Adeno-associated virus vectors and methods of using them for reducing the risk of metastasis, treatment, and prevention

Abstract

The present disclosure provides a recombinant adeno-associated virus (rAAV) vector that expresses a bispecific fusion protein that binds to GD2 and CD3, and methods of using it for reducing the risk of metastasis, prevention, or treatment. The present disclosure also provides an rAAV vector for expressing a bispecific fusion protein. TIFF2025525035000061.tif110170

Description

Technical Field

[0001] Cross-reference This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 391,967, filed Jul. 25, 2022, which is hereby incorporated by reference in its entirety.

[0002] Field The present disclosure generally relates to adeno-associated virus (AAV) vectors for delivering transgene sequences encoding bispecific fusion proteins comprising GD2 binding sites and CD3 binding sites. The present disclosure further relates to methods of killing circulating tumor cells, thereby reducing the risk of cancer and metastatic disease, delaying its onset, and preventing it.

Background Art

[0003] Background Cancer remains a major global health problem and is the second leading cause of death in the United States. Current treatment options for cancer are not effective for all patients and often can be associated with significant adverse side effects.

[0004] Cancer immunotherapy is a promising modality for treatment because it exhibits higher specificity than conventional chemotherapy and can promote the destruction of tumor cells by inducing the patient's own immune system. Bispecific T cell engager proteins are recombinant fusion proteins that have been described in the prior art and bind to both tumor cells and T cells, thereby stimulating the destruction of tumor cells.

[0005] Adeno-associated virus (AAV) has been used as a gene therapy vector to achieve long-term and consistent blood levels for cancer immunotherapy. For example, AAV encoding a bispecific αCD19-αCD3 protein achieved persistence in the bloodstream and antitumor effects for over one year in a CD19+ lymphoma model (Cripe et al., Science Advances (in press)).

[0006] Considering that metastasis is thought to occur from circulating tumor cells that can be considered as the "leukemic compartment" of solid tumors, long-term, persistent immunological pressure targeting cancer can be effectively used to prevent the occurrence of metastasis. Since circulating tumor cells are present outside the immunosuppressive solid tumor microenvironment, they may be more vulnerable to immunotherapy.

[0007] Disialoganglioside GD2 (GD2) is a dilsialoganglioside that is expressed at low levels in normal tissues but overexpressed in a wide range of tumors. GD2 is involved in tumor development and malignant phenotypes through enhancing cell proliferation, motility, migration, adhesion, and invasion, depending on the tumor type. GD2 is highly expressed by almost all neuroblastomas, most melanomas and retinoblastomas, and many Ewing sarcomas. To varying degrees, GD2 is expressed by small cell lung cancer, gliomas, osteosarcomas, and soft tissue sarcomas. SUMMARY OF THE INVENTION

[0008] Summary The present disclosure relates to methods of use and compositions of adeno-associated virus vectors for expressing bispecific fusion proteins for cancer and metastasis risk reduction, prevention, and treatment.

[0009] The present disclosure relates to methods of use and compositions of adeno-associated virus vectors for expressing bispecific fusion proteins for cancer and metastasis risk reduction, prevention, and treatment.

[0010] In some aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector that, from 5' to 3', (a) a 5' AAV inverted terminal repeat (ITR); (b) a promoter; (c) the following: (i) A GD2-binding site comprising a light chain variable region (VL) containing the complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3) sequences of anti-GD2 antibodies SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75 or SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, respectively, and a heavy chain variable region (VH) containing the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; (ii) A linker peptide, and (iii) A CD3-binding site comprising the VH and VL of an anti-CD3 antibody An introduced gene encoding a bispecific fusion protein comprising; (d) A modified RNA stability regulatory element (MRE); and (e) A 3’ AAV ITR Provided is a recombinant adeno-associated virus (rAAV) vector comprising. In some embodiments, the promoter is selected from the group consisting of the chicken β-actin promoter, the elongation factor 1α (EF1α) promoter, the simian virus 40 (SV40) promoter, or the CAG promoter. In some embodiments, the promoter is the CAG promoter. In some embodiments, the promoter comprises a sequence that is at least 95% identical to SEQ ID NO: 66. In some embodiments, the anti-GD2 antibody VL and VH each comprise a sequence that is at least 95% identical to SEQ ID NO: 2 and SEQ ID NO: 1, respectively. In some embodiments, the GD2 binding site is a single-chain variable region fragment (scFv). In some embodiments, the anti-GD2 antibody VL is fused to the anti-GD2 antibody VH using an scFv linker peptide comprising SEQ ID NO: 25. In some embodiments, the anti-GD2 antibody VL is fused to the anti-GD2 antibody VH by an scFv linker peptide comprising the sequence of SEQ ID NO: 20. In some embodiments, the anti-GD2 antibody VL and VH each comprise a sequence that is at least 95% identical to SEQ ID NO: 4 and SEQ ID NO: 3, respectively. In some embodiments, the GD2 binding site is a single-chain variable region fragment (scFv). In some embodiments, the anti-GD2 antibody VL is fused to the anti-GD2 antibody VH by an scFv linker peptide comprising SEQ ID NO: 20. In some embodiments, the GD2 binding site comprises a sequence that is at least 95% identical to SEQ ID NO: 7. In some embodiments, the anti-CD3 antibody VH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively, and the anti-CD3 antibody VL comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively. In some embodiments, the anti-CD3 antibody VH and VL each comprise a sequence that is at least 95% identical to SEQ ID NO: 14 and SEQ ID NO: 15, respectively.In some embodiments, the CD3 binding site is a single-chain variable region fragment (scFv). In some embodiments, the anti-CD3 antibody VH is fused to the anti-CD3 antibody VL by an scFv linker peptide comprising the same sequence as SEQ ID NO: 25. In some embodiments, the CD3 binding site comprises a sequence that is at least 95% identical to SEQ ID NO: 16. In some embodiments, the anti-CD3 antibody VH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 91, SEQ ID NO: 92, and SEQ ID NO: 93, respectively, and the anti-CD3 antibody VL comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively. In some embodiments, the anti-CD3 antibody VH and VL each comprise a sequence that is at least 95% identical to SEQ ID NO: 18 and SEQ ID NO: 19, respectively. In some embodiments, the CD3 binding site is a single-chain variable region fragment (scFv). In some embodiments, the anti-CD3 antibody VH is fused to the anti-CD3 antibody VL by an scFv linker peptide comprising the same sequence as SEQ ID NO: 25. In some embodiments, the CD3 binding site comprises a sequence that is at least 95% identical to SEQ ID NO: 17. In some embodiments, the bispecific fusion protein comprises an N-terminal signal peptide comprising a sequence that is at least 95% identical to SEQ ID NO: 26. In some embodiments, the bispecific fusion protein comprises a sequence that is at least 95% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. In some embodiments, the transgene comprises a sequence that is at least 95% identical to SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45. In some embodiments, the transgene further comprises a regulatory element 5' or 3' of the sequence encoding the bispecific fusion protein.In some embodiments, the regulatory element is located 3' of the sequence encoding the bispecific fusion protein. In some embodiments, the regulatory element is derived from the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and comprises a sequence that is at least 95% identical to SEQ ID NO: 64. In some embodiments, the transgene further comprises a Kozak sequence. In some embodiments, the vector further comprises a polyadenylation sequence 3' of the transgene sequence and 5' of the 3' AAV ITR. In some embodiments, the polyadenylation sequence is a bovine growth hormone (BGH) polyadenylation sequence that is at least 95% identical to SEQ ID NO: 65. In some embodiments, the 3' AAV ITR comprises a sequence that is at least 95% identical to SEQ ID NO: 59. In some embodiments, the vector further comprises an antibiotic resistance gene sequence. In some embodiments, the antibiotic resistance gene is a kanamycin resistance gene. In some embodiments, the vector comprises a sequence that is at least 95% identical to SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 57.

[0011] In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a sequence that is at least 90% identical to SEQ ID NO: 11.

[0012] In some aspects, the disclosure provides a method of reducing the risk of metastatic disease in a patient, comprising administering to the patient an effective amount of the recombinant adeno-associated virus (rAAV) vector or a pharmaceutical formulation thereof described herein.

[0013] In some aspects, the disclosure provides a method of delaying the onset of metastatic disease in a patient, comprising administering to the patient an effective amount of the recombinant adeno-associated virus (rAAV) vector or a pharmaceutical formulation thereof described herein.

[0014] In some aspects, the present disclosure provides a method of preventing metastatic disease in a patient, comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector described herein or a pharmaceutical formulation thereof.

[0015] In some aspects, the present disclosure provides a method of promoting T cell-mediated killing of circulating tumor cells in a patient, comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector described herein or a pharmaceutical formulation thereof. In some embodiments, the rAAV or its pharmaceutical formulation is administered concurrently with treatment of the primary tumor. In some embodiments, treatment of the primary tumor includes surgical resection, radiation therapy, chemotherapy, or immunotherapy.

[0016] In some aspects, the present disclosure provides a method of preventing cancer in a patient having a predisposition to develop GD2+ tumors, comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector described herein or a pharmaceutical formulation thereof.

[0017] In some aspects, the present disclosure provides a method of preventing cancer recurrence in a patient in remission from GD2+ cancer, comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector described herein or a pharmaceutical formulation thereof. In some embodiments, the AAV or its pharmaceutical formulation is administered in combination with a checkpoint inhibitor selected from the group consisting of CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors. In some embodiments, the checkpoint inhibitor is selected from the group consisting of pembrolizumab, ipilimumab, nivolumab, and atezolizumab.

[0018] In some aspects, the present disclosure provides a pharmaceutical formulation comprising a recombinant adeno-associated virus (rAAV) vector described herein and a pharmaceutically acceptable carrier.

[0019] In some aspects, the present disclosure provides a method of reducing the risk of metastatic disease in a patient, comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector or a pharmaceutical formulation thereof. In some aspects, the rAAV vector comprises, from 5' to 3', a 5' AAV inverted terminal repeat (ITR); a promoter; a transgene comprising a sequence encoding a bispecific fusion protein; and a 3' AAV ITR. In some aspects, the bispecific fusion protein comprises a GD2 binding site comprising a variable light chain (VL) and variable heavy chain (VH) of an anti-GD2 antibody; a linker peptide; and a CD3 binding site comprising a VH and VL of an anti-CD3 antibody.

[0020] In some aspects, the present disclosure provides a method of delaying the onset of metastatic disease in a patient, comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector or a pharmaceutical formulation thereof. In some aspects, the rAAV comprises, from 5' to 3', a 5' AAV inverted terminal repeat (ITR); a promoter; a transgene comprising a sequence encoding a bispecific fusion protein; and a 3' AAV ITR. In some aspects, the bispecific fusion protein comprises a GD2 binding site comprising a variable light chain (VL) and variable heavy chain (VH) of an anti-GD2 antibody; a linker peptide; and a CD3 binding site comprising a VH and VL of an anti-CD3 antibody.

[0021] In some aspects, the present disclosure provides a method of preventing metastatic disease in a patient, comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector or a pharmaceutical formulation thereof. In some aspects, the rAAV comprises, from 5' to 3', a 5' AAV inverted terminal repeat (ITR); a promoter; a transgene comprising a sequence encoding a bispecific fusion protein; and a 3' AAV ITR. In some aspects, the bispecific fusion protein comprises a GD2 binding site comprising a variable light chain (VL) and variable heavy chain (VH) of an anti-GD2 antibody; a linker peptide; and a CD3 binding site comprising a VH and VL of an anti-CD3 antibody.

[0022] In some aspects, the present disclosure provides a method for promoting T cell-mediated killing of circulating tumor cells in a patient, comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector or a pharmaceutical formulation thereof. In some aspects, the rAAV comprises, from 5' to 3', a 5' AAV inverted terminal repeat (ITR); a promoter; a transgene comprising a sequence encoding a bispecific fusion protein; and a 3' AAV ITR. In some aspects, the bispecific fusion protein comprises a GD2 binding site comprising the variable light chain (VL) and variable heavy chain (VH) of an anti-GD2 antibody; a linker peptide; and a CD3 binding site comprising the VH and VL of an anti-CD3 antibody.

[0023] In some aspects, the rAAV or a pharmaceutical formulation thereof is administered concurrently with the treatment of the primary tumor. In some aspects, the treatment of the primary tumor comprises surgical resection, radiation therapy, chemotherapy, or immunotherapy.

[0024] In some aspects, the present disclosure provides a method for preventing cancer in a patient having a predisposition to develop GD2+ tumors, comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector or a pharmaceutical formulation thereof. In some aspects, the rAAV comprises, from 5' to 3', a 5' AAV inverted terminal repeat (ITR); a promoter; a transgene comprising a sequence encoding a bispecific fusion protein; and a 3' AAV ITR. In some aspects, the bispecific fusion protein comprises a GD2 binding site comprising the variable light chain (VL) and variable heavy chain (VH) of an anti-GD2 antibody; a linker peptide; and a CD3 binding site comprising the VH and VL of an anti-CD3 antibody.

[0025] In some aspects, the present disclosure provides a method for preventing cancer recurrence in a patient in remission from GD2+ cancer, comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector or a pharmaceutical formulation thereof. In some aspects, the rAAV comprises, from 5' to 3', a 5' AAV inverted terminal repeat (ITR); a promoter; a transgene comprising a sequence encoding a bispecific fusion protein; and a 3' AAV ITR. In some aspects, the bispecific fusion protein comprises a GD2 binding site comprising a variable light chain (VL) and a variable heavy chain (VH) of an anti-GD2 antibody; a linker peptide; and a CD3 binding site comprising a VH and a VL of an anti-CD3 antibody.

[0026] In some aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3', a 5' AAV inverted terminal repeat (ITR); a promoter; a transgene comprising a sequence encoding a bispecific fusion protein having at least 95% identity to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13; and a 3' AAV ITR.

[0027] In some aspects, the rAAV or a pharmaceutical formulation thereof is administered in combination with a checkpoint inhibitor selected from the group consisting of a CTLA-4 inhibitor, a PD-1 inhibitor, and a PD-L1 inhibitor. In some aspects, the checkpoint inhibitor is selected from the group consisting of pembrolizumab, ipilimumab, nivolumab, and atezolizumab.

[0028] In some aspects, the 5' AAV ITR comprises a sequence having at least 95% identity to SEQ ID NO: 58. In some aspects, the 3' AAV ITR comprises a sequence having at least 95% identity to SEQ ID NO: 59.

[0029] In some aspects, the promoter is selected from the chicken β-actin promoter, elongation factor 1α (EF1α) promoter, simian virus 40 (SV40) promoter, and CAG promoter. In some aspects, the promoter is the CAG promoter. In some aspects, the promoter comprises a sequence that is at least 95% identical to SEQ ID NO: 66.

[0030] In some aspects, the anti-GD2 antibody VL of the GD2 binding site has complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) sequences of SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, respectively, and the anti-GD2 antibody VH of the GD2 binding site has CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively. In some aspects, the anti-GD2 antibody VL and VH have sequences that are at least 95% identical to SEQ ID NO: 2 and SEQ ID NO: 1, respectively. In some aspects, the GD2 binding site is a single-chain variable region fragment (scFv). In some aspects, the anti-GD2 antibody VL of the GD2 binding site is fused to the anti-GD2 antibody VH of the GD2 binding site by an scFv linker peptide comprising the sequence of SEQ ID NO: 20. In some aspects, the GD2 binding site comprises a sequence that is at least 95% identical to SEQ ID NO: 5.

[0031] In some aspects, the anti-GD2 antibody VL of the GD2 binding site has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, respectively, and the anti-GD2 antibody VH of the GD2 binding site has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively. In some aspects, the anti-GD2 antibody VL and VH of the GD2 binding site have sequences that are at least 95% identical to SEQ ID NO: 4 and SEQ ID NO: 3, respectively. In some aspects, the GD2 binding site is a single-chain variable region fragment (scFv). In some aspects, the anti-GD2 antibody VL of the GD2 binding site is fused to the anti-GD2 antibody VH of the GD2 binding site by an scFv linker peptide comprising SEQ ID NO: 20. In some aspects, the scFv comprises a sequence that is at least 95% identical to SEQ ID NO: 7.

[0032] In some aspects, the linker peptide comprises a sequence identical to SEQ ID NO: 25 or SEQ ID NO: 20.

[0033] In some aspects, the anti-CD3 antibody VH of the CD3 binding site has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively, and the anti-CD3 antibody VL has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively.

[0034] In some aspects, the VH and VL of the anti-CD3 antibody of the CD3 binding site each contain a sequence that is at least 95% identical to SEQ ID NO: 14 and SEQ ID NO: 15, respectively. In some aspects, the CD3 binding site is a single-chain variable region fragment (scFv). In some aspects, the VL of the anti-CD3 antibody of the CD3 binding site is fused to the VH of the anti-CD3 antibody of the CD3 binding site by an scFv linker peptide containing the same sequence as SEQ ID NO: 25. In some aspects, the CD3 binding site contains a sequence that is at least 95% identical to SEQ ID NO: 16.

[0035] In some aspects, the VH of the anti-CD3 antibody of the CD3 binding site has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 91, SEQ ID NO: 92, and SEQ ID NO: 93, respectively, and the VL of the anti-CD3 antibody has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively.

[0036] In some aspects, the VH and VL of the anti-CD3 antibody of the CD3 binding site each contain a sequence that is at least 95% identical to SEQ ID NO: 18 and SEQ ID NO: 19, respectively. In some aspects, the CD3 binding site is a single-chain variable region fragment (scFv). In some aspects, the VL of the anti-CD3 antibody of the CD3 binding site is fused to the VH of the anti-CD3 antibody of the CD3 binding site by an scFv linker peptide containing the same sequence as SEQ ID NO: 25. In some aspects, the CD3 binding site contains a sequence that is at least 95% identical to SEQ ID NO: 17.

[0037] In some aspects, the bispecific fusion protein contains a sequence that is at least 95% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

[0038] In some embodiments, the bispecific fusion protein has an N-terminal signal peptide comprising a sequence that is at least 95% identical to SEQ ID NO: 26.

[0039] In some embodiments, the transgene comprises a sequence that is at least 95% identical to SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45.

[0040] In some embodiments, the transgene further has a regulatory element 5' or 3' of the sequence encoding the bispecific fusion protein. In some embodiments, the regulatory element is 3' of the sequence encoding the bispecific fusion protein. In some embodiments, the regulatory element is derived from the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and comprises a sequence that is at least 95% identical to SEQ ID NO: 64.

[0041] In some embodiments, the transgene further has a Kozak sequence.

[0042] In some embodiments, the vector further has a polyadenylation sequence 3' of the transgene sequence and 5' of the 3' AAV ITR. In some embodiments, the polyadenylation sequence is a bovine growth hormone (BGH) polyadenylation sequence that is at least 95% identical to SEQ ID NO: 65.

[0043] In some embodiments, the vector further has an antibiotic resistance gene sequence. In some embodiments, the antibiotic resistance gene is a kanamycin resistance gene.

[0044] In some embodiments, the vector comprises a sequence that is at least 95% identical to SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 57.

[0045] In some aspects, the present disclosure provides a recombinant AAV vector that follows any one of the above aspects.

[0046] In some aspects, the present disclosure provides a pharmaceutical formulation comprising a recombinant adeno-associated virus (rAAV) vector that follows any one of the above aspects and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0047]

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Mode for Carrying Out the Invention

[0048] Detailed Description The present disclosure provides a recombinant adeno - associated virus (rAAV) vector comprising a nucleic acid encoding a bispecific fusion protein comprising the heavy - chain variable region (VH) and light - chain variable region (VL) of an anti - GD2 antibody and the VH and VL of an anti - CD3 antibody.

[0049] The present disclosure also provides methods for using the rAAVs described herein to reduce the risk of metastases in patients, prevent them, or treat them.

[0050] To facilitate understanding of the present disclosure, several terms and phrases are defined below.

[0051] As used herein, the terms "a" and "an" mean "one or more" and include the plural form, unless the context is inappropriate.

[0052] The terms "nucleic acid", "nucleotide", or "oligonucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specifically limited, this term encompasses nucleic acids including known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly recited sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0053] The term "gene" can refer to a segment of DNA involved in the production or coding of a polypeptide chain. It can include regions before and after the coding region (leader and trailer), as well as intervening sequences (introns) between individual coding segments (exons).

[0054] A "promoter" is defined as one or more nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes a nucleic acid sequence near the transcription start site. A promoter can also optionally include distal enhancer or repressor elements that can be located thousands of base pairs away from the transcription start site.

[0055] An "regulatory element" as used herein refers to a nucleic acid sequence that can regulate the transcription of a gene (e.g., a transgene) and / or the stability or translation of the transcribed mRNA product. In some embodiments, the regulatory element can regulate the tissue-specific transcription of a gene. The regulatory element can include at least one transcription factor binding site, e.g., a transcription factor binding site for a muscle-specific transcription factor. A regulatory element as used herein increases or enhances promoter-driven gene expression when compared to the transcription of a gene from a promoter alone in the absence of the regulatory element. A regulatory element as used herein can occur at any distance (i.e., proximal or distal) relative to the transgene it regulates. A regulatory element as used herein can include a part of a larger sequence involved in transcriptional control, e.g., a part of a promoter sequence. However, a regulatory element alone is typically not sufficient to initiate transcription by itself and requires the presence of a promoter.

[0056] A nucleic acid is "functionally linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is functionally linked to a coding sequence if it affects the transcription of the coding sequence; or a ribosome binding site is functionally linked to a coding sequence if it is positioned to facilitate translation.

[0057] As used herein, the term "array of equivalent coding potential" refers to a nucleic acid sequence that has functional equivalence to another reference nucleic acid. An array of equivalent coding potential may or may not have the same primary nucleotide sequence. For example, with respect to a reference nucleic acid encoding an expressed polypeptide, an array of equivalent coding potential can functionally encode the same expressed polypeptide and may contain the same primary nucleotide sequence as the reference nucleic acid, or may contain one or more alternative codons compared to the reference nucleic acid. For example, an endogenous nucleic acid sequence encoding a polypeptide can be modified by codon optimization to yield a sequence that encodes the same polypeptide. A codon-optimized sequence can be one in which codons in a polynucleotide encoding a polypeptide have been substituted to modify the activity, expression, and / or stability of the polynucleotide. For example, codon optimization can be used to vary the degree of sequence similarity of an array of equivalent coding potential compared to an endogenous gene sequence while retaining the potential to encode the protein product of the endogenous gene.

[0058] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, these terms encompass amino acid chains of any length, including full-length proteins and functional fragments thereof, wherein the amino acid residues are linked by covalent peptide bonds.

[0059] The terms "variable domain" (e.g., VH domain or VL domain) and "variable region" are used interchangeably and refer to the portions of an antibody or immunoglobulin domain that exhibit variability in sequence and are involved in determining the specificity and binding affinity of a particular antibody. The variability is not evenly distributed throughout the entire variable domain of the antibody; rather, it is concentrated in specific subdomains of each of the heavy and light chain variable regions. These subdomains are called "hypervariable regions" or "complementary determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domain are called "framework" regions (FRM or FR) and provide a scaffold for the six CDRs in three-dimensional space to form the antigen-binding surface.

[0060] As used herein, the terms "complementary" or "complementarity" refer to specific base pairing between nucleotides or nucleic acids. Complementary nucleotides are generally A and T (or A and U), and G and C.

[0061] As used herein, the term "transgene" refers to an exogenous gene artificially introduced into the genome of a cell, or an endogenous gene artificially introduced into a non-native locus in the genome of a cell. A transgene can refer to a segment of DNA involved in the production or coding of a polypeptide chain. A transgene can include regions (leaders and trailers) before and after the coding region, as well as intervening sequences (introns) between individual coding segments (exons).

[0062] As used herein, the terms "introduce" or "deliver" in the context of a nucleic acid, e.g., an AAV vector, refer to the transfer of a nucleic acid from outside the cell into the interior of the cell, e.g., a muscle cell. In some cases, introduction refers to the transfer of a nucleic acid from outside the cell into the interior of the cell nucleus. Various methods of such transfer are contemplated, including, but not limited to, electroporation, contact with nanowires or nanotubes, receptor-mediated internalization, transfer via cell-penetrating peptides, liposome-mediated transfer, etc.

[0063] As used herein, the terms "packaged" or "encapsulated" refer to the formation of AAV particles by including an AAV vector within a viral capsid.

[0064] The terms "substantial identity" or "substantially identical", when used in the context of polynucleotide or polypeptide sequences, refer to a sequence having at least 60% sequence identity to a reference sequence. Alternatively, the percent identity can be any integer from 60% to 100%. Exemplary embodiments, as described below, when compared to a reference sequence using the programs described herein, preferably BLAST, using standard parameters, include at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. One of ordinary skill in the art will recognize that these values can be appropriately adjusted to determine the corresponding identity of the proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[0065] For sequence comparison, typically one sequence functions as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, sub-sequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters.

[0066] Algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating a search to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence as long as the cumulative alignment score increases. The cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatch residue; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the word hits in each direction stops when the cumulative alignment score decreases by an amount X from its reached maximum value; when the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, a word size (W) of 28, an expectation value (E) of 10, M = 1, N = -2, and a comparison of both strands.In the case of amino acid sequences, the BLASTP program, by default, uses a word size (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0067] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that a match between two nucleotide sequences or amino acid sequences occurs by chance. For example, in the comparison of a test nucleic acid with a reference nucleic acid, when the minimum total probability is less than about 0.01, more preferably less than about 10 -5 less, and most preferably less than about 10 -20 less, the nucleic acid is considered to be similar to the reference sequence.

[0068] The terms "recipient", "individual", "subject", "host", and "patient" are used interchangeably herein and, in some embodiments, refer to any mammalian subject, particularly a human, for whom diagnosis, treatment, or therapy is desired. "Mammal" for treatment purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. In some embodiments, the mammal is a human. None of these terms require medical practitioner monitoring and / or a diagnosis of cancer or an ongoing cancer treatment.

[0069] As used herein, the term "efficient delivery" or "delivering efficiently" refers to the administration of a recombinant adeno-associated virus vector encoding a transgene that results in the expression of the transgene in a desired cell or tissue.

[0070] As used herein, the term "effective amount" refers to the amount of a substance (e.g., a recombinant adeno-associated virus of the present disclosure) sufficient to produce a beneficial or desired result (e.g., expression of a protein, or a desired prophylactic or therapeutic effect). The effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treating" includes any effect, e.g., alleviation, mitigation, modulation, improvement or elimination, that results in the amelioration of a condition, disease, disorder, etc., or improvement of their symptoms.

[0071] Throughout this specification, when a composition is described as having, comprising, or including a particular component, or when a process and method are described as having, comprising, or including a particular step, it is further contemplated that there exist compositions of the present disclosure consisting essentially of, or consisting of, the recited components, and that there exist processes and methods according to the present disclosure consisting essentially of, or consisting of, the recited processing steps.

[0072] 1. Recombinant adeno-associated virus (AAV) vector As used herein, a "recombinant adeno-associated virus (rAAV) vector" refers to a vector (e.g., a nucleic acid vector) comprising a promoter and one or more transgenes, or polynucleotides of interest, flanked by AAV inverted terminal repeat (ITR) sequences. The rAAV vectors described herein can be replicated and packaged into viral particles when introduced into a host cell further comprising one or more vectors encoding the rep and cap gene products.

[0073] Inverted terminal repeat The inverted terminal repeat (ITR) is a 145-nucleotide palindromic sequence adjacent to the transgene. The 5’ and 3’ ITRs of the recombinant adeno-associated virus (rAAV) vector are necessary for both the integration of the transgene into the host cell genome (e.g., human chromosome 19) and the encapsulation into AAV particles.

[0074] In some embodiments, the rAAV vectors of the present disclosure include ITR sequences derived from any one AAV serotype, such as AAVrh.74, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13. In preferred embodiments, the recombinant AAV vectors disclosed herein include the 5’ and 3’ ITR sequences of AAV2. In some embodiments, the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, or derivatives thereof. In some embodiments, the recombinant AAV vectors disclosed herein include the AAV2 5’ and 3’ ITR sequences. In some embodiments, the recombinant AAV vectors disclosed herein include the AAV8 5’ and 3’ ITR sequences.

[0075] In some embodiments, the recombinant AAV vectors described herein comprise a 5' AAV2 ITR having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 58 (see Table 1A). In some embodiments, the 5' AAV2 ITR comprises a sequence having at least about 80% identity to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises a sequence having at least about 85% identity to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises a sequence having at least about 90% identity to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises a sequence having at least about 95% identity to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises a sequence having at least about 96% identity to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises a sequence having at least about 97% identity to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises a sequence having at least about 98% identity to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises a sequence having at least about 99% identity to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises a sequence having 100% identity to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR consists of SEQ ID NO: 58.

[0076] In some embodiments, the recombinant AAV vectors described herein comprise a 3’ AAV2 ITR having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 59 (see Table 1A). In some embodiments, the 3’ AAV2 ITR comprises a sequence having at least about 80% identity to SEQ ID NO: 59. In some embodiments, the 3’ AAV2 ITR comprises a sequence having at least about 85% identity to SEQ ID NO: 59. In some embodiments, the 3’ AAV2 ITR comprises a sequence having at least about 90% identity to SEQ ID NO: 59. In some embodiments, the 3’ AAV2 ITR comprises a sequence having at least about 95% identity to SEQ ID NO: 59. In some embodiments, the 3’ AAV2 ITR comprises a sequence having at least about 96% identity to SEQ ID NO: 59. In some embodiments, the 3’ AAV2 ITR comprises a sequence having at least about 97% identity to SEQ ID NO: 59. In some embodiments, the 3’ AAV2 ITR comprises a sequence having at least about 98% identity to SEQ ID NO: 59. In some embodiments, the 3’ AAV2 ITR comprises a sequence having at least about 99% identity to SEQ ID NO: 59. In some embodiments, the 3’ AAV2 ITR comprises a sequence having 100% identity to SEQ ID NO: 59. In some embodiments, the 3’ AAV2 ITR comprises SEQ ID NO: 59. In some embodiments, the 3’ AAV2 ITR consists of SEQ ID NO: 59.

[0077] (Table 1A) AAV ITR Sequences TIFF2025525035000002.tif45160

[0078] Promoter Promoters drive the expression of the AAV vector-introduced gene and are typically located upstream (or 5') of the introduced gene whose expression they regulate.

[0079] In some embodiments, the recombinant AAV vectors of the disclosure include mammalian promoters, such as human, non-human primate (e.g., cynomolgus monkey), mouse, horse, cow, pig, cat, and dog promoters. In some embodiments, the recombinant AAV vectors disclosed herein include strong and constitutively active promoters to drive high-level expression of the introduced gene. For example, the promoter is a CAG promoter (cytomegalovirus immediate enhancer fused with the chicken β-actin promoter), a cytomegalovirus (CMV) promoter / enhancer, an elongation factor 1α (EF1α) promoter, a simian virus 40 (SV40) promoter, or a chicken β-actin promoter.

[0080] In some embodiments, the promoter described herein comprises a CAG promoter having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 66. In some embodiments, the CAG promoter comprises a sequence having at least about 80% identity to SEQ ID NO: 66. In some embodiments, the CAG promoter comprises a sequence having at least about 85% identity to SEQ ID NO: 66. In some embodiments, the CAG promoter comprises a sequence having at least about 90% identity to SEQ ID NO: 66. In some embodiments, the CAG promoter comprises a sequence having at least about 95% identity to SEQ ID NO: 66. In some embodiments, the CAG promoter comprises a sequence having at least about 96% identity to SEQ ID NO: 66. In some embodiments, the CAG promoter comprises a sequence having at least about 97% identity to SEQ ID NO: 66. In some embodiments, the CAG promoter comprises a sequence having at least about 98% identity to SEQ ID NO: 66. In some embodiments, the CAG promoter comprises a sequence having at least about 99% identity to SEQ ID NO: 66. In some embodiments, the CAG promoter comprises a sequence having 100% identity to SEQ ID NO: 66.

[0081] CAG promoter sequence: TIFF2025525035000003.tif189134

[0082] SV40 intron In some embodiments, the recombinant AAV vector of the disclosure comprises an SV40 intron. The SV40 intron is a regulatory element commonly used in gene therapy vectors and enhances the translation and stability of the expressed RNA transcript.

[0083] In some embodiments, the SV40 intron is downstream (i.e., 3') of the promoter and upstream (i.e., 5') of the transgene. In other embodiments, the SV40 intron can be downstream (i.e., 3') of the transgene.

[0084] In some embodiments, the SV40 intron comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises SEQ ID NO: 67. In some embodiments, the SV40 intron consists of SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence having at least about 80% identity to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence having at least about 85% identity to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence having at least about 90% identity to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence having at least about 95% identity to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence having at least about 96% identity to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence having at least about 97% identity to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence having at least about 98% identity to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence having at least about 99% identity to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence having 100% identity to SEQ ID NO: 67.

[0085] SV40 intron sequence: TIFF2025525035000004.tif19132

[0086] Polyadenylation sequence In some embodiments, the recombinant AAV vectors of the disclosure include a sequence encoding a polyadenylation sequence, such as the bovine growth hormone (BGH) polyadenylation sequence (SEQ ID NO: 65) or the SV40 polyadenylation sequence (SEQ ID NO: 68). Polyadenylation sequences are nucleic acid elements commonly used in gene therapy vectors that assist in the transport of RNA from the nucleus, the translation of RNA, and the stability of RNA.

[0087] In some embodiments, the recombinant AAV vector of the disclosure comprises a sequence encoding a BGH poly(A) tail having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 65. In some embodiments, the BGH poly(A) tail comprises a sequence having at least about 80% identity to SEQ ID NO: 65. In some embodiments, the BGH poly(A) tail comprises a sequence having at least about 85% identity to SEQ ID NO: 65. In some embodiments, the BGH poly(A) tail comprises a sequence having at least about 90% identity to SEQ ID NO: 65. In some embodiments, the BGH poly(A) tail comprises a sequence having at least about 95% identity to SEQ ID NO: 65. In some embodiments, the BGH poly(A) tail comprises a sequence having at least about 96% identity to SEQ ID NO: 65. In some embodiments, the BGH poly(A) tail comprises a sequence having at least about 97% identity to SEQ ID NO: 65. In some embodiments, the BGH poly(A) tail comprises a sequence having at least about 98% identity to SEQ ID NO: 65. In some embodiments, the BGH poly(A) tail comprises a sequence having at least about 99% identity to SEQ ID NO: 65. In some embodiments, the BGH poly(A) tail comprises a sequence having 100% identity to SEQ ID NO: 65. In some embodiments, the BGH poly(A) tail comprises a sequence according to SEQ ID NO: 65. In some embodiments, the BGH poly(A) tail consists of a sequence according to SEQ ID NO: 65.

[0088] In some embodiments, the recombinant AAV vector of the present disclosure comprises a sequence encoding an SV40 poly(A) tail having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 68. In some embodiments, the SV40 poly(A) tail comprises a sequence having at least about 80% identity to SEQ ID NO: 68. In some embodiments, the SV40 poly(A) tail comprises a sequence having at least about 85% identity to SEQ ID NO: 68. In some embodiments, the SV40 poly(A) tail comprises a sequence having at least about 90% identity to SEQ ID NO: 68. In some embodiments, the SV40 poly(A) tail comprises a sequence having at least about 95% identity to SEQ ID NO: 68. In some embodiments, the SV40 poly(A) tail comprises a sequence having at least about 96% identity to SEQ ID NO: 68. In some embodiments, the SV40 poly(A) tail comprises a sequence having at least about 97% identity to SEQ ID NO: 68. In some embodiments, the SV40 poly(A) tail comprises a sequence having at least about 98% identity to SEQ ID NO: 68. In some embodiments, the SV40 poly(A) tail comprises a sequence having at least about 99% identity to SEQ ID NO: 68. In some embodiments, the SV40 poly(A) tail comprises a sequence having 100% identity to SEQ ID NO: 68. In some embodiments, the SV40 poly(A) tail comprises a sequence according to SEQ ID NO: 68. In some embodiments, the SV40 poly(A) tail consists of a sequence according to SEQ ID NO: 68.

[0089] BGH poly(A) tail sequence: TIFF2025525035000005.tif29128

[0090] SV40 poly(A) tail sequence: TIFF2025525035000006.tif14128

[0091] Enhancer In some embodiments, the recombinant AAV vector of the present disclosure comprises one or more enhancer sequences. The enhancer sequence can increase the transcription level of the transgene, for example, by functioning as a binding site for transcription factors and co-regulatory factors that assist DNA looping and recruitment of the transcriptional machinery to the promoter.

[0092] In some embodiments, the enhancer is downstream (i.e., 3') of the 5' ITR and upstream (i.e., 5') of the promoter. In some embodiments, the enhancer is downstream (i.e., 3') of the promoter and upstream (i.e., 5') of the transgene. In some embodiments, the enhancer is downstream (i.e., 3') of the transgene and upstream (i.e., 5') of the 3' UTR.

[0093] Antibiotic resistance gene In some embodiments, the recombinant AAV vector of the present disclosure comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, zeocin, or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin.

[0094] In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 85% (e.g., 85%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 97. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 97. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 97. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 97. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 97. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 97. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 97. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having the nucleic acid sequence of SEQ ID NO: 97.

[0095] In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 85% (e.g., 85%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 122. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 122. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 122. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 122. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 122. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 122. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 122. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having the nucleic acid sequence of SEQ ID NO: 122.

[0096] Kanamycin Variant 1 TIFF2025525035000007.tif97128

[0097] Kanamycin Variant 2 TIFF2025525035000008.tif97128

[0098] Kozak sequence In some embodiments, the recombinant AAV vectors of the present disclosure include a Kozak sequence. In some embodiments, the Kozak sequence is the AAV2 Kozak sequence. In some embodiments, the Kozak sequence is the AAV8 Kozak sequence. In some embodiments, the Kozak sequence is the AAV-rh74 Kozak sequence. Exemplary Kozak sequences are found in Table 1B below.

[0099] (Table 1B) Exemplary Kozak sequences TIFF2025525035000009.tif127128

[0100] In some embodiments, the Kozak sequence includes a sequence having at least 85% (e.g., 85%, 90%, 95%, 97%, 98%, or 99%) sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 98-121. In some embodiments, the Kozak sequence includes a sequence having at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 98-121. In some embodiments, the Kozak sequence includes a sequence having at least 90% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 98-121. In some embodiments, the Kozak sequence includes a sequence having at least 95% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 98-121. In some embodiments, the Kozak sequence includes a sequence having at least 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 98-121. In some embodiments, the Kozak sequence includes a sequence having at least 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 98-121. In some embodiments, the Kozak sequence includes a sequence having at least 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 98-121. In some embodiments, the Kozak sequence includes a sequence having any one of the nucleic acid sequences of SEQ ID NOs: 98-121.

[0101] αGD2-αCD3 transgene In some embodiments, the transgene of the present disclosure is a nucleic acid sequence encoding a bispecific fusion protein having a GD2 binding site and a CD3 binding site. In some embodiments, the GD2 binding site comprises the heavy chain variable region (VH) and the light chain variable region (VL) of an anti-GD2 antibody, and the CD3 binding site comprises the VH and VL of an anti-CD3 antibody.

[0102] In some embodiments, the transgene may be integrated into the genome of the cell or expressed episomally.

[0103] GD2 binding site The GD2 binding site may comprise a polypeptide or a complex of two or more polypeptides that specifically binds to a dicyclo ganglioside having the following structure. TIFF2025525035000010.tif58128

[0104] In some embodiments, the GD2 binding site comprises a heavy chain variable region (VH) and a light chain variable region (VL). Table 2A lists the VH and VL domains of anti-GD2 antibodies, as well as their corresponding complementarity-determining regions (CDRs), that can specifically bind to GD2 in combination. Table 2B lists the corresponding nucleotide sequences of the VH and VL domains of the anti-GD2 antibodies.

[0105] (Table 2A) αGD2 VH / VL and CDR amino acid sequences TIFF2025525035000011.tif172170

[0106] (Table 2B) αGD2 VH / VL nucleotide sequences TIFF2025525035000012.tif167169

[0107] In some embodiments, the GD2 binding site comprises VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences selected from the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences listed in Table 2A, determined under Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), the IMGT unique numbering scheme, Chothia (see, for example, Chothia C & Lesk A M, (1987), J. Mol. Biol. 196: 901-917), MacCallum (see MacCallum R M et al., (1996) J. Mol. Biol. 262: 732-745), or any other CDR determination method known in the art.

[0108] Unless otherwise indicated, the CDR sequences provided in Table 2A are determined under the Kabat numbering scheme.

[0109] In some embodiments, the GD2 binding site comprises: (i) VL CDR1 comprising the amino acid sequence of SEQ ID NO: 73; (ii) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 74; (iii) VL CDR3 comprising the amino acid sequence of SEQ ID NO: 75; (iv) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 70; (v) VH CDR2 comprising the amino acid sequence of SEQ ID NO: 71; and (vi) VH CDR3 comprising the amino acid sequence of SEQ ID NO: 72.

[0110] In some embodiments, the GD2 binding site comprises: (i) VL CDR1 comprising the amino acid sequence of SEQ ID NO: 79; (ii) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 80; (iii) VL CDR3 comprising the amino acid sequence of SEQ ID NO: 81; (iv) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 76; (v) VH CDR2 comprising the amino acid sequence of SEQ ID NO: 77; and (vi) VH CDR3 comprising the amino acid sequence of SEQ ID NO: 78.

[0111] Table 2A further lists the amino acid sequences of exemplary VH and VL domains that can specifically bind to GD2 in combination. In some embodiments, the GD2 binding site of the present disclosure comprises a VH and a VL having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences listed in Table 2A. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3; and a VL comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3; and a VL comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3; and a VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3; and a VL comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 4.In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3; and a VL comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3; and a VL comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3; and a VL comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3; and a VL comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the GD2 binding site comprises a VH comprising the amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3; and a VL comprising the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 4.

[0112] In some embodiments, the GD2 binding site of the present disclosure comprises (i) a VH having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 1; and (ii) a VL having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 2. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 1; and a VL comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 2. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 1; and a VL comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 2. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 1; and a VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 2. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 1; and a VL comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 2.In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 1; and a VL comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 2. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 1; and a VL comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 2. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 1; and a VL comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 2. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 1; and a VL comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 2. In some embodiments, the GD2 binding site comprises a VH comprising the amino acid sequence according to SEQ ID NO: 1; and a VL comprising the amino acid sequence according to SEQ ID NO: 2.

[0113] In some embodiments, the GD2 binding site of the present disclosure comprises: (i) a VH having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 3; and (ii) a VL having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 4. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 3; and a VL comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 4. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 3; and a VL comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 4. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 3; and a VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 4. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 3; and a VL comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 4.In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 3; and a VL comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 4. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 3; and a VL comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 4. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 3; and a VL comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 4. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 3; and a VL comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 4. In some embodiments, the GD2 binding site comprises a VH comprising the amino acid sequence according to SEQ ID NO: 3; and a VL comprising the amino acid sequence according to SEQ ID NO: 4.

[0114] In some embodiments, the GD2 binding site includes, but is not limited to, a single-chain variable region fragment (scFv), an antibody, a Fab, a Fab’, an F(ab’)2, a minibody, or a nanobody (VHH). For example, in some embodiments, the bispecific fusion protein of the disclosure comprises an scFv polypeptide that specifically binds to GD2.

[0115] In some embodiments, the GD2 binding site of the present disclosure is in scFv format. In some embodiments, the GD2-binding scFv of the present disclosure comprises an scFv linker polypeptide that functionally connects the VH domain and the VL domain. For example, the GD2-binding scFv comprises, from the N-terminus to the C-terminus, the VL domain of an anti-GD2 antibody, the scFv linker polypeptide, and the VH domain of an anti-GD2 antibody. In other embodiments, the GD2-binding scFv comprises, from the N-terminus to the C-terminus, the VH domain of an anti-GD2 antibody, the scFv linker polypeptide, and the VL domain of an anti-GD2 antibody.

[0116] In some embodiments, the scFv linker polypeptide comprises a sequence selected from the linker sequences in Table 3A.

[0117] (Table 3A) scFv linker peptide sequences TIFF2025525035000013.tif57136

[0118] In some embodiments, the GD2-binding scFv of the present disclosure comprises a spacer peptide fused to the N-terminus of the scFv linker peptide at the C-terminus of the VH region or at the C-terminus of the VL domain. In some embodiments, the spacer peptide comprises a sequence selected from the spacer sequences listed in Table 3B.

[0119] (Table 3B) Spacer peptide sequences TIFF2025525035000014.tif37136

[0120] Table 4A lists the amino acid sequences of exemplary GD2-binding scFvs. In some embodiments, the bispecific fusion proteins of the present disclosure include a GD2-binding scFv having a sequence that is at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the scFv sequences listed in Table 4A. Table 4B lists the corresponding nucleotide sequences of exemplary GD2-binding scFvs.

[0121] (Table 4A) GD2-binding scFv amino acid sequences TIFF2025525035000015.tif133166* Italic with underline The letters indicate the scFv linker sequence.

[0122] (Table 4B) GD2-binding scFv nucleotide sequences TIFF2025525035000016.tif171167* With underline The letters indicate the scFv linker sequence.

[0123] In some embodiments, the bispecific fusion protein of the present disclosure comprises an scFv that specifically binds to GD2 and has an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 5. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 85% identity to SEQ ID NO: 5. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 90% identity to SEQ ID NO: 5. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 95% identity to SEQ ID NO: 5. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 96% identity to SEQ ID NO: 5. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 97% identity to SEQ ID NO: 5. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 98% identity to SEQ ID NO: 5. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 99% identity to SEQ ID NO: 5. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having 100% identity to SEQ ID NO: 5.

[0124] In some embodiments, the scFv that specifically binds to GD2 comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 85% identity to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 90% identity to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 95% identity to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 96% identity to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 97% identity to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 98% identity to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 99% identity to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having 100% identity to SEQ ID NO: 6.

[0125] In some embodiments, the bispecific fusion protein of the present disclosure comprises a scFv that specifically binds to GD2 and that comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 7 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%). In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 85% identity to SEQ ID NO: 7. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 90% identity to SEQ ID NO: 7. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 95% identity to SEQ ID NO: 7. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 96% identity to SEQ ID NO: 7. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 97% identity to SEQ ID NO: 7. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 98% identity to SEQ ID NO: 7. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 99% identity to SEQ ID NO: 7. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having 100% identity to SEQ ID NO: 7.

[0126] In some embodiments, the bispecific fusion protein of the present disclosure comprises a scFv that specifically binds to GD2 and has an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 8. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 85% identity to SEQ ID NO: 8. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 90% identity to SEQ ID NO: 8. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 95% identity to SEQ ID NO: 8. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 96% identity to SEQ ID NO: 8. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 97% identity to SEQ ID NO: 8. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 98% identity to SEQ ID NO: 8. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having at least about 99% identity to SEQ ID NO: 8. In some embodiments, the scFv that specifically binds to GD2 comprises a sequence having 100% identity to SEQ ID NO: 8.

[0127] CD3 binding site The bispecific fusion proteins of the present disclosure can include a polypeptide or a complex of two or more polypeptides that specifically binds to CD3 on the surface of T cells. In some embodiments, the bispecific fusion proteins of the present disclosure bind to CD3 expressed on mature T lymphocytes, such as αβ T cells, γδ T cells, NK-T cells, mucosal-associated invariant T (MAIT) cells, and phenotypic subsets thereof. In some embodiments, binding of CD3 induces activation of T cells when cross-linked to GD2.

[0128] As used herein, the CD3 binding site is, in some embodiments, a polypeptide or a complex of two or more polypeptides that specifically binds to CD3 (SEQ ID NO: 69). For example, the CD3 binding site binds to the CD3ε chain.

[0129] CD3 sequence (ε chain): TIFF2025525035000017.tif27144

[0130] In some embodiments, the CD3 binding site comprises a VH (VH) and a light chain variable region (VL). Table 5A lists the VH and VL regions of anti-CD3 antibodies, and their corresponding complementarity-determining regions (CDRs), that can specifically bind to CD3 in combination. Table 5B lists the corresponding nucleotide sequences of the VH and VL regions of anti-CD3 antibodies.

[0131] (Table 5A) αCD3 VH / VL sequences and CDRs TIFF2025525035000018.tif177160

[0132] (Table 5B) αCD3 VH / VL nucleotide sequences TIFF2025525035000019.tif40160TIFF2025525035000020.tif245160TIFF2025525035000021.tif138160

[0133] In some embodiments, the CD3 binding site comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences listed in Table 5A, determined under the IMGT unique numbering scheme, Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (e.g., see Chothia C & Lesk A M, (1987), J. Mol. Biol. 196: 901-917), MacCallum (see MacCallum R M et al., (1996) J. Mol. Biol. 262: 732-745), or any other CDR determination method known in the art.

[0134] Unless otherwise indicated, the CDR sequences provided in Table 5A are determined under the Kabat numbering scheme.

[0135] In some embodiments, the CD3 binding site comprises: (i) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 85; (ii) VH CDR2 comprising the amino acid sequence of SEQ ID NO: 86; (iii) VH CDR3 comprising the amino acid sequence of SEQ ID NO: 87; (iv) VL CDR1 comprising the amino acid sequence of SEQ ID NO: 88; (v) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 89; and (vi) VL CDR3 comprising the amino acid sequence of SEQ ID NO: 90.

[0136] In some embodiments, the CD3 binding site comprises: (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 91; (ii) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 92; (iii) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 93; (iv) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 94; (v) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 95; and (vi) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 96.

[0137] Table 5A further lists the amino acid sequences of exemplary VH and VL domains that can specifically bind to CD3 in combination. In some embodiments, the CD3 binding site of the present disclosure comprises a VH and a VL having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH and VL sequences listed in Table 5A.

[0138] In some embodiments, the CD3 binding site of the present disclosure comprises (i) a VH having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 14; and (ii) a VL having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 15. In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 14; and a VL comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 15. In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 14; and a VL comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 15. In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 14; and a VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 15. In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 14; and a VL comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 15.In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 14; and a VL comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 15. In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 14; and a VL comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 15. In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 14; and a VL comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 15. In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 14; and a VL comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 15. In some embodiments, the CD3 binding site comprises a VH comprising the amino acid sequence according to SEQ ID NO: 14; and a VL comprising the amino acid sequence according to SEQ ID NO: 15.

[0139] In some embodiments, the CD3 binding site of the present disclosure comprises (i) a VH having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18; and (ii) a VL having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 19. In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 18; and a VL comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 19. In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 18; and a VL comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 19. In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 18; and a VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 19. In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 18; and a VL comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 19.In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 18; and a VL comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 19. In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 18; and a VL comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 19. In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 18; and a VL comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 19. In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 18; and a VL comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 19. In some embodiments, the CD3 binding site comprises a VH comprising the amino acid sequence according to SEQ ID NO: 18; and a VL comprising the amino acid sequence according to SEQ ID NO: 19.

[0140] In some embodiments, the CD3 binding site comprises, but is not limited to, a single-chain variable region fragment (scFv), an antibody, a Fab, a Fab’, an F(ab’)2, a minibody, or a nanobody (VHH). For example, in some embodiments, the bispecific fusion protein of the present disclosure comprises an scFv polypeptide that specifically binds to CD3.

[0141] In some embodiments, the CD3 binding site of the present disclosure is in scFv format. In some embodiments, the CD3 binding scFv of the present disclosure comprises an scFv linker polypeptide that functionally connects the VH domain and the VL domain. For example, in some embodiments, the CD3 binding scFv comprises, from the N-terminus to the C-terminus, the VL domain of an anti-CD3 antibody, an scFv linker polypeptide, and the VH domain of an anti-CD3 antibody. In other embodiments, the CD3 binding scFv comprises, from the N-terminus to the C-terminus, the VH domain of an anti-CD3 antibody, an scFv linker polypeptide, and the VL domain of an anti-CD3 antibody.

[0142] In some embodiments, the scFv linker polypeptide comprises a sequence selected from the linker sequences in Table 3A.

[0143] Table 6A lists the amino acid sequences of exemplary CD3 binding scFvs. In some embodiments, the bispecific fusion protein of the present disclosure comprises a CD3 binding scFv having a sequence that is at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the scFv sequences listed in Table 6A. Table 6B lists the corresponding nucleotide sequences of exemplary CD3 binding scFvs.

[0144] (Table 6A) αCD3 scFv Amino Acid Sequences TIFF2025525035000022.tif65160* With underline The letters indicate the scFv linker sequence.

[0145] (Table 6B) αCD3 scFv Nucleotide Sequences TIFF2025525035000023.tif79160TIFF2025525035000024.tif245160TIFF2025525035000025.tif128160* With underline The letters indicate the scFv linker sequence.

[0146] In some embodiments, the bispecific fusion protein of the present disclosure comprises a scFv that specifically binds to CD3 and that comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 16. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 85% identity to SEQ ID NO: 16. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 90% identity to SEQ ID NO: 16. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 95% identity to SEQ ID NO: 16. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 96% identity to SEQ ID NO: 16. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 97% identity to SEQ ID NO: 16. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 98% identity to SEQ ID NO: 16. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 99% identity to SEQ ID NO: 16. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having 100% identity to SEQ ID NO: 16.

[0147] In some embodiments, the bispecific fusion protein of the present disclosure comprises a scFv that specifically binds to CD3 and comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 17. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 85% identity to SEQ ID NO: 17. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 90% identity to SEQ ID NO: 17. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 95% identity to SEQ ID NO: 17. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 96% identity to SEQ ID NO: 17. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 97% identity to SEQ ID NO: 17. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 98% identity to SEQ ID NO: 17. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 99% identity to SEQ ID NO: 17. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having 100% identity to SEQ ID NO: 17.

[0148] Exemplary αGD2-αCD3 bispecific fusion protein Enumerated below are examples of the bispecific fusion proteins of the present disclosure that comprise a GD2 binding site fused to a CD3 binding site via a linker peptide.

[0149] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences selected from the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences listed in Table 2A; (ii) a linker peptide comprising a sequence selected from Table 7; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences listed in Table 5A. In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences listed in Table 5A; (ii) a linker peptide comprising a sequence selected from Table 7; and (iii) a GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences selected from the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences listed in Table 2A. The order of the GD2 binding site and the CD3 binding site is not limited. For example, the GD2 binding site is at the amino terminus of the bispecific fusion protein and the CD3 binding site is at the carboxy terminus of the bispecific fusion protein. In some embodiments, the CD3 binding site is at the amino terminus of the bispecific fusion protein and the GD2 binding site is at the carboxy terminus of the bispecific fusion protein.

[0150] (Table 7) Linker Peptide TIFF2025525035000026.tif27136

[0151] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively.

[0152] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively.

[0153] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively.

[0154] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively.

[0155] In some embodiments, the bispecific fusion protein of the disclosure comprises: (i) a GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences respectively listed in Table 2A; (ii) a linker peptide comprising a sequence selected from Table 7; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences listed in Table 5A.

[0156] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 2 and SEQ ID NO: 1, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0157] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 2 and SEQ ID NO: 1, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0158] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 4 and SEQ ID NO: 3, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0159] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a GD2 binding site comprising VL and VH domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL and VH domain sequences corresponding to SEQ ID NO: 4 and SEQ ID NO: 3, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising VH and VL domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH and VL domain sequences corresponding to SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0160] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a GD2 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the scFv sequences listed in Table 4A; (ii) a linker peptide comprising a sequence selected from Table 7; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the scFv sequences listed in Table 6A.

[0161] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a GD2 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 5; (ii) a linker peptide comprising the sequence of SEQ ID NO: 20; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 17.

[0162] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a GD2 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 5; (ii) a linker peptide comprising the sequence of SEQ ID NO: 25; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 16.

[0163] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a GD2 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 7; (ii) a linker peptide comprising the sequence of SEQ ID NO: 20; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 17.

[0164] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a GD2 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 7; (ii) a linker peptide comprising the sequence of SEQ ID NO: 20; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 16.

[0165] In some embodiments, the bispecific fusion protein of the present disclosure comprises a spacer peptide fused to the N-terminus of an scFv linker peptide at the C-terminus of the VH domain of the GD2 scFv, at the C-terminus of the VL domain of the GD2 scFv, at the C-terminus of the VH domain of the CD3 scFv, and / or at the C-terminus of the VL domain of the CD3 scFv. In some embodiments, the spacer peptide comprises a sequence selected from the spacer sequences listed in Table 3B.

[0166] In some embodiments, the bispecific fusion protein of the present disclosure has an amino acid sequence corresponding to the sequences listed in Table 8A. Table 8B lists the corresponding nucleotide sequences of the bispecific fusion protein.

[0167] (Table 8A) Bispecific fusion protein amino acid sequences TIFF2025525035000027.tif69160TIFF2025525035000028.tif254160* Italic with underline The letters indicate the scFv linker sequence; With underline The letters indicate the linker sequence; the bold letters indicate the spacer sequence.

[0168] (Table 8B) Bispecific fusion protein nucleotide sequences TIFF2025525035000029.tif197160TIFF2025525035000030.tif245160TIFF2025525035000031.tif245160TIFF2025525035000032.tif245160

[0169] In addition to the sequences presented in Table 8A, the bispecific fusion protein of the present disclosure may further comprise a signal peptide fused to the N-terminus. It is understood that the mature form of the protein with the signal peptide cleaved is expressed.

[0170] For example, the bispecific fusion protein of the present disclosure further comprises a signal peptide comprising an amino acid sequence that is at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 26.

[0171] Signal peptide sequence: TIFF2025525035000033.tif12128

[0172] In some embodiments, the bispecific fusion protein of the present disclosure has an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 9. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 9. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 9. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 9. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 9. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 9. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 9. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 9. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to SEQ ID NO: 9.

[0173] In some embodiments, the bispecific fusion protein of the present disclosure has an amino acid sequence with at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 10. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 10. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 10. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 10. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 10. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 10. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 10. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 10. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to SEQ ID NO: 10.

[0174] In some embodiments, the bispecific fusion protein of the present disclosure has an amino acid sequence with at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 11. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 11. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 11. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 11. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 11. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 11. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 11. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 11. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to SEQ ID NO: 11.

[0175] In some embodiments, the bispecific fusion protein of the present disclosure has an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 12. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 12. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 12. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 12. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 12. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 12. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 12. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 12. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to SEQ ID NO: 12.

[0176] In some embodiments, the bispecific fusion protein of the present disclosure has an amino acid sequence with at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 13. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 13. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 13. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 13. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 13. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 13. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 13. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 13. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to SEQ ID NO: 13.

[0177] Exemplary rAAV vectors In some embodiments, the rAAV vectors of the present disclosure comprise a transgene nucleotide sequence corresponding to any one of the sequences listed in Table 8B. In some embodiments, the transgene comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 41-45. In some embodiments, the transgene comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 41-45. In some embodiments, the transgene comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 41-45. In some embodiments, the transgene comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 41-45. In some embodiments, the transgene comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 41-45. In some embodiments, the transgene comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 41-45. In some embodiments, the transgene comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 41-45. In some embodiments, the transgene comprises a sequence having 100% identity to any one of SEQ ID NOs: 41-45.

[0178] In some embodiments, the rAAV vectors of the present disclosure comprise a transgene sequence that is at least 85% sequence identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 41.

[0179] In some embodiments, the rAAV vectors of the present disclosure comprise a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 42 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0180] In some embodiments, the rAAV vectors of the present disclosure comprise a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 43 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0181] In some embodiments, the rAAV vectors of the present disclosure comprise a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 44 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0182] In some embodiments, the rAAV vectors of the present disclosure comprise a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 45 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0183] In some embodiments, the rAAV vectors of the present disclosure include one or more regulatory elements. In some embodiments, the one or more regulatory elements are 5' to the sequence encoding the bispecific fusion protein. In some embodiments, the one or more regulatory elements are 3' to the sequence encoding the bispecific fusion protein. For example, in some embodiments, the regulatory element is 3' to the sequence encoding the bispecific fusion protein and is derived from the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the regulatory element is at least 85% identical to SEQ ID NO: 64 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%). In some embodiments, the WPRE comprises a sequence having at least about 85% identity to SEQ ID NO: 64. In some embodiments, the WPRE comprises a sequence having at least about 90% identity to SEQ ID NO: 64. In some embodiments, the WPRE comprises a sequence having at least about 95% identity to SEQ ID NO: 64. In some embodiments, the WPRE comprises a sequence having at least about 96% identity to SEQ ID NO: 64. In some embodiments, the WPRE comprises a sequence having at least about 97% identity to SEQ ID NO: 64. In some embodiments, the WPRE comprises a sequence having at least about 98% identity to SEQ ID NO: 64. In some embodiments, the WPRE comprises a sequence having at least about 99% identity to SEQ ID NO: 64. In some embodiments, the WPRE comprises a sequence having 100% identity to SEQ ID NO: 64.

[0184] Regulatory element derived from WPRE: TIFF2025525035000034.tif68134

[0185] In some embodiments, the regulatory element is 3’ to the sequence encoding the bispecific fusion protein and is a modified RNA stability regulatory element (MRE). In some embodiments, the regulatory element is at least 85% identical to SEQ ID NO: 123 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%). In some embodiments, the MRE comprises a sequence having at least about 85% identity to SEQ ID NO: 123. In some embodiments, the MRE comprises a sequence having at least about 90% identity to SEQ ID NO: 123. In some embodiments, the MRE comprises a sequence having at least about 95% identity to SEQ ID NO: 123. In some embodiments, the MRE comprises a sequence having at least about 96% identity to SEQ ID NO: 123. In some embodiments, the MRE comprises a sequence having at least about 97% identity to SEQ ID NO: 123. In some embodiments, the MRE comprises a sequence having at least about 98% identity to SEQ ID NO: 123. In some embodiments, the MRE comprises a sequence having at least about 99% identity to SEQ ID NO: 123. In some embodiments, the MRE comprises a sequence having 100% identity to SEQ ID NO: 123.

[0186] In some embodiments, the MRE comprises a sequence having at least about 85% identity to SEQ ID NO: 124. In some embodiments, the MRE comprises a sequence having at least about 90% identity to SEQ ID NO: 124. In some embodiments, the MRE comprises a sequence having at least about 95% identity to SEQ ID NO: 124. In some embodiments, the MRE comprises a sequence having at least about 96% identity to SEQ ID NO: 124. In some embodiments, the MRE comprises a sequence having at least about 97% identity to SEQ ID NO: 124. In some embodiments, the MRE comprises a sequence having at least about 98% identity to SEQ ID NO: 124. In some embodiments, the MRE comprises a sequence having at least about 99% identity to SEQ ID NO: 124. In some embodiments, the MRE comprises a sequence having 100% identity to SEQ ID NO: 124.

[0187] Modified RNA stability regulatory element (MRE) variant 1 TIFF2025525035000035.tif68134

[0188] Modified RNA stability regulatory element (MRE) variant 2 TIFF2025525035000036.tif68134

[0189] In some embodiments, the rAAV vector of the present disclosure has a nucleotide sequence that is at least 85% sequence identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the nucleotide sequences listed in Table 9.

[0190] (Table 9) Exemplary AAV2 vector sequences TIFF2025525035000037.tif201161TIFF2025525035000038.tif244161TIFF2025525035000039.tif244161TIFF2025525035000040.tif244161TIFF2025525035000041.tif244161TIFF2025525035000042.tif244161TIFF2025525035000043.tif244161TIFF2025525035000044.tif244161TIFF2025525035000045.tif244161TIFF2025525035000046.tif244161TIFF2025525035000047.tif244161TIFF2025525035000048.tif244161TIFF2025525035000049.tif244161TIFF2025525035000050.tif244161TIFF2025525035000051.tif244161TIFF2025525035000052.tif244161TIFF2025525035000053.tif157161

[0191] In some embodiments, the rAAV vector comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 53-57. In some embodiments, the rAAV vector comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 53-57. In some embodiments, the rAAV vector comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 53-57. In some embodiments, the rAAV vector comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 53-57. In some embodiments, the rAAV vector comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 53-57. In some embodiments, the rAAV vector comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 53-57. In some embodiments, the rAAV vector comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 53-57. In some embodiments, the rAAV vector comprises a sequence having 100% identity to any one of SEQ ID NOs: 53-57.

[0192] In some embodiments, the rAAV vector of the present disclosure has a nucleotide sequence that is at least 85% sequence identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 53.

[0193] In some embodiments, the rAAV vectors of the present disclosure have a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO: 54 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0194] In some embodiments, the rAAV vectors of the present disclosure have a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO: 55 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0195] In some embodiments, the rAAV vectors of the present disclosure have a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO: 56 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0196] In some embodiments, the rAAV vectors of the present disclosure have a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO: 57 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0197] In some embodiments, the rAAV vectors of the present disclosure include one or more components (e.g., regulatory elements, transgenes) that contain reduced CpG dinucleotides and / or increased methylation of CpG dinucleotides compared to parental equivalents. In some embodiments, the CpG dinucleotides are reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% compared to parental equivalents. In some embodiments, the CpG dinucleotides are reduced in the range of about 5% to about 90%, about 10% to about 80%, about 15% to about 75%, about 20% to about 70%, about 25% to about 65%, or about 30% to about 60%. In some embodiments, the methylation of CpG dinucleotides is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% compared to parental equivalents. In some embodiments, the methylation of CpG dinucleotides is increased in the range of about 5% to about 90%, about 10% to about 80%, about 15% to about 75%, about 20% to about 70%, about 25% to about 65%, or about 30% to about 60%.

[0198] 2. Production of Recombinant Adeno-Associated Virus (AAV) Vectors Recombinant AAV particles can be produced by any standard method (e.g., WO 2001 / 083692, which is hereby incorporated by reference in its entirety; Masic et al. 2014. Molecular Therapy, 22(11):1900-1909; Carter, 1992, Current Opinions in Biotechnology, 1533-539; Muzyczka, 1992, Curr. Topics in Microbial, and Immunol., 158:97-129); Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al, J. Virol, 62: 1963 (1988); and Lebkowski et al, Mol. Cell. Biol, 7:349 (1988). Samulski et al, J. Virol., 63:3822-3828 (1989); U.S. Patent No. 5,173,414; WO 95 / 13365; U.S. Patent No. 5,658.776; WO95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US 96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. Vaccine 13: 1244-1250 (1995); Paul et al. Human Gene Therapy 4:609-615 (1993); Clark et al. Gene Therapy 3: 1124-1132 (1996); U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent No. 6,258,595).For example, in some embodiments, the rAAV vectors described herein are transformed into Escherichia coli to scale up DNA production, purified using any standard method (e.g., Maxi-Prep K, Thermo Scientific), and verified by restriction digestion or sequencing. The purified rAAV vector can then be combined with a plasmid containing the AAV rep and AAV cap genes, as well as an AAV helper plasmid, and transfected into a suitable packaging cell line (e.g., HEK293, HeLa, Sf9, PerC.6, MRC-5, WI-38, Vera, or FRhL-2 cells) using standard methods (e.g., calcium phosphate transfection, liposomes, polyethyleneimine, electroporation, etc.). The AAV rep and cap genes can be derived from any AAV serotype and may be the same as or different from those of the recombinant AAV vector ITR, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. In one embodiment, the recombinant AAV described herein contains AAV rep and cap genes derived from AAV2 and AAV9, respectively. The AAV helper plasmid can be derived from any AAV serotype and may be the same as or different from those of the recombinant AAV vector ITR, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. In one embodiment, the recombinant AAV described herein contains a plasmid having a helper gene derived from AAV2.

[0199] In some embodiments, the AAV rep and cap genes are derived from AAVrh.74. In some embodiments, the rep and cap genes comprise a nucleotide sequence having at least 85% (e.g., 85%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the rep and cap genes comprise a nucleotide sequence having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the rep and cap genes comprise a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the rep and cap genes comprise a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the rep and cap genes comprise a nucleotide sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the rep and cap genes comprise a nucleotide sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the rep and cap genes comprise a nucleotide sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the rep and cap genes comprise a nucleotide sequence having the nucleic acid sequence of SEQ ID NO: 125.

[0200] (Table 10) Rep and Cap Sequences TIFF2025525035000054.tif123161TIFF2025525035000055.tif244161TIFF2025525035000056.tif244161TIFF2025525035000057.tif244161TIFF2025525035000058.tif244161TIFF2025525035000059.tif244161TIFF2025525035000060.tif59161

[0201] In some embodiments, the recombinant AAVs described herein are harvested from packaging cells and purified by standard methods in the art, such as by cesium chloride ultracentrifugation gradient or column chromatography (e.g., Clark et al, Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69: 427-443 (2002); U.S. Patent No. 6,566,118 and WO 98 / 09657, which are incorporated herein by reference in their entirety).

[0202] In some embodiments, the rAAV of the disclosure comprises a nucleotide sequence that is at least 85% sequence identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the nucleotide sequences listed in Table 9.

[0203] In some embodiments, the rAAV of the present disclosure is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, or derivatives thereof. In some embodiments, the rAAV is AAV2, or a derivative thereof. In some embodiments, the rAAV is AAV8, or a derivative thereof. In some embodiments, the rAAV is AAV-rh74, or a derivative thereof.

[0204] 3. Pharmaceutical Composition The recombinant AAV vectors described herein can be used in the manufacture of pharmaceutical compositions. In some embodiments, the pharmaceutical compositions disclosed herein comprise the recombinant AAV vectors of the present disclosure and a pharmaceutically acceptable carrier, and optionally, other agents, pharmaceuticals, stabilizers, buffers, carriers, adjuvants, diluents, etc. "Pharmaceutically acceptable" means a material that is not toxic or otherwise undesirable, i.e., the material can be administered to a subject without causing undesirable biological effects.

[0205] In some embodiments, the pharmaceutical composition includes sterile aqueous and non-aqueous injection solutions, which are optionally isotonic with the blood of the subject to which the pharmaceutical composition is to be delivered. The pharmaceutical composition can contain antioxidants, buffers, bacteriostatic agents, and solutes, which render the composition isotonic with the blood of the intended subject to which the composition is administered. Aqueous and non-aqueous sterile suspensions, solutions, and emulsions can include suspending and thickening agents. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters for injection such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including physiological saline and buffered media. In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable vehicle and can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oil. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0206] In some embodiments, the pharmaceutical composition can be presented in unit / dose or multi-dose containers, such as sealed ampoules and vials, and can also be stored in a freeze-dried state that requires only the addition of a sterile liquid carrier, such as physiological saline or water for injection, immediately prior to use.

[0207] In some embodiments, the pharmaceutical compositions disclosed herein can be formulated for intravenous, intramuscular, intrathecal, or intraventricular administration.

[0208] 4. Treatment Methods The recombinant AAV (rAAV) vectors or pharmaceutical compositions containing them of the present disclosure can be administered to a subject in need thereof by any delivery mode including, but not limited to, intravenous, intraperitoneal, and intramuscular administration.

[0209] In some embodiments, the recombinant AAV vectors of the present disclosure or pharmaceutical compositions containing the same can be administered in one, two, three, four, five, or more doses. In some embodiments, when multiple doses are administered, the doses can be administered to the subject in need thereof simultaneously or at intervals.

[0210] This application provides methods for reducing the risk of metastases, preventing them, and treating them by administering to a patient an rAAV as described herein, or a pharmaceutical formulation thereof.

[0211] In other embodiments, the recombinant AAV vectors of the present disclosure or pharmaceutical compositions containing the same can be administered as a single intravenous dose or a divided intravenous dose. In some embodiments, the dose for intravenous delivery is 1 X 10 10 ~1 X 10 13 vg / kg, 2 X 10 10 ~1 X 10 13 vg / kg, 3 X 10 10 ~1 X 10 13 vg / kg, 4 X 10 10 ~1 X 10 13 vg / kg, 5 X 10 10 ~1 X 10 13 vg / kg, 6 X 10 10 ~1 X 10 13 vg / kg, 7 X 10 10 ~1 X 10 13 vg / kg, 8 X 10 10 ~1 X 10 13 vg / kg, 9 X 10 10 ~1 X 10 13 vg / kg, 1 X 10 11 ~1 X 10 13 vg / kg, 2 X 10 11 ~1 X 10 13 vg / kg, 3 X 10 11 ~1 X 10 13 vg / kg, 4 X 10 11 ~1 X 10 13 vg / kg, 5 X 1011 ~1 × 10 13 vg / kg, 6 × 10 11 ~1 × 10 13 vg / kg, 7 × 10 11 ~1 × 10 13 vg / kg, 8 × 10 11 ~1 × 10 13 vg / kg, 9 × 10 11 ~1 × 10 13 vg / kg, 1 × 10 12 ~1 × 10 13 vg / kg, 2 × 10 12 ~1 × 10 13 vg / kg, 3 × 10 12 ~1 × 10 13 vg / kg, 4 × 10 12 ~1 × 10 13 vg / kg, 5 × 10 12 ~1 × 10 13 vg / kg, 6 × 10 12 ~1 × 10 13 vg / kg, 7 × 10 12 ~1 × 10 13 vg / kg, 8 × 10 12 ~1 × 10 13 vg / kg, 9 × 10 12 ~1 × 10 13 vg / kg, 1 × 10 10 ~1 × 10 12 vg / kg, 2 × 10 10 ~1 × 10 12 vg / kg, 3 × 10 10 ~1 × 10 12 vg / kg, 4 × 10 10 ~1 × 10 12 vg / kg, 5 × 10 10 ~1 × 10 12 vg / kg, 6 × 10 10 ~1 × 10 12 vg / kg, 7 × 10 10 ~1 × 10 12 vg / kg, 8 × 10 10 ~1 × 10 12 vg / kg, 9 × 10 10~1 X 10 12 vg / kg, 1 X 10 11 ~1 X 10 12 vg / kg, 2 X 10 11 ~1 X 10 12 vg / kg, 3 X 10 11 ~1 X 10 12 vg / kg, 4 X 10 11 ~1 X 10 12 vg / kg, 5 X 10 11 ~1 X 10 12 vg / kg, 6 X 10 11 ~1 X 10 12 vg / kg, 7 X 10 11 ~1 X 10 12 vg / kg, 8 X 10 11 ~1 X 10 12 vg / kg, 9 X 10 11 ~1 X 10 12 vg / kg, 1 X 10 10 ~1 X 10 11 vg / kg, 2 X 10 10 ~1 X 10 11 vg / kg, 3 X 10 10 ~1 X 10 11 vg / kg, 4 X 10 10 ~1 X 10 11 vg / kg, 5 X 10 10 ~1 X 10 11 vg / kg, 6 X 10 10 ~1 X 10 11 vg / kg, 7 X 10 10 ~1 X 10 11 vg / kg, 8 X 10 10 ~1 X 10 11 vg / kg, or 9 X 10 10 ~1 X 10 11 (which may be virus genome (vg) / kilogram (kg) (vg / kg)). In some embodiments, the recombinant AAV vector of the present disclosure or a pharmaceutical composition containing the same is administered as a single intravenous dose of 1 X 10 11 vg / kg. In some embodiments, the recombinant AAV vector of the present disclosure or a pharmaceutical composition containing the same is 3 X 1011 It is administered as a single intravenous dose of vg / kg. In some embodiments, the recombinant AAV vector of the present disclosure or a pharmaceutical composition comprising the same is 1 X 10 12 It is administered as a single intravenous dose of vg / kg. In some embodiments, the recombinant AAV vector of the present disclosure or a pharmaceutical composition comprising the same is 3 X 10 12 It is administered as a single intravenous dose of vg / kg. In some embodiments, the recombinant AAV vector of the present disclosure or a pharmaceutical composition comprising the same is 5 X 10 12 It is administered as a single intravenous dose of vg / kg.

[0212] In some embodiments, the present application provides a method for treating cancer in a patient by administering to the patient an effective amount of the rAAV vector described herein or a pharmaceutical formulation thereof. In some embodiments, the cancer is neuroblastoma, melanoma, retinoblastoma, Ewing's sarcoma, small cell lung tumor, glioma, osteosarcoma, or soft tissue sarcoma. For example, in some embodiments, the cancer is neuroblastoma.

[0213] Method for reducing the risk of metastasis In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having: (i) A GD2-binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) A CD3-binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively. In some embodiments, the patient is not diagnosed with cancer. In some embodiments, the patient has not received cancer treatment.

[0214] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively.

[0215] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively.

[0216] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having: (i) a GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively.

[0217] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having: (i) A GD2-binding site comprising VL and VH domains having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL and VH domain sequences corresponding to SEQ ID NO: 2 and SEQ ID NO: 1, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a CD3-binding site comprising VH and VL domains having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH and VL domain sequences corresponding to SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0218] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 2 and SEQ ID NO: 1, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0219] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 4 and SEQ ID NO: 3, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0220] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2-binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 4 and SEQ ID NO: 3, respectively; (ii) a linker peptide comprising the sequence corresponding to SEQ ID NO: 25; and (iii) a CD3-binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0221] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2-binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 5; (ii) a linker peptide comprising the sequence of SEQ ID NO: 20; and (iii) a CD3-binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 17.

[0222] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having: (i) a GD2 binding site comprising a scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 5; (ii) a linker peptide comprising the sequence of SEQ ID NO: 25; and (iii) a CD3 binding site comprising a scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 16.

[0223] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having: (i) A GD2 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 7 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%); (ii) a linker peptide comprising the sequence of SEQ ID NO: 20; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 17 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0224] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 7 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%); (ii) a linker peptide comprising the sequence of SEQ ID NO: 25; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 16 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0225] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 9, or a pharmaceutical formulation thereof.

[0226] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 10, or a pharmaceutical formulation thereof.

[0227] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 11, or a pharmaceutical formulation thereof.

[0228] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 12, or a pharmaceutical formulation thereof.

[0229] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 13, or a pharmaceutical formulation thereof.

[0230] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 41, or a pharmaceutical formulation thereof.

[0231] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 42 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0232] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 43 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0233] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 44 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0234] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 45 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0235] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 53.

[0236] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 54.

[0237] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 55.

[0238] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 56.

[0239] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 57.

[0240] Method for preventing metastasis In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively. In some embodiments, the patient is not diagnosed with cancer. In some embodiments, the patient has not received cancer treatment.

[0241] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively.

[0242] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2-binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; (ii) A linker peptide comprising the sequence corresponding to SEQ ID NO: 20; and (iii) A CD3-binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively.

[0243] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2-binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; (ii) A linker peptide comprising the sequence corresponding to SEQ ID NO: 25; and (iii) A CD3-binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively.

[0244] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 2 and SEQ ID NO: 1, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0245] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising VL and VH domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL and VH domain sequences corresponding to SEQ ID NO: 2 and SEQ ID NO: 1, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising VH and VL domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH and VL domain sequences corresponding to SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0246] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 4 and SEQ ID NO: 3, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0247] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 4 and SEQ ID NO: 3, respectively; (ii) a linker peptide comprising the sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0248] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 5; (ii) a linker peptide comprising the sequence of SEQ ID NO: 20; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 17.

[0249] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having: (i) a GD2 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 5; (ii) a linker peptide comprising the sequence of SEQ ID NO: 25; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 16.

[0250] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having: (i) A GD2 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 7 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%); (ii) a linker peptide comprising the sequence of SEQ ID NO: 20; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 17 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0251] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 7 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%); (ii) a linker peptide comprising the sequence of SEQ ID NO: 25; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 16 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0252] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 9, or a pharmaceutical formulation thereof.

[0253] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 10, or a pharmaceutical formulation thereof.

[0254] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 11, or a pharmaceutical formulation thereof.

[0255] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 12, or a pharmaceutical formulation thereof.

[0256] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 13, or a pharmaceutical formulation thereof.

[0257] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 41.

[0258] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 42.

[0259] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 43.

[0260] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 44.

[0261] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 45.

[0262] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 53.

[0263] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 54.

[0264] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 55.

[0265] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 56.

[0266] In some embodiments, the present application provides methods for preventing metastasis by administering to a patient an effective amount of rAAV that contains at least 85% sequence identity to SEQ ID NO: 57 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0267] Methods for Treating Metastases In some embodiments, the present application provides methods for treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein, or a pharmaceutical formulation thereof, having: (i) a GD2-binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively. The CD3 binding site includes the VL CDR2 and VL CDR3 sequences.

[0268] In some embodiments, the present application provides methods for treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein, or a pharmaceutical formulation thereof, having: (i) A GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively.

[0269] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively.

[0270] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having: (i) a GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively.

[0271] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having: (i) A GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 2 and SEQ ID NO: 1, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0272] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising VL and VH domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL and VH domain sequences corresponding to SEQ ID NO: 2 and SEQ ID NO: 1, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising VH and VL domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH and VL domain sequences corresponding to SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0273] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 4 and SEQ ID NO: 3, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0274] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 4 and SEQ ID NO: 3, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 4 and SEQ ID NO: 3, respectively; 15. A CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to 15.

[0275] In some embodiments, the present application provides methods for treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein, or a pharmaceutical formulation thereof, having: (i) a GD2 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 5; (ii) a linker peptide comprising the sequence of SEQ ID NO: 20; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 17.

[0276] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having: (i) a GD2 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 5; (ii) a linker peptide comprising the sequence of SEQ ID NO: 25; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 16.

[0277] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having: (i) a GD2 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 7 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%); (ii) a linker peptide comprising the sequence of SEQ ID NO: 20; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 17 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0278] In some embodiments, the present application provides methods for treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein, or a pharmaceutical formulation thereof, having: (i) a GD2 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 7; (ii) a linker peptide comprising the sequence of SEQ ID NO: 25; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 16.

[0279] In some embodiments, the present application provides methods for treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence with at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 9, or a pharmaceutical formulation thereof.

[0280] In some embodiments, the present application provides methods for treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence with at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 10, or a pharmaceutical formulation thereof.

[0281] In some embodiments, the present application provides methods for treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence with at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 11, or a pharmaceutical formulation thereof.

[0282] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 12, or a pharmaceutical formulation thereof.

[0283] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 13, or a pharmaceutical formulation thereof.

[0284] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 41, or a pharmaceutical formulation thereof.

[0285] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 42 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0286] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 43 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0287] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 44 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0288] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 45 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0289] In some embodiments, the present application provides methods for treating metastasis by administering to a patient an effective amount of an rAAV that contains at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 53.

[0290] In some embodiments, the present application provides methods for treating metastasis by administering to a patient an effective amount of an rAAV that contains at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 54.

[0291] In some embodiments, the present application provides methods for treating metastasis by administering to a patient an effective amount of an rAAV that contains at least 85% sequence identity to SEQ ID NO: 55 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0292] In some embodiments, the present application provides methods for treating metastasis by administering to a patient an effective amount of an rAAV that contains at least 85% sequence identity to SEQ ID NO: 56 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0293] In some embodiments, the present application provides methods for treating metastasis by administering to a patient an effective amount of an rAAV that contains at least 85% sequence identity to SEQ ID NO: 57 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0294] Methods for enhancing T cell-mediated killing of circulating tumor cells - Patent Application 20070122999 In some embodiments, the present application provides methods for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein, or a pharmaceutical formulation thereof, having: (i) a GD2-binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively. The CD3 binding site includes the VL CDR2 and VL CDR3 sequences.

[0295] In some embodiments, the present application provides methods for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein, or a pharmaceutical formulation thereof, having: (i) A GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively.

[0296] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively.

[0297] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a GD2-binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively. The CD3 binding site includes the VL CDR2 and VL CDR3 sequences.

[0298] In some embodiments, the present application provides methods for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein, or a pharmaceutical formulation thereof, having: (i) A GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 2 and SEQ ID NO: 1, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0299] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 2 and SEQ ID NO: 1, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0300] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 4 and SEQ ID NO: 3, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0301] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 4 and SEQ ID NO: 3, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0302] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 5; (ii) A linker peptide comprising the sequence of SEQ ID NO: 20; and (iii) A CD3 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 17.

[0303] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 5; (ii) A linker peptide comprising the sequence of SEQ ID NO: 25; and (iii) A CD3 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 16.

[0304] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a GD2 binding site comprising a scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 7; (ii) a linker peptide comprising the sequence of SEQ ID NO: 20; and (iii) a CD3 binding site comprising a scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 17.

[0305] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 7; (ii) a linker peptide comprising the sequence of SEQ ID NO: 25; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 16.

[0306] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 9, or a pharmaceutical formulation thereof.

[0307] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 10, or a pharmaceutical formulation thereof.

[0308] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 11, or a pharmaceutical formulation thereof.

[0309] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 12, or a pharmaceutical formulation thereof.

[0310] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 13, or a pharmaceutical formulation thereof.

[0311] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 41.

[0312] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 42.

[0313] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 43.

[0314] In some embodiments, the present application provides methods for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector containing a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 44 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0315] In some embodiments, the present application provides methods for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector containing a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 45 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0316] In some embodiments, the present application provides methods for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV that contains at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 53.

[0317] In some embodiments, the present application provides methods for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV that contains at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 54.

[0318] In some embodiments, the present application provides methods for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV that contains at least 85% sequence identity to SEQ ID NO: 55 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0319] In some embodiments, the present application provides methods for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV that contains at least 85% sequence identity to SEQ ID NO: 56 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0320] In some embodiments, the present application provides methods for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV that contains at least 85% sequence identity to SEQ ID NO: 57 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0321] Methods for preventing cancer in patients predisposed to developing tumors In some embodiments, the present application provides methods of preventing cancer in a patient predisposed to developing a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein, or a pharmaceutical formulation thereof, having: (i) A GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively.

[0322] In some embodiments, the present application provides a method of preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively.

[0323] In some embodiments, the present application provides a method of preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively.

[0324] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively.

[0325] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 2 and SEQ ID NO: 1, respectively; (ii) a linker peptide comprising the sequence corresponding to SEQ ID NO: 20; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0326] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising VL and VH domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL and VH domain sequences corresponding to SEQ ID NO: 2 and SEQ ID NO: 1, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising VH and VL domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH and VL domain sequences corresponding to SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0327] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 4 and SEQ ID NO: 3, respectively; (ii) a linker peptide comprising the sequence corresponding to SEQ ID NO: 20; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0328] In some embodiments, the present application provides a method of preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising a VL domain and a VH domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 4 and SEQ ID NO: 3, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0329] In some embodiments, the present application provides a method of preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 5 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%); (ii) a linker peptide comprising the sequence of SEQ ID NO: 20; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 17 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0330] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 5 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%); (ii) a linker peptide comprising the sequence of SEQ ID NO: 25; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 16 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0331] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a GD2 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 7; (ii) a linker peptide comprising the sequence of SEQ ID NO: 20; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 17.

[0332] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a GD2 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 7; (ii) a linker peptide comprising the sequence of SEQ ID NO: 25; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 16.

[0333] In some embodiments, the application provides methods of preventing cancer in a patient predisposed to developing a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence with at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 9, or a pharmaceutical formulation thereof.

[0334] In some embodiments, the present application provides methods of preventing cancer in a patient predisposed to developing a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence with at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 10, or a pharmaceutical formulation thereof.

[0335] In some embodiments, the present application provides a method of preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 11, or a pharmaceutical formulation thereof.

[0336] In some embodiments, the present application provides a method of preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 12, or a pharmaceutical formulation thereof.

[0337] In some embodiments, the present application provides a method of preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 13, or a pharmaceutical formulation thereof.

[0338] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 41 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0339] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 42 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0340] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 43 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0341] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 44.

[0342] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 45.

[0343] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of an rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 53.

[0344] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 54.

[0345] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 55.

[0346] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 56.

[0347] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 57.

[0348] Method for preventing cancer recurrence In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively.

[0349] In some embodiments, the present application provides a method of preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a GD2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively.

[0350] In some embodiments, the present application provides a method of preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2-binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) A CD3-binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively.

[0351] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2-binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) A CD3-binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively.

[0352] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising VL and VH domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL and VH domain sequences corresponding to SEQ ID NO: 2 and SEQ ID NO: 1, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a CD3 binding site comprising VH and VL domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH and VL domain sequences corresponding to SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0353] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising VL and VH domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL and VH domain sequences corresponding to SEQ ID NO: 2 and SEQ ID NO: 1, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising VH and VL domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH and VL domain sequences corresponding to SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0354] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having: (i) A GD2 binding site comprising VL and VH domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL and VH domain sequences corresponding to SEQ ID NO: 4 and SEQ ID NO: 3, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 20; and (iii) a CD3 binding site comprising VH and VL domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH and VL domain sequences corresponding to SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0355] In some embodiments, the present application provides a method of preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising VL and VH domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL and VH domain sequences corresponding to SEQ ID NO: 4 and SEQ ID NO: 3, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 25; and (iii) a CD3 binding site comprising VH and VL domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH and VL domain sequences corresponding to SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0356] In some embodiments, the present application provides a method of preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 5 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%); (ii) a linker peptide comprising the sequence of SEQ ID NO: 20; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 17 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0357] In some embodiments, the present application provides a method of preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A GD2 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 5 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%); (ii) a linker peptide comprising the sequence of SEQ ID NO: 25; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity to SEQ ID NO: 16 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0358] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a GD2 binding site comprising a scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 90; (ii) a linker peptide comprising the sequence of SEQ ID NO: 20; and (iii) a CD3 binding site comprising a scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 17.

[0359] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 7, which is a GD2 binding site; (ii) a linker peptide containing the sequence of SEQ ID NO: 25; and (iii) an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 16, which is a CD3 binding site.

[0360] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 9, or a pharmaceutical formulation thereof.

[0361] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 10, or a pharmaceutical formulation thereof.

[0362] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 11, or a pharmaceutical formulation thereof.

[0363] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 12, or a pharmaceutical formulation thereof.

[0364] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 13, or a pharmaceutical formulation thereof.

[0365] In some embodiments, the present application provides a method for preventing cancer recurrence (e.g., GD2+ cancer) in a patient in remission by administering to the patient an effective amount of an rAAV vector comprising a transgene sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 41.

[0366] In some embodiments, the present application provides a method for preventing cancer recurrence (e.g., GD2+ cancer) in a patient in remission by administering to the patient an effective amount of an rAAV vector comprising a transgene sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 42.

[0367] In some embodiments, the present application provides a method for preventing cancer recurrence (e.g., GD2+ cancer) in a patient in remission by administering to the patient an effective amount of an rAAV vector comprising a transgene sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 43.

[0368] In some embodiments, the present application provides a method for preventing cancer recurrence (e.g., GD2+ cancer) in a patient in remission by administering to the patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 44 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0369] In some embodiments, the present application provides a method for preventing cancer recurrence (e.g., GD2+ cancer) in a patient in remission by administering to the patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 45 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0370] In some embodiments, the present application provides a method for preventing cancer recurrence (e.g., GD2+ cancer) in a patient in remission by administering to the patient an effective amount of an rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 53.

[0371] In some embodiments, the present application provides a method for preventing cancer recurrence (e.g., GD2+ cancer) in a patient in remission from cancer by administering to the patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 54.

[0372] In some embodiments, the present application provides a method for preventing cancer recurrence (e.g., GD2+ cancer) in a patient in remission from cancer by administering to the patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 55.

[0373] In some embodiments, the present application provides a method for preventing cancer recurrence (e.g., GD2+ cancer) in a patient in remission from cancer by administering to the patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 56.

[0374] In some embodiments, the present application provides a method for preventing cancer recurrence (e.g., GD2+ cancer) in a patient in remission from cancer by administering to the patient an effective amount of rAAV comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 57.

[0375] Combined treatment In another aspect of the present disclosure, the rAAV vector, or a pharmaceutical formulation thereof, is administered concurrently with the treatment of the primary tumor.

[0376] In some embodiments of the present disclosure, the rAAV vector, or a pharmaceutical formulation thereof, is administered concurrently with surgical resection, radiation therapy, chemotherapy, or immunotherapy of the primary tumor.

[0377] In some embodiments, the rAAV vector, or a pharmaceutical formulation thereof, as described herein, is administered in combination with a checkpoint inhibitor selected from CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors.

[0378] In some embodiments, the rAAV vector, or a pharmaceutical formulation thereof, as described herein, is administered in combination with a CTLA-4 inhibitor selected from ipilimumab and tremelimumab. For example, in some embodiments, the rAAV, or a pharmaceutical formulation thereof, as described herein, is administered in combination with ipilimumab.

[0379] In some embodiments, rAAV as described herein, or a pharmaceutical formulation thereof, is administered in combination with a PD-1 inhibitor selected from pembrolizumab, nivolumab, semipramab, dostarlimab, JTZ-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012, AMP-224, and AMP-514. For example, in some embodiments, an rAAV vector as described herein, or a pharmaceutical formulation thereof, is administered in combination with pembrolizumab or nivolumab.

[0380] In some embodiments, rAAV vector as described herein, or a pharmaceutical formulation thereof, is administered in combination with a PD-L1 inhibitor selected from atezolizumab, avelumab, durvalumab, KN035, CK-301, AUNP12, CA-170, or BMS-986189. For example, in some embodiments, an rAAV vector as described herein, or a pharmaceutical formulation thereof, is administered in combination with atezolizumab.

[0381] This application provides a method for reducing the risk of metastasis, preventing metastasis, or treating metastasis, wherein an rAAV vector as described herein, or a pharmaceutical formulation thereof, is administered concurrently with the treatment of the primary tumor. In some embodiments, the primary tumor is neuroblastoma, melanoma, retinoblastoma, Ewing sarcoma, small cell lung tumor, glioma, osteosarcoma, or soft tissue sarcoma. For example, in some embodiments, the primary tumor is neuroblastoma.

[0382] In some embodiments, rAAV as described herein, or a pharmaceutical formulation thereof, is administered in combination with one or more AAVs encoding different bispecific fusion proteins targeting different tumor-associated antigens.

Example

[0383] The present disclosure, which is generally described now, can be more easily understood by referring to the following examples, which are included merely for the purpose of explaining certain aspects and embodiments of the present disclosure and are not intended to limit the present disclosure.

[0384] Example 1: Molecular Cloning of AAV Transgene Construct From 5' to 3', a bispecific fusion protein transgene was synthesized by functionally linking (i) a codon-optimized nucleotide sequence encoding the VL domain of an anti-GD2 antibody; (ii) a nucleotide sequence encoding a first scFv linker peptide; (iii) a codon-optimized nucleotide sequence encoding the VH domain of an anti-GD2 antibody; (iv) a nucleotide sequence encoding a linker peptide; (v) a codon-optimized nucleotide sequence encoding the VH domain of an anti-CD3 antibody; (vi) a nucleotide sequence encoding a second scFv linker peptide; and (vii) a codon-optimized nucleotide sequence encoding the VL domain of an anti-CD3 antibody.

[0385] The transgene cassette was synthesized by functionally linking a CAG promoter sequence, the bispecific fusion protein transgene, and a bovine growth hormone (BGH) polyadenylation sequence. The transgene cassette was cloned into a suitable cloning vector (e.g., pUC) and confirmed by DNA sequencing. The confirmed construct was restriction digested and gel purified for subsequent cloning into a suitable AAV8 backbone vector containing AAV2 ITR sites and a kanamycin resistance gene.

[0386] After ligation, the DNA was transformed into Escherichia coli (e.g., VB UltraStable™, Vector Builder, Chicago, IL), grown on kanamycin selection medium, and purified. The ligated gene construct that was successful was identified by restriction digestion.

[0387] The clones were then scaled up by bacterial transformation into E. coli. The correct plasmid sequence was reconfirmed by restriction digestion.

[0388] Transient transfection and virus packaging Using standard calcium phosphate transfection methods (e.g., as described in Vandendriessche et al. (2007. J Thromb Haemost 5:16-24), incorporated herein by reference), the confirmed AAV vectors were transiently transfected into HEK293 cells in combination with an adenovirus helper plasmid and a packaging construct delivering the AAV rep and cap genes. Two days after transfection, AAV particles were harvested, purified by two successive cesium chloride density gradient ultracentrifugations, and titers were measured.

[0389] Example 2: Binding of bispecific fusion proteins to GD2 and CD3 Purified AAV particles as described in Example 1 were used to transduce producer cells (e.g., HEK293 cells) to express the bispecific fusion proteins. The bispecific fusion proteins were collected and purified from culture supernatants and / or cell lysates and assayed for GD2 and CD3 binding.

[0390] The binding of the bispecific fusion protein to GD2 was assayed using human cancer cell lines expressing GD2 (e.g., cell lines with high levels of GD2 expression or cell lines engineered to express exogenous GD2). Various concentrations of the bispecific fusion protein were incubated with GD2-expressing cells, and binding was detected using a fluorophore-conjugated secondary antibody. The cells were analyzed by flow cytometry and compared to binding of cells incubated with a GD2-binding control antibody.

[0391] Similarly, T cell lines or peripheral blood mononuclear cells (PBMCs) were used to assay the binding of bispecific fusion proteins to CD3. Bispecific fusion proteins at various concentrations were incubated with T cell lines or PBMCs, and binding was detected using a fluorophore-conjugated secondary antibody. Cells were analyzed by flow cytometry and compared to the binding of cells incubated with an anti-CD3 control antibody.

[0392] Standard co-culture cytotoxicity assays were also performed using a dilution titration of the purified bispecific fusion protein in culture medium alone and in 100% mouse or pooled human serum. After 48 hours, the concentrations of bispecific fusion protein required to kill 10% (EC10), 50% (EC50), and 90% (EC90) of target cells under standard conditions were determined. ED10 / 50 / 90 were determined for 10 different cell lines with different levels of GD2 expression (e.g., T98G, U87MG, C3c GBM cells).

[0393] Example 3: Bispecific fusion proteins induce T cell-mediated cytotoxicity in vitro PBMCs were isolated from human peripheral blood buffy coats using density gradient centrifugation. PBMCs were then co-cultured with human cancer cells expressing GD2 in the presence of bispecific fusion proteins or control α-GD2 antibody at various concentrations. After co-culture, cells were lysed and analyzed using a commercially available cytotoxicity assay (e.g., CytoTox96® Non-Radioactive Cytotoxicity Assay, Promega, Madison, WI) according to the manufacturer's instructions.

[0394] Example 4: Dose determination To determine vector doses and serum levels for administration to human patients, mice were given different doses of AAV in semi-logarithmic increments (1 x 10 11 、3 x 10 11 、1 x 10 12 、and 3 x 10 12They were administered at (vg / kg), and serum levels were analyzed from retro-orbital bleeds at a time point predicted to be well after steady state (e.g., 28 days). Vector doses in animal experiments were selected that achieved serum levels exceeding the EC90 for most cell lines, as determined in Example 2.

[0395] Example 5: AAV8 delivery of bispecific fusion protein prevents metastasis To determine the efficacy of AAV8-delivered bispecific fusion protein in preventing metastasis, mouse treatment groups consisted of (1) no local control, (2) oncolytic HSV1, (3) surgical resection, and (4) radiotherapy. Each treatment group included its own vehicle control (i.e., without bispecific fusion protein) group for comparison.

[0396] GD2+ human cancer cells (e.g., GFP+ or CD45-CD56+, CD81+, NB84+, GD2+ cells) were administered at various concentrations to the test groups at either flank of mice, and then the mice were administered purified AAV8 or control for in vivo expression of the bispecific fusion protein. The primary tumor size and the presence of metastatic lesions were monitored by measuring the bioluminescence of the engrafted tumors and satellite metastases (if present) at various time points. Metastases were confirmed and / or counted at necropsy. Metastatic tissues (e.g., lung and liver) were analyzed for T cell infiltration, activation, and / or exhaustion by immunohistochemistry and / or flow cytometry. Peripheral T cells were also analyzed by flow cytometry for activation and checkpoint marker expression every two weeks. Flow cytometry was also used to monitor circulating tumor cells every two weeks.

[0397] Alternatively, use any one of a number of neuroblastoma xenograft models (e.g., TH-MYCN; Braekeveldt and Bexell, Cell Tissue Res. 2018; 372(2):233-243; Ornell and Coburn, BMC Biomedical Engineering. 2019; 33; Weiss et al. Embo J. 1997;16(11):2985-95). For example, implant GD2+ human cancer cells expressing luciferase or green fluorescent protein (GFP) subcutaneously into mice (e.g., Balb / c). Grow the tumors to an appropriate size and then intravenously administer purified AAV8 particles or a control to the mice for in vivo expression of the bispecific fusion protein. Monitor the primary tumor size and the presence of metastatic lesions by measuring the bioluminescence of the engrafted tumors and satellite metastases (if present) at various time points. Confirm and / or count metastases by necropsy. Analyze metastatic tissues (e.g., lung and liver) for T cell infiltration, activation, and / or exhaustion by immunohistochemistry and / or flow cytometry. Also analyze peripheral T cells by flow cytometry for activation and checkpoint marker expression every two weeks. Use flow cytometry also to monitor circulating tumor cells every two weeks.

[0398] Example 6: Determination of Immunogenicity and Loss of Expression Over Time of AAV8-Delivered Bispecific Fusion Protein To determine whether the AAV8-delivered bispecific fusion protein induces an immune response, administer the AAV intravenously to immunocompetent C57Bl / 6 mice. Monitor the mice by weekly blood tests for the first three months to measure the levels of the bispecific fusion protein and then monthly for the next nine months. Monitor body weight at each blood draw as a safety / toxicity readout.

[0399] Example 7: Evaluation of the Effect of Anti-GD2 / anti-CD3ε Fusion Protein on GD2-Expressing Cell Lines AAV constructs were prepared, each encoding one of five anti-GD2 / anti-CD3ε bispecific fusion proteins, each containing one of two anti-GD2 scFvs (hu3F8V5 and 14G2a) and one of two anti-CD3 scFvs (OKT3 and L2K-07) linked by a linker. One hu3F8V5 / OKT3 protein (1169) containing an additional spacer moiety was prepared. The AAV construct design is detailed in Figures 2A-2B. The constructs in Figure 2B were tested in vitro and in vivo and were found to generate dimer proteins, which caused some killing in vitro (data not shown). In a flank tumor model, the construct in Figure 2B failed to control flank tumors.

[0400] Manufacturability was assessed by measuring protein production in 293T cells and binding to the GD2-expressing neuroblastoma cell lines SK-N-Be(2)-CD19, CHP-134-CD19, and SK-N-AS-CD19 (Figure 3). These results indicate that protein 1172 (hu3F8V5 / L2K-07) showed the best protein production in 293T cells.

[0401] To evaluate the effect of the anti-GD2 / anti-CD3ε bispecific fusion protein on GD2-expressing neuroblastoma cells, supernatants were harvested from 293T cells engineered to produce one of the control or anti-GD2 / anti-CD3ε bispecific fusion proteins (1169, 1170, 1172, 1173, or 1175) detailed above by AAV transduction. SK-N-Be(2)-CD19, CHP-134-CD19, and SK-N-AS-CD19 cells were incubated at an effector:target (E:T) ratio of 10:1 with supernatants from human peripheral blood mononuclear cells (huPBMC) and 293T cells, respectively. After 48 hours of incubation, target cell viability was measured (Figure 4). The results showed that co-treatment of the two expressing target cells with huPBMC and bispecific fusion proteins 1169, 1170, 1172, and 1175 increased target cell killing of SK-N-AS-CD19, but not of SK-N-Be(2)-CD19 or CHP-134-CD19 cells. Target cells were analyzed for GD2 expression (Figures 5A-5B), which showed that both of the tested SK-N-Be(2)-CD19 and CHP-134-CD19 cells had lost surface GD2 expression. These results indicate that the anti-GD2 / anti-CD3ε bispecific fusion protein can induce target-specific cell killing.

[0402] To evaluate the relative cell killing ability of various anti-GD2 / anti-CD3ε bispecific fusion proteins against GD2-expressing neuroblastoma cells, SK-N-AS, SK-N-Be(2), and CHP-134, as well as a CD19-expressing prototype, were incubated at an E:T ratio of 10:1 with 293T supernatant containing the anti-GD2 / anti-CD3ε bispecific fusion protein (1172, huOKT3 / 5F11-HDD, or CAG-193 / dCGMRE) and huPBMC. After 48 hours of incubation, the target cell viability was measured (Figure 6). These results indicate that protein 1172 induced superior cell killing compared to the other test proteins, and a decreased effect was seen with CHP-134. Target cells were analyzed for GD2 expression (Figure 7), which showed that both the CHP-134 parental and CD19 cells tested had lost surface GD2 expression. These results demonstrate that anti-GD2 / anti-CD3ε bispecific fusion proteins can induce target-specific cell killing.

[0403] Prior to further experiments, SK-N-AS, SK-N-Be(2), and CHP-134 neuroblastoma cells were evaluated for GD2 expression. 5×10 5 cells of each strain were stained with an anti-GD2 antibody, and GD2 staining was evaluated by flow cytometry (Figure 8). The results show that both normal BT474 cells and clone 5 cells expressed high levels of GD2. These results indicate that SK-N-Be(2) and CHP-134 retained GD2 expression. To evaluate the cell killing ability of the anti-GD2 / anti-CD3ε bispecific fusion protein 1172, CHP-134, SK-N-AS, and SK-N-SH cells were incubated at an E:T ratio of 10:1 with 293T supernatant containing protein 1172 at concentrations of 0 pM, 10 pM, 100 pM, and 1000 pM and huPBMC. After 48 hours of incubation, the target cell viability (upper panel) and GD2 expression (lower panel) were measured (Figure 9).

[0404] To further evaluate the target-specific cell-killing ability of the anti-GD2 / anti-CD3ε bispecific fusion protein 1172, various neuroblastoma cells were evaluated for GD2 surface expression by flow cytometry (Figures 10A-10B). The cells were then incubated with 293T supernatants and huPBMCs containing protein 1172 at concentrations of 0 pM, 100 pM, and 1000 pM at an E:T ratio of 10:1. After 48 hours of incubation, target cell viability was measured (Figure 10C). These results indicate that the cell-killing ability of the anti-GD2 / anti-CD3ε bispecific fusion protein 1172 correlates with the surface expression of GD2 on target cells.

[0405] To evaluate the potential effect of human serum on the target-specific cell-killing ability of anti-GD2 / anti-CD3ε bispecific fusion protein 1172, various neuroblastoma cell lines were incubated with 293T supernatant containing protein 1172 at concentrations of 0 pM, 50 pM, 100 pM, 500 pM, 1000 pM, and 5000 pM; human serum at concentrations of 10%, 50%, and 100%; and huPBMCs at an E:T ratio of 10:1. After 48 hours of incubation, target cell viability was measured (Figure 11). These results indicate that the presence of human serum had limited effect on the target-specific cell-killing ability of anti-GD2 / anti-CD3ε bispecific fusion protein 1172.

[0406] To evaluate the target-specific cell-killing ability of anti-GD2 / anti-CD3ε bispecific fusion protein 1172 against GD2-expressing lung cancer cells, H446, H446-Luc, and H2228 cells were incubated with 293T supernatants and huPBMCs containing protein 1172 at concentrations of 0 pM, 100 pM, and 1000 pM at an E:T ratio of 10:1. After 48 hours of incubation, target cell viability was measured (Figure 12). These results indicate that GD2 / anti-CD3ε bispecific fusion protein 1172 successfully mediated the killing of GD2-expressing lung cancer cells.

[0407] Example 8: In Vivo Evaluation of the Effect of Anti-GD2 / Anti-CD3ε Fusion Protein To evaluate the antitumor effect of the prototype AAV8-anti-GD2 / anti-CD3ε bispecific fusion protein (GD2 scFv: 5F11) (with and without co-treatment with the oncolytic virus talimogene laherparepvec (TVEC)), 5 × 10 12 genome copies / kilogram (gc / kg) of the AAV vector (anti-GD2 / anti-CD3ε or GFP control) were injected into SK-N-Be(2)-CD19 / Luc tumor-bearing huPBMC-NSGS mice, and the groups were co-treated with TVEC. All mice were co-injected with a single dose of huPBMC. The experimental outline is provided in Figure 13A, and GD2 surface staining is shown in Figure 13B. Images of the mouse tumors on the indicated days are shown in Figure 13C. Tumor growth measured by luminescence is shown in Figure 13D. Survival for each group is shown in Figure 13E. Overall mouse weight is shown in Figure 13F. The results also showed toxicity in TVEC-treated mice regardless of the other treatments. Loss of GD2 expression on SKNBe(2)-CD19 / Luc tumor cells was revealed in subsequent tests.

[0408] To evaluate the antitumor effect of rAAV8-CAG-234-MRE anti-GD2 / anti-CD3ε bispecific fusion protein in two neuroblastoma xenograft models, 5 × 10 12Mice were injected with 1 gc / kg AAV vector or control. An overview of the experiment is provided in Figure 14A, and surface staining of GD2 on tumor cells is shown in Figure 14B. Images of SK-N-Be(2)-Luc tumor-bearing mice on the indicated days are shown in Figure 14C. Tumor growth measured by luminescence and tumor volume is shown in Figures 14D and 14E, respectively. Survival for each group is shown in Figure 14F. The concentration of the anti-GD2 / anti-CD3ε bispecific fusion protein is shown in Figure 14G. A standard curve of the anti-GD2 / anti-CD3ε bispecific fusion protein concentration is shown in Figure 14H. Total mouse weight is shown in Figure 14I. Images of CHP134-Luc tumor-bearing mice on the indicated days are shown in Figure 14J. Tumor growth measured by luminescence and tumor volume is shown in Figures 14K and 14L, respectively. Survival for each group is shown in Figure 14M. The concentration of the anti-GD2 / anti-CD3ε bispecific fusion protein is shown in Figure 14N. A standard curve of the anti-GD2 / anti-CD3ε bispecific fusion protein concentration is shown in Figure 14O. Total mouse weight is shown in Figure 14P. These results showed a significant increase in survival observed for anti-GD2 / anti-CD3ε bispecific fusion protein-treated mice in both models. Serum analysis further showed appropriate levels of circulating anti-GD2 / anti-CD3ε bispecific fusion protein.

[0409] To evaluate the antitumor effect of rAAV8-CAG-234-MRE anti-GD2 / anti-CD3ε bispecific fusion protein in combination with anti-PDL1 or oncolytic herpes virus (HSV1716), CHP134-LUC tumor-bearing huPBMC-NSGS mice were injected with 5×10 12 gc / kg AAV vector or control. huPBMC were administered three times on days 14, 21, and 28 after tumor engraftment. A subset of mice received five intraperitoneal administrations of 100 μg anti-PDL1 (on days 18, 21, 25, 28, 32, 35) or 1×10 on days 18, 25, 32 after tumor engraftment. 5HSV1716 of pfu was administered intratumorally three times. An overview of the experiment is provided in Fig. 15A. Images of tumor-bearing mice treated with AAV vector or control + anti-PDL1 on the indicated days are shown in Fig. 15B. Tumor growth measured by luminescence and tumor volume is shown in Figs. 15C and 15D, respectively. Survival for each group is shown in Fig. 15E. The concentration of the anti-GD2 / anti-CD3ε bispecific fusion protein is shown in Fig. 15F. A standard curve of the anti-GD2 / anti-CD3ε bispecific fusion protein concentration is shown in Fig. 15G. The overall mouse weight is shown in Fig. 15H. Images of tumor-bearing mice treated with AAV vector, HSV1716, or AAV vector + HSV1716 on the indicated days are shown in Fig. 15I. Tumor growth measured by luminescence and tumor volume is shown in Figs. 15J and 15K, respectively. Survival for each group is shown in Fig. 15L. The concentration of the anti-GD2 / anti-CD3ε bispecific fusion protein is shown in Fig. 15M. A standard curve of the anti-GD2 / anti-CD3ε bispecific fusion protein concentration is shown in Fig. 15N. The overall mouse weight is shown in Fig. 15O. These results show a significant benefit with HSV1716 treatment alone. Two HSV1716 control mice without measurable primary tumors developed metastatic disease, but metastatic disease was not observed in the anti-GD2 / anti-CD3ε bispecific fusion protein + HSV1716 combination therapy mice.

[0410] To evaluate the pharmacokinetics of the anti-GD2 / anti-CD3ε bispecific fusion protein, NSGS mice were given 10 μg (0.4 mg / kg) of the anti-GD2 / anti-CD3ε bispecific fusion protein or 5×10 12Mice were injected with 5×10

[0411] gc / kg of AAV vector. Serum was collected at the indicated time points. The concentration of the purified anti-GD2 / anti-CD3ε bispecific fusion protein after injection is shown in Figure 16A. The concentration of the AAV-expressed anti-GD2 / anti-CD3ε bispecific fusion protein is shown in Figure 16B. These results indicate that while the level of the purified anti-GD2 / anti-CD3ε bispecific fusion protein drops below the detection threshold within 5 hours of injection, the AAV-expressed anti-GD2 / anti-CD3ε bispecific fusion protein becomes detectable for the first time at 72 hours and reaches a steady-state concentration within 14 days. The sustained level of the anti-GD2 / anti-CD3ε bispecific fusion protein was detected up to 200 days after injection. 12 To evaluate the pharmacokinetics of the anti-GD2 / anti-CD3ε bispecific fusion protein in immunocompetent mice, BALB / c mice were injected with 5×10

[0412] gc / kg AAV vector and serum was collected at the indicated time points. The serum concentration of the anti-GD2 / anti-CD3ε bispecific fusion protein at the indicated time points is shown in Figure 17. These results indicate that a steady-state concentration was achieved within 14 days. The sustained level of the anti-GD2 / anti-CD3ε bispecific fusion protein was detected on day 125 after injection. 12 To develop a mouse CHLA255-luc metastatic neuroblastoma model for a preclinical trial of AAV8-anti-GD2 / anti-CD3ε bispecific fusion protein therapy for disseminated disease, NSG-SGM3 mice were retroorbitally injected with 5×10 6 gc / kg of AAV vector or GFP control. On day 25 after rAAV8 injection, mice were injected with 5×10 6It was established by injecting individual human CHLA255-Luc neuroblastoma cells. Regardless of the reagent, all injections were prepared at a total volume of 100 μL using sterile PBS as a diluent. For serum pharmacokinetic analysis, a small amount of blood was collected from the retro-orbital sinus of each mouse on day 21 after rAAV8 injection. The mice were monitored for tumor growth with Xenogen IVIS Spectrum, and the disease progression was analyzed with Living Image 4.4 software (Caliper Life Sciences, Hopkinton, MA). Mouse imaging was started 7 days after tumor cell injection and then performed weekly thereafter until the experimental endpoint criteria were met. When the criteria were met, the animals were humanely euthanized. All surviving animals were sacrificed on day 42 for histological analysis. Some evidence of graft-versus-host disease was observed in both the treatment group and the control group. The experimental outline is provided in Figure 18A. Images of tumor-bearing mice on the indicated days are shown in Figure 18B. Tumor growth measured by luminescence is shown in Figure 18C. The overall mouse weight is shown in Figure 18D. The concentration of anti-GD2 / anti-CD3ε bispecific fusion protein is shown in Figure 18E. Images of mouse organs extracted by autopsy are shown in Figure 18F. Fluorescent images of brain samples and liver samples are shown in Figures 18G and 18H, respectively. Comparative fluorescent images of brain, liver, and lung tissues are shown in Figure 18I. Measurement of circulating immune cells is shown in Figure 18J. These results showed that anti-GD2 / anti-CD3ε bispecific fusion protein therapy prevented or controlled the growth of metastatic disease compared to the control.

[0413] To develop a mouse CHLA255-luc metastatic neuroblastoma model for the efficacy of AAV8-anti-GD2 / anti-CD3ε bispecific fusion protein therapy, NSG-SGM3 mice were retro-orbitally injected with 5 × 10 12 genome copies per kilogram (gc / kg) of AAV vector or GFP control. On day 25 after rAAV8 injection, the mice were injected with 5 × 10 6 individual huPBMCs, and then four weekly injections (retro-orbital or tail vein) of huPBMCs were performed. Metastatic disease was induced 4 hours after the first huPBMC injection by injecting 1 × 10 5or 5×10 5 established by injecting 5×10 human CHLA255-Luc neuroblastoma cells. Regardless of the reagent, all injections were prepared in a total volume of 100 μL using sterile PBS as a diluent. For serum pharmacokinetic analysis, a small amount of blood was collected from the retroorbital sinus of each mouse on day 21 after rAAV8 injection. Mice were monitored for tumor growth with Xenogen IVIS Spectrum, and disease progression was analyzed with Living Image 4.4 software (Caliper Life Sciences, Hopkinton, MA). Mouse imaging was started 7 days after tumor cell injection and then performed weekly thereafter until the experimental endpoint criteria were met, at which point the animals were humanely euthanized. An overview of the experiment is provided in Figure 19A. Images of mice injected with 5×10 5 on the indicated day are shown in Figure 19B. The overall survival of the mice is shown in Figure 19C, and the overall mouse weight is shown in Figure 18D. Images of mice injected with 1×10 5 on the indicated day are shown in Figure 19E. The overall survival of the mice is shown in Figure 19F, and the overall mouse weight is shown in Figure 19G. These results indicate that AAV8-anti-GD2 / anti-CD3ε bispecific fusion protein therapy prevented or controlled the growth of metastatic disease compared to the control.

[0414] To test the efficacy of AAV8-anti-GD2 / anti-CD3ε bispecific fusion protein therapy in an established metastatic neuroblastoma model, NSG-SGM3 mice were retroorbitally injected with 5 ×10 12 gc / kg of AAV vector or GFP control. Metastatic disease was established by injecting 1×10 6 human CHLA255-Luc neuroblastoma cells into the tail vein of these mice. Seven days after tumor engraftment, the mice were injected with 5×10 6Individual huPBMCs were injected, followed by three weekly injections (retro-orbital or tail vein) of huPBMCs. In this study, a total of 20 mice were used, equally divided between genders. The treatment and control groups consisted of 5 male mice and 5 female mice each. Regardless of the reagent, all injections were prepared at a total volume of 100 μL using sterile PBS as a diluent. For serum pharmacokinetic analysis, a small amount of blood was collected from the retro-orbital sinus of each mouse on day 21 after rAAV8 injection. The mice were monitored for tumor growth using Xenogen IVIS Spectrum, and the disease progression was analyzed using Living Image 4.4 software (Caliper Life Sciences, Hopkinton, MA). Mouse imaging was initiated 7 days after tumor cell injection and then performed weekly thereafter until the experimental endpoint criteria were met, at which point the animals were humanely euthanized. An overview of the experiment is provided in Figure 20A. Images of tumor-bearing mice on the indicated days are shown in Figure 20B. Tumor growth measured by luminescence is shown in Figure 20C. The concentration of the anti-GD2 / anti-CD3ε bispecific fusion protein is shown in Figure 20D. The overall survival of the mice is shown in Figure 20E, and the overall mouse weight is shown in Figure 20F. These results showed that female mice treated with AAV8-anti-GD2 / anti-CD3ε bispecific fusion protein had slightly delayed tumor growth, while 4 out of 5 males had no tumors and ultimately died due to GvHD. Also, male mice were found to have approximately twice as much anti-GD2 / anti-CD3ε bispecific fusion protein in their serum compared to female mice.

[0415] To test the efficacy of AAV8-anti-GD2 / anti-CD3ε bispecific fusion protein therapy in preventing the growth of CHLA255-luc metastatic nodules immediately after tumor seeding, NSG-SGM3 mice were injected retro-orbitally with 5×10 12 gc / kg of AAV vector or GFP control. On day 25 after rAAV8 injection, metastatic disease was established by injecting 1×10 6 individual human CHLA255-Luc neuroblastoma cells into the tail vein of these mice. Four hours after tumor engraftment, the mice were given 1×10 6Individual huPBMCs were injected, followed by four weekly injections (postorbital or tail vein) of huPBMCs. Regardless of the reagent, all injections were prepared in a total volume of 100 μL using sterile PBS as a diluent. For serum pharmacokinetic analysis, a small amount of blood was collected from the retroorbital sinus of each mouse on day 21 after rAAV8 injection. Mice were monitored for tumor growth with Xenogen IVIS Spectrum, and disease progression was analyzed with Living Image 4.4 software (Caliper Life Sciences, Hopkinton, MA). Mouse imaging was initiated 7 days after tumor cell injection and then performed weekly thereafter until the experimental endpoint criteria were met, at which point the animals were humanely euthanized. All surviving animals were sacrificed on day 42 for histological analysis. Some evidence of graft-versus-host disease was observed in both the treatment and control groups. The experimental outline is provided in Figure 21A. Images of tumor-bearing mice on the indicated days are shown in Figure 21B. Tumor growth measured by luminescence is shown in Figure 21C. The overall survival of the mice is shown in Figure 21D, and the overall mouse weight is shown in Figure 21E. The concentration of the anti-GD2 / anti-CD3ε bispecific fusion protein is shown in Figure 21F.

[0416] To test the efficacy of AAV8-anti-GD2 / anti-CD3ε bispecific fusion protein therapy in a GD2-expressing lung cancer model, male mice were injected with 1×10 12 gc / kg of the AAV vector or GFP control by retroorbital injection, while female mice were injected with 2×10 12 , 3×10 12 , or 1×10 13 gc / kg of the AAV vector or GFP control (n = 5 / group). All rAAV8 injections were performed 25 days prior to tail vein injection of 1×10 6 individual H446-Luc small cell lung cancer cells. huPBMCs were administered 3 days after tumor engraftment and then continued weekly for a total of 4 injections. 2×10 12Female mice at the gc / kg rAAV8 dosage level were injected with an equivalent volume of PBS (without huPBMC) to serve as a control. The experimental overview is provided in Figure 22A. Images of tumor-bearing mi...

Claims

1. Recombinant adeno-associated virus (rAAV) vector, with a 5' to 3' position. (a) 5' AAV reverse terminal repeat sequence (ITR); (b) Promoter; (c) Below: (i) A GD2 binding site of an anti-GD2 antibody comprising a light chain variable region (VL) containing complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3) sequences of SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, or SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, respectively, and a heavy chain variable region (VH) containing CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, or SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively; (ii) Linker peptide, and (iii) CD3 binding site including VH and VL of the anti-CD3 antibody A transgene encoding a bispecific fusion protein containing the following: (d) Modified RNA stability modifier elements (MREs); and (e) 3' AAV ITR A recombinant adeno-associated virus (rAAV) vector containing the above.

2. The rAAV vector according to claim 1, wherein the promoter is selected from the group consisting of the chicken β-actin promoter, the elongation factor 1α (EF1α) promoter, the monkey virus 40 (SV40) promoter, or the CAG promoter.

3. The rAAV vector according to claim 1, wherein the promoter contains a sequence that is at least 95% identical to SEQ ID NO:

66.

4. (a) Anti-CD3 antibody VH comprises CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, respectively, and anti-CD3 antibody VL comprises CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90, respectively, and / or (b) The anti-CD3 antibody VH contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 91, SEQ ID NO: 92, and SEQ ID NO: 93, respectively, and the anti-CD3 antibody VL contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively. The rAAV vector according to claim 1.

5. (i) (a) Anti-GD2 antibodies VL and VH each contain sequences that are at least 95% identical to SEQ ID NO: 2 and SEQ ID NO: 1, or (b) Anti-GD2 antibodies VL and VH contain sequences that are at least 95% identical to SEQ ID NO: 4 and SEQ ID NO: 3, respectively, and / or (ii) (a) Anti-CD3 antibodies VH and VL contain sequences that are at least 95% identical to SEQ ID NO: 14 and SEQ ID NO: 15, respectively, or (b) Anti-CD3 antibodies VH and VL contain sequences that are at least 95% identical to SEQ ID NO: 18 and SEQ ID NO: 19, respectively. The rAAV vector according to claim 1.

6. The rAAV vector according to claim 5, wherein the GD2 binding site or CD3 binding site is a single-chain variable region fragment (scFv).

7. (i) (a) Anti-GD2 antibody VL is fused to anti-GD2 antibody VH using an scFv linker peptide containing the amino acid sequence of SEQ ID NO: 25, or (b) Anti-GD2 antibody VL is fused to anti-GD2 antibody VH by an scFv linker peptide containing the amino acid sequence of SEQ ID NO: 20, and / or (ii) Anti-CD3 antibody VH is fused to anti-CD3 antibody VL by an scFv linker peptide containing the same sequence as SEQ ID NO:

25. The rAAV vector according to claim 6.

8. (i) The GD2 binding site comprises a sequence that is at least 95% identical to SEQ ID NO: 7, and / or (ii) (a) The CD3 binding site contains a sequence that is at least 95% identical to SEQ ID NO: 16, and / or (b) The CD3 binding site contains a sequence that is at least 95% identical to SEQ ID NO: 1, The rAAV vector according to claim 4.

9. (a) The bispecific fusion protein is (i) An N-terminal signal peptide containing at least 95% identical sequence to SEQ ID NO: 26, and / or (ii) Sequences that are at least 95% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO:

13. Includes, (b) The introduced gene is (i) A sequence that is at least 95% identical to SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, or SEQ ID NO: 45, and / or (ii) A regulatory element at the 5' or 3' position of the sequence encoding the bispecific fusion protein, and / or (iii) Kossack sequence Includes, (c) The regulatory element is derived from a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and contains a sequence that is at least 95% identical to SEQ ID NO: 64, and / or (d) The 3' AAV ITR contains a sequence that is at least 95% identical to SEQ ID NO:

59. The rAAV vector according to claim 1.

10. The vector is (a) A polyadenylated sequence at 3' and 5' of the transgene sequence and / or (b) Antibiotic resistance gene sequences The rAAV vector according to claim 1, further comprising:

11. (a) The polyadenylated sequence is a bovine growth hormone (BGH) polyadenylated sequence that is at least 95% identical to SEQ ID NO: 65, and / or (b) The antibiotic resistance gene is the kanamycin resistance gene, The rAAV vector according to claim 10.

12. The rAAV vector according to claim 1, comprising a sequence that is at least 95% identical to one of SEQ ID NO: 53 to 57.

13. A recombinant adeno-associated virus (rAAV) vector containing a sequence at least 90% identical to SEQ ID NO:

11.

14. A pharmaceutical formulation comprising an rAAV vector according to any one of claims 1 to 13, or an rAAV vector according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier, for reducing the risk of metastatic disease in a patient, delaying the onset of said disease, or preventing said disease.

15. A pharmaceutical formulation comprising an rAAV vector according to any one of claims 1 to 13, or an rAAV vector according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier, for promoting T cell-mediated killing of circulating tumor cells in a patient.

16. The rAAV vector or the pharmaceutical formulation according to claim 15, or a pharmaceutical formulation comprising the rAAV vector, wherein the rAAV vector or the pharmaceutical formulation is administered concurrently with treatment of a primary tumor.

17. The rAAV vector according to claim 16, or a pharmaceutical formulation comprising the rAAV vector, wherein treatment of the primary tumor includes surgical resection, radiotherapy, chemotherapy, or immunotherapy.

18. A pharmaceutical formulation comprising the rAAV vector according to any one of claims 1 to 13, or the rAAV vector according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier, for preventing cancer or cancer recurrence in patients with a predisposition to develop GD2+ tumors.

19. The rAAV vector or the pharmaceutical formulation according to claim 14, wherein the rAAV vector or the pharmaceutical formulation is administered together with a checkpoint inhibitor selected from the group consisting of a CTLA-4 inhibitor, a PD-1 inhibitor, and a PD-L1 inhibitor.

20. The rAAV vector according to claim 19, or a pharmaceutical formulation comprising an rAAV vector, wherein the checkpoint inhibitor is selected from the group consisting of pembrolizumab, ipilimumab, nivolumab, and atezolizumab.

21. A pharmaceutical formulation comprising a recombinant adeno-associated virus (rAAV) vector according to any one of claims 1 to 13, and a pharmaceutically acceptable carrier.

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