Targeted degradation of Anti-AAV antibodies to enable AAV-based gene therapy

Abstract

A composition of matter including a binding moiety that binds to antibodies to AAV, a cellular receptor-binding moiety that binds to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) on the surface of hepatocytes or other degrading cells in a subject or patient, and optionally, a linker moiety connecting the binding moiety that binds to antibodies to AAV and the cellular receptor-binding moiety, wherein the composition of matter is useful for removing antibodies to AAV in a subject or patient.

Description

TARGETED DEGRADATION OF ANTI-AAV ANTIBODIES TO ENABLE AAV-BASED GENE THERAPYFIELD OF THE INVENTION

[0001] The invention generally relates to medicinal preparations characterized by the non-active ingredients used, e.g., carriers or inert additives, targeting or modifying agents chemically bound to the active ingredient, the non-active ingredient being chemically bound to the active ingredient, e.g., polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g., an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a ceil surface antigen or a ceil surface determinant, and particularly to bifunctional molecules which contain a circulating protein-binding moiety linked through a linker group to a cellular receptorbinding moiety to treat enable and enhance AAV-based gene therapy hindered by anti-AAV antibodies present in individuals.BACKGROUND OF THE INVENTION

[0002] Adeno-associated virus (AAV) vectors are useful biomedical tools for in vivo gene therapy. However, their homology with wild-type adeno-associated viruses, which often infect humans naturally, poses limitations in terms of immune responses associated with this gene therapy vector platform. Both humoral immunity and cell-mediated immunity to wild-type AAV are known in the biomedical art to be present in many (30-60%) healthy subjects. Preexisting anti-AAV antibodies can have a negative impact on the outcome of the subject's gene therapy because the antibodies interfere with gene transfer by blocking transduction. Thus, preexisting anti-AAV antibodies limit the use of AAV-based gene therapy. Patients needing gene therapy treatment are often excluded from gene therapy clinical trials based on pre-existing anti-AAV antibodies.

[0003] Subjects and patients of adeno-associated virus gene therapy may develop further anti-AAV immune responses, becoming ineligible for subsequent gene therapy and posing safety concerns. Thus, there remains a need in the biomedical art for new medicines capable of neutralizing or removing anti- AAV antibodies to enable and enhance AAV-based gene therapy.SUMMARY OF THE INVENTION

[0004] The invention provides targeted degradation of anti-AAV antibodies to enable or enhance AAV-based gene therapy. The removal of anti-AAV antibody enables AAV viral transduction.

[0005] In one embodiment, the invention provides a composition of matter (agent, TRAP™) comprising: a binding moiety that can bind to antibodies to AAV, a cellular receptor-binding moiety that can bind to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface of hepatocytes or other degrading cells in a subject or patient, and a linker moiety connecting the antibodies to AAV-binding moiety and the cellular receptorbinding moiety. This composition of matter (an agent) is a bifunctional modality that drives ASGPR- dependent degradation of anti-AAV neutralizing antibodies.

[0006] In some embodiments, the selective ASGPR-dependent bifunctional degrader degrades anti- AAV antibodies across isotypes.

[0007] In other embodiments, the selective ASGPR-dependent bifunctional degrader degrades anti- AAV antibodies to AAV serotypes relevant to current clinical use, such as AAV1, AAV2, AAV5, AAV8, AAV9, AAV rhlO, or AAV rh74.

[0008] In some embodiments, the invention provides a composition of matter (an agent, a TRAP™) having a structure of:RCN-(Xaa)y-Rcc,[AGN104], or a salt thereof, wherein ABT is the binding moiety, and TBT is the cellular receptor-binding moiety.

[0009] In some embodiments, the cellular receptor-binding moiety comprises an ASGPR binding group according to the chemical structure:[TBT102], wherein the cellular receptor-binding moiety has additional elements described in this specification.

[0010] In several embodiments, the binding moieties are adeno-associated virus capsids. An advantage of an adeno-associated virus capsid as the binding moiety is that the binding moiety engages many of the subject or patient antibodies with a single binding moiety that is epitope agnostic. In one embodiment, the binding moiety is produced from unmodified AAV VPS capsids obtained from purified AAV capsid components. The purified AAV capsid components are verified for purity and stoichiometry of capsid assembly. In another embodiment, the binding moiety is produced from commercially- available AAV capsids.

[0011] In a particular embodiment, the composition of matter (agent, TRAP™) is AGN301. This composition of matter has an antibody-binding moiety comprising the polypeptide sequence of SEQ ID NO: 5. This composition of matter can be synthesized by chemical conjugation.

[0012] In a particular embodiment, the composition of matter is AGN302. This composition of matter has an antibody-binding moiety comprising the polypeptide sequence of SEQ ID NO: 6. This composition of matter can be synthesized by enzymatic conjugation.

[0013] In a particular embodiment, the AAV capsids are functionalized with an ASGPR ligand on AAV capsid protein lysine residues. In other particular embodiments, the binding moiety isfunctionalized with an ASGPR ligand on AAV capsid protein cysteine residues, by fusion tags and enzymatic conjugation, or by other techniques known to persons having ordinary skill in the biomedical art. In a particular embodiment, the functionalization stoichiometry is about one ASGPR ligand per capsid. In another particular embodiment, the functionalization stoichiometry is more than one ASGPR ligand per capsid.

[0014] In several embodiments, the binding moieties are adeno-associated virus capsid subunits. An advantage of an adeno-associated virus capsid subunit or an antigenically functional fragment thereof as the binding moiety is that the binding moiety provides a productive AAV epitope presentation, including select interfaces. In a particular embodiment, the binding moiety is produced from sub-capsid complexes. The assembly and homogeneity of sub-capsid complexes can be verified by techniques known to persons having ordinary skill in the biomedical art. In a particular embodiment, the binding moiety is produced from AAV9 capsid subunits.

[0015] In a particular embodiment, the AAV capsids are functionalized with an ASGPR ligand on AAV capsid protein lysine residues or by other functionalization techniques known to persons having ordinary skill in the biomedical art.

[0016] In several embodiments, the binding moieties are peptides that specifically bind to an antibody that binds to an adeno-associated virus. The peptides can be linear peptides or constrained peptides. Advantages of an adeno-associated virus peptide as the binding moiety are manufacturability and the ability to establish a proof of concept for the composition of matter (agent, TRAP™).

[0017] In several embodiments, the binding moiety is a peptide comprising the sequence QAQAQT (SEQ ID NO: 4) as described by Giles et al., J. Virol., 92(20), e01011-18 (September 26, 2018).

[0018] In several embodiments, the binding moiety is a peptide selected from a commercially- available library, such as one available from GeneScript, Cat. No. RP30243, which provides a pool of 182 peptides derived from a peptide scan (15mers with 11 amino acid overlap) through Capsid protein (Swiss-Prot ID: Q6JC40) of Adeno-Associated Virus (AAV) - Type 9.

[0019] In several embodiments, the binding moiety is a peptide identified or confirmed by a process to identify epitopes to produce effective anti-AAV antibody bifunctional degraders. In a particular embodiment, the identification or confirmation process comprises the steps of (1) enriching anti-AAV antibodies from subject or patient samples using immobilized AAV capsids, such as by the method used by Orlowski et al., Molecular Therapy-Methods & Clinical Development. 16, 192-203 (March 13, 2020), (2) selecting dominant epitopes using a peptide microarray, such as the commercially- available PEPperCHIP® Pan-AAV Capsid Protein Microarray, (3) enriching capsid peptide fragments insolution and identify by mass spectroscopy (MS), (4) obtaining the protein sequence the antibodies themselves, then (4) leveraging B cell single-cell sequencing data, after enriching for B cell producing anti-AAV antibodies. In a particular embodiment, the AAV serotype is AAV9.

[0020] In a particular embodiment, the binding moiety is an empty viral capsid formed by individual VP3 proteins, for example sixty capsid proteins, each VPS protein, comprising about sixty ABT301 VPS proteins, each having the polypeptide sequence of SEQ ID NO: 5. In similar embodiments, the VPS proteins have a polypeptide sequence similar to SEQ ID NO: 5 but with conservative substitutions, such that the VPS proteins can assemble to form an AAV capsid. Such assembled capsids would have a structure that could be recognized by anti-AAV antibodies.

[0021] In another particular embodiment, the binding moiety is an empty viral capsid formed by individual VP3-IT proteins, for example sixty capsid proteins, each VP3-IT protein, comprising about sixty ABT302 VP3-IT proteins, each having the polypeptide sequence of SEQ ID NO: 6. In similar embodiments, the VP3-IT proteins have a polypeptide sequence similar to SEQ ID NO: 6 but with conservative substitutions, such that the VP3-IT proteins can assemble to form an AAV capsid. Such assembled capsids would have a structure that could be recognized by anti-AAV antibodies.

[0022] In another embodiment, the invention provides a method of making the composition of matter (agent, TRAP™). In yet another embodiment, the method comprises a step of chemical conjugation. In yet another embodiment, the method comprises a step of enzymatic conjugation.

[0023] In another embodiment, the invention provides a method of removing from a subject in need thereof by administering the ASGPR-dependent bifunctional degrader to the subject or patient. The method of removing is the induction of liver-directed, ASGPR-dependent removal of anti-AAV antibodies. The route of administration can be by any therapeutically-effective method, e.g., intravenously. In another embodiment, the need thereof is elevated neutralizing anti-AAV antibody titers in subject or patient candidates for AAV gene therapy. In one embodiment, the subject or patient in need thereof is a person excluded from gene therapy or therapies because of elevated anti-AAV antibody titers. In one embodiment, the subject or patient in need thereof is a gene therapy-treated patient. In one embodiment, the dosage schedule begins before the delivery of gene therapy. In one embodiment, the treatment duration is concurrent with the gene therapy schedule. In one embodiment, the efficacy is measured by an increased gene therapy vector transduction.

[0024] In another embodiment, the invention provides a method of treating a disease state or condition associated with the presence of anti-AAV antibodies in a subject by administering an effective amount of the agent to the subject or patient. In a particular embodiment, the agent is administered tothe subject at least twenty-four hours before administering an AAV-derived gene therapy vector to the subject.

[0025] In another embodiment, the invention provides a composition including the agent, and at least one additional agent comprising a moiety capable of binding to that forms the antibody moiety of the first compound.

[0026] In another embodiment, the invention provides a composition including the agent and at least one pharmaceutically acceptable excipient.

[0027] In one advantageous aspect, the invention optimizes therapeutic AAV-derived gene vector uptake and gene expression.

[0028] In another advantageous aspect, the invention provides a single composition of matter that targets all immunoglobulin subclasses and isotypes. Use of the composition of matter results in the removal of IgG and IgM antibodies and other factors directed against AAV and related viral vectors.

[0029] In another advantageous aspect, the invention provides some compositions of matter where the antibody-binding moiety is a capsid comprising only one VP3 or VP3-derived protein.

[0030] Several objects, features, aspects, and advantages of the invention will become more apparent from the following detailed description of embodiments of the invention, along with the accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] For illustration, some embodiments of the invention are shown in the drawings described below. Like numerals in the drawings indicate like elements throughout. The invention is not limited to the precise arrangements, dimensions, and instruments shown.

[0032] FIG. 1 shows the structure of one embodiment of the bifunctional degrader (agent, TRAP™).

[0033] FIG. 2 shows the chemical structure of a TBT moiety (maleimide PGN3; TBT103) for engineered cysteine-based conjugation.

[0034] FIG. 3 shows the chemical structure of a TBT moiety (aGN3 with polyglycine; TBT104) for sortase-based conjugation.

[0035] FIG. 4 is a line graph showing AAV9 capsid-bait TRAP™ degrader AGN302 binds an anti-AAV antibody by surface plasmon resonance.

[0036] FIG. 5 is a line graph showing AAV9 capsid-bait TRAP™ degrader binds antibody and ASGPR. Anti-AAV TRAP™ binds ASGPR by surface plasmon resonance.

[0037] FIG. 6 shows the chemical structure of AGN301.

[0038] FIG. 7 shows the chemical structure of AGN302. The peptide DIPATYEFTDGKHYITNEPIPPK was inserted internally in the VP3-IT peptide ABT302, i.e., not at the N-terminus or C-terminus, in each of the sixty VPS proteins that collectively constitute an individual capsid.

[0039] FIG. 8 is a line graph showing cellular internalization into the lysosome by AGN301 as compared to ABT301, demonstrating that an ASGPR-dependent mechanism of internalization.

[0040] FIG. 9 is a line graph showing cellular internalization into the lysosome by AGN302 as compared to ABT302, demonstrating that an ASGPR-dependent mechanism of internalization.DETAILED DESCRIPTION OF THE INVENTION

[0041] The following detailed description is provided to aid persons having ordinary skill in the biomedical art. Exemplary embodiments are described, but these embodiments are only exemplary. This disclosure is not limited thereto but is defined by the scope of the appended claims. Persons having ordinary skill in the biomedical art may make modifications and variations in the embodiments described in this specification without departing from the spirit or scope of this disclosure.Industrial Applicability

[0042] The invention provides a medically useful composition of matter (agent, TRAP™) for enabling or enhancing AAV-based gene therapy otherwise hindered by anti-AAV antibodies.

[0043] Adeno-associated viruses possess features useful as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. AAV infects many mammalian cells allowing the possibility of targeting many tissues in vivo. AAV transduces slowly dividing and nondividing cells and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element).

[0044] However, 30-60% of individuals have anti-AAV antibodies from natural infection limiting the impact of AAV gene therapy (GTx). Recipients of AAV gene therapy and vaccines can develop anti-AAV antibodies and become ineligible for subsequent AAV gene therapy. Patients in need are often excluded from AAV gene therapy clinical trials based on pre-existing anti-AAV antibodies.

[0045] ASGPR-dependent bifunctional degraders can be a therapy for the selective degradation of anti-AAV antibodies across isotypes, thus the applicability, effectiveness, and potentially safety of GTx for patients with pre-existing anti-AAV antibodies.

[0046] This specification describes the functional proof-of-concept, which is the use of bifunctional antibody degraders comprising an empty AAV capsid to remove anti-AAV antibodies, including amonoclonal anti-AAV9 antibody, and promote transduction of an AAV9 virus expressing green fluorescent protein (GFP). This specification shows that an anti-AAV9 antibody is internalized in cells, and routed to the lysosomal compartment, by an ASGPR-dependent anti-AAV antibody degrader. Thus, neutralization of AAV9-GFP transduction by anti-AAV antibodies is removed.

[0047] This specification also describes the use of an IgG degrader to remove a monoclonal anti- AAV9 antibody and promote transduction of an AAV9 virus expressing green fluorescent protein (GFP). This specification shows that an anti-AAV9 antibody and an IgGl control are internalized by an ASGPR- dependent pan IgG degrader, thus, AAV9-GFP transduction is neutralized by an anti-AAV9 antibody but restored to non-neutralizing levels by an IgG degrader treatment.

[0048] IgG degraders are described, for example, in International Publication No. WO 2019 / 199621 published October 17, 2019, International Publication No. WO 2019 / 199634 published October 17, 2019, International Publication No. WO 2021 / 155317 published August 5, 2021, International Publication No. WO 2022 / 235699 published November 10, 2022, International Publication No. WO 2024 / 227119 published October 31, 2024, the content of which applications are incorporated herein in their entireties by reference.Treatment rationale.

[0049] After the bifunctional degrader and the bound antibodies to AAV9 protein are endocytosed, they are released from the ASGPR through calcium depletion from the endosome and changes in binding site amino acid protonation due to decreased pH. The ASGPR is recycled back to the hepatocyte surface. Endocytosed proteins are trafficked to late endosomes, which are fused with lysosomes. Lysosomal proteases then degrade endocytosed proteins, permanently removing them from circulation.

[0050] To demonstrate the safety and efficacy of the composition of matter (agent, TRAP™), these assays, disclosed in this specification or known to persons having ordinary skill in the biomedical art, can be performed.

[0051] (1) Demonstrate agent binding to dominant AAV-neutralizing antibodies.

[0052] (2) Demonstrate ASGPR-dependent endocytosis of anti-AAV antibodies through degraders composed of AAV capsids linked to ASGPR ligands in a cell model.

[0053] (3) Demonstrate ASGPR-dependent endocytosis of monoclonal neutralizing antibodies using bifunctional capsid-based degraders in a cell model.

[0054] (4) Demonstrate neutralization of a reporter AVV virus in transduction assays.

[0055] (5) Demonstrate reversal of anti-AAV neutralization in cells.

[0056] (6) Demonstrate AAV transduction in transduction assays.

[0057] (7) Demonstrate endocytosis of antibodies.

[0058] (8) Demonstrate selective anti-AAV depletion in mice.

[0059] (9) Demonstrate reversal of anti-AAV neutralization in mice.

[0060] (10) Demonstrate activity in non-human primates.

[0061] (11) Demonstrate activity in humans.

[0062] (12) Demonstrate removal of anti-AAV antibodies in humans.

[0063] (13) Demonstrate improved gene therapy in humans.Definitions

[0064] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims are listed below. Unless stated otherwise or implicit from context, these terms and phrases shall have the meanings below. These definitions aid in describing embodiments but are not intended to limit the claimed invention.

[0065] As used in this application, except as otherwise expressly provided in this specification, each term shall have the meaning set forth below. Additional definitions are set forth throughout the application. Where a term is not specifically defined in this specification, it is given a biomedical art- recognized meaning, applying that term in context to its use in describing the invention.

[0066] The articles "a" and "an" have the plain meaning of one or more than one, i.e., at least one, of the grammatical object of the article unless the context indicates otherwise. For example, "an element" means one element or more than one element.

[0067] The term "ABT" is the moiety to which an antibody specifically binds. In some embodiments of this specification, the ABT binds to the antibodies to AAV or variants thereof. In some embodiments of this specification, the ABT binds to the antibodies to AAV9.

[0068] The term "active Ingredient" has the United States Food & Drug Administration-provided meaning of any component that provides pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or any function of a human body or an animal body.

[0069] The term "adeno-associated virus" (AAV) has the biomedical art-recognized meaning of a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length, including two nucleotide inverted terminal repeats (ITRs). The term adeno-associated virus can refer to the virus itself or its derivatives, including all subtypes of naturally occurring or recombinant forms. Theadeno-associated virus can be selected from the Markush group of viruses consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, AAVTT or Anc80, AAV7m8 and their derivatives. The several serotypes of AAV are each associated with a specific clade, the members of which share serologic and functional similarities. AAV9 sequences are "clade F" sequences. See Gao et al., J. Viral., 78, 6381-6388 (2004). The nucleotide sequences of the genomes of the AAV serotypes are known to persons having skill in the biomedical art. The complete genome of AAV-1 is in GenBank Accession No. NC_002077. The complete genome of AAV- 2 is in GenBank Accession No. NC_001401 and Srivastava et al., J. Viral., 45, 555-564 (1983). The complete genome of AAV-3 is in GenBank Accession No. NC_1829. The complete genome of AAV-4 is in GenBank Accession No. NC_001829. The AAV-5 genome is in GenBank Accession No. AF085716. The complete genome of AAV-6 is in GenBank Accession No. NC_00 1862. At least portions of AAV- 7 and AAV-8 genomes are in GenBank Accession Nos. AX753246 and AX753249, respectively. The AAV-9 genome is in Gao et al., J. Viral., 78, 6381-6388 (2004). The AAV-10 genome is in De et al., Mol. Then, 13(1), 67-76 (2006). The AAV-11 genome is in Mori et al., Virology, 330(2), 375-383 (2004). Portions of the AAV-12 genome are in GenBank Accession No. DQ813647. Portions of the AAV-13 genome are in GenBank Accession No. EU285562. The sequence of the AAV rh.74 genome is in U.S. Pat. No. 9,434,928. The sequence of the AAV-B1 genome is in Choudhury et al., Mol. Then, 24(7), 1247-1257 (2016). Anc80 is an AAV vector of AAV1, AAV2, AAV8 and AAV9. The sequence of Anc80 is in Zinn et al., Cell Reports 12, 1056-1068, 2015 and Inti. Pat. Publ. WO 2015 / 054653, both incorporated by reference herein, in their entirety and GenBank Accession Nos. KT235804-KT235812. Cis-acting sequences directing viral DNA replication, encapsidation / packaging, and host cell chromosome integration are contained within the ITRs. Three AAV promoters (named p5, pl9, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The cap gene encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and nonconsensus translational start sites are responsible for the production of the three related capsid proteins. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158, 97-129 (1992).

[0070] The term "agent" has the biomedical art-recognized meaning of a composition of matter useful for performing a function. Several biomedically useful functions are described in this specification.

[0071] The term "alleviate" has the biomedical art-recognized meaning of a process by which the severity of a sign or symptom of a disorder is decreased. A sign or symptom can be alleviated without being eliminated. The administration of compositions or pharmaceutical compositions of the inventionmay or can lead to the elimination of a sign or symptom. However, elimination is not required. Effective dosages should be expected to decrease the severity of a sign or symptom.

[0072] The term "asialoglycoprotein receptor (ASGPR) binding group" has the biomedical art- recognized meaning of a binding group that binds to a hepatocyte asialoglycoprotein receptor. The ASGPR binding group selectively binds to hepatocyte asialoglycoprotein receptors on the surface of hepatocytes. In several embodiments of this specification, an ASGPR binding group is a component of a bifunctional agent as a cellular receptor-binding moiety covalently bound to the antibody-binding moiety through a linker group or directly. It is through this ASGPR moiety that bifunctional agents complexed with a circulating protein, e.g., bind to hepatocytes. After the bifunctional agent complexed with a circulating protein is bound to a hepatocyte or other cell, the circulating protein is taken into the hepatocyte or other cell via a phagocytosis mechanism, wherein the circulating protein is degraded through lysosomal degradation.

[0073] The term "asialoglycoprotein receptor (ASGPR) has the biomedical art-recognized meaning of lectins, which bind asialoglycoprotein and glycoproteins from which a sialic acid has been removed to expose galactose residues. These cellular receptors are located on mammalian hepatocytes and other cells, such as glandular cells of the gallbladder and the stomach. ASGPR removes target glycoproteins from circulation.

[0074] The term "at least one of," when preceding a list of elements, modifies the entire list of elements and does not modify the individual elements of the list.

[0075] The term "cell-mediated immunity" has the biomedical art-recognized meaning of the activation of phagocytes, antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen, rather than antibody generation.

[0076] The term "cellular receptor-binding moiety" has the biomedical art-recognized meaning. In several embodiments of this specification, the cellular receptor-binding moiety is an asialoglycoprotein receptor (ASGPR) binding group.

[0077] The term "cellular receptor" has the biomedical art-recognized meaning of a protein on the surface of a cell that binds to a compound, e.g., a ligand, e.g., a protein, in solution or on another cell. Generally, ligand-receptor binding induces one or more biological responses. In this specification, an asialoglycoprotein receptor (ASGPR) is a cellular receptor on the surface of hepatocytes or other cells that binds to an asialoglycoprotein or a derivative thereof.

[0078] The term "conservative substitution" has the biomedical art-recognized meaning. For example, sequence identity can be measured using sequence analysis software, for example, SequenceAnalysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs. Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, or other modifications. Conservative substitutions include substitutions within these groups: glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine. A residue having similar physiochemical characteristics can replace an amino acid, e.g., substituting one aliphatic residue for another, such as He, Vai, Leu, or Ala for one another,, or substitution of one polar residue for another, such as between Lys and Arg, Glu and Asp, or Gin and Asn. Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any of the assays to confirm that the desired activity, e.g., the ability of AAV capsid proteins to assemble to AAV capsids.

[0079] The term "constrained peptide" has the biomedical art-recognized meaning of peptides whose conformation is restricted, frequently to a peptide with a conformation that the ligand assumes upon target binding or to a subset of structures occupied by a flexible unconstrained peptide.

[0080] The term "efficacy" has the biomedical art-recognized meaning. The efficacy of the bifunctional degrader can be measured by an increased gene therapy vector transduction.

[0081] The term "gene therapy" has the biomedical art-recognized meaning of a medical technology that aims to produce a therapeutic effect through the manipulation of gene expression or through altering the biological properties of living cells.

[0082] The term "hepatocyte" has the biomedical art-recognized meaning of a cell of the main parenchymal tissue of the liver. Hepatocytes make up 55-65% of the liver's mass.

[0083] The term "humoral immunity" has the biomedical art-recognized meaning of the aspects of immunity mediated by macromolecules located in extracellular fluids, such as secreted antibodies, complement proteins, and antimicrobial peptides.

[0084] The term "IC5o" has the biomedical art-recognized meaning of an amount, concentration, or dosage of a particular test compound that achieves a 50% inhibition of a maximal response in an assay that measures such response.

[0085] The term "IgG" antibody has the biomedical art-recognized meaning. Each IgG molecule consists of the basic four-chain immunoglobulin structure— two y (gamma) heavy chains and two identical light chains (either kappa or lambda)— and carries two identical antigen-binding sites. Thereare four subclasses of IgG, each with minor differences in its H chains but with distinct biological properties.

[0086] The term "IgGl" antibody has the biomedical art-recognized meaning of an IgG antibody where the Ig gamma-1 chain C region is a protein encoded by the IGHG1 gene in humans.

[0087] The term "lgG2" antibody has the biomedical art-recognized meaning of an IgG antibody where the Ig gamma-2 chain C region is a protein encoded by the IGHG2 gene in humans.

[0088] The term "lgG4" antibody has the biomedical art-recognized meaning of an IgG antibody where the Ig gamma-4 chain C region is a protein encoded by the IGHG4 gene in humans. lgG4 has little effector function. lgG4 cannot fix complement.

[0089] The term "I VIG" has the biomedical art-recognized meaning of administering intravenous immunoglobulin (I VIG).

[0090] The term "KD" has the biomedical art-recognized meaning of the measured equilibrium dissociation constant between a compound or ligand and a protein or binding domain of a protein.

[0091] The term "linker moiety" has the biomedical art-recognized meaning of a moiety of a chemical compound that links one moiety of the chemical compound to another moiety of the same compound.

[0092] The term "moiety" has the biomedical meaning of a defined chemical group or entity with a particular structure or activity. A moiety generally refers to a part of a molecule. In some embodiments, a binding moiety maintains one or more desired structural features, properties, functions, or properties, e.g., 3-dimension structure, antigen specificity, antigen-binding capacity, or immunological functions, etc., comparable to its corresponding binding protein, e.g., an antibody. In some embodiments, a moiety is monovalent. In some embodiments, a moiety is bivalent. In other embodiments, a moiety is polyvalent.

[0093] The term "monotherapy" is a biomedical art-recognized term for administering a single active or therapeutic compound to a subject or patient in need. Monotherapy usually involves administering a therapeutically effective amount of an active composition.

[0094] The term "Multimodal Antibody Therapy Enhancer (MATE®)" has the proprietary meaning. See International Pat. Publ. WO 2021 / 102052 (Kleo Pharmaceuticals).

[0095] The term "neutralizing antibody" has the biomedical art-recognized meaning of an antibody that defends against an infectious particle by neutralizing its biological effect, rendering the infectious particle no longer infectious or pathogenic.

[0096] The term "neutralizing assays" has the biomedical art-recognized meaning and includes the use of techniques such as plaque reduction (which compares counts of virus plaques in control wells with those in inoculated cultures), microneutralization (which is performed in microtiter plates filled with small amounts of sera), and colorimetric assays (which depend on biomarkers indicating metabolic inhibition of the virus).

[0097] The term "on" has the plain meaning. When an element is called being on another element, it can be directly in contact with the other element, or intervening elements may be present therebetween. When an element is called being "directly on" another element, no intervening elements are present.

[0098] The term "or" as used in this specification, means "or." The term "or" as used in this specification, includes all combinations of one or more of the associated listed items.

[0099] The term "other degrading cells" has the biomedical art-recognized meaning. Asialoglycoprotein receptors (ASGPRs) are found on hepatocytes and the glandular cells of the gallbladder and stomach.

[0100] The term "pharmaceutically acceptable excipient" has the biomedical art-recognized meaning of an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use and human pharmaceutical use. A "pharmaceutically acceptable excipient," as used in the specification and claims, includes both one and more than one such excipient. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences 23rd edition (Elsevier, 2020).

[0101] The term "pharmaceutically acceptable" has the biomedical art-recognized meaning of those compounds, anions, cations, materials, compositions, carriers, or dosage forms that are, within the scope of sound medical judgment, suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0102] The term "protein-binding moiety" has the biomedical art-recognized meaning of a region of a chemical composition, e.g., a polypeptide region of a chemical composition, that specifically binds to a protein, e.g., a specific protein.

[0103] The term "ROC" has the biomedical art-recognized meaning of the receiver operating characteristic curve.

[0104] The term "seroprevalence" has the biomedical art-recognized meaning of the percentage of people in a population with antibodies in their blood that show they have been exposed to a virus or other infectious agent. Studying the seroprevalence of antibodies to a specific virus can show how many people have been infected with that virus.

[0105] The term "TBT" is the moiety that binds to a cellular receptor-binding moiety. In some embodiments of this specification, the TBT binds to ASGPR.

[0106] The term "titer" has the biomedical art-recognized meaning of measurement of the amount or concentration of a substance in a solution, such as the amount of antibodies found in a subject or patient blood.

[0107] The term "total antibody assay" has the biomedical art-recognized meaning of an assay to measure the total antibody in an item of interest. The antibody may be a subject, patient, or sample thereof.

[0108] The term "TRAP" has the proprietary meaning described in this specification of a targeted removal of aberrant protein. A TRAP is a bifunctional degrader.

[0109] The term "transduction inhibition assay" has the biomedical art-recognized meaning of a cell-based functional assay that measures the level of transduction inhibition by neutralizing antibodies or non-antibody neutralizing factors. The assays can use subject or patient samples to measure their ability to reduce the transduction of a cell line by an adeno-associated virus vector.

[0110] The term "universal antibody-binding moiety" has the biomedical art-recognized meaning of a polypeptide region of an antibody-binding protein that binds a class of antibodies rather than a specific set of antibodies.

[0111] The term "VP3" has the meaning in this specification of a protein derived from AAV, used to reconstitute viral capsids. See SEQ ID NO: 5.

[0112] The term "VP3-IT" has the meaning in this specification of a version of VP3 containing an internal peptide tag for enzymatic conjugation of the ASGPR ligand. This used the technology described in Buldun et al, J. Am. Chem. Soc. (2018). See SEQ ID NO: 6.

[0113] The terms "an effective amount" and "a therapeutically effective amount" have the biomedical art-recognized meaning of an amount effective to achieve its intended purpose. The effect can be detected by any assay method known in the art. The precise effective amount for a subject depends on the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically effectiveamounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.

[0114] The terms "combination therapy" and "co-therapy" have the biomedical art-recognized meaning of the administration of a composition described in this specification and at least a second agent as part of a specific treatment regimen intended to provide the beneficial effect from the coaction of these therapeutic agents. The beneficial effect of the combination may include but is not limited to pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents. These therapeutic agents are typically administered in combination over a defined time, usually minutes, hours, days, or weeks, depending on the combination selected. The term combination therapy" includes the administration of the therapeutic agents described above combined with other biologically active ingredients and non-drug therapies, e.g., surgery or radiation treatment. Where the combination therapy further comprises a non-drug treatment, the non-drug treatment may be conducted at any suitable time if a beneficial effect from the co-action of the combination of the therapeutic agents is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.

[0115] The terms "comprises," "comprising," "includes," and "including" specify the presence of stated features, regions, integers, steps, operations, elements, or components but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, or groups thereof.

[0116] The terms "first," "second," "third," etc., have the plain meaning of describing several elements, components, regions, layers, or sections. These terms should not limit these elements, components, regions, layers, or sections. These terms are only used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. A first element, component, region, layer, or section could be called a second element, component, region, layer, or section without departing from the teachings of the present embodiments.

[0117] The terms "subject" and "patient" have the biomedical art-recognized meanings. The term "patient" includes human and other mammalian subjects receiving prophylactic or therapeutic treatment.

[0118] The terms "treating" and "treat" have the biomedical art-recognized meaning of managing and caring for a patient to combat a disease, condition, or disorder. Treating includes administering acomposition described in this specification to alleviate the symptoms or complications of a disease, condition, or disorder or to eliminate the disease, condition, or disorder.

[0119] Some embodiments are described below by referring to structures and schemes to explain aspects of the description.

[0120] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by persons having ordinary skill in the biomedical art.

[0121] This specification does not concern a process for cloning humans, methods for modifying the germ line genetic identity of humans, uses of human embryos for industrial or commercial purposes, or procedures for modifying the genetic identity of animals likely to cause them suffering with no substantial medical benefit to humans or animals resulting from these processes.Methods of removal of antibodies from a subject or patient.

[0122] How to administer. The best mode of administration depends on where treatment is taking place, whether a hospital or outpatient. In one embodiment, the method of administration is by subcutaneous administration to a subject of the composition of matter (agent, TRAP™) In another embodiment, the method of administration is by intravenous administration to a subject of the composition of matter.

[0123] The removal of antibodies from a subject or patient can be measured by methods known to persons having ordinary skill in the art.The chemical structure of the composition of matter.

[0124] In an embodiment, the invention provides a composition of matter (agent, TRAP™) comprising a binding moiety that can bind to anti-AAV antibodies, a cellular receptor-binding moiety that binds to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface of degrading cells in a subject or patient, and a linker moiety linking the antibody moiety and the cellular receptor-binding moiety.

[0125] In some embodiments, the invention provides a composition of matter (an agent) having a structure selected from the Markush group of structures consisting of:RCN-(Xaa)y-Rcc,[AGN104] or a pharmaceutically acceptable salt thereof. In these structures, a, b, and c may independently be an integer of 1 or greater. In some embodiments, each cellular receptor-binding moiety independently has the structure of -(RCN-(Xaa)y-Rcc) or salt form thereof.

[0126] In some embodiments, an agent comprises one and no more than one binding moiety. In some embodiments, one or no more than one binding moiety is bound to a linker moiety. In some embodiments, a is 1. In some embodiments, a is 2 or more. In some embodiments, one and no more than one cellular receptor-binding moiety is bonded to a linker moiety. In some embodiments, b is 1. In some embodiments, two or more cellular receptor-binding moieties are bonded to a single linker moiety. In some embodiments, b is 2 or more. In some embodiments, an agent comprises one and no more than one cellular receptor-binding moiety. In some embodiments, c is 1. In some embodiments, b is 1 and c is 1. In some embodiments, a is 1, b is 1 and c is 1. In some embodiments, an agent comprises two or more moieties that bind to anti-AAV antibodies. In some embodiments, b is 2 or more and c is 1. In some embodiments, b is 2 or more and c is 2 or more. In some embodiments, b is 1, and c is 2 or more.

[0127] In some embodiments, c is 1-20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, c is selected from the Markush group of size ranges, where c is 1-15, c is 1-10, c is 1-9, c is 1-8, c is 1-7, c is 1-6, c is 1-5, c is 1-4, c is 1-3, and c is 1-2. In some embodiments, c is a size selected from the Markush group of sizes consisting of 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0128] In some embodiments, each cellular receptor-binding moiety in an agent is the same. In some embodiments, each linker moiety connecting a cellular receptor-binding moiety to an antibodymoiety is the same. In some embodiments, the TBT in agents is the same. In some embodiments, the -L-(TBT)b are the same.

[0129] As known by persons having ordinary skill in the biomedical art, an antibody agent may comprise more than one site, e.g., one on each of the more than one chain, e.g., one or each heavy chain. In some embodiments, an antibody moiety comprises two heavy chains, and one or both amino acid residues or amino acid residues corresponding thereto are each independently connected to a cellular receptor-binding moiety optionally through a linker. In some embodiments, one and no more than one is connected. In some embodiments, c is 1. In some embodiments, both are connected. In some embodiments, c is 2. In some embodiments, both antibodies to AAV-binding moieties or both linker moieties (if any) are the same.

[0130] In some embodiments, an agent comprises one and no more than one binding moiety. In some embodiments, one or no more than one binding moiety is bound to a linker moiety. In some embodiments, a is 1. In some embodiments, an agent comprises two or more moieties. In some embodiments, two or more moieties are bound to a single linker moiety. In some embodiments, a is 2 or more. In some embodiments, one and no more than one cellular receptor-binding moiety is bonded to a linker moiety. In some embodiments, b is 1. In some embodiments, two or more cellular receptorbinding moieties are bonded to a single linker moiety. In some embodiments, b is 2 or more. In some embodiments, an agent comprises one and no more than one cellular receptor-binding moiety. In some embodiments, c is 1. In some embodiments, b is 1 and c is 1. In some embodiments, a is 1, b is 1, and c is 1. In some embodiments, an agent comprises two or more antibodies to AAV-binding moieties. In some embodiments, b is 2 or more and c is 1. In some embodiments, b is 2 or more and c is 2 or more. In some embodiments, b is 1 and c is 2 or more.

[0131] In some embodiments, c is 1-20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, c is selected from the Markush group of size ranges, where c is 1-15, c is 1-10, c is 1-9, c is 1-8, c is 1-7, c is 1-6, c is 1-5, c is 1-4, c is 1-3, and c is 1-2. In some embodiments, c is a size selected from the Markush group of sizes consisting of 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0132] In some embodiments, each cellular receptor-binding moiety in an agent is the same. In some embodiments, each linker moiety connecting a cellular receptor-binding moiety to an antibody moiety is the same. In some embodiments, the TBT in agents is the same. In some embodiments, -L-(TBT)b is the same.

[0133] As known by persons having ordinary skill in the biomedical art, an antibody agent may comprise more than one site, e.g., one on each of the more than one chain, e.g., one or each heavychain. In some embodiments, an antibody moiety comprises two heavy chains, and one or both amino acid residues or amino acid residues corresponding thereto are each independently connected to a cellular receptor-binding moiety optionally through a linker. In some embodiments, one and no more than one is connected. In some embodiments, c is 1. In some embodiments, both are connected. In some embodiments, c is 2. In some embodiments, both antibodies to AAV-binding moieties or both linker moieties (if any) are the same.Binding moiety.

[0134] In some embodiments, the AAV antibody-binding moiety comprises a moiety selected from the Markush group consisting of one or more amino acid residues, a peptide moiety, a cyclic peptide moiety, a peptide comprising one or more natural amino acid residues, and a peptide comprising one or more unnatural natural amino acid residues.

[0135] In a particular moiety, the binding moiety is a reconstituted, empty AAV capsid formed by the assembly of individual AAV9 VPS proteins, each VPS protein having the polypeptide sequence of SEQ ID NO: 5. In similar embodiments, the VPS proteins have a polypeptide sequence similar to SEQ ID NO: 5 but with conservative substitutions, such that the VPS proteins can assemble to form an AAV capsid. Such assembled capsids would have a structure that could be recognized by anti-AAV antibodies.

[0136] In a particular moiety, the binding moiety is a reconstituted, empty AAV capsid formed by the assembly of individual AAV9 VP3-IT proteins, each VPIT protein having the polypeptide sequence of SEQ ID NO: 6. In similar embodiments, the VP3-IT proteins have a polypeptide sequence similar to SEQ ID NO: 6 but with conservative substitutions, such that the VP3-IT proteins can assemble to form an AAV capsid. Such assembled capsids would have a structure that could be recognized by anti-AAV antibodies. Antibody-binding moiety.

[0137] Several antibody-binding moieties, including universal antibody-binding moieties, can be used following the teachings of this specification. Certain antibody-binding moieties and technologies for identifying or assessing antibody-binding moieties are described in WQ2019 / 023501 and WO2019 / 136442, each of which is incorporated in this specification in its entirety by reference. Persons having ordinary skill in the biomedical art know that additional technologies in the biomedical art may be suitable for identifying or assessing antibody-binding moieties in accordance with this specification. In some embodiments, an antibody-binding moiety comprises one or more amino acid residues, each independently natural or unnatural.

[0138] In some embodiments, a peptide unit comprises a functional group in an amino acid residue that can react with a functional group of another amino acid residue. In some embodiments, a peptideunit comprises an amino acid residue with a side chain which comprises a functional group that can react with another functional group of the side chain of another amino acid residue to form a linkage, e.g., see moieties described in TABLE 2 of the International Patent Publication WO 2024 / 228935 (Biohaven Therapeutics Ltd.). In some embodiments, one functional group of one amino acid residue is connected to a functional group of another amino acid residue to form a linkage (or bridge). Linkages are bonded to backbone atoms of peptide units and comprise no backbone atoms. In some embodiments, a peptide unit comprises a linkage formed by two side chains of non-neighboring amino acid residues. In some embodiments, a linkage is bonded to two backbone atoms of two nonneighboring amino acid residues. In some embodiments, both backbone atoms bonded to a linkage are carbon atoms.

[0139] Persons having ordinary skill in the biomedical art know that an amino acid residue may be replaced by another amino acid residue having similar properties, e.g., one XaaH, e.g., Vai, Leu, etc., may be replaced with another XaaH, e.g., Leu, He, Ala, etc., one XaaAmay be replaced with another XaaA, one Xaapmay be replaced with another Xaap, one XaaNmay be replaced with another XaaN, one XaaLmay be replaced with another XaaL, etc.

[0140] In some embodiments, antibody-binding moieties, e.g., antibody-binding moieties, and useful technologies for developing or assessing these moieties are described in, e.g., Alves, Langmuir, 28, 9640-9648 (2012), Choe et al., Materials, 9, 994 (2016), Gupta et al., Nature Biomedical Engineering, 3, 917-929 (2019), Muguruma et al., ACS Omega, 4, 14390-14397 (2019), Yamada et al., Angewandte Chemie Int., Ed Engl.; 58(17), 5592-5597 (April 16, 2019), Kruljec et al., Bioconjugate Chem., 28(8): 2009- 2030 (2017), e.g., Fabsorbent, triazines, etc.; Kruljec et al., Bioconjugate Chem., 29(8), 2763-2775 (2018), W02012017021A2, etc., the binding moieties, e.g., antibody-binding moieties) of each of which is incorporated in this specification in its entirety by reference.

[0141] In some embodiments, an antibody-binding moiety, e.g., a protein-binding moiety, e.g., an antibody-binding moiety, is an affinity substance described in Australian Patent Publication 2018259856 or International Patent Publication WO 2018 / 199337, the affinity substance of each of which is incorporated in this specification by reference.

[0142] In some embodiments, an antibody-binding moiety comprises an adapter protein agent, e.g., as described by Hui et al., Bioconjugate Chem., 26, 1456-1460 (2015). In some embodiments, when used in accordance with this specification, adapter proteins do not require reactive residues, e.g., BPA, to achieve one or more advantages.

[0143] In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, is or comprises a triazine moiety, e.g., one described in US 2009 / 0286693. In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, is of such a structure that its corresponding compound is a compound described in US 2009 / 0286693, the compounds of which are independently incorporated in this specification by reference. In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, is ABT. In some embodiments, ABT is of such a structure that H-ABT is a compound described in US 2009 / 0286693, the compounds of which are independently incorporated in this specification by reference. In some embodiments, this compound can bind to an antibody. In some embodiments, this compound can bind to the Fc region of an antibody.

[0144] In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, is or comprises a triazine moiety, e.g., one described in Teng et al., J. Mol. Recognition, 12, 67-75 (1999). In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, is of such a structure that its corresponding compound is a compound described in Teng, the compounds of which are independently incorporated in this specification by reference. In some embodiments, an antibodybinding moiety, e.g., an antibody-binding moiety, is of such a structure that H-ABT is a compound described in Teng, the compounds of which are independently incorporated in this specification by reference. In some embodiments, this compound can bind to an antibody. In some embodiments, this compound can bind to the Fc region of an antibody.

[0145] In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, is a triazine moiety, e.g., one described by Uttamchandani et al., J. Comb. Chem., 6(6), 862-8 (November- December 2004). In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, is of such a structure that its corresponding compound is a compound described in Uttamchandani, the compounds of which are independently incorporated in this specification by reference. In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, is of such a structure that H-ABT is a compound described by Uttamchandani, which is independently incorporated in this specification by reference. In some embodiments, this compound can bind to an antibody. In some embodiments, this compound can bind to the Fc region of an antibody.

[0146] In some embodiments, an antibody-binding moiety binds to one or more binding sites of a protein selected from the Markush group of proteins consisting of protein A, protein G, protein L, protein Z, protein LG, protein LA, and protein AG. In some embodiments, an antibody-binding moiety is described in Choe, Durgannavar, & Chung, Materials, 9(12) (2016).11

[0147] Other useful technologies are described in Mustafaoglu et al., Analyst, 141(24), 6571-6582 (November 28, 2016).

[0148] In some embodiments, an amino acid has the structure of the formula LNK101:NH(Ral)-Lal-C(Ra2)(Ra3)-La2-COOH[LNK101]Amino acids

[0149] In some embodiments, an amino acid has the structure of the formula LNK101:NH(Ral)-Lal-C(Ra2)(Ra3)-La2-COOH,[LNK101] or a salt thereof, wherein: each of Ral, Ra2, Ra3is independently -La-R'; each of Laland La2is independently La; each Lais independently a covalent bond, or an optionally substituted bivalent group selected from Ci-C20aliphatic or Ci-C20heteroaliphatic having 1-5 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with -C(R')2-, -Cy-, -O-, -S-, -S-S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2- -S(O)2N(R')-, -C(O)S-, or -C(O)O-; each -Cy- is independently an optionally substituted bivalent monocyclic, bicyclic, or polycyclic group wherein each monocyclic ring is independently selected from a C3.2o cycloaliphatic ring, a Ce-2o aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; each R' is independently -R, -C(O)R, -CO2R, or -SO2R; each R is independently -H, or an optionally substituted group selected from Ci.3Oaliphatic, Ci.3Oheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, Ce-3o aryl, Ce-3o arylaliphatic, Ce-3o arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, or two R groups are optionally and independently taken together to form a covalent bond, or:two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; or two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon.

[0150] In some embodiments, an amino acid residue has the structure of -N(Ral)-Lal-C(Ra2)(Ra3)-La2-COO- or a salt form thereof.Linker moieties.

[0151] In some embodiments, provided compounds and agents may comprise one or more amino acid moieties, e.g., antibody-binding moieties, linker moieties, etc. Amino acid moieties can either be those of natural amino acids or unnatural amino acids. In some embodiments, an amino acid has the structure of the formula LNK101:NH(Ral)-Lal-C(Ra2)(Ra3)-La2-COOH,[LNK101] or a salt thereof, wherein: each of Ral, Ra2, and Ra3is independently -La-R' or an amino acid side chain; each of Laland La2is independently La; each Lais independently a covalent bond, or an optionally substituted bivalent group selected from C1-C20 aliphatic or C1-C20 heteroaliphatic having 1-5 heteroatoms, wherein one or more methylene units of the group are optionally and independently replaced with -C(R')z-, -Cy-, -O-, -S-, -S-S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2- -S(O)2N(R')-, -C(O)S-, or -C(O)O-; each -Cy- is independently an optionally substituted bivalent monocyclic, bicyclic, or polycyclic group wherein each monocyclic ring is independently selected from a C3-20 cycloaliphatic ring, a Ce-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; each R' is independently -R, -C(O)R, -CO2R, or -SO2R; each R is independently -H, or an optionally substituted group selected from C1-30 aliphatic, C1-30heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, C6-3o aryl, C6-3o arylaliphatic, C6-3o arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or two R groups are optionally and independently taken together to form a covalent bond, or: two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; or two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic, or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon.In some embodiments, an amino acid residue, e.g., of an amino acid having the structure of formula LNK101, has the structure of -N(Ral)-Lal-C(Ra2)(Ra3)-La2-CO-. In some embodiments, each amino acid residue in a peptide independently has the structure of -N(Ral)-Lal-C(Ra2)(Ra3)-La2-CO-.

[0152] In some embodiments, the invention provides a derivative of an amino acid of formula LNK101 or a salt thereof. In some embodiments, a derivative is an ester. In some embodiments, the invention provides a composition of matter of formula NH(Ral)-Lal-C(Ra2)(Ra3)-La2-COORCTor salt thereof, wherein RCTis R' and each other variable is independently as described in this specification. In some embodiments, RCTis R. In some embodiments, RCTis optionally substituted aliphatic. In some embodiments, RCTis t-butyl.

[0153] In some embodiments, Lalis a covalent bond. In some embodiments, a composition of matter of formula LNK101 is of the structure NH(Ral)-C(Ra2)(Ra3)-La2-COOH. In some embodiments, La2is -CH2SCH2-.

[0154] In some embodiments, La2is a covalent bond. In some embodiments, a composition of matter of formula LNK101 is of the structure NH(Ral)-Lal-C(Ra2)(Ra3)-COOH. In some embodiments, an amino acid residue has the structure of -N(Ral)-Lal-C(Ra2)(Ra3)-CO-. In some embodiments, Lalis -CH2CH2S-. In some embodiments, Lalis -CH2CH2S-, wherein the CH2is bonded to NH(Ral).

[0155] In some embodiments, Lalis a covalent bond, and La2is a covalent bond. In some embodiments, a composition of matter of formula LNK101 is of the structure NH(Ral)-C(Ra2)(Ra3)-COOH. In some embodiments, a composition of matter of formula LNK101 is of the structure NH(Ral)-CH(Ra2)-COOH. In some embodiments, a composition of matter of formula LNK101 has a structure selected from the Markush group of peptides consisting of NH(Ral)-CH(Ra3)-COOH, NH2-CH(Ra2)-COOH, NH2-CH(Ra3)-COOH, -N(Ral)-C(Ra2)(Ra3)-CO-, -N(Ral)-CH(Ra2)-CO- -N(Ral)-CH(Ra3)-CO-, -NH-CH(Ra2)-CO-, and -NH-CH(Ra3)-CO-

[0156] In some embodiments, Lais a covalent bond. In some embodiments, Lais optionally substituted by Ci-6bivalent aliphatic. In some embodiments, Lais optionally substituted for Ci_6alkylene. In some embodiments, Lais -CH2-. In some embodiments, Lais -CH2CH2-. In some embodiments, Lais -CH2CH2CH2-

[0157] In some embodiments, moieties are optionally connected through linker moieties. In some embodiments, a reactive group, e.g., RG, is connected to a cellular receptor-binding moiety, e.g., TBT, through a linker, e.g., LRM. In some embodiments, a moiety, e.g., LG, may also comprise one or more linkers, e.g., LLG1, LLG2, LLG3, LLG4, etc., to link several portions. In some embodiments, LLGis a linker moiety described in this specification. In some embodiments, LLG1is a linker moiety described in this specification. In some embodiments, LLG2is a linker moiety described in this specification. In some embodiments, LLG3is a linker moiety described in this specification. In some embodiments, LLG4is a linker moiety described in this specification. In some embodiments, LRMis a linker moiety described in this specification. In some embodiments, LPMis L. In some embodiments, LPMis a linker moiety described in this specification. In some embodiments, LPMis L.

[0158] Linker moieties of several types or for several purposes, e.g., those used in antibody-drug conjugates, etc., may be used in accordance with this specification.

[0159] Linker moieties can be bivalent or polyvalent, depending on how they are used. In some embodiments, a linker moiety is bivalent. In some embodiments, a polyvalent linker connects more than two moieties.

[0160] Other linker moieties are disclosed in International Patent Publication WO 2024 / 155750 , which is incorporated herein in its entirety by reference.

[0161] In some embodiments, where a particular protecting group (PG), leaving group (LG), or transformation condition is depicted, persons having ordinary skill in the biomedical art know that other protecting groups, leaving groups, and transformation conditions are also suitable and are contemplated. These groups and transformations are described in Smith & March, March's AdvancedOrganic Chemistry: Reactions, Mechanisms, and Structure, 5thedition (John Wiley & Sons, 2001), Larock, Comprehensive Organic Transformations, 2ndedition (John Wiley & Sons, 1999), and Greene's Protecting Groups in Organic Synthesis, Wuts, editor (John Wiley & Sons, 2014), the entirety of each of which is incorporated in this specification by reference.

[0162] In some embodiments, an oxygen-protecting group includes carbonyl and hydroxylprotecting groups, etc. Hydroxyl-protecting groups and amino-protecting groups are well known in the biomedical art and include those described in Greene's Protecting Groups in Organic Synthesis, Wuts, editor (John Wiley & Sons, 2014), the entirety of which is incorporated in this specification by reference.

[0163] Persons having ordinary skill in the biomedical art know that provided agents may contain one or more stereocenters and may be present as a racemic or diastereomeric mixture. They know that there are many methods known in the biomedical art for the separation of isomers to obtain stereoenriched or stereopure isomers of those compounds, including but not limited to HPLC, chiral HPLC, fractional crystallization of diastereomeric salts, kinetic enzymatic resolution, e.g., by fungal- derived, bacterial- derived, or animal-derived lipases or esterases), and formation of covalent diastereomeric derivatives using an enantioenriched reagent.

[0164] Persons having ordinary skill in the biomedical art know that several functional groups present in compounds of this specification, such as aliphatic groups, alcohols, carboxylic acids, esters, amides, aldehydes, halogens, and nitriles, can be interconverted by techniques well known in the biomedical art including, but not limited to reduction, oxidation, esterification, hydrolysis, partial oxidation, partial reduction, halogenation, dehydration, partial hydration, and hydration. See Smith & March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5thedition (John Wiley & Sons, 2001), which is incorporated in this specification by reference. These interconversions may require one or more of the aforementioned techniques, and certain methods for synthesizing compounds of this specification are described below in the Exemplification.

[0165] As known by persons having ordinary skill in the biomedical art, reaction partners are generally contacted with each other under conditions and for a time sufficient for producing the desired results, e.g., formation of product agents and compositions thereof to desired extents. Many reaction conditions / reaction times may be assessed and used if they are suitable for desired purposes in accordance with this specification.

[0166] In some embodiments, the invention provides products of provided processes, which have low levels of damage to antibody moieties compared to processes comprising steps performed for antibody-binding moiety removal but not for substantial conjugation of moieties of interest, e.g.,antibodies to AAV9-binding moieties. In some embodiments, provided product agent compositions have high homogeneity.

[0167] In some embodiments, the invention provides a product agent, which is an agent comprising an antibody-binding moiety, a cellular receptor-binding moiety, and optionally a linker moiety linking an antibody-binding moiety and a cellular receptor-binding moiety. In some embodiments, the invention provides compositions of such agents.

[0168] In some embodiments, the invention provides a composition comprising a plurality of agents, wherein each agent independently comprises an antibody-binding moiety, a cellular receptorbinding moiety, and optionally a linker moiety linking an antibody-binding moiety and a cellular receptor-binding moiety.

[0169] In some embodiments, the invention provides a composition comprising a plurality of agents, each of which independently comprises an antibody-binding moiety, a cellular receptor-binding moiety, and optionally a linker moiety linking the antibody moiety and the cellular receptor-binding moiety; wherein antibody moieties of agents of the plurality comprise a common amino acid sequence, and agents of the plurality share a common cellular receptor-binding moiety independently of at least one common amino acid residue of the common amino acid sequence; and wherein about l%-100% of all agents that comprise an antibody moiety that comprises the common amino acid sequence and the cellular receptor-binding moiety are agents of the plurality.

[0170] In some embodiments, the invention provides a composition comprising a plurality of agents, each of which independently comprises an antibody-binding moiety, a cellular receptor-binding moiety, and optionally a linker moiety linking an antibody moiety and a cellular receptor-binding moiety; wherein agents of the plurality share the same or substantially the same antibody moiety and a cellular receptor-binding moiety at least one common location; and wherein about l%-100% of all agents that comprise the antibody moiety and the cellular receptor-binding moiety are agents of the plurality.Reactive Group

[0171] In some embodiments, provided agents, compounds, e.g., those useful as reaction partners such as first agents, comprise reactive groups, e.g., RG. In some embodiments, reactive groups are located between antibody-binding moieties and moieties of interest. Reactive groups are optionally and independently linked to antibody-binding moieties and moieties of interest via linkers. In some embodiments, RG is a reaction group, as described in this specification.

[0172] In some embodiments, reactive groups, when used in agents that comprise no antibodybinding moieties, react slowly and provide a low level of, in some embodiments, substantially noconjugation of moieties of interest with target agents. As shown in this specification, a combination of reactive groups with antibody-binding moieties in the same agents, e.g., as in compounds of formula AGN301 or salts thereof, can promote reactions between reactive groups and target agents, enhance reaction efficiency, reduce side reactions, or improve reaction selectivity, e.g., in terms of target sites wherein conjugation of moieties of interest with target agents occurs.

[0173] In some embodiments, a reactive group, e.g., RG, is or comprises an ester group. In some embodiments, a reactive group, e.g., RG, is or comprises an electrophilic group, e.g., a Michael acceptor.

[0174] In some embodiments, a reactive group, e.g., RG, is or comprises — LRG1— l_RG2— , wherein each of LRG1and LRG2is independently L. In some embodiments, a reactive group, e.g., RG, is or comprises -LLG4-LRG1-LRG2-, wherein each variable is as described in this specification. In some embodiments, a reactive group, e.g., RG, is or comprises -LLG3-LLG4-LRG1-LRG2-, wherein each variable is as described in this specification. In some embodiments, a reactive group, e.g., RG, is or comprises -LLG2-LLG3-LLG4-LRG1-LRG2-, wherein each variable is as described in this specification. In some embodiments, a reactive group, e.g., RG, is or comprises -LLG4-LRG2-, wherein each variable is as described in this specification. In some embodiments, a reactive group, e.g., RG, is or comprises -LLG3-LLG4-LRG2-, wherein each variable is as described in this specification. In some embodiments, a reactive group, e.g., RG, is or comprises -LLG2-LLG3-LLG4-LRG2-, wherein each variable is as described in this specification.

[0175] In some embodiments, LLG4is -O-. In some embodiments, LLG4is -N(R)-. In some embodiments, LLG4is -NH-.

[0176] In some embodiments, LLG3is or comprises an optionally substituted aryl ring. In some embodiments, LLG3is or comprises a phenyl ring. In some embodiments, an aryl or phenyl ring is substituted. In some embodiments, a substituent is an electron-withdrawing group as described in this specification, e.g., -NO2, -F, etc.

[0177] In some embodiments, LRG1is a covalent bond. In some embodiments, LRG1is not a covalent bond. In some embodiments, LRG1is -S(O)2-.

[0178] In some embodiments, LRG2is -C(O)-. In some embodiments, a reactive group comprises LLG4-C(O), wherein each variable is described in this specification. In some embodiments, a reactive group comprises -LLG3-LLG4-C(O)-, wherein each variable is as described in this specification. In some embodiments, a reactive group comprises -LLG2-LLG3-LLG4-C(O)-, wherein each variable is as described in this specification.

[0179] In some embodiments, LRG2is -LRG3-C(=CRRG1RKG2)-CRRG3RRG4-, wherein each of RRG1, RRG2, RRG3and RRG4is independently -L-R', and LRG3is -C(O)-, -C(O)O- -C(O)N(R')-, -S(O)-, -S(O)2- -P(O)(OR')-, -P(O)(SR')-, or -P(O)(N(R')2)-. In some embodiments, each of RRG1, RRG2, RRG3, and RRG4is independently R'. In some embodiments, one or more of RRG1, RRG2, RRG3and RRG4is independently -H. In some embodiments, LRG3is -C(O)-. In some embodiments, LRG3is -C(O)O-. In some embodiments, -O-, -N(R')-, etc. of LRG3is bonded to LPM.

[0180] In some embodiments, RRG1is -H. In some embodiments, RRG3is -H.

[0181] In some embodiments, LRG2is optionally substituted -LRG3-C(=CHRRG2)-CHRRG4-, wherein each variable is as described in this specification.

[0182] In some embodiments, RRG2and RRG4are taken together with their intervening atoms to form an optionally substituted ring as described in this specification. In some embodiments, a formed ring is an optionally substituted 3-10 membered monocyclic or bicyclic ring having 0-5 heteroatoms. In some embodiments, a formed ring is an optionally substituted 3-10-membered cycloaliphatic ring. In some embodiments, a formed ring is selected from the Markush group consisting of optionally substituted cycloaliphatic rings consisting of a 3-8 membered cycloaliphatic ring, a 5-8 membered cycloaliphatic ring., a 5-membered cycloaliphatic ring, a 6-membered cycloaliphatic ring, and a 7- membered cycloaliphatic ring. In some embodiments, a formed ring is substituted. In some embodiments, a formed ring is not substituted. In some embodiments, a formed ring contains no additional unsaturation besides the double bond in C(=CHRRG2) or C(=CRRG1RRG2).

[0183] In some embodiments, a reactive group, e.g., -LLG2-LLG3-LLG4-LRG1-LRG2- or _LLG2_LLG3_LLG4_LRGI_is a structureselected from TABLE 3 of the International Patent Publication WO 2024 / 228935 (Biohaven Therapeutics Ltd.).

[0184] In some embodiments, -LLG4-LRG2- is -O-C(O)- or -S-C(O)-. In some embodiments, _LLG4_LRGI_LRG2_IS_S-C(O)_

[0185] In some embodiments, -LLG4-LRG2- is -N(-)-C(O)-, wherein N is a ring atom of an optionally substituted heteroaryl ring. In some embodiments, -LLG4-LRG2- is -N(-)-C(O)-, wherein N is a ring atom of LLG4, which is or comprises an optionally substituted heteroaryl ring. In some embodiments, -LLG4-LRG2- is -N(-)-C(O)-O-, wherein N is a ring atom of LLG4, which is or comprises an optionally substituted heteroaryl ring.

[0186] In some embodiments, LRG2is optionally substituted -CH2-C(O)-, wherein -CH2- is bonded to an electron-withdrawing group comprising or connected to an antibody-binding moiety. In some embodiments, LRG2is optionally substituted -CH2- bonded to an electron-withdrawing group comprisingor connected to an antibody-binding moiety. In some embodiments, LRG1is an electron-withdrawing group. In some embodiments, LRG1is selected from the Markush group consisting of -C(O)-, -S(O)-, -S(O)2-, -P(O(OR)-, -P(O(SR)-, -P(O(N(R)2)-, -OP(O(OR)-, -OP(O(SR)-, and -OP(O(N(R)2)-.

[0187] In some embodiments, LRG2is optionally substituted -CH2-C(O)-, wherein -CH2- is bonded to a leaving group comprising or connected to an antibody-binding moiety. In some embodiments, LRG2is optionally substituted -CH2- bonded to a leaving group comprising or connected to an antibody-binding moiety. In some embodiments, LRG1is selected from the Markush group consisting of -O-C(O)-, -OS(O)2-, -OP(O(OR)-, -OP(O(SR)-, and -OP(O(N(R)2)-.

[0188] In some embodiments, a reactive group reacts with an amino group of a target agent. In some embodiments, an amino group is -NH2of the side chain of a lysine residue.

[0189] In some embodiments, a target agent is a protein agent. In some embodiments, a target agent is an antibody agent. In some embodiments, a reactive group reacts with an amino acid residue of this protein or antibody agent. In some embodiments, an amino acid residue is a lysine residue. In some embodiments, a reactive group reacts with -NH2of the side chain of a lysine residue. In some embodiments, a reactive group is or comprises -C(O)-O- reacts with -NH2, e.g., of the side chain of a lysine residue) and forms an amide group -C(O)-O- with the -NH2.

[0190] In some embodiments, reactive groups, e.g., a first reactive group, a second reactive group, etc., are located at terminal locations. In some embodiments, the first agents comprise the first reactive groups linked to TARGET-binding moieties optionally through linker moieties, and they do not contain antibody-binding moieties.

[0191] In some embodiments, the invention provides methods for preparing a composition comprising a plurality of agents, wherein each agent independently comprises an antibody-binding moiety, a cellular receptor-binding moiety, and optionally a linker moiety linking an antibody-binding moiety and a cellular receptor-binding moiety; which method comprises contacting a plurality of agents, each independently comprising a reactive group with a plurality of antibody agents.

[0192] In some embodiments, an agent comprising a reactive group comprises an antibody-binding moiety, a cellular receptor-binding moiety, and optionally a linker. In some embodiments, agents comprising a reactive group share the same cellular receptor-binding moiety. In some embodiments, agents comprising a reactive group share the same structure. In some embodiments, antibody molecules are of such structures, properties, or activities to provide antibody moieties in agents described in this specification. In some embodiments, a plurality of antibody molecules comprises two or more IgG subclasses. In some embodiments, a plurality of antibody molecules comprises IgGl. Insome embodiments, a plurality of antibody molecules comprises lgG2. In some embodiments, a plurality of antibody molecules comprises lgG4. In some embodiments, a plurality of antibody molecules comprises IgGl and lgG2. In some embodiments, a plurality of antibody molecules comprises IgGl, lgG2, and lgG4. In some embodiments, a plurality of antibody molecules comprises IgGl, lgG2, lgG3 and lgG4. In some embodiments, a plurality of antibody molecules is I VIG antibody molecules.

[0193] Other reactive moieties are disclosed in International Patent Publication WO 2024 / 155750 , which is incorporated herein in its entirety by reference.Cellular receptor-binding moiety.

[0194] According to embodiments of the present invention, several receptor-binding moieties are described in International Pat. Publ. WO 2019 / 199621, published October 17, 2019, WO 2019 / 199634, published October 17, 2019, and International Pat. Publ. WO 2021 / 072246, published April 15, 2021, each of which is incorporated in this specification in its entirety by reference.

[0195] In an embodiment, the cellular receptor-binding moiety may include an asialoglycoprotein receptor (ASGPR) binding group connected through an amine group to the linker moiety.

[0196] The amine group may be a primary alkyl amine group or a secondary alkyl amine group, each of which is optionally substituted on the amine group with a C1-C3 alkyl group.

[0197] The cellular receptor-binding moiety may include an ASGPR binding group according to the chemical structure disclosed in Inti. Pat. Publ. WO 2019 / 199621.

[0198] The cellular receptor-binding moiety may have the following structure:where RAis a C1-C3 alkyl group optionally substituted with 1-5 halo groups (preferably RAis a methyl or ethyl group optionally substituted with from 1-3 fluoro groups);ZAis -(CH2)IM, -O-(CH2)IM, S-(CH2)IM, NRM-(CH2)IM, C(O)-(CH2)IM- a PEG group containing from 1 to 8 preferably 1-4 ethylene glycol residues or a -C(O)(CH2)IMNRM group (preferably a PEG containinggroup comprising from 1 to 8 ethylene glycol, preferably 2-4 ethylene glycol residues) where IM and RMare the same as above; andZBis absent, (CH2)IM, C(O)-(CH2)IM- or C(O)-(CH2)|M-NRM, where IM and RMare the same as above.

[0199] In an embodiment, RAmay be a methyl or ethyl group optionally substituted with one to three fluoro groups.

[0200] In an embodiment, ZAis a PEG group containing 1 to 4 ethylene glycol residues.

[0201] In an embodiment, the methyl or ethyl group may be substituted with from 1-3 fluoro groups.

[0202] In an embodiment, the ASGPR binding group may be N-acetyl-D-galactosamine.

[0203] In an embodiment, the cellular receptor-binding moiety may be a low-density lipoprotein receptor-related protein 1 (LRP1), a low-density lipoprotein receptor (LDLR), a FcyRI binding group, a FcRn binding group, a transferrin receptor-binding group, or a macrophage scavenger receptor-binding group.Pharmaceutically acceptable excipients.

[0204] Formulations suitable for parenteral administration, such as by intraarticular (in the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally. Parenteral, oral, and intravenous are the preferred administration methods. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.

[0205] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include these components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicities such as sodium chloride or dextrose. The pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.Statistical analyses.

[0206] Normal distribution quantitative variables can be expressed as means and SDs and compared by an independent-sample t-test. The inventors used median and interquartile ranges for non-normally distributed variables and analyzed them with the Mann-Whitney U test. Categorical data is summarized by percentages. A two-sided p-value <0.05 is considered statistically significant. Statistical tests are performed using SPSS version 16.0.Characterizing antibody-binding moieties.

[0207] Many technologies are available for identifying, assessing, or characterizing antibodybinding moieties, including protein-binding moieties, e.g., antibody-binding moieties such as universal antibody-binding moieties, or their use in provided technologies, e.g., those described in International Pat. Publ. WO 2019 / 023501 (Kleo Pharmaceuticals, Inc.), the technologies of which are incorporated in this specification by reference. In some embodiments, an antibody-binding moiety is a moiety, e.g., a small molecule, peptide, nucleic acid, etc., that can selectively bind to IgG and provide or stimulate ADCC or ADCP. In some embodiments, peptide display technologies, e.g., phase display, non-cellular display, etc., can identify antibody-binding moieties. In some embodiments, an antibody-binding moiety is a moiety, e.g., small molecule moiety, peptide moiety, nucleic acid moiety, etc., that can bind to IgG and optionally can compete with known antibody binders, e.g., protein A, protein G, protein L, etc.

[0208] Persons having ordinary skill in the biomedical art know that antibody-binding moieties described in this specification target antibodies with several properties and activities, e.g., antibodies recognizing different antigens, having optional modifications, etc. In some embodiments, these antibodies include antibodies administered to a subject, e.g., for therapeutic purposes. In some embodiments, antibody-binding moieties described in this specification may bind antibodies toward different antigens and are useful for conjugating moieties of interest with several antibodies.

[0209] In some embodiments, an antibody-binding moiety comprises a meditope agent moiety. A meditope agent is described in, e.g., U.S. Pat. Publ. 2019 / 0111149.

[0210] In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, can bind to human IgG. In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, can bind to an antibody selected from the Markush group of antibodies consisting of rabbit IgG, IgGl, lgG2, lgG3, and lgG4. In some embodiments, an antibody-binding moiety, e.g., an antibody-binding moiety, binds to IgGl, lgG2, and lgG4.Structure of recombinant ligand constructs.

[0211] Biophysical / biochemical potency assay to measure target engagement. A biomolecule coupled to the surface of the sensor chip as a ligand. As the analyte flows in solution over an immobilized ligand, binding to the sensor chip surface induces a change in refractive index proportional to bound mass.

[0212] An assay can be done using a Biacore S200 instrument. This machine has a high sensitivity and low-medium throughput.

[0213] ASGPR-dependent uptake assay. A HEK293 bioluminescent cell-based assay can be used to assess ternary complex formation uptake by cells in vitro. This on-mechanism endocytosis assay measures the accumulation in HEK293 cells.

[0214] Surface plasmon resonance. The binding ability of the antibodies to AAV9-binding moiety can be measured by surface plasmon resonance (Biacore™ and IBIS-MX96 systems) and bio-layer interferometry (ForteBio™ Octet™ systems).

[0215] Screening cascade plans-. SPR target engagement, ternary complex formation, cell-based assays, and endocytosis assays. Additional bridging assays to confirm degradation.Manufacturing the agent.

[0216] VHH production has a good titer (500mg / L) using CHO cells as a host. The 500 ml scale is useful because recovery is '“50-60% after purification to get 100 mg protein.

[0217] The manufacturing risks dimer / aggregation during the purification.Methods of making several agents.

[0218] Agents of this specification may be prepared or isolated by synthetic or semi-synthetic methods or recombinant methods in accordance with this specification. In some embodiments, polypeptide agents, e.g., cellular receptor-binding moiety peptide agents, may be prepared using biological expression systems. In some embodiments, provided agents are prepared synthetically. In some embodiments, provided agents are prepared using certain technologies described in W02019 / 023501, which is incorporated in this specification by reference.

[0219] In some embodiments, the invention provides a method of synthesis comprising the steps of contacting a first agent comprising a cellular receptor-binding moiety linked to a first reactive group optionally through a first linker with a second agent comprising an antibody moiety linked to a second reactive group optionally through a second linker, wherein the first reactive group reacts with a secondreactive group, and forming a product agent comprising a cellular receptor-binding moiety and an antibody-binding moiety optionally through a linker.

[0220] In some embodiments, the invention provides a method of synthesis comprising the steps of contacting a first composition comprising a plurality of first agents, each independently comprising a cellular receptor-binding moiety linked to a first reactive group optionally through a first linker moiety, with a second composition comprising a plurality of second agents, each independently comprising an antibody moiety optionally linked to a second reactive group, optionally through a second linker moiety, wherein a product composition comprising a plurality of product agents, each independently comprising a cellular receptor-binding moiety and an antibody-binding moiety optionally through a linker, is formed.EXAMPLES

[0221] The invention is further illustrated by non-limited EXAMPLES.EXAMPLE 1Capsid-bait designs for identifying antibody-binding moieties and manufacturing TRAP™ agents

[0222] Step Cl. The inventors produced unmodified VPS capsids from purified components. Verify purity and monodispersity after capsid assembly. Optimize purity and yields.

[0223] Step C2. The inventors produced AAV9 capsids functionalized with an ASGPR ligand on lysine residues. Use commercially available capsids or those produced in Aim Cl.

[0224] Step C3. The inventors demonstrated ASGPR-dependent endocytosis of capsids in a cell model. Use unmodified commercially available capsids or produced in Step Cl, or functionalized tools produced in Step C2.

[0225] Step C4. The inventors demonstrated ASGPR-dependent endocytosis of monoclonal neutralizing antibodies using bifunctional capsid-based degraders in a cell model. Incorporate neutralization from human sera or AAV transduction assays to validate the same.

[0226] Step C5. Persons having ordinary skill in the biomedical art can demonstrate that AAVR binding can be removed while maintaining anti-AAV binding. One can determine this experimentally by direct binding and confirm endocytosis with mutant capsids.Functiongl proof-of-concept

[0227] The inventors used a generic IgG bifunctional degrader to remove a monoclonal anti-AAV9 antibody and promote the transduction of an AAV9 virus (GFP).

[0228] Result 1 An anti-AAV9 antibody and IgGl control are internalized by an ASGPR-dependent pan IgG degrader. See FIG. 7.

[0229] Result 2-. AAV9-GFP transduction is neutralized by an anti-AAV9 antibody but restored to non-neutralizing levels with IgG bifunctional degrader treatment. See FIG. 8.EXAMPLE 2Empty AAV9 VP3 capsid production

[0230] Expression plasmids for AAV9 VPS proteins and AAP2 were co-transfected at 1:1 ratio into HEK 293F cells. Harvested cell pellets were re-suspended in Lysis buffer (20 mM Tris, 50 mM NaCI, pH 7.5) and lysed using a microfluidizer. Soluble intracellular proteins were enriched by centrifugation and the supernatants filtered (0.2 pM) prior to affinity chromatography using POROS Capture Select AAV9 Affinity resin. Protein was eluted in 0.1 M glycine, pH 3.0, and affinity chromatography elution fractions were pooled and subjected to size exclusion chromatography using a HiLoad (26 / 600) Superdex 200 pg column in phosphate-buffered saline, pH 7.4 buffer. Peak fractions containing AAV9 VP3 capsid were pooled, concentrated, and the purity and homogeneity of the reconstituted capsids was confirmed by SDS-PAGE, liquid chromatography-mass spectrometry, and size exclusion high performance liquid chromatography analysis on a TSK gel G3000 SWXL 7.8 X 300 mm column following standard procedures.

[0231] These methods produced an AAV9 VP3-only capsid, ABT301. The theoretical molecular weight was calculated to be 59,822 Da. The molecular weight was confirmed by SDS-PAGE.

[0232] Characterization by size exclusion chromatography (SEC) produced these results under the following conditions: Column = TSK gel G3000 SWXL 7.8 X 300 mm, buffer = 0.4 M Na-per chlorate pH 6.3, and flow rate = 1 mL / min:

[0234] Characterization by liquid chromatography-mass spectrometry (LCMS) showed a predominate single peaks under reduced and non-reduced conditions.

[0235] Dynamic light scattering (DLS) assays showed these results.

[0236] These methods also produced an AAV9 VP3-only capsid were each VP3 monomer includes an internal tag (VP3-IT) for directed enzymatic conjugation, ABT302. The theoretical molecular weight was calculated to be 63,316 Da. The molecular weight was confirmed by SDS-PAGE.

[0237] Characterization by size exclusion chromatography (SEC) produced these results under the following conditions: Column = TSK gel G3000 SWXL 7.8 X 300 mm, buffer = 0.4 M Na-per chlorate pH 6.3, and flow rate = 1 mL / min.

[0238] The standard curve was:EXAMPLE 3Surface plasmon resonance (SPR)

[0241] Capsid-based degraders were immobilized on a CM5 chip (Cytiva, BR100530). Samples were diluted to 9.5 pg / ml in 10 mM sodium acetate pH 4.0 / 5.0 and injected for 130-200 s at 10 pl / min flow rate. Immobilization levels ranging from 900RU-1300RU were obtained. For anti-AAV9 antibody HL2368 (Millipore, MABF2307), HL2374 (Millipore, MABF2326) and ADK9-hl (Progen, 692378) interactions, twofold concentration dilutions of antibody were prepared in assay buffer HBS-EP+ (Cytiva, BR100669) and injected on the immobilised capsid surface in a single cycle kinetics method with sixty second contact time, 120 seconds dissociation time, flow rate 30 pl / min at 25°C temperature. Surface was regenerated using 10 mM glycine pH 1.5 injected for twenty seconds at 30 pl / min flow rate. For ASGPR-Hlbinteractions, the same method was followed using assay buffer HBS-P+ (Cytiva, BR100671)SPR testing of enzymatically conjugated bifunctional degraders.

[0245] The inventors demonstrated by surface plasmon resonance assays the binding of the capsiddegrader AGN302 to antibody and ASGPR. See FIG. 5, anti-AAV antibody binding by SPR (TRAP™ binds anti-AAV antibody by SPR) and FIG. 6, ASGPR binding by SPR (anti-AAV TRAP™ binds ASGPR by SPR).

[0246] The inventors obtained similar results for surface plasmon resonance assays for enzymatically conjugated capsid degrader AGN302 and the chemically conjugated capsid degrader AGN301 for both the anti-AAV antibody binding by SPR and the ASGPR binding by SPR.EXAMPLE 4AAV9-GFP deneutralization

[0247] HEK-ASGPR cells were maintained in DMEM low glucose (Thermo Fisher, 11885076) supplemented with 10% fetal bovine serum (Thermo Fisher, 10082147), 1% PenStrep (Thermo Fisher, 15140122), and 200 pg / ml G418 (Thermo Fisher, 10131027) in an incubator set at 37°C with 5% CO2. 10,000 cells were seeded in a 384-well assay plate for overnight attachment. Capsid reagents were added to serum from six donor samples which were neutralizing against AAV9 and prepared at a 1:10 dilution in DMEM supplemented with 10% fetal bovine serum. Capsid-serum or serum only preparations were added to the assay plate with HEK-ASGPR cells. An AAV9-eGFP reporter virus solution (fully assembled AAV9 capsids containing an eGFP reporter gene controlled by a CMV promoter; Progen, 66V09) was added to the assay plate. The assay plate was moved to an IncuCyte S5 (Sartorius) instrument and images from individual wells were collected every six hours to monitor confluence and green fluorescence (a marker of AAV9-eGFP reporter virus transduction) over a forty-eight-hour timeframe.

[0248] The results were visualized using Prism (GraphPad Software) and reported for the forty- eight-hour timepoint.A AV 9 VP3 only capsid is a decoy for polyclonal anti-AAV neutralizing antibodies.

[0249] These assays showed that purified AAV9 capsids bind to polyclonal anti-AAV antibodies in human serum to enable AAV9-GFP transduction.

[0250] The assay tested three conditions. In the first condition, AAV9-GFP only (viral vector expressing green fluorescent protein) was used to transduce cells in vitro in the presence of neutralizing serum from healthy human donors. The serum in six different assays was from six individual human donors whose serum contained Neutralizing anti-AAV antibodies. No fluorescent GFP expression was observed in most of these assays. TheAAV9-GFP was neutralized by the donor sera.

[0251] In the second condition, AAV9-GFP plus ABT301 decoy capsid was used to transduce cells in vitro in the presence of neutralizing serum from same six healthy human donors described above. GFP expression was observed for all these assays indicating a binding and decoy function of ABT301 for neutralizing anti-AAV antibodies in human serum.

[0252] In the third condition, AAV9-GFP plus ABT302 modified decoy capsid was used to transduce cells in vitro in the presence of neutralizing serum from same six healthy human donors described above. GFP expression was observed for all these assays indicating a binding and decoy function ofABT302 for neutralizing anti-AAV antibodies in human serum. Thus, purified AAV9 capsids bind to polyclonal anti-AAV antibodies in human serum to enable AAV9-GFP transduction.AAV9 VP3 capsid is a decoy for polyclonal anti-AAV neutralizing antibodies - quantification.

[0253] Results: VPS (ABT301)-containing and VP3-IT (ABT302)-containing capsids can be used to created ASGPR- dependent degraders. These capsids bind to polyclonal anti-AAV antibodies in human serum, serving as decoy and enabling AAV9-GFP transduction, as shown by an increased green fluorescence total area.EXAMPLE 5AAV9 VP3 capsid functionalization for chemically conjugated capsid AGN301

[0254] This method was used to create a chemically conjugated AAV-capsid based degrader.

[0255] Purified AAV9 VPS capsid ABT301 (SEQ ID NO: 5) was concentrated to 1.33 pM and supplemented with freshly dissolved DBCO-PEG4-NHS ester (BroadPharm, BP-22288) at 120-fold excess molar equivalent and incubated for approximately sixteen hours at room temperature. Reactions were terminated by desalting using a Hi-prep 26 / 10 Desalting column (Sephadex G-25) in phosphate-buffered saline, pH 7.4 buffer. The reaction product was supplemented with freshly dissolved tri-N- acetylgalactosamine azide at fifty-fold excess molar equivalent and incubated for approximately sixteen hours at room temperature. Reactions were terminated by desalting using a Hi-prep 26 / 10 Desalting column (Sephadex G-25) in phosphate-buffered saline, pH 7.4 buffer and analyzed by SDS-PAGE and liquid chromatography-mass spectrometry, following standard procedures.Chemical conjugation of VPS capsids to make a degrader.

[0256] AGN301 was assayed after conjugation. Representative liquid chromatography / mass spectroscopy (LC-MS) results were as follows:

[0257] SDS-PAGE analysis of VP3 agent (GN3 conjugated capsids) showed a predominate single peak.EXAMPLE 6AAV9 VP3 capsid functionalization for the enzymatically conjugated capsid AGN302

[0258] This method was used to create an enzymatically conjugated AAV-capsid based degrader, AGN302.

[0259] Purified AAV9 VP3-IT capsid ABT302 (SEQ ID NO: 6) was buffer exchanged to Tris Borate buffer pH 7.4 and concentrated to 6.0 mg / mL. A ligase enzyme was prepared in the same buffer and concentrated to 5.0 mg / mL. A peptide functionalized with tri-N-acetylgalactosamine was prepared at 0.5 mM in 50% acetonitrile. Enzymatic conjugation reactions were prepared with 20 pM AAV9 VP3-IT capsid, 50 pM peptide and 200 pM ligase enzyme, and incubated for approximately eighteen hours at room temperature, before being terminated by gel-filtration chromatography. The reaction mixture was injected onto a Hi Load 16 / 600 Superdex 200 pg column for gel-filtration chromatography and analysed by SDS-PAGE and liquid chromatography-mass spectrometry, following standard procedures.Design of the VP3-IT construct.

[0260] The inventors performed a site-specific functionalization of AAV9-capsid at a protruding loop. The inventors inserted a peptide tag (DogTag, DIPATYEFTDGKHYITNEPIPPK, SEQ ID NO: 7) within a protruding loop on the AAV9 capsid. This insertion enables site-specific functionalization. SnoopLigase drives isopeptide bond formation between two peptides: DogTag and SnoopTagJr. (KLGSIEFIKVNK, SEQ ID NO: 8) GN3 functionalization can be performed at the N terminus or the C terminus. Insertion of a peptide tag (DogTag) within a protruding loop on the AAV9 capsid enables site-specific functionalization. See Buldun et al., J. Am. Chem. Soc., Vol. 140, Issue 8, pages 3008-3018 (2018).

[0261] AGN302 was assayed after conjugation. Representative liquid chromatography / mass spectroscopy (LC-MS) results were as follows:

[0262] SDS-PAGE analysis of VP3-IT (control and Snoop Tag-GN3 conjugated capsids) showed a predominate single peak at six time points.EXAMPLE 7Capsid-degrader internalization methods for the chemically conjugated capsid AGN301

[0263] HEK-ASGPR cells were maintained in DMEM low glucose (Thermo Fisher, 11885076) supplemented with 10% fetal bovine serum (Thermo Fisher, 10082147), 1% PenStrep (Thermo Fisher, 15140122), and 200 pg / ml G418 (Thermo Fisher, 10131027) in an incubator set at 37°C with 5% CO2. 25,000 cells were seeded in a 96-well assay plate for overnight attachment. Internalization assay was performed using ADK9-hl, an Anti-AAV9 antibody (Progen, 692378) labelled with LysoLight deep red dye (Thermo Fisher, L36001) according to the manufacturer protocol. For assay set up, the cell culture media was replaced with opti-MEM (Gibco, 31985070) containing labelled ADK9-hl (0.3 pg / ml) followed by addition of Vp3 capsid degraders. AlO-point concentration response curve was generated using a twofold serial dilution with a top concentration of 10 nM. The unconjugated VP3 capsid was used as negative control. The assay plate was moved to an IncuCyte S5 (Sartorius) instrument and images from individual wells were collected every two hours to monitor confluence and red fluorescence area (for LysoLight deep red fluorescence) over a seventy-two-hour timeframe.

[0264] Results were visualized using Prism (GraphPad Software).Chemically conjugated capsid. Cellular internalization into the lysosome.

[0265] Results: AAV9-VP3-GN3 (AGN301) facilitates a dose-dependent degradation of monoclonal anti-AAV9 ADK9hl antibody as measured by the LysoLight dye that fluoresces upon cathepsin cleavage in the lysosome. See Holly et al., bioRxiv (2024). Unconjugated AAV9-VP3 capsid does not result in degradation of ADK9hl, suggesting an ASGPR-dependent mechanism of internalization. See FIG. 8, EC5o 0.52 nM, as averaged from three independent experiments at the twenty-four hour time point. Individual experiment ECsovalues = 0.27, 0.70, and 0.51 nM.

[0266] The specific assay conditions for endocytosis cellular assay which results are shown in FIG. 8 (compiled data) were: HEK293-ASGPR1 cell line, ADK9hl directly conjugated to LysoLight Deep Red dye, FA = fluorescence area, and EC5o fitting limited to [Ligand] < 10 nM. The data points are mean ± SEM from eight technical replicates across three independent assays.EXAMPLE 8Capsid-degrader internalization methods for the enzymatically conjugated capsid AGN302

[0267] HEK-ASGPR cells were maintained in DMEM low glucose (Thermo Fisher, 11885076) supplemented with 10% fetal bovine serum (Thermo Fisher, 10082147), 1% PenStrep (Thermo Fisher, 15140122), and 200 pg / ml G418 (Thermo Fisher, 10131027) in an incubator set at 37°C with 5% CO2.25,000 cells were seeded in a 96-well assay plate for overnight attachment. Internalization assay was performed using HL2374-lgGl (Abeam, ab315826) labelled with LysosLight deep red dye (Thermo Fisher, L36001) as per manufacturer protocol. For assay set up, the cell culture media was replaced with opti- MEM (Gibco, 31985070) containing labelled antibody (lpg / ml) followed by addition of VP3-IT capsid degraders. AlO-point concentration response curve was generated using a two-fold serial dilution with a top concentration of 10 nM. The unconjugated VP3-IT capsids and peptide were used as negative controls. The assay plate was moved to an IncuCyte S5 (Sartorius) instrument and images from individual wells were collected every two hours to monitor confluence and red fluorescence area (for LysoLight deep red fluorescence) over a seventy-two-hour timeframe.

[0268] Results were visualized using Prism (GraphPad Software).Enzymatically conjugated capsid. Cellular internalization into the lysosome.

[0269] Results: AAV9-VP3-IT-GN3 (AGN302) facilitates a dose-dependent degradation of monoclonal anti-AAV9 HL2374 antibody as measured by the LysoLight dye. Unconjugated AAV9-VP3-IT capsid does not result in degradation of HL2374, suggesting an ASGPR-dependent mechanism of internalization. See FIG. 9, EC5o = 6.47 nM, as averaged from three independent experiments at the twenty-four hour timepoint. Individual experiment EC5o values = 6.85, 6.60, and 5.38 nM.

[0270] The specific assay conditions for endocytosis cellular assay which results are shown in FIG. 9 (compiled data) were: HEK293-ASGPR1 cell line, HL2374 directly conjugated to LysoLight Deep Red dye, FA = fluorescence area, and EC5o fitting limited to [Ligand] < 10 nM. The data points are mean ± SEM from nine technical replicates across three independent assays.EQUIVALENTS

[0271] Persons having ordinary skill in the biomedical art will recognize or be able to determine using no more than routine experimentation many equivalents to the specific procedures described in this specification. These equivalents are within the scope of this invention and are covered by the following claims. For example, pharmaceutically acceptable salts other than those specifically disclosed in the description and Examples in this specification can be employed. Furthermore, it is intended that specific items within lists of items, or subset groups of items within larger groups of items, can be combined with other specific items, subset groups of items, or larger groups of items whether there is a specific disclosure in this specification identifying such a combination.

[0272] Some embodiments of the invention can be practiced according to the following numbered paragraphs:

[0273] 1. A composition of matter comprising: (a) an immune antibody-binding moiety that binds to antibodies to an adeno-associated virus (AAV), (b) a cellular receptor-binding moiety capable of binding to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on surface degrading cells, and (c) a linker moiety (optionally a single peptide linkage) connecting the antibodies to AAV-binding moiety and the cellular receptor-binding moiety.

[0274] 2. The composition of matter of embodiment 1, wherein the adeno-associated virus is AAV9.

[0275] 3. The composition of matter of embodiment 1, wherein the binding immune antibodybinding moiety is an AAV capsid.

[0276] 4. The composition of matter of embodiment 1, wherein the binding immune antibodybinding moiety is an AAV capsid subunit or a fragment thereof.

[0277] 5. The composition of matter of embodiment 1, wherein the immune antibody-binding moiety is a peptide that specifically binds to an antibody that binds to an adeno-associated virus.

[0278] 6. The composition of matter of embodiment 5, wherein the peptide is a linear peptide.

[0279] 7. The composition of matter of embodiment 5, wherein the peptide is a constrained peptide.

[0280] 8. The composition of matter of embodiment 5, further comprising a VHH moiety conjugated to the linker moiety, wherein one or two peptides are conjugated to one or each of the protein chains of the VHH moiety.

[0281] 9. The composition of matter of embodiment 1, wherein the VHH moiety contains a LALA peptide substitution corresponding to a heavy chain sequence comprising a LALA peptide substitution at sites L234A and L235A.

[0282] 10. The composition of matter of embodiment 1, wherein the VHH moiety contains a LALA-PA peptide substitution corresponding to a heavy chain sequence comprising a LALA-PA peptide substitution at sites L234A, L235A, and P329A.

[0283] 11. The composition of matter of embodiment 1, having a structure of:pharmaceutically acceptable salt thereof, wherein: each of a and b is independently an integer of 1 or greater; each ABT isa antibodies to AAV-binding moiety or a fragment thereof; L is a linker moiety; and each TBT is independently a cellular receptor-binding moiety that binds to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on the surface degrading cells in a subject or patient.

[0284] 12. The composition of matter of embodiment 11, wherein a is 1, b is 3, and each TBT comprises an N-acetyl-D-galactosamine (GalNAc) moiety.

[0285] 13. The composition of matter of embodiment 1, wherein the cellular receptor-binding moiety comprises an ASGPR binding group connected through an amine group.

[0286] 14. The composition of matter of embodiment 1, wherein the cellular receptor-binding moiety comprises an ASGPR binding group according to the chemical structure:pharmaceutically acceptable salt, stereoisomer, solvate, or polymorph thereof.

[0287] 15. The composition of matter of embodiment 1, wherein the cellular receptor-binding moiety has the following structure:group optionally substituted with 1-5 halo groups (preferably RAis a methyl or ethyl group optionally substituted with from 1-3 fluoro groups); ZAis -(CH2)IM, -O-(CH2)IM, S-(CH2)IM, NRM-(CH2)IM, C(O)-(CH2)IM- a PEG group containing from 1 to 8 preferably 1-4 ethylene glycol residues or a -C(O)(CH2)|MNRMgroup (preferably aPEG containing group comprising from 1 to 8 ethylene glycol, preferably 2-4 ethylene glycol residues) where IM and RMare the same as above; and ZBis absent, (CH2)IM, C(O)-(CH2)IM- or C(O)-(CH2)|M-NRM, where IM and RMare the same as above.

[0288] 16. The composition of matter of embodiment 1, wherein the ASGPR binding group is N- acetyl-D-galactosamine.

[0289] 17. A pharmaceutical composition comprising a composition of matter any of the preceding embodiments 1-16 and a pharmaceutically acceptable excipient.

[0290] 18. A method of removing antibodies to AAV in a subject comprising administering to the subject an agent of any of the preceding embodiments 1-16.

[0291] 19. A method of treating a disease state or condition associated with elevated levels of antibodies to AAV in a patient by administering to the patient an effective amount of an agent of any of the preceding embodiments 1-16.REFERENCES

[0292] Persons having ordinary skill in the biomedical art can use these patents, patent applications, and scientific references to guide them to predictable results when making and using the invention.Patent literature

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[0302] U.S. Pat. Publ. 2019 / 0111149 (Gardiner et al.), Anti-EGFR antibody drug conjugate.Non-patent literature

[0303] Buldun et al., SnoopLigase catalyzes peptide-peptide locking and enables solid-phase conjugate isolation. Journal of the American Chemical Society 140(8), 3008-3018 (2018).

[0304] Caianiello et al., Bifunctional small molecules that mediate the degradation of extracellular proteins. Nature Chemical Biology, 17(9), 947-953 (2021) describes the ASGPR-dependent mechanism of Molecular Degraders of Extracellular targets (MoDE)-induced degradation.

[0305] Choe, Durgannavar, & Chung, Fc-binding ligands of immunoglobulin G: An overview of high affinity proteins and peptides. Materials, 9(12) (2016).

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[0307] Cobb et al., A combination of two human neutralizing antibodies prevents SARS-CoV-2 infection in rhesus macaques. bioRxiv, 2021-09 (2021).

[0308] Day et al., Adeno-associated virus serotype 9 antibodies in patients screened for treatment with onasemnogene abeparvovec. Mol. Ther. Methods Clin. Dev., 21, 76-82 (February 24, 2021).

[0309] De et al., High levels of persistent expression of al-antitrypsin mediated by the nonhuman primate serotype rh. 10 adeno-associated virus despite preexisting immunity to common human adeno- associated viruses. Mol. Ther., 13(1), 67-76 (2006).

[0310] DeLano et al., Convergent solutions to binding at a protein-protein interface. Science, 287, 1279-1283 (2000).

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[0312] GenBank Accession No. AF085716. The genome of AAV-5.

[0313] GenBank Accession No. AX753246. The genome of AAV-7.

[0314] GenBank Accession No. AX753249. The genome of AAV-8.

[0315] GenBank Accession No. DQ813647. The genome of AAV-12.

[0316] GenBank Accession No. EU285562. The genome of AAV-13.

[0317] GenBank Accession No. NC_00 1862. The genome of AAV-6.

[0318] GenBank Accession No. NC_001401. The genome of AAV-2.

[0319] GenBank Accession No. NC_001829. The genome of AAV-4.

[0320] GenBank Accession No. NC_002077. The genome of AAV-1.

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[0322] GenBank Accession Nos. KT235804-KT235812.

[0323] Giles et al., Mapping an adeno-associated Virus 9-specific neutralizing epitope to develop next-generation gene delivery vectors. J. Virol., 92(20), e01011-18 (September 26, 2018).

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[0326] Kabat et al., Sequences of Proteins of Immunological Interest. (U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, 1991), providing the Kabat CDR definitions and numbering used for biologies.

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[0329] Lefranc et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Developmental & Comparative Immunology, 27(1), 55-77 (2003), providing the IMGT CDR definitions and numbering used for biologies.

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[0336] Orlowski & Weber, Selective anti-AAV antibody depletion by hemapheresis and immunoadsorption. In Cardiac Gene Therapy: Methods and Protocols (New York, NY: Springer US, 2022), pages 235-248.

[0337] Orlowski et al., Successful transduction with AAV vectors after selective depletion of anti- AAV antibodies by immunoadsorption. Molecular Therapy-Methods & Clinical Development. 16, 192- 203 (March 13, 2020).

[0338] Penaud-Budloo et al., Pharmacology of Recombinant Adeno-associated Virus Production. Molecular Therapy-Methods and Clinical Development (Nature Publishing Group, 2018), pp.166-180.

[0339] Saxena & Wu, Advances in therapeutic Fc engineering-modulation of IgG-associated effector functions and serum half-life. Frontiers in immunology, 7, 580 (2016) (review).

[0340] Schulz et al., Binding and neutralizing anti-AAV antibodies: Detection and implications for rAAV-mediated gene therapy, Molecular Therapy, 31(3), 616-630 (2023).

[0341] Shields et al., High resolution mapping of the binding site on human IgGl for FcyRI, FcyRII, FcyRIII, and FcRn and design of IgGl variants with improved binding to the FcyR. Journal of Biological Chemistry 276(9), 6591-6604 (2001).

[0342] Srivastava et al., Nucleotide sequence and organization of the adeno-associated virus 2 genome. J. Viral., 45, 555-564 (1983).

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[0345] Teng et al., A strategy for the generation of biomimetic ligands for affinity chromatography. Combinatorial synthesis and biological evaluation of an IgG binding ligand, J. Mol. Recognition, 12, 67- 75 (1999).

[0346] Tilman et al., Novel human IgGl and lgG4 Fc-engineered antibodies with completely abolished immune effector functions. Protein Engineering, Design and Selection, Volume 29, Issue 10, pages 457-466 (October 2016). This paper shows that even LALA itself abolishes clq binding. P329A alone is tested, abolishes clq binding, and reduces FcgR binding. They show that P329G / LALA further reduces FcgR binding beyond LALA alone.

[0347] Uttamchandani et al., Microarrays of tagged combinatorial triazine libraries in the discovery of small-molecule ligands of human IgG, J. Comb. Chem., 6(6), 862-8 (November-December 2004).

[0348] Watanabe et al., Human soluble phospholipase A2 receptor is an inhibitor of the integrin- mediated cell migratory response to collagen. Am. J. Physiol. Cell Physiol., 315, C398-C408 (2018).

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[0350] Zinn et al., In silico reconstruction of the viral evolutionary lineage yields a potent gene therapy vector. Cell Reports 12, 1056-1068 (2015).Textbooks and technical references

[0351] Current Protocols in Molecular Biology (CPMB), Ausubel, editor (John Wiley and Sons, Inc., 2014).

[0352] Janeway's Immunobiology, Murphy, Mowat, & Weaver, editors (Taylor & Francis Limited, 2014).

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[0354] Molecular Cloning: A Laboratory Manual, 4th ed., Michael Richard Green, and Joseph Sambrook, (2012). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (ISBN 1936113414).

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[0358] Throughout this application, several publications are referenced by author name and date or by patent or patent publication number. The disclosures of these publications are incorporated in their entireties by reference into this application to describe the state of the art more fully as known to persons having ordinary skill in the biomedical art as of the date of the invention described and claimed in this specification. However, the citation of a reference in this specification should not be construed as an acknowledgment that this reference is prior art to the present invention.

[0359] All patents and publications cited throughout this specification are incorporated by reference to disclose and describe the materials and methods that might be used with the technologies described in this specification. The publications discussed are provided only for their disclosure before the filing date. They should not be construed as an admission that the inventors may not antedate this disclosure under prior invention or for any other reason. If there is an apparent discrepancy between a prior patent or publication and the description provided in this specification, the specification (including any definitions) and claims shall control. All statements about the date or contents of these documents are based on the information available to the applicants. These statements are no admission to the correctness of the dates or contents of these documents. The publication dates in this specification may differ from the actual publication dates. If there is an apparent discrepancy between a publication date in this specification and the actual publication date supplied by the publisher, the actual publication date shall control.SEQUENCE LISTINGSequence Number (ID) : 1 Length: 330Molecule Type: AA Features Location / Quali tiers : source, 1. .330 mol type, protein organism, Homo sapiens Residues :ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPBPVTVS WNSGALTSGV HTFPAVLQSS 60GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG 120PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 180STYRWSVLT VLHQDWINGK EYKCRVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 240LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 300QQGNVFSCSV MHEATHNHYT QKSLSLSPGK 330Sequence Number (ID) : 2 Length: 326Molecule Type: AA Features Location / Quali tiers : source, 1. .326 mol type, protein organism, Homo sapiens Residues :ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPBPVTVS WNSGALTSGV HTFPAVLQSS 60GLYSLSSWT VPSSNFGTQT YTCNVDHKPS NTKVDKTVER KCCVECPPCP APPVAGPSVF 120LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVQFNWYVDG VEVHNAKTKP REBQFNSTFR 180WSVLTVLHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG OPREPQVYTL PPSREEMTKN 240QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN 300VFSCSVMHEA THNHYTQKSL SLSPGK 326Sequence Number (ID) : 3 Length: 327Molecule Type: AA Features Location / Quali tiers : source, 1. .327 mol type, protein organism, Homo sapiens Residues :ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPBPVTVS WNSGALTSGV HTFPAVLQSS 60GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KTGPPCPSCP APEFLGGPSV 120FLFPPKPKDT LMISRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQ FNSTY 180RWSVLTVLH QDWINGKEYK CKVSNKGLPS SIEKTISKAK GOPREPQVYT LPPSQEEMTK 240NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG 300NVFSCSVMHE ATHNHYTQKS LSLSLGK 327Sequence Number (ID) :Length: 6 Molecule Type: AAFeatures Location / Quali tiers : source, 1. .6 mol type, protein organism, synthetic construct Residues :QAQAQT 6Sequence Number (ID) : 5 Length: 534Molecule Type: AA Features Location / Quali tiers : source, 1. .534 mol type, protein organism, synthetic constructResidues :MASGGGAPVA DNNEGADGVG SSSGNWHCDS QWLGDRVITT STRTWALPTY NNHLYKQISN 60STSGGSSNDN AYFGYSTPWG YFDFNRFHCH FSPRDWQRLI NNNWGFRPKR LNFKLFNIQV 120KEVTDNNGVK TIANNLTSTV QVFTDSDYQL PYVLGSAHEG CLPPFPADVF MIPQYGYLTL 180NDGSQAVGRS SFYCLEYFPS QMLRTGNNFQ FSYEFENVPF HSSYAHSQSL DRLMNPLIDQ 240YLYYLSKTIN GSGQNQQTLK FSVAGPSNMA VQGRNYIPGP SYRQQRVSTT VTQNNNSEFA 300WPGASSWALN GRNSLMNPGP AMASHKEGED RFFPLSGSLI FGKQGTGRDN VDADKVMITN 360EEEIKTTNPV ATESYGQVAT NHQSAQAQAQ TGWVQNQGIL PGMVWQDRDV YLQGPIWAKI 420PHTDGNFHPS PLMGGFGMKH PPPQILIKNT PVPADPPTAF NKDKLNSFIT QYSTGQVSVE 480IEWELQKENS KRWNPEIQYT SNYYKSNNVE FAVNTEGVYS EPRPIGTRYL TRNL 534Sequence Number ( ID ) : 6Length : 569Molecule Type : AAFeatures Location / Quali tiers : source , 1 . . 569 mol type, protein organi sm, synthetic construct- REGION, 387 . . 421> note , DogTag sequence was described by Buldun et al J Am Chem Soc 2018 .Residues :MASGGGAPVA DNNEGADGVG SSSGNWHCDS QWLGDRVITT STRTWALPTY NNHLYKQI SN 60STSGGSSNDN AYFGYSTPWG YFDFNRFHCH FSPRDWQRLI NNNWGFRPKR LNFKLFNIQV 120KEVTDNNGVK TIANNLTSTV QVFTDSDYQL PYVLGSAHEG CLPPFPADVF MI PQYGYLTL 180NDGSQAVGRS SFYCLEYFPS QMLRTGNNFQ FSYEFENVPF HSSYAHSQSL DRLMNPLIDQ 240YLYYLSKTIN GSGQNQQTLK FSVAGPSNMA VQGRNYIPGP SYRQQRVSTT VTQNNNSEFA 300WPGASSWALN GRNSLMNPGP AMASHKEGED RFFPLSGSLI FGKQGTGRDN VDADKVMITN 360EEEIKTTNPV ATESYGQVAT NHQSAQGSGS GSDIPATYEF TDGKHYITNE PI PPKGSGSG 420SAQAQTGWVQ NQGILPGMVW QDRDVYLQGP IWAKIPHTDG NFHPSPLMGG FGMKHPPPQI 480LIKNTPVPAD PPTAFNKDKL NSFITQYSTG QVSVEIEWEL QKENSKRWNP EIQYTSNYYK 540SNNVEFAVNT EGVYSEPRPI GTRYLTRNL 569Sequence Number ( ID ) : 7 Length : 23Molecule Type : AAFeatures Location / Quali tiers : source , 1 . . 23 mol type, protein organi sm, synthetic construct - CARBOHYD, 17> note , Linkage site Residues :DIPATYEFTD GKHYITNEPI PPK 23Sequence Number (ID) : 8 Length: 12Molecule Type: AA Features Location / Quali tiers : source, 1. .12 mol type, protein organism, synthetic constructResidues :KLGSIEFIKV NK 12

Claims

1. CLAIMS1. A composition of matter comprising: an antibody-binding moiety that binds to antibodies to an adeno-associated virus (AAV), a cellular receptor-binding moiety capable of binding to hepatocytes or other degrading cells through asialoglycoprotein receptors (ASGPR) of hepatocytes or other cell receptors on surface degrading cells, and a linker moiety (optionally a single peptide linkage) connecting the antibodies to AAV-binding moiety and the cellular receptor-binding moiety.

2. The composition of matter of Claim 1, wherein the adeno-associated virus is AAV9.

3. The composition of matter of Claim 1, antibody-binding moiety is selected from the group consisting of an AAV capsid, an AAV capsid subunit or a fragment thereof, or a peptide that specifically binds to an antibody that binds to an adeno-associated virus.

4. The composition of matter of Claim 1, wherein the antibody-binding moiety is an AAV capsid comprising polypeptides having the structure of SEQ ID NO: 5 or a variant of SEQ ID NO: 5 comprising conservative amino acid substitutions.

5. The composition of matter of Claim 1, wherein the composition of matter is AGN301.

6. The composition of matter of Claim 1, wherein the antibody-binding moiety is an AAV capsid comprising polypeptides having the structure of SEQ ID NO: 6 or a variant of SEQ ID NO: 6 comprising conservative amino acid substitutions.

7. The composition of matter of Claim 1, wherein the composition of matter is AGN302.

8. The composition of matter of Claim 1, wherein the cellular receptor-binding moiety has the following structure:where RAis a C1-C3 alkyl group optionally substituted with 1-5 halo groups (preferably RAis a methyl or ethyl group optionally substituted with from 1-3 fluoro groups);ZAis -(CH2)IM, -O-(CH2)IM, S-(CH2)IM, NRM-(CH2)IM, C(O)-(CH2)IM- a PEG group containing from 1 to 8 preferably 1-4 ethylene glycol residues or a -C(O)(CH2)IMNRM group (preferably a PEG containing group comprising from 1 to 8 ethylene glycol, preferably 2-4 ethylene glycol residues) where IM and RMare the same as above; andZB is absent, (CH2)IM, C(O)-(CH2)IM- or C(O)-(CH2)IM-NRM, where IM and RM are the same as above.

9. The composition of matter of Claim 1, wherein the ASGPR binding group is N-acetyl-D- galactosamine.

10. The composition of matter of Claim 1, for use in removing antibodies to AAV from a subject.

11. The composition of matter of Claim 1, for use in enhancing AAV-based gene therapy in a subject by removing antibodies to AAV from the subject.

12. A pharmaceutical composition comprising a composition of matter any of the preceding Claims 1-10 and a pharmaceutically acceptable excipient.

13. A method of removing antibodies to AAV in a subject comprising administering to the subject an agent of any of the preceding Claims 1-9.

14. A method enhancing AAV-based gene therapy in a patient by administering to the patient an effective amount of an agent of any of the preceding Claims 1-9.

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