (hereinafter "Schenk"). Multiple independently derived clone-derived sequences can be compared to ensure that no changes have been introduced during amplification. The VH region sequence can also be determined or confirmed by sequencing the VH fragment obtained by 5'RACE RT-PCR and 3'g2b-specific primers.

[0225] The light chain variable VL region of anti-Aβ CPA can be cloned in a similar manner. In one approach, a consensus primer set is designed for amplification of the VL region using a 5' primer designed to hybridize to the VL region containing the translation start codon and a 3' primer specific to the Ck region downstream of the VJ junction. In another approach, the cDNA encoding the VL is cloned using 5'RACE RT-PCR. Exemplary primers are described in Schenk (above). The cloned sequence is then combined with a sequence encoding a human (or other non-human species) constant region.

[0226] This specification also provides vectors comprising one of the nucleic acids described herein operably ligated to one or more regulatory sequences to bring about the expression of any of the cell permeabilizing agents described herein in mammalian cells.

[0227] The Specified also provides vectors comprising nucleic acids encoding mature heavy chain variable domains (e.g., any of the heavy chain variable domains described herein, with or without CMIP) and light chain variable domains (e.g., any of the heavy chain variable domains described herein, with or without CMIP) operably linked to one or more regulatory sequences to result in the expression of any of the antibodies or antigen-binding fragments described herein in mammalian cells.

[0228] In one approach, the heavy and light chain variable regions are re-engineered to encode splice donor sequences downstream of their respective VDJ or VJ junctions, and cloned into mammalian expression vectors such as pCMV-hyl for the heavy chain and pCMV-Mcl for the light chain. These vectors encode the human γl and Ck constant regions as exon fragments downstream of the inserted variable region cassette. After sequence validation, the heavy and light chain expression vectors can be co-transfected into CHO cells to produce chimeric antibodies. 48 hours after transfection, the conditioned medium is collected and assayed by Western blotting for antibody production or ELISA for antigen binding. The chimeric antibodies are humanized as described above.

[0229] Chimeric, veneerated, humanized, and human antibodies are typically produced by recombinant expression, which can be used for the production of anti-Aβ CPAs of this disclosure. Recombinant polynucleotide constructs typically include an expression regulatory sequence operably linked to the coding sequence of an antibody chain, which may include innately related or heterologous expression regulatory elements such as a promoter. The expression regulatory sequence may be a promoter system in a vector capable of transforming or transfecting eukaryotic or prokaryotic host cells. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high levels of nucleotide sequence expression and collection and purification of cross-reactive antibodies.

[0230] Accordingly, this specification provides host cells transformed with any of the vectors described herein. This specification also provides host cells containing any of the nucleic acids described herein.

[0231] Expression vectors are typically replicable within a host organism, either as episomes or as an integral part of host chromosomal DNA. Generally, expression vectors contain a selection marker, such as ampicillin resistance or hygromycin resistance, to enable the detection of cells transformed with the desired DNA sequence.

[0232] E. coli is a useful prokaryotic cell host for expressing antibodies, particularly antibody fragments. Microorganisms such as yeast are also useful for expression. Saccharomyces is a yeast host that optionally has suitable vectors containing expression regulatory sequences, origins of replication, termination sequences, etc. Typical promoters include 3-phosphoglycerate kinase and other glycosphagocytes. Inducible yeast promoters include, in particular, alcohol dehydrogenase, isocytochrome C, and promoters derived from enzymes that utilize maltose and galactose.

[0233] Mammalian cells can be used to express nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). Numerous suitable host cell lines capable of secreting intact heterologous proteins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myeloma cells including Sp2 / 0 and NS0. The cells may be non-human. Expression vectors for these cells may contain expression regulatory sequences such as origins of replication, promoters, and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Examples of expression regulatory sequences include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, etc. See Co et al., J.Immunol. 148:1149 (1992). In some embodiments, the promoter is a eukaryotic promoter.

[0234] Alternatively, antibody coding sequences may be incorporated into transgenes for introduction into the genome of transgenic animals and subsequent expression in the milk of transgenic animals (see, for example, U.S. Patents 5,741,957, 5,304,489, and 5,849,992). Suitable transgenes include light and / or heavy chain coding sequences operably linked to promoters and enhancers derived from mammary gland-specific genes such as casein or beta-lactoglobulin.

[0235] Vectors containing the target DNA segment can be transferred into host cells in a manner appropriate to the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, bioristic, or virus-based transfection can be used for other cell hosts. Other methods used to transform mammalian cells include the use of polyblens, protoplast fusion, liposomes, electroporation, and microinjection. For the production of transgenic animals, the transgene can be microinjected into fertilized oocytes or incorporated into the genome of embryonic stem cells, and the nuclei of such cells can be transferred into enucleated oocytes.

[0236] After introducing a vector(s) encoding antibody heavy and light chains into a cell culture, the cell pool can be screened for growth productivity and product quality in serum-free medium. The top-performing cell pool can then be subjected to FACS-based single-cell cloning to generate monoclonal cell lines. Specific productivity exceeding 50 pg or 100 pg per cell per day can be used, corresponding to product titers exceeding 7.5 g / L culture. Antibodies produced by single-cell clones can also be tested for turbidity, filtration characteristics, PAGE, IEF, UV scan, HPSEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assays such as ELISA or Biacore. Selected clones can then be banked in multiple vials and cryopreserved for subsequent use.

[0237] After expression, the antibody can be purified according to standard procedures in the art, including protein A capture, HPLC purification, column chromatography, and gel electrophoresis (see, in general, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0238] Methods for the commercial production of antibodies may be used for the production of anti-Aβ CPA of the present disclosure, including codon optimization, promoter selection, transcription element selection, terminator selection, serum-free single-cell cloning, cell banking, use of selection markers for copy number amplification, CHO terminator, or improvement of protein titer (see, for example, U.S. Patents 5,786,464, 6,114,148, 6,063,598, 7,569,339, W02004 / 050884, W02008 / 012142, W02005 / 019442, W02008 / 107388, W02009 / 027471, and U.S. Patent 5,888,809).

[0239] DNA can be delivered in its naked form (i.e., colloidal or without inclusion material). Alternatively, several viral vector systems can be used, including retrovirus systems (see Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993)), adenovirus vectors (e.g., see Bett et al., J. Virol. 67, 591 1 (1993)), adeno-associated virus vectors (e.g., see Zhou et al., J. Exp. Med. 179, 1867 (1994)), viral vectors derived from the pox family, including vaccinia virus and tripoxvirus, and viral vectors derived from the alphavirus genus, such as those derived from Sindbis and Semliki forest viruses (e.g., Dubensky et al.) See al., J. Virol. 70, 508-519 (1996), Venezuelan encephalitis virus (see U.S. Patent No. 5,643,576), and rhabdoviruses, such as vesicular stomatitis virus (see WO96 / 34625) and papillomavirus (Ohe et al., Human Gene Therapy 6:325-333 (1995), Woo et al., WO94 / 12629 and Xiao & Brandsma, Nucleic Acids. Res. 24:2630-2622 (1996)).

[0240] DNA encoding an immunogen, or a vector containing it, can be packaged in liposomes. Suitable lipids and related analogues are described in U.S. Patents 5,208,036, 5,264,618, 5,279,833, and 5,283,185. Vectors and immunogen-encoding DNA can be adsorbed or associated with particulate carriers, examples of which include polymethyl methacrylate polymers, as well as polylactic acid and poly(lactic acid-co-glycolide) (see, e.g., McGee et al., J. Micro Encap. 1996).

[0241] V. Additional Conjugates The cell permeabilizers of this disclosure, comprising conjugate antibodies and antigen-binding antibody fragments that specifically bind to antigens such as Aβ (e.g., human Aβ), are useful for detecting the presence of Aβ and for monitoring and evaluating the effectiveness of therapeutic agents used to treat patients diagnosed with inclusion body myositis (IBM) or sporadic inclusion body myositis (sIBM).

[0242] The cell permeabilizing agents described herein may be further conjugated with other therapeutic moieties, other proteins, other antibodies, and / or detectable labels. See WO03 / 057838, U.S. Patent No. 8,455,622. Such therapeutic moieties may be any agents that can be used to treat, suppress, alleviate, prevent, or improve an undesirable condition or disease in a patient, such as inclusion body myositis (IBM).

[0243] For example, such cell permeabilisants can be linked to radioactive isotopes. Examples of radioactive isotopes include yttrium. 90 (90Y), Indium 111 (111In) 131 1. 99 mTc, radioactive silver-111, radioactive silver-199, and bismuth 213 The following are examples. Linking of radioisotopes to antibodies or antigen-binding antibody fragments can be carried out using conventional bifunctional chelates. For linking of radioactive silver-111 and radioactive silver-199, sulfur-based linkers may be used. See Hazra et al., Cell Biophys. 24-25:1-7 (1994). Linking of silver radioisotopes may involve reducing immunoglobulins with ascorbic acid. For radioisotopes such as 111In and 90Y, ibritumomabutiuxetane may be used, which react with these isotopes to form 111In-ibritumomabutiuxetane and 90Y-ibritumomabutiuxetane, respectively. See Witzig, Cancer Chemother. Pharmacol., 48(Suppl l):S91-S95 (2001).

[0244] Some such antibody or antigen-binding antibody fragments can be conjugated to other therapeutic parts. Such therapeutic parts may be, for example, cytotoxic, cell proliferation inhibitory, immunomodulatory, neurotrophic, or neuroprotective. For example, an antibody or antigen-binding antibody fragment can be conjugated with a toxic chemotherapeutic agent such as meitansine or geldanamycin, a tubulin inhibitor such as a tubulin conjugate (e.g., auristatin), or a minor groove conjugate such as calicheamicin. Other representative therapeutic parts include agents known to be useful in the treatment, management, or relief of inclusion body myositis.

[0245] Antibodies or antigen-binding antibody fragments can also be conjugated with detectable labels. Such antibodies and antigen-binding antibody fragments can be used, for example, to diagnose inclusion body myositis. Typical detectable labels that can be conjugated or linked to antibodies or antigen-binding antibody fragments include various enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic groups such as streptavidin / biotin and avidin / biotin; fluorescent substances such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent substances such as luminol; bioluminescent substances such as luciferase, luciferin, and echoline; radioactive silver-111, radioactive silver-199, and bismuth. 213 , iodine ( 131 I, 125 I, 123 I, 121 I), carbon ( 14 C), sulfur ( 5 S), trilithium ( 3 H), Indium ( 115 In, 113 In, 112 In, 111 In), technetium ( 99 Tc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103Pd), molybdenum ( 9 9Mo), Xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 PM, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Enjoy, 57 Co, 65 ZN, 85 SR, 32 P, 153 Gd, 169 Yb, 51 CR, 54 Mn, 75 Se, 113 Sn, and 117 Examples include radioactive materials such as Sn, positron-emitting metals used in various positron emission tomography (PET) imaging techniques, non-radioactive paramagnetic metal ions, and molecules bound to radioactive labels or specific radioisotopes.

[0246] The therapeutic portion, other proteins, other antibodies, and / or detectable labels may be conjugated to the antibody or antigen-binding antibody fragment of the present invention, either directly or indirectly via an intermediate (e.g., a linker). For example, Arnon et al., “Monoclonal Antibodies For Immunotargeting of Drugs in Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985), Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (2 ndSee Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987), Thorpe, “Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review,” in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985), “Analysis, Results, and Future Prospective of The Therapeutic Use of Radio-labeled Antibody in Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., Immunol. Rev., 62: 119-58 (1982). Appropriate linkers include, for example, cleavable and non-cleavable linkers. Various linkers can be used that release a bound therapeutic moiety, protein, antibody, and / or detectable label under acidic conditions, reducing conditions, exposure to a specific protease, or other defined conditions.

[0247] In some embodiments, the cell permeabilisant is also conjugated to a therapeutic agent, cytotoxic agent, cell proliferation inhibitor, immunomodulator, neurotrophic agent, or neuroprotective agent as described herein. For example, an antibody present in the cell permeabilisant may be conjugated (i.e., formed a heteroconjugate) with a therapeutic moiety, such as a cytotoxic agent, radiotherapy agent, immunomodulator, or secondary antibody. Typical therapeutic moieties include agents known to be useful in treating, managing, or alleviating Aβ-related disease or the symptoms of Aβ-related disease.

[0248] The therapeutic portion and / or detectable substance may be directly conjugated or linked to any of the mouse, chimeric, or humanized antibodies described herein via an intermediate (e.g., a linker) using techniques known in the art. For example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985), Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (2 nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987), Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985), “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al. al., Immunol. Rev., 1982, 62:119-58.

[0249] The cell permeabilizers used in the disclosed formulations may also include modified forms of mouse, chimeric, or humanized antibodies that have an increased in vivo half-life compared to the corresponding unmodified antibody. Such modified forms may be prepared, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protective / blocking groups, proteolytic cleavage, or linkage to cell ligands or other proteins. Typical methods for extending the half-life of antibodies are described in PCT International Publication No. WO02 / 060919.

[0250] VI. Pharmaceutical Compositions This disclosure also provides pharmaceutical compositions and products. Accordingly, this specification provides pharmaceutical compositions comprising any of the cell permeabilizing agents described herein and a pharmaceutically acceptable carrier or diluent.

[0251] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dose forms (i.e., single doses). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. For injection, the cell permeabilizer may be formulated in an aqueous solution, preferably (to reduce discomfort at the injection site) Hanks' solution, Ringer's solution, or a physiologically compatible buffer such as saline or acetate buffer. The solution may contain formulations such as suspensions, stabilizers, and / or dispersants. Alternatively, the cell permeabilizer may be in a lyophilized form for preparation before use with a suitable vehicle, e.g., sterile pyrogen-free water.

[0252] The anti-Aβ cell permeabilizing agents described herein may be present in any pharmaceutically acceptable excipient or carrier. For example, the anti-Aβ cell permeabilizing agents described herein may be present in a buffer solution. The buffer solution may have a pH of about 6 to about 7. Typically, the formulations are sterile, as achieved, for example, by sterile filtration using a 0.2 μm or 0.22 μm filter. The formulations disclosed herein are generally stable to freeze and thaw.

[0253] In some embodiments, it may be desirable to use a pharmaceutical composition containing one of the cell permeabilisants described herein in an ex vivo or in vitro manner. For example, the method may be for non-diagnostic and / or non-therapeutic purposes. In such cases, a sample such as cells, tissues, and / or organs removed from a patient is exposed to a pharmaceutical composition containing one of the cell permeabilisants described herein.

[0254] For preventive purposes, cell permeabilizers (e.g., nucleic acids or vectors encoding either or any of the cell permeabilizers) or their pharmaceutical compositions are administered in regimes (dosage, frequency of administration, and route of administration) that are effective in reducing the risk, mitigating the severity, or delaying the onset of at least one sign or symptom of amyloidogenic disease (e.g., IBM) in patients who are susceptible to or otherwise at risk of disease such as IBM. In particular, the regimes are preferably effective in inhibiting or delaying Aβ aggregates (e.g., human Aβ aggregates) in muscle tissue and / or inhibiting or delaying their toxic effects and / or inhibiting / delaying the onset of motor impairment.

[0255] For therapeutic purposes, cell permeabilisers are administered to patients suspected of having a disease (e.g., IBM) or already suffering from the disease, in a regime (dose, frequency of administration, and route of administration) that is effective in improving at least one sign or symptom of the disease, or at least inhibiting its further exacerbation. In particular, the regime is preferably effective in reducing, or at least inhibiting, further increases in cytoplasmic levels, associated toxicity, and / or motor impairment of Aβ (e.g., human Aβ) and / or aggregates formed therefrom.

[0256] A regime is considered therapeutically or prophylactically effective if individual treated patients achieve a more favorable outcome than the mean outcome in a control group of equivalent patients not treated by the methods disclosed herein.

[0257] VII. Treatment regimens As used herein, the terms “to treat” and “treatment” mean the alleviation or remission of one or more symptoms or effects associated with a disease, the prevention, inhibition or delay of the onset of one or more symptoms or effects of a disease, a reduction in the severity or frequency of one or more symptoms or effects of a disease, and / or an increase or tendency toward a desired outcome as described herein.

[0258] The desired outcomes of the treatments disclosed herein vary according to the patient profile and are readily determinable to those skilled in the art. Desired outcomes include improvements in the patient's health status. Generally, desired outcomes include measurable indicators such as a reduction or clearance of pathological Aβ in IBM disease.

[0259] This specification provides a method for delivering an antibody that specifically binds to Aβ into a cell, comprising contacting the cell with one of the anti-Aβ CPAs described herein, thereby resulting in the internalization of at least an antigen-binding fragment of the antibody into the cell. In some embodiments, the method comprises at least the transfer of the antigen-binding fragment of the antibody into the cell's cytosol.

[0260] This specification also provides a method for binding to an intracellular Aβ protein in a cell, the method comprising contacting the cell with one of the anti-Aβ CPAs described herein, thereby resulting in at least internalization and transfer of the antigen-binding fragment of the antibody into the cytosol. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are in vitro. In some embodiments, the cells are within a subject.

[0261] This specification also provides a method for inhibiting or reducing the aggregation of Aβ in a subject having or at risk of developing an Aβ-related disease, comprising administering an effective amount of one of the anti-Aβ CPAs described herein to the subject, thereby inhibiting or reducing the aggregation of Aβ in the subject.

[0262] This specification also provides a method for treating or preventing an Aβ-related disease in a subject, comprising administering a therapeutically effective amount of one of the cell-permeable agents described herein, thereby treating or preventing an Aβ-related disease.

[0263] In some embodiments, AB-related disease is IBM.

[0264] This specification also provides a method for detecting Aβ deposition in subjects who have or are at risk of developing an Aβ-related disease, comprising administering one of the cell permeabilizing agents described herein to the subject and detecting antibodies bound to Aβ in the subject.

[0265] This specification also provides a method for detecting Aβ in a sample obtained from a patient having or at risk of developing an Aβ-related disease, comprising contacting the sample with one of the cell permeabilisers described herein and detecting the binding of an antibody to Aβ in the sample. In some embodiments, the cell permeabiliser is administered by intravenous injection into the body of the subject. In some embodiments, the cell permeabiliser or the antibody in the cell permeabiliser is labeled. In some embodiments, the cell permeabiliser is labeled by fluorescent, paramagnetic, or radioactive labeling. In some embodiments, the radioactive labeling is detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

[0266] Cellular permeabilis agents are administered in an effective regime, meaning a dosage, route of administration, and frequency that delays the onset of the disorder during treatment, reduces its severity, prevents further deterioration, and / or improves at least one sign or symptom. If the patient already has the disorder, the regime may be called a therapeutically effective regime. If the patient is at higher risk of the disorder than the general population but has not yet experienced symptoms, the regime may be called a preventively effective regime. In some cases, the effectiveness of treatment or prevention in an individual patient can be observed compared to past controls or past experiences of the same patient. In other cases, the effectiveness of treatment or prevention can be demonstrated in preclinical or clinical trials in the treated population compared to a control population of untreated patients.

[0267] Administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, subarachnoid, intraperitoneal, topical, intranasal, or intramuscular. Some cell permeabilisants can be administered into the systemic circulation by intravenous or subcutaneous administration.

[0268] Cell permeabilizing agents can be administered intravenously or subcutaneously in doses ranging from approximately 0.5 mg / kg to approximately 30 mg / kg relative to the host's body weight. For example, doses may be approximately 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 5.0 mg / kg, 8.0 mg / kg, 10 mg / kg, 15 mg / kg, 16 mg / kg, 20 mg / kg, 24 mg / kg, 25 mg / kg, or 30 mg / kg relative to body weight. The dose may also be approximately 0.5 mg / m² depending on body surface area. 2 ~about 500mg / m 2 For example, 0.5, 5, 10, 50, 100, 250 or 500 mg / m² 2 It can be administered by: For intravenous administration, a sufficient amount of the cell permeabilizing agent formulation to achieve the desired dose for an individual patient is transferred from one or more vials to one or more intravenous bags containing a liquid (e.g., saline solution) and administered to the patient.

[0269] Cell permeabilis agents are usually administered in multiple doses. The frequency of administration depends, in particular, on the half-life of the circulating cell permeabilis agent, the patient's condition, and the route of administration. Depending on the patient's condition or the progression of the disorder being treated, the frequency may be daily, weekly, monthly, every three months, or at irregular intervals.

[0270] Exemplary treatment regimens involve administration every two weeks, once a month, or once every three to six months. The frequency of administration depends on whether the disorder is acute or chronic and the disorder's response to treatment. The frequency of administration can be adjusted according to the pharmacokinetic profile of the antibody preparation in the patient. For example, the half-life of the cell permeabilisant may ensure an administration frequency of two weeks. In some embodiments disclosed herein, the cell permeabilisant is administered to the patient for at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, five years, ten years, or over the patient's lifetime.

[0271] Normal levels of Aβ (e.g., human Aβ) can be measured in the muscle of representative samples of individuals in the general population who have not been diagnosed with IBM and are not considered to have an increased risk of developing such disease (e.g., representative samples of disease-free individuals over 50 years of age). Alternatively, normal levels may be recognized in individual patients if the PET signal obtained by the method of the present invention in muscle known to develop Aβ aggregates (e.g., human Aβ aggregates) is no different (within the range of measurement accuracy) from the signal from muscle known not to normally develop such depositions. Elevated levels in an individual may be recognized by comparison with normal levels (e.g., outside the mean and variance of the standard deviation), or simply by an elevation of the signal in muscle associated with Aβ aggregates (e.g., human Aβ aggregates) that exceeds the experimental error compared to areas not known to be associated with deposition. For the purpose of comparing the levels of Aβ aggregates (e.g., human Aβ aggregates) in individuals and populations, Aβ aggregates should preferably be measured in the same region(s) of muscle, which includes at least one region (e.g., in the cytoplasm) known to form Aβ aggregates associated with a particular disease (e.g., IBM).

[0272] Patients with elevated levels of Aβ aggregates (e.g., human Aβ aggregates) are candidates for initiating immunotherapy. After initiating immunotherapy, a decrease in Aβ3 aggregate levels (e.g., human Aβ aggregates) can first be seen as an indicator that the treatment is having the desired effect. The observed decrease may range, for example, from 1–100%, 1–50%, or 1–25% of the baseline value. Such an effect may be measured in one or more muscles where deposition is known to form, or from the mean of such areas. The overall effect of the treatment can be estimated by adding the percentage decrease (%) compared to baseline to the increase in Aβ aggregates (e.g., human Aβ aggregates) that would occur in an average untreated patient without treatment.

[0273] Maintaining Aβ aggregates (e.g., human Aβ aggregates) at a nearly constant level, or even a slight increase in Aβ aggregates (e.g., human Aβ aggregates), can be an indicator of the response to treatment, although it represents a suboptimal response. By comparing such a response to the time course of Aβ aggregate levels (e.g., human Aβ aggregates) in untreated patients with a specific disease (e.g., IBM), it is possible to determine whether immunotherapy has the effect of inhibiting further increases in Aβ aggregates (e.g., human Aβ aggregates).

[0274] VIII. Kit This disclosure further provides a kit (e.g., a container) comprising any of the cell permeabilisants described herein and related materials, e.g., instructions for use (e.g., a package insert). The instructions for use may include, for example, instructions for administration of the cell permeabilisant and optionally one or more additional agents. The container for the cell permeabilisant may be a unit dose, a bulk package (e.g., a multi-dose package), or a dose less than a unit dose.

[0275] IX. Detection Method In some embodiments, the cell permeabilizers of the Disclosure further provide a method for detecting Aβ in a sample. For example, in some embodiments, the Disclosure provides a method for detecting Aβ in a sample, comprising contacting the sample with the cell permeabilizer of the Disclosure and detecting the binding of the cell permeabilizer or antibody to Aβ. For example, such a method may be ex vivo or in vitro. In some embodiments, the sample is a biological sample derived from a subject (e.g., a human subject). In some embodiments, the subject is a human. In some embodiments, the subject is a patient who has or is at risk of having an Aβ-related disease. In such cases, a sample such as cells, tissues, and / or organs removed from the patient is exposed to the antibody or antigen-binding fragment described herein. In some embodiments, the sample comprises cells derived from the patient, and the cells are lysed before administration of the antibody or antigen-binding fragment described herein. [Examples]

[0276] The following embodiments are included to illustrate the mechanisms disclosed herein. Certain aspects of the following embodiments are described in terms of techniques and procedures that the co-inventors of the Invention find or intend to work well in the implementations disclosed herein. In light of this disclosure and the general level of those skilled in the art, those skilled in the art will understand that the following embodiments are for illustrative purposes only and that many changes, modifications, and alterations can be adopted without departing from the scope of this disclosure.

[0277] Example 1: Intracellular integration of an anti-GFP cell permeabilizing agent containing selected CMIP To demonstrate the ability of the CMIPs of this disclosure to be internalized into cells, a panel of exemplary CMIPs (shown in Table 5 below) were evaluated for internalization. A panel of anti-GFP cell permeabilizers was expressed, and each CPA contained an anti-GFP antibody having the CMIPs of Table 5, conjugated to the C-terminus of the light chain via a polypeptide linker having a GGSSS sequence (SEQ ID NO: 256). The anti-GFP antibodies contained the heavy-chain and light-chain variable regions of ScFv disclosed in WO2022 / 053642A1.

[0278] HEK cells were transiently transfected with Htt-exon1-Q103-eGFP to express GFP in the cell cytosol. After 24 hours, the cells were incubated with 11 ug / ml anti-GFP CPA for another 24 hours (n=2 / CPA). The cells were then washed, fixed / permeabilized, and stained with AF647 conjugate anti-human secondary antibody to stain intracellular CPA. The stained cells were imaged using high-content imaging (Operetta system) with a 40x water immersion objective lens (30 fields / well). The amount of intracellular CPA detected via the secondary antibody was quantified, and the number was normalized to the observed value for CPA including CMIP4 (SEQ ID NO: 143). Quantitative analysis was performed using Harmony software.

[0279] Table 6 provides the relative internalization efficiency (normalized relative to CMIP4) for the exemplary CMIPs of this disclosure. This internalization efficiency was evaluated using the method described above. [Table 6-1] [Table 6-2] [Table 6-3]

[0280] Example 2: Immunogenicity of CMIP To understand the therapeutic potential of the cell permeabilis agents of this disclosure, the immunogenicity of exemplary CMIPs was evaluated. Qualitative immunogenicity profiles for CMIP sequences were generated using the EpiQuest program, which provides a matrix of predicted probabilities of CTL epitopes for each CMIP sequence analyzed. Using the EpiQuest software, each amino acid residue in a sequence is assigned a position along the X-axis, and each amino acid residue is assigned a predicted immunogenicity (PI) value as an integer along the Y-axis. A positive PI value indicates the probability of an immunogenic epitope containing that amino acid residue at that position, while a negative PI value indicates a low probability of an immunogenic epitope containing that amino acid residue at that position. Combining these values ​​across all amino acid residues in the CMIP sequence yields an immunogenicity profile for each CMIP sequence analyzed.

[0281] To compare potential immunogenicity across CMIP, immunogenicity profiles generated by the EpiQuest program were converted to an IU scale of 0 to 10 immunogenicity units (IU). EpiQuest immunogenicity profiles were generated using default EpiQuest thresholds and converted to IU values ​​as follows: Firstly, each residue is assigned an IU value based on the PI value provided by the EpiQuest program. For each residue with a positive PI value, an immunogenicity unit (IU) score of 0.2 is assigned for each shaded tile from 0 to 5 (for example, if all five vertical tiles of a residue are shaded from 0 to 5, an IU of 1 is assigned to that residue). In addition to the vertical tiles from 1 to 5, any vertical tiles shaded from 6 to 10 are assigned an additional IU score of 0.4 per shaded tile. Similarly, any shaded vertical tiles from 11 to 15 are assigned an IU score of 0.6 per shaded tile. For example, if an amino acid residue has 12 PI values ​​(indicated by 12 vertically shaded tiles), it would be assigned an IU score of 4.2 IU (by adding 0.2 for each of the five shaded tiles 1-5 to total 1.0, adding 0.4 for each of the five shaded tiles 6-10 to total 2.0, and adding 0.6 for each of the two shaded tiles 11-12 to total 1.2).

[0282] Secondly, a penalty score is added to the IU score based on the number of consecutive amino acid residues that are assessed to have a positive PI value. A penalty of 1 IU is imposed for three or more consecutive amino acid residues with a PI value of 1 to 5 (indicated by positively shaded tiles for three or more consecutive residues). No additional penalty is imposed even if all adjacent tiles have a PI value of 5. A penalty score of 2 is imposed for three or more consecutive amino acid residues with a PI value of 6 to 10. In addition, a penalty score of 3 is imposed for three or more consecutive amino acid residues with a PI value of 11 to 15. Even if the calculated score is higher, the numerator is assigned a maximum score of 10.

[0283] Table 7 provides the IU scores for exemplary CMIPs of this disclosure. These IU scores were calculated using the method described above. [Table 7-1] [Table 7-2] [Table 7-3]

[0284] Example 3. M-lycotoxin cell permeabilizer reduces the area and number of Aβ foci. First, HEK cells were transfected with APP770sw[K670N-M671L_Swedish], a model for analyzing Aβ-related diseases. After 24 hours, the cells were subjected to separate 24-hour incubations with cell permeabilizers (h2931_ML (L17E ligated to the C-terminus of the light chain)) at concentrations of 1, 10, and 100 μg / ml, respectively. Subsequently, the cells were washed, fixed, permeabilized, and stained with m3D6 anti-Aβ antibody and AF647 conjugate anti-mouse secondary antibody. Stained cells were imaged using high-content imaging (Operetta system) with a 40x water immersion objective lens. Quantitative analysis was performed using Harmony software.

[0285] Figure 1A shows the total Aβ focus area (px 2 Figure 1B is a graph showing the results. The data demonstrate that incubation with the h2931-M-lycotoxin cell permeabilizer (e.g., anti-Aβ cell permeabilizer) reduced the total Aβ focus area in a concentration-dependent manner. The data also showed a decrease in the total Aβ focus area at each concentration compared to the control sample. Figure 1B is a graph showing the number of Aβ foci, which shows that incubation with the h2931-M-lycotoxin cell permeabilizer reduced the number of Aβ foci in a concentration-dependent manner compared to the control. Figure 1C is a graph showing the average focus area of ​​Aβ foci in cells treated with h2931 or the h2931 cell permeabilizer. Thus, Figures 1A-C demonstrate that the h2931 cell permeabilizer (i.e., cell permeabilizer) interferes with Aβ aggregation in a concentration-dependent manner.

[0286] Example 4. The M-lycotoxin cell permeabilizer reduces the area and number of Aβ foci compared to the isotype control. HEK cells were transiently transfected with APP770sw[K670N-M671L_Swedish]. After 24 hours, the cells were incubated for 24 hours with either 10 μg / ml or 100 μg / ml of h2931_ML (L17E ligated to the C-terminus of the light chain), or 100 ug / ml of untagged h2931 antibody. The cells were then washed, fixed, permeabilized, and stained with m3D6 anti-Aβ antibody and AF647 conjugate anti-mouse secondary antibody. The stained cells were imaged using high-content imaging (Operetta system) with a 40x water immersion objective lens. Quantitative analysis was performed using Harmony software.

[0287] Figure 2A is a graph showing the total Aβ focus area. The data demonstrate that incubation with the h2931-M-lycotoxin cell permeabilizer (e.g., anti-Aβ cell permeabilizer) resulted in a concentration-dependent reduction in the total Aβ focus area compared to untreated and IgG isotype controls. Figure 2B is a graph showing the number of Aβ foci, demonstrating that incubation with the h2931-M-lycotoxin cell permeabilizer resulted in a concentration-dependent decrease in the number of Aβ foci compared to untreated and IgG isotype controls. Figure 2C is a graph showing the average focus area of ​​Aβ foci in cells treated with h2931 or the h2931 cell permeabilizer. In summary, Figures 2A-C demonstrate that the h2931 cell permeabilizer interferes with Aβ aggregation in a concentration-dependent manner.

[0288] Furthermore, HEK cells were transiently transfected with APP770sw[K670N-M671L_Swedish]. After 24 hours, transfected HEK cells were incubated with five different concentrations of h2931-M-lycotoxin (L17E ligated to the C-terminus of the light chain) cell permeabilizer (e.g., anti-Aβ cell permeabilizer) at 1, 2.5, 5, 10, and 100 μg / ml. Cells treated with 100 ug / ml of untagged h2931 were used as a control. The cells were then fixed, permeabilized, and stained with m3D6 anti-Aβ antibody and AF647 conjugate anti-mouse secondary antibody. Stained cells were imaged using high-content imaging (Operetta system) with a 40x water immersion objective lens. Quantitative analysis was performed using Harmony software.

[0289] Figure 3A is a graph showing the total Aβ focus area. The data demonstrate that incubation with the h2931-M-lycotoxin cell permeabilizer (e.g., anti-Aβ cell permeabilizer) resulted in a concentration-dependent reduction in the total Aβ focus area compared to untreated and IgG isotype controls. Figure 3B is a graph showing the number of Aβ foci, demonstrating that incubation with the h2931-M-lycotoxin cell permeabilizer resulted in a concentration-dependent decrease in the number of Aβ foci compared to untreated and IgG isotype controls. Figure 3C is a graph showing the average focus area of ​​Aβ foci in cells treated with either h2931 (i.e., untagged) or the h2931 cell permeabilizer. Therefore, Figures 3A-C demonstrate that the h2931 cell permeabilizer interferes with Aβ aggregation in a concentration-dependent manner.

[0290] Example 5. The M-lycotoxin cell permeabilizer disrupts Aβ deposition in the absence of functional FcK or FcRn regions. HEK cells were transiently transfected with APP770sw[K670N-M671L_Swedish]. After 24 hours, the transfected HEK cells were incubated for another 24 hours with various h2931-CMIP4 cell permeabilizers, each containing h2931 (i.e., untagged antibody), h2931 with disrupted Fcγ function (L234A / L325A mutation), or h2931 antibody with mutations in the FcRn region (H310A / H435Q mutation). For h2931_CMIP4, h2931_CMIP4_LALA, and h2931_CMIP4_H310A_H435Q, CMIP4 was ligated to the C-terminus of the light chain. Subsequently, the cells were fixed, permeabilized, and stained with primary m3D6 anti-Aβ antibody, followed by secondary antibodies AF647 conjugate anti-mouse and AF568 conjugate anti-human. The stained cells were imaged using high-content imaging (Operetta system) with a 40x water immersion objective lens. Quantitative analysis was performed using Harmony software.

[0291] Figure 4A is a graph showing the total Aβ focus area per cell. The data demonstrate that incubation with the h2931 cell permeabilizer reduced the total Aβ focus area per cell compared to untreated and IgG isotype controls, regardless of Fcγ or FcRn function. In other words, the h2931 cell permeabilizer (e.g., anti-Aβ cell permeabilizer) can disrupt intracellular Aβ deposition without the function of either the FcK domain or the FcRn region. Figure 4B is a graph showing the number of Aβ focus areas per cell, demonstrating that incubation with the h2931 cell permeabilizer resulted in a lower number of Aβ foci per cell compared to untreated and IgG isotype controls, regardless of Fcγ or FcRn function. Figure 4C is a graph showing antibody (i.e., anti-Aβ cell permeabilizer) internalization per cell, demonstrating that incubation with the h2931 cell permeabilizer increased antibody internalization per cell compared to untagged h2931 antibody, regardless of Fcγ or FcRn function. Therefore, Figures 4A-4C demonstrate that the h2931 cell permeabilizer interferes with intracellular Aβ deposition even when Fcγ or FcRn function is suppressed by mutation.

[0292] Example 6. CMIP-4 cell permeabilizer reduces the area and number of Aβ foci. HEK cells were transiently transfected with APP770sw[K670N-M671L_Swedish]. After 24 hours, transfected HEK cells were incubated for another 24 hours with either 100 μg / ml of untagged mouse-derived 2931 antibody (mzd-2931) or 100 μg / ml of mzd-2931-CMIP4 cell permeabilizer (i.e., anti-Aβ cell permeabilizer). For mzd-2931_CMIP4, CMIP4 was ligated to the C-terminus of the light chain. Cells were fixed, permeabilized, and stained with primary m3D6 anti-Aβ antibody, followed by secondary antibodies AF647 conjugate anti-mouse and AF568 conjugate anti-human. Stained cells were imaged using high-content imaging (Operetta system) with a 40x water immersion objective lens. Quantitative analysis was performed using Harmony software.

[0293] Figure 5A is a graph showing the total Aβ focus area per cell, demonstrating that incubation with the mzd-2931-CMIP4 cell permeabilizer reduced the total Aβ focus area per cell compared to cells treated with untagged mzd-2931 antibody (i.e., without the intracellular integration module). Figure 5B is a graph showing the number of Aβ foci per cell, demonstrating that incubation with the mzd-2931-CMIP4 cell permeabilizer resulted in fewer Aβ focus areas per cell compared to cells treated with untagged mzd-2931 antibody. Figure 5C is a graph showing the internalization of the antibody or cell permeabilizer per cell. The data demonstrate that incubation with the mzd-2931-CMIP4 cell permeabilizer increased internalization into these cells compared to cells treated with untagged mzd-2931. Therefore, Figures 5A-C demonstrate that the cell permeabilizer of this disclosure is internalized by disrupting Aβ aggregation, even when it contains a mouse-derived antibody.

[0294] Example 7. Anti-Aβ cell permeability agent HEK cells were transiently transfected with APP770sw[K670N-M671L_Swedish]. After the first 24 hours, the transfected HEK cells were exposed to different treatments for a further 24 hours. These treatments included untagged h2931 antibody, h2931-CMIP4, and h2931 conjugated with a CMIP4-derived peptide, each at a concentration of 100 μg / ml. For each of h2931_CMIP4-1 to h2931-CMIP4-25, CMIP4 was ligated at the C-terminus of its light chain. Subsequently, the cells were fixed, permeabilized, and stained with m3D6 anti-Aβ antibody along with AF647 conjugated anti-mouse and AF568 conjugated anti-human secondary antibodies. The stained cells were then imaged using a high-content imaging Operetta system with a 40x water immersion objective lens. Quantitative analysis was performed using Harmony software.

[0295] Figure 6A is a graph showing the total Aβ focus area per cell. The data demonstrate that the cell permeabilizers of this disclosure significantly reduced the total Aβ focus area per cell compared to untreated cells and cells treated with either an IgG isotype or an untagged h2931 antibody (i.e., without the intracellular integration module). Figure 6B is a graph showing the average number of Aβ focus areas per cell, which demonstrates that the cell permeabilizers of this disclosure (e.g., cell permeabilizers) significantly reduced the number of Aβ foci per cell compared to untreated cells and cells treated with either an IgG isotype or an untagged h2931 antibody. Figure 6C is a graph showing the internalization of the antibody or cell permeabilizer per cell. The data demonstrate that the cell permeabilizers of this disclosure were readily internalized in transfected HEK cells and resulted in higher signaling compared to untreated cells and cells treated with either an IgG isotype or an untagged h2931 antibody. Therefore, Figures 6A-C demonstrate that cell permeabilizers containing anti-Aβ antibodies effectively internalize and bind to Aβ peptides, effectively disrupting intracellular Aβ aggregates. Figures 6A-C further show that this effect is observed with various cell permeabilizers containing CMIPs.

[0296] Example 8. Anti-Aβ cell permeabilizing agents reduce Aβ aggregates in Aβ model cells. HEK cells were transduced with a lentiviral vector containing APP770sw[K670N-M671L_Swedish]. After 48 hours, the transduced HEK cells were subjected to a further 24-hour incubation with either untagged h2931 antibody or h2931-CMIP4 antibody at a concentration of 100 μg / ml each. After incubation, the cells were fixed, permeabilized, and stained with m3D6 anti-Aβ antibody along with AF647 conjugate anti-mouse and AF568 conjugate anti-human secondary antibodies. The stained cells were then imaged using a high-content imaging Operetta system with a 40x water immersion objective lens. Quantitative analysis was performed using Harmony software.

[0297] Figure 7A is an image of untreated HEK cells expressing APPsw after lentiviral transfection. Cells are shown in gray, and Aβ aggregates are shown in white. Figure 7A demonstrates substantial Aβ production and aggregation in untreated HEK cells. Figure 7B is an image of HEK cells expressing APPsw after lentiviral transfection and incubation with untagged h2931 antibody. Cells are shown in gray, and Aβ aggregates are shown in white. Figure 7B demonstrates a reduction in Aβ aggregation compared to h2931-treated HEK cells. Figure 7C is an image of HEK cells expressing APPsw after lentiviral transfection and incubation with h2931-CMIP4 cell permeabilizer. CMIP4 is ligated to the C-terminus of the light chain. Cells are shown in gray, and Aβ aggregates are shown in white. Figure 7C demonstrates a significant reduction in Aβ aggregation compared to h2931-CMIP4 cell permeabilizer-treated HEK cells.

[0298] Example 9. The CMIP-4 cell permeabilizer reduces the area and number of Aβ foci and is internalized by HEK cells. HEK cells were transduced with a lentiviral vector containing APP770sw[K670N-M671L_Swedish] at an MOI (Multiple Infection Intake) of 10. After 48 hours, the transduced HEK cells were subjected to a further 24-hour incubation with either untagged h2931 antibody or h2931-CMIP4 antibody at a concentration of 100 μg / ml, respectively. CMIP4 was ligated to the C-terminus of the light chain. After incubation, the cells were fixed, permeabilized, and stained with m3D6 anti-Aβ antibody along with AF647 conjugate anti-mouse and AF568 conjugate anti-human secondary antibodies. The stained cells were then imaged using a high-content imaging Operetta system with a 40x water immersion objective lens. Quantitative analysis was performed using Harmony software.

[0299] Figure 8A is a graph showing the total Aβ focus area per cell, demonstrating that incubation with the h2931-CMIP4 cell permeabilizer reduced the total Aβ focus area per cell in lentiAPPsw-transduced HEK-293 cells compared to untreated cells and cells treated with untagged h2931 antibody. Figure 8B is a graph showing the number of Aβ foci per cell, demonstrating that incubation with the h2931-CMIP4 cell permeabilizer reduced the number of Aβ focus areas per cell compared to untreated cells and cells treated with untagged h2931 antibody. Figure 8C is a graph showing the internalization of antibody or cell permeabilizer per cell, demonstrating that incubation with the h2931-CMIP4 cell permeabilizer increased internalization compared to untreated cells and cells treated with untagged h2931 antibody alone.

[0300] Example 10. HEK cells are Aβ 1-42 It stably expresses and forms Aβ aggregates. First, HEK cells were transduced with a lentiviral vector containing APP770sw[K670N-M671L_Swedish] at a MOI (Multiple Infection Intake) of 10. After transduction, non-transduced cells were eliminated using a dose-dependent puromycin selection process, and cells successfully incorporated into the lentiviral construct were enriched. To achieve stable expression of APPsw, lentiviral transduction and puromycin selection were performed two more times on viable cells. Subsequently, the cells were fixed, permeabilized, and stained with primary antibodies m3D6 anti-Aβ, CT695, and LN27 anti-APP. Secondary antibodies AF647 conjugate anti-mouse and AF568 conjugate anti-rabbit were used to conjugate the primary antibodies. High-content imaging was performed using the Operetta system with a 40x water immersion objective lens, and quantitative analysis was performed using Harmony software.

[0301] Figure 9A shows a high-content image of stable APPsw-HEK cells stained with either anti-Aβ antibody m3D6 (left) or anti-Aβ antibody CT695 (right). Cell bodies are shown as gray, rounded structures, Aβ aggregates are shown as diffused gray structures, and exemplary aggregates are identified by white arrows. Figure 9A shows that stable APPsw-HEK cells express a large amount of Aβ, resulting in aggregation. Figure 9B is a graph showing the percentage of stable APPsw-HEK cells with Aβ aggregates compared to untransfected HEK cells (control), as measured by either anti-Aβ antibody m3D6 (left) or anti-Aβ antibody CT695 (right). Under both measurements, approximately 80% of stable APPsw-HEK cells contain Aβ1-42 aggregates. Therefore, Figures 9A-B show that stable APPsw-HEK cells express Aβ and have substantial Aβ 1-42 This demonstrates that it leads to aggregation.

[0302] Example 11. The mouse-type CMIP-4 cell permeabilizer reduces the area and number of Aβ foci and is internalized by HEK cells. First, HEK cells were transduced with a lentiviral vector containing APP770sw[K670N-M671L_Swedish] at a MOI (Multiple Infection Intake) of 10. After transduction, non-transduced cells were eliminated using a dose-dependent puromycin selection process, and cells successfully incorporated into the lentiviral construct were enriched. To achieve stable expression of APPsw, lentiviral transduction and puromycin selection were performed two more times on viable cells. Subsequently, stable APPsw cells were separately exposed to untagged mzd-2931 and mzd-2931-CMIP4 cell permeabilizers, each incubated at a concentration of 100 μg / ml for 24 hours. The cells were then fixed, permeabilized, and stained with h2931 anti-Aβ antibody along with AF647 conjugate anti-mouse and AF568 conjugate anti-human secondary antibodies. Stained cells were imaged using high-content imaging (Operetta system) with a 40x water immersion objective lens. Quantitative analysis was performed using Harmony software.

[0303] Figure 10A is a graph showing the total Aβ focus area per cell, demonstrating that incubation with the mzd2931-CMIP4 cell permeabilizer (e.g., anti-Aβ cell permeabilizer) resulted in a smaller total Aβ focus area per cell in lentiAPPsw-transduced HEK-293 cells compared to untreated cells and cells treated with untagged mzd2931 antibody. Figure 10B is a graph showing the number of Aβ foci per cell, demonstrating that incubation with the mzd2931-CMIP4 cell permeabilizer resulted in a smaller number of Aβ focus areas per cell compared to untreated cells and cells treated with untagged mzd2931 antibody. Figure 10C is a graph showing the internalization of the cell permeabilizer per cell, demonstrating that incubation with the mzd2931-CMIP4 cell permeabilizer resulted in increased internalization of the cell permeabilizer compared to untreated cells and cells treated with untagged mzd2931 antibody. CMIP4 is ligated to the C-terminus of the light chain.

[0304] All cited publications (including GenBank accession numbers, UniProtKB / Swiss-Prot accession numbers, etc.), patents, and patent applications are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, and patent application were specifically and individually indicated to be incorporated by reference in their entirety for all purposes. Where there are any differences in the sequence relating to Genbank and UniProtKB / Swiss-Prot accession numbers, etc., this application refers to the sequence relating to the cited accession numbers as of the effective filing date of the application, which means the actual filing date, or the earlier date of the priority application disclosing the relevant accession numbers. Unless otherwise specified, any feature, step, element, embodiment, or aspect of this disclosure may be used in combination with any other. Although this disclosure has been described in detail with examples and illustrations for the purposes of clarity and understanding, it is clear that certain changes and modifications can be made within the scope of the appended claims.

[0305] Array Appendix Sequence ID 1: DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQRPGQSPQRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPWTFGQGTKVEIK Sequence ID 2: DVVMTQSPLSLPVTLGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIK Sequence ID 40: huIgG1 stationary ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 41: Hu Kappa Stationary RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 42: h2726_VH (variable weight) nucleotide sequence GAAGTGCAGCTTCTGGAGAGCGGGGGCGGCCTGGTGCAGCCGGGCGGATCCCTGAGACTGTCCTGTGCCGCGTCCGGTTTTACCTTCTCCAACTACGGAATGTCATGGGTCCGCCAAGCACCCGGAAAGGGATTGGAATGGGTGGCTTCGATCCGGTCCGGCTCGGGACGGACCTACT ACTCCGATAACGTCAAGGGCAGATTCACTATTAGCCGGGACAACAGCAAGAATACCCTGTACCTCCAAATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTATTACTGCGTGCGCTACGACCACTACTCGGGTTCCTCTGATTACTGGGGACAGGGGACCCTCGTGACTGTGTCAAGC Sequence ID 43: h2726_VL (Variable Light) Nucleotide Sequence GATGTCGTGATGACCCAGTCACCACTGTCCCTTCCTGTGACTCCCGGAGAACCGGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGCTCGATTATGACGGAAAGACCTACCTGAACTGGTTGCTCCAAAAGCCTGGGCAGAGCCCCCAGAGACTGATCTACAAAGTGTCCAACAGGGACTCGGGCGTGCCGGACCGCTTCTCGGGGTCCGGTTCCGGTACCGACTTTACGCTGAAGATCTCACGGGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGCACTCACTTCCCGCGGACCTTCGGACAAGGCACCAAGGTCGAGATCAAG SEQ ID NO: 44: h2931_VH (variable heavy) nucleotide sequence GAAGTGCAGCTCCTGGAGTCCGGGGGTGGACTGGTGCAGCCCGGGGGCAGCCTGAGGCTGAGCTGCGCCGCGTCAGGATTCACCTTCTCCAACTTCGGAATGTCCTGGGTCAGACAGGCCCCGGGAAAGGGCCTTGAATGGGTGGCTAGCGTGCGCTCCGGTTCCGGACGGACCTACTACTCGGACAACGTGAAGGGCCGGTTTACTATCTCCCGGGACAATTCGAAGAACACCCTGTACCTCCAAATGAACTCCTTGCGCGCCGAGGATACCGCAGTGTATTACTGCGTGCGCTACGACCACTACTCTGGCACTAGCGATTACTGGGGCCAGGGAACTCTGGTCACCGTGTCGTCA SEQ ID NO: 45: h2931_VL (variable light) nucleotide sequence GATGTCGTGATGACTCAGTCACCTCTGTCCCTGCCTGTGACCCTTGGGGAACCCGCCTCGATCTCGTGCAAGAGCTCCCAGAGCCTGCTCGACTATGATGGAAAGACCTACCTGAACTGGTTGCTCCAAAAGCCGGGCCAGAGCCCCCAGAGGCTGATCTACCGCGTGACCAACCGCGACACCGGGGTGCCGGACCGGTTCTCCGGATCCGGCAGCGGCACTGACTTCACCCTGAAAATTTCCAGAGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGTACTCACTTTCCACGGTCCTTCGGTCAAGGAACCAAGGTCGAGATCAAG SEQ ID NO: 46: h2731_VH (variable heavy) nucleotide sequence GAAGTGCAGCTTCTGGAGAGCGGGGGCGGCCTGGTGCAGCCGGGCGGATCCCTGAGACTGTCCTGTGCCGCGTCCGGTTTTACCTTCTCCAACTACGGAATGTCATGGGTCCGCCAAGCACCCGGAAAGGGATTGGAATGGGTGGCTTCGATCCGGTCCGGCTCGGGACGGACCTACTACTCCGATAACGTCAAGGGCAGATTCACTATTAGCCGGGACAACAGCAAGAATACCCTGTACCTCCAAATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTATTACTGCGTGCGCTACGACCACTACTCGGGTTCCTCTGATTACTGGGGACAGGGGACCCTCGTGACTGTGTCAAGC SEQ ID NO: 47: h2731_VL (variable light) nucleotide sequence GATGTCGTGATGACTCAGTCACCTCTGTCCCTGCCTGTGACCCTTGGGGAACCCGCCTCGATCTCGTGCAAGAGCTCCCAGAGCCTGCTCGACTATGATGGAAAGACCTACCTGAACTGGTTGCTCCAAAAGCCGGGCCAGAGCCCCCAGAGGCTGATCTACCGCGTGACCAACCGCGACACCGGGGTGCCGGACCGGTTCTCCGGATCCGGCAGCGGCACTGACTTCACCCTGAAAATTTCCAGAGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGTACTCACTTTCCACGGTCCTTCGGTCAAGGAACCAAGGTCGAGATCAAG SEQ ID NO: 48: h2831_VH (variable heavy) nucleotide sequence GAAGTGCAGCTGCTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGGGGATCCCTGCGGCTTTCCTGCGCCGCATCCGGCTTCACCTTTTCAAACTTCGGAATGTCGTGGGTCAGACAGGCCCCGGGAAAGGGTCTGGAATGGGTGGCCTCAGTGCGGTCCGGATCGGGTAGAACCTACTACAGCGATAACGTGAAGGGCCGGTTCACGATCTCCCGCGACAACTCCAAGAACACCCTGTACTTGCAAATGAATAGCCTCAGGGCTGAGGATACCGCGGTCTACTACTGTGTGCGCTATGACCACTACACTGGAACTAGCGACTACTGGGGCCAGGGGACCCTCGTGACTGTGTCGTCC SEQ ID NO: 49: h2831_VL (variable light) nucleotide sequence GATGTCGTGATGACTCAGTCACCTCTGTCCCTGCCTGTGACCCTTGGGGAACCCGCCTCGATCTCGTGCAAGAGCTCCCAGAGCCTGCTCGACTATGATGGAAAGACCTACCTGAACTGGTTGCTCCAAAAGCCGGGCCAGAGCCCCCAGAGGCTGATCTACCGCGTGACCAACCGCGACACCGGGGTGCCGGACCGGTTCTCCGGATCCGGCAGCGGCACTGACTTCACCCTGAAAATTTCCAGAGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGTACTCACTTTCCACGGTCCTTCGGTCAAGGAACCAAGGTCGAGATCAAG SEQ ID NO: 50: h2926_VH (variable heavy) nucleotide sequence GAAGTGCAGCTCCTGGAGTCCGGGGGTGGACTGGTGCAGCCCGGGGGCAGCCTGAGGCTGAGCTGCGCCGCGTCAGGATTCACCTTCTCCAACTTCGGAATGTCCTGGGTCAGACAGGCCCCGGGAAAGGGCCTTGAATGGGTGGCTAGCGTGCGCTCCGGTTCCGGACGGACCTACTACTCGGACAACGTGAAGGGCCGGTTTACTATCTCCCGGGACAATTCGAAGAACACCCTGTACCTCCAAATGAACTCCTTGCGCGCCGAGGATACCGCAGTGTATTACTGCGTGCGCTACGACCACTACTCTGGCACTAGCGATTACTGGGGCCAGGGAACTCTGGTCACCGTGTCGTCA SEQ ID NO: 51: h2926_VL (variable light) nucleotide sequence GATGTCGTGATGACCCAGTCACCACTGTCCCTTCCTGTGACTCCCGGAGAACCGGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGCTCGATTATGACGGAAAGACCTACCTGAACTGGTTGCTCCAAAAGCCTGGGCAGAGCCCCCAGAGACTGATCTACAAAGTGTCCAACAGGGACTCGGGCGTGCCGGACCGCTTCTCGGGGTCCGGTTCCGGTACCGACTTTACGCTGAAGATCTCACGGGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGCACTCACTTCCCGCGGACCTTCGGACAAGGCACCAAGGTCGAGATCAAG SEQ ID NO: 52: h4921G VH (variable heavy) nucleotide sequence GAGGTGCAGCTGCTGGAGTCGGGGGGGGGACTCGTGCAGCCCGGGGGCTCCCTGAGACTCTCTTGTGCCGCCTCCGGCTTCACTTTTTCAAACTTCGGAATGTCCTGGGTCCGCCAAGCACCGGGAAAGGGTCTGGAATGGGTCGCCAGCGTGCGGTCCGGCGGCGGACGGACTTACTACTCCGACAACGTGAAGGGCCGGTTCACCATCTCAAGGGATAACTCCAAGAATACTCTGTACTTGCAAATGAACTCGCTGCGCGCTGAAGATACCGCGGTGTACTATTGCGTGCGCTACGACCACTACTCCGGTACCAGCGACTACTGGGGACAGGGAACCCTTGTGACCGTGTCGAGC SEQ ID NO: 53: h4921G VL (variable light) nucleotide sequence GATGTCGTGATGACTCAGTCGCCCCTCTCCCTGCCTGTGACTCTGGGGGAACCCGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGTTGGACTCAGACGGAAAGACCTACCTTAACTGGCTGCTGCAAAAGCCAGGACAGAGCCCGCAGAGGCTGATCTACCGCGTGACCAACCGGGATACGGGAGTGCCGGACAGATTCAGCGGCTCGGGTTCCGGCACCGACTTCACCCTCAAAATCTCCCGCGTCGAGGCCGAGGACGTGGGCGTGTATTACTGTTGGCAGGGAACCCACTTTCCTCGGACCTTCGGTCAAGGGACTAAGGTCGAAATCAAG SEQ ID NO: 54: h2826 VH (variable heavy) nucleotide sequence GAAGTGCAGCTGCTGGAGTCTGGCGGCGGACTGGTGCAGCCCGGGGGATCCCTGCGGCTTTCCTGCGCCGCATCCGGCTTCACCTTTTCAAACTTCGGAATGTCGTGGGTCAGACAGGCCCCGGGAAAGGGTCTGGAATGGGTGGCCTCAGTGCGGTCCGGATCGGGTAGAACCTACTACAGCGATAACGTGAAGGGCCGGTTCACGATCTCCCGCGACAACTCCAAGAACACCCTGTACTTGCAAATGAATAGCCTCAGGGCTGAGGATACCGCGGTCTACTACTGTGTGCGCTATGACCACTACACTGGAACTAGCGACTACTGGGGCCAGGGGACCCTCGTGACTGTGTCGTCC SEQ ID NO: 55: h2826 VL (variable light) nucleotide sequence GATGTCGTGATGACCCAGTCACCACTGTCCCTTCCTGTGACTCCCGGAGAACCGGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGCTCGATTATGACGGAAAGACCTACCTGAACTGGTTGCTCCAAAAGCCTGGGCAGAGCCCCCAGAGACTGATCTACAAAGTGTCCAACAGGGACTCGGGCGTGCCGGACCGCTTCTCGGGGTCCGGTTCCGGTACCGACTTTACGCTGAAGATCTCACGGGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGCACTCACTTCCCGCGGACCTTCGGACAAGGCACCAAGGTCGAGATCAAG SEQ ID NO: 56: h2929 VH (variable heavy) nucleotide sequence GAAGTGCAGCTCCTGGAGTCCGGGGGTGGACTGGTGCAGCCCGGGGGCAGCCTGAGGCTGAGCTGCGCCGCGTCAGGATTCACCTTCTCCAACTTCGGAATGTCCTGGGTCAGACAGGCCCCGGGAAAGGGCCTTGAATGGGTGGCTAGCGTGCGCTCCGGTTCCGGACGGACCTACTACTCGGACAACGTGAAGGGCCGGTTTACTATCTCCCGGGACAATTCGAAGAACACCCTGTACCTCCAAATGAACTCCTTGCGCGCCGAGGATACCGCAGTGTATTACTGCGTGCGCTACGACCACTACTCTGGCACTAGCGATTACTGGGGCCAGGGAACTCTGGTCACCGTGTCGTCA SEQ ID NO: 57: h2929 VL (variable light) nucleotide sequence GATGTCGTGATGACCCAAAGCCCCCTGTCCCTCCCTGTGACTCCTGGAGAGCCGGCGTCCATTTCCTGCCGGTCAAGCCAGTCCTTGGTGGACTACGACGGAAAGACCTACCTCAACTGGCTGCTGCAGCGCCCCGGGCAGTCGCCGCAGCGGCTTATCTACAAAGTGTCCAACCGCGACTCGGGCGTGCCGGATAGGTTTTCGGGTTCCGGAAGCGGCACCGACTTCACCCTGAAAATCTCCAGAGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGTTCTCACTTCCCACGGTCATATGGCCAAGGGACTAAGGTCGAAATCAAG SEQ ID NO: 58: h3818G VH (variable heavy) nucleotide sequence GAAGTGCAGCTCCTGGAGTCCGGCGGTGGACTGGTGCAGCCGGGCGGATCCCTGAGACTGTCCTGCGCCGCGTCGGGCTTTACTTTCGCAAATTACGGCATGAGCTGGGTCAGACAGGCCCCCGGGAAGGGTCTGGAATGGGTGGCCAGCGTCCGGAGCGGGGGATCCCGGACCTATTACTCCGACAACGTGAAGGGCCGCTTCACCATCTCAAGGGACAACTCCAAGAACACCCTGTACTTGCAAATGAACAGCCTTCGGGCTGAGGATACTGCCGTGTACTACTGCGTGCGCTACGACCACTACTCCGGATCCTCGGATTACTGGGGACAGGGAACCCTCGTGACCGTGTCATCG SEQ ID NO: 59: h3818G VL (variable light) nucleotide sequence GATGTCGTGATGACTCAGTCGCCCCTCTCCCTGCCTGTGACTCTGGGGGAACCCGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGATGGACACCGACGGAAAGACCTACCTTAACTGGCTGCTGCAAAAGCCAGGACAGAGCCCGCAGAGGCTGATCTACAAAGTGTCAAACCGGGAGTCCGGAGTGCCGGACAGATTCAGCGGCTCGGGTTCCGGCACCGACTTCACCCTCAAAATCTCCCGCGTCGAGGCCGAGGACGTGGGCGTGTATTACTGTTGGCAGGGAACCCACTTTCCTCGGACCTTCGGTCAAGGGACTAAGGTCGAAATCAAG SEQ ID NO: 60: h2927 VH (variable heavy) nucleotide sequence GAAGTGCAGCTCCTGGAGTCCGGGGGTGGACTGGTGCAGCCCGGGGGCAGCCTGAGGCTGAGCTGCGCCGCGTCAGGATTCACCTTCTCCAACTTCGGAATGTCCTGGGTCAGACAGGCCCCGGGAAAGGGCCTTGAATGGGTGGCTAGCGTGCGCTCCGGTTCCGGACGGACCTACTACTCGGACAACGTGAAGGGCCGGTTTACTATCTCCCGGGACAATTCGAAGAACACCCTGTACCTCCAAATGAACTCCTTGCGCGCCGAGGATACCGCAGTGTATTACTGCGTGCGCTACGACCACTACTCTGGCACTAGCGATTACTGGGGCCAGGGAACTCTGGTCACCGTGTCGTCA SEQ ID NO: 61: h2927 VL (variable light) nucleotide sequence GATGTCGTGATGACTCAGTCACCGCTCTCCCTCCCTGTGACCCCGGGCGAACCAGCGTCGATCTCCTGCAAGAGCAGCCAATCATTGCTGGACTACGACGGAAAGACCTATCTTAACTGGCTGCTGCAGAAGCCCGGGCAGAGCCCGCAGCGCCTGATCTACAAAGTGTCCAACAGAGACTCCGGAGTGCCTGATAGGTTCTCGGGTTCCGGCTCCGGTACCGACTTCACTCTGAAAATTTCCCGGGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGCACCCACTTCCCCCGGTCGTTTGGACAAGGGACCAAGGTCGAGATCAAG SEQ ID NO: 62: h49K3G VH (variable heavy) nucleotide sequence GAGGTGCAGCTGCTGGAGTCGGGGGGGGGACTCGTGCAGCCCGGGGGCTCCCTGAGACTCTCTTGTGCCGCCTCCGGCTTCACTTTTTCAAACTTCGGAATGTCCTGGGTCCGCCAAGCACCGGGAAAGGGTCTGGAATGGGTCGCCAGCGTGCGGTCCGGCGGCGGACGGACTTACTACTCCGACAACGTGAAGGGCCGGTTCACCATCTCAAGGGATAACTCCAAGAATACTCTGTACTTGCAAATGAACTCGCTGCGCGCTGAAGATACCGCGGTGTACTATTGCGTGCGCTACGACCACTACTCCGGTACCAGCGACTACTGGGGACAGGGAACCCTTGTGACCGTGTCGAGC SEQ ID NO: 63: h49K3G VL (variable light) nucleotide sequence GATGTCGTGATGACTCAGTCGCCCCTCTCCCTGCCTGTGACTCTGGGGGAACCCGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGTTGGACTCAGACGGAAAGACCTACCTTAACTGGCTGCTGCAAAAGCCAGGACAGAGCCCGCAGAGGCTGATCTACAAAGTGTCAAACCGGGATTCCGGAGTGCCGGACAGATTCAGCGGCTCGGGTTCCGGCACCGACTTCACCCTCAAAATCTCCCGCGTCGAGGCCGAGGACGTGGGCGTGTATTACTGTTGGCAGGGAACCCACTTTCCTCGGACCTTCGGTCAAGGGACTAAGGTCGAAATCAAG SEQ ID NO: 64: h4917G VH (variable heavy) nucleotide sequence GAGGTGCAGCTGCTGGAGTCGGGGGGGGGACTCGTGCAGCCCGGGGGCTCCCTGAGACTCTCTTGTGCCGCCTCCGGCTTCACTTTTTCAAACTTCGGAATGTCCTGGGTCCGCCAAGCACCGGGAAAGGGTCTGGAATGGGTCGCCAGCGTGCGGTCCGGCGGCGGACGGACTTACTACTCCGACAACGTGAAGGGCCGGTTCACCATCTCAAGGGATAACTCCAAGAATACTCTGTACTTGCAAATGAACTCGCTGCGCGCTGAAGATACCGCGGTGTACTATTGCGTGCGCTACGACCACTACTCCGGTACCAGCGACTACTGGGGACAGGGAACCCTTGTGACCGTGTCGAGC SEQ ID NO: 65: h4917G VL (variable light) nucleotide sequence GATGTCGTGATGACTCAGTCGCCCCTCTCCCTGCCTGTGACTCTGGGGGAACCCGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGTTGGACTCAGACGGAAAGACCTACCTTAACTGGCTGCTGCAAAAGCCAGGACAGAGCCCGCAGAGGCTGATCTACAAAGTGACCAACCGGGAGTCCGGAGTGCCGGACAGATTCAGCGGCTCGGGTTCCGGCACCGACTTCACCCTCAAAATCTCCCGCGTCGAGGCCGAGGACGTGGGCGTGTATTACTGTTGGCAGGGAACCCACTTTCCTCGGTCATTCGGTCAAGGGACTAAGGTCGAAATCAAG SEQ ID NO: 66: h2727 VH (variable heavy) nucleotide sequence GAAGTGCAGCTTCTGGAGAGCGGGGGCGGCCTGGTGCAGCCGGGCGGATCCCTGAGACTGTCCTGTGCCGCGTCCGGTTTTACCTTCTCCAACTACGGAATGTCATGGGTCCGCCAAGCACCCGGAAAGGGATTGGAATGGGTGGCTTCGATCCGGTCCGGCTCGGGACGGACCTACTACTCCGATAACGTCAAGGGCAGATTCACTATTAGCCGGGACAACAGCAAGAATACCCTGTACCTCCAAATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTATTACTGCGTGCGCTACGACCACTACTCGGGTTCCTCTGATTACTGGGGACAGGGGACCCTCGTGACTGTGTCAAGC SEQ ID NO: 67: h2727 VL (variable light) nucleotide sequence GATGTCGTGATGACTCAGTCACCGCTCTCCCTCCCTGTGACCCCGGGCGAACCAGCGTCGATCTCCTGCAAGAGCAGCCAATCATTGCTGGACTACGACGGAAAGACCTATCTTAACTGGCTGCTGCAGAAGCCCGGGCAGAGCCCGCAGCGCCTGATCTACAAAGTGTCCAACAGAGACTCCGGAGTGCCTGATAGGTTCTCGGGTTCCGGCTCCGGTACCGACTTCACTCTGAAAATTTCCCGGGTGGAAGCCGAGGACGTGGGAGTGTACTACTGTTGGCAGGGCACCCACTTCCCCCGGTCGTTTGGACAAGGGACCAAGGTCGAGATCAAG SEQ ID NO: 68: h4918G VH (variable heavy) nucleotide sequence GAGGTGCAGCTGCTGGAGTCGGGGGGGGGACTCGTGCAGCCCGGGGGCTCCCTGAGACTCTCTTGTGCCGCCTCCGGCTTCACTTTTTCAAACTTCGGAATGTCCTGGGTCCGCCAAGCACCGGGAAAGGGTCTGGAATGGGTCGCCAGCGTGCGGTCCGGCGGCGGACGGACTTACTACTCCGACAACGTGAAGGGCCGGTTCACCATCTCAAGGGATAACTCCAAGAATACTCTGTACTTGCAAATGAACTCGCTGCGCGCTGAAGATACCGCGGTGTACTATTGCGTGCGCTACGACCACTACTCCGGTACCAGCGACTACTGGGGACAGGGAACCCTTGTGACCGTGTCGAGC SEQ ID NO: 69: h4918G VL (variable light) nucleotide sequence GATGTCGTGATGACTCAGTCGCCCCTCTCCCTGCCTGTGACTCTGGGGGAACCCGCGTCCATTTCGTGCAAGAGCAGCCAGTCCCTGATGGACACCGACGGAAAGACCTACCTTAACTGGCTGCTGCAAAAGCCAGGACAGAGCCCGCAGAGGCTGATCTACAAAGTG TCAAACCGGGAGTCCGGAGTGCCGGACAGATTCAGCGGCTCGGGTTCCGGCACCGACTTCACCCTCAAAATCTCCCGCGTCGAGGCCGAGGACGTGGGCGTGTATTACTGTTGGCAGGGAACCCACTTTCCTCGGACCTTCGGTCAAGGGACTAAGGTCGAAATCAAG Sequence ID 70: Aducanumab heavy chain: QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWFDGTKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDV WGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 71: Aducanumab light chain: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 72: Bapineuzumab HC (heavy chain) EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 73: Bapineuzumab VH (variable weight) EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASIRSGGGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYSGSSDYWGQGTLVTVSS Sequence ID 16: VH CDR1 GFTFSNYGMS Sequence ID 17: VH CDR2 SIRSGGGRTYYSNDYNVKG Sequence ID 18: VH CDR3 YDHYSGSSDY Sequence ID 77: Bapineuzumab LC (light chain) DVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 78: Bapineuzumab VL (Variable Mild) DVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPRTFGQGTKVEIK Sequence ID 26: VL CDR1 KSSQSLLDSDGKTYLN Sequence ID 27: VL CDR2 LVSSKLDS Sequence ID 28: VL CDR3 WQGTHFPRT Sequence ID 82: Gantenerumab HC amino acid sequence: QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFD VWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 83: Gantenerumab LC amino acid sequence: DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 84: Amyloid beta (Aβ) 1-42: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA Sequence ID 85: Amyloid-beta (Aβ) precursor protein: MLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHMNVQNGKWDSDPSGTKTCIDTKEGILQYCQEVYPELQITNVVEANQPVTIQNWCKRGRKQCKTHPHFVIPYRCLVGEFVSDALLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGVEFVCCPLAEE SDNVDSADAEEDDSDVWWGGADTDYADGSEDKVVEVAEEEEVAEVEEEEADDDEDDEDGDEVEEEAEEPYEEATERTTSIATTTTTTTESVEEVVREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSAMSQSLLKTTQEPLARDPVKLPTTAASTPDAVDKYLETPGDE NEHAHFQKAKERLEAKHRERMSQVMREWEEAERQAKNLPKADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLNDRRRLALENYITALQAVPPRPRHVFNMLKKYVRAEQKDRQHTLKHFEHVRMVDPKKAAQIRSQVMTHLRVIYERMNQSLSLLYNVPAVAEEIQDEVDELLQKEQNYSDDVL ANMISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPANTENENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQQNGYENPTYKFFEQMQN Sequence ID 86:huIgG1 constant nucleotide sequence GCCAGCACTAAGGGGCCTAGCGTCTTTCCGCTGGCCCCGTCCTCCAAGTCCACTTCGGGTGGAACCGCGGCACTGGGGTGCCTCGTGAAGGACTACTTCCCCGAGCCGGTCACCGTGTCCTGGAACTCGGGAGCCCTGACCTCCGGAGTGCATACTTTCCCTGCGGTGCTGCAGTCCTCCGGGCTCTACTCGCTGTCAAGCGTGGTCACCGTCCCGAGCTCATCCCTGGGTACTCAGACCTACATTTGCAACGTGAACCACAAACCTTCCAACACCAAGGTCGACAAGAAAGTGGAGCCTAAGAGCTGCGACAAGACCCACACCTGTCCCCCGTGTCCCGCCCCTGAGCTGCTGGGCGGCCCCAGCGTGTTCCTCTTCCCGCCTAAGCCGAAGGACACTCTGATGATCTCGAGAACCCCTGAAGTGACCTGTGTGGTGGTGGATGTGTCCCACGAGGATCCGGAAGTGAAGTTCAATTGGTACGTGGACGGAGTGGAAGTCCATAACGCCAAGACCAAGCCCCGCGAGGAACAGTACAACTCAACTTACCGGGTGGTGTCAGTGCTGACCGTGCTGCACCAAGATTGGCTGAACGGGAAGGAGTACAAGTGCAAAGTCTCCAACAAGGCGCTGCCGGCCCCCATTGAAAAGACCATCAGCAAGGCTAAGGGCCAGCCCCGGGAACCACAGGTCTACACCTTGCCCCCTTCCCGGGAGGAAATGACCAAGAACCAAGTGTCGCTGACGTGCCTGGTCAAGGGCTTTTATCCATCTGACATCGCCGTGGAGTGGGAAAGCAACGGCCAGCCGGAAAACAACTACAAGACTACCCCGCCTGTGCTGGACTCCGACGGCTCGTTCTTCCTGTATTCCAAGCTCACCGTGGATAAGTCCAGATGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGATGCATGAGGCCCTGCACAACCACTACACTCAGAAATCACTGTCCCTTTCCCCCGGAAAGTAA Sequence ID 87: Hu-Kappa constant nucleotide sequence CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA SEQ ID NO: 347 h2931 untagged antibody heavy chain [ka] Sequence ID 348 h2931 untagged antibody light chain [ka] Sequence ID 349 h2931_Ab_LC-Ct-CMIP4 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSNFGMSWVRQAPGKGLEWVASVRSGSGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYTGTSDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 350 h2931_Ab_LC-Ct-CMIP4 light chain [ka] Sequence ID 351 h2931_Ab_LC-Ct-L17E-M Lycotoxin Heavy Chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSNFGMSWVRQAPGKGLEWVASVRSGSGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYTGTSDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 352 h2931_Ab_LC-Ct-L17E-M lycotoxin light chain [ka] Sequence ID 353 h2931_Ab_LC-Ct-cycR8 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSNFGMSWVRQAPGKGLEWVASVRSGSGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYTGTSDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 354 h2931_Ab_LC-Ct-cycR8 light chain [ka] Sequence ID 355 h2931_Ab_LC-Ct-cycTAT heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSNFGMSWVRQAPGKGLEWVASVRSGSGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYTGTSDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 356 h2931_Ab_LC-Ct-cycTAT light chain [ka] Sequence ID 357 h2931_Ab_LC-Ct-R8 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSNFGMSWVRQAPGKGLEWVASVRSGSGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYTGTSDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 358 h2931_Ab_LC-Ct-R8 light chain [ka] Sequence ID 359 h2931_Ab_LC-Ct-TAT heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSNFGMSWVRQAPGKGLEWVASVRSGSGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYTGTSDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 360 h2931_Ab_LC-Ct-TAT light chain [ka] Sequence ID 361 h2931_Ab_HC-H433A_LC-Ct-CMIP4 (Effector Function Null Cell Permeation Agent) Heavy Chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSNFGMSWVRQAPGKGLEWVASVRSGSGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYTGTSDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALANHYTQKSLSLSPGK Sequence ID 362 h2931_Ab_HC-H433A_LC-Ct-CMIP4 (Effector Function Null Cell Permeation Agent) Light Chain [ka] Sequence ID 363: mzd-2931-CMIP4 heavy chain EVQLVESGGGLVKPGGSLKLSCAASGFTFSNFGMSWVRQAPEKGLEWVASVRSGSGRTYYSDNVKGRFTISRDNAKNTLFLQMTSLRSEDTALYYCVRYDHYTGTSDYWGQG TSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIK PCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIER TISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK Sequence ID 364 mzd-2931-CMIP4 light chain [ka] Sequence ID 365 h2931-CMIP4-LALA heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSNFGMSWVRQAPGKGLEWVASVRSGSGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYTGTSDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* Sequence ID 366 h2931-CMIP4-LALA Light Chain [ka] Sequence ID 367 h2931-CMIP4-H310A_H435Q Heavy Chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSNFGMSWVRQAPGKGLEWVASVRSGSGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYTGTSDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPGK* Sequence ID 368 h2931-CMIP4-H310A_H435Q Light Chain [ka] Sequence ID 369 h2931-CMIP4-1 Light Chain DVVMTQSPLSLPVTLGEPA [ka] Sequence ID 370 h2931-CMIP4-2 Light Chain [ka] Sequence ID 371 h2931-CMIP4-3 Light Chain [ka] Sequence ID 372 h2931-CMIP4-4 Light Chain [ka] Sequence ID 373 h2931-CMIP4-5 Light Chain [ka] Sequence ID 374 h2931-CMIP4-6 Light Chain [ka] Sequence ID 375 h2931-CMIP4-7 Light Chain [ka] Sequence ID 376 h2931-CMIP4-8 Light Chain [ka] Sequence ID 377 h2931-CMIP4-9 Light Chain [ka] Sequence ID 378 h2931-CMIP4-10 Light Chain [ka] Sequence ID 379 h2931-CMIP4-11 Light Chain [ka] Sequence ID 380 h2931-CMIP4-12 Light Chain [ka] Sequence ID 381 h2931-CMIP4-13 Light Chain [ka] Sequence ID 382 h2931-CMIP4-14 Light Chain [ka] Sequence ID 383 h2931-CMIP4-15 Light Chain [ka] Sequence ID 384 h2931-CMIP4-16 Light Chain [ka] Sequence ID 385 h2931-CMIP4-17 Light Chain [ka] Sequence ID 386 h2931-CMIP4-18 Light Chain [ka] Sequence ID 387 h2931-CMIP4-19 Light Chain [ka] Sequence ID 388 h2931-CMIP4-20 Light Chain [ka] Sequence ID 389 h2931-CMIP4-21 Light Chain [ka] Sequence ID 390 h2931-CMIP4-22 Light Chain [ka] Sequence ID 391 h2931-CMIP4-23 Light Chain [ka] Sequence ID 392 h2931-CMIP4-24 Light Chain [ka] Sequence ID 393 h2931-CMIP4-25 Light Chain [ka] SEQ ID NO: 394 H2931_Heavy Chain (Common to all CMIP4-1-25 (SEQ ID NOs: 369-393)) EVQLLESGGGLVQPGGSLRLSCAASGFTFSNFGMSWVRQAPGKGLEWVASVRSGSGRTYYSDNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRYDHYTGTSDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The following applies to sequence numbers 347-394. The linker array is shown in bold font. The intracellular relocation module is indicated by a double underline. The steady-state domain is indicated by the underline. Sequence ID 395 Formula IA IX 2 -X 3 -TALX 7 -FX 9 -GX 11-AAX 14 -KX 16 -EAX 19 -QFLX 23 -X 24 -L Sequence ID 396 Formula IB IWX 3 -X 4 -ALX 7 -FX 9 -GX 11 -X 12 -X 13 -AX 15 -AEAX 19 -X 20 -FX 22 -SX 24 -L Sequence ID 397 Type IC X 1 -X 2 -LTX 5 -LKX 8 -SX 10 -KAAAX 15 -AEAKQX 21 -LSX 24 -L Sequence ID 398 IWLTX 5 -X 6 -KFSX 10 -KX 12 -AAKAX 17 -X 18 -KQFLX 23 -X 24 -X 25 Sequence ID 399, Formula (IIA) IX 2 -X 3 -TALX 7 -FX 9 -GX 11 -AAX 14 -KX 16 -EAX 19 -QFLX 23 -X 24 -L Sequence ID 400, Equation (IIB) IWX 3 -X 4 -ALX 7 -FX 9 -GX 11 -X 12 -X 13 -AX 15 -AEAX 19 -QFX 22 -SX 24 -L Sequence ID 401 (Formula IIC) [ka]

Claims

1. An anti-amyloid-beta peptide (Aβ) cell permeabilizer (CPA) comprising (i) an intracellular location module (CIM), and (ii) an antibody that specifically binds to an amyloid-beta (Aβ) peptide.

2. The anti-Aβ CPA according to claim 1, wherein the CIM comprises a cell membrane internalized peptide (CMIP).

3. The aforementioned CMIP is given by formulas (IA) to (ID): I-X ２ -X ３ -T-A-L-X ７ -F-X ９ -G-X １１ -A-A-X １４ -K-X １６ -E-A-X １９ -Q-F-L-X ２３ -X ２４ -L(I-A) (SEQ ID NO: 395), I-W-X ３ -X ４ -A-L-X ７ -F-X ９ -G-X １１ -X １２ -X １３ -A-X １５ -A-E-A-X １９ -X ２０ -F-X ２２ -S-X ２４ -L(I-B) (Sequence ID 396), X １ -X ２ -L-T-X ５ -L-K-X ８ -S-X １０ -K-A-A-A-X １５ -A-E-A-K-Q-X ２１ -L-S-X ２４ -L(I-C)(SEQ ID NO: 397), I-W-L-T-X ５ -X ６ -K-F-S-X １０ -K-X １２ -A-A-K-A-X １７ -X １８ -K-Q-F-L-X ２３ -X ２４ -X ２５ An amino acid sequence selected from (I-D) (SEQ ID NO: 398), During the ceremony, X １ However, these are hydrophobic amino acid residues or positively charged amino acid residues. X ２ However, these are aromatic amino acid residues, positively charged amino acid residues, or negatively charged amino acid residues. X ３ However, these are aromatic amino acid residues, hydrophobic amino acid residues, positively charged amino acid residues, or negatively charged amino acid residues. X ４ However, it is a neutral hydrophilic amino acid residue or an aromatic amino acid residue. X ５ However, it is a hydrophobic amino acid residue or an aromatic amino acid residue, X ６ However, it is a hydrophobic amino acid residue, X ７ However, these are hydrophobic amino acid residues or positively charged amino acids. X ８ However, it is an aromatic amino acid residue, X ９ However, these are neutral hydrophilic amino acid residues, aromatic amino acid residues, or positively charged amino acid residues. X １０ However, these are neutral hydrophilic amino acid residues, aromatic amino acid residues, or positively charged amino acid residues. X １１ However, these are neutral hydrophilic amino acid residues, hydrophobic amino acid residues, or positively charged amino acid residues. X １２ However, these are neutral hydrophilic amino acid residues, hydrophobic amino acid residues, or positively charged amino acid residues. X １３ However, it is a neutral hydrophilic amino acid residue or a hydrophobic amino acid residue. X １４ However, these are hydrophobic amino acid residues or positively charged amino acids. X １５ However, these are aromatic amino acid residues, positively charged amino acid residues, or negatively charged amino acid residues. X １６ However, these are hydrophobic amino acid residues, aromatic amino acid residues, positively charged amino acid residues, or negatively charged amino acid residues. X １７ However, these are positively charged amino acid residues or negatively charged amino acid residues. X １８ However, these are hydrophobic amino acid residues, aromatic amino acid residues, or positively charged amino acid residues. X １９ However, these are hydrophobic amino acid residues or positively charged amino acid residues. X ２０ However, these are neutral hydrophilic amino acid residues or negatively charged amino acid residues. X ２１ However, it is a hydrophobic amino acid residue or an aromatic amino acid residue, X ２２ However, it is absent, hydrophobic amino acid residue, aromatic amino acid residue, or positively charged amino acid residue. X ２３ However, it is absent, a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively charged amino acid residue. X ２４ However, it is absent, an aromatic amino acid residue, or a positively charged amino acid residue. X ２５ The anti-Aβ CPA according to claim 2, comprising the amino acid sequence, or a pharmaceutically acceptable salt thereof, wherein the amino acid is absent or hydrophobic.

4. The anti-Aβ CPA according to claim 1 or 2, wherein the CIM comprises a wild-type M-lycotoxin peptide.

5. The anti-Aβ CPA according to claim 1 or 2, wherein the CIM comprises an M-lycotoxin derivative.

6. The anti-Aβ CPA according to claim 1 or 2, wherein the CIM comprises a polyarginine amino acid sequence.

7. The anti-Aβ CPA according to any one of claims 1, 2, and 6, wherein the CIM comprises a plurality of polyarginine amino acid sequences.

8. The anti-Aβ CPA according to claim 1 or 2, wherein the CIM comprises a TAT amino acid sequence.

9. The anti-Aβ CPA according to any one of claims 1, 2, and 8, wherein the CIM comprises a plurality of TAT amino acid sequences.

10. The anti-Aβ CPA according to any one of claims 1 to 9, wherein the CIM comprises a macrocyclic molecule.

11. The anti-Aβ CPA according to claim 10, wherein the macrocyclic molecule is formed by a covalent bond between two amino acid residues of the CIM.

12. The anti-Aβ CPA according to claim 10 or 11, wherein the macrocyclic molecule is formed by a disulfide bond between two cysteine ​​residues of the CIM.

13. The anti-Aβ according to any one of claims 1 to 2 or 4 to 12, wherein the CIM comprises a polypeptide having an amino acid sequence selected from any one of sequence numbers 101 to 107, 110, and 116 to 338.

14. The anti-Aβ CPA according to any one of claims 1 to 13, wherein the CIM further comprises one or more spacer regions.

15. The anti-Aβ CPA according to claim 14, wherein at least one of the one or more spacer regions comprises one or more amino acid residues.

16. The anti-Aβ CPA according to claim 14 or 15, wherein at least one of the one or more spacer regions comprises one or more glycine residues.

17. The anti-Aβ CPA according to claim 1, wherein the CIM is a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 101 to 338.

18. The anti-Aβ CPA according to any one of claims 1 to 17, wherein the CMIP is covalently bound to the antibody.

19. The anti-Aβ CPA according to any one of claims 1 to 17, wherein the CMIP is non-covalently bound to the antibody.

20. The anti-Aβ CPA according to any one of claims 1 to 19, wherein the CPA further comprises a linker that connects the CIM to the antibody.

21. The anti-Aβ CPA according to claim 20, wherein the linker is covalently bound to both the CIM and the antibody.

22. The anti-Aβ CPA according to claim 20 or 21, wherein the linker is a severable linker.

23. The anti-Aβ CPA according to claim 20 or 21, wherein the linker is an inseparable linker.

24. The anti-Aβ CPA according to any one of claims 20 to 23, wherein the linker comprises a polypeptide.

25. The anti-Aβ CPA according to any one of claims 20 to 24, wherein the linker comprises one or more glycine residues.

26. The anti-Aβ CPA according to any one of claims 20 to 25, wherein the linker is a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 343 to 346.

27. The anti-Aβ CPA according to any one of claims 1 to 26, wherein the antibody is linked to the C-terminus of the CIM.

28. The anti-Aβ CPA according to any one of claims 1 to 26, wherein the antibody is linked to the N-terminus of the CIM.

29. The anti-Aβ CPA according to any one of claims 1 to 28, wherein the antibody comprises a heavy chain or a portion thereof.

30. The anti-Aβ CPA according to claim 29, wherein the CIM is covalently bonded to the C-terminus of the heavy chain.

31. The anti-Aβ CPA according to claim 29, wherein the CIM is covalently bonded to the N-terminus of the heavy chain.

32. The anti-Aβ CPA according to any one of claims 1 to 31, wherein the antibody comprises a light chain or a portion thereof.

33. The anti-Aβ CPA according to claim 32, wherein the CIM is covalently bonded to the C-terminus of the light chain.

34. The anti-Aβ CPA according to claim 32, wherein the CIM is covalently bonded to the N-terminus of the light chain.

35. The antibody competes for binding to a reference antibody comprising heavy chain variable domains including heavy chain CDR1, CDR2, and CDR3, and light chain variable domains including light chain CDR1, CDR2, and CDR3, wherein the heavy chains CDR1, CDR2, and CDR3, and the light chains CDR1, CDR2, and CDR3 are as shown for one of the antibodies in Tables 1A and 1B, according to any one of claims 1 to 34.

36. The anti-Aβ CPA according to any one of claims 1 to 35, wherein the antibody comprises a heavy chain variable domain including heavy chain CDR1, CDR2, and CDR3, and a light chain variable domain including light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR1, CDR2, and CDR3, and the light chain CDR1, CDR2, and CDR3 are as shown for one of the antibodies in Tables 1A and 1B.

37. The anti-Aβ CPA according to any one of claims 1 to 36, wherein the antibody comprises a heavy chain variable domain as shown for one of the antibodies in Table 1A and Table 1B.

38. The anti-Aβ CPA according to any one of claims 1 to 37, wherein the antibody comprises a light chain variable domain as shown for one of the antibodies in Table 1A and Table 1B.

39. The antibody comprises a heavy chain variable domain including heavy chain CDR1, CDR2, and CDR3, and a light chain variable domain including light chain CDR1, CDR2, and CDR3, where, The heavy chain CDR1 includes one of sequence numbers 16, 19, or 20, The heavy chain CDR2 includes one of sequence numbers 20, 21, 22, or 23, The heavy chain CDR3 comprises one of sequence numbers 18, 24, or 25. The light chain CDR1 includes one of sequence numbers 26, 29, 31, or 32. The light chain CDR2 includes one of sequence numbers 33, 34, 35, or 36. The anti-Aβ CPA according to any one of claims 1 to 35, wherein the light chain CDR3 comprises one of sequence numbers 28, 38, or 39.

40. The anti-Aβ CPA according to claim 35 or claim 39, wherein the heavy chain variable domain is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, 6, and 7, excluding the CDR, and the light chain variable domain is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15, excluding the CDR.

41. The anti-Aβ CPA according to claim 40, wherein the heavy chain variable domain is at least 98% identical to an amino acid sequence selected from SEQ ID NOs: 3, 4, 5, 6, and 7, excluding the CDR, and the light chain variable domain is at least 98% identical to an amino acid sequence selected from SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, and 15, excluding the CDR.

42. The anti-Aβ CPA according to claim 41, wherein the heavy chain variable domain is selected from sequence numbers 3, 4, 5, 6, and 7, and the light chain variable domain is selected from sequence numbers 8, 9, 10, 11, 12, 13, 14, and 15.

43. The antibody comprises a heavy chain variable domain including heavy chain CDR1, CDR2, and CDR3, and a light chain variable domain including light chain CDR1, CDR2, and CDR3, where, The heavy chain CDR1 has the amino acid sequence GTFFSNX Ａ GMS (wherein, X Ａ (is Y or F) (Sequence ID 88) The heavy chain CDR2 has the amino acid sequence SX Ａ RSGSGRTYYSDNVKG (in the formula, Ａ (is I or V) (Sequence ID 89) The heavy chain CDR3 has the amino acid sequence YDHYX Ａ GX Ｂ SDY (in the formula, X Ａ is S or T, X Ｂ (is S or T) (Sequence ID 90) The light chain CDR1 comprises the amino acid sequence KSSQSLLDYDGKTYLN (SEQ ID NO: 91), The light chain CDR2 has amino acid sequence X Ａ VX Ｂ NRDX Ｃ (In the formula, X Ａ is K or R, and X Ｂ is S or T, X Ｃ (is S or T) (Sequence ID 92) The light chain CDR3 has the amino acid sequence WQGTHFPRX Ａ (In the formula, X Ａ The anti-Aβ CPA according to any one of claims 1 to 35, comprising (Sequence ID 93) (where is S or T).

44. The light chain CDR3 is WQGTHFPRX Ａ FX Ｂ (In the formula, X Ａ is S or T, X Ｂ The anti-Aβ CPA according to claim 43, comprising (where is F or Y) (Sequence ID 94).

45. The antibody comprises a heavy chain variable domain including heavy chain CDR1, CDR2, and CDR3, and a light chain variable domain including light chain CDR1, CDR2, and CDR3, where, The heavy chain CDR1 has the amino acid sequence GFTFX Ａ NX Ｂ GMS (wherein, X Ａ is S or A, X Ｂ (is Y or F) (Sequence ID 95) said heavy chain CDR2 comprises the amino acid sequence SX Ａ RSGX Ｂ X Ｃ RTYYSDNVKG (wherein X Ａ is I or V, X Ｂ is S or G, and X Ｃ is S or G) (SEQ ID NO: 96), said heavy chain CDR3 is the amino acid sequence YDHYX Ａ GX Ｂ SDY (wherein X Ａ is S or T, and X Ｂ is S or T) (SEQ ID NO: 90) The light chain CDR1 has amino acid sequence X Ａ SSQSLX Ｂ DX Ｃ DGKTYLN (wherein, X Ａ is K or R, and X Ｂ is V, M, or L, X Ｃ (is Y, T, or S) (Sequence ID 97) The light chain CDR2 has amino acid sequence X Ａ VX Ｂ NRX Ｃ X Ｄ (In the formula, X Ａ is K or R, and X Ｂ is S or T, X Ｃ is E or D, X Ｄ (is S or T) (Sequence ID 98) The light chain CDR3 has the amino acid sequence WQGX Ａ HFPRX Ｂ (In the formula, X Ａ is S or T, X Ｂ The anti-Aβ CPA according to any one of claims 1 to 35, comprising (Sequence ID 99) (where is S or T).

46. The light chain CDR3 is WQGTHFPRX Ａ FX Ｂ X Ｃ (In the formula, X Ａ is S or T, X Ｂ is S or T, X Ｃ The anti-Aβ CPA according to claim 45, comprising (where is F or Y) (Sequence ID 100).

47. The anti-Aβ CPA according to any one of claims 1 to 46, wherein the antibody is humanized.

48. The anti-Aβ CPA according to any one of claims 1 to 47, wherein the antibody is a human IgG1 isotype.

49. The anti-Aβ CPA according to any one of claims 1 to 48, wherein the antibody is a complete antibody, a chimeric antibody, a CDR-transplanted antibody, or a recombinant antibody.

50. The anti-Aβ CPA according to any one of claims 1 to 48, wherein the fragment is Fab, Fab', F(ab')2, Fabc, or Fv.

51. The anti-Aβ CPA according to any one of claims 1 to 50, wherein the antibody further comprises a heavy chain constant domain having an amino acid sequence at least 95% identical to that of SEQ ID NO:

40.

52. The anti-Aβ CPA according to any one of claims 1 to 51, wherein the antibody further comprises a light chain constant domain having an amino acid sequence at least 95% identical to that of SEQ ID NO:

41.

53. The anti-Aβ CPA according to any one of claims 1 to 52, wherein the heavy chain variable domain is fused to the heavy chain steady domain, and the light chain variable domain is fused to the light chain steady domain.

54. The anti-Aβ CPA according to claim 55, wherein the heavy chain constant domain is a variant of the natural human heavy chain constant domain, and has reduced binding to the Fcγ receptor compared to the natural heavy chain constant domain.

55. The anti-Aβ CPA according to claim 52 or 53, wherein the heavy chain constant domain is an IgG1 isotype.

56. The anti-Aβ CPA according to any one of claims 1 to 54, wherein the antibody has at least one mutation in its constant domain.

57. The anti-Aβ CPA according to claim 55, wherein the at least one mutation reduces complement binding or activation by the constant domain.

58. The anti-Aβ CPA according to claim 57, wherein the at least one mutation is located at one or more of the following EU numbering positions: 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331.

59. The anti-Aβ CPA according to claim 57, wherein the antibody has alanine at positions 318, 320, and 322 in EU numbering.

60. The anti-Aβ CPA according to any one of claims 1 to 59, wherein the antibody specifically binds to an epitope having an amino acid sequence including three or more amino acid positions of amino acids 1 to 7 of Aβ.

61. The anti-Aβ CPA according to any one of claims 1 to 34, wherein the antibody is selected from aducanumab, lecanumab, bapineuzumab, solanezumab, ganterelumab, crenezumab, and ponezumab.

62. The anti-Aβ CPA according to claim 1, wherein the anti-Aβ CPA comprises a polypeptide that is at least 95% identical to a sequence selected from any one of SEQ ID NOs: 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, and 394.

63. The anti-Aβ CPA according to claim 1 or 62, wherein the anti-Aβ CPA comprises a polypeptide that is at least 98% identical to a sequence selected from any one of SEQ ID NOs: 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, and 394.

64. The anti-Aβ CPA according to any one of claims 1, 62, or 63, wherein the anti-Aβ CPA comprises a polypeptide sequence selected from any one of SEQ ID NO: 349, SEQ ID NO: 351, SEQ ID NO: 353, SEQ ID NO: 355, SEQ ID NO: 357, SEQ ID NO: 359, SEQ ID NO: 361, SEQ ID NO: 363, SEQ ID NO: 365, SEQ ID NO: 367, and SEQ ID NO:

394.

65. The anti-Aβ CPA according to claim 1 or 62-64, wherein the anti-Aβ CPA comprises a polypeptide that is at least 95% identical to a sequence selected from any one of SEQ ID NOs: 350, 352, 354, 356, 358, 360, 362, 364, 366, and 368-393.

66. The anti-Aβ CPA according to claim 1 or 62 to 65, wherein the anti-Aβ CPA comprises a polypeptide that is at least 98% identical to a sequence selected from any one of SEQ ID NOs: 350, 352, 354, 356, 358, 360, 362, 364, 366, and 368 to 393.

67. The anti-Aβ CPA according to claim 1 or 62 to 66, wherein the anti-Aβ CPA comprises a polypeptide sequence selected from any one of SEQ ID NOs: 350, 352, 354, 356, 358, 360, 362, 364, 366, and SEQ ID NOs: 368 to 393.

68. The anti-Aβ CPA comprises a first polypeptide and a second polypeptide, further, The first polypeptide contains a sequence that is at least 95% identical to sequence number 349, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 350, The first polypeptide contains a sequence that is at least 95% identical to sequence number 351, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 352, The first polypeptide contains a sequence that is at least 95% identical to sequence number 353, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 354, The first polypeptide contains a sequence that is at least 95% identical to sequence number 355, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 356, The first polypeptide contains a sequence that is at least 95% identical to sequence number 357, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 358, The first polypeptide contains a sequence that is at least 95% identical to sequence number 359, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 360, The first polypeptide contains a sequence that is at least 95% identical to sequence number 361, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 362, The first polypeptide contains a sequence that is at least 95% identical to sequence number 363, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 364, The first polypeptide contains a sequence that is at least 95% identical to sequence number 365, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 366, The first polypeptide contains a sequence that is at least 95% identical to sequence number 367, and the second polypeptide contains a sequence that is at least 95% identical to sequence number 368, or The anti-Aβ CPA according to claim 1, wherein the first polypeptide comprises a sequence that is at least 95% identical to sequence number 394, and the second polypeptide comprises a sequence that is at least 95% identical to any one of sequence numbers 369 to 393.

69. The anti-Aβ CPA comprises a first polypeptide and a second polypeptide, further, The first polypeptide includes SEQ ID NO: 349, and the second polypeptide includes SEQ ID NO: 350, The first polypeptide includes SEQ ID NO: 351, and the second polypeptide includes SEQ ID NO: 352, The first polypeptide includes SEQ ID NO: 353, and the second polypeptide includes SEQ ID NO: 354, The first polypeptide includes SEQ ID NO: 355, and the second polypeptide includes SEQ ID NO: 356, The first polypeptide includes SEQ ID NO: 357, and the second polypeptide includes SEQ ID NO: 358, The first polypeptide includes SEQ ID NO: 359, and the second polypeptide includes SEQ ID NO: 360, or The first polypeptide includes SEQ ID NO: 361, and the second polypeptide includes SEQ ID NO: 362, The first polypeptide includes sequence number 363, and the second polypeptide includes sequence number 364, The first polypeptide includes SEQ ID NO: 365, and the second polypeptide includes SEQ ID NO: 366, The first polypeptide includes SEQ ID NO: 367, and the second polypeptide includes SEQ ID NO: 368, or The anti-Aβ CPA according to claim 1, wherein the first polypeptide comprises SEQ ID NO: 394, and the second polypeptide comprises any one of SEQ ID NOs: 369 to 393.

70. The anti-Aβ CPA according to any one of claims 1 to 69, wherein the antibody is conjugated with a therapeutic agent, a cytotoxic agent, a cell proliferation inhibitor, an immunomodulator, a neurotrophic agent, or a neuroprotective agent.

71. The cell permeabilizing agent according to any one of claims 1 to 70, wherein the heavy chain does not contain a C-terminal lysine residue.

72. A pharmaceutical composition comprising an anti-Aβ CPA according to any one of claims 1 to 71, and a pharmaceutically acceptable carrier or diluent.

73. A nucleic acid encoding at least a portion of the anti-Aβ CPA according to any one of claims 1 to 71.

74. The nucleic acid according to claim 73, wherein the nucleic acid encodes the CIM or a portion thereof.

75. The nucleic acid according to claim 73 or 74, wherein the nucleic acid encodes the CIM or a portion thereof, and at least one of the heavy chain variable domain and the light chain variable domain of the antibody.

76. The nucleic acid according to any one of claims 73 to 75, wherein the nucleic acid encodes the CIM or a part thereof, and the heavy chain variable domain of the antibody or a part thereof.

77. The nucleic acid according to any one of claims 73 to 75, wherein the nucleic acid encodes the CIM or a part thereof, and the light chain variable domain of the antibody or a part thereof.

78. A vector comprising the nucleic acid according to any one of claims 73 to 77, operably linked to one or more regulatory sequences to bring about the expression of the cell permeabilizing agent according to any one of claims 1 to 71 in mammalian cells.

79. The vector according to claim 78, wherein the one or more regulatory sequences include one or more of an enhancer, a ribosome binding site, a transcription termination signal, and a promoter, and optionally the promoter is a eukaryotic promoter.

80. The vector according to claim 78 or 79, wherein the nucleic acid is codon-optimized for expression in a host cell.

81. A host cell transformed with the vector described in any one of claims 78 to 80.

82. A host cell comprising the nucleic acid described in any one of claims 73 to 77.

83. A method for delivering an antibody that specifically binds to Aβ into a cell, comprising contacting the cell with an anti-Aβ CPA according to any one of claims 1 to 71, thereby resulting in the internalization of at least an antigen-binding fragment of the antibody into the cell.

84. The method according to claim 83, wherein the method further comprises, at a minimum, the transfer of the antigen-binding fragment of the antibody into the cytosol of the cell.

85. A method for binding to intracellular Aβ protein in cells, wherein the method is The method comprising contacting the cells with an anti-Aβ CPA according to any one of claims 1 to 71, thereby resulting in at least internalization and transfer to the cytosol of an antigen-binding fragment of the antibody.

86. The method according to any one of claims 83 to 85, wherein the cells are mammalian cells.

87. The method according to any one of claims 83 to 86, wherein the cells are in vitro.

88. The method according to any one of claims 83 to 86, wherein the cells are located within the target area.

89. A method for inhibiting or reducing the aggregation of Aβ in a subject having or at risk of developing an Aβ-related disease, comprising administering an effective amount of the anti-Aβ CPA described in any one of claims 1 to 71 to the subject, thereby inhibiting or reducing the aggregation of Aβ in the subject.

90. A method for treating or preventing an Aβ-related disease in a subject, comprising administering a therapeutically effective amount of a cell permeable agent according to any one of claims 1 to 71, thereby treating or preventing the Aβ-related disease.

91. The method according to claim 90, wherein the Aβ-related disease is inclusion body myositis.

92. A method for detecting Aβ deposition in a subject having or at risk of developing an Aβ-related disease, comprising administering a cell permeabilizing agent according to any one of claims 1 to 71 to the subject, and detecting the antibody bound to Aβ in the subject.

93. A method for detecting Aβ in a sample obtained from a patient having or at risk of developing an Aβ-related disease, comprising contacting the sample with a cell permeabilizing agent according to any one of claims 1 to 71, and detecting the binding of the antibody to Aβ in the sample.

94. The method according to claim 92 or 93, wherein the antibody is labeled.

95. The method according to claim 94, wherein the antibody is labeled with a fluorescent label, a paramagnetic label, or a radioactive label.

96. The method according to claim 95, wherein the radioactive label is detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

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