AAV viral particles retargeted to EGFR-expressing cancer cells

Abstract

Provided herein are compositions and methods for retargeting viral particles, e.g., adeno-associated virus (AAV) particles, to cells expressing EGFR or a variant thereof using EGFR-targeting ligands. Also provided herein are methods of treating a disease or disorder associated with a cell (over)expressing EGFR or a variant thereof, e.g., cancer.

Description

AAV VIRAL PARTICLES RETARGETED TO EGFR-EXPRESSING CANCER CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to U.S. Provisional Application No. 63 / 675,679, filed July 25, 2024, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The official copy of the sequence listing is submitted electronically via EFS- Web with a file named, “11881WO01.xml,” created on July 25, 2025, and having a size of 463 kilobytes, and is filed concurrently with the specification. The sequence listing contained in the XML formatted document is part of the specification and is herein incorporated by reference in its entirety.TECHNICAL FIELDS

[0003] The disclosure herein relates to methods of making and using viral particles, e.g., AAV particles, comprising capsid proteins retargeted to an Epidermal Growth Factor Receptor (EGFR), e.g., cancer-associated mutant variants of EGFR, useful for targeting of EGFR-expressing cells, e.g., tumor cells.BACKGROUND OF THE INVENTION

[0004] The delivery of genes into particular target cells has become one of the most important technologies in modern medicine for the potential treatment of a variety of chronic and genetic diseases. Ideally, a gene delivery vehicle is able to stably introduce genetic material into desired cells and avoid introducing genetic material into non-target cells.

[0005] Viral particles, particularly those based on adeno-associated viruses (AAV), as gene delivery vehicles have been the focus of much research since AAVs are capable of transducing a wide range of primate species and tissues in vivo with no evidence of toxicity or pathogenicity. (Muzyczka, et al. (1992) Current Topics in Microbiology and Immunology, 158:97-129). Moreover, AAV safely transduces postmitotic tissues. Although the virus can occasionally integrate into host chromosomes, it does so very infrequently into a safe-harbor locus in human chromosome 19, and only when the replication (Rep) proteins are supplied intrans. AAV genomes rapidly circularize and concatemerize in infected cells, and exist in a stable, episomal state in infected cells to provide long-term stable expression of their payloads.

[0006] Additionally, manipulating and redirecting AAV infection to specific cells has been achieved in recent years. Many of the advances in targeted gene therapy using viral particles may be summarized as non-recombinatorial (non-genetic) or recombin atori al (genetic) modification of the viral particle, which result in the pseudotyping, expanding, and / or retargeting of the natural tropism of the viral particle. (Reviewed in Nicklin and Baker (2002) Curr. Gene Ther. 2:273-93; Verheiji and Rottier (2012) Advances Virol 2012: 1-15).

[0007] In a direct recombinatorial targeting approach, a targeting ligand is directly inserted into, or coupled to, a viral capsid, i.e., protein viral capsid genes are modified to express capsid proteins comprising a heterologous targeting ligand. The targeting ligand then redirects, e.g., binds, a receptor or marker preferentially or exclusively expressed on a target cell. (Stachler et al. (2006) Gene Ther. 13:926-931; White et al. (2004) Circulation 109:513- 519; see also Park et al., (2007) Frontiers in Bioscience 13:2653-59; Girod et al. (1999) Nature Medicine 5:1052-56; Grifman et al. (2001) Molecular Therapy 3:964-75; Shi et al. (2001) Human Gene Therapy 12:1697-1711 ; Shi and Bartlett (2003) Molecular Therapy 7:515-525).

[0008] In indirect recombinatorial approaches, a viral capsid is modified with a heterologous “scaffold”, which then links to an adaptor that includes a targeting ligand. The adaptor binds to the scaffold and the target cell. (Arnold et al. (2006) Mol. Ther. 5:125-132; Ponnazhagen et al. (2002) J. Virol. 76:12900-907; see also WO 97 / 05266) Scaffolds such as (1) Fc binding molecules (e.g., Fc receptors, Protein A, etc.), which bind to the Fc of antibody adaptors, (2) (strept)avidin, which binds to biotinylated adaptors, (3) biotin, which binds to adaptors fused with (strept)avidin, (4) a detectable label, which is useful for detection and / or isolation of viral particles, bound by a bispecific adaptor able to non- covalently bind the detectable label and target molecule, and recently (5) proteimprotein binding pairs that form isopeptide bonds have been described for a variety of viral particles. (See, e.g., Gigout et al. (2005) Molecular Therapy 11 :856-865; Stachler et al. (2008) Molecular Therapy 16:1467-1473; Quetglas et al. (2010) Virus Research 153:179-196;Ohno et al. (1997) Nature Biotechnology 15:763-767; Klimstra et al. (2005) Virology 338:9- 21).

[0009] With the advances providing the ability to direct AAV infection, there remains a need to discover targets for the specific transfer of nucleic acid molecules to a cell, e.g., an EGFR-expressing cell, e.g., a tumor cell.SUMMARY OF THE INVENTION

[0010] It is shown herein that an AAV capsid protein may be modified to allow for the targeted introduction of a polynucleotide into mammalian cells expressing Epidermal Growth Factor Receptor (EGFR) or a variant thereof, e.g., tumor cells expressing EGFRvIII.

[0011] Viral particles as described herein are particularly suited for the targeted and specific introduction of a polynucleotide into a cell expressing EGFR or a variant thereof, e.g., a mutant variant associated with cancer, e.g., EGFRvIII, since the viral capsid or viral capsid protein(s) described herein are retargeted with (e.g., comprise, display and / or is associated with, etc.) a targeting ligand that binds EGFR or a variant thereof, e.g., EGFRvIII. In some embodiments, a viral capsid or viral capsid protein comprises a first member of a proteimprotein binding pair, which is optionally associated with its cognate second member of the proteimprotein binding pair, wherein the second member comprises and / or is linked to (e.g., fused to) a targeting ligand that binds a tumor cell-specific surface protein, e.g., EGFR or a variant thereof. In some embodiments, the targeting ligand is operably linked to the second member, e.g., fused to the second member, optionally via a linker. In some embodiments, a targeting ligand may be a binding moiety, e.g., a natural ligand, antibody or antigen binding portion thereof, a multispecific binding molecule, etc. In some embodiments, the targeting ligand is an antibody or antigen binding portion thereof. In some embodiments, the targeting ligand is an antibody or antigen binding portion thereof comprising a variable domain that binds EGFR or a variant thereof, e.g., on a tumor cell, and a heavy chain constant domain. In some embodiments, the targeting ligand is an antibody or antigen binding portion thereof comprising a variable domain that binds EGFR or a variant thereof on a target cell and an IgG heavy chain constant domain. In some embodiments, the targeting ligand is an antibody or antigen binding portion thereof comprising a variable domain that binds a tumor cell-specific surface protein (e.g., EGFR, e.g., EGFRvIII) on a target cell and an IgG heavychain constant domain, wherein the IgG heavy chain constant domain is operably linked, e.g., via a linker, to a protein (e.g., a second member of a proteimprotein binding pair) that forms an isopeptide covalent bond with the first member of the proteimprotein binding pair. In some embodiments, a capsid protein described herein comprises a first member comprising SpyTag operably linked to the viral capsid protein, and covalently linked to the SpyTag, a second member comprising SpyCatcher linked to a targeting ligand comprising an antibody variable domain and an IgG heavy chain domain, wherein SpyCatcher and the IgG heavy chain domain are linked via an amino acid linker, e.g., GSGESG. (SEQ ID NO: 388). In some embodiments, the targeting ligand binds a mammalian EGFR or a variant thereof, e.g., human EGFR, e.g., human EGFRvIII. In some embodiments, the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 comprising an amino acid sequence of a heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1 , LCDR2, LCDR3, and / or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 as set forth in or encoded by a sequence or sequences as set forth in Table 1 and / or any one of SEQ ID NOs: 228-371.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 illustrates an example of covalent isopeptide-based binding of monoclonal antibodies to liver-detargeted AAV capsid mutants with the non-limiting bacteria-derived SpyTag-SpyCatcher system. Liver-detargeted AAV capsid mutants are generated via the use of AAV Cap gene plasmids containing well-described mutations that ablate the binding to glycans that naturally serve as AAV receptors. AAV capsid mutants are then subsequently engineered via insertion of the SpyTag peptide into a variable loop, e.g., variable loop IV or variable loop VIII of the AAV structural proteins VP1 , VP2 and VP3. “Mosaic” SpyTagged AAV capsids are then generated by mixing AAV Cap gene plasmids carrying the liver-detargeting mutations with AAV Cap gene plasmids carrying both the liver-detargeting mutation and the SpyTag insertion at different ratios. SpyCatcher-fused monoclonal antibodies are generated by genetic fusion of the antibody CDH3 cDNAsequence with cDNA encoding the SpyCatcher protein. Antibody-retargeted AAV can be then produced by conjugating Mosaic SpyTagged AAV capsids with SpyCatcher-fused monoclonal antibodies in various pH and temperature conditions.

[0013] Figure 2 demonstrates that conjugation of EGFR antibodies confers gain of transduction to AAV9 W503A mutants in several mouse tumor cell lines overexpressing human EGFRvITI. Figure 2 shows representative luciferase expression (relative light units; RLU) as measured in mouse tumor cell lines that overexpress (A) human EGFRvIII: B16F1, (B) B16F10.9, and (C) GL261, after transduction at various multiplicities of infection (“MOI”; 1 x 102to 1 x 10svg / ml) with the following viruses: (i) AAV9 wildtype (WT), (ii) liver-detargeted mutant AAV9 W5O3A, (iii) liver-detargeted AAV9 W503A with SpyTag- SpyCatcher conjugation to a representative anti-EGFRvIII antibody, H1H163N2, and (iv) liver-detargeted AAV9 W5O3A with SpyTag-SpyCatcher conjugation to a representative anti-EGFRvIII antibody, H1H10126P2. Figure 2 also shows bar graphs showing representative luciferase expression (RLU) as measured in mouse tumor cell lines that overexpress (D) human EGFRvIII: B16F1, (E) B16F10.9, and (F) GL261, after transduction at the single MOI of 1 x 105vg / ml with the following viruses: (i) AAV9 WT, (ii) liver- detargeted mutant AAV9 W503A, (iii) liver-detargeted AAV9 W503A with SpyTag- SpyCatcher conjugation to a representative anti-EGFRvIII antibody, H1H163N2, and (iv) liver-detargeted AAV9 W5O3A with SpyTag-SpyCatcher conjugation to a representative anti-EGFRvIII antibody, H1H10126P2.

[0014] Figure 3 demonstrates that conjugation of EGFR antibodies confers gain of transduction to AAV9 W503A mutants in several human tumor cell lines overexpressing EGFRvIII. Figure 3 shows representative luciferase expression (RLU) as measured in cell lines that overexpress human EGFRvIII: engineered human glioblastoma cell lines (A) U-87 and (B) U-251, as well as (C) HEK293 cells, after transduction at various MOI (1 x 102to 1 x 105vg / ml) with the following viruses: (i) AAV9 WT, (ii) liver-detargeted mutant AAV9 W503A, (iii) liver-detargeted AAV9 W5O3A with SpyTag-SpyCatcher conjugation to a representative anti-EGFRvIII antibody, H1H163N2, and (iv) liver-detargeted AAV9 W503A with SpyTag-SpyCatcher conjugation to a representative anti-EGFRvIII antibody, H1H10126P2. Figure 3 also shows bar graphs showing representative luciferase expression (RLU) as measured in cell lines that overexpress human EGFRvIII: engineered humanglioblastoma cell lines (D) U-87 and (E) U-251, as well as (F) HEK293, after transduction at the single MOI of 1 x 105vg / ml with the following viruses: (i) AAV9 WT, (ii) liver- detargeted mutant AAV9 W503A, (iii) liver-detargeted AAV9 W503A with SpyTag- SpyCatcher conjugation to a representative anti-EGFRvIII antibody, H1H163N2, and (iv) liver-detargeted AAV9 W5O3A with SpyTag-SpyCatcher conjugation to a representative anti-EGFRvIII antibody, H1 H10126P2.

[0015] Figure 4 shows a representative flow cytometry quantitation of eGFP positive glioblastoma tumor cells (U251 / EGFRvIII) after injection with different MOIs of wildtype AAV2 or AAV9 particles encapsulating a nucleotide of interest encoding eGFP under the control of a CAG promoter; AAV2 or AAV9 particles comprising one or more detargeting mutation(s) (e.g., R585A and R588A (HBM), or W503A, respectively), retargeted with a representative anti-EGFR antibody designated as H1H10126P, and encapsulating a nucleotide of interest encoding eGFP under the control of a CAG promoter.

[0016] Figure 5 demonstrates that AAV9 W503A mutants retargeted with EGFR antibodies specifically transduce syngeneic tumors overexpressing human EGFRvIII in vivo, while maintaining detargeting from the liver. Ten days following xenografting with syngeneic tumors overexpressing human EGFRvIII, model mice (expressing human EGFR) were injected with 1 x 1011vg / mL of AAV9 WT, AAV9 W503A, or AAV9 W503A conjugated to two different representative hEGFR antibodies, designated as H1863N2 and H10126P. Seven days following injection, tumors and livers were harvested and ex vivo bioluminescence quantification was assessed via IVIS Imaging. The top panel depicts the timeline of the experiment, the leftmost bar graph depicts the fold change in bioluminescence over liver-detargeted AAV9 W503 A in tumor tissue, while the rightmost bar graph depicts that fold change in bioluminescence over AAV9 WT in liver tissue.

[0017] Figures 6A-6B depict representative immunohistochemistry images of eGFP expression (Figure 6A) or EGFRvIII expression (Figure 6B) in glioblastoma tumors (U251 / EGFRvIII), or livers of mice bearing U251 / EGFRvIII xenografts. The top panel of Figure 6A depicts a timeline of the experiment. Tumors and livers from male SCID mice were harvested 21 days after injection with 2 x 1011vg / mL of: AAV2 WT or AAV9 WT particles encapsulating a nucleotide of interest encoding eGFP under the control of a CAG promoter; AAV2 or AAV9 particles comprising one or more detargeting mutation(s) (e.g.,HBM, or W503A, respectively), retargeted with a representative anti-EGFR antibody (Ab) or non-binding Control Ab, and encapsulating a nucleotide of interest encoding eGFP under the control of a CAG promoter; or vehicle (PBS / 0.001% Pluronic acid). Tumors and livers harvested from AAV-treated mice were assessed for GFP expression (Figure 6A; middle and bottom panels), and tumors and livers from vehicle-treated mice were assessed for EGFRvIII expression (Figure 6B).DETAILED DESCRIPTION

[0018] Epidermal Growth Factor Receptor (EGFR; also known as ErbBl or HER1) is a member of the ErB / HER family of receptor tyrosine kinases (RTKs). EGFR is a single-pass transmembrane protein comprising an extracellular (EC) domain, a transmembrane (TM) domain, an intracellular RTK domain, and C-terminal tail. The EC domain can be further subdivided into Domains 1, II, III and IV, also referred to as ligand binding domain 1 (LI), cystine rich domain 1 (CR1), L2 and CR2, respectively. Ligand binding to wildtype EGFR triggers auto- and trans- phosphorylation of the C-terminal tail and recruitment of proteins that initiate signaling cascades involved with growth and proliferation. Overexpression of EGFR or aberrant signaling of mutant EGFR can result in unchecked growth and proliferation leading to cancer.

[0019] EGFRvIII is one example of a mutant variant of EGFR associated with cancer. EGFRvIII is a truncated variant of EGFR comprising (a) a deletion of 267 amino acids (amino acids 6-273 of wildtype human EGFR), comprising parts of domains I (LI) and II (CR1) of the EC domain, as well as (b) the insertion of a new glycine residue. EGFRvIII is a result of gene amplification and the in-frame deletion of 801 base pairs of the EGFR gene spanning exons 2-7, creating a junction site between exons 1 and 8 that encodes the new glycine residue. The resulting EGFRvIII protein is incapable of binding any known ligand and has constitutive signaling activity that drives oncogenesis. Notably, evidence suggests that EGFRvIII is a tumor-specific receptor with normal tissues being largely devoid of EGFRvIII expression.

[0020] The human EGFR gene is located on the short arm of chromosome 7, comprises 28 coding exons and is approximately 9,905 nucleotides in length. A representative human EGFR nucleotide sequence is comprised within NCBI ReferenceSequence No. NM_005228.5 or set forth as SEQ ID NO: 391 (mature) or SEQ ID NO: 396 (precursor). It should be noted that an EGFR transcript, e.g., EGFR nucleotide sequence, as used herein may be interchangeably referred to as EGFR transcript variant 1. A representative human EGFR amino acid sequence is set forth as NCBI Reference Sequence No. NP_005219.2 or SEQ ID NO: 392 (mature) or SEQ ID NO: 397 (precursor). It should be noted that an EGFR polypeptide, e.g., wildtype EGFR, as used herein may be interchangeably referred to as EGFR isoform a. A representative EGFRvIH nucleotide sequence is comprised within NCBI Reference Sequence No. NM_001346941.2 and set forth as SEQ ID NO: 393 (mature) or SEQ ID NO: 397 (precursor). It should be noted that the EGFRvIH transcript, e.g., EGFRvIII nucleotide sequence, may be interchangeably referred to herein as EGFR transcript variant EGFRvIII. A representative human EGFRvIII amino acid sequence is set forth as NCBI Reference Sequence No. NP_001333870.1 or SEQ ID NO: 394 (mature) or SEQ ID NO: 398 (precursor). It should be noted that an EGFRvIII polypeptide as used herein may be interchangeably referred to as EGFR isoform i. Mature human EGFR and human EGFRvIII polypeptides are the same as their respective precursor polypeptides, except that they lack the signal peptide set forth as SEQ ID NO: 399.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0022] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0023] The term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.

[0024] The "percent (%) identity" or the like may be readily determined for amino acid or nucleotide sequences, over the full-length of a protein, or a portion thereof. A portionmay be at least about 5 amino acids or 24 nucleotides, respectively, in length, and may be up to about 700 amino acids or 2100 nucleotides, respectively. Generally, when referring to "identity", "homology", or "similarity" between two different adeno-associated viruses, "identity", "homology" or "similarity" is determined in reference to "aligned" sequences. "Aligned" sequences or "alignments" refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence.

[0025] Alignments may be performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Sequence alignment programs are available for amino acid sequences, e.g., the "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME", and "Match-Box" programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

[0026] Multiple sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include, "Clustal W", "CAP Sequence Assembly", "MAP", and "MEME", which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using FASTA™, a program in GCG Version 6.1. Fasta™ provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA™ with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference.

[0027] “Significant identity” encompasses amino acid or nucleic acid sequences alignments that are at least 90%, e.g., at least 93%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, or e.g., at least 100% identical.

[0028] The term “chimeric” encompasses a functional gene or polypeptide comprising nucleic acid sequences or amino acid sequences, respectively, from at least two different AAV serotype, e.g., portions of a gene or polypeptide of at least a first and second AAV, wherein the at least first and second portions are operably linked to form a functional chimeric AAV nucleic acid that encodes a functional amino acid. Unless specified as chimeric, nucleotide sequences, genes, polypeptides, and amino acids are considered nonchimeric in that the nucleotide sequences, genes, polypeptides, and amino acids comprise a nucleic acid sequence or amino acid sequence having significant identity to a nucleic acid sequence or amino acid sequence, respectively, of a single AAV serotype.

[0029] The term "antibody” encompasses immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable domain (VH) and a heavy chain constant region (CH). The heavy chain constant region comprises at least three domains, CHI, CH2, CH3 and optionally CH4. Each light chain comprises a light chain variable domain (CH) and a light chain constant region (CL). The heavy chain and light chain variable domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each heavy and light chain variable domain comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3. Typical tetrameric antibody structures comprise two identical antigen-binding domains, each of which formed by association of the VH and VL domains, and each of which together with respective CH and CL domains form the antibody Fv region. Single domain antibodies comprise a single antigen-binding domain, e.g., a VH or a VL. The “paratope” of an antibody comprises an antigen-binding portion of the antibody, e.g., the part of the antibody that recognizes and binds to a first member of a specific proteimprotein binding pair to which the antibody is the second member. It is a small region (of 5 to 10 amino acids) of an antibody's Fv region, part of the fragment antigen-binding (Fab region), and may contain parts of the antibody’s heavy and / or light chains. A paratope specifically binds a first member of a specific proteimprotein binding pair when the paratope binds thefirst member of a specific binding pair with a high affinity. The term “high affinity” antibody refers to an antibody that has a KD with respect to its target first member of a specific binding pair about of 10"9M or lower (e.g., about 1 x 10'9M, 1 x IO10M, 1 x 10"11M, or about 1 x IO12M). In one embodiment, KD is measured by surface plasmon resonance, e.g., BIACORE™; in another embodiment, KD is measured by ELISA.

[0030] The term "antibody", as used herein, also encompasses antigen-binding portions or fragments of full antibody molecules. The terms “antigen-binding fragment” of an antibody, “antigen-binding portion” of an antibody, “antigen-binding domain” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0031] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.

[0032] An antigen-binding portion of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0033] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Nonlimiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) VH- CHI ; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (V) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (X) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2- CH ; (xiii) VL-CH2-CH3; and (xiv) VL-CL- In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g. , 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Moreover, an antigenbinding fragment of an antibody of the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and / or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).

[0034] As with full antibody molecules, antigen-binding portions may be monospecific or multispecific e.g., bispecific). A bispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the non-limiting example formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art.

[0035] The phrase “complementarity determining region,” or the term “CDR,” includes an amino acid sequence encoded by a nucleic acid sequence of an organism’ s immunoglobulin genes that normally (i.e. , in a wild-type animal) appears between two framework regions in a variable region of a light or a heavy chain of an immunoglobulin molecule (e.g., an antibody or a T cell receptor). A CDR can be encoded by, for example, a germ line sequence or a rearranged or unrearranged sequence, and, for example, by a naive or a mature B cell or a T cell. A CDR can be somatically mutated (e.g., vary from a sequence encoded in an animal’s germ line), humanized, and / or modified with amino acid substitutions, additions, or deletions. In some circumstances (e.g., for a CDR3), CDRs can be encoded by two or more sequences (e.g., germ line sequences) that are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in a B cell nucleic acid sequence, e.g., as the result of splicing or connecting the sequences (e.g., V-D-J recombination to form a heavy chain CDR3).

[0036] The phrase “heavy chain,” or “immunoglobulin heavy chain” includes an immunoglobulin heavy chain sequence, including immunoglobulin heavy chain constant region sequence, from any organism. Heavy chain variable domains include three heavy chain CDRs and four FR regions, unless otherwise specified. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has, following the variable domain (from N-terminal to C-terminal), a CHI domain, a hinge, a CH2 domain, and a CH3 domain. A functional fragment of a heavy chain includes a fragment that is capable of specifically recognizing an first member of a specific binding pair (e.g., recognizing the first member of a specific binding pair with a KD in the micromolar, nanomolar, or picomolar range), that is capable of expressing and secreting from a cell, and that comprises at least one CDR. Heavy chain variable domains are encoded by variable region nucleotide sequence, which generally comprises VH, DH, and Ju segments derived from a repertoire of VH, DH, and JH segments present in the germline. Sequences, locations and nomenclature for V, D, and J heavy chain segments for various organisms can be found in IMGT database, which is accessible via the internet on the world wide web (www) at the URL “imgt.org.”

[0037] The term "heavy chain only antibody," "heavy chain only antigen binding protein," "single domain antigen binding protein," "single domain binding protein" or the like refers to a monomeric or homodimeric immunoglobulin molecule comprising an immunoglobulin-like chain comprising a variable domain operably linked to a heavy chain constant region, that is unable to associate with a light chain because the heavy chain constant region typically lacks a functional CHI domain. Accordingly, the term "heavy chain only antibody," "heavy chain only antigen binding protein," "single domain antigen binding protein," "single domain binding protein" or the like encompasses a both (i) a monomeric single domain antigen binding protein comprising one of the immunoglobulin- like chain comprising a variable domain operably linked to a heavy chain constant region lacking a functional Cnl domain, or (ii) a homodimeric single domain antigen binding protein comprising two immunoglobulin-like chains, each of which comprising a variable domain operably linked to a heavy chain constant region lacking a functional Cnl domain. In various aspects, a homodimeric single domain antigen binding protein comprises two identical immunoglobulin-like chains, each of which comprising an identical variable domain operably linked to an identical heavy chain constant region lacking a functional CHI domain. Additionally, each immunoglobulin-like chain of a single domain antigen binding protein comprises a variable domain, which may be derived from heavy chain variable region gene segments (e.g., Vn, Dn, Jn), light chain gene segments (e.g., Vi„ JT ), or a combination thereof, linked to a heavy chain constant region (CH) gene sequence comprising a deletion or inactivating mutation in a CHI encoding sequence (and, optionally, a hinge region) of a heavy chain constant region gene, e.g., IgG, IgA, IgE, IgD, or a combination thereof. A single domain antigen binding protein comprising a variable domain derived from heavy chain gene segments may be referred to as a "VH- single domain antibody" or "Vu-single domain antigen binding protein”, see, e.g., U.S. Patent No. 8,754,287; U.S. Patent Publication Nos. 20140289876; 20150197553; 20150197554; 20150197555; 20150196015; 20150197556 and 20150197557, each of which is incorporated in its entirety by reference. A single domain antigen binding protein comprising a variable domain derived from light chain gene segments may be referred to as a or "Vn-single domain antigen binding protein," see, e.g., U.S. Publication No. 20150289489, incorporated in its entirety by reference.

[0038] The phrase “light chain” includes an immunoglobulin light chain sequence from any organism, and unless otherwise specified includes human kappa (K) and lambda (X) light chains and a VpreB, as well as surrogate light chains. Light chain variable domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain includes, from amino terminus to carboxyl terminus, a variable domain that includes FR1-CDR1 -FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region amino acid sequence. Light chain variable domains are encoded by the light chain variable region nucleotide sequence, which generally comprises light chain VL and light chain JL gene segments, derived from a repertoire of light chain V and J gene segments present in the germline. Sequences, locations and nomenclature for light chain V and J gene segments for various organisms can be found in IM GT database, which is accessible via the internet on the world wide web (www) at the URL “imgt.org.” Light chains include those, e.g., that do not selectively bind either a first or a second first member of a specific binding pair selectively bound by the first member of a specific binding pair-binding protein in which they appear. Light chains also include those that bind and recognize, or assist the heavy chain with binding and recognizing, one or more first member of a specific binding pairs selectively bound by the first member of a specific binding pair-binding protein in which they appear. Light chains also include those that bind and recognize, or assist the heavy chain with binding and recognizing, one or more first member of a specific binding pairs selectively bound by the first member of a specific binding pair-binding protein in which they appear. Common or universal light chains include those derived from a human VK1-39JK5 gene or a human VK3-20JK1 gene, and include somatically mutated e.g., affinity matured) versions of the same. Exemplified human VL segments include a human VK1-39 gene segment, a human VK3-20 gene segment, a human VX 1 -40 gene segment, a human V T-44 gene segment, a human VZ.2-8 gene segment, a human V .2-14 gene segment, and human VX3-21 gene segment, and include somatically mutated (e.g., affinity matured) versions of the same. Light chains can be made that comprise a variable domain from one organism (e.g., human or rodent, e.g., rat or mouse; or bird, e.g., chicken) and a constant region from the same or a different organism (e.g., human or rodent, e.g., rat or mouse; or bird, e.g., chicken).

[0039] The phrase "operably linked", as used herein, includes a physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact, directly or indirectly with one another, or otherwise coordinate with each other to participate in a biological event, which juxtaposition achieves or permits such interaction and / or coordination. To give but one example, a regulatory element (e.g., an expression control sequence) in a nucleic acid is said to be "operably linked" to a coding sequence when it is located relative to the coding sequence such that its presence or absence impacts expression and / or activity of the coding sequence. In many embodiments, “operable linkage” involves covalent linkage of relevant components or elements with one another. Those skilled in the art will readily appreciate that, in some embodiments, covalent linkage is not required to achieve effective operable linkage. For example, proteins operably linked together may be associated with each other, e.g., via a covalent bond or a non-covalent bond. As a nonlimiting example, a capsid protein as described herein may be operably linked to a targeting ligand, where the capsid protein is non-covalently bound to the targeting ligand, or covalently bound to the targeting ligand, optionally with or without a scaffold and / or adaptor between the capsid protein and the targeting ligand. As another example, in some embodiments, nucleic acid regulatory elements that are operably linked with coding sequences that they control are contiguous with the nucleotide. Alternatively, or additionally, in some embodiments, one or more such regulatory elements acts in trans or at a distance to control a coding sequence. In some embodiments, the term "regulatory element" as used herein refers to polynucleotide sequences which are necessary and / or sufficient to affect the expression and processing of coding sequences to which they are ligated. In some embodiments, a regulatory element may be or comprise appropriate transcription initiation, termination, promoter and / or enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and / or, in some embodiments, sequences that enhance protein secretion. In some embodiments, one or more regulatory elements are preferentially or exclusively active in a particular host cell or organism, or type thereof. To give but one example, in prokaryotes, regulatory elements may typically include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, in many embodiments, regulatory elements maytypically include promoters, enhancers, and / or transcription termination sequences. Those of ordinary skill in the art will appreciate from context that, in many embodiments, the term "regulatory elements" refers to components whose presence is essential for expression and processing, and in some embodiments includes components whose presence is advantageous for expression (including, for example, leader sequences, targeting sequences, and / or fusion partner sequences).

[0040] “Retargeting” or “redirecting” may include a scenario in which the wildtype particle targets several cells within a tissue and / or several organs within an organism, and general targeting of the tissue or organs is reduced or abolished by insertion of the heterologous amino acid, and retargeting to more a specific cell in the tissue or a specific organ in the organism is achieved with the targeting ligand (e.g., via a targeting ligand) that binds a marker expressed by the specific cell. Such retargeting or redirecting may also include a scenario in which the wildtype particle targets a tissue, and targeting of the tissue is reduced to or abolished by insertion of the heterologous amino acid, and retargeting to a completely different tissue is achieved with the targeting ligand.

[0041] “Specific binding pair,” “binding pair,” “proteimprotein binding pair” and the like includes two members (e.g., a first member (e.g., a first polypeptide) and a second cognate member (e.g., a second polypeptide)) that interact to form a bond (e.g., a non- covalent bond between a first member epitope and a second member antigen-binding portion of an antibody that recognizes the epitope; a covalent bond between e.g., proteins capable of forming isopeptide bonds; split inteins that recognize each other and, through the process of protein trans-splicing, mediate ligation of the flanking proteins and their own removal, etc.). In some embodiments, the term "cognate" refers to components that typically function together, e.g., biomolecules that typically interact (e.g., a receptor and its ligand). Epitopes and cognate antibodies thereto, particularly epitopes that may also act as a detectable label (e.g., c-myc) are well-known in the art. Specific proteimprotein binding pairs that typically interact to form a covalent isopeptide bond are reviewed in Veggiani et al. (2014) Trends Biotechnol. 32:506, and include peptide:peptide binding pairs such as SpyTag:SpyCatcher, SpyTag002:SpyCatcher002; SpyTag:KTag; isopeptag:pilin C, SnoopTag:SnoopCatcher, etc., and variants thereof, e.g., SpyTag003:SpyCatcher003. Generally, a first member of a protein: protein binding pair refers to member of a proteimprotein binding pair, which isgenerally less than 30 amino acids in length, and which typically interacts (e.g., forms a spontaneous bond, e.g., a covalent isopeptide, a non-covalent bond, etc.) with the second cognate protein, typically wherein the second cognate protein is generally larger, but may also be less than 30 amino acids in length such as in the SpyTag:KTag system.

[0042] The term "isopeptide bond" refers to an amide bond between a carboxyl or carboxamide group and an amino group at least one of which is not derived from a protein main chain or alternatively viewed is not part of the protein backbone. An isopeptide bond may form within a single protein or may occur between two peptides or a peptide and a protein. Thus, an isopeptide bond may form intramolecularly within a single protein or intermolecularly i.e., between two peptide / protein molecules, e.g. between two peptide linkers. Typically, an isopeptide bond may occur between a lysine residue and an asparagine, aspartic acid, glutamine, or glutamic acid residue or the terminal carboxyl group of the protein or peptide chain or may occur between the alpha-amino terminus of the protein or peptide chain and an asparagine, aspartic acid, glutamine or glutamic acid. Each residue of the pair involved in the isopeptide bond is referred to herein as a reactive residue. In preferred embodiments of the invention, an isopeptide bond may form between a lysine residue and an asparagine residue or between a lysine residue and an aspartic acid residue. Particularly, isopeptide bonds can occur between the side chain amine of lysine and carboxamide group of asparagine or carboxyl group of an aspartate.

[0043] The SpyTag:SpyCatcher system is described in U.S. Patent No. 9,547,003 and Zaveri et al. (2012) PNAS 109:E690-E697, each of which is incorporated herein in its entirety by reference, and is derived from the CnaB2 domain of the Streptococcus pyogenes fibronecting-binding protein FbaB. By splitting the domain, Zakeri et al. obtained a peptide “SpyTag” having the sequence AHIVMVDAYKPTK (SEQ ID NO: 372) which forms an amide bond to its cognate protein “SpyCatcher,” an 112 amino acid polypeptide having the amino acid sequence set forth in SEQ ID NO: 373. (Zakeri (2012), supra). An additional specific binding pair derived from CnaB2 domain is SpyTag:KTag, which forms an isopeptide bond in the presence of SpyLigase (SEQ ID NO: 382). (Fierer (2014) PNAS 111:E1176-1181) SpyLigase was engineered by excising the strand from SpyCatcher that contains a reactive lysine, resulting in KTag, 10-residue first member of a proteimprotein binding pair having the amino acid sequence ATHIKFSKRD (SEQ ID NO: 374). TheSpyTag002:SpyCatcher002 system is described in Keeble et al (2017) Angew Chem Int Ed Engl 56: 16521-25, incorporated herein in its entirety by reference. SpyTag002 has the amino acid sequence VPTIVMVDAYKRYK, set forth as SEQ ID NO: 376, and binds SpyCatcher002 (SEQ ID NO: 377). SpyTag003 has the amino acid sequence RGVPHIVMVDAYKRYK, set forth as SEQ ID NO: 378, and binds SpyCatcher003 (SEQ ID NO: 379).

[0044] The SnoopTag:SnoopCatcher system is described in Veggiani (2016) PNAS 113:1202-07. The D4 Ig-like domain of RrgA, an adhesion from Streptococcus pneumoniae, was split to form SnoopTag (residues 734-745; SEQ ID NO: 380) and SnoopCatcher (residues 749-860; SEQ ID NO: 381). Incubation of SnoopTag and SnoopCatcher results in a spontaneous isopeptide bond that is specific between the complementary proteins. Veggiani (2016)), supra.

[0045] The isopeptag:pilin-C specific binding pair was derived from the major pilin protein Spy0128 from Streptococcus pyogenes. (Zakeir and Howarth (2010) J. Am. Chem. Soc. 132:4526-27). Isopeptag has the amino acid sequence TDKDMTITFTNKKDAE, set forth as SEQ ID NO: 375, and binds pilin-C (residues 18-299 of Spy0128). Incubation of Isopeptag and pilin-C results in a spontaneous isopeptide bond that is specific between the complementary proteins. Zakeir and Howarth (2010), supra.

[0046] Other systems to facilitate retargeting can be based upon the splitting and engineering of RegA domain 4. These have led to SnoopTagIr:SnoopCatcher, DogTag: DogCatcher and Snoop Ligase. Other systems include Isopeptag:Pilin-N, SdyTg:SdyCatcher, Jo:In, 3kptTag: 3kptCatcher, 4oqlTaq / 4oql Catcher, NGTag / Catcher, Rumtrunk / Mooncake,GalacTag, Cpe, Ececo, Corio and all others based upon isopeptide bond-forming binding pairs. Also encompassed by the first and / or second members of the proteimprotein binding pairs as described herein are functional portions or fragments of known isopeptide bond- forming pairs, i.e., portions or fragments of binding pairs that retain the ability to form an isopeptide bond.

[0047] The term “detectable label” includes a polypeptide sequence that is a member of a specific binding pair, e.g., that specifically binds via a non-covalent bond with another polypeptide sequence, e.g., an antibody paratope, with high affinity. Exemplary and nonlimiting detectable labels include hexahistidine tag, FLAG tag, Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, and the myc tag from c-myc (SEQ ID NO: 389). (Reviewed in Zhao et al. (2013) J. Analytical Meth. Chem. 1-8; incorporated herein by reference). A common detectable label for primate AAV is the Bl epitope (SEQ ID NO: 390). Some AAV capsid proteins described herein, which do not naturally comprise the B l epitope, may be modified herein to comprise a Bl epitope. Generally, AAV capsid proteins described herein may comprise a sequence with substantial homology to the B 1 epitope within the last 10 amino acids of the capsid protein. Accordingly, in some embodiments, a non-primate AAV capsid protein of the invention may be modified with one but less than five point mutations within the last 10 amino acids of the capsid protein such that the AAV capsid protein comprises a B 1 epitope.

[0048] The term "target cells" includes any cells in which expression of a nucleotide is desired. Preferably, target cells exhibit a receptor on their surface that allows the cell to be targeted with a targeting ligand, as described below.

[0049] The term "transduction" or “infection” or the like refers to the introduction of a nucleic acid into a target cell nucleus by a viral particle. The term efficiency in relation to transduction or the like, e.g., “transduction efficiency” refers to the fraction (e.g., percentage) of cells expressing a nucleotide after incubation with a set number of viral particles comprising the nucleotide. Well-known methods of determining transduction efficiency include flow cytometry of cells transduced with a fluorescent reporter gene, RT-PCR for expression of the nucleotide, etc.

[0050] Generally, “reference” viral capsid protein / capsid / particle are identical to test viral capsid protein / capsid / particle but for the change for which the effect is to be tested. For example, to determine the effect, e.g., on transduction efficiency, of inserting a first member of a specific binding pair into a test viral particle, the transduction efficiencies of the test viral particle (in the absence or presence of an appropriate targeting ligand) can be compared to the transduction efficiencies of a reference viral particle (in the absence or presence of an appropriate targeting ligand if necessary) which is identical to the test viral particle in every instance (e.g., additional point mutations, nucleotide, numbers of viral particles and target cells, etc.) except for the presence of a first member of a specific binding pair. In some embodiments, a reference viral capsid protein is one that is able to form a capsid with asecond viral capsid protein modified to comprise at least a first member of a proteimprotein binding pair, where the reference viral capsid protein does not comprise the first member of a proteimprotein binding pair, preferably wherein the capsid formed by the reference viral capsid protein and the modified viral capsid protein is a mosaic capsid.Adeno-associated viruses (AAV)

[0051] AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof. AAVs are small, non-enveloped, single-stranded DNA viruses. Generally, a wildtype AAV genome is 4.7 kb and is characterized by two inverted terminal repeats (ITR) and two open reading frames (ORFs), rep and cap. The wildtype rep reading frame encodes four proteins of molecular weight 78 kD (“Rep78”), 68 kD (“Rep68”), 52 kD (“Rep52”) and 40 kD (“Rep 40”). Rep78 and Rep68 are transcribed from the p5 promoter, and Rep52 and Rep40 are transcribed from the pl9 promoter. These proteins function mainly in regulating the transcription and replication of the AAV genome. The wildtype cap reading frame encodes three structural (capsid) viral proteins (VPs) having molecular weights of 83-85 kD (VP1), 72-73 kD (VP2) and 61-62 kD (VP3). More than 80% of total proteins in an AAV virion (capsid) comprise VP3; in mature virions VP1, VP2 and VP3 are found at relative abundance of approximately 1 : 1: 10, although ratios of 1 : 1 :8 have been reported. Padron et al. (2005) J. Virology 79:5047-58.

[0052] The genomic sequences of various serotypes of AAV, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC_002077 (AAV1), AF063497 (AAV1), NC001401 (AAV-2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8); the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences. See also, e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221 :208; Shade et al., (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology33:375-383; US Patent Publication 20170130245; international patent publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Pat. No. 6,156,303, each of which is incorporated by reference in its entirety by reference. Table 6 herein provides sequences of various non-primate AAV.

[0053] “AAV” encompasses all subtypes and both naturally occurring and modified forms that are well-known in the art. AAV includes primate AAV (e.g., AAV type 1 (AAV1), primate AAV type 2 (AAV2), primate AAV type 3 (AAV3B), primate AAV type 4(AAV4), primate AAV type 5 (AAV5), primate AAV type 6 (AAV6), primate AAV type 7(AAV7), primate AAV type 8 (AAV8), primate AAV type 9 (AAV9), AAV10, AAV11,AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, primate AAV type rhlO (AAV rhlO), AAV type hlO (AAV hlO), AAV type hul l (AAV hul 1), AAV type rh32.33 (AAV rh32.33), AAV retro (AAV retro), AAV PHP.B, AAV PHP.eB, AAV PHP.S, AAV2 / 8, etc., non-primate animal AAV (e.g., avian AAV (AAAV)) and other non-primate animal AAV such as mammalian AAV (e.g., bat AAV, sea lion AAV, bovine AAV, canine AAV, equine AAV, caprine AAV, and ovine AAV etc.), squamate AAV (e.g., snake AAV, bearded dragon AAV), and engineered variants thereof, etc. "Primate AAV" refers to AAV generally isolated from primates. Similarly, "non-primate animal AAV" refers to AAV isolated from non-primate animals.

[0054] As used herein, “of a [specified] AAV” in relation to a gene (e.g., rep, cap, etc.), capsid protein (e.g., a VP1 capsid protein, a VP2 capsid protein, a VP3 capsid protein, etc.), region of a capsid protein of a specified AAV (e.g., PLA2 region, VPl-u region, VP1 / VP2 common region, VP3 region), nucleotide sequence (e.g., ITR sequence), e.g., a cap gene or capsid protein of AAV etc., encompasses, in addition to the gene or the polypeptide respectively comprising a nucleic acid sequence or amino acid sequence set forth herein for the specified AAV, also variants of the gene or polypeptide, including variants comprising the least number of nucleotides or amino acids required to retain one or more biological functions. As used herein, a variant gene or a variant polypeptide comprises a nucleic acid sequence or amino acid sequence that differs from the nucleic acid sequence or amino acid sequence set forth herein for the gene or polypeptide of a specified AAV, wherein the difference(s) does not generally alter at least one biological function of the gene or polypeptide, and / or the phylogenetic characterization of the gene or polypeptide, e.g., wherethe difference(s) may be due to degeneracy of the genetic code, isolate variations, length of the sequence, etc. For example, rep gene and the cap gene as used here may encompass rep and cap genes that differ from the wildtype gene in that the genes may encode one or more Rep proteins and Cap proteins, respectively. In some embodiments, a Rep gene encodes at least Rep78 and / or Rep68. In some embodiments, cap gene includes those may differ from the wildtype in that one or more alternative start codons or sequences between one or more alternative start codons are removed such that the cap gene encodes only a single Cap protein, e.g., wherein the VP2 and / or VP3 start codons are removed or substituted such that the cap gene encodes a functional VP1 capsid protein but not a VP2 capsid protein or a VP3 capsid protein. Accordingly, as used herein, a rep gene encompasses any sequence that encodes a functional Rep protein. A cap gene encompasses any sequence that encodes at least one functional cap gene.

[0055] It is well-known that the wildtype cap gene expresses all three VP1, VP2, and VP3 capsid proteins from a single open reading frame of the cap gene under control of the p40 promoter found in the rep ORF. The term "capsid protein,” “Cap protein” and the like includes a protein that is part of the capsid of the virus. For adeno- associated viruses, the capsid proteins are generally referred to as VP1, VP2 and / or VP3, and may be encoded by the single cap gene. For AAV, the three AAV capsid proteins are produced in nature an overlapping fashion from the cap ORF alternative translational start codon usage, although all three proteins use a common stop codon. The ORF of a wildtype cap gene encodes from 5’ to 3’ three alternative start codons: “the VP1 start codon,” “the VP2 start codon,” and “the VP3 start codon”; and one “common stop codon”. The largest viral protein, VP1, is generally encoded from the VP1 start codon to the “common stop codon.” VP2 is generally encoded from the VP2 start codon to the common stop codon. VP3 is generally encoded from the VP3 start codon to the common stop codon. Accordingly, VP1 comprises at its N- terminus sequence that it does not share with the VP2 or VP3, referred to as the VP 1 -unique region (VPl-u). The VPl-u region is generally encoded by the sequence of a wildtype cap gene starting from the VP1 start codon to the “VP2 start codon.” VPl-u comprises a phospholipase A2 domain (PLA2), which may be important for infection, as well as nuclear localization signals which may aid the virus in targeting to the nucleus for uncoating and genome release. The VP1, VP2, and VP3 capsid proteins share the same C-terminalsequence that makes up the entirety of VP3, which may also be referred to herein as the VP3 region. The VP3 region is encoded from the VP3 start codon to the common stop codon. VP2 has an additional ~ 60 amino acids that it shares with the VP1. This region is called the VP1 / VP2 common region.

[0056] In some embodiments, one or more of the Cap proteins of the invention may be encoded by one or more cap genes having one or more ORFs. In some embodiments, the VP proteins of the invention may be expressed from more than one ORF comprising nucleotide sequence encoding any combination of VP1, VP2, and / or VP3 by use of separate nucleotide sequences operably linked to at least one expression control sequence for expression in packaging cell, each producing one or more of VP1 , VP2, and / or VP3 capsid proteins of the invention. In some embodiments, a VP capsid protein of the invention may be expressed individually from an ORF comprising nucleotide sequence encoding any one of VP1, VP2, or VP3 by use of separate nucleotide sequences operably linked to one expression control sequence for expression in a viral replication cell, each producing only one of VP1, VP2, or VP3 capsid protein. In another embodiment, VP proteins may be expressed from one ORF comprising nucleotide sequences encoding VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in a viral replication cell, each producing VP1, VP2, and VP3 capsid protein. Accordingly, although amino acid positions provided herein may be provided in relation to the VP1 capsid protein of the referenced AAV, a skilled artisan would be able to respectively and readily determine the position of that same amino acid within the VP2 and / or VP3 capsid protein of the AAV, and the corresponding position of amino acids among different AAV.

[0057] The phrase “Inverted terminal repeat” or “ITR” includes symmetrical nucleic acid sequences in the genome of adeno-associated viruses required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. The ITRs serve as the origins of replication for viral DNA synthesis and are essential cis components for generating AAV particles, e.g., packaging into AAV particles.

[0058] AAV ITR comprise recognition sites for replication proteins Rep78 or Rep68. A"D" region of the ITR comprises the DNA nick site where DNA replication initiates and provides directionality to the nucleic acid replication step. An AAV replicating in a mammalian cell typically comprises two ITR sequences.

[0059] A single ITR may be engineered with Rep binding sites on both strands of the “A” regions and two symmetrical D regions on each side of the ITR palindrome. Such an engineered construct on a double-stranded circular DNA template allows Rep78 or Rep68 initiated nucleic acid replication that proceeds in both directions. A single ITR is sufficient for AAV replication of a circular particle. In methods of producing an AAV viral particle of the invention, the rep encoding sequence encodes a Rep protein or Rep protein equivalent that is capable of binding an ITR comprised on the transfer plasmid.

[0060] The Cap proteins of the invention, when expressed with appropriate Rep proteins by a packaging cell, may encapsidate a transfer plasmid comprising a nucleotide and an even number of two or more ITR sequences. In some embodiments, a transfer plasmid comprises one ITR sequence. In some embodiments, a transfer plasmid comprises two ITR sequences.

[0061] Either Rep78 and / or Rep68 bind to unique and known sites on the sequence of the ITR hairpin, and act to break and unwind the hairpin structures on the end of an AAV genome, thereby providing access to replication machinery of the viral replication cell. As is well-known, Rep proteins may be expressed from more than one ORF comprising nucleotide sequence encoding any combination of Rep78, Rep68, Rep 52 and / or Rep40 by use of separate nucleotide sequences operably linked to at least one expression control sequence for expression in a viral replication cell, each producing one or more of Rep78, Rep68, Rep 52 and / or Rep40 Rep proteins. Alternatively, Rep proteins may be expressed individually from an ORF comprising a nucleotide sequence encoding any one of Rep78, Rep68, Rep 52, or Rep40 by use of separate nucleotide sequences operably linked to one expression control sequence for expression in a packaging cell, each producing only one Rep78, Rep68, Rep 52, or Rep40 Rep protein. In another embodiment, Rep proteins may be expressed from one ORF comprising nucleotide sequences encoding Rep78 and Rep52 Rep proteins operably linked to at least one expression control sequence for expression in a viral replication cell each producing Rep78 and Rep52 Rep protein.

[0062] In a method of producing an AAV virion, e.g., viral particle, of the invention, a rep encoding sequence and a cap gene of the invention may be provided a single packaging plasmid. However, a skilled artisan will recognize that such proviso is not necessary. Such viral particles may or may not include a genome.

[0063] A “chimeric AAV capsid protein” includes an AAV capsid protein that comprises amino acid sequences, e.g., portions, from two or more different AAV and that is capable of forming and / or forms an AAV viral capsid / viral particle. A chimeric AAV capsid protein is encoded by a chimeric AAV capsid gene, e.g., a chimeric nucleotide comprising a plurality, e.g., at least two, nucleic acid sequences, each of which plurality is identical to a portion of a capsid gene encoding a capsid protein of distinct AAV, and which plurality together encodes a functional chimeric AAV capsid protein. Association of a chimeric capsid protein to a specific AAV indicates that the capsid protein comprises one or more portions from a capsid protein of that AAV and one or more portions from a capsid protein of a different AAV. For example, a chimeric AAV2 capsid protein includes a capsid protein comprising one or more portions of a VP1, VP2, and / or VP3 capsid protein of AAV2 and one or more portions of a VP1, VP2, and / or VP3 capsid protein of a different AAV.

[0064] The term “portion” refers to at least 5 amino acids or at least 15 nucleotides, but less than the full-length polypeptide or nucleic acid molecule, with 100% identity to a sequence from which the portion is derived, see Penzes (2015) J. General Virol. 2769. A “portion” encompasses any contiguous segment of amino acids or nucleotides sufficient to determine that the polypeptide or nucleic acid molecule form which the portion is derived is “of a [specified] AAV” or has “significant identity” to a particular AAV, e.g., a non-primate animal AAV or remote AAV. In some embodiments, a portion comprises at least 5 amino acids or 15 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 10 amino acids or 30 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 15 amino acids or 45 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 20 amino acids or 60 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 25 amino acids or 75 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 30 amino acids or 90 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 35 amino acids or 105 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 40 amino acids or 120nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 45 amino acids or 135 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 50 amino acids or 150 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 60 amino acids or 180 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 70 amino acids or 210 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 80 amino acids or 240 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 90 amino acids or 270 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 100 amino acids or 300 nucleotides with 100% identity to a sequence associated with the specified AAV.Modified virus capsid proteins, viral particles, viral nucleic acids

[0065] In some embodiments, a Cap protein, e.g., a VP1 capsid protein as described herein, a VP2 capsid protein as described herein, and / or a VP3 capsid protein as described herein, is modified to comprise any one or combination of e.g., insertion of a targeting ligand, a chemical modification, a first member of a binding pair, a detectable label, point mutation, etc.

[0066] Generally, modification of gene or a polypeptide of a specified AAV, or variants thereof, results in nucleic acid sequence or an amino acid sequence that differs from the nucleic acid sequence or amino acid sequence set forth herein for the specified AAV, wherein the modification alters, confers, or removes one or more biological functions, but does not change the phylogenetic characterization of, the gene or polypeptide as an AAV gene or AAV polypeptide. Modifications may include any one or a combination of: substitution of sequences of a first AAV serotype with sequences of a second AAV serotype to create chimerism; chemical modification; an insertion of: a first member of a binding pair, and / or a point mutation; etc., such that the natural tropism of the capsid protein is reduced to abolished, the tropism of the capsid protein may be more easily redirected, and / or such thatthe capsid protein comprises a detectable label. Modifications as described herein generally do not alter and preferably decrease the low to no recognition of the modified capsid by preexisting antibodies found in the general population that were produced during the course of infection with another AAV, e.g., infection with serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, virions based on such serotypes, virions from currently used AAV gene therapy modalities, or a combination thereof.Targeting ligands

[0067] Modifications described herein may pertain to the association (e.g., display, operable linkage, binding, etc.) of a targeting ligand to a modified capsid protein and / or capsid comprising a modified capsid protein. Generally, a targeting ligand as described herein binds a surface protein expressed by a mammalian tumor cell, e.g., a mammalian tumor cell-specific surface protein. In certain embodiments, the tumor cell-specific surface protein comprises EGFR.

[0068] In some embodiments, a modified capsid protein and / or modified capsid comprises a targeting ligand that binds mammalian EGFR. In some embodiments, the mammalian EGFR is a mutant variant EGFR, e.g., a mutant variant that (i) does not bind to one or more ligands that bind to a wildtype mammalian EGFR and / or (ii) has constitutive signaling activity. In some embodiments, the mutant variant of the mammalian EGFR is a mutant variant of a human EGFR. In some embodiments, the mutant variant of human EGFR comprises a deletion of amino acids 6-273 relative to wildtype human EGFR and / or the introduction of a new glycine residue. In some embodiments, the mutant variant of human EGFR is encoded by an EGFR gene comprising a deletion of exons 2-7, wherein the fusion junction of exons 1 and 8 encodes a new glycine residue. In some embodiments, the mutant variant EGFR comprises an exon 19 deletion, an EGFR L858R mutation, an EGFR exon20 insertion, exon 18 / 21 atypical mutations, other activating EGFR mutations. In some embodiments, the mutant variant of human EGFR is human EGFRvIII. In some embodiments, the targeting ligand binds to human EGFR comprising an amino acid sequence set forth as SEQ ID NO: 392. In some embodiments, the targeting ligand binds to human EGFRvIII comprising an amino acid sequence set forth as SEQ ID NO: 394. In some embodiments, the targeting ligand preferentially binds to human EGFRvIII over wildtypehuman EGFR. In some embodiments, the targeting ligand binds human EGFRvIII and does not detectably bind wildtype human EGFR.

[0069] Antibodies to EGFRvIII, such as Hl 863, are well-known in the art, see, e.g., WO2015138460A 1 , incorporated herein in its entirety by reference. Other non- limiting examples of antibodies to EGFRvIII include ABT-806 and humanized derivatives thereof, and antibodies used in the antibody drug conjugate AMG 595. Table 1 provides a summary of the SEQ ID NO for each binding portion (e.g., heavy chain variable domain (HCVR), light chain variable domain (LCVR), and CDR1, CDR2, and CDR3) and the full heavy chain (HC) and light chain (LC) sequences of non-limiting and exemplifying anti-EGFR monoclonal antibodies (Antibody Designation) that may be used to redirect an AAV capsid as described herein. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human EGFR or a variant thereof, e.g., human EGFRvIII, wherein the targeting ligand comprises heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence(s) at least 90% identical to, respectively, an amino acid sequence of a heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in or encoded by a sequence or sequences as set forth in Table 1 and / or any one of SEQ ID NOs: 228-371. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human EGFR or a variant thereof, e.g., human EGFRvIII, wherein the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, HCDR1 , HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence at least 95% identical to, respectively, amino acid sequence(s) of a heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in or encoded by a sequence or sequences as set forth in Table 1 and / or any one of SEQ ID NOs: 228-371. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that bindshuman EGFR or a variant thereof, e.g., human EGFRvIII, wherein the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence at least 97% identical to amino acid sequence(s) of a heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or set of HCDR1-HCDR2- HCDR3-LCDR1-LCDR2-LCDR3 set forth in or encoded by a sequence or sequences as set forth in Table 1 and / or any one of SEQ ID NOs: 228-371. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human EGFR or a variant thereof, e.g., human EGFRvIII, wherein the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or set of HCDR1- HCDR2-HCDR3-LCDRI-LCDR2-LCDR3 amino acid sequence(s) at least 98% identical to amino acid sequence(s) of a heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in or encoded by a sequence or sequences as set forth in Table 1 and / or any one of SEQ ID NOs: 228-371. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human EGFR or a variant thereof, e.g., human EGFRvIII, wherein the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, CDR3, LCDR1, LCDR2, LCDR3, and / or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences 99% identical to amino acid sequences of a heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or set of HCDR1- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in or encoded by a sequence or sequences as set forth in Table 1 and / or any one of SEQ ID NOs: 228-371. Also described herein are antibodies, or antigen-binding fragments thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR / LCVR amino acid sequence pair as defined by any of the exemplified anti-EGFR antibodies listed inTable 1. In some embodiments, a targeting ligand as described herein comprises the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 229 / 237, 245 / 253, 261 / 269, 277 / 285, 293 / 301, 309 / 317, 325 / 333, 341 / 349, 357 / 365, and 405 / 413. In some embodiments, a targeting ligand as described herein comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 229 / 237, 245 / 253, 261 / 269, 277 / 285, 293 / 301, 309 / 317, 325 / 333, 341 / 349, 357 / 365, and 405 / 413.Table 1. SEQ ID NOs of Amino Acid Sequences of Domains in Antibodies or Antigenbinding Fragments (e.g., Fabs or scFv Molecules) that may be used to retarget AAV to human EGFR or a variant thereof. SEQ ID NOs for corresponding DNA sequences are presented in parentheses below each amino acid sequence.

[0070] Additionally, the heavy chain (HC) and light chain (EC) nucleotide (DNA) and amino acid (AA) sequences for H1863N2 and H10126P are as follows: H1863N2 HCDNA = SEQ ID NO: 400, H1863N2 HC AA = SEQ ID NO: 401, H1863N2 LC DNA = SEQ ID NO: 402, H1863N2 LC AA = SEQ ID NO: 403, H10126P HC DNA = SEQ ID NO: 420, H10126P HC AA = SEQ ID NO: 421, H10126P LC DNA = SEQ ID NO: 422, and H10126P LC AA = SEQ ID NO: 423.

[0071] Antibodies may be referred to herein according to the following nomenclature: Fc prefix (e.g. "H1H," "H2M," "H3M," etc.), followed by a numerical identifier (e.g. "2194," "2195," "1863," etc.), followed by a "P" or "N" suffix, as shown in Table 1. Thus, according to this nomenclature, an antibody may be referred to herein as, e.g., "H1H2194N," "H2M191 IN," "H3M1913N," etc. The H1H, H2M and H3M prefixes on the antibody designations used herein indicate the species of the variable domains and the particular Fc region isotype of the antibody. For example, an "H1H" antibody has a human IgGl Fc, an "H2M" antibody has a mouse IgG2 Fc, and an "H3M" antibody has a mouse IgG3 Fc (all variable regions are fully human as denoted by the first “H” in the antibody designation). As will be appreciated by a person of ordinary skill in the art, an antibody having a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a mouse IgGl Fc can be converted to an antibody with a human lgG4, etc.), but in any event, the variable domains (including the CDRs) - which are indicated by the numerical identifiers shown in Table 1 - will remain the same, and the binding properties are expected to be identical or substantially similar regardless of the nature of the Fc domain. Additionally, an antibody or antigen-binding fragment thereof as described herein may designated by its variable domains alone and without specifying the Fc region. For example, H2M1863N2 as disclosed in Table 1 has the same variable domains as H1863N2.

[0072] Non-limiting examples of targeting ligands that bind human EGFR or a variant thereof, e.g., human EGFRvIII, include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain- specific antibodies, single domain antibodies, domain- deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.),small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "targeting ligand," as used herein. In non-limiting embodiments, an anti-EGFR targeting ligand that binds human EGFR or a variant thereof, e.g., human EGFRvIII, useful for retargeting viral capsids as described herein comprise comprises an scFv. As a non-limiting example, an scFv sequences in VL-(Gly4Ser)3-Vn format useful for retargeting viral capsids as described herein may comprise a heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 that is 90%, 95%, 97%, 98%, 99% or 100% identical, respectively, to any one of the amino acid sequences of a heavy chain variable domain, light chain variable domain, heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 as set forth in or encoded by a sequence or sequences set forth in Table 1 and / or any one of SEQ ID NOs: 228-371.

[0073] In some embodiments, the targeting ligand comprises an amino acid sequence or is encoded by a polynucleotide sequence selected from any of the HCDR1 amino acid or nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least about 70-99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9%) sequence identity thereto.

[0074] In some embodiments, the targeting ligand comprises an amino acid sequence or is encoded by a polynucleotide sequence selected from any of the HCDR2 amino acid or nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least about 70-99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9%) sequence identity thereto.

[0075] In some embodiments, the targeting ligand comprises an amino acid sequence or is encoded by a polynucleotide sequence selected from any of the HCDR3 amino acid or nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least about 70-99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9%) sequence identity thereto.

[0076] In some embodiments, the targeting ligand comprises a set of HCDR1- HCDR2-HCDR3 amino acid sequences comprising or encoded by a set of HCDR1-HCDR2-HCDR3 amino acid or nucleic acid sequences listed in Table 1, or substantially similar sequences thereof having at least about 70-99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9%) sequence identity thereto.

[0077] In some embodiments, the targeting ligand comprises an amino acid sequence or is encoded by a polynucleotide sequence selected from any of the LCDR1 amino acid or nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least about 70-99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9%) sequence identity thereto.

[0078] In some embodiments, the targeting ligand comprises an amino acid sequence or is encoded by a polynucleotide sequence selected from any of the LCDR2 amino acid or nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least about 70-99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9%) sequence identity thereto. In some embodiments, the LCDR2 amino acid sequence is selected from the group consisting WAS (SEQ ID NO: 241), AAS (SEQ ID NO: 257), AAS (SEQ ID NO: 273), KAS (SEQ ID NO: 289), GAS (SEQ ID NO: 305), KVS (SEQ ID NO: 321), ATS (SEQ ID NO: 337), AAS (SEQ ID NO: 353), ATS (SEQ ID NO: 369), and AAS (SEQ ID NO: 417).

[0079] In some embodiments, the targeting ligand comprises an amino acid sequence or is encoded by a polynucleotide sequence selected from any of the LCDR3 amino acid or nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least about 70-99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9%) sequence identity thereto.

[0080] In some embodiments, the targeting ligand comprises a set of LCDR1- LCDR2-LCDR3 amino acid sequences comprising or encoded by a set of LCDR1-LCDR2- LCDR3 amino acid or nucleic acid sequences listed in Table 1, or substantially similar sequences thereof having at least about 70-99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9%) sequence identity thereto.

[0081] In some embodiments, the targeting ligand comprises a set of HCDR1- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences comprising or encoded bya set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid or nucleic acid sequences listed in Table 1, or substantially similar sequences thereof having at least about 70-99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9%) sequence identity thereto.

[0082] In some embodiments, the targeting ligand comprises an amino acid sequence or is encoded by a polynucleotide sequence selected from any of the HCVR amino acid or nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least about 70-99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9%) sequence identity thereto.

[0083] In some embodiments, the targeting ligand comprises an amino acid sequence or is encoded by a polynucleotide sequence selected from any of the LCVR amino acid or nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least about 70-99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9%) sequence identity thereto.

[0084] In some embodiments, the targeting ligand comprises an amino acid sequence or is encoded by a polynucleotide sequence selected from any of the heavy chain (HC) amino acid or nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least about 70-99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9%) sequence identity thereto.

[0085] In some embodiments, the targeting ligand comprises an amino acid sequence or is encoded by a polynucleotide sequence selected from any of the light chain (LC) amino acid or nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least about 70-99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9%) sequence identity thereto.Bispecific Targeting Ligands

[0086] In some embodiments, the targeting ligand comprises a multispecific binding molecule, e.g., a bispecific antigen-binding molecule, e.g., a bispecific antibody. In some embodiments, the multispecific antigen-binding molecule comprises a first antigen-binding domain and a second antigen-binding domain.

[0087] In some embodiments, the first antigen-binding domain binds to a surface protein expressed by a mammalian tumor cell, e.g., EGFR or a variant thereof, e.g., EGFRvIII, , e.g., as described herein.

[0088] In some embodiments, the second antigen-binding domain binds to a first member of a proteimprotein binding pair, e.g., a detectable label, comprised within, displayed by, etc., an AAV capsid protein, e.g., at a variable loop of an AAV capsid.

[0089] In some embodiments, the second antigen-binding domain of a multispecific targeting ligand described herein binds to a first member of a proteimprotein binding pair, wherein the first member of the proteimprotein binding pair is heterologous to the AAV capsid protein. “Heterologous” in this context means heterologous as compared to the AAV serotype from which the capsid protein is derived. In some embodiments, the second antigen-binding domain specifically binds to a c-myc epitope, e.g., comprising an amino acid sequence set forth as SEQ ID NO: 389. In some embodiments, the second antigen-binding domain comprises an antibody paratope that specifically binds to a c-myc epitope, e.g., comprising an amino acid sequence set forth as SEQ ID NO: 389. Thus, the present disclosure includes bispecific antigen-binding molecules, e.g., antibodies, wherein one domain of antigen-binding domain binds to a heterologous detectable label displayed by an AAV capsid (e.g., c-myc, B l, etc.), and the other antigen-binding domain binds to a surface protein expressed by a mammalian tumor cell, e.g., EGFR or a variant thereof, e.g., EGFRvIII. Accordingly, in some embodiments, the multispecific antigen-binding domain comprising the first and second antigen-binding domains retargets an AAV particle comprising an AAV capsid protein comprising the heterologous epitope to a mammalian tumor cell.

[0090] In some embodiments, the second antigen-binding domain of a multispecific targeting ligand described herein binds to a first member of a proteimprotein binding pair, wherein the first member of a proteimprotein binding pair comprises one or more epitopes of a capsid of an AAV particle. Thus, the present disclosure includes bispecific antigen-binding molecules, e.g., antibodies, wherein one domain of antigen-binding domain binds to a capsid of an AAV particle (e.g., a capsid comprising a wild-type and / or non- wild-type AAV capsid protein(s)), and the other antigen-binding domain binds to a surface protein expressed by a mammalian tumor cell, e.g., EGFR or a variant thereof, e.g., EGFRvIII. Accordingly, in someembodiments, the multispecific antigen-binding domain comprising the first and second antigen-binding domains as described herein retargets an AAV particle (i.e., comprising an AAV capsid protein comprising one or more epitopes, e.g., epitopes comprised within a wildtype AAV capsid protein, recognized by at least one of the antigen-binding domains) to a cell expressing EGFR or a variant thereof, e.g., mammalian tumor cell.

[0091] In some embodiments, the epitope of the capsid of the AAV particle may comprise any of various serotype- specific or serotype-non-specific (i.e., “universal”) epitopes understood by one of ordinary skill in the art.

[0092] In some embodiments, the one or more epitope(s) of the capsid of the AAV particle are comprises of any amino acid residues of a capsid protein VP1, VP2 or VP3, or a combination thereof. The AAV particle can be derived from any AAV serotype described herein. In some embodiments, the one or more epitope(s) comprise any variable region of a capsid, or any combination thereof, e.g., a variable region comprised of a capsid protein VP1, VP2 or VP3, or a combination thereof (e.g., variable region 1, 11, Ill, IV, V, VI, Vll, VIII, IX, or a combination thereof, as described in Emmanuel, et al., Journal of Virology, 2022, 96, 3).

[0093] As a non-limiting example, one or more epitope(s) of an AAV 1 capsid may comprise any amino acid residue or combination thereof of a variable region I, II, III, IV, V, VI, VII, VIII, IX, or combination thereof. In some embodiments, the one or more AAV1 epitope(s) comprise any of residues 456-459, 492-499, 582, 583, 588-591, 593-595, 597, or any combination thereof, as described in Tseng, et al., Front Immunol. 2014, 5: 9.

[0094] As a non-limiting example, one or more epitope(s) of an AAV2 capsid may comprise any amino acid residue or combination thereof of a variable region I, II, III, IV, V, VI, VII, VIII, IX, or combination thereof. In some embodiments, the one or more AAV2 epitope(s) comprise any of residues 253, 254, 258, 261-264, 272-281, 369-378, 381, 384, 385, 474-483, 492-502, 534, 548, 556, 560-573, 585-589, 601-610, 658-660, 708, 717, or any combination thereof, as described in Tseng, et al., Front Immunol. 2014, 5: 9.

[0095] As a non-limiting example, one or more epitope(s) of an AAV5 capsid may comprise any amino acid residue or combination thereof of a variable region I, II, III, IV, V, VI, VII, VIII, IX, or combination thereof. In some embodiments, the one or more AAV5 epitope(s) comprise any of residues 246, 254-261, 374, 375, 483, 485-492, 494, 496, 499-501, 530, 532-538, 653, 654, 656, 657, 704-708, or any combination thereof as described in Tseng, et al., Front Immunol. 2014, 5: 9.

[0096] As a non-limiting example, one or more epitope(s) of an AAV8 capsid may comprise any amino acid residue or combination thereof of a variable region I, II, III, IV, V, VI, VII, VIII, IX, or combination thereof. In some embodiments, the one or more AAV8 epitope(s) comprise any of residues 586-591 or any combination thereof as described in Tseng, et al., Front Immunol. 2014, 5: 9.

[0097] As a non-limiting example, one or more epitope(s) of an AAV9 capsid may comprise any amino acid residue or combination thereof of a variable region I, II, III, IV, V, VI, VII, VIII, IX, or combination thereof. In some embodiments, the one or more AAV8 epitope(s) comprise any of residues 221, 228, 246-248, 250-256, 258-260, any of residues 262-273 of variable region I, any of residues 274, 275, 278, 291, 293, 324, 325, any of residues 327-333 of variable region II, any of residues 334, 335, 338, 341, 363-365, 369-373, 375, 376, any of residues 382-387, 389, 391 of variable region Ill, any of residues 438-441, 443, any of residues 446, 448, 449, 451-460, 462, 464-473 of variable region IV, any of residues 488, 491-501, 503-506, 510-515 of variable region V, any of residues 528-534, 545 of variable region VI, any of residues 547-560 of variable region VII, residue 572, any of residues 579, 584, 587-590 of variable region VIII, any of residues 651-653, 655-676, 678, any of residues 701-720, 722, 724-728 of variable region IX, or any of residues 730, 731, or any combination thereof, as described in Emmanuel, et al., Journal of Virology, 2022, 96, 3.

[0098] Sequence identifiers corresponding to exemplified anti- AAV antibodies provided herein are listed in Table 2 and Table 3. Table 2 sets forth the amino acid sequence identifiers of the heavy chain variable regions (HC VRs) and light chain variable regions (LCVRs), heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), as well as heavy chain (HC) and light chain (LC) of the exemplary anti- AAV antibodies and antigen-binding fragments. Table 3 sets forth the sequence identifiers of the nucleic acid molecules encoding the HCVRs, LCVRs, HCDR1 , HCDR2, HCDR3, LCDR1, LCDR2 LCDR3, HC and LC of the exemplary anti- AAV antibodies and antigen-binding fragments described herein. Corresponding Fab and “standard mAb” (i.e., mAb comprising a constant region derived from IgG4 subclass) pairs are listed consecutively within Table 2 andTable 3. Non-limiting examples of amino acid sequences and nucleotide sequences of the anti- AAV antibodies are also listed below.Table 2. Amino Acid Sequence Identifiers for Anti- AAV AntibodiesTable 3. Nucleic Acid Sequence Identifiers for Anti-AAV AntibodiesREGN13876LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 13)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 14)REGN13877 (NAC67604)LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 13)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 14)REGN13878LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 13)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 14)REGN13879LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 13)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 14)REGN13880 (NAC67600)LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 13)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 14)REGN13881LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 13)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 14)REGN13882LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 13)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 14)REGN13883LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 13)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 14)REGN13884LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 13)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 14)REGN13885LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 13)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 14)REGN13070LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 122)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 123)REGN13071LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 122)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 123)REGN13072LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 122)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 123)REGN13073LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 122)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 123)REGN13074LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 122)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 123)REGN13075LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 122)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 123)REGN13076LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 122)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 123)REGN13220LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 122)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 123)REGN13221LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 122)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 123)REGN13284LCDR2 DNA SequenceGCTGCATCC(SEQ ID NO: 122)LCDR2 Amino Acid SequenceAAS(SEQ ID NO: 123)

[0099] In some embodiments, a bispecific antigen-binding molecule as disclosed herein comprises a first antigen-binding domain that binds EGFRvIII and a second antigen binding domain that binds one or more epitopes of a capsid of an AAV particle. In someembodiments, the first antigen binding domain and the second antigen binding domain are selected from any one of the bispecific anti- AAV x anti-EGFRvIII antigen-binding molecules as set forth in Table 9 herein, e.g., any one of BSID1, BSID2, BSID3, BSID4, BSID5, BSID6, BSID7, BSID8, BSID9, BSID10, BSID11, BSID12, BSID13, BSID14, BSID15, BSID16, BSID17, BSID18, BSID19, and BSID20.

[0100] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 401, or SEQ ID NO: 403, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 1 15, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, or SEQ ID NO: 129, respectively.

[0101] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 401, or SEQ ID NO: 403, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 139, or SEQ ID NO: 129, respectively.

[0102] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 261, SEQ ID NO: 263, SEQID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 401, or SEQ ID NO: 403, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 119, SEQ ID NO: 121 , SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 149, or SEQ ID NO: 129, respectively.

[0103] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 401, or SEQ ID NO: 403, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 159, or SEQ ID NO: 129, respectively.

[0104] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 2T , SEQ ID NO: 275, SEQ ID NO: 401, or SEQ ID NO: 403, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 169, or SEQ ID NO: 129, respectively.

[0105] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR,the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 401, or SEQ ID NO: 403, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR 1 , the HCDR2, the HCDR3, the LCVR, the LCDR 1 , the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 145, SEQ ID NO: 176, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 178, or SEQ ID NO: 129, respectively.

[0106] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 2T , SEQ ID NO: 275, SEQ ID NO: 401, or SEQ ID NO: 403, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 188, or SEQ ID NO: 129, respectively.

[0107] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 401, or SEQ ID NO: 403, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 198, or SEQ ID NO: 129, respectively.

[0108] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 401 , or SEQ ID NO: 403, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 208, or SEQ ID NO: 129, respectively.

[0109] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 401, or SEQ ID NO: 403, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 218, or SEQ ID NO: 129, respectively.

[0110] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 405, SEQ ID NO: 407, SEQ ID NO: 409, SEQ ID NO: 411, SEQ ID NO: 413, SEQ ID NO: 415, SEQ ID NO: 417, SEQ ID NO: 419, SEQ ID NO: 421, or SEQ ID NO: 423, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 111, SEQ IDNO: 113, SEQ ID NO: 1 15, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, or SEQ ID NO: 129, respectively.

[0111] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 405, SEQ ID NO: 407, SEQ ID NO: 409, SEQ ID NO: 411, SEQ ID NO: 413, SEQ ID NO: 415, SEQ ID NO: 417, SEQ ID NO: 419, SEQ ID NO: 421, or SEQ ID NO: 423, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 139, or SEQ ID NO: 129, respectively.

[0112] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 405, SEQ ID NO: 407, SEQ ID NO: 409, SEQ ID NO: 411, SEQ ID NO: 413, SEQ ID NO: 415, SEQ ID NO: 417, SEQ ID NO: 419, SEQ ID NO: 421, or SEQ ID NO: 423, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 149, or SEQ ID NO: 129, respectively.

[0113] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 405, SEQ ID NO: 407, SEQ ID NO: 409, SEQ ID NO: 411, SEQ ID NO: 413, SEQ ID NO: 415, SEQ ID NO: 417, SEQ ID NO: 419, SEQ ID NO: 421, or SEQ ID NO: 423, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprisingthe HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 159, or SEQ ID NO: 129, respectively.

[0114] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 405, SEQ ID NO: 407, SEQ ID NO: 409, SEQ ID NO: 411, SEQ ID NO: 413, SEQ ID NO: 415, SEQ ID NO: 417, SEQ ID NO: 419, SEQ ID NO: 421, or SEQ ID NO: 423, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 169, or SEQ ID NO: 129, respectively.

[0115] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 405, SEQ ID NO: 407, SEQ ID NO: 409, SEQ ID NO: 411, SEQ ID NO: 413, SEQ ID NO: 415, SEQ ID NO: 417, SEQ ID NO: 419, SEQ ID NO: 421, or SEQ ID NO: 423, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 145, SEQ ID NO: 176, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 178, or SEQ ID NO: 129, respectively.

[0116] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 405, SEQ ID NO: 407, SEQ ID NO: 409, SEQ ID NO: 411, SEQ ID NO: 413, SEQ ID NO: 415, SEQ ID NO: 417, SEQID NO: 419, SEQ ID NO: 421, or SEQ ID NO: 423, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 188, or SEQ ID NO: 129, respectively.

[0117] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 405, SEQ ID NO: 407, SEQ ID NO: 409, SEQ ID NO: 411, SEQ ID NO: 413, SEQ ID NO: 415, SEQ ID NO: 417, SEQ ID NO: 419, SEQ ID NO: 421, or SEQ ID NO: 423, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 198, or SEQ ID NO: 129, respectively.

[0118] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 405, SEQ ID NO: 407, SEQ ID NO: 409, SEQ ID NO: 411, SEQ ID NO: 413, SEQ ID NO: 415, SEQ ID NO: 417, SEQ ID NO: 419, SEQ ID NO: 421, or SEQ ID NO: 423, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 208, or SEQ ID NO: 129, respectively.

[0119] In some embodiments, a bispecific antigen-binding molecule of the disclosure comprises: (a) a first antigen-binding domain that binds to EGFRvIII comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, theHC, or the LC amino acid sequence as set forth in SEQ ID NO: 405, SEQ ID NO: 407, SEQ ID NO: 409, SEQ ID NO: 411, SEQ ID NO: 413, SEQ ID NO: 415, SEQ ID NO: 417, SEQ ID NO: 419, SEQ ID NO: 421, or SEQ ID NO: 423, respectively, and (b) a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle comprising the HCVR, the HCDR1, the HCDR2, the HCDR3, the LCVR, the LCDR1, the LCDR2, the LCDR3, the HC, or the LC amino acid sequence as set forth in SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 218, or SEQ ID NO: 129, respectively.

[0120] The first antigen-binding domain and the second antigen-binding domain of a bispecific antigen-binding molecule, as described herein, may be directly or indirectly connected to one another to form the bispecific antigen-binding molecule. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be connected to a separate multimerizing domain. The association of one multimerizing domain with another multimerizing domain facilitates the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a "multimerizing domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or constitution. For example, a multimerizing domain may be a polypeptide comprising an immunoglobulin CH3 domain. A non-limiting example of a multimerizing component is an Fc portion of an immunoglobulin (comprising a CH2-CH3 domain), e.g., an Fc domain of an IgG selected from the isotypes IgGl, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0121] Bispecific antigen-binding molecules as disclosed herein will typically comprise two multimerizing domains, e.g., two Fc domains that are each individually part of a separate antibody heavy chain. The first and second multimerizing domains may be of the same IgG isotype such as, e.g., IgGl / IgGl, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second multimerizing domains may be of different IgG isotypes such as, e.g., IgGl / IgG2, IgGl / IgG4, IgG2 / IgG4, etc.

[0122] In certain embodiments, the multimerizing domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine residues. In other embodiments, the multimerizing domain is a cysteine residue, or a shortcysteine-containing peptide. Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0123] Any bispecific antibody format or technology may be used to make a bispecific antigen-binding molecule as disclosed herein. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigenbinding specificity to produce a bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in the context of the present invention include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgGl / IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats; see also Brinkmann and Konterman (2017) mAbs 9:182-212; each of which is incorporated by reference in its entirety).

[0124] A targeting ligand that binds human EGFR or a variant thereof, e.g., human EGFRvIII, may be associated with (e.g., displayed by, operably linked to, bound to) a modified AAV capsid protein and resulting AAV capsids according to well-known methods, e.g., a direct approach in which the targeting ligand is directly inserted into (e.g., using recombinatorial methods) according to well-known methods. See, e.g. , Stachler et al. (2006), supra', White et al. (2004), supra', Girod et al. (1999), supra', Grifman et al. (2001), supra', Shi et al. (2001), supra', Shi and Bartlett (2003), supra. A targeting ligand that binds human EGFR or a variant thereof, e.g., human EGFRvIII, may be coupled to a modified AAV capsid protein and resulting AAV capsids using well-known chemical linkers, e.g., wherein the AAV capsid protein may be chemically modified to comprise a dibenzocycootyne group or an azide group, and optionally wherein a targeting ligand as described herein is attached to the dibenzocycootyne group or the azide group, see, e.g. , U.S. 2022 / 028234, incorporated herein by reference in its entirety; wherein targeting ligand is covalently linked to a primary amino acid group of an AAV capsid protein, e.g,. via a -CSNH- bond, etc. In some embodiments, a modified capsid as described herein comprises a targeting ligand, e.g., ananti-EGFR antibody or binding portion thereof, directly inserted into or coupled to it according to well-known direct recombinatorial methods.

[0125] In some embodiments, multispecific, e.g., bispecific, binding molecules of the present disclosure may two or more (e.g., three, four) antigen-binding domains. In some embodiments, the two or more (e.g., three, four) antigen-binding domains may independently be in a Fab or an scFv format.

[0126] Single chain Fv or “scFv” antibody fragments comprise the VH and VL domains of an antibody in a single polypeptide chain, are capable of being expressed as a single chain polypeptide and retain the specificity of the intact antibodies from which they are derived. Generally, an scFv polypeptide may further comprise a polypeptide linker between the VH and VL domain that enables the scFv to form the desired structure for target binding. Examples of linkers suitable for connecting the VH and VL chains of an scFV are the linkers are described herein.

[0127] Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise Vi -linker-Vn or may comprise VH-linker-VL.

[0128] The scFv can comprise VL and VH sequences from any suitable species, such as murine, human or humanized VH and VL sequences.

[0129] To create an scFv-encoding nucleic acid, the VL and Vu-encoding DNA fragments are operably linked to another fragment encoding a linker, e.g., encoding any of the linkers described herein, such that the VL and VH sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see, e.g., Bird et al., 1988, Science 242:423- 426; Huston et ai, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et ai, 1990, Nature 348:552-554).

[0130] The multispecific binding molecules of the disclosure may comprise at least one Fab domain. Fab domains were traditionally produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In the multispecific binding molecules of the disclosure, the Fab domains are recombinantly expressed as part of a larger molecule.

[0131] The Fab domains can comprise constant domain and variable region sequences from any suitable species, and thus can be murine, chimeric, human or humanized.

[0132] Fab domains typically comprise a CHI domain attached to a VH domain which pairs with a CL domain attached to a VL domain. In a wild-type immunoglobulin, the VH domain is paired with the VL domain to constitute the Fv region, and the CHI domain is paired with the CL domain to further stabilize the binding module. A disulfide bond between the two constant domains can further stabilize the Fab domain.

[0133] For the multispecific binding molecules of the disclosure, particularly when the light chain is not a common or universal light chain, it is advantageous to use Fab heterodimerization strategies to permit the correct association of Fab domains belonging to the same antigen-binding domain and minimize aberrant pairing of Fab domains belonging to different antigen-binding domains. For example, the Fab heterodimerization strategies shown in Table 4 below can be used:Table 4. Fab Heterodimerization Strategies

[0134] Accordingly, in certain embodiments, correct association between the two polypeptides of a Fab is promoted by exchanging the VL and VH domains of the Fab for each other or exchanging the CHI and CL domains for each other, e.g., as described in WO 2009 / 080251.

[0135] Correct Fab pairing can also be promoted by introducing one or more amino acid modifications in the CHI domain and one or more amino acid modifications in the CL domain of the Fab and / or one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain. The amino acids that are modified are typically part of the VH:VL and CHI :CL interface such that the Fab components preferentially pair with each other rather than with components of other Fabs.

[0136] In one embodiment, the one or more amino acid modifications are limited to the conserved framework residues of the variable (VH, VL) and constant (CHI, CL) domains as indicated by the Kabat numbering of residues. Almagro, 2008, Frontiers In Bioscience 13:1619-1633 provides a definition of the framework residues on the basis of Kabat, Chothia, and IMGT numbering schemes.

[0137] In one embodiment, the modifications introduced in the VH and CHI and / or VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interface can be achieved on the basis of steric and hydrophobic contacts, electrostatic / charge interactions or a combination of the variety of interactions. The complementarity between protein surfaces is broadly described in the literature in terms of lock and key fit, knob into hole, protrusion and cavity, donor and acceptor etc., all implying the nature of structural and chemical match between the two interacting surfaces.

[0138] In one embodiment, the one or more introduced modifications introduce a new hydrogen bond across the interface of the Fab components. In one embodiment, the one or more introduced modifications introduce a new salt bridge across the interface of the Fab components. Exemplary substitutions are described in WO 2014 / 150973 and WO 2014 / 082179, the contents of which are hereby incorporated by reference,

[0139] In some embodiments, the Fab domain comprises a 192E substitution in the CHI domain and 114A and 137K substitutions in the CL domain, which introduces a saltbridge between the CFM and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199- 211).

[0140] In some embodiments, the Fab domain comprises a 143Q and 188V substitutions in the CHI domain and 113T and 176V substitutions in the CL domain, which serves to swap hydrophobic and polar regions of contact between the CHI and CL domain (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0141] In some embodiments, the Fab domain can comprise modifications in some or all of the VH, CHI, VL, CL domains to introduce orthogonal Fab interfaces which promote correct assembly of Fab domains (Lewis et al., 2014 Nature Biotechnology 32:191-198). In an embodiment, 39K, 62E modifications are introduced in the VH domain, H172A, F174G modifications are introduced in the CHI domain, 1 R, 38D, (36F) modifications are introduced in the VL domain, and L135Y, S176W modifications are introduced in the CL domain. In another embodiment, a 39 Y modification is introduced in the V domain and a 38R modification is introduced in the VL domain.

[0142] Fab domains can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond, thereby increasing the efficiency of Fab component pairing. For example, an engineered disulfide bond can be introduced by introducing a 126C in the CHI domain and a 121 C in the CL domain (see, e.g., Mazor et al., 2015, MAbs 7:377- 89).

[0143] Fab domains can also be modified by replacing the CHI domain and CL domain with alternative domains that promote correct assembly. For example, Wu et al., 2015, Mabs 7:364-76, describes substituting the CHI domain with the constant domain of a T cell receptor and substituting the CL domain with the b domain of the T cell receptor, and pairing these domain replacements with an additional charge-charge interaction between theVL and VH domains by introducing a 38D modification in the Vi. domain and a 39K modification in the VH domain.

[0144] In lieu of, or in addition to, the use of Fab heterodimerization strategies to promote correct VH-VL pairings, the VL of common light chain (also referred to as a universal light chain) can be used for each Fab VL region of a MBM of the disclosure. In various embodiments, employing a common light chain as described herein reduces the number of inappropriate species of MBMs as compared to employing original cognate VLS. In various embodiments, the VL domains of the MBMs are identified from monospecific antibodies comprising a common light chain. In various embodiments, the VH regions of the MBMs comprise human heavy chain variable gene segments that are rearranged in vivo within mouse B cells that have been previously engineered to express a limited human light chain repertoire, or a single human light chain, cognate with human heavy chains and, in response to exposure with an antigen of interest, generate an antibody repertoire containing a plurality of human VHS that are cognate with one or one of two possible human VLs, wherein the antibody repertoire specific for the antigen of interest. Common light chains are those derived from a rearranged human VK1-39JK5 sequence or a rearranged human VK3-20JK1 sequence, and include somatically mutated (e.g., affinity matured) versions. See, for example, U.S. Patent No. 10,412,940.

[0145] The multispecific binding molecules disclosed herein can comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived.

[0146] Provided herein are antibodies, and antigen-binding fragments thereof, e.g., bispecific antigen binding molecules, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”), and having weak or no detectable binding to a AAV capsid antigen or a cell surface molecule antigen.

[0147] The antibodies and antigen-binding molecules, e.g., bispecific antigen-binding molecules, of the present disclosure may comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the individual antigen-binding domains were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The antigen-binding molecules of the present disclosure may comprise antigen-binding domains which are derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues within the VH and / or VL domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antigen-binding domain was originally derived).

[0148] Furthermore, the antigen-binding domains may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g. , wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence.Once obtained, antigen-binding domains that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Bispecific antigen-binding molecules comprising one or more antigen-binding domains obtained in this general manner are encompassed within the present disclosure.Linkers

[0149] In certain aspects, the present disclosure provides multispecific binding molecules, e.g., bispecific antigen-binding molecules, in which two or more components of an antigen-binding domain (e.g., a Vu and a Vi. of an scFv), two or more antigen-binding domains (e.g., an scFv and a Fab, or a Fab and a Fab), or an antigen-binding domain and a non- antigen-binding domain component (e.g., an Fc region) are connected to one another by a peptide linker.

[0150] A peptide linker can range from 2 amino acids to 60 or more amino acids, and in certain aspects a peptide linker ranges from 3 amino acids to 50 amino acids, from 4 to 30 amino acids, from 5 to 25 amino acids, from 10 to 25 amino acids, 10 amino acids to 60 amino acids, from 12 amino acids to 20 amino acids, from 20 amino acids to 50 amino acids, or from 25 amino acids to 35 amino acids in length.

[0151] In particular aspects, a peptide linker, e.g., a peptide linker separating an scFv domain and a heavy chain constant region, is at least 5 amino acids, at least 6 amino acids or at least 7 amino acids in length and optionally is up to 30 amino acids, up to 40 amino acids, up to 50 amino acids or up to 60 amino acids in length.

[0152] In some embodiments of the foregoing, the linker ranges from 5 amino acids to 50 amino acids in length, e.g., ranges from 5 to 50, from 5 to 45, from 5 to 40, from 5 to 35, from 5 to 30, from 5 to 25, or from 5 to 20 amino acids in length. In other embodiments of the foregoing, the linker ranges from 6 amino acids to 50 amino acids in length, e.g., ranges from 6 to 50, from 6 to 45, from 6 to 40, from 6 to 35, from 6 to 30, from 6 to 25, or from 6 to 20 amino acids in length. In yet other embodiments of the foregoing, the linker ranges from 7 amino acids to 50 amino acids in length, e.g., ranges from 7 to 50, from 7 to 45, from 7 to 40, from 7 to 35, from 7 to 30, from 7 to 25, or from 7 to 20 amino acids in length.

[0153] Charged (e.g., charged hydrophilic linkers) and / or flexible linkers are particularly preferred.

[0154] Examples of flexible linkers that can be used in the multispecific binding molecules of the disclosure include those disclosed by Chen et ai, 2013, Adv Drug Deliv Rev. 65(10): 1357-1369 and Klein et a / ., 2014, Protein Engineering, Design & Selection 27(10): 325-330. Particularly useful flexible linkers are or comprise repeats of glycines and serines, e.g., a monomer or multimer of GnS (SEQ ID NO: 424) or SGn(SEQ ID NO: 425), where n is an integer from 1 to 10, e.g., 1 2, 3, 4, 5, 6, or 7, 8, 9 or 10. In one embodiment, the linker is or comprises a monomer or multimer of repeat of G4S (SEQ ID NO: 425), e.g., (GGGGS)n (SEQ ID NO: 427), where n is an integer from 1 to 10, e.g., 1 , 2, 3, 4, 5, 6, or 7, 8, 9 or 10. In some embodiments, the linker is or comprises, e.g., (GGGGS)s (SEQ ID NO: 428). In some embodiments, the linker is or comprises, e.g., (GGGGS)4 (SEQ ID NO: 429).

[0155] Polyglycine linkers can suitably be used in the multispecific binding molecules of the disclosure. In some embodiments, the peptide linker, e.g., a peptide linker separating an scFv domain and a heavy chain such as the scFv domain of a first antigenbinding domain and the heavy chain variable region of a second antigen-binding domain, comprises two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO: 430), five consecutive glycines (5Gly) (SEQ ID NO: 431), six consecutive glycines (6Gly) (SEQ ID NO: 432), seven consecutive glycines (7Gly) (SEQ ID NO: 433), eight consecutive glycines (8Gly) (SEQ ID NO: 434), or nine consecutive glycines (9Gly) (SEQ ID NO: 435).

[0156] In particular embodiments, the linker, e.g., a peptide linker separating an scFv domain and a heavy chain constant region, is composed of both G4S (SEQ ID NO: 425) or a multimer thereof and one or more additional glycines, e.g., 2Gly, 3Gly or 4Gly (SEQ ID NO: 430). Examples of such linkers include G4S (SEQ ID NO: 425), GG, 4xG4S GG (SEQ ID NO: 436), and 7xG4S GG (SEQ ID NO: 437).Constant Regions

[0157] In some embodiments, multispecific binding molecules of the disclosure comprise constant regions (e.g., CHI, hinge, CH2, CH3, CL) derived from any suitable class of antibody. In some embodiments, the constant regions are derived from a human antibody.

[0158] In some embodiments, the constant regions can be derived from any suitable class of antibody, including IgA (including subclasses IgAl and IgA2), IgD, IgE, IgG (including subclasses IgGl, IgG2, IgG3 and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgGl, IgG2, IgG3 or IgG4. In one embodiment the constant region is derived from IgGl. In one embodiment the Fc domain is derived from IgG4.

[0159] In some embodiments, a heavy chain constant region of a human IgGl which may be used in accordance with the disclosure may comprise the amino acid sequence as set forth in SEQ ID NO: 438, which may be encoded by the nucleic acid sequence as set forth in SEQ ID NO: 439.

[0160] In some embodiments, a heavy chain constant region of a human IgGl which may be used in accordance with the disclosure may comprise the amino acid sequence as set forth in SEQ ID NO: 440, which may be encoded by the nucleic acid sequence as set forth in SEQ ID NO: 441).

[0161] In some embodiments, a heavy chain constant region of a human IgGl which may be used in accordance with the disclosure may comprise the amino acid sequence as set forth in SEQ ID NO: 442, which may be encoded by the nucleic acid sequence as set forth in SEQ ID NO: 443 or SEQ ID NO: 444.

[0162] In some embodiments, a heavy chain constant region of a human IgGl which may be used in accordance with the disclosure may comprise the amino acid sequence as set forth in SEQ ID NO: 445, which may be encoded by the nucleic acid sequence as set forth in SEQ ID NO: 446 or SEQ ID NO: 447.

[0163] In some embodiments, a heavy chain constant region of a human IgG4, h!gG4us, which may be used in accordance with the disclosure may comprise the amino acid sequence as set forth in SEQ ID NO: 448, which may be encoded by the nucleic acid sequence as set forth in SEQ ID NO: 449.Hinge Regions

[0164] The multispecific binding molecules of the disclosure can also comprise hinge regions, e.g., connecting an antigen-binding domain to an Fc region. The hinge region can be a native or a modified hinge region. Hinge regions are typically found at the N-termini of Fc regions.

[0165] A native hinge region is the hinge region that would normally be found between Fab and Fc domains in a naturally occurring antibody. A modified hinge region is any hinge that differs in length and / or composition from the native hinge region. Such hinges can include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama or goat hinge regions. Other modified hinge regions may comprise a complete hinge region derived from an antibody of a different class or subclass from that of the heavy chain Fc region. Alternatively, the modified hinge region may comprise part of a natural hinge or a repeating unit in which each unit in the repeat is derived from a natural hinge region. In a further alternative, the natural hinge region may be altered by converting one or more cysteine or other residues into neutral residues, such as serine or alanine, or by converting suitably placed residues into cysteine residues. By such means the number of cysteine residues in the hinge region may be increased or decreased. Other modified hinge regions may be entirely synthetic and may be designed to possess desired properties such as length, cysteine composition and flexibility.

[0166] A number of modified hinge regions have already been described for example, in U.S. Patent No. 5,677,425, WO9915549, W02005003170, W02005003169, W02005003170, WO9825971 and W02005003171 and these are incorporated herein by reference.

[0167] In one embodiment, the Fc region of one or both chains of the multispecific binding molecules of the disclosure possess an intact hinge region, e.g., a hinge domain, at its N-terminus. In some embodiments, “hinge domain” refers to the sequence from about Glu216 or about Cys226 to about Pro230 of human IgGl (Burton, 1985 Molec. Immunol. 22:161- 206), or the corresponding sequence in another antibody class or isotype.

[0168] In various embodiments, positions 233-236 within a hinge domain may be G, G, G and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, with positions numbered by EU numbering.

[0169] In some embodiments, the multispecific binding molecules of the disclosure comprise a modified hinge domain that reduces binding affinity for an Fey receptor relative to a wild-type hinge domain of the same isotype (e.g., human IgGl or human lgG4).

[0170] In one embodiment, the Fc region of one or both heavy chains of the multispecific binding molecules of disclosure possesses an intact hinge domain at its N- terminus.

[0171] In one embodiment both the Fc region and the hinge region of an the multispecific binding molecule of the disclosure are derived from IgG4 and the hinge region comprises the modified sequence CPPC (SEQ ID NO: 450). The core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO: 451) compared to IgGl that contains the sequence CPPC (SEQ ID NO: 450).

[0172] The serine residue which may be present in the IgG4 sequence can lead to increased flexibility in this region, and therefore a proportion of molecules form disulfide bonds within the same protein chain (an intrachain disulfide) rather than bridging to the other heavy chain in the IgG molecule to form the interchain disulfide (Angel et ai, 1993, Mol Immunol 30(1 ): 105-108). Changing the serine residue to a proline to give the same core sequence as IgGl allows complete formation of inter-chain disulfides in the lgG4 hinge region, thus reducing heterogeneity in the purified product.

[0173] The hinge region can be a chimeric hinge region.

[0174] For example, a chimeric hinge may comprise an “upper hinge” sequence, derived from a human IgGl, a human IgG2 or a human IgG4 hinge region, combined with a “lower hinge” sequence, derived from a human IgGl, a human IgG2 or a human IgG4 hinge region.

[0175] In various embodiments, a chimeric hinge region may be as described in WO2014 / 121087, which is incorporated by reference in its entirety herein. In various embodiments, a chimeric hinge region comprises the amino acid sequence as set forth in SEQ ID NO: 452, or SEQ ID NO: 453. Such chimeric hinge sequences can be suitably linked to an IgG4 CH2 region (for example by incorporation into an IgG4 Fc domain, for example a human or murine Fc domain), which can be further modified in the CH2 and / or CH3 domain to reduce effector function.

[0176] In further embodiments, the hinge region can be modified to reduce effector function, for example as described in W02016161010, which is incorporated by reference in its entirety herein. In various embodiments, the positions 233-236 of the modified hinge region are G, G, G and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied,unoccupied, and unoccupied; or all unoccupied, with positions numbered by EU numbering (as shown in FIG. 1 of W02016161010A2). These segments can be represented as GGG-, GG-, G — or - with representing an unoccupied position.

[0177] Position 236 is unoccupied in canonical human IgG2 but is occupied by in other canonical human IgG isotypes. Positions 233-235 are occupied by residues other than G in all four human isotypes (as shown in FIG. 1 of W02016161010A2).

[0178] The hinge modification within positions 233-236 can be combined with position 228 being occupied by P. Position 228 is naturally occupied by P in human IgGl and IgG2 but is occupied by S in human IgG4 and R in human IgG3. An S228P mutation in an IgG4 antibody is advantageous in stabilizing an IgG4 antibody and reducing exchange of heavy chain light chain pairs between exogenous and endogenous antibodies. Preferably positions 226-229 are occupied by C, P, P and C respectively.

[0179] Exemplary hinge regions have residues 226-236, sometimes referred to as middle (or core) and lower hinge, occupied by the modified hinge sequences designated GGG-(233-236), GG-(233-236), G— (233-236) and no G(233-236). Optionally, the hinge domain amino acid sequence comprises CPPCPAPGGG-GPSVF (SEQ ID NO: 454), CPPCPAPGG-GPSVF (SEQ ID NO: 455), CPPCPAPG— GPSVF (SEQ ID NO: 456), or CPPCPAP — GPSVF (SEQ ID NO: 457).

[0180] The modified hinge regions described above can be incorporated into a heavy chain constant region, which typically include CH2 and CH3 domains, and which may have an additional hinge segment (e.g., an upper hinge) flanking the designated region. Such additional constant region segments present are typically of the same isotype, preferably a human isotype, although can be hybrids of different isotypes. The isotype of such additional human constant regions segments is preferably human IgG4 but can also be human IgGl, IgG2, or IgG3 or hybrids thereof in which domains are of different isotypes. Exemplary sequences of human IgGl, IgG2 and IgG4 are shown in FIGS. 2-4 of W02016161010A2.

[0181] In specific embodiments, the modified hinge sequences can be linked to an IgG4 CH2 region (for example by incorporation into an IgG4 Fc domain, for example a human or murine Fc domain, which can be further modified in the CH2 and / or CH3 domain to reduce effector function).Fc Domains

[0182] The multispecific binding molecules of the disclosure can include an Fc region derived from any suitable species. In one embodiment the Fc region is derived from a human Fc domain.

[0183] In some embodiments, multispecific binding molecules disclosed herein can have, e.g., fully human variable regions but can have mouse constant regions (e.g., a mouse IgGl Fc or a mouse IgG2 Fc (a or b isotype)) or human constant regions (e.g., a human IgGl Fc or a human IgG4 Fc). As will be appreciated by a person of ordinary skill in the art, a multispecific binding molecule having a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a mouse IgGl Fc can be converted to an antibody with a human IgG4, etc.), but in any event, the variable domains (including the CDRs) will remain the same, and the binding properties to antigen are expected to be identical or substantially similar regardless of the nature of the constant domain.

[0184] The Fc domain can be derived from any suitable class of antibody, including IgA (including subclasses IgAl and lgA2), IgD, IgE, IgG (including subclasses IgGl, IgG2, IgG3 and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgGl, IgG2, IgG3 or IgG4. In one embodiment the Fc domain is derived from IgGl. In one embodiment the Fc domain is derived from IgG4.

[0185] The two Fc domains within the Fc region can be the same or different from one another. In a native antibody the Fc domains are typically identical, but for the purpose of producing multispecific binding molecules of the disclosure, the Fc domains might advantageously be different to allow for heterodimerization.

[0186] In native antibodies, the heavy chain Fc domain of IgA, IgD and IgG is composed of two heavy chain constant domains (CH2 and CH3) and that of IgE and IgM is composed of three heavy chain constant domains (CH2, CH3 and CH4). These dimerize to create an Fc region.

[0187] In multispecific binding molecules of the present disclosure, the Fc region, and / or the Fc domains within it, can comprise heavy chain constant domains from one or more different classes of antibody, for example one, two or three different classes.

[0188] In some embodiments, the Fc region comprises CH2 and CH3 domains derived from IgGl .

[0189] In some embodiments, the Fc region comprises CH2 and CH3 domains derived from IgG2.

[0190] In some embodiments, the Fc region comprises CH2 and CH3 domains derived from IgG3.

[0191] In some embodiments, the Fc region comprises CH2 and CH3 domains derived from IgG4.

[0192] In some embodiments, the Fc region comprises a CH4 domain from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

[0193] In some embodiments, the Fc region comprises CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.

[0194] It will be appreciated that the heavy chain constant domains for use in producing an Fc region for the multispecific binding molecules of the present disclosure may include variants of the naturally occurring constant domains described above. Such variants may comprise one or more amino acid variations compared to wild type constant domains. In one example the Fc region of the present disclosure comprises at least one constant domain that varies in sequence from the wild-type constant domain. It will be appreciated that the variant constant domains may be longer or shorter than the wild-type constant domain. Preferably the variant constant domains are at least 60% identical or similar to a wild-type constant domain. In another example the variant constant domains are at least 70% identical or similar. In another example the variant constant domains are at least 80% identical or similar. In another example the variant constant domains are at least 90% identical or similar. In another example the variant constant domains are at least 95% identical or similar.

[0195] IgM and IgA occur naturally in humans as covalent multimers of the common H2L2 antibody unit. IgM occurs as a pentamer when it has incorporated a J-chain, or as a hexamer when it lacks a J-chain. IgA occurs as monomer and dimer forms. The heavy chains of IgM and IgA possess an 18 amino acid extension to the C-terminal constant domain, known as a tailpiece. The tailpiece includes a cysteine residue that forms a disulfide bond between heavy chains in the polymer and is believed to have an important role in polymerization. The tailpiece also contains a glycosylation site. In certain embodiments, the MBMs of the present disclosure do not comprise a tailpiece.

[0196] The Fc domains that are incorporated into the multispecific binding molecules of the present disclosure may comprise one or more modifications that alter the functional properties of the proteins, for example, binding to Fc-receptors such as FcRn or leukocyte receptors, binding to complement, modified disulfide bond architecture, or altered glycosylation patterns.

[0197] The Fc domains can also be altered to include modifications that improve manufacturability of asymmetric multispecific binding molecules, for example by allowing heterodimerization, which is the preferential pairing of non-identical Fc domains over identical Fc domains. Heterodimerization permits the production of multispecific binding molecules in which different antigen-binding domains are connected to one another by an Fc region containing Fc domains that differ in sequence.

[0198] It will be appreciated that any of the modifications mentioned above can be combined in any suitable manner to achieve the desired functional properties and / or combined with other modifications to alter the properties of the multispecific binding molecules.

[0199] In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduces binding to an Fc receptor and / or effector function.

[0200] In a particular embodiment the Fc receptor is an Fey receptor. In one embodiment the Fc receptor is a human Fc receptor. In one embodiment the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIla, most specifically human FcyRIIIa. In one embodiment the effector function is one or more selected from the group of complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion. In a particular embodiment, the effector function is ADCC.

[0201] In some embodiments, the Fc region comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numberings according to Kabat EU index). In a more specific embodiment, the Fc region comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numberings according to Kabat EU index). In some embodiments, the Fc region comprises the amino acid substitutions L234A and L235A (numberings according to Kabat EU index).In one such embodiment, the Fc region is an IgD Fc region, particularly a human IgD Fc region. In one embodiment, the Fc region comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numberings according to Kabat EU index). In one embodiment, the Fc region comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numberings according to Kabat EU index). In a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular embodiments, the Fc region comprises amino acid substitutions at positions P329, L234 and L235 (numberings according to Kabat EU index). In more particular embodiments, the Fc region comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or “LALAPG”).

[0202] Typically, the same one or more amino acid substitution is present in each of the two Fc domains of an Fc region. Thus, in a particular embodiment, each Fc domain of the Fc region comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e. in each of the first and the second Fc domains in the Fc region the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A) and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to Kabat EU index).

[0203] In one embodiment, the Fc domain is an IgGl Fc domain, particularly a human IgGl Fc domain.

[0204] Typically, the same one or more amino acid substitution is present in each of the two Fc domains of an Fc region. Thus, in a particular embodiment, each Fc domain of the Fc region comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e. in each of the first and the second Fc domains in the Fc region the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A) and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to Kabat EU index).

[0205] In one embodiment, the Fc domain is an IgG 1 Fc domain, particularly a human IgGl Fc domain. In some embodiments, the IgGl Fc domain is a variant IgGl comprising D265A, N297A mutations (EU numbering) to reduce effector function.

[0206] In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to Fc receptors. Exemplary IgG4 Fc domains with reduced binding to Fc receptors may comprise an amino acid sequence selected from Table 5 below. In some embodiments, the Fc domain includes only the bolded portion of the sequences shown below.Table 5. IgG4 Fc domains

[0207] In a particular embodiment, the IgG4 with reduced effector function comprises the bolded portion of the amino acid sequence of SEQ ID NO: 461, sometimes referred to herein as IgG4s or hlgG4s.

[0208] For heterodimeric multispecific binding molecules, it is possible to incorporate a combination of the variant IgG4 Fc sequences set forth above, for example an Fc regioncomprising a combination of SEQ ID NO: 460 (or the bolded portion thereof) and SEQ ID NO: 462 (or the bolded portion thereof) or an Fc region comprising a combination of SEQ ID NO: 461 (or the bolded portion thereof) and SEQ ID NO: 463 (or the bolded portion thereof).

[0209] Many multispecific molecule formats entail dimerization between two Fc domains that, unlike a native immunoglobulin, are operably linked to non-identical antigenbinding domains (or portions thereof, e.g., a VH or VH-CH1 of a Fab). Inadequate heterodimerization of two Fc regions to form an Fc domain has can be an obstacle for increasing the yield of desired multispecific molecules and represents challenges for purification. A variety of approaches available in the art can be used in for enhancing dimerization of Fc domains that might be present in the multispecific binding molecules of the disclosure, for example as disclosed in EP 1870459A1 ; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441 ; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No.2006204493A1; and PCT Publication No. W02009 / 089004A1.

[0210] The present disclosure provides multispecific binding molecules comprising Fc heterodimers, i.e., Fc regions comprising heterologous, non-identical Fc domains. Heterodimerization strategies are used to enhance dimerization of Fc regions operably linked to different antigen-binding domains (or portions thereof, e.g., a VH or VH-CH1 of a Fab) and reduce dimerization of Fc domains operably linked to identical antigen-binding domains. Typically, each Fc domain in the Fc heterodimer comprises a CH3 domain of an antibody. The CH3 domains are derived from the constant region of an antibody of any isotype, class or subclass, and preferably of IgG (IgGl, IgG2, IgG3 and IgG4) class, as described in the preceding section.

[0211] Heterodimerization of the two different heavy chains at CH3 domains give rise to the desired multispecific binding molecule, while homodimerization of identical heavy chains will reduce yield of the desired multispecific binding molecule. Thus, in a preferred embodiment, the two heavy chains that associate to form an multispecific binding molecule of the disclosure will contain CH3 domains with modifications that favor heterodimeric association relative to unmodified chains.

[0212] In a specific embodiment said modification promoting the formation of Fc heterodimers is a so-called “knob-into-hole” or “knob-in-hole” (KiH) modification,comprising a “knob” modification in one of the Fc domains and a “hole” modification in the other Fc domain. The knob-into-hole technology is described e.g. in U.S. Patent No.5,731,168; US 7,695,936; Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7-15. Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).

[0213] Accordingly, in some embodiments, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable. Preferably said amino acid residue having a larger side chain volume is selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably said amino acid residue having a smaller side chain volume is selected from alanine (A), serine (S), threonine (T), and valine (V). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by sitespecific mutagenesis, or by peptide synthesis. An exemplary substitution is Y470T.

[0214] In a specific such embodiment, in the first Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to Kabat EU index). In a further embodiment, in the first Fc domain additionally the serineresidue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced with a cysteine residue), and in the second Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numbering according to Kabat EU index). In a particular embodiment, the first Fc domain comprises the amino acid substitutions S354C and T366W, and the second Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index).

[0215] In some embodiments, electrostatic steering (e.g., as described in Gunasekaran et al., 2010, J Biol Chem 285(25): 19637-46) can be used to promote the association of the first and the second subunit of the Fc domain.

[0216] As an alternative, or in addition, to the use of Fc domains that are modified to promote heterodimerization, an Fc domain can be modified to allow a purification strategy that enables selections of Fc heterodimers. In one such embodiment, one heavy chain comprises a modified Fc domain that abrogates its binding to Protein A, thus enabling a purification method that yields a heterodimeric protein. See, for example, U.S. Patent No. 8,586,713. As such, the multispecific binding molecules comprise a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the multispecific binding molecule to Protein A as compared to a corresponding multispecific binding molecule lacking the amino acid difference. In one embodiment, the first CH3 domain binds Protein A and the second CH3 domain contains a mutation / modification that reduces or abolishes Protein A binding such as an H95R modification (by 1MGT exon numbering; H435R by EU numbering). The second CH3 may further comprise a Y96F modification (by IM GT; Y436F by EU). This class of modifications is referred to herein as “star” mutations.

[0217] According to certain embodiments of the present disclosure, anti-AAV antigen-binding molecules and anti-AAV x anti-EGFR, e.g., anti-EGFRvIII, molecule antigen-binding molecules, are provided comprising an Fc domain comprising one or more mutations. Non-limiting examples of Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., E or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., Sor T), and 256 (e.g., S / R / Q / E / D or T); or a modification at position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or a modification at position 250 and / or 428; or a modification at position 307 or 308 e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428E (e.g., M428E) and 434S (e.g., N434S) modification; a 428E, 2591 (e.g., V259I), and 308F (e.g., V3O8F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428E modification (e.g., T250Q and M428E); and a 307 and / or 308 modification (e.g., 308F or 308P).

[0218] In certain embodiments of the disclosure, anti- AAV x anti-EGFR, e.g., anti- EGFRvIII, multispecific antibodies provided herein are human antibodies. The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and, in particular, CDR3. However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0219] The antibodies of the disclosure may, in some embodiments, be recombinant human antibodies. The term “recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created, or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the aminoacid sequences of the VH and Vi. regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0220] Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via interchain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.

[0221] The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30: 105) to levels typically observed using a human IgGl hinge. The instant disclosure encompasses antibodies having one or more mutations in the hinge, CH2 or CH3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form.

[0222] The antibodies of the disclosure may be isolated antibodies. An “isolated antibody,” as used herein, means an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an "isolated antibody" for purposes of the present disclosure. An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0223] The anti- AAV x anti-EGFR, e.g., anti-EGFRvIII, multispecific antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies werederived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present disclosure includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues within the VH and / or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived).

[0224] Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc.Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.

[0225] Provided herein are anti-AAV x anti-EGFR, e.g., anti-EGFRvIII, multispecific antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes anti-AAV x anti-EGFR, e.g., anti-EGFRvITI, multispecific antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 3 or fewer, 2, or 1 conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences set forth in Table 1 or Table 2 herein.

[0226] Accordingly, in some embodiments, provided herein is an anti-AAV x anti- EGFRvIII bispecific antibody comprising (a) a first antigen-binding domain comprising a set of three HCDRs (HCDR1-HCDR2-HCDR3), a set of three LCDRs (LCDR1-LCDR2- LCDR3), a set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3), an HCVR, an LCVR, and / or an HCVR / LCVR pair of an anti-AAV antibody disclosed Table 2 and / or SEQ ID NOs: 1-227; and (b) a second antigen-binding domain comprising a set of three HCDRs (HCDR1-HCDR2-HCDR3), a set of three LCDRs (LCDR1-LCDR2-LCDR3), a set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3), an HCVR, an LCVR, and / or an HCVR / LCVR pair of an anti-EGFRvIII antibody disclosed in Table 1 and / or set forth in SEQ ID NOs: 228-371.Scaffolded Targeting Ligands

[0227] In some embodiments, a targeting ligand that binds human EGFR or a variant thereof, e.g., human EGFRvIII, may be associated with (e.g., displayed by, operably linked to, bound to) a modified AAV capsid protein and resulting AAV capsids according to indirect recombinatorial approaches, wherein the AAV capsid protein is modified to comprise a first member of a binding pair (e.g., a heterologous scaffold), and optionally wherein the first member of the binding pair is linked to (e.g., covalently or non-covalently bound to) a second cognate member of the binding pair (e.g., an adaptor), further optionally wherein the second cognate member of the binding pair is fused to the targeting ligand. Non- limiting andexemplary binding pairs are listed in Buning and Srivastava (2019) Mol. Ther. Methods Clin Dev 12:248-265.

[0228] Accordingly, in some embodiments, modifications of a capsid protein as described herein include those that generally result from modifications at the genetic level, e.g., via modification of a cap gene, such as modifications that insert first member of a binding pair (e.g., a proteimprotein binding pair, a protein ucleic acid binding pair), a detectable label, etc., for display by the Cap protein.

[0229] In some embodiments, the first member forms a binding pair with an immunoglobulin constant domain. In some embodiments, the first member forms a binding pair with a metal ion, e.g., Ni2+, Co2+, Cu2+, Zn2+, Fe3+, etc. In some embodiments, the first member is selected from the group consisting of Streptavidin, Strep II, HA, L14, 4C-RGD, LH, and Protein A.

[0230] In some embodiments, the binding pair comprises an enzyme: nucleic acid binding pair. In some embodiments, the first member comprises a HUH-endonuclease or HUH-tag and the second member comprises a nucleic acid binding domain. In some embodiments, the first member comprises a HUH tag. See, e.g., U.S. 2021 / 0180082, incorporated herein in its entirety by reference.

[0231] In some embodiments, a capsid protein of the invention comprises at least a first member of a peptide:peptide binding pair.

[0232] In some embodiments, each of a first member and a second member of a peptide:peptide binding pair comprises an intein. See, e.g., Wagner et al., (2021) Adv. Sci. 8: 2004018 (1 of 22); Muik et al. (2017) Biomaterials 144: 84, each of which is incorporated herein in its entirety by reference.

[0233] In some embodiments, a first member is a B cell epitope, e.g., is between about 1 amino acid and about 35 amino acids in length, and forms a binding pair with an antibody paratope, e.g., an immunoglobulin variable domain. In some embodiments, a capsid protein of the invention may be modified to comprise a detectable label as a first member of a binding pair. Many detectable labels are known in the art. (See, e.g.: Nilsson et al. (1997) “Affinity fusion strategies for detection, purification, and immobilization of modified proteins”; Protein Expression and Purification 11: 1-16, Terpe et al. (2003); “Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems”Applied Microbiology and Biotechnology 60:523-533; and references therein). Detectable labels include, but are not limited to, a polyhistidine detectable labels (e.g., a His-6, His-8, or His-10) that binds immobilized divalent cations (e.g., Ni2+), a biotin moiety (e.g., on an in vivo biotinylated polypeptide sequence) that binds immobilized avidin, a GST (glutathione S- transferase) sequence that binds immobilized glutathione, an S tag that binds immobilized S protein, an antigen that binds an immobilized antibody or domain or fragment thereof (including, e.g., T7, myc, FLAG, and B tags that bind corresponding antibodies), a FLASH Tag (a high detectable label that couples to specific arsenic based moieties), a receptor or receptor domain that binds an immobilized ligand (or vice versa), protein A or a derivative thereof (e.g., Z) that binds immobilized IgG, maltose-binding protein (MBP) that binds immobilized amylose, an albumin-binding protein that binds immobilized albumin, a chitin binding domain that binds immobilized chitin, a calmodulin binding peptide that binds immobilized calmodulin, and a cellulose binding domain that binds immobilized cellulose. Another exemplary detectable label is a SNAP-tag. In some embodiments, a detectable label disclosed herein comprises a detectable label recognized by an antibody paratope, wherein the detectable label and the antibody paratope form a proteimprotein binding pair.

[0234] In some embodiments, a capsid protein of the invention comprises a first member of a proteimprotein binding pair comprising a detectable label, which may also be used for the detection and / or isolation of the Cap protein and / or as a first member of a protein :protein binding pair. In some embodiments, a detectable label acts as a first member of a proteimprotein binding pair for the binding of a targeting ligand comprising a multispecific binding protein that may bind both the detectable label and a target expressed by a cell. In some embodiments, a Cap protein of the invention comprises a first member of a protein :protein binding pair comprising c-myc (EQKLISEEDL; SEQ ID NO: 389). Use of a detectable label as a first member of a protein :protein binding pair is described in, e.g., W02019006043, incorporated herein in its entirety by reference.

[0235] In some embodiments, the first member comprises a Bl epitope (SEQ ID NO: 390). In some embodiments, a capsid protein is modified to comprise a Bl epitope in the VP3 region. In some embodiments, the first member is selected from the group consisting of FLAG, HA and c-myc (EQKLISEEDL; SEQ ID NO: 389).

[0236] In some embodiments, a capsid protein comprises a first member of a protein :protein binding pair, wherein the proteimprotein binding pair forms a covalent isopeptide bond. In some embodiments, the first member of a peptide:peptide binding pair is covalently bound via an isopeptide bond to a cognate second member of the peptide:peptide binding pair, and optionally wherein the cognate second member of the peptide:peptide binding pair is fused with a targeting ligand, which targeting ligand binds a target expressed by a cell. In some embodiments, the proteimprotein binding pair may be selected from the group consisting of SpyTag:SpyCatcher, SpyTag002:SpyCatcher002, SpyTagOO3:SpyCatcherOO3, SpyTag:KTag, Isopeptag:pilin-C, and SnoopTag:SnoopCatcher. In some embodiments, wherein the first member is SpyTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyCatcher (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is SpyTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is KTag (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is KTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyTag (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is SnoopTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is SnoopCatcher (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is Isopeptag (or a biologically active portion or variant thereof) and the protein (second cognate member) is Pilin-C (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is SpyTag002 (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyCatcher002 (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is SpyTag003 (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyCatcher003 (or a biologically active portion or variant thereof). In some embodiments, a Cap protein of the invention comprises a SpyTag, or a biologically active portion or variant thereof. Use of a first member of a proteimprotein binding pair is described in WO2019006046, incorporated herein in its entirety.

[0237] In some embodiments, a first member of a protein: protein binding pair and / or detectable label is operably linked to (translated in frame with, chemically attached to, and / ordisplayed by) a Cap protein of the invention via a first or second linker, e.g., an amino acid spacer that is at least one amino acid in length. In some embodiments, the first member of a proteimprotein binding pair is flanked by a first and / or second linker, e.g., a first and / or second amino acid spacer, each of which spacer is at least one amino acid in length.

[0238] In some embodiments, the first and / or second linkers are not identical. In some embodiments, the first and / or second linker is each independently one or two amino acids in length. In some embodiments, the first and / or second linker is each independently one, two or three amino acids in length. In some embodiments, the first and / or second linker is each independently one, two, three, or four amino acids in length. In some embodiments, the first and / or second linker is each independently one, two, three, four, or five amino acids in length. In some embodiments, the first and / or second linker are each independently one, two, three, four, or five amino acids in length. In some embodiments, the first and / or second linker is each independently one, two, three, four, five, or six amino acids in length. In some embodiments, the first and / or second linker is each independently one, two, three, four, five, six, or seven amino acids in length. In some embodiments, the first and / or second linker is each independently one, two, three, four, five, six, seven, or eight amino acids in length. In some embodiments, the first and / or second linker is each independently one, two, three, four, five, six, seven, eight or nine amino acids in length. In some embodiments, the first and or second linker is each independently one, two, three, four, five, six, seven, eight, nine, or ten amino acids in length. In some embodiments, the first and or second linker is each independently one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids in length.

[0239] In some embodiments, the first and second linkers are identical in sequence and / or in length and are each one amino acid in length. In some embodiments, the first and second linkers are identical in length, and are each one amino acid in length. In some embodiments, the first and second linkers are identical in length, and are each two amino acids in length. In some embodiments, the first and second linkers are identical in length, and are each three amino acids in length. In some embodiments, the first and second linkers are identical in length, and are each four amino acids in length, e.g., the linker is GLSG (SEQ ID NO: 383). In some embodiments, the first and second linkers are identical in length, and are each five amino acids in length. In some embodiments, the first and second linkers areidentical in length, and are each six amino acids in length, e.g., the first and second linkers each comprise a sequence of GLSGSG (SEQ ID NO: 384). In some embodiments, the first and second linkers are identical in length, and are each seven amino acids in length. In some embodiments, the first and second linkers are identical in length, and are each eight amino acids in length, e.g., the first and second linkers each comprise a sequence of GLSGLSGS (SEQ ID NO: 385). In some embodiments, the first and second linkers are identical in length, and are each nine amino acids in length. In some embodiments, the first and second linkers are identical in length, and are each ten amino acids in length, e.g., the first and second linkers each comprise a sequence of GLSGLSGLSG (SEQ ID NO: 386) or GLSGGSGLSG (SEQ ID NO: 387). In some embodiments, the first and second linkers are identical in length, and are each more than ten amino acids in length.

[0240] Generally, a first member of a proteimprotein binding pair amino acid sequence as described herein, e.g., comprising a first member of a specific binding pair by itself or in combination with one or more linkers, is between about 5 amino acids to about 50 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is at least 5 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 6 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 7 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 8 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 9 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 10 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 11 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 12 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 13 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 14 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 15 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 16 amino acids in length. In some embodiments, the first member of a proteimproteinbinding pair amino acid sequence is 17 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 18 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 19 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 20 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 21 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 22 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 23 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 24 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 25 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 26 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 27 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 28 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 29 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 30 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 31 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 32 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 33 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 34 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 35 amino acids in length.In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 36 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 37 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 38 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 39 amino acids in length. In some embodiments, the first member of aprotein :protein binding pair amino acid sequence is 40 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 41 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 42 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 43 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 44 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 45 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 46 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 47 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 48 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 49 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 50 amino acids in length.Modified Capsids Comprising Modified Capsid Proteins

[0241] In some embodiments a viral capsid comprising a modified viral capsid protein as described herein is a mosaic capsid, e.g., comprises at least two sets of VP1, VP2, and / or VP3 proteins, each set of which is encoded by a different cap gene. A mosaic capsid herein generally refers to a mosaic of a first viral capsid protein modified to comprise a first member of a binding pair and a second corresponding viral capsid protein lacking the first member of a binding pair. In relation to a mosaic capsid, the second viral capsid protein lacking the first member of a binding pair may be referred to as a reference capsid protein encoded by a reference cap gene. In some mosaic capsid embodiments, preferably when the VP1, VP2, and / or VP3 capsid proteins modified with a first member of proteimprotein pair is not a chimeric capsid protein, a VP 1 , VP2, and / or VP3 reference capsid protein may comprise an amino acid sequence identical to that of the viral VP1, VP2, and / or VP3 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of a binding pair. In some mosaic capsid embodiments, a VP1, VP2, and / or VP3 reference capsid protein corresponds to the viral VP1, VP2, and / or VP3capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of a binding pair. In some embodiments, a VP1 reference capsid protein corresponds to the viral VP1 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of a binding pair. In some embodiments, a VP2 reference capsid protein corresponds to the viral VP2 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of a binding pair. In some embodiments, a VP3 reference capsid protein corresponds to the viral VP3 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of a binding pair. In some mosaic capsid embodiments comprising a chimeric VP1, VP2, and / or VP3 capsid protein further modified to comprise a first member of a binding pair, a reference protein may be a corresponding capsid protein from which portions thereof form part of the chimeric capsid protein. As a non-limiting example in some embodiments, mosaic capsid comprising a chimeric AAV2 / AAAV VP1 capsid protein modified to comprise a first member of a binding pair may further comprise as a reference capsid protein: an AAV2 VP1 capsid protein lacking the first member, an A AAV VP1 capsid protein lacking the first member, a chimeric AAV2 / AAAV VP1 capsid protein lacking the first member. Similarly, in some embodiments, a mosaic capsid comprising a chimeric AAV2 / AAAV VP2 capsid protein modified to comprise a first member of a binding pair may further comprise as a reference capsid protein: an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, a chimeric AAV2 / AAAV VP2 capsid protein lacking the first member. In some embodiments, a mosaic capsid comprising a chimeric AAV2 / AAAV VP3 capsid protein modified to comprise a first member of a binding pair may further comprise as a reference capsid protein: an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, a chimeric AAV2 / AAAV VP3 capsid protein lacking the first member. In some mosaic capsid embodiments, a reference capsid protein may be any capsid protein so long as it that lacks the first member of the binding pair and is able to form a capsid with the first capsid protein modified with the first member of a binding pair.

[0242] Generally, mosaic particles may be generated by transfecting mixtures of the modified and reference Cap genes into production cells at the indicated ratios. The proteinsubunit ratios, e.g., modified VP proteimunmodified VP protein ratios, in the particle may, but do not necessarily, stoichiometrically reflect the ratios of the at least two species of the cap gene encoding the first capsid protein modified with a first member of a binding pair and the one or more reference cap genes, e.g., modified cap gene:reference cap gene(s) transfected into packaging cells. In some embodiments, the protein subunit ratios in the particle do not stoichiometrically reflect the modified cap gene reference cap gene(s) ratio transfected into packaging cells.

[0243] In some mosaic viral particle embodiments, the protein subunit ratio ranges from about 1:59 to about 59:1. In some mosaic viral particle embodiments, the protein subunit is at least about 1: 1 (e.g., the mosaic viral particle comprises about 30 modified capsid proteins and about 30 reference capsid protein). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 :2 (e.g., the mosaic viral particle comprises about 20 modified capsid proteins and about 40 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 3:5. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:3 (e.g., the mosaic viral particle comprises about 15 modified capsid proteins and about 45 reference capsid proteins) . In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:4 (e.g., the mosaic viral particle comprises about 12 modified capsid proteins and 48 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:5 (e.g., the mosaic viral particle comprises 10 modified capsid proteins and 50 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:6. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 :7. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 :8. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:9 (e.g., the mosaic viral particle comprises about 6 modified capsid proteins and about 54 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:10. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1: 11 (e.g., the mosaic viral particle comprises about 5 modified capsid proteins and about 55 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:12. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1: 13. Insome mosaic viral particle embodiments, the protein subunit ratio is at least about 1: 14 (e.g., the mosaic viral particle comprises about 4 modified capsid proteins and about 56 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:15. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:19 (e.g., the mosaic viral particle comprises about 3 modified capsid proteins and about 57 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:29 (e.g., the mosaic viral particle comprises about 2 modified capsid proteins and about 58 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:59. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 2: 1 (e.g., the mosaic viral particle comprises about 40 modified capsid proteins and about 20 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 5:3. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 3: 1 (e.g., the mosaic viral particle comprises about 45 modified capsid proteins and about 15 reference capsid proteins) . In some mosaic viral particle embodiments, the protein subunit ratio is at least about 4:1 (e.g., the mosaic viral particle comprises about 48 modified capsid proteins and 12 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 5: 1 (e.g., the mosaic viral particle comprises 50 modified capsid proteins and 10 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 6:1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 7:1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 8:1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 9:1 (e.g., the mosaic viral particle comprises about 54 modified capsid proteins and about 6 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 10:1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 11:1 (e.g., the mosaic viral particle comprises about 55 modified capsid proteins and about 5 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 12:1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 13:1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 14:1 (e.g., the mosaic viral particle comprises about 56 modified capsid proteins and about 4 referencecapsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 15:1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 19:1 (e.g., the mosaic viral particle comprises about 57 modified capsid proteins and about 3 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 29: 1 (e.g., the mosaic viral particle comprises about 58 modified capsid proteins and about 2 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 59:1.

[0244] In some non-mosaic viral particle embodiments, the protein subunit ratio may be 1:0 wherein each capsid protein of the non-mosaic viral particle is modified with a first member of a binding pair. In some non-mosaic viral particle embodiments, the protein subunit ratio may be 0:1 wherein each capsid protein of the non-mosaic viral particle is not modified with a first member of a binding pair.Insertion sites

[0245] Due to the high conservation of at least large stretches and the large member of closely related family members, the corresponding insertion sites for AAV other than the enumerated AAV can be identified by performing an amino acid alignment or by comparison of the capsid structures. See, e.g., Rutledge et al. (1998) J. Virol. 72:309-19; Mietzsch et al. (2019) Viruses 11, 362, 1-34, and U.S. Patent No. 9,624,274 for exemplary alignments of different AAV capsid proteins, each of which is incorporated herein by reference in its entirety. For example, Mietzcsh et al. (2019) provide an overlay of ribbons from different dependoparvovirus, depicting the variable regions VR I to VR IX. Using such structural analysis as described therein, and sequence analysis, a skilled artisan may determine which amino acids within the variable region correspond to amino acid sequence of AAV that can accommodate the insertion of, e.g., a targeting ligand as described herein, a first member of a binding pair and / or detectable label.

[0246] Generally, the targeting ligand, first member of a binding pair, and / or detectable label may be inserted into a variable region or variable loop of an AAV capsid protein, a GH loop of an AAV capsid protein, etc. In some embodiments, the targeting ligand, first member of a binding pair, and / or detectable label is inserted into variable loop IV and / or variable loop VIII of the AAV structural proteins VP1, VP2 and VP3.

[0247] In some embodiments, the first member of a binding pair and / or detectable label is inserted in a VP1 capsid protein of a non-primate animal AAV after an amino acid position corresponding with an amino acid position selected from the group consisting of G453 of AAV2 capsid protein VP1, N587 of AAV2 capsid protein VP1, G453 of AAV9 capsid protein VP1, and A589 of AAV9 capsid protein VP1. In some embodiments, the first member of a binding pair and / or detectable label is inserted in a VP1 capsid protein of a non- primate animal AAV between amino acids that correspond with N587 and R588 of an AAV2 VP1 capsid. Additional suitable insertion sites of a non-primate animal VP1 capsid protein include those corresponding to I- 1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-459, 1-471, 1-520, 1-534, 1-570, 1-573, 1-584, 1-587, 1-588, 1-591, 1-657, 1-664, 1-713 and I- 716 of the VP1 capsid protein of AAV2 (Wu et al. (2000) J. Virol. 74:8635-8647). A modified virus capsid protein as described herein may be a non-primate animal capsid protein comprising a first member of a binding pair and / or detectable label inserted into a position corresponding with a position of an AAV2 capsid protein selected from the group consisting of 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-459, 1-471, 1-520, 1-534, 1- 570, 1-573, 1-584, 1-587, 1-588, 1-591, 1-657, 1-664, 1-713, 1-716, and a combination thereof. Additional suitable insertion sites of a non-primate animal AAV that include those corresponding to 1-587 or 1-590 of AAV1, 1-589 of AAV1, 1-585 of AAV3, 1-584 or 1-585 of AAV4, and 1-575 or 1-585 of AAV5. In some embodiments, a modified virus capsid protein as described herein may be a non-primate animal capsid protein comprising a targeting ligand, first member of a binding pair and / or detectable label inserted into a position corresponding with a position selected from the group consisting of 1-587 (AAV1), 1-589 (AAV1), 1-585 (AAV3), 1-585 (AAV4), 1-585 (AAV5), and a combination thereof.

[0248] In some embodiments, the first member of a binding pair and / or detectable label is inserted in a VP1 capsid protein of a non-primate animal AAV after an amino acid position corresponding with an amino acid position selected from the group consisting of 1444 of an avian AAV capsid protein VP1, 1580 of an avian AAV capsid protein VP1, 1573 of a bearded dragon AAV capsid protein VP1, 1436 of a bearded dragon AAV capsid protein VP1, 1429 of a sea lion AAV capsid protein VP1, 1430 of a sea lion AAV capsid protein VP1, 1431 of a sea lion AAV capsid protein VP1, 1432 of a sea lion AAV capsid protein VP1, 1433 of a sea lion AAV capsid protein VP1, 1434 of a sea lion AAV capsid protein VP1, 1436 of asea lion AAV capsid protein VP1, 1437 of a sea lion AAV capsid protein VP1, and 1565 of a sea lion AAV capsid protein VP1.

[0249] The nomenclature I-###, I# or the like herein refers to the insertion site (I) with ### naming the amino acid number relative to the VP1 protein of an AAV capsid protein, however such the insertion may be located directly N- or C-terminal, preferably C- terminal of one amino acid in the sequence of 5 amino acids N- or C-terminal of the given amino acid, preferably 3, more preferably 2, especially 1 amino acid(s) N- or C-terminal of the given amino acid. Additionally, the positions referred to herein are relative to the VP1 protein encoded by an AAV capsid gene, and corresponding positions (and point mutations thereof) may be easily identified for the VP2 and VP3 capsid proteins encoding by the capsid gene by performing a sequence alignment of the VP1, VP2 and VP3 proteins encoded by the appropriate AAV capsid gene.

[0250] Accordingly, an insertion into the corresponding position of the coding nucleic acid of one of these sites of the cap gene leads to an insertion into VP1, VP2 and / or VP3, as the capsid proteins are encoded by overlapping reading frames of the same gene with staggered start codons. Therefore, for AAV2, for example, according to this nomenclature insertions between amino acids 1 and 138 are only inserted into VP1, insertions between 138 and 203 are inserted into VP1 and VP2, and insertions between 203 and the C-terminus are inserted into VP1, VP2 and VP3, which is of course also the case for the insertion site 1-587. Therefore, the present invention encompasses structural genes of AAV with corresponding insertions in the VP1, VP2 and / or VP3 proteins.

[0251] Also provided herein are nucleic acids that encode a VP3 capsid protein of the invention. AAV capsid proteins may be, but are not necessarily, encoded by overlapping reading frames of the same gene with staggered start codons. In some embodiments, a nucleic acid that encodes a VP3 capsid protein of the invention does not also encode a VP2 capsid protein or VP1 capsid protein of the invention. In some embodiments, a nucleic acid that encodes a VP3 capsid protein of the invention may also encode a VP2 capsid protein of the invention but does not also encode a VP1 capsid of the invention. In some embodiments, a nucleic acid that encodes a VP3 capsid protein of the invention may also encode a VP2 capsid protein of the invention and a VP1 capsid of the invention.

[0252] In some embodiments, a viral capsid comprising the modified viral capsid protein comprising the first and second members of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.) is able to infect a specific cell, e.g., has an enhanced capacity to target and bind a specific cell compared to that of a control viral capsid that is identical to the modified viral capsid protein except that it lacks either or both the first and second members of a binding pair, e.g., comprises a control capsid protein. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a detectable transduction efficiency compared to the undetectable transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 10% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 20% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 30% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 40% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 50% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 60% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein asdescribed herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 70% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 75% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 80% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 85% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 90% greater than the transduction efficiency of a control capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 95% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 99% greater than the transduction efficiency of a control viral capsid.

[0253] In some embodiments, a viral capsid comprising the modified viral capsid protein comprising the first and second members of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.) is able to infect a specific cell, e.g., has an enhanced capacity to target and bind a specific cell compared to that of a control viral capsid that is identical to the modified viral capsid protein except that it lacks either or both the first and second members of a binding pair, e.g., comprises a control capsid protein. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and secondmembers of a binding pair linked to a targeting ligand exhibits a detectable transduction efficiency compared to the undetectable transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 10% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 20% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 30% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 40% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 50% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 60% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 70% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 75% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to atargeting ligand exhibits a transduction efficiency that is 80% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 85% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 90% greater than the transduction efficiency of a control capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 95% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 99% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 1.5-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 2-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 3 -fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 4-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits atransduction efficiency that is at least 5 -fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 6-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 7-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 8-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 9-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 10-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 20-fold greater than the transduction efficiency of a control capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 30-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 40-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked toa targeting ligand exhibits a transduction efficiency that is at least 50-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 60-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 70-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 80-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 90-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 100-fold greater than the transduction efficiency of a control viral capsid In some embodiments, a viral particle of the invention comprising a viral capsid protein comprising an amino acid sequence of a capsid protein of a non-primate animal AAV, a remote AAV, or a combination thereof, and optionally comprising a first and second members of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.) is better able to evade neutralization by pre-existing antibodies in serum isolated from a human patient compared to an appropriate control viral particle (e.g., comprising a viral capsid of an AAV serotype from which a portion is included in the viral capsid of the invention, e.g., as part of the viral capsid protein comprising an amino acid sequence of a capsid protein of a non-primate animal AAV, a remote AAV, or a combination thereof), which also optionally comprises a first and second members of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.). In some embodiments, a viral particle of the invention comprising a viral capsid protein comprising an amino acid sequenceof a capsid protein of a non-primate animal AAV, a remote AAV, or a combination thereof requires at least 2-fold more total IVIG or IgG for neutralization (e.g., 50% or more infection inhibition) compared to an appropriate control viral particle, e.g., (e.g., a viral particle of the invention has an IC50 value that is at least 2-fold that of a control virus particle).

[0254] In some embodiments of the invention comprising a detectable label, a targeting ligand comprises a multispecific binding molecule, e.g., a bispecific antibody, comprising (i) an antibody paratope that specifically binds the detectable label and (ii) a second binding domain that specifically binds a receptor, which may be conjugated to the surface of a bead (e.g., for purification) or expressed by a target cell. Accordingly, a multispecific binding molecule comprising (i) an antibody paratope that specifically binds the detectable label and (ii) a second binding domain that specifically binds a receptor targets the viral particle. Such “targeting” or “directing” may include a scenario in which the wildtype viral particle targets several cells within a tissue and / or several organs within an organism, which broad targeting of the tissue or organs is reduced to abolished by insertion of the detectable label, and which retargeting to more specific cells in the tissue or more specific organ in the organism is achieved with the multispecific binding molecule. Such retargeting or redirecting may also include a scenario in which the wildtype viral particle targets a tissue, which targeting of the tissue is reduced to abolished by insertion of the detectable label, and which retargeting to a completely different tissue is achieved with the multispecific binding molecule. An antibody paratope as described herein generally comprises at a minimum a complementarity determining region (CDR) that specifically recognizes the detectable label, e.g., a CDR3 region of a heavy and / or light chain variable domain. In some embodiments, a multispecific binding molecule comprises an antibody (or portion thereof) that comprises the antibody paratope that specifically binds the detectable label. For example, a multispecific binding molecule may comprise a single domain heavy chain variable region or a single domain light chain variable region, wherein the single domain heavy chain variable region or single domain light chain variable region comprises an antibody paratope that specifically binds the detectable label. In some embodiments, a multispecific binding molecule may comprise an Fv region, e.g., a multispecific binding molecule may comprise an scFv, that comprises an antibody paratope that specifically binds the detectable label. In someembodiments, a multispecific binding molecule as described herein comprises an antibody paratope that specifically binds c-myc (SEQ ID NO: 389).

[0255] In some embodiments, the modified capsid may further comprise a mutation, e.g., a substitution, insertion, or deletion of an amino acid, that reduces or abolishes the natural tropism of the capsid protein. In some embodiments, the modified capsid protein is derived from an AAV9 serotype, and the further mutation comprises a W50 A mutation. In some embodiments, the modified capsid protein is derived from an AAV6 serotype, and the further mutation comprises a K531A or K531E mutation. In some embodiments, the modified capsid protein is derived from an AAV2 serotype, and the further mutation comprises a R484A, R487A, R487G, K532A, K532D, R585A, R585S, R585Q, R585A, R588A, or R588T mutation. In some embodiments, the AAV2 serotype comprises the heparin binding mutations (HBM), R585A and R588A.

[0256] One embodiment of the present invention is a multimeric structure comprising a modified viral capsid protein of the present invention. A multimeric structure comprises at least 5, preferably at least 10, more preferably at least 30, most preferably at least 60 modified viral capsid proteins comprising a first member of a specific binding pair as described herein. They can form regular viral capsids (empty viral particles) or viral particles (capsids encapsidating a nucleotide). The formation of viral particles comprising a viral genome is a highly preferred feature for use of the modified viral capsids described herein.

[0257] A further embodiment of the present invention is the use of at least one modified viral capsid protein and / or a nucleic acid encoding same, preferably at least one multimeric structure (e.g., viral particle) for the manufacture of and use in transfer of a nucleotide to a target cell.Methods of Use and Making

[0258] A further embodiment of the modified viral capsids described herein is their use for delivering a polynucleotide, e.g., a reporter gene or a therapeutic gene, to a target cell. Generally, packaging of a polynucleotide comprises replacing an AAV genome between AAV ITR sequences with a gene to create a transfer plasmid, which is then encapsulated in an AAV capsid according to well-known methods Thus, a modified viral capsid as described herein may encapsulate a transfer plasmid and / or a polynucleotide, which may generallycomprise 5’ and 3' inverted terminal repeat (ITR) sequences flanking a gene, e.g., reporter gene(s) or therapeutic gene(s), or a portion of the gene (which may be under the control of a viral or non-viral promoter). According to well-known methods of packaging AAV viral particles, the modified viral capsids, the 5’ ITR, and the 3’ ITR need not be of the same AAV serotype. In one embodiment, a transfer plasmid and / or polynucleotide comprises from 5 ’ to 3’ : a 5’ ITR, a promoter, a gene (e.g., a reporter and / or therapeutic gene) and a 3 TR.

[0259] A consideration for AAV transfer plasmid design is that a wildtype AAV genome is ~4.7kb. Thus, included herein are the well-known strategies that provide for packaging nucleotides that exceed the packaging capacity of an individual AAV. Such strategies include, but are not limited to, dual-vector strategies that exploit ITR-mediated recombination to express genes that are larger than a wildtype AAV genome by way of transcript splicing across intermolecularly recombined ITRs from two complementary vector genomes, vector recombination by homology, RNA trans-splicing, and / or protein “transsplicing” via split intein designs. See, e.g., Nakai, H. et al. (2000) Nat. Biotechnol. 18:527- 532; Sun, L. (2000) Nat. Med. 6: 599-602 (2000); Ghosh, A., et al. (2008) Mol. Ther.16:124-130 (2008); Lai, Y (2005) Nat. Biotechnol. 23:1435-1439; Chew, W. L. et al. (2016) Nat. Methods 13:868-874; Li, J. (2008) Hum. Gene Ther. 19:958-964, each of which reference is incorporated herein in its entirety by reference.

[0260] Dual AAV vector strategies to transfer of a large gene into target cells have been described, which rely on different mechanisms including, but not limited to, trans- splicing, including overlapping regions in the dual vectors, and a hybrid of the two. Tomabene and Trapani (2020) Human Gene Ther. 31:47-56; see also U.S. Patent No. 8,236,557, each of which is incorporated herein by reference in its entirety.

[0261] A trans-splicing approach takes advantage of the ability of AAV ITR sequences to concatemerized to reconstitute full-length genomes, wherein each of two or more viral capsids respectively encapsulate one of two or more transfer plasmids, each of which transfer plasmid comprises a portion of the gene. For example, in a dual vector approach, the two transfer plasmids may be designed as follows: the 5 ’-transfer plasmid comprises the promoter, the 5 ’ portion of the coding sequence of the gene, and a splicing donor (SD) signal; the 3 ’-transfer plasmid comprises a splicing acceptor (SA) signal, the 3’ portion of the gene, and the polyA signal. Upon tail-to-head ITR-mediated concatemerizationof the two AAV genomes, the SD and SA signals will allow splicing of the recombined genome.

[0262] A large gene is also split when taking an overlapping region approach. In the overlapping region approach, the 5’ and 3’ portions (and thus the 5’ transfer plasmid and 3’ transfer plasmid) share a recombinogenic sequence, e.g., region of homology, e.g., each portion comprises an overlapping sequence. The gene is made whole in a targeted cell via homologous recombination mediated by the recombinogenic sequence, e.g., homology / overlapping region.

[0263] In a hybrid approach, the 5 ’-transfer plasmid and 3 ’-transfer plasmid each comprise a highly recombinogenic sequence, wherein the recombinogenic sequence is placed downstream of an SD signal of a 5’ portion of the coding sequence of the gene and upstream of an SA signal of a 3’ portion of the coding sequence of the gene. In this hybrid system, the gene may be made whole either via ITR-mediated concatemerization and splicing and / or by homologous recombination.

[0264] Trans-splicing at the RNA or protein levels may also be utilized. In an RNA trans-splicing approach, two transfer plasmids may respectively encode for 5’ and 3’ fragments of the pre-mRNA of a large gene and share an intronic hybridization domain that can favor trans-splicing, leading to joining of the two half-transcripts into an intact full-length mRNA.

[0265] Protein trans-splicing occurs post-translationally and is catalyzed by intervening proteins called split-inteins. Split-inteins are expressed as two independent polypeptides (N-intein and C-intein) at the extremities of two host proteins. The N-intein and C-intein polypeptides remain catalytically inactive until they encounter each other. Upon encountering each other, each intein precisely excises itself from the host protein while mediating ligation of the N- and C- host polypeptides via a peptide bond. Split-intein use has been used in AAV-based delivery of therapeutic genes in muscle, liver, and retinal diseases. For example, on co-delivery of two halves of the mini-dystrophin cDNA fused to N- and C- intein coding sequences, efficient production of the two polypeptides was shown. Li et al. (2008) Hum Gene Ther 19:958-64. Similarly, AAV-split-inteins have been widely used for the expression and ligation of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nuclease.

[0266] The above dual vector approaches are well-known in the art. See, e.g., Tomabene and Trapani (2020), supra; U.S. Patent No. 8,236,557. Thus, in some embodiments, a modified viral capsid described herein encapsulates a nucleotide, wherein the nucleotide comprises a portion of a gene. In some embodiments, a nucleotide comprising a portion of a gene further comprises a splicing donor signal or a splicing acceptor signal and / or a recombinogenic sequence. In some embodiments, a nucleotide comprising a portion of a gene comprises an intronic hybridization domain encoding sequence. In some embodiments, a nucleotide comprising a portion of a gene comprises a N-intein or C-intein encoding sequence.

[0267] Design of the transfer plasmid / nucleotide includes including one or more regulatory elements, e.g., promoter and / or enhancer elements, that will control expression of the gene. Non-limiting examples of useful promoters include, e.g., cytomegalovirus (CMV)- promoter, the spleen focus forming virus (SFFV)-promoter, the elongation factor 1 alpha (EFl a) -promoter (the 1.2 kb EFla-promoter or the 0.2 kb EFla-promoter), the chimeric EF 1 a / IF4-promoter, and the phospho-glycerate kinase (PGK)-promoter. An internal enhancer may also be present in the viral construct to increase expression of the gene. For example, the CMV enhancer (Karasuyama et al. 1989. J. Exp. Med. 169:13, which is incorporated herein by reference in its entirety) may be used. In some embodiments, the CMV enhancer can be used in combination with the chicken [3-actin promoter, e.g., CBA. In some embodiments, the regulatory element further comprises a chimeric chicken |3-actin promoter / minute virus of mice (MVM) intron and a bovine growth hormone (BGH) poly adenylation sequence. In some embodiments, the regulatory element comprises a CBh regulatory element (See, Gray, et al., Hum Gene Ther. 2011 Sep; 22(9): 1143-1 153, incorporated herein by reference in its entirety). In some embodiments, tissue specific regulatory elements, e.g., a nervous system specific promoter and / or regulatory element, may be used to drive the expression of the gene.

[0268] In some embodiments, bidirectional promoter vectors have also been employed for delivery of dual therapeutic gene cassettes. An example of this is the bidirectional chicken -actin ubiquitous promoter that drives the simultaneous expression of the hexosaminidase a- and -subunits of the HexA enzyme, the two respective genes involved in Tay-Sachs and Sandhoff diseases. Lahey, et al. (2020) Mol. Ther. 28: 2150-2160, incorporated herein in its entirety by reference. In some embodiments, a transfer plasmidand / or nucleotide herein comprises a bidirectional promoter, wherein the bidirectional promoter drives the expression of two different genes.

[0269] A variety of reporter genes (or detectable moieties) can be encapsidated in a multimeric structure comprising the modified viral capsid proteins described herein.Exemplary reporter genes include, for example, 0-galactosidase (encoded lacZ gene), Green Fluorescent Protein (GFP), enhanced Green Fluorescent Protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof. The methods described herein demonstrate the construction of targeting particles that employ the use of a reporter gene that encodes green fluorescent protein, however, persons of skill upon reading this disclosure will understand that the viral capsids described herein can be generated in the absence of a reporter gene or with any reporter gene known in the art.

[0270] A variety of therapeutic genes can also be encapsidated in a multimeric structure comprising the modified viral capsid proteins described herein, e.g., as part of a transfer particle. Non-limiting examples of a therapeutic gene include those that encode a toxin (e.g., a suicide gene), a therapeutic antibody or fragment thereof, a bispecific T-cell engager, a CRISPR / Cas system or portion(s) thereof, antisense RNA, siRNA, shRNA, etc.

[0271] In some embodiments, the therapeutic gene encodes a nucleic acid molecule or protein that inhibits an oncogenic gene or gene product, e.g., oncogene inhibitors, cell cycle inhibitors, etc. Tn some embodiments, the gene encodes a therapeutic protein, a cytokine, an immune checkpoint protein, a programmed cell death protein, a suicide gene, a toxic protein or peptide, an antibody or a fragment thereof, a bispecific T-cell engager, a chimeric antigen receptor (CAR), a T cell receptor (TCR), a gene editing system or a component(s) thereof, an antisense oligonucleotide, a ribozyme, a microRNA, a short hairpin RNA, or an RNAi molecule. In some embodiments, the therapeutic gene may be a gene editing ribonucleoprotein complex or a component(s) thereof. In various embodiments, the therapeutic gene may comprise, for example, a Cas protein, e.g., Cas9 protein, and / or a guide RNA (gRNA) specific to a specified gene.

[0272] A further embodiment of the present invention is a process for the preparation of a modified capsid protein, the method comprising the steps of:(a) expressing a nucleic acid molecule encoding the modified capsid protein under suitable conditions, and(b) isolating the expressed capsid protein of step (a).

[0273] In some embodiments, a viral particle as described herein comprises a mosaic capsid, e.g., a capsid comprising capsid proteins genetically modified as described herein (in the absence or presence of a covalent bond with a targeting ligand) in a certain ratio with reference capsid proteins. A method for making such a mosaic viral particle comprises(a) expressing a nucleic acid encoding the modified capsid protein and a nucleotide encoding a reference capsid protein at a ratio (wt / wt) of at least about 60:1 to about 1:60, e.g., 2:1, 1 :1, 3:5 ,1:2, 1:3, etc. under suitable conditions, and(b) isolating the expressed capsid protein of step (a).

[0274] In some embodiments, a composition described herein comprises, or a method described herein combines, a modified cap gene: reference cap gene (or combination of reference cap genes) at a ratio that ranges from at least about 1 :60 to about 60:1, e.g., 2:1, 1 :1, 3:5, 1 :2, 1:3, etc. In some embodiments, the ratio is at least about 1:2. In some embodiments, the ratio is at least about 1 :3. In some embodiments, the ratio is at least about 1 :4. In some embodiments, the ratio is at least about 1:5. In some embodiments, the ratio is at least about 1 :6. In some embodiments, the ratio is at least about 1 :7. In some embodiments, the ratio is at least about 1 :8. In some embodiments, the ratio is at least about 1 :9. In some embodiments, the ratio is at least about 1:10. In some embodiments, the ratio is at least about 1 : 11. In some embodiments, the ratio is at least about 1 : 12. In some embodiments, the ratio is at least about 1 :13. In some embodiments, the ratio is at least about 1 :14. In some embodiments, the ratio is at least about 1:15. In some embodiments, the ratio is at least about 1 : 16. In some embodiments, the ratio is at least about 1 : 17. In some embodiments, the ratio is at least about 1 : 18. In some embodiments, the ratio is at least about 1 : 19. In some embodiments, the ratio is at least about 1 :20. In some embodiments, the ratio is at least about 1 :25. In some embodiments, the ratio is at least about 1 :30. In some embodiments, the ratio is at least about 1 :35. In some embodiments, the ratio is at least about 1 :40. In some embodiments, the ratio is at least about 1 :45. In some embodiments, the ratiois at least about 1 :50. In some embodiments, the ratio is at least about 1 :55. In some embodiments, the ratio is at least about 1 :60. In some embodiments, the ratio is at least about 2:1. In some embodiments, the ratio is at least about 3:1. In some embodiments, the ratio is at least about 4:1. In some embodiments, the ratio is at least about 5:1. In some embodiments, the ratio is at least about 6:1. In some embodiments, the ratio is at least about 7:1 . In some embodiments, the ratio is at least about 8: 1. In some embodiments, the ratio is at least about 9:1. In some embodiments, the ratio is at least about 10: 1. In some embodiments, the ratio is at least about 11:1. In some embodiments, the ratio is at least about 12:1. In some embodiments, the ratio is at least about 13:1. In some embodiments, the ratio is at least about 14: 1. In some embodiments, the ratio is at least about 15: 1. In some embodiments, the ratio is at least about 16:1. In some embodiments, the ratio is at least about 17:1. In some embodiments, the ratio is at least about 18: 1. In some embodiments, the ratio is at least about 19:1. In some embodiments, the ratio is at least about 20:1. In some embodiments, the ratio is at least about 25:1. In some embodiments, the ratio is at least about 30: 1. In some embodiments, the ratio is at least about 35: 1. In some embodiments, the ratio is at least about 40:1. In some embodiments, the ratio is at least about 45:1. In some embodiments, the ratio is at least about 50:1. In some embodiments, the ratio is at least about 55:1. In some embodiments, the ratio is at least about 60: 1.

[0275] In some embodiments, VP protein subunit ratios in the mosaic viral particle may, but do not necessarily, stoichiometrically reflect the ratios of modified cap gene:reference cap gene. As a non-limiting exemplary embodiment, a mosaic capsid formed according to the method may be considered to, but does not necessarily, have a modified capsid proteimreference capsid protein ratio similar to the ratio (wt:wt) of nucleic acids encoding same used to produce the mosaic capsid. In some embodiments, a mosaic capsid comprises a protein subunit ratio of about 1:59 to about 59: 1.

[0276] Further embodiments of the present invention is a method for altering the tropism of a virus, the method comprising the steps of: (a) inserting a nucleic acid encoding an amino acid sequence into a nucleic acid sequence encoding an viral capsid protein to form a nucleotide sequence encoding a genetically modified capsid protein comprising the amino acid sequence and / or (b) culturing a packaging cell in conditions sufficient for the production of viral particles, wherein the packaging cell comprises the nucleic acid. A furtherembodiment of the present invention is a method for displaying a targeting ligand on the surface of a capsid protein, the method comprising the steps of: (a) expressing a nucleic acid encoding a modified viral capsid protein as described herein (and optionally with a nucleotide encoding a reference capsid protein) under suitable conditions, wherein the nucleic acid encodes a capsid protein comprising a first member of a specific binding pair, (b) isolating the expressed capsid protein comprising a first member of a specific binding pair of step (a) or capsid comprising same, and (c) incubating the capsid protein or capsid with a second cognate member of the specific binding pair under conditions suitable for allowing the formation of an isopeptide bond between the first and second member, wherein the second cognate member of the specific binding pair is fused with a targeting ligand.

[0277] In some embodiments, the packaging cell further comprises a helper plasmid and / or a transfer plasmid comprising a polynucleotide. In some embodiments, the methods further comprise isolating self-complementary adeno-associated viral particles from culture supernatant. In some embodiments, the methods further comprise lysing the packaging cell and isolating single- stranded adeno-associated viral particles from the cell lysate. In some embodiments, the methods further comprise (a) clearing cell debris, (b) treating the supernatant containing viral particles with nucleases, e.g., DNase I and MgCh, (c) concentrating viral particles, (d) purifying the viral particles, and (e) any combination of (a)- (d).

[0278] Packaging cells useful for production of the viral particles described herein include, e.g., animal cells permissive for the virus, or cells modified to be permissive for the virus; or the packaging cell construct, for example, with the use of a transformation agent such as calcium phosphate. Non-limiting examples of packaging cell lines useful for producing viral particles described herein include, e.g., human embryonic kidney 293 (HEK- 293) cells (e.g., American Type Culture Collection [ATCC] No. CRL-1573), HEK-293 cells that contain the SV40 Large T-antigen (HEK-293T or 293T), HEK293T / 17 cells, human sarcoma cell line HT-1080 (CCL-121), lymphoblast-like cell line Raji (CCL-86), glioblastoma-astrocytoma epithelial-like cell line U87-MG (HTB-14), T-lymphoma cell line HuT78 (TIB-161), NIH / 3T3 cells, Chinese Hamster Ovary cells (CHO) (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), HeLa cells (e.g., ATCC No. CCL-2), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12I l lcells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, CAP cells, CAP-T cells, and the like.

[0279] L929 cells, the FLY viral packaging cell system outlined in Cosset et al (1995)J Virol 69,7430-7436, NSO (murine myeloma) cells, human amniocytic cells (e.g., CAP, CAP-T), yeast cells (including, but not limited to, S. cerevisiae, Pichia pastoris), plant cells (including, but not limited to, Tobacco NT1 , BY-2), insect cells (including but not limited to SF9, S2, SF21, Tni (e.g. High 5)) or bacterial cells (including, but not limited to, E. coli).

[0280] For additional packaging cells and systems, packaging techniques and particles for packaging the nucleic acid genome into the pseudotyped viral particle see, for example, Polo, et al, Proc Natl Acad Sci USA, (1999) 96:4598-4603. Methods of packaging include using packaging cells that permanently express the viral components, or by transiently transfecting cells with plasmids. For methods of detecting and evaluating viruses, e.g., AAV, see US20240168018A1, incorporated by reference herein in its entirety.

[0281] Further embodiments include methods comprising contacting a modified Cap protein as described herein with the targeting vector in conditions sufficient to operably link the modified Cap protein with the targeting vector, e.g., in conditions sufficient to promote association of the targeting vector to the modified Cap protein, e.g., via chemical linkage and / or association of first and second members of a specific binding pair, wherein the first member is inserted into the modified Cap protein the first member and the targeting vector is fused to the second member of the specific binding pair.

[0282] Further embodiments include methods of redirecting a virus and / or delivering a reporter or therapeutic gene to a target cell, the method comprising a method for transducing cells in vitro (e.g., ex vivo) or in vivo, the method comprising the steps of: contacting the target cell with a viral particle comprising a capsid described herein, wherein the capsid comprises a targeting ligand that specifically binds a receptor expressed by the target cell. In some embodiments, the target cell is in vitro (e.g., ex vivo). In other embodiments, the target cell is in vivo in a subject, e.g., a human.Target Cells

[0283] Once a particular population of target cells is identified in which expression of a nucleotide is desired, a target cell surface protein, e.g., a receptor, is selected that isspecifically expressed on that population of target cells. The target cell surface protein may be expressed exclusively on that population of cells or to a greater extent on that population of cells than on other populations of cells. The more specific the expression, the more specifically delivery can be directed to the target cells. Depending on the context, the desired amount of specificity of the marker (and thus of the gene delivery) may vary. For example, for introduction of a toxic gene, a high specificity is most preferred to avoid killing nontargeted cells. For expression of a protein for harvest, or expression of a secreted product where a global impact is desired, less marker specificity may be needed.

[0284] As discussed above, the target cell surface protein may be any receptor for which a targeting ligand can be identified or created. Preferably the target receptor is a peptide or polypeptide, such as a receptor. However, in other embodiments the target receptor may be a carbohydrate or other molecule that can be recognized by a binding partner. If a binding partner, e.g., ligand, for the target receptor is already known, it may be used as the affinity molecule. However, if a binding molecule is not known, antibodies to the target receptor may be generated using standard procedures. The antibodies can then be used as a targeting ligand.

[0285] Thus, target cells may be chosen based on a variety of factors, including, for example, (1) the application (e.g., therapy, expression of a protein to be collected, and conferring disease resistance) and (2) expression of a marker with the desired amount of specificity.

[0286] Target cells are not limited in any way and include both germline cells and cell lines and somatic cells and cell lines. When the target cells are germline cells, the target cells are preferably selected from the group consisting of single-cell embryos and embryonic stem cells (ES).

[0287] In some embodiments, the target cell is a cancerous cell, e.g., a tumor cell. In some embodiments, the cancer is a EGFR-expressing cancer. In some embodiments, the cancer is selected from esophageal carcinoma, lung squamous cell carcinoma, lung adenocarcinoma, cervical cancer (including cervical squamous cell carcinoma), endometrial adenocarcinoma, bladder cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer (NSCLC), colorectal cancer (such as microsatellite-stable colorectal cancer), sigmoid colon adenocarcinoma, rectal cancer, endometrial cancer, skin cancer, head & neck squamouscell carcinoma, brain cancer, glioblastoma multiforme, non-CNS tumor, cutaneous squamous cell carcinoma, breast cancer, gastric cancer, gastroesophageal cancer, gastroesophageal adenocarcinoma, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, nasopharyngeal carcinoma, anal carcinoma, mesothelioma, renal cell carcinoma, gallbladder / cholangiocarcinoma, pancreatic carcinoma, penile squamous cell carcinoma, or vulvovaginal carcinoma. Tn certain embodiments, the cancer is one or more of the following cancers: glioblastoma, renal cell carcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer (ductal or intraductal), or melanoma.

[0288] In some embodiments, the target cell comprises a cell expressing EGFR or a variant thereof. In some embodiments, the target cell overexpresses EGFR. In some embodiments, the target cell expresses a mutant variant of EGFR. In some embodiments, the mutant variant of EGFR initiates intracellular signaling independent of ligand binding to the EC domain, e.g., has constitutive signaling. In some embodiments, the mutant variant of EGFR does not bind to one or more ligands that bind to a wildtype EGFR. In embodiments, the mutant variant of EGFR comprises one or more variant selected from the group consisting of EGFRvIII, or an EGFR encoded by an EGFR gene comprising an EGFR Exon 19 deletion, EGFR L858R mutation, EGFR exon20 insertion, exon 18 / 21 atypical mutations, other activating EGFR mutations. In some embodiments, the mutant variant of human EGFR comprises a deletion of amino acids 6-273 relative to wildtype human EGFR and / or the introduction of a new glycine residue. In some embodiments, the mutant variant of human EGFR is encoded by an EGFR gene comprising a deletion of exons 2-7, wherein the fusion junction of exons 1 and 8 encodes a new glycine residue. In some embodiments, the mutant variant of EGFR comprises EGFRvIII.Pharmaceutical compositions, dosage forms and administration

[0289] A further embodiment provides a medicament comprising at least one modified viral capsid protein and appropriate targeting ligand, e.g., that binds EGFR or avariant thereof, e.g., EGFRvIII, according to this invention and / or a nucleic acid according to this invention. Preferably such medicament is useful as a gene transfer particle.

[0290] Also disclosed herein are pharmaceutical compositions comprising the viral particles described herein and a pharmaceutically acceptable carrier and / or excipient. In addition, disclosed herein are pharmaceutical dosage forms comprising the viral particle described herein.

[0291] As discussed herein, the viral particles described herein can be used for various therapeutic applications (in vivo and ex vivo) and as research tools.

[0292] Pharmaceutical compositions based on the viral particles disclosed herein can be formulated in any conventional manner using one or more physiologically acceptable carriers and / or excipients. The viral particles may be formulated for administration by, for example, injection, inhalation or insulation (either through the mouth or the nose) or by oral, buccal, parenteral or rectal administration, or by administration directly to a tumor.

[0293] The pharmaceutical compositions can be formulated for a variety of modes of administration, including systemic, topical or localized administration. Techniques and formulations can be found in, for example, Remington’s Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For the purposes of injection, the pharmaceutical compositions can be formulated in liquid solutions, preferably in physiologically compatible buffers, such as Hank's solution or Ringer's solution. In addition, the pharmaceutical compositions may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms of the pharmaceutical composition are also suitable.

[0294] For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycolate); or wetting agents (e.g. sodium lauryl sulfate). The tablets can also be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups orsuspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g. oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

[0295] The pharmaceutical compositions can be formulated for parenteral administration by injection, e.g. by bolus injection or continuous infusion. Formulations for injection can be presented in a unit dosage form, e.g. in ampoules or in multi-dose containers, with an optionally added preservative. The pharmaceutical compositions can further be formulated as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain other agents including suspending, stabilizing and / or dispersing agents.

[0296] Additionally, the pharmaceutical compositions can also be formulated as a depot preparation. These long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Other suitable delivery systems include microspheres, which offer the possibility of local noninvasive delivery of drugs over an extended period of time. This technology can include microspheres having a precapillary size, which can be injected via a coronary catheter into any selected part of an organ without causing inflammation or ischemia. The administered therapeutic is men slowly released from the microspheres and absorbed by the surrounding cells present in the selected tissue.

[0297] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts, and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration can occur using nasal sprays or suppositories. For topical administration, the viral particles described herein can be formulated into ointments, salves, gels, or creams as generally knownin the art. A wash solution can also be used locally to treat an injury or inflammation in order to accelerate healing.

[0298] Pharmaceutical forms suitable for injectable use can include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid. It must be stable under the conditions of manufacture and certain storage parameters (e.g., refrigeration and freezing) and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0299] If formulations disclosed herein are used as a therapeutic to boost an immune response in a subject, a therapeutic agent can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0300] A carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents known in the art. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0301] Sterile injectable solutions can be prepared by incorporating the active compounds or constructs in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.

[0302] Upon formulation, solutions can be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but slow-release capsules or microparticles and microspheres and the like can also be employed.

[0303] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intratumorally, intramuscular, subcutaneous and intraperitoneal administration. In this context, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion.

[0304] The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. For example, a subject may be administered viral particles described herein on a daily or weekly basis for a time period or on a monthly, bi- yearly or yearly basis depending on need or exposure to a pathogenic organism or to a condition in the subject (e.g., cancer).

[0305] In addition to the compounds formulated for parenteral administration, such as intravenous, intratumorally, intradermal or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; liposomal formulations; time release capsules; biodegradable and any other form currently used.

[0306] One may also use intranasal or inhalable solutions or sprays, aerosols or inhalants. Nasal solutions can be aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions can be prepared so that they are similar in many respects to nasal secretions. Thus, the aqueous nasal solutions usually are isotonic and slightly buffered to maintain a pH of 5.5 to 7.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, and appropriate drug stabilizers, if required, may be included in the formulation. Various commercial nasal preparations are known and can include, for example, antibiotics and antihistamines and are used for asthma prophylaxis.

[0307] Oral formulations can include excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. In certain defined embodiments, oral pharmaceutical compositions will include an inert diluent or assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0308] The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.

[0309] Further embodiments disclosed herein can concern kits for use with methods and compositions. Kits can also include a suitable container, for example, vials, tubes, mini- or microfuge tubes, test tube, flask, bottle, syringe or other container. Where an additional component or agent is provided, the kit can contain one or more additional containers into which this agent or component may be placed. Kits herein will also typically include a means for containing the viral particles and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Optionally, one or more additional active agents such as, e.g., anti-inflammatory agents, anti-viral agents, anti-fungal or anti-bacterial agents or anti-tumor agents may be desired for compositions described.

[0310] Compositions disclosed herein may be administered by any means known in the art. For example, compositions may include administration to a subject intravenously, intrasy novially, intratumorally, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarec tally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion, via a catheter, via a lavage, in a cream, or in a lipid composition.

[0311] Compositions may be administered to a subject intravenously, intratumorally, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, intracistemally, intracerebroventricularly, intraparenchymal, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion, via a catheter, via a lavage, in a cream, or in a lipid composition.

[0312] Any method known to one skilled in the art may be used for large scale production of viral particles, packaging cells and particle constructs described herein. For example, master and working seed stocks may be prepared under GMP conditions in qualified primary CEFs or by other methods. Packaging cells may be plated on large surface area flasks, grown to near confluence and viral particles purified. Cells may be harvested and viral particles released into the culture media isolated and purified, or intracellular viral particles released by mechanical disruption (cell debris can be removed by large-pore depth filtration and host cell DNA digested with endonuclease). Virus particles may be subsequently purified and concentrated by tangential-flow filtration, followed by diafiltration. The resulting concentrated bulk may be formulated by dilution with a buffer containing stabilizers, filled into vials, and lyophilized. Compositions and formulations may be stored for later use. For use, lyophilized viral particles may be reconstituted by addition of diluent.

[0313] Certain additional agents used in the combination therapies can be formulated and administered by any means known in the art.

[0314] Compositions as disclosed herein can also include adjuvants such as aluminum salts and other mineral adjuvants, tensoactive agents, bacterial derivatives, vehicles and cytokines. Adjuvants can also have antagonizing immunomodulating properties. For example, adjuvants can stimulate Thl or Th2 immunity. Compositions and methods as disclosed herein can also include adjuvant therapy.Methods of and Compositions for use in Treatment / Prevention

[0315] Further provided herein are methods of treatment and / or prevention of a disease or disorder associated with a cell (over)expressing EGFR or a variant thereof, e.g., EGFRvIII, the method comprising administering a composition as described herein, e.g., an AAV particle comprising am EGFR-targeting ligand, to a subject in need thereof. Also provided are compositions, e.g., AAV particles comprising an EGFR-targeting ligand and pharmaceutical compositions thereof, for use in, e.g., the manufacture of a medicament for, treating and / or preventing a disease or disorder associated with a cell (over)expressing EGFR or a variant thereof, e.g., EGFRvIII.

[0316] As used herein “a disease or disorder associated with a cell (over)expressing EGFR or a variant thereof’ refers to a disease or disorder associated with EGFR overexpression, expression of a mutant, e.g., deleterious, variant of EGFR, or a combination thereof, resulting in dysregulated EGFR signaling. For example, as a consequence of gene amplification, EGFR overexpression can result in dysregulated EGFR signaling that contributes to cancer progression. Mutation of the EGFR gene can also result in the expression of a variant EGFR with dysregulated signaling that contributes to cancer progression.

[0317] Generally, methods of treating a disease or disorder associated with a cell (over)expressing EGFR or a variant thereof, e.g., cancer, as disclosed herein comprise administering to a subject having or at risk of developing such disease or disorder a viral particle, e.g., an AAV particle, or pharmaceutical composition as described herein, wherein the viral particle comprises:(i) a viral capsid, e.g., AAV capsid, modified to comprise a first member of a proteimprotein binding pair,(ii) a polynucleotide encapsidated within the viral capsid, and(iii) a second member of the proteimprotein binding pair, wherein the second member of the proteimprotein binding pair comprises a targeting ligand that binds EGFR or a variant thereof , wherein the first member of the proteimprotein binding pair and the second member of the proteimprotein binding pair are associated to direct the tropism of the viral capsid to a cell expressing EGFR or a variant thereof in the subject in need thereof.

[0318] In some embodiments, administering comprises intravenous, intratumoral, intradermal, intraarterial, intraperitoneal, intralesion, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, topical, intramuscular, intrathecal, intracistem, intracerebroventricular, intraparenchymal, subcutaneous, subconjunctival, intra vesicular, mucosal, intrapericardial, intraumbilical, intraocular, and / or oral administration.

[0319] Also provided herein is the use of an AAV particle as described herein or a pharmaceutical composition comprising the same as described herein for, e.g., use in the manufacture of a medicament for, treating a disease or disorder associated with a cell (over)expressing EGFR or a variant thereof, wherein the AAV particle comprises a polynucleotide encapsidated within the AAV particle, and wherein the polynucleotide encodes a therapeutic protein, a cytokine, an immune checkpoint protein, a programmed cell death protein, a suicide gene, an antibody or a fragment thereof, a bispecific T-cell engager, a CRISPR / Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or a shRNA molecule.

[0320] Also provided herein is an AAV particle as described herein or a pharmaceutical composition comprising the same as described herein for use in, e.g., the manufacture of a medicament for, treating a disease or disorder associated with a cell (over)expressing EGFR or a variant thereof, wherein the AAV particle comprises a polynucleotide encapsidated within the AAV particle, and wherein the polynucleotide encodes a therapeutic protein, a cytokine, an immune checkpoint protein, a programmed cell death protein, a suicide gene, an antibody or afragment thereof, a bispecific T-cell engager, a CRISPR / Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or a shRNA molecule.

[0321] In some embodiments, the disease or disorder associated with a cell (over)expressing EGFR or a variant thereof is cancer, and the cell is a tumor cell. In some embodiments, the tumor cell overexpresses wildtype mammalian, e.g., human, EGFR. In some embodiments, the tumor cell expresses a mutant variant of mammalian, e.g., human, EGFR, e.g., EGFRvIII. In some embodiments, the tumor cell overexpresses wildtype mammalian, e.g., human, EGFR and (over)expresses a mutant variant of mammalian, e.g., human, EGFR, e.g., EGFRvIII. As used herein, a “cancer associated with EGFRvIII” refers to a cancer that is known to be related to, e.g., caused by, the expression of EGFRvIII.

[0322] Non-limiting examples of diseases or disorders associated with a cell (over)expressing EGFR or a variant thereof include: esophageal carcinoma, lung squamous cell carcinoma, lung adenocarcinoma, cervical cancer (including cervical squamous cell carcinoma), endometrial adenocarcinoma, bladder cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer (NSCLC), colorectal cancer (such as microsatellite-stable colorectal cancer), sigmoid colon adenocarcinoma, rectal cancer, endometrial cancer, skin cancer, head & neck squamous cell carcinoma, brain cancer, glioblastoma multiforme, non- CNS tumor, cutaneous squamous cell carcinoma, breast cancer, gastric cancer, gastroesophageal cancer, gastroesophageal adenocarcinoma, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, nasopharyngeal carcinoma, anal carcinoma, mesothelioma, renal cell carcinoma, gallbladder / cholangiocarcinoma, pancreatic carcinoma, penile squamous cell carcinoma, or vulvovaginal carcinoma. In certain embodiments, the cancer is one or more of the following cancers: glioblastoma, renal cell carcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer (ductal or intraductal), or melanoma.

[0323] Non-limiting examples of cancers associated with EGFRvIII include: glioblastoma, renal cell carcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovariancancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer (ductal or intraductal), or melanoma.

[0324] In some embodiments the polynucleotide encodes a therapeutic protein, a cytokine, an immune checkpoint protein, a programmed cell death protein, a suicide gene, an antibody or a fragment thereof, a bispecific T-cell engager, a CRISPR / Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or a shRNA molecule. For example, in some embodiments, the therapeutic polynucleotide encodes a nucleic acid molecule or protein that inhibits an oncogenic gene or gene product. In some embodiments, the oncogenic gene or gene product comprises a mammalian EGFR gene, a mammalian EGFR mRNA molecule or a mammalian EGFR polypeptide. In some embodiments, the therapeutic gene encodes an oncolytic gene product.

[0325] Non-limiting embodiments are described below.Embodiment 1. An adeno-associated virus (AAV) particle comprising:(i) an AAV capsid protein, and(ii) a targeting ligand that binds a mammalian Epidermal Growth Factor Receptor (EGFR) or variant thereof, wherein the AAV capsid protein is operably linked with the targeting ligand.Embodiment 2. The AAV particle of embodiment 1 , wherein:(a) the AAV capsid protein comprises a first member and a second member of a protein: protein binding pair, and(b) the second member of the protei protein binding pair comprises the targeting ligand that binds the mammalian EGFR or variant thereof, wherein the first member of the proteimprotein binding pair and the second member of the proteimprotein binding pair are associated to direct the tropism of the AAV particle to a cell expressing the mammalian EGFR or variant thereof.Embodiment 3. The AAV particle of embodiment 1 or embodiment 2, wherein the mammalian EGFR is a mutant variant of a mammalian EGFR.Embodiment 4. The AAV particle of embodiment 3, wherein the mutant variant of the mammalian EGFR: (i) does not bind to one or more ligands that bind to a wildtype mammalian EGFR and / or (ii) has constitutive signaling activity.Embodiment 5. The AAV particle of embodiment 3 or embodiment 4, wherein the mutant variant of the mammalian EGFR is a mutant variant of a human EGFR.Embodiment 6. The AAV particle of embodiment 5, wherein the mutant variant of the human EGFR comprises a deletion of amino acids 6-273 relative to a wildtype human EGFR and / or the introduction of a new glycine residue.Embodiment 7. The AAV particle of embodiment 5 or embodiment 6, wherein the mutant variant of the human EGFR is encoded by an EGFR gene comprising a deletion of exons 2-7, wherein the fusion junction of exons 1 and 8 encodes a new glycine residue.Embodiment 8. The AAV particle of any one of embodiments 5-7, wherein the mutant variant of the human EGFR is EGFRvIII.Embodiment 9. The AAV particle of any one of embodiments 2-8, wherein the cell expressing the mammalian EGFR or variant thereof is a tumor cell.Embodiment 10. The AAV particle of embodiment 9, wherein the tumor cell is selected from the group consisting of: esophageal carcinoma, lung squamous cell carcinoma, lung adenocarcinoma, cervical cancer (including cervical squamous cell carcinoma), endometrial adenocarcinoma, bladder cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer (NSCLC), colorectal cancer (such as microsatellite-stable colorectal cancer), sigmoid colon adenocarcinoma, rectal cancer, endometrial cancer, skin cancer, head & neck squamous cell carcinoma, brain cancer, glioblastoma multiforme, non-CNS tumor, cutaneous squamous cell carcinoma, breast cancer, gastric cancer, gastroesophageal cancer, gastroesophageal adenocarcinoma, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, nasopharyngeal carcinoma, anal carcinoma, mesothelioma, renal cell carcinoma,gallbladder / cholangiocarcinoma, pancreatic carcinoma, penile squamous cell carcinoma, or vulvovaginal carcinoma.Embodiment 11. The AAV particle of any one of embodiments 1-10, wherein the cell expressing the mammalian EGFR or variant thereof is a tumor cell, further wherein the tumor cell is selected from the group consisting of: glioblastoma, renal cell carcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer (ductal or intraductal), or melanoma.Embodiment 12. The AAV particle of any one of embodiments 1-11, wherein the AAV particle is in vitro.Embodiment 13. The AAV particle of any one of embodiments 1-11, wherein the AAV particle is in vivo.Embodiment 14. The AAV particle of any one of embodiments 1-13, wherein the targeting ligand is an antibody or a portion thereof.Embodiment 15. The AAV particle of any one of embodiments 1-14, wherein the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2, an LCDR3, and / or a set of HCDR1-HCDR2-HCDR3- LCDR1-LCDR2-LCDR3 amino acid sequence(s) at least 90% identical to, respectively, an amino acid sequence of a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2, an LCDR3, and / or a set of HCDR1-HCDR2-HCDR3- LCDR1-LCDR2-LCDR3 as set forth in or encoded by any one of SEQ ID NOs: 228-371.Embodiment 16. The AAV particle of any one of embodiments 1-15, wherein the targeting ligand comprises the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 229 / 237, 245 / 253, 261 / 269, 277 / 285, 293 / 301, 309 / 317, 325 / 333, 341 / 349, 357 / 365, and 405 / 413.Embodiment 17. The AAV particle of any one of embodiments 1-16, wherein the targeting ligand comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 229 / 237, 245 / 253, 261 / 269, 277 / 285, 293 / 301, 309 / 317, 325 / 333, 341 / 349, 357 / 365, and 405 / 413.Embodiment 18. The AAV particle of any one of embodiments 2-17, wherein:(a) (i) the first member of the protei protein binding pair comprises SpyTag, Isopeptag, SnoopTag, SpyTag002, SpyTag003, or any biologically active portions or variants thereof, (ii) the second member of the proteimprotein binding pair comprises SpyCatcher, KTag, pilin Embodiment 19. The AAV particle of embodiment 18(a), wherein Embodiment 20. The AAV particle of any one of embodiments 2-19, comprising a first and / or second linker operably linking the first member of the protein: protein binding pair to the AAV capsid protein.Embodiment 21. The AAV particle of embodiment 20, wherein the first and second linker are not identical, or the first and second linker are identical.Embodiment 22. The AAV particle of embodiment 20 or embodiment 21 , wherein the first linker is 10 amino acids in length and / or the second linker is 10 amino acids in length.Embodiment 23. The AAV particle of any one of embodiments 1-22, wherein the AAV capsid protein comprises a modified VP 1 capsid protein, a modified VP2 capsid protein, and / or a modified VP3 capsid protein, andwherein the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein comprises an insertion of a first member of a proteimprotein binding pair and / or the targeting ligand, and and wherein a portion of the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein, that comprises the insertion of a first member of a protein :protein binding pair and / or the targeting ligand, further comprises an amino acid sequence at least 90% identical to a corresponding capsid protein of a wild-type AAV.Embodiment 24. The AAV particle of embodiment 23, wherein the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein further comprises, in addition to the insertion of a first member of a proteimprotein binding pair and / or the targeting ligand:(i) a substitution, insertion, or deletion of an amino acid,(ii) a chimeric amino acid sequence, or(iii) any combination of (i) and (ii).Embodiment 25. The AAV particle of embodiment 24, wherein the substitution, insertion, or deletion of an amino acid reduces the natural tropism of the AAV particle and / or creates a detectable label.Embodiment 26. The AAV particle of any one of embodiments 1-25, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, a non-primate animal AAV listed in Table 6, and any chimera thereof.Embodiment 27. The AAV particle of any one of embodiments 1-26, wherein the AAV is AAV2.Embodiment 28. The AAV particle of any one of embodiments 1-27, wherein the AAV particle comprises a modified AAV2 VP1 capsid protein that comprises a first member of aprotein :protein binding pair inserted at an amino acid position 1-453 and / or 1-587, and optionally linked to the AAV sequence via a linker on one or both sides.Embodiment 29. The AAV particle of embodiment 28, wherein the AAV particle comprises a modified AAV2 VP1 capsid protein that comprises the first member of the protein :protein binding pair inserted, optionally via a linker, at position G453, optionally wherein the modified AAV2 VP1 capsid protein further comprises a mutation selected from R585A, R588A, R484A, R487A, K532A, and any combination thereof.Embodiment 30. The AAV particle of embodiment 28 or embodiment 29, wherein the AAV particle comprises a mosaic AAV capsid comprising a second set of AAV2 VP1 capsid proteins lacking the first member of the proteimprotein binding pair, optionally wherein the second set of AAV2 VP1 capsid proteins comprises a mutation selected from R585A, R588A, R484A, R487A, K532A and any combination thereof.Embodiment 31. The AAV particle of any one of embodiments 1-26, wherein the AAV is AAV9.Embodiment 32. The AAV particle of embodiment 31 , wherein the AAV capsid protein comprises a modified AAV9 VP1 capsid protein that comprises a first member of a proteimprotein binding pair inserted, optionally via a linker, at position 1-453 or 1-589.Embodiment 33. The AAV particle of embodiment 32, wherein the AAV particle comprises a modified AAV9 VP1 capsid protein that comprises a first member of a proteimprotein binding pair inserted, optionally via a linker, at position G453, optionally wherein the modified AAV9 VP1 capsid protein further comprises a mutation selected from N272A, W503A, and a combination thereof.Embodiment 34. The AAV particle of embodiment 32 or embodiment 33, wherein the AAV particle comprises a mosaic AAV capsid comprising a second set of AAV9 VP1 capsid proteins lacking the first member of the proteimprotein binding pair,optionally wherein the second set of AAV9 VP1 capsid proteins comprises a mutation selected from N272A, W503A, and a combination thereof.Embodiment 35. The AAV particle any one of embodiments 1-26, wherein the AAV is a non-primate animal AAV.Embodiment 36. The AAV particle of embodiment 35, wherein the non-primate animal AAV is an avian AAV (AAAV).Embodiment 37. The AAV particle of embodiment 36, wherein the AAV capsid protein comprises a modified AAAV VP1 capsid protein that comprises the first member of the proteimprotein binding pair inserted, optionally via a linker, at position 1-444 or 1-580.Embodiment 38. The AAV particle of embodiment 35, wherein the non-primate animal AAV is a squamate AAV.Embodiment 39. The AAV particle of embodiment 38, wherein the squamate AAV is a bearded dragon AAV.Embodiment 40. The AAV particle of embodiment 39, wherein the AAV capsid protein comprises a modified bearded dragon AAV VP1 capsid protein that comprises the first member of the proteimprotein binding pair inserted, optionally via a linker, at position 1-573 or 1-436.Embodiment 41. The AAV particle of embodiment 35, wherein the non-primate animal AAV is a non-primate mammalian AAV.Embodiment 42. The AAV particle of embodiment 41 , wherein the non-primate mammalian AAV is a sea lion AAV.Embodiment 43. The AAV particle of embodiment 42, wherein the modified AAV capsid protein comprises a modified sea lion AAV VP1 capsid protein that comprises the first member of the proteimprotein binding pair inserted, optionally via a linker, at a position selected from the group consisting of 1-429, 1-430, 1-431, 1-432, 1-433, 1-434, 1-436, 1-437, and 1-565.Embodiment 44. The AAV particle of any one of embodiments 1-43, wherein the AAV particle comprises a mosaic AAV capsid, optionally wherein the mosaic AAV capsid comprises (i) a first plurality of reference AAV capsid proteins, each of which is not associated with the targeting ligand, and (ii) a second plurality of AAV capsid proteins, each of which is associated with the targeting ligand, optionally wherein the mosaic AAV capsid comprises the first plurality of reference AAV capsid proteins and the second plurality of AAV capsid proteins at a ratio of 1 :7.Embodiment 45. The AAV particle of any one of embodiments 1-44, further comprising a polynucleotide encapsidated within the AAV particle.Embodiment 46. The AAV particle of embodiment 45, wherein the polynucleotide is a reporter gene.Embodiment 47. The AAV particle of embodiment 45 or embodiment 46, wherein the polynucleotide encodes 0-galactosidase, green fluorescent protein (GFP), enhanced Green Fluorescent Protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T- Sapphire, luciferase, alkaline phosphatase, or a combination thereof.Embodiment 48. The AAV particle of embodiment 45, wherein the polynucleotide encodes a therapeutic protein, a cytokine, an immune checkpoint protein, a programmed cell death protein, a suicide gene, an antibody or a fragment thereof, a bispecific T-cell engager, aCRISPR / Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or a shRNA molecule.Embodiment 49. The AAV particle of embodiment 48, wherein the polynucleotide (i) encodes a nucleic acid molecule or protein that inhibits an oncogenic gene or gene product, and / or (ii) encodes an oncolytic gene product.Embodiment 50. The AAV particle of embodiment 49, wherein the oncogenic gene or gene product comprises a mammalian EGFR gene, a mammalian EGFR mRNA molecule or a mammalian EGFR polypeptide.Embodiment 51. The AAV particle of embodiment 49, wherein the oncolytic gene product comprises: a therapeutic protein, a suicide gene, a toxic protein or peptide, an antibody or a fragment thereof, a chimeric antigen receptor (CAR), a T cell receptor (TCR), a gene editing system or a component(s) thereof, an antisense oligonucleotide, a ribozyme, a microRNA, a short hairpin RNA, an RNAi molecule, a Cas protein, e.g., Cas9 protein, and / or a guide RNA (gRNA) specific to a gene of interest.Embodiment 52. A pharmaceutical composition comprising (a) the AAV particle according to any one of embodiments 45-51, and (b) a pharmaceutically acceptable carrier or excipient.Embodiment 53. A method of delivering a polynucleotide to a cell expressing the mammalian EGFR or variant thereof, comprising contacting the cell expressing the mammalian EGFR or variant thereof with (a) the AAV particle according to any one of embodiments 44-51, or (b) the pharmaceutical composition of embodiment 52.Embodiment 54. The method of embodiment 53, wherein the contacting is performed ex vivo.Embodiment 55. The method of embodiment 53, wherein the contacting is performed in a subject.Embodiment 56. The method of embodiment 55, wherein the subject is a primate animal, optionally wherein the primate animal is a human.Embodiment 57. The method of any one of embodiments 53-56, wherein the cell expressing the mammalian EGFR or variant thereof is a tumor cell.Embodiment 58. The method of embodiment 57, wherein the tumor cell is selected from the group consisting of: esophageal carcinoma, lung squamous cell carcinoma, lung adenocarcinoma, cervical cancer (including cervical squamous cell carcinoma), endometrial adenocarcinoma, bladder cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer (NSCLC), colorectal cancer (such as microsatellite-stable colorectal cancer), sigmoid colon adenocarcinoma, rectal cancer, endometrial cancer, skin cancer, head and neck squamous cell carcinoma, brain cancer, glioblastoma multiforme, non-CNS tumor, cutaneous squamous cell carcinoma, breast cancer, gastric cancer, gastroesophageal cancer, gastroesophageal adenocarcinoma, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, nasopharyngeal carcinoma, anal carcinoma, mesothelioma, renal cell carcinoma, gallbladder / cholangiocarcinoma, pancreatic carcinoma, penile squamous cell carcinoma, and vulvovaginal carcinoma.Embodiment 59. The method of embodiment 57, wherein the mammalian EGFR or variant thereof is EGFRvIII, further wherein the tumor cell is selected from the group consisting of: glioblastoma, renal cell carcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer (ductal or intraductal), or melanoma.Embodiment 60...

Claims

CLAIMSWhat is claimed is:

1. An adeno- associated virus (AAV) particle comprising:(i) an AAV capsid protein, and(ii) a targeting ligand that hinds a mammalian Epidermal Growth Factor Receptor (EGFR) or variant thereof, wherein the AAV capsid protein is operably linked with the targeting ligand.

2. The AAV particle of claim 1, wherein:(a) the AAV capsid protein comprises a first member and a second member of a proteimprotein binding pair, and(b) the second member of the proteimprotein binding pair comprises the targeting ligand that binds the mammalian EGFR or variant thereof, wherein the first member of the proteimprotein binding pair and the second member of the proteimprotein binding pair are associated to direct the tropism of the AAV particle to a cell expressing the mammalian EGFR or variant thereof.

3. The AAV particle of claim 2, wherein the mammalian EGFR is a mutant variant of a mammalian EGFR.

4. The AAV particle of claim 3, wherein the mutant variant of the mammalian EGFR: (i) does not bind to one or more ligands that bind to a wildtype mammalian EGFR and / or (ii) has constitutive signaling activity.

5. The AAV particle of claim 4, wherein the mutant variant of the mammalian EGFR is a mutant variant of a human EGFR.

6. The AAV particle of claim 5, wherein the mutant variant of the human EGFR comprises a deletion of amino acids 6-273 relative to a wildtype human EGFR and / or the introduction of a new glycine residue.

7. The AAV particle of claim 6, wherein the mutant variant of the human EGFR is encoded by an EGFR gene comprising a deletion of exons 2-7, wherein the fusion junction of exons 1 and 8 encodes a new glycine residue.

8. The AAV particle of claim 7, wherein the mutant variant of the human EGFR is EGFRvIII.

9. The AAV particle of claim 8, wherein the cell expressing the mammalian EGFR or variant thereof is a tumor cell.

10. The AAV particle of claim 9, wherein the tumor cell is selected from the group consisting of: esophageal carcinoma, lung squamous cell carcinoma, lung adenocarcinoma, cervical cancer (including cervical squamous cell carcinoma), endometrial adenocarcinoma, bladder cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer (NSCLC), colorectal cancer (such as microsatellite-stable colorectal cancer), sigmoid colon adenocarcinoma, rectal cancer, endometrial cancer, skin cancer, head & neck squamous cell carcinoma, brain cancer, glioblastoma multiforme, non-CNS tumor, cutaneous squamous cell carcinoma, breast cancer, gastric cancer, gastroesophageal cancer, gastroesophageal adenocarcinoma, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, nasopharyngeal carcinoma, anal carcinoma, mesothelioma, renal cell carcinoma, gallbladder / cholangiocarcinoma, pancreatic carcinoma, penile squamous cell carcinoma, or vulvovaginal carcinoma.1 1. The AAV particle of claim 8, wherein the cell expressing the mammalian EGFR or variant thereof is a tumor cell, further wherein the tumor cell is selected from the group consisting of: glioblastoma, renal cell carcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer(e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer (ductal or intraductal), or melanoma.

12. The AAV particle of claim 8, wherein the AAV particle is in vitro.

13. The AAV particle of claim 8, wherein the AAV particle is in vivo.

14. The AAV particle of claim 8, wherein the targeting ligand is an antibody or a portion thereof.

15. The AAV particle of claim 8, wherein the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2, an LCDR3, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence(s) at least 90% identical to, respectively, an amino acid sequence of a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2, an LCDR3, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 as set forth in or encoded by any one of SEQ ID NOs: 228-371.

16. The AAV particle of claim 15, wherein the targeting ligand comprises the HCDR1- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 229 / 237, 245 / 253, 261 / 269, 277 / 285, 293 / 301, 309 / 317, 325 / 333, 341 / 349, 357 / 365, and 405 / 413.

17. The AAV particle of claim 16, wherein the targeting ligand comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 229 / 237, 245 / 253, 261 / 269, 277 / 285, 293 / 301, 309 / 317, 325 / 333, 341 / 349, 357 / 365, and 405 / 413.

18. The AAV particle of claim 8, wherein:(a) (i) the first member of the proteimprotein binding pair comprises SpyTag, Isopeptag, SnoopTag, SpyTag002, SpyTag003, or any biologically active portions or variants thereof, (ii) the second member of the proteimprotein binding pair comprises SpyCatcher, KTag, pilin-C, SnoopCatcher, SpyCatcher002, SpyCatcher003, or any biologically active portions or variants thereof, and the targeting ligand that binds the mammalian EGFR or variant thereof, and (iii) the first member of the proteimprotein binding pair and the second member of the proteimprotein binding pair are linked by an isopeptide bond, or(b) (i) the first member of the proteimprotein binding pair comprises an epitope of the AAV capsid protein, (ii) the second member of the proteimprotein binding pair comprises a multispecific antigen-binding protein comprising a first antigen-binding domain that binds the epitope of the AAV capsid protein, optionally wherein the first antigen-binding domain comprises an amino acid sequence as set forth in or encoded by any one of SEQ ID NOs: 1- 227, and a second antigen-binding domain comprising the targeting ligand that binds the mammalian EGFR or variant thereof, and (iii) the first member and the second member are operably linked by a non-covalent bond between the epitope of the AAV capsid protein and the first antigen-binding domain, optionally wherein the first member comprises a sequence encoding a detectable label or portion thereof, or an AAV capsid protein or portion thereof.

19. The AAV particle of claim 18(a), wherein:(a) the first member of the proteimprotein binding pair comprises SpyTag, or any biologically active portion or variant thereof, and(b) the second member of the proteimprotein binding pair comprises SpyCatcher, or any biologically active portions or variants thereof, fused to the targeting ligand that binds the mammalian EGFR or variant thereof.

20. The AAV particle of claim 8, comprising a first and / or second linker operably linking the first member of the proteimprotein binding pair to the AAV capsid protein.

21. The AAV particle of claim 20, wherein the first and second linker are not identical, or the first and second linker are identical.

22. The AAV particle of claim 21, wherein the first linker is 10 amino acids in length and / or the second linker is 10 amino acids in length.

23. The AAV particle of claim 8, wherein the AAV capsid protein comprises a modified VP1 capsid protein, a modified VP2 capsid protein, and / or a modified VP3 capsid protein, and wherein the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein comprises an insertion of a first member of a proteimprotein binding pair and / or the targeting ligand, and and wherein a portion of the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein, that comprises the insertion of a first member of a proteimprotein binding pair and / or the targeting ligand, further comprises an amino acid sequence at least 90% identical to a corresponding capsid protein of a wild-type AAV.

24. The AAV particle of claim 23, wherein the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein further comprises, in addition to the insertion of a first member of a proteimprotein binding pair and / or the targeting ligand:(i) a substitution, insertion, or deletion of an amino acid,(ii) a chimeric amino acid sequence, or(iii) any combination of (i) and (ii).

25. The AAV particle of claim 24, wherein the substitution, insertion, or deletion of an amino acid reduces the natural tropism of the AAV particle and / or creates a detectable label.

26. The AAV particle of claim 8, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, a non-primate animal AAV listed in Table 6, and any chimera thereof.

27. The AAV particle of claim 26, wherein the AAV is AAV2.

28. The AAV particle of claim 27, wherein the AAV particle comprises a modified AAV2 VP1 capsid protein that comprises a first member of a protein: protein binding pair inserted at an amino acid position 1-453 and / or 1-587, and optionally linked to the AAV sequence via a linker on one or both sides.

29. The AAV particle of claim 28, wherein the AAV particle comprises a modified AAV2 VP1 capsid protein that comprises the first member of the protein: protein binding pair inserted, optionally via a linker, at position G453, optionally wherein the modified AAV2 VP1 capsid protein further comprises a mutation selected from R585A, R588A, R484A, R487A, K532A, and any combination thereof.

30. The AAV particle or composition of claim 29, wherein the AAV particle comprises a mosaic AAV capsid comprising a second set of AAV2 VP1 capsid proteins lacking the first member of the proteimprotein binding pair, optionally wherein the second set of AAV2 VP1 capsid proteins comprises a mutation selected from R585A, R588A, R484A, R487A, K532A and any combination thereof.

31. The AAV particle of claim 26, wherein the AAV is AAV9.

32. The AAV particle of claim 31, wherein the AAV capsid protein comprises a modified AAV9 VP1 capsid protein that comprises a first member of a protein :protein binding pair inserted, optionally via a linker, at position 1-453 or 1-589.

33. The AAV particle of claim 32, wherein the AAV particle comprises a modified AAV9 VP1 capsid protein that comprises a first member of a protein :protein binding pair inserted, optionally via a linker, at position G453, optionally wherein the modified AAV9 VP1 capsid protein further comprises a mutation selected from N272A, W503A, and a combination thereof.

34. The AAV particle of claim 33, wherein the AAV particle comprises a mosaic AAV capsid comprising a second set of AAV9 VP1 capsid proteins lacking the first member of the protein: protein binding pair, optionally wherein the second set of AAV9 VP1 capsid proteins comprises a mutation selected from N272A, W503A, and a combination thereof.

35. The AAV particle of claim 26, wherein the AAV is a non-primate animal AAV.

36. The AAV particle of claim 35, wherein the non-primate animal AAV is an avian AAV (AAAV).

37. The AAV particle of claim 36, wherein the AAV capsid protein comprises a modified AAAV VP1 capsid protein that comprises the first member of the proteimprotein binding pair inserted, optionally via a linker, at position 1-444 or 1-580.

38. The AAV particle of claim 35, wherein the non-primate animal AAV is a squamate AAV.

39. The AAV particle of claim 38, wherein the squamate AAV is a bearded dragon AAV.

40. The AAV particle of claim 39, wherein the AAV capsid protein comprises a modified bearded dragon AAV VP1 capsid protein that comprises the first member of the protein :protein binding pair inserted, optionally via a linker, at position 1-573 or 1-436.

41. The AAV particle of claim 35, wherein the non-primate animal AAV is a non-primate mammalian AAV.

42. The AAV particle of claim 41, wherein the non-primate mammalian AAV is a sea lion AAV.

43. The AAV particle of claim 42, wherein the modified AAV capsid protein comprises a modified sea lion AAV VP1 capsid protein that comprises the first member of the proteimprotein binding pair inserted, optionally via a linker, at a position selected from the group consisting of 1-429, 1-430, 1-431, 1-432, 1-433, 1-434, 1-436, 1-437, and 1-565.

44. The AAV particle of claim 8, wherein the AAV particle comprises a mosaic AAV capsid, optionally wherein the mosaic AAV capsid comprises (i) a first plurality of reference AAV capsid proteins, each of which is not associated with the targeting ligand, and (ii) a second plurality of AAV capsid proteins, each of which is associated with the targeting ligand, optionally wherein the mosaic AAV capsid comprises the first plurality of reference AAV capsid proteins and the second plurality of AAV capsid proteins at a ratio of 1:7.

45. The AAV particle of claim 8, further comprising a polynucleotide encapsidated within the AAV particle.

46. The AAV particle of claim 45, wherein the polynucleotide is a reporter gene.

47. The AAV particle of claim 46, wherein the polynucleotide encodes P-galactosidase, green fluorescent protein (GFP), enhanced Green Fluorescent Protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof.

48. The AAV particle of claim 45, wherein the polynucleotide encodes a therapeutic protein, a cytokine, an immune checkpoint protein, a programmed cell death protein, a suicide gene, an antibody or a fragment thereof, a bispecific T-cell engager, a CRISPR / Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule.

49. The AAV particle of claim 48, wherein the polynucleotide (i) encodes a nucleic acid molecule or protein that inhibits an oncogenic gene or gene product, and / or (ii) encodes an oncolytic gene product.

50. The AAV particle of claim 49, wherein the oncogenic gene or gene product comprises a mammalian EGFR gene, a mammalian EGFR mRNA molecule or a mammalian EGFR polypeptide.

51. The AAV particle of claim 49, wherein the oncolytic gene product comprises: a therapeutic protein, a suicide gene, a toxic protein or peptide, an antibody or a fragment thereof, a chimeric antigen receptor (CAR), a T cell receptor (TCR), a gene editing system or a component(s) thereof, an antisense oligonucleotide, a ribozyme, a microRNA, a short hairpin RNA, an RNAi molecule, a Cas protein, e.g., Cas9 protein, and / or a guide RNA (gRNA) specific to a gene of interest.

52. A pharmaceutical composition comprising (a) the AAV particle according to claim 45, and (b) a pharmaceutically acceptable carrier or excipient.

53. A method of delivering a polynucleotide to a cell expressing the mammalian EGFR or variant thereof, comprising contacting the cell expressing the mammalian EGFR or variant thereof with (a) the AAV particle according to any one of claims 45-51, or (b) the pharmaceutical composition of claim 52.

54. The method of claim 53, wherein the contacting is performed ex vivo.

55. The method of claim 53, wherein the contacting is performed in a subject.

56. The method of claim 55, wherein the subject is a primate animal, optionally wherein the primate animal is a human.

57. The method of claim 53, wherein the cell expressing the mammalian EGFR or variant thereof is a tumor cell.

58. The method of claim 57, wherein the tumor cell is selected from the group consisting of: esophageal carcinoma, lung squamous cell carcinoma, lung adenocarcinoma, cervical cancer (including cervical squamous cell carcinoma), endometrial adenocarcinoma, bladder cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer (NSCLC), colorectal cancer (such as microsatellite-stable colorectal cancer), sigmoid colon adenocarcinoma, rectal cancer, endometrial cancer, skin cancer, head and neck squamous cell carcinoma, brain cancer, glioblastoma multiforme, non-CNS tumor, cutaneous squamous cell carcinoma, breast cancer, gastric cancer, gastroesophageal cancer, gastroesophageal adenocarcinoma, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, nasopharyngeal carcinoma, anal carcinoma, mesothelioma, renal cell carcinoma, gallbladder / cholangiocarcinoma, pancreatic carcinoma, penile squamous cell carcinoma, and vulvovaginal carcinoma.

59. The method of claim 57, wherein the mammalian EGFR or variant thereof is EGFRvIII, further wherein the tumor cell is selected from the group consisting of: glioblastoma, renal cell carcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer (ductal or intraductal), or melanoma.

60. The method of claim 53, wherein the polynucleotide encodes a therapeutic protein, a cytokine, an immune checkpoint protein, a programmed cell death protein, a cytokine, an immune checkpoint protein, a programmed cell death protein, a suicide gene, an antibody or a fragment thereof, a bispecific T-cell engager, a CRISPR / Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule.

61. The method of claim 60, wherein the polynucleotide (i) encodes a nucleic acid molecule or protein that inhibits an oncogenic gene or gene product, and / or (ii) encodes an oncolytic gene product.

62. The method of claim 61 , wherein the oncogenic gene or gene product comprises a mammalian EGFR gene, a mammalian EGFR mRNA molecule or a mammalian EGFR polypeptide.

63. The method of claim 61 , wherein the oncolytic gene product comprises: a therapeutic protein, a suicide gene, a toxic protein or peptide, an antibody or a fragment thereof, a chimeric antigen receptor (CAR), a T cell receptor (TCR), a gene editing system or a component(s) thereof, an antisense oligonucleotide, a ribozyme, a microRNA, a short hairpin RNA, an RNAi molecule, a Cas protein, e.g., Cas9 protein, and / or a guide RNA (gRNA) specific to a gene of interest.

64. A method of treating a disease or disorder associated with a cell (over)expressing EGFR or a variant thereof in a subject in need thereof comprising: administering to the subject (a) an AAV particle according to any one of claims 48- 51, or (b) the pharmaceutical composition of claim 52, wherein the AAV particle comprises a polynucleotide encapsidated within the AAV particle, and wherein the polynucleotide encodes a therapeutic protein, a cytokine, an immune checkpoint protein, a programmed cell death protein, a cytokine, an immune checkpoint protein, a programmed cell death protein, a suicide gene, an antibody or a fragment thereof, a bispecific T-cell engager, a CRISPR / Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule.

65. The method of claim 64, wherein the administering comprises intravenous, intratumoral, intradermal, intraarterial, intraperitoneal, intralesion, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, topical, intramuscular, intrathecal, intracistem, intracerebroventricular, intraparenchymal,subcutaneous, subconjunctival, intra vesicular, mucosal, intrapericardial, intraumbilical, intraocular, and / or oral administration.

66. The method of claim 64, wherein the disease or disorder associated with a cell (over)expressing EGFR or a variant thereof is a cancer selected from the group consisting of: esophageal carcinoma, lung squamous cell carcinoma, lung adenocarcinoma, cervical cancer (including cervical squamous cell carcinoma), endometrial adenocarcinoma, bladder cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer (NSCLC), colorectal cancer (such as microsatellite-stable colorectal cancer), sigmoid colon adenocarcinoma, rectal cancer, endometrial cancer, skin cancer, head and neck squamous cell carcinoma, brain cancer, glioblastoma multiforme, non-CNS tumor, cutaneous squamous cell carcinoma, breast cancer, gastric cancer, gastroesophageal cancer, gastroesophageal adenocarcinoma, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, nasopharyngeal carcinoma, anal carcinoma, mesothelioma, renal cell carcinoma, gallbladder / cholangiocarcinoma, pancreatic carcinoma, penile squamous cell carcinoma, and vulvovaginal carcinoma.

67. The method of claim 64, wherein the disease or disorder associated with a cell (over)expressing EGFR or a variant thereof is a cancer associated with EGFRvIII.

68. The method of claim 67, wherein the cancer associated with EGFRvIII comprises a cancer selected from the group consisting of: glioblastoma, renal cell carcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer (ductal or intraductal), or melanoma.

69. The method of claim 64, wherein the polynucleotide (i) encodes a nucleic acid molecule or protein that inhibits an oncogenic gene or gene product, and / or (ii) encodes an oncolytic gene product.

70. The method of claim 69, wherein the oncogenic gene or gene product comprises a mammalian EGFR gene, a mammalian EGFR mRNA molecule or a mammalian EGFR polypeptide.

71. The method of claim 69, wherein the oncolytic gene or gene product comprises: a therapeutic protein, a suicide gene, a toxic protein or peptide, an antibody or a fragment thereof, a chimeric antigen receptor (CAR), a T cell receptor (TCR), a gene editing system or a component(s) thereof, an antisense oligonucleotide, a ribozyme, a microRNA, a short hairpin RNA, an RNAi molecule, a Cas protein, e.g., Cas9 protein, and / or a guide RNA (gRNA) specific to a gene of interest.

72. Use of (a) the AAV particle of any one of claims 48-51, or (b) the pharmaceutical composition of claim 52 for treating a disease or disorder associated with a cell (over)expressing EGFR or a variant thereof, wherein the AAV particle comprises a polynucleotide encapsidated within the AAV particle, and wherein the polynucleotide encodes a therapeutic protein, a cytokine, an immune checkpoint protein, a programmed cell death protein, a suicide gene, an antibody or a fragment thereof, a bispecific T-cell engager, a CRISPR / Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule.

73. The use of claim 72, wherein the disease or disorder associated with a cell (over)expressing EGFR or a variant thereof is selected from the group consisting of: esophageal carcinoma, lung squamous cell carcinoma, lung adenocarcinoma, cervical cancer (including cervical squamous cell carcinoma), endometrial adenocarcinoma, bladder cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer (NSCLC), colorectal cancer (such as microsatellite-stable colorectal cancer), sigmoid colon adenocarcinoma, rectal cancer, endometrial cancer, skin cancer, head and neck squamous cell carcinoma, brain cancer, glioblastoma multiforme, non-CNS tumor, cutaneous squamous cell carcinoma, breast cancer, gastric cancer, gastroesophageal cancer, gastroesophageal adenocarcinoma, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, nasopharyngeal carcinoma,anal carcinoma, mesothelioma, renal cell carcinoma, gallbladder / cholangiocarcinoma, pancreatic carcinoma, penile squamous cell carcinoma, and vulvovaginal carcinoma.

74. The use of claim 72, wherein the disease or disorder associated with a cell (over)expressing EGFR or a variant thereof is a cancer associated with EGFRvIII.

75. The use of claim 74, wherein the cancer associated with EGFRvIII comprises a cancer selected from the group consisting of: glioblastoma, renal cell carcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer (ductal or intraductal), or melanoma.

76. The use of claim 72, wherein the polynucleotide (i) encodes a nucleic acid molecule or protein that inhibits an oncogenic gene or gene product, and / or (ii) encodes an oncolytic gene product.

77. The use of claim 76, wherein the oncogenic gene or gene product comprises a mammalian EGFR gene, a mammalian EGFR mRNA molecule or a mammalian EGFR polypeptide.

78. The use of claim 76, wherein the oncolytic gene or gene product comprises: a therapeutic protein, a suicide gene, a toxic protein or peptide, an antibody or a fragment thereof, a chimeric antigen receptor (CAR), a T cell receptor (TCR), a gene editing system or a component(s) thereof, an antisense oligonucleotide, a ribozyme, a microRNA, a short hairpin RNA, an RNAi molecule, a Cas protein, e.g., Cas9 protein, and / or a guide RNA (gRNA) specific to a gene of interest.

79. The AAV particle of any one of claims 48-51, or the pharmaceutical composition of claim 52, for use in treating a disease or disorder associated with a cell (over)expressing EGFR or a variant thereof,wherein the AAV particle comprises a polynucleotide encapsidated within the AAV particle, and wherein the polynucleotide encodes a therapeutic protein, a cytokine, an immune checkpoint protein, a programmed cell death protein, a suicide gene, an antibody or a fragment thereof, a bispecific T-cell engager, a CRISPR / Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule.

80. The AAV particle or pharmaceutical composition thereof of claim 79, wherein the disease or disorder associated with a cell (over)expressing EGFR or a variant thereof is selected from the group consisting of: esophageal carcinoma, lung squamous cell carcinoma, lung adenocarcinoma, cervical cancer (including cervical squamous cell carcinoma), endometrial adenocarcinoma, bladder cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer (NSCLC), colorectal cancer (such as microsatellite-stable colorectal cancer), sigmoid colon adenocarcinoma, rectal cancer, endometrial cancer, skin cancer, head and neck squamous cell carcinoma, brain cancer, glioblastoma multiforme, non-CNS tumor, cutaneous squamous cell carcinoma, breast cancer, gastric cancer, gastroesophageal cancer, gastroesophageal adenocarcinoma, pancreatic cancer, prostate cancer, ovarian cancer, melanoma, nasopharyngeal carcinoma, anal carcinoma, mesothelioma, renal cell carcinoma, gallbladder / cholangiocarcinoma, pancreatic carcinoma, penile squamous cell carcinoma, and vulvovaginal carcinoma.

81. The AAV particle or pharmaceutical composition thereof of claim 79, wherein the disease or disorder associated with a cell (over)expressing EGFR or a variant thereof is a cancer associated with EGFRvIII.

82. The AAV particle or pharmaceutical composition thereof of claim 81 , wherein the cancer associated with EGFRvIII comprises a cancer selected from the group consisting of: glioblastoma, renal cell carcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovariancancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer (ductal or intraductal), or melanoma.

83. The AAV particle or pharmaceutical composition thereof of claim 79, wherein the polynucleotide (i) encodes a nucleic acid molecule or protein that inhibits an oncogenic gene or gene product, and / or (ii) encodes an oncolytic gene product.

84. The AAV particle or pharmaceutical composition thereof of claim 83, wherein the oncogenic gene or gene product comprises a mammalian EGFR gene, a mammalian EGFR mRNA molecule or a mammalian EGFR polypeptide.

85. The AAV particle or pharmaceutical composition thereof of claim 83, wherein the oncolytic gene product comprises: a therapeutic protein, a suicide gene, a toxic protein or peptide, an antibody or a fragment thereof, a chimeric antigen receptor (CAR), a T cell receptor (TCR), a gene editing system or a component(s) thereof, an antisense oligonucleotide, a ribozyme, a microRNA, a short hairpin RNA, an RNAi molecule, a Cas protein, e.g., Cas9 protein, and / or a guide RNA (gRNA) specific to a gene of interest.

86. The AAV particle of any one of claims 1-17 or 23-51, the pharmaceutical composition of claim 52, the method of any one of claims 53-71, the use of any one of claims 72-78, or the AAV particle or pharmaceutical composition for use according to any one of claims 79-85, wherein the targeting ligand comprises a bispecific antigen-binding molecule comprising a first antigen-binding domain that binds to EGFRvIII and a second antigenbinding domain that binds to one or more epitopes of a capsid of an AAV particle.

87. The AAV particle, the pharmaceutical composition, the method, the use, or the AAV particle or pharmaceutical composition for use according to claim 86, wherein the bispecific antigen-binding molecule comprises a first antigen binding domain and a second antigen binding domain of a bispecific antigen-binding molecule as set forth in Table 9 herein.

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