Expression of antigen-binding proteins in the nervous system

Abstract

The present invention provides recombinant vectors that express bivalent binding members; and methods for using such vectors to modify cells of the nervous system to express such binding members in the brains of patients with neurodegenerative diseases such as neurodegenerative diseases.

Description

[Technical Field] The present invention provides recombinant vectors that express bivalent binding members; and methods for using such vectors to modify cells of the nervous system to express such binding members in the brains of patients with neurodegenerative diseases such as neurodegenerative diseases. [Previous Technology] Alzheimer's disease (AD) is characterized by progressive neurodegeneration, leading to memory loss and cognitive decline. Its pathological features include the accumulation of extracellular amyloid plaques and intraneuronal tau fibrils. Therapies targeting amyloid β (Aβ) have been actively investigated for many years because of its genetic and pathological involvement in AD (Tcw and Goate, Cold Spring Harb Perspect Med. (2017) 7(6): pii a024539). Although elevated levels of amyloid precursor protein (APP) and Aβ are associated with the pathogenesis of AD, Aβ peptides exist in different conformations and fibrillary states, and it is unclear which substance should be targeted for therapeutic benefit (Benilova et al., Nat Neurosci. (2012) 15:349-57). Despite these uncertainties, passive immunotherapy has been extensively tested clinically against different forms of Aβ; however, these approaches have been hampered by other issues. First, the blood-brain barrier (BBB) ​​restricts the transport of biomolecules, requiring high doses injected peripherally to achieve therapeutically relevant levels in the brain. At high doses, several anti-Aβ antibodies have caused adverse reactions in clinical trials, notably amyloid-associated imaging abnormalities (ARIA); these adverse reactions are thought to be caused by antibody accumulation at vascular amyloid sites, triggering local inflammation via Fc-dependent effector function (Mo et al., Ann Clin Transl Neu. (2017) 4:931-42). Second, there is a need to maintain levels above the minimum therapeutic dose, which necessitates long-term passive immunotherapy, patient involvement and compliance, and significant commercial costs. Transferring genes into the central nervous system (CNS) allows the production of therapeutic proteins within neuronal cells, thus bypassing the BBB. Attempts have been made to mediate the expression of intact immunoglobulins (IgG) or single-chain variable fragments (scFv) in the CNS using AAV, but both approaches have inherent limitations (Sudol et al., Mol Ther. (2009) 17:2031-40; Ryan et al., Mol Ther. (2010) 18:1471-81; Levites et al., J Neurosci. (2006) 26:11923-28; Levites et al., J Neurosci. (2015) 35:6265-76; Kou et al., JAD. (2011) 27:23-38; Fukuchi et al., Neurobio Dis. (2006) 23:502-11; Liu et al., J Neurosci. (2016) 36:12425-35). In the CNS, the heavy and light chains of IgG are expressed only through self-cleavage of the F2A sequence, generating two chains from a single promoter cassette. The F2A peptide maintains attachment to either the heavy or light chain and possesses potential immunogenicity (Saunders et al., J Vir. (2015) 89:8334-45). On the other hand, gene-based delivery of scFV proteins often results in a significant loss of affinity due to the loss of titer. Removal of the Fc region also leads to loss of FcRn binding, resulting in shorter peripheral half-lives and reduced antigen (Ag) binding of scFv, which is then outflowed from the brain via retrograde endocytosis (Deane et al., J Neurosci. (2005) 25:11495-503; Boado, et al., Bioconjug Chem. (2007) 18:447-55; Zhang et al., J Neuroimm. (2001) 114:168-72; Schlachetzki et al., J Neurochem. (2002) 81:203-6). Therefore, antibody therapy holds promise for CNS diseases such as Alzheimer's disease, but is limited by the challenge of delivering therapeutic proteins to the diseased brain. Thus, for antibody-based therapies, there is a need to improve central nervous system access. [Summary of the Invention] The present invention provides a method for expressing a divalent binding member in cells of the nervous system, comprising introducing an expression cartridge encoding a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody light chain variable domain (VL), and an IgG Fc region into the cells, wherein VH and VL form antigen-binding sites that specifically bind to the target protein, and after expression in the cells, the two molecules of the polypeptide form a disulfide-bonded homodimeric divalent binding member that is specific to the target protein. In some specific examples, the cells of the nervous system are neurons, glial cells, ependymal cells, or brain epithelial cells. In further specific examples, glial cells are selected from oligodendritic cells, astrocytes, exocarp cells, Schwann cells, and microglia. In some specific examples, the cells are human cells, such as cells in the brain of a human patient. In some specific examples, the target protein is a protein that is expressed in the brain, and can be an amyloid β-peptide (Aβ), tau, SOD-1, TDP-43, ApoE, or α-synuclein. In some specific examples, the polypeptide comprises, from the N-terminus to the C-terminus, (i) VH, a peptide linker, and VL; or VL, a peptide linker, and VH; and (ii) an IgG Fc region. In further specific examples, the peptide linker comprises the sequence GGGGS (SEQ ID NO: 3); for example, the peptide linker has the sequence [G4S]3 (SEQ ID NO: 2). In some specific examples, the bivalent binding member of the present invention binds to the neonatal Fc receptor (FcRn), but does not bind to the Fcγ receptor due to one or more mutations in the IgG Fc region. In some specific examples, the method of the present invention includes administering a viral vector containing an expression capsule. The viral vector may be a recombinant virus. In further specific examples, the recombinant virus is introduced into the patient's brain via intracranial injection, intrathecal injection, or intracranial injection. Other recombinant viruses may be, for example, recombinant adeno-associated virus (rAAV), such as rAAV serotype 1 or 2. In some specific cases, the expression of a polypeptide is transcribedly controlled by a persistently activated promoter or an inducible promoter. The method of the present invention can be used to treat patients suffering from neurodegenerative diseases such as Alzheimer's disease, amyloid cerebrovascular disease, synucleinosis, tau disease, or amyotrophic lateral sclerosis (ALS). In another embodiment, the present invention provides a method for treating neurodegenerative diseases, comprising administering to a patient in need a therapeutically effective amount of a composition comprising a viral vector disclosed herein, the viral vector representing a divalent binding member of the present invention. In another embodiment, the present invention provides a bivalent binding member for treating a patient in need; and a bivalent binding member for use in manufacturing a medicament for treating a patient in need, wherein the patient suffers from, for example, a neurodegenerative disease, such as Alzheimer's disease, amyloidosis, synucleinosis, tau disease, or ALS. Other features, objects, and advantages of the invention will become apparent in the following detailed description. However, it should be understood that while the detailed description points out specific examples and embodiments of the invention, it is provided by way of illustration only and not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art based on the detailed description.

Implementation Method

Claims

1. Use of a recombinant adeno-associated virus (AAV) in the preparation of a medicament for treating human patients in need, wherein the recombinant AAV is tropist for neural tissue and comprises an expression cassette encoding a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody light chain variable domain (VL), and an IgG Fc region, wherein VH and VL form scFV antigen-binding sites that specifically bind to a target protein in the central nervous system, wherein the IgG Fc region is the C-terminus of scFV and comprises a portion of an immunoglobulin (Ig) heavy chain that begins in a hinge region upstream of a papain cleavage site and terminates at the C-terminus of the Ig heavy chain; wherein, upon expression in the cells of the neural system of the human patient, the two molecules of the polypeptide form a disulfide-bonded homodimeric divalent binding member specific to the target protein; and wherein the divalent binding member binds to the neonatal Fc receptor (FcRn), but due to IgG One or more mutations in the Fc region selected from C220S, C226S, C229S, N297Q, N297A, N297G, E233P, L234V, L234F, L234A, L235A, L235E, P331S, and P238S (EU number) do not bind to the Fcγ receptor.

2. As claimed in claim 1, wherein the cells of the nervous system are neurons; glial cells selected from oligodendritic cells, astrocytes, exocarp cells, Schwann cells and microglia; ependymal cells; and brain epithelial cells.

3. As requested in claim 2, wherein the cell is a human cell.

4. As requested in claim 3, wherein the cell is in the brain of the human patient.

5. The use of any of claims 1 to 4, wherein the target protein is a protein expressed in the brain.

6. As claimed in claim 5, wherein the protein is an amyloid β-peptide (Aβ), tau, SOD-1, TDP-43, ApoE, or α-synuclein.

7. As claimed in any of claims 1 to 4, wherein the polypeptide comprises, from the N-terminus to the C-terminus, (i) VH, a peptide linker, and VL; or (ii) VL, a peptide linker, and VH.

8. As claimed in claim 7, wherein the peptide linker comprises the sequence GGGGS (SEQ ID NO: 3).

9. As claimed in claim 1, wherein the recombinant AAV is introduced into the brain of the human patient via intracranial injection, intrathecal injection, or intracisional injection.

10. As requested in any of claims 1 to 4, wherein the recombinant AAV is serotype 1.

11. As requested in any of claims 1 to 4, wherein the recombinant AAV is serotype 2.

12. As claimed in any of claims 1 to 4, wherein the expression of the polypeptide is transcribedly controlled by a persistently activated promoter or an inducible promoter.

13. For any of the uses of claims 1 to 4, wherein the human patient has a neurodegenerative disease.

14. As claimed in claim 13, wherein the human patient has Alzheimer's disease, amyloidosis, synucleinosis, tau disease, or amyotrophic lateral sclerosis (ALS).

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