Adeno-associated virus vectors and methods for reducing risk of metastasis, treating and preventing metastasis thereof

Abstract

The present disclosure provides recombinant adeno-associated virus (rAAV) vectors that express bispecific fusion proteins that bind to GD2 and CD3, and methods of using the same to reduce the risk of, prevent or treat metastasis. The present disclosure also provides rAAV vectors for the expression of bispecific fusion proteins.

Description

[0001] Cross-references

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 391,967, filed on July 25, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure generally relates to adeno-associated viral (AAV) vectors for delivering transgenic sequences encoding bispecific fusion proteins comprising a GD2 binding site and a CD3 binding site. The present disclosure also relates to methods for killing circulating tumor cells, thereby reducing the risk of cancer and metastatic disease, delaying the onset of cancer and metastatic disease, and preventing cancer and metastatic disease. Background Art

[0004] Cancer remains a significant health problem worldwide and is the second leading cause of death in the U.S. Current treatment options for cancer are not effective for all patients and can often be associated with significant adverse side effects.

[0005] Cancer immunotherapies are a promising treatment modality because they exhibit greater specificity than conventional chemotherapeutics and can promote tumor cell destruction by priming the patient's own immune system. Bispecific T cell engager proteins are recombinant fusion proteins that bind to both tumor cells and T cells, thereby stimulating tumor cell destruction.

[0006] Adeno-associated virus (AAV) has been used as a gene therapy vector to achieve long-term, consistent bloodstream levels of cancer immunotherapy. For example, AAV encoding a bispecific αCD19-αCD3 protein achieved persistence in the bloodstream for more than a year and anti-tumor efficacy in a CD19+ lymphoma model (Cripe et al., Science Advances, in press).

[0007] Given that metastases are thought to arise from circulating tumor cells, which can be considered the "leukemic compartment" of solid tumors, long-term, sustained immune pressure targeting cancer could be effectively used to prevent the development of metastases. Because circulating tumor cells exist outside the immunosuppressive solid tumor microenvironment, they may be more susceptible to immunotherapy.

[0008] The disaloganglioside GD2 (GD2) is a disialoganglioside with limited expression in normal tissues but overexpressed in a wide range of tumors. Depending on the tumor type, GD2 has been implicated in tumorigenesis and malignant phenotypes by enhancing cell proliferation, motility, migration, adhesion, and invasion. GD2 is highly expressed by nearly all neuroblastomas, most melanomas and retinoblastomas, and many Ewing sarcomas. To a more variable degree, GD2 is expressed by small cell lung cancers, gliomas, osteosarcomas, and soft tissue sarcomas. Summary of the Invention

[0009] The present disclosure relates to compositions and methods for using adeno-associated viral vectors to express bispecific fusion proteins to reduce the risk of, prevent, and treat cancer and metastasis.

[0010] The present disclosure relates to compositions and methods for using adeno-associated viral vectors to express bispecific fusion proteins to reduce the risk of, prevent, and treat cancer and metastasis.

[0011] In some aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3', (a) a 5' AAV inverted terminal repeat (ITR); (b) a promoter; and (c) a transgene encoding a bispecific fusion protein comprising: (i) a GD2 binding site comprising a light chain variable region (VL) and a heavy chain variable region (VH) of an anti-GD2 antibody, the light chain variable region (VL) comprising the complementary determining region 1 (CDR1), complementary determining region 2 (CDR2), and complementary determining region 3 (CDR3) sequences of SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, respectively, or SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81, respectively, and the heavy chain variable region (VH) comprising SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively, SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively. NO:78; (ii) a linker peptide, and (iii) a CD3 binding site comprising the VH and VL of an anti-CD3 antibody; (d) a modified RNA stability regulatory element (MRE) and (e) a 3' AAV ITR. In some embodiments, the promoter is selected from a chicken β-actin promoter, an elongation factor 1α (EF1α) promoter, a simian virus 40 (SV40) promoter, or a CAG promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter comprises a sequence at least 95% identical to SEQ ID NO:66. In some embodiments, the anti-GD2 antibody VL and VH comprise sequences 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 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 via a scFv linker peptide comprising the sequence of SEQ ID NO: 20. In some embodiments, the anti-GD2 antibody VL and VH comprise sequences 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 fragment (scFv). In some embodiments, the anti-GD2 antibody VL is fused to the anti-GD2 antibody VH via a scFv linker peptide comprising the sequence of SEQ ID NO: 20. In some embodiments, the GD2 binding site comprises a sequence 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 comprise sequences 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 fragment (scFv). In some embodiments, the anti-CD3 antibody VH is fused to the anti-CD3 antibody VL via an scFv linker peptide comprising a sequence identical to SEQ ID NO: 25. In some embodiments, the CD3 binding site comprises a sequence 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 comprise sequences 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 fragment (scFv). In some embodiments, the anti-CD3 antibody VH is fused to the anti-CD3 antibody VL via an scFv linker peptide comprising a sequence identical to SEQ ID NO: 25. In some embodiments, the CD3 binding site comprises a sequence 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 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' to the sequence encoding the bispecific fusion protein. In some embodiments, the regulatory element is 3' to the sequence encoding the bispecific fusion protein.In some embodiments, the regulatory element is derived from a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and comprises a sequence 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' to the transgene sequence and 5' to the 3' AAV ITR. In some embodiments, the polyadenylation sequence is a bovine growth hormone (BGH) polyadenylation sequence at least 95% identical to SEQ ID NO:65. In some embodiments, the 3' AAV ITR comprises a sequence 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 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.

[0012] In some aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a sequence at least 90% identical to SEQ ID NO:11.

[0013] 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 viral (rAAV) vector described herein, or a pharmaceutical formulation thereof.

[0014] 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 viral (rAAV) vector described herein, or a pharmaceutical formulation thereof.

[0015] 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 viral (rAAV) vector described herein or a pharmaceutical formulation thereof.

[0016] 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 as described herein or a pharmaceutical preparation thereof. In some embodiments, rAAV or its pharmaceutical preparation is administered simultaneously with the treatment of the primary tumor. In some embodiments, the treatment of the primary tumor comprises surgical resection, radiotherapy, chemotherapy or immunotherapy.

[0017] In some aspects, the present disclosure provides a method of preventing cancer in a patient prone to developing GD2+ tumors, comprising administering to the patient an effective amount of a recombinant adeno-associated viral (rAAV) vector described herein or a pharmaceutical formulation thereof.

[0018] In some aspects, the present disclosure provides a method for preventing cancer recurrence in patients in remission of GD2+ cancer, comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector as described herein or a pharmaceutical preparation thereof. In some embodiments, AAV or its pharmaceutical preparation is administered together with a checkpoint inhibitor selected from the following: CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors. In some embodiments, the checkpoint inhibitor is selected from: pembrolizumab, ipilimumab, nivolumab, and atezolizumab.

[0019] In some aspects, the present disclosure provides pharmaceutical formulations comprising a recombinant adeno-associated virus (rAAV) vector described herein and a pharmaceutically acceptable carrier.

[0020] In some aspects, the present disclosure provides a method for 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 terminal inverted 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 light chain variable region (VL) and a heavy chain variable region (VH) of an anti-GD2 antibody; a linker peptide; and a CD3 binding site comprising VH and VL of an anti-CD3 antibody.

[0021] In some aspects, the present disclosure provides a method for delaying the onset of metastatic disease in a patient, comprising administering to the patient an effective amount of an 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 light chain variable region (VL) and a heavy chain variable region (VH) of an anti-GD2 antibody; a linker peptide; and a CD3 binding site comprising VH and VL of an anti-CD3 antibody.

[0022] In some aspects, the present disclosure provides a method for 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 terminal inverted 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 light chain variable region (VL) and a heavy chain variable region (VH) of an anti-GD2 antibody; a linker peptide; and a CD3 binding site comprising VH and VL of an anti-CD3 antibody.

[0023] 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 terminal inverted 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 light chain variable region (VL) and a heavy chain variable region (VH) of an anti-GD2 antibody; a linker peptide; and a CD3 binding site comprising VH and VL of an anti-CD3 antibody.

[0024] In some aspects, rAAV or a pharmaceutical formulation thereof is administered concurrently with treatment of the primary tumor. In some aspects, treatment of the primary tumor comprises surgical resection, radiation therapy, chemotherapy, or immunotherapy.

[0025] In some aspects, the present invention provides a method for preventing cancer in a patient prone to developing 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 a light chain variable region (VL) and a heavy chain variable region (VH) of an anti-GD2 antibody; a linker peptide; and a CD3 binding site comprising the VH and VL of an anti-CD3 antibody.

[0026] In some aspects, the present invention provides a method for preventing cancer recurrence in a patient in remission of a 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 terminal inverted 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 light chain variable region (VL) and a heavy chain variable region (VH) of an anti-GD2 antibody; a linker peptide; and a CD3 binding site comprising the VH and VL of an anti-CD3 antibody.

[0027] 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 comprising a sequence 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; and a 3' AAV ITR.

[0028] In some aspects, rAAV or a pharmaceutical formulation thereof is administered 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.

[0029] In some aspects, the 5' AAV ITR comprises a sequence at least 95% identical to SEQ ID NO: 58. In some aspects, the 3' AAV ITR comprises a sequence at least 95% identical to SEQ ID NO: 59.

[0030] In some aspects, the promoter is selected from the group consisting of a chicken β-actin promoter, an elongation factor 1α (EF1α) promoter, a simian virus 40 (SV40) promoter, and a CAG promoter. In some aspects, the promoter is a CAG promoter. In some aspects, the promoter comprises a sequence that is at least 95% identical to SEQ ID NO: 66.

[0031] In some aspects, the anti-GD2 antibody VL of the GD2 binding site has the complementary determining region 1 (CDR1), complementary determining region 2 (CDR2), and complementary 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 the 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 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 via an scFv linker peptide comprising the sequence of SEQ ID NO: 20. In some aspects, the GD2 binding site comprises a sequence at least 95% identical to SEQ ID NO:5.

[0032] 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 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 via 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.

[0033] In some aspects, the linker peptide comprises a sequence identical to SEQ ID NO:25 or SEQ ID NO:20.

[0034] 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.

[0035] In some aspects, the anti-CD3 antibody VH and VL of the CD3 binding site comprise sequences 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 fragment (scFv). In some aspects, the anti-CD3 antibody VL of the CD3 binding site is fused to the anti-CD3 antibody VH of the CD3 binding site via an scFv linker peptide comprising a sequence identical to SEQ ID NO: 25. In some aspects, the CD3 binding site comprises a sequence at least 95% identical to SEQ ID NO: 16.

[0036] In some aspects, the anti-CD3 antibody VH 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 anti-CD3 antibody VL has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:94, SEQ ID NO:95, and SEQ ID NO:96, respectively.

[0037] In some aspects, the anti-CD3 antibody VH and VL of the CD3 binding site comprise sequences 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 fragment (scFv). In some aspects, the anti-CD3 antibody VL of the CD3 binding site is fused to the anti-CD3 antibody VH of the CD3 binding site via an scFv linker peptide comprising a sequence identical to SEQ ID NO: 25. In some aspects, the CD3 binding site comprises a sequence at least 95% identical to SEQ ID NO: 17.

[0038] In some aspects, the bispecific fusion protein comprises a sequence 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.

[0039] In some aspects, the bispecific fusion protein has an N-terminal signal peptide comprising a sequence at least 95% identical to SEQ ID NO:26.

[0040] In some aspects, 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.

[0041] In some aspects, the transgene further comprises a regulatory element 5' or 3' to the sequence encoding the bispecific fusion protein. In some aspects, the regulatory element is 3' to the sequence encoding the bispecific fusion protein. In some aspects, the regulatory element is derived from the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and comprises a sequence at least 95% identical to SEQ ID NO: 64.

[0042] In some aspects, the transgene also has a Kozak sequence.

[0043] In some aspects, the vector further has a polyadenylation sequence 3' to the transgene sequence and 5' to the 3' AAV ITR. In some aspects, the polyadenylation sequence is a bovine growth hormone (BGH) polyadenylation sequence that is at least 95% identical to SEQ ID NO:65.

[0044] In some aspects, the vector also has an antibiotic resistance gene sequence. In some aspects, the antibiotic resistance gene is a kanamycin resistance gene.

[0045] In some aspects, 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.

[0046] In some aspects, the present disclosure provides a recombinant AAV vector according to any of the above aspects.

[0047] In some aspects, the present disclosure provides a pharmaceutical preparation comprising the recombinant adeno-associated virus (rAAV) vector of any aspect described above and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figures 1A-1E Figure 1 is a vector map of an AAV2 plasmid encoding a bispecific fusion protein that binds GD2 and CD3. Figure 1A The vector map encodes a bispecific fusion protein comprising the VL and VH of hu3F8V5 and the VH and VL of an anti-CD3 antibody. Figure 1B The vector map encodes a bispecific fusion protein comprising the VL and VH of hu3F8V5 and the VH and VL of anti-CD3. Figure 1C

[00145] Figure 1 is a vector map encoding an alternative bispecific fusion protein comprising the VL and VH of hu3F8V5 and the VH and VL of anti-CD3. Figure 1D The vector map encodes a bispecific fusion protein comprising the VL and VH of an anti-GD2 antibody (14G2a) and the VH and VL of an anti-CD3 antibody. Figure 1E

[00145] The following is a map of vectors encoding alternative bispecific fusion proteins comprising the VL and VH of an anti-GD2 antibody (14G2a) and the VH and VL of an anti-CD3 antibody.

[0049] Figures 2A-2B Depicted are the construct designs of various constructs encoding bispecific anti-GD2 / anti-CD3ε bispecific fusion proteins.

[0050] Figure 3 Depicted are the results of a manufacturability analysis of anti-GD2 / anti-CD3ε bispecific fusion proteins as measured by binding to the indicated cell lines (upper panel) based on a standard curve for the control antibody belintoquinone (lower panel).

[0051] Figure 4 Depicted are graphs of the viability of the indicated cell lines following incubation with T cells and supernatants containing the indicated anti-GD2 / anti-CD3ε bispecific fusion proteins.

[0052] Figures 5A-5B Quantification of surface GD2 and CD19 expression in the indicated cell lines is depicted. Figure 5A Depicted are histograms of staining for GD2 and CD19 in the indicated cell lines. Figure 5B Depicted are the interpolated amounts of GD2 or CD19 surface molecules in the indicated cell lines based on flow cytometry results.

[0053] Figure 6 Depicted are graphs of the viability of the indicated cell lines (parental or CD19-expressing) following incubation with T cells and supernatants containing the indicated anti-GD2 / anti-CD3ε bispecific fusion proteins.

[0054] Figure 7 Depicted are quantification of surface GD2 expression in the indicated cell lines (upper and lower left panels) and interpolated numbers of GD2 surface molecules in the indicated cell lines (lower right panel).

[0055] Figure 8 Depicted are quantification of surface GD2 expression in the indicated parental (upper panels) or luciferase reporter (lower panels) cell lines.

[0056] Figure 9 Graphs depicting viability of the indicated cell lines following incubation with human peripheral blood mononuclear cells (huPBMCs) and the indicated concentrations of anti-GD2 / anti-CD3ε bispecific fusion protein (upper panel) and GD2 expression in the assayed cell lines (lower panel).

[0057] Figures 10A-10B Depicted are the ( Figure 10A ) and as molecular quantification of equivalent soluble fluorescent dyes ( Figure 10B) Quantification of surface GD2 expression in the indicated cell lines. Figure 10C Graph depicting viability of the indicated cell lines following incubation with huPBMCs and the indicated concentrations of anti-GD2 / anti-CD3ε bispecific fusion protein.

[0058] Figure 11 Graph depicting viability of the indicated cell lines following incubation with huPBMCs and the indicated concentrations of anti-GD2 / anti-CD3ε bispecific fusion protein.

[0059] Figure 12 Graph depicting viability of the indicated cell lines following incubation with huPBMCs and the indicated concentrations of anti-GD2 / anti-CD3ε bispecific fusion protein.

[0060] Figures 13A-13F Depicted are the results of a murine model of the antitumor effect of an anti-GD2 / anti-CD3ε bispecific fusion protein with and without co-treatment with the oncolytic virus talimogene laherparepvec (TVEC). Figure 13A Depicts the experimental overview. Figure 13B Shown are images of GD2 surface staining in tumor cells to be transplanted. Figure 13C Images of mouse tumors on the indicated days are depicted. Figure 13D Tumor size measured by luminescence is depicted. Figure 13E Graphs measuring the survival rate of each group are shown. Figure 13F A graph depicting overall mouse body weight is shown.

[0061] Figures 14A-14P Depicted are the results of a murine model demonstrating the anti-tumor effects of an anti-GD2 / anti-CD3ε bispecific fusion protein in two neuroblastoma xenograft models. Figure 14A Depicts the experimental overview. Figure 14B Shown are images of GD2 surface staining in tumor cells to be transplanted. Figure 14C Images of mouse tumors on the indicated days are depicted. Figure 14D Tumor size measured by luminescence is depicted. Figure 14E Tumor size, measured by tumor volume, is depicted. Figure 14F Graphs measuring the survival rate of each group are shown. Figure 14G Depicted are graphs showing the levels of anti-GD2 / anti-CD3ε bispecific fusion protein in mouse serum. Figure 14H A standard curve of anti-GD2 / anti-CD3ε bispecific fusion protein concentrations in mouse serum was plotted. Figure 14I is a graph depicting total mouse body weight. Figure 14J Images of mouse tumors on the indicated days are depicted. Figure 14K Tumor size measured by luminescence is depicted. Figure 14LTumor size, measured by tumor volume, is depicted. Figure 14M Graphs measuring the survival rate of each group are shown. Figure 14N Depicted are graphs showing the levels of anti-GD2 / anti-CD3ε bispecific fusion protein in mouse serum. Figure 14O A standard curve of anti-GD2 / anti-CD3ε bispecific fusion protein concentrations in mouse serum was plotted. Figure 14P is a graph depicting total mouse body weight.

[0062] Figures 15A-15O Depicted are the results of a murine model of the anti-tumor effect of an anti-GD2 / anti-CD3ε bispecific fusion protein in combination with anti-PDL1 or oncolytic herpes virus (HSV1716). Figure 15A Depicts the experimental overview. Figure 15B Depicted are images of mouse tumors on the indicated days in mice treated with anti-GD2 / anti-CD3ε bispecific fusion protein in combination with control or anti-PDL1. Figure 15C Depicted are tumor sizes measured by luminescence in mice treated with anti-GD2 / anti-CD3ε bispecific fusion protein in combination with control or anti-PDL1. Figure 15D Depicted are tumor sizes measured by tumor volume in mice treated with anti-GD2 / anti-CD3ε bispecific fusion protein in combination with control or anti-PDL1. Figure 15E Shown are graphs measuring the survival rate of each group of mice treated with anti-GD2 / anti-CD3ε bispecific fusion protein in combination with control or anti-PDL1. Figure 15F Depicted are graphs showing the levels of anti-GD2 / anti-CD3ε bispecific fusion protein in the sera of mice treated with the anti-GD2 / anti-CD3ε bispecific fusion protein in combination with control or anti-PDL1. Figure 15G A standard curve of the concentration of anti-GD2 / anti-CD3ε bispecific fusion protein in the sera of mice treated with the anti-GD2 / anti-CD3ε bispecific fusion protein in combination with control or anti-PDL1 is depicted. Figure 15H is a graph depicting changes in total body weight of mice treated with anti-GD2 / anti-CD3ε bispecific fusion protein in combination with control or anti-PDL1. Figure 15I Depicted are images of mouse tumors on the indicated days in mice treated with anti-GD2 / anti-CD3ε bispecific fusion protein in combination with control or HSV1716. Figure 15J Depicted are tumor sizes measured by luminescence in mice treated with anti-GD2 / anti-CD3ε bispecific fusion protein in combination with control or HSV1716. Figure 15K Depicted are tumor sizes measured by tumor volume in mice treated with anti-GD2 / anti-CD3ε bispecific fusion protein in combination with control or HSV1716. Figure 15LShown are graphs measuring the survival rate of each group of mice treated with anti-GD2 / anti-CD3ε bispecific fusion protein in combination with control or HSV1716. Figure 15M Depicted are graphs showing the levels of anti-GD2 / anti-CD3ε bispecific fusion protein in the sera of mice treated with anti-GD2 / anti-CD3ε bispecific fusion protein in combination with control or HSV1716. Figure 15N A standard curve of the concentration of anti-GD2 / anti-CD3ε bispecific fusion protein in the sera of mice treated with the anti-GD2 / anti-CD3ε bispecific fusion protein in combination with control or HSV1716 is depicted. Figure 15O is a graph depicting changes in total body weight of mice treated with anti-GD2 / anti-CD3ε bispecific fusion protein in combination with control or HSV1716.

[0063] Figures 16A-16B Depicted are the results of pharmacokinetic analysis of anti-GD2 / anti-CD3ε bispecific fusion protein in mouse serum. Figure 16A Depicted are the concentrations of anti-GD2 / anti-CD3ε bispecific fusion proteins in mouse serum at the indicated times following direct injection. Figure 16B Depicted are the concentrations of anti-GD2 / anti-CD3ε bispecific fusion protein in mouse serum at the indicated times of administration of AAV constructs encoding the anti-GD2 / anti-CD3ε bispecific fusion protein.

[0064] Figure 17 Depicted are the concentrations of anti-GD2 / anti-CD3ε bispecific fusion protein in mouse serum at the indicated times of administration of AAV constructs encoding the anti-GD2 / anti-CD3ε bispecific fusion protein.

[0065] Figures 18A-18J Depicted are the results of a murine metastatic neuroblastoma model used for preliminary testing of AAV8-anti-GD2 / anti-CD3ε bispecific fusion protein therapy for disseminated disease. Figure 18A Depicts the experimental overview. Figure 18B Images of mouse tumors on the indicated days are depicted. Figure 18C Tumor size measured by luminescence is depicted. Figure 18D Graphs depicting overall mouse body weight changes are depicted. Figure 18E Shown are graphs illustrating the levels of anti-GD2 / anti-CD3ε bispecific fusion protein in mouse serum. Figure 18F Depicted are images of mouse organs after necropsy. Figure 18G Depicted are fluorescent images of post-mortem mouse brain samples. Figure 18H Depicted are fluorescent images of post-necropsy mouse liver samples. Figure 18I Fluorescent images of post-mortem mouse brain (top), liver (middle), and lung (bottom) samples. Figure 18JDepicted are measurements of the proportions of the indicated circulating immune cells.

[0066] Figures 19A-19G Depicted are the results of a murine model demonstrating the anti-tumor effects of an anti-GD2 / anti-CD3ε bispecific fusion protein in the murine CHLA255-luc metastatic neuroblastoma model. Figure 19A Depicts the experimental overview. Figure 19B The image shows the injection of 5×10 5 Images of mouse tumors on the indicated days in mice with 100 tumor cells. Figure 19C Shown is a depiction of the injection of 5 × 10 5 Figure 3 is a graph showing the overall survival rate of mice in each group with 10 tumor cells. Figure 19D Depicted is the description of the injection of 5 × 10 5 Figure 3 shows the overall body weight changes of mice with 10 tumor cells. Figure 19E Depicted is the injection of 1×10 5 Images of mouse tumors on the indicated days in mice with 100 tumor cells. Figure 19F Shown is a depiction of the injection of 1×10 5 Figure 3 is a graph showing the overall survival rate of mice in each group with 10 tumor cells. Figure 19G Depicted is the description of the injection of 1×10 5 Figure 3 shows the overall body weight changes of mice with 10 tumor cells.

[0067] Figures 20A-20F Depicted are the results of a murine model demonstrating the anti-tumor effects of an anti-GD2 / anti-CD3ε bispecific fusion protein in an established metastatic neuroblastoma model. Figure 20A Depicts the experimental overview. Figure 20B Images of mouse tumors on the indicated days are depicted. Figure 20C Tumor size measured by luminescence is depicted. Figure 20D Depicted are graphs showing the levels of anti-GD2 / anti-CD3ε bispecific fusion protein in mouse serum. Figure 20E Graphs measuring the survival rate of each group are shown. Figure 20F is a graph depicting changes in overall body weight of mice.

[0068] Figures 21A-21F Depicted are results from a murine model of AAV8-anti-GD2 / anti-CD3ε bispecific fusion protein therapy that prevented CHLA255-luc metastatic nodule growth shortly after tumor inoculation. Figure 21A Depicts the experimental overview. Figure 21B Images of mouse tumors at the indicated times are depicted. Figure 21C Tumor size measured by luminescence is depicted. Figure 21D Graphs measuring the survival rate of each group are shown. Figure 21E is a graph depicting changes in overall body weight of mice. Figure 21FDepicted are graphs showing the levels of anti-GD2 / anti-CD3ε bispecific fusion protein in mouse serum.

[0069] Figures 22A-22F Results from a GD2-expressing lung cancer model depicting the efficacy of AAV8-anti-GD2 / anti-CD3ε bispecific fusion protein therapy. Figure 22A Depicts the experimental overview. Figure 22B Images of mouse tumors on the indicated days are depicted. Figure 22C Tumor size measured by luminescence is depicted. Figure 22D Graphs measuring the survival rate of each group are shown. Figure 22E is a graph depicting changes in overall body weight of mice. Figure 22F Depicted are graphs showing the levels of anti-GD2 / anti-CD3ε bispecific fusion protein in mouse serum. DETAILED DESCRIPTION

[0070] 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.

[0071] The present disclosure also provides methods of using the rAAV described herein to reduce the risk of, prevent, or treat metastasis in a patient.

[0072] To facilitate understanding of this disclosure, a number of terms and phrases are defined below.

[0073] As used herein, the terms "a" and "an" mean "one or more" and include the plural unless the context is inappropriate.

[0074] The term "nucleic acid," "nucleotide" or "oligonucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed-base 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)).

[0075] The term "gene" may refer to a DNA segment involved in producing or encoding a polypeptide chain. It may include regions before and after the coding region (leader and trailer sequences) and intervening sequences (introns) between individual coding segments (exons).

[0076] "Promoter" is defined as one or more nucleic acid control sequences that direct the transcription of a nucleic acid. As used herein, a promoter includes nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements, which can be located up to several thousand base pairs from the start site of transcription.

[0077] " regulatory element " used herein refers to the nucleic acid sequence that can regulate the transcription of gene (such as transgenic) and / or regulate the stability of transcribed mRNA product or translation.In some embodiments, regulatory element can regulate the tissue-specific transcription of gene. Regulatory element can comprise at least one transcription factor binding site, such as the transcription factor binding site of muscle-specific transcription factor. Compared with the gene transcription from a separate promoter in the absence of regulatory element, regulatory element as used herein increases or enhances promoter-driven gene expression. Regulatory element as used herein can be present at any distance (that is, proximal or distal) from the transgenic that they regulate. Regulatory element as used herein can comprise the part of the larger sequence that participates in transcriptional control, such as the part of promoter sequence. However, a separate regulatory element is usually not enough to initiate transcription itself, but requires the presence of a promoter.

[0078] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation.

[0079] As used herein, the term "sequence of equivalent coding potential" refers to a nucleic acid sequence that is functionally equivalent to another reference nucleic acid. A sequence with equivalent coding potential can have or not have the same primary nucleotide sequence. For example, for a reference nucleic acid encoding an expressed polypeptide, a sequence with equivalent coding potential can functionally encode the same expressed polypeptide and can comprise the same primary nucleotide sequence as the reference nucleic acid, or can comprise one or more optional codons compared to the reference nucleic acid. For example, the endogenous nucleic acid sequence encoding the polypeptide can be changed by codon optimization to obtain a sequence encoding the same polypeptide. A codon-optimized sequence can be a sequence in which the codons in the polynucleotide encoding the polypeptide have been replaced to change the activity, expression and / or stability of the polynucleotide. For example, codon optimization can be used to change the sequence similarity degree of a sequence with equivalent coding potential compared to an endogenous gene sequence while retaining the potential for the protein product of the endogenous gene encoding the polypeptide.

[0080] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the term includes amino acid chains of any length, including full-length proteins and functional fragments thereof, in which the amino acid residues are linked by covalent peptide bonds.

[0081] The terms "variable domain" (e.g., VH domain or VL domain) and "variable region" are used interchangeably and refer to the portion of an antibody or immunoglobulin domain that exhibits sequence variability and is involved in determining the specificity and binding affinity of a particular antibody. Variability is not evenly distributed throughout the variable domain of an antibody; it is concentrated in the subdomains of each of the heavy and light chain variable regions. These subdomains are called "hypervariable regions" or "complementarity determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domain are called "framework" regions (FRMs or FRs) and provide a scaffold for the six CDRs in three-dimensional space to form an antigen-binding surface.

[0082] As used herein, the term "complementary" or "complementarity" refers to specific base pairing between nucleotides or nucleic acids. Complementary nucleotides are typically A and T (or A and U), and G and C.

[0083] 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 DNA segment that is involved in producing or encoding a polypeptide chain. A transgene can include regions preceding and following the coding region (leader and trailer sequences) as well as intervening sequences (introns) between individual coding segments (exons).

[0084] As used herein, in the context of nucleic acids (e.g., AAV vectors), the term "introducing" or "delivering" refers to the translocation of a nucleic acid from an extracellular space into a cell (e.g., a muscle cell). In some cases, introducing refers to the translocation of a nucleic acid from an extracellular space into the nucleus of a cell. Various methods of such translocation are contemplated, including but not limited to electroporation, contact with nanowires or nanotubes, receptor-mediated internalization, translocation via cell-penetrating peptides, liposome-mediated translocation, and the like.

[0085] As used herein, the term "packaged" or "encapsidated" refers to the inclusion of an AAV vector within a viral capsid to form an AAV particle.

[0086] The terms "substantial identity" or "substantially identical" as used in the context of polynucleotide or polypeptide sequences refer to sequences that have at least 60% sequence identity to a reference sequence. Alternatively, the percent identity can be any integer from 60% to 100%. Exemplary embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. One skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc.

[0087] For sequence comparison, a sequence is usually used as a reference sequence compared to a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, and if necessary, subsequence coordinates are specified, 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 sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters.

[0088] The algorithm that is suitable for determining sequence identity and sequence similarity percentage is BLAST and BLAST 2.0 algorithm, and it is described in Altschul etc. (1990) J.Mol.Biol.215:403-410 and Altschul etc. (1977) Nucleic Acids Res.25:3389-3402.The software for carrying out BLAST analysis can be publicly obtained by the National Center for Biotechnology Information (NCBI) website.This algorithm relates to first identifying high-scoring sequence pairs (HSP) by determining the short word of length W in the query sequence, when with the word of same length in the database sequence, described short word mates or meets some positive value threshold score T.T is referred to as neighborhood word score threshold value (Altschul etc., the same).These initial neighborhood word hits serve as the seed for starting search to find the longer HSP containing them.Then word hits extend in two directions along each sequence, until can increase cumulative comparison score. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of the word hit in each direction is terminated when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score goes to zero or below zero due to the accumulation of one or more negative-scoring residue alignments; or 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 the defaults word length (W) of 28, expectation (E) of 10, M=1, N=-2, and a comparison of both chains. For amino acid sequences, the BLASTP program uses the defaults word length (W) of 3, expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0089] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, if the smallest sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.01, more preferably less than about 10, then the match is considered to be a likely match. -5 , most preferably less than about 10 -20 , the nucleic acid is considered similar to the reference sequence.

[0090] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and, in some embodiments, refer to any mammalian subject, particularly a human, in need of diagnosis, treatment, or therapy. "Mammal," for therapeutic purposes, refers to any animal classified as a mammal, including humans, livestock and farm animals, and laboratory, zoo, sports, or pet animals such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, and the like. In some embodiments, the mammal is a human. None of these terms require medical supervision and / or cancer diagnosis or current cancer treatment.

[0091] As used herein, the term "effective delivery" or "effectively delivers" refers to the administration of a recombinant adeno-associated viral vector encoding a transgene, resulting in expression of the transgene in the desired cells or tissues.

[0092] As used herein, the term "effective amount" refers to an amount of a substance (e.g., a recombinant adeno-associated virus of the present disclosure) sufficient to achieve a beneficial or desired result (e.g., expression of a protein, or a desired prophylactic or therapeutic effect). An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a specific formulation or route of administration. As used herein, the term "treat" includes any effect that results in improvement of a condition, disease, disorder, or the like, or improvement of its symptoms, such as alleviation, reduction, regulation, improvement, or elimination.

[0093] Throughout this specification, where compositions are described as having, including, or comprising particular components, or where processes and methods are described as having, including, or comprising particular steps, it is contemplated that there are additionally compositions of the disclosure consisting essentially of, or consisting of, the recited components, as well as processes and methods according to the disclosure consisting essentially of, or consisting of, the recited process steps.

[0094] 1. Recombinant adeno-associated virus (AAV) vector

[0095] As used herein, "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 replicate and, when introduced into a host cell that also comprises one or more vectors encoding the rep and cap gene products, can be packaged into viral particles.

[0096] Inverted terminal repeats

[0097] Inverted terminal repeats (ITRs) are palindromic 145-nucleotide sequences that flank the transgene. Both the 5' and 3' ITRs of recombinant adeno-associated virus (rAAV) vectors are essential for integration of the transgene into the host cell genome (e.g., chromosome 19 in humans) and for encapsidation into AAV particles.

[0098] In some embodiments, the rAAV vectors of the present disclosure comprise ITR sequences from any one of the AAV serotypes, such as AAVrh.74, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13. In preferred embodiments, the recombinant AAV vectors described herein comprise AAV2 5' and 3' ITR sequences. 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, and AAV-rh10. L65, 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-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, or derivatives thereof. In some embodiments, the recombinant AAV vectors disclosed herein comprise AAV2 5' and 3' ITR sequences. In some embodiments, the recombinant AAV vectors disclosed herein comprise AAV8 5' and 3' ITR sequences.

[0099] In some embodiments, the recombinant AAV vectors described herein comprise a 5' AAV2 ITR that has 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 that is at least about 80% identical to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises a sequence that is at least about 85% identical to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises a sequence that is at least about 90% identical to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises a sequence that is at least about 95% identical to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises a sequence that is at least about 96% identical to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises a sequence that is at least about 97% identical to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises a sequence that is at least about 98% identical to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises a sequence that is at least about 99% identical to SEQ ID NO: 58. In some embodiments, the 5' AAV2 ITR comprises a sequence that is 100% identical 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.

[0100] In some embodiments, the recombinant AAV vectors described herein comprise a 3' AAV2 ITR that has 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 that is at least about 80% identical to SEQ ID NO: 59. In some embodiments, the 3' AAV2 ITR comprises a sequence that is at least about 85% identical to SEQ ID NO: 59. In some embodiments, the 3' AAV2 ITR comprises a sequence that is at least about 90% identical to SEQ ID NO: 59. In some embodiments, the 3' AAV2 ITR comprises a sequence that is at least about 95% identical to SEQ ID NO: 59. In some embodiments, the 3' AAV2 ITR comprises a sequence that is at least about 96% identical to SEQ ID NO: 59. In some embodiments, the 3' AAV2 ITR comprises a sequence that is at least about 97% identical to SEQ ID NO: 59. In some embodiments, the 3' AAV2 ITR comprises a sequence that is at least about 98% identical to SEQ ID NO: 59. In some embodiments, the 3' AAV2 ITR comprises a sequence that is at least about 99% identical to SEQ ID NO: 59. In some embodiments, the 3' AAV2 ITR comprises a sequence that is 100% identical 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.

[0101] Table 1A - AAV ITR sequences

[0102]

[0103] promoter

[0104] Promoters drive expression of AAV vector transgenes and are typically located upstream (or 5' to) the transgene whose expression they regulate.

[0105] In some embodiments, the recombinant AAV vectors disclosed herein comprise 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 comprise a strong constitutively active promoter to drive high-level expression of the transgene. For example, the promoter is a CAG promoter (cytomegalovirus early enhancer fused to a chicken β-actin promoter), a cytomegalovirus (CMV) promoter / enhancer, an elongation factor 1 alpha (EF1 alpha) promoter, a simian virus 40 (SV40) promoter, or a chicken β-actin promoter.

[0106] In some embodiments, the promoters described herein comprise 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 that is at least about 98% identical to SEQ ID NO: 66. In some embodiments, the CAG promoter comprises a sequence that is at least about 99% identical to SEQ ID NO: 66. In some embodiments, the CAG promoter comprises a sequence that is 100% identical to SEQ ID NO: 66.

[0107] CAG promoter sequence:

[0108]

[0109] (SEQ ID NO:66)

[0110] SV40 intron

[0111] In some embodiments, the recombinant AAV vectors of the present disclosure comprise an SV40 intron, which is a commonly used regulatory element in gene therapy vectors and enhances the translation and stability of expressed RNA transcripts.

[0112] In certain embodiments, the SV40 intron is downstream (ie, 3') of the promoter and upstream (ie, 5') of the transgene. In other embodiments, the SV40 intron can be downstream (ie, 3') of the transgene.

[0113] In some embodiments, the SV40 intron comprises a sequence that has 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 that has at least about 80% identity to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence that has at least about 85% identity to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence that has at least about 90% identity to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence that has at least about 95% identity to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence that is at least about 96% identical to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence that is at least about 97% identical to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence that is at least about 98% identical to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence that is at least about 99% identical to SEQ ID NO: 67. In some embodiments, the SV40 intron comprises a sequence that is 100% identical to SEQ ID NO: 67.

[0114] SV40 intron sequence:

[0115] GTAAGTTTAGTCTTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGTCCCTCAGTCGATGTTGCCTTTACTTCTAG

[0116] (SEQ ID NO:67)

[0117] polyadenylation sequence

[0118] In some embodiments, the recombinant AAV vectors of the present disclosure comprise 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 commonly used nucleic acid elements in gene therapy vectors that facilitate RNA export from the cell nucleus, RNA translation, and RNA stability.

[0119] In some embodiments, the recombinant AAV vectors of the present disclosure comprise 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 that is at least about 97% identical to SEQ ID NO: 65. In some embodiments, the BGH poly(A) tail comprises a sequence that is at least about 98% identical to SEQ ID NO: 65. In some embodiments, the BGH poly(A) tail comprises a sequence that is at least about 99% identical to SEQ ID NO: 65. In some embodiments, the BGH poly(A) tail comprises a sequence that is 100% identical 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.

[0120] In some embodiments, the recombinant AAV vectors of the present disclosure comprise 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 that is at least about 97% identical to SEQ ID NO: 68. In some embodiments, the SV40 poly(A) tail comprises a sequence that is at least about 98% identical to SEQ ID NO: 68. In some embodiments, the SV40 poly(A) tail comprises a sequence that is at least about 99% identical to SEQ ID NO: 68. In some embodiments, the SV40 poly(A) tail comprises a sequence that is 100% identical 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.

[0121] BGH poly(A) tail sequence:

[0122] CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAGAATAGCAGGCATGCTGGGGA

[0123] (SEQ ID NO:65)

[0124] SV40 poly(A) tail sequence:

[0125] AACTTGTTTATTGCAGCTTAATGGTTACAAATAAAGCAATAGCATCACAAATTTCAAAATAAAGCATTTTTTTCACTGC

[0126] (SEQ ID NO:68)

[0127] enhancer

[0128] In some embodiments, the recombinant AAV vectors of the present disclosure comprise one or more enhancer sequences. Enhancer sequences can increase the transcription level of a transgene, for example, by serving as binding sites for transcription factors and co-regulators that assist in DNA looping and recruit the transcription machinery to the promoter.

[0129] In some embodiments, the enhancer is downstream of the 5'ITR (i.e., 3' side) and upstream of the promoter (i.e., 5' side). In some embodiments, the enhancer is downstream of the promoter (i.e., 3' side) and upstream of the transgene (i.e., 5' side). In some embodiments, the enhancer is downstream of the transgene (i.e., 3' side) and upstream of the 3'UTR (i.e., 5' side).

[0130] Antibiotic resistance genes

[0131] 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.

[0132] 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.

[0133] 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.

[0134] Kanamycin variant 1

[0135] ATGGCTAAAATGAGAATATCACCGGAATTGAAAAAACTGATCGAAAAATACCGCTGCGTAAAAGATACGGAAGGAATGTCTCCTGCTAAGGTATATAAGCTGGTGGGAGAAAATGAAAACCTATATTTAAAAATGACGGACAGCCGGTATAAAGGGACCACCTATGATGTGGAACGGGAAAAGGACATGATGCTATGGCTGGAAGGAAAGCTGCCTGTTCCAAAGGTCCTGCACTTTGAACGGCATGATGGCTGGAGCAATCTGCTCATGAGTGAGGCCGATGGCGTCCTTTGCTCGGAAGAGTATGAAGATGAACAAAGCCCTGAAAAGATTATCGAGCTGTATGCGGAGTGCATCAGGCTCTTTCACTCCATCGACATATCGGATTGTCCCTATACGAATAGCTTAGACAGCCGCTTAGCCGAATTGGATTACTTACTGAATAACGATCTGGCCGATGTGGATTGCGAAAACTGGGAAGAAGACACTCCATTTAAAGATCCGCGCGAGCTGTATGATTTTTTAAAGACGGAAAAGCCCGAAGAGGAACTTGTCTTTTCCCACGGCGACCTGGGAGACAGCAACATCTTTGTGAAAGATGGCAAAGTAAGTGGCTTTATTGATCTTGGGAGAAGCGGCAGGGCGGACAAGTGGTATGACATTGCCTTCTGCGTCCGGTCGATCAGGGAGGATATCGGGGAAGAACAGTATGTCGAGCTATTTTTTGACTTACTGGGGATCAAGCCTGATTGGGAGAAAATAAAATATTATATTTTACTGGATGAATTGTTTTAG

[0136] (SEQ ID NO:97)

[0137] Kanamycin Variant 2

[0138] ttagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcat

[0139] (SEQ ID NO:122)

[0140] Kozak sequence

[0141] In some embodiments, the recombinant AAV vectors of the present disclosure comprise a Kozak sequence. In some embodiments, the Kozak sequence is an AAV2 Kozak sequence. In some embodiments, the Kozak sequence is an AAV8 Kozak sequence. In some embodiments, the Kozak sequence is an AAV-rh74 Kozak sequence. Exemplary Kozak sequences are shown in Table 1B below.

[0142] Table 1B. Exemplary Kozak sequences

[0143]

[0144]

[0145] In some embodiments, the Kozak sequence comprises a sequence having at least 85% (e.g., 85%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 98-121. In some embodiments, the Kozak sequence comprises a sequence having at least 85% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 98-121. In some embodiments, the Kozak sequence comprises a sequence having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 98-121. In some embodiments, the Kozak sequence comprises a sequence having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 98-121. In some embodiments, the Kozak sequence comprises a sequence having at least 97% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 98-121. In some embodiments, the Kozak sequence comprises a sequence having at least 98% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 98-121. In some embodiments, the Kozak sequence comprises a sequence having at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 98- 121. In some embodiments, the Kozak sequence comprises a sequence having the nucleic acid sequence of any one of SEQ ID NOs: 98-121.

[0146] αGD2-αCD3 transgenic

[0147] 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 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.

[0148] In some embodiments, the transgene is incorporated into the genome of the cell or can be expressed episomally.

[0149] GD2 binding site

[0150] The GD2 binding site may comprise a polypeptide or a complex of two or more polypeptides that specifically binds to disialoganglioside having the structure shown below.

[0151] GD2

[0152]

[0153] 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, and their corresponding complementarity determining regions (CDRs), which, in combination, can specifically bind to GD2. Table 2B lists the corresponding nucleotide sequences of the VH and VL domains of anti-GD2 antibodies.

[0154] Table 2A - αGD2 VH / VL and CDR amino acid sequences

[0155]

[0156]

[0157] Table 2B - αGD2 VH / VL nucleotide sequences

[0158]

[0159]

[0160] In some embodiments, the GD2 binding site comprises a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence selected from the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences listed in Table 2A as determined by Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunology Interest, NIH Publication No. 91-3242, Bethesda), the IMGT unique numbering scheme, Chothia (see, e.g., Chothia C & Lesk AM (1987), J. Mol. Biol. 215:495-495), MacCallum (see MacCallum RM et al., (1996) J. Mol. Biol. 196:901-917), or any other CDR determination method known in the art.

[0161] Unless otherwise indicated, the CDR sequences provided in Table 2A are identified according to the Kabat numbering scheme.

[0162] In some embodiments, the GD2 binding site comprises: (i) a VLCDR1 comprising the amino acid sequence of SEQ ID NO:73; (ii) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:74; (iii) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:75; (iv) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:70; (v) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:71; and (vi) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:72.

[0163] In some embodiments, the GD2 binding site comprises: (i) a VLCDR1 comprising the amino acid sequence of SEQ ID NO:79; (ii) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:80; (iii) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:81; (iv) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:76; (v) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:77; and (vi) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:78.

[0164] Table 2A further lists the amino acid sequences of exemplary VH and VL domains that can be combined to specifically bind to GD2. In some embodiments, the GD2 binding sites of the present disclosure comprise VH and VL domains that have 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 the VH and VL domain sequences listed in Table 2A. In some embodiments, the GD2 binding site comprises a VH comprising an amino acid sequence that has at least 80% sequence identity with the amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3; and a VL comprising an amino acid sequence that has at least 80% sequence identity with 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 an 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 an 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 an 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 an 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 an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3; and a VL comprising an amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 4.

[0165] In some embodiments, a 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, a 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% 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 an amino acid sequence according to SEQ ID NO: 1; and a VL comprising an amino acid sequence according to SEQ ID NO: 2.

[0166] In some embodiments, a 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, a 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 an amino acid sequence according to SEQ ID NO: 3; and a VL comprising an amino acid sequence according to SEQ ID NO: 4.

[0167] In some embodiments, the GD2 binding site includes, but is not limited to, a single-chain variable fragment (scFv), an antibody, Fab, Fab', F(ab')2, a minibody, or a nanobody (VHH). For example, in some embodiments, the bispecific fusion proteins of the present disclosure comprise scFv polypeptides that each specifically bind to GD2.

[0168] In some embodiments, the GD2-binding sites disclosed herein are in the form of scFvs. In some embodiments, the GD2-binding scFvs disclosed herein comprise an scFv linker polypeptide operably linking a VH domain and a VL domain. For example, a GD2-binding scFv comprises, from N-terminus to C-terminus, the VL domain of an anti-GD2 antibody, an scFv linker polypeptide, and the VH domain of an anti-GD2 antibody. In other embodiments, a GD2-binding scFv comprises, from N-terminus to C-terminus, the VH domain of an anti-GD2 antibody, an scFv linker polypeptide, and the VL domain of an anti-GD2 antibody.

[0169] In some embodiments, the scFv linker polypeptide comprises a sequence selected from the linker sequences in Table 3A.

[0170] Table 3A-scFv linker peptide sequences

[0171]

[0172]

[0173] 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.

[0174] Table 3B - Spacer Peptide Sequences

[0175]

[0176] Table 4A lists the amino acid sequences of exemplary GD2-binding scFvs. In some embodiments, the bispecific fusion proteins of the present disclosure comprise a GD2-binding scFv comprising 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.

[0177] Table 4A - GD2 binding scFv amino acid sequences

[0178]

[0179]

[0180] * Underlined italics Text indicates scFv linker sequence.

[0181] Table 4B - GD2 binding scFv nucleotide sequences

[0182]

[0183]

[0184] * Underline Text indicates scFv linker sequence.

[0185] In some embodiments, the bispecific fusion proteins of the present disclosure comprise a scFv that specifically binds to GD2, the scFv 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: 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 GD2 comprises a sequence that is at least about 97% identical to SEQ ID NO: 5. In some embodiments, the scFv that specifically binds GD2 comprises a sequence that is at least about 98% identical to SEQ ID NO: 5. In some embodiments, the scFv that specifically binds GD2 comprises a sequence that is at least about 99% identical to SEQ ID NO: 5. In some embodiments, the scFv that specifically binds GD2 comprises a sequence that is 100% identical to SEQ ID NO: 5.

[0186] In some embodiments, a scFv that specifically binds to GD2 comprises an amino acid sequence that has 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, a scFv that specifically binds to GD2 comprises a sequence that has at least about 85% identity to SEQ ID NO: 6. In some embodiments, a scFv that specifically binds to GD2 comprises a sequence that has at least about 90% identity to SEQ ID NO: 6. In some embodiments, a scFv that specifically binds to GD2 comprises a sequence that has at least about 95% identity to SEQ ID NO: 6. In some embodiments, a scFv that specifically binds to GD2 comprises a sequence that has at least about 96% identity to SEQ ID NO: 6. In some embodiments, a scFv that specifically binds to GD2 comprises a sequence that has at least about 97% identity to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds GD2 comprises a sequence that is at least about 98% identical to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds GD2 comprises a sequence that is at least about 99% identical to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds GD2 comprises a sequence that is 100% identical to SEQ ID NO: 6.

[0187] In some embodiments, the bispecific fusion proteins of the present disclosure comprise a scFv that specifically binds to GD2, the scFv 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: 7. 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 GD2 comprises a sequence that is at least about 97% identical to SEQ ID NO: 7. In some embodiments, the scFv that specifically binds GD2 comprises a sequence that is at least about 98% identical to SEQ ID NO: 7. In some embodiments, the scFv that specifically binds GD2 comprises a sequence that is at least about 99% identical to SEQ ID NO: 7. In some embodiments, the scFv that specifically binds GD2 comprises a sequence that is 100% identical to SEQ ID NO: 7.

[0188] In some embodiments, the bispecific fusion proteins of the present disclosure comprise a scFv that specifically binds to GD2, the scFv 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: 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 GD2 comprises a sequence that is at least about 97% identical to SEQ ID NO: 8. In some embodiments, the scFv that specifically binds GD2 comprises a sequence that is at least about 98% identical to SEQ ID NO: 8. In some embodiments, the scFv that specifically binds GD2 comprises a sequence that is at least about 99% identical to SEQ ID NO: 8. In some embodiments, the scFv that specifically binds GD2 comprises a sequence that is 100% identical to SEQ ID NO: 8.

[0189] CD3 binding site

[0190] The bispecific fusion proteins of the present disclosure may 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 (e.g., αβT cells, γδT cells, NK-T cells, mucosal-associated invariant T (MAIT) cells, and phenotypic subsets thereof). In some embodiments, when bridged to GD2, binding of CD3 induces activation of T cells.

[0191] As used herein, in some embodiments, a CD3 binding site is a polypeptide that specifically binds to CD3 (SEQ ID NO: 69) or a complex of two or more polypeptides. For example, a CD3 binding site binds to the CD3 epsilon chain.

[0192] CD3 sequence (ε chain):

[0193] MRWNTFWGILCLSLLAVGTCQDDAENIEYKVSISGTSVELTCPLDSDENLKWEKNGQELPQKHDKHLVLQDFSEVEDSGYYVCYTPASNKNTYLYLKARVCEYCVEVDLTAVAIIIIVDICITLGLLMVIYYWSKNRKAKAKPVTRGTGAGSRPRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRAV

[0194] (SEQ ID NO:69)

[0195] In some embodiments, the CD3 binding site comprises a heavy chain variable region (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), which in combination can specifically bind to CD3. Table 5B lists the corresponding nucleotide sequences of the VH and VL regions of anti-CD3 antibodies.

[0196] Table 5A - αCD3 VH / VL sequences and CDRs

[0197]

[0198]

[0199] Table 5B - αCD3 VH / VL nucleotide sequences

[0200]

[0201]

[0202]

[0203] In some embodiments, the CD3 binding site comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequence selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences listed in Table 5A as determined according to the IMGT unique numbering scheme, Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk AM, (1987), J. Mol. Biol. 196:901-917), MacCallum (see MacCallum RM et al., (1996) J. Mol. Biol. 262:732-745), or any other CDR determination method known in the art.

[0204] Unless otherwise indicated, the CDR sequences provided in Table 5A are identified according to the Kabat numbering scheme.

[0205] In some embodiments, the CD3 binding site comprises: (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 85; (ii) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 86; (iii) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 87; (iv) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 88; (v) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 89; and (vi) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 90.

[0206] 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.

[0207] 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 sites of the present disclosure comprise VH and VL that have 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.

[0208] In some embodiments, a 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, a CD3 binding site comprises a VH comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence according to SEQ ID NO: 14; and a VL comprising an amino acid sequence having at least 80% sequence identity to an 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 an amino acid sequence according to SEQ ID NO: 14; and a VL comprising an amino acid sequence according to SEQ ID NO: 15.

[0209] In some embodiments, a 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, a CD3 binding site comprises a VH comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence according to SEQ ID NO: 18; and a VL comprising an amino acid sequence having at least 80% sequence identity to an 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 an amino acid sequence according to SEQ ID NO: 18; and a VL comprising an amino acid sequence according to SEQ ID NO: 19.

[0210] In some embodiments, the CD3 binding site includes, but is not limited to, a single-chain variable fragment (scFv), an antibody, Fab, Fab', F(ab')2, a miniantibody, or a nanoantibody (VHH). For example, in some embodiments, the bispecific fusion proteins of the present disclosure comprise scFv polypeptides that each specifically bind to CD3.

[0211] In some embodiments, the CD3 binding site of the present disclosure is in the form of an scFv. In some embodiments, the CD3 binding scFv of the present disclosure comprises an scFv linker polypeptide operably connecting a VH domain and a VL domain. For example, in some embodiments, the CD3 binding scFv comprises, from N-terminus to 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 N-terminus to C-terminus, the VH domain of an anti-CD3 antibody, an scFv linker polypeptide, and the VL domain of an anti-CD3 antibody.

[0212] In some embodiments, the scFv linker polypeptide comprises a sequence selected from the linker sequences in Table 3A.

[0213] Table 6A lists the amino acid sequences of exemplary CD3-binding scFvs. In some embodiments, the bispecific fusion proteins of the present disclosure comprise a CD3-binding scFv comprising 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.

[0214] Table 6A - αCD3 scFv amino acid sequence

[0215]

[0216] * Underline Text indicates scFv linker sequence.

[0217] Table 6B - αCD3 scFv nucleotide sequence

[0218]

[0219]

[0220]

[0221] * Underline The text of represents the scFv linker sequence.

[0222] In some embodiments, the bispecific fusion proteins of the present disclosure comprise a scFv that specifically binds to CD3, 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: 16. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence that is at least about 85% identical to SEQ ID NO: 16. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence that is at least about 90% identical to SEQ ID NO: 16. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence that is at least about 95% identical to SEQ ID NO: 16. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence that is at least about 96% identical to SEQ ID NO: 16. In some embodiments, the scFv that specifically binds CD3 comprises a sequence that is at least about 97% identical to SEQ ID NO: 16. In some embodiments, the scFv that specifically binds CD3 comprises a sequence that is at least about 98% identical to SEQ ID NO: 16. In some embodiments, the scFv that specifically binds CD3 comprises a sequence that is at least about 99% identical to SEQ ID NO: 16. In some embodiments, the scFv that specifically binds CD3 comprises a sequence that is 100% identical to SEQ ID NO: 16.

[0223] In some embodiments, the bispecific fusion proteins of the present disclosure comprise a scFv that specifically binds to CD3, 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: 17. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence that is at least about 85% identical to SEQ ID NO: 17. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence that is at least about 90% identical to SEQ ID NO: 17. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence that is at least about 95% identical to SEQ ID NO: 17. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence that is at least about 96% identical to SEQ ID NO: 17. In some embodiments, the scFv that specifically binds CD3 comprises a sequence that is at least about 97% identical to SEQ ID NO: 17. In some embodiments, the scFv that specifically binds CD3 comprises a sequence that is at least about 98% identical to SEQ ID NO: 17. In some embodiments, the scFv that specifically binds CD3 comprises a sequence that is at least about 99% identical to SEQ ID NO: 17. In some embodiments, the scFv that specifically binds CD3 comprises a sequence that is 100% identical to SEQ ID NO: 17.

[0224] Exemplary αGD2-αCD3 bispecific fusion proteins

[0225] Listed below are examples of bispecific fusion proteins of the present disclosure comprising a GD2 binding site fused to a CD3 binding site via a linker peptide.

[0226] In some embodiments, the bispecific fusion proteins of the present disclosure comprise (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence selected from the group consisting of 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 the group consisting of Table 7; and (iii) a CD3 binding site comprising a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequence selected from the group consisting of 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 proteins of the present disclosure comprise (i) a CD3 binding site comprising a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequence selected from the group consisting of 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 a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence selected from the group consisting of 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 intended to be limiting. For example, the GD2 binding site is located at the amino terminus of the bispecific fusion protein, and the CD3 binding site is located at the carboxyl terminus of the bispecific fusion protein. In some embodiments, the CD3 binding site is located at the amino terminus of the bispecific fusion protein, and the GD2 binding site is located at the carboxyl terminus of the bispecific fusion protein.

[0227] Table 7 - Linker Peptides

[0228] connector sequence Linker 1 (SEQ ID NO: 20) GGGGSGGGGSGGGGS Linker 6 (SEQ ID NO: 25) GGGGS

[0229] In some embodiments, the bispecific fusion proteins of the present disclosure comprise: (i) a GD2 binding site comprising the 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the group consisting of: CDR3 sequence.

[0230] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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; (iii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VH CDR3 sequence selected from the group consisting of: CDR3, VL CDR1, VL CDR2 and VL CDR3 sequences.

[0231] In some embodiments, the bispecific fusion proteins of the present disclosure comprise: (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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the group consisting of 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. CDR3 sequence.

[0232] In some embodiments, the bispecific fusion proteins of the present disclosure comprise: (i) a GD2 binding site comprising the 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the group consisting of: 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. CDR3 sequence.

[0233] In some embodiments, the bispecific fusion proteins of the present disclosure comprise: (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 listed in Table 2A, respectively. domain; (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.

[0234] In some embodiments, the bispecific fusion proteins of the present disclosure comprise: (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 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; The VH domain and VL domain sequences of NO:19 have 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.

[0235] In some embodiments, the bispecific fusion proteins of the present disclosure comprise: (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 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; The VH domain and VL domain sequences of NO:15 have 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.

[0236] In some embodiments, the bispecific fusion proteins of the present disclosure comprise: (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 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; The VH domain and VL domain sequences of NO:19 have 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.

[0237] In some embodiments, the bispecific fusion proteins of the present disclosure comprise: (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 VL domain and a VH domain corresponding to SEQ ID NO: 14 and SEQ ID NO: 3, respectively. The VH domain and VL domain sequences of NO:15 have 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.

[0238] In some embodiments, the bispecific fusion proteins of the present disclosure comprise: (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 an scFv sequence 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 an scFv sequence listed in Table 6A.

[0239] In some embodiments, the bispecific fusion proteins of the present disclosure comprise: (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.

[0240] In some embodiments, the bispecific fusion proteins of the present disclosure comprise: (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.

[0241] In some embodiments, the bispecific fusion proteins of the present disclosure comprise: (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.

[0242] In some embodiments, the bispecific fusion proteins of the present disclosure comprise: (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: 16.

[0243] In some embodiments, the bispecific fusion proteins of the present disclosure comprise a spacer peptide fused to the N-terminus of the 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.

[0244] In some embodiments, the bispecific fusion proteins of the present disclosure have amino acid sequences corresponding to the sequences listed in Table 8 A. Table 8B lists the corresponding nucleotide sequences of the bispecific fusion proteins.

[0245] Table 8A - Bispecific fusion protein amino acid sequences

[0246]

[0247]

[0248]

[0249] * Underlined italics Text indicates scFv linker sequence; Underline Text indicates linker peptide sequence; bold text indicates spacer sequence.

[0250] Table 8B - Bispecific fusion protein nucleotide sequences

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257] In addition to the sequences shown in Table 8A, the bispecific fusion proteins of the present disclosure may also comprise a signal peptide fused to the N-terminus. It should be understood that the mature form of the protein from which the signal peptide is cleaved is expressed.

[0258] 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.

[0259] Signal peptide sequence:

[0260] MWWRLWWLLLLLLLLWPMVWAA

[0261] (SEQ ID NO:26)

[0262] In some embodiments, the bispecific fusion protein of the present disclosure 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: 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 that is at least about 98% identical to SEQ ID NO: 9. In some embodiments, the bispecific fusion protein comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 9. In some embodiments, the bispecific fusion protein comprises an amino acid sequence that is 100% identical to SEQ ID NO: 9.

[0263] In some embodiments, the bispecific fusion proteins of the present disclosure comprise 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. 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 that is at least about 98% identical to SEQ ID NO: 10. In some embodiments, the bispecific fusion protein comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 10. In some embodiments, the bispecific fusion protein comprises an amino acid sequence that is 100% identical to SEQ ID NO: 10.

[0264] In some embodiments, the bispecific fusion proteins of the present disclosure comprise 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. 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 that is at least about 98% identical to SEQ ID NO: 11. In some embodiments, the bispecific fusion protein comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 11. In some embodiments, the bispecific fusion protein comprises an amino acid sequence that is 100% identical to SEQ ID NO: 11.

[0265] In some embodiments, the bispecific fusion proteins of the present disclosure comprise 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 that is at least about 98% identical to SEQ ID NO: 12. In some embodiments, the bispecific fusion protein comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 12. In some embodiments, the bispecific fusion protein comprises an amino acid sequence that is 100% identical to SEQ ID NO: 12.

[0266] In some embodiments, the bispecific fusion proteins of the present disclosure comprise 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. 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 that is at least about 98% identical to SEQ ID NO: 13. In some embodiments, the bispecific fusion protein comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO: 13. In some embodiments, the bispecific fusion protein comprises an amino acid sequence that is 100% identical to SEQ ID NO: 13.

[0267] Exemplary rAAV vectors

[0268] 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 that is 100% identical to any one of SEQ ID NOs: 41-45.

[0269] In some embodiments, the rAAV vectors of the present disclosure comprise 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.

[0270] In some embodiments, the rAAV vectors of the present disclosure comprise 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.

[0271] In some embodiments, the rAAV vectors of the present disclosure comprise 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.

[0272] In some embodiments, the rAAV vectors of the present disclosure comprise 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:44.

[0273] In some embodiments, the rAAV vectors of the present disclosure comprise 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:45.

[0274] In some embodiments, the rAAV vector of the present disclosure comprises one or more regulatory elements. In some embodiments, one or more regulatory elements are on the 5' side of the sequence encoding the bispecific fusion protein. In some embodiments, one or more regulatory elements are on the 3' side of the sequence encoding the bispecific fusion protein. For example, in some embodiments, the regulatory element is on the 3' side of 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%, at least 100%). In some embodiments, WPRE comprises a sequence with at least about 85% identity to SEQ ID NO: 64. In some embodiments, WPRE comprises a sequence with at least about 90% identity to SEQ ID NO: 64. In some embodiments, the WPRE comprises a sequence that is at least about 95% identical to SEQ ID NO: 64. In some embodiments, the WPRE comprises a sequence that is at least about 96% identical to SEQ ID NO: 64. In some embodiments, the WPRE comprises a sequence that is at least about 97% identical to SEQ ID NO: 64. In some embodiments, the WPRE comprises a sequence that is at least about 98% identical to SEQ ID NO: 64. In some embodiments, the WPRE comprises a sequence that is at least about 99% identical to SEQ ID NO: 64. In some embodiments, the WPRE comprises a sequence that is 100% identical to SEQ ID NO: 64.

[0275] Regulatory elements of WPRE origin:

[0276] TAACGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGG

[0277] (SEQ ID NO:64)

[0278] In some embodiments, the regulatory element is at the 3' side of 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 with at least about 85% identity to SEQ ID NO: 123. In some embodiments, the MRE comprises a sequence with at least about 90% identity to SEQ ID NO: 123. In some embodiments, the MRE comprises a sequence with at least about 95% identity to SEQ ID NO: 123. In some embodiments, the MRE comprises a sequence with at least about 96% identity to SEQ ID NO: 123. In some embodiments, the MRE comprises a sequence with at least about 97% identity to SEQ ID NO: 123. In some embodiments, the MRE comprises a sequence that is at least about 98% identical to SEQ ID NO: 123. In some embodiments, the MRE comprises a sequence that is at least about 99% identical to SEQ ID NO: 123. In some embodiments, the MRE comprises a sequence that is 100% identical to SEQ ID NO: 123.

[0279] In some embodiments, the MRE comprises a sequence that is at least about 85% identical to SEQ ID NO: 124. In some embodiments, the MRE comprises a sequence that is at least about 90% identical to SEQ ID NO: 124. In some embodiments, the MRE comprises a sequence that is at least about 95% identical to SEQ ID NO: 124. In some embodiments, the MRE comprises a sequence that is at least about 96% identical to SEQ ID NO: 124. In some embodiments, the MRE comprises a sequence that is at least about 97% identical to SEQ ID NO: 124. In some embodiments, the MRE comprises a sequence that is at least about 98% identical to SEQ ID NO: 124. In some embodiments, the MRE comprises a sequence that is at least about 99% identical to SEQ ID NO: 124. In some embodiments, the MRE comprises a sequence that is 100% identical to SEQ ID NO: 124.

[0280] Modified RNA stability regulatory element (MRE) variant 1

[0281] CGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGG

[0282] (SEQ ID NO:123)

[0283] Modified RNA stability regulatory element (MRE) variant 2

[0284] GAGCATCTTACCGCCATTTATAACCCATATTTGTTCTGTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAACAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGTAATTACTAGTTCAAGGTGTATTGCCACAAG ACAAACATGTTAAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATATTCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCTGCTTTATAGCCTCT GTATCTAGCTATTGCTTCCCGTACGGCTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTAGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGCGTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGC TGGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTTCGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGGGCACTGATAATTCCGTGGTGTTGTC

[0285] (SEQ ID NO: 124)

[0286] In some embodiments, the rAAV vectors of the present disclosure have a nucleotide sequence that has 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 nucleotide sequences listed in Table 9.

[0287] Table 9 - Exemplary AAV2 vector sequences

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310] In some embodiments, the rAAV vector comprises a sequence that is at least about 85% identical to any one of SEQ ID NOs: 53-57. In some embodiments, the rAAV vector comprises a sequence that is at least about 90% identical to any one of SEQ ID NOs: 53-57. In some embodiments, the rAAV vector comprises a sequence that is at least about 95% identical to any one of SEQ ID NOs: 53-57. In some embodiments, the rAAV vector comprises a sequence that is at least about 96% identical to any one of SEQ ID NOs: 53-57. In some embodiments, the rAAV vector comprises a sequence that is at least about 97% identical to any one of SEQ ID NOs: 53-57. In some embodiments, the rAAV vector comprises a sequence that is at least about 98% identical to any one of SEQ ID NOs: 53-57. In some embodiments, the rAAV vector comprises a sequence that is at least about 99% identical to any one of SEQ ID NOs: 53-57. In some embodiments, the rAAV vector comprises a sequence that is 100% identical to any one of SEQ ID NOs: 53-57.

[0311] In some embodiments, the rAAV vectors of the present disclosure have a nucleotide 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:53.

[0312] In some embodiments, the rAAV vectors of the present disclosure have a nucleotide 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:54.

[0313] In some embodiments, the rAAV vectors of the present disclosure have a nucleotide 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:55.

[0314] In some embodiments, the rAAV vectors of the present disclosure have a nucleotide 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:56.

[0315] In some embodiments, the rAAV vectors of the present disclosure have a nucleotide 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:57.

[0316] In some embodiments, the rAAV vector of the present disclosure includes one or more components (e.g., regulatory elements, transgenes) that include reduced CpG dinucleotides and / or increased CpG dinucleotide methylation compared to parent equivalents. In some embodiments, 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 parent equivalents. In some embodiments, CpG dinucleotides are reduced by 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 a CpG dinucleotide 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 the parental equivalent. In some embodiments, the methylation of a CpG dinucleotide 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%.

[0317] 2. Recombinant Adeno-Associated Virus (AAV) Vector Production

[0318] Recombinant AAV particles can be produced by any standard method (see, for example, WO 2001 / 083692; 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; WO 95 / 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., HumanGene 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, which are incorporated herein by reference in their entireties). For example, in some embodiments, the rAAV vectors described herein can be transformed into E. 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 and an AAV helper plasmid to be transfected into an appropriate 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 from any AAV serotype and can be the same or different from the serotype of the recombinant AAV vector ITRs, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. In certain embodiments, the recombinant AAV described herein comprises AAV rep and cap genes derived from AAV2 and AAV9, respectively. The AAV helper plasmid can be from any AAV serotype and can be the same or different serotype as 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 certain embodiments, the recombinant AAV described herein comprises a plasmid having helper genes derived from AAV2.

[0319] In some embodiments, the AAV rep and cap genes are from AAVrh.74. In some embodiments, the rep and cap genes comprise nucleotide sequences that have 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 nucleotide sequences that have at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the rep and cap genes comprise nucleotide sequences that have at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the rep and cap genes comprise nucleotide sequences that have at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the rep and cap genes comprise nucleotide sequences that have at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 125. In some embodiments, the rep and cap genes comprise nucleotide sequences that have 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.

[0320] Table 10. Rep and Cap sequences

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330] In some embodiments, the recombinant AAV described herein is harvested from packaging cells and purified by standard methods in the art (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. Pat. No. 6,566,118 and WO 98 / 09657, the entire contents of which are incorporated herein by reference), such as by cesium chloride ultracentrifugation gradients or column chromatography.

[0331] In some embodiments, the rAAV of the present disclosure comprises a nucleotide 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 the nucleotide sequences listed in Table 9.

[0332] 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- 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.

[0333] 3. Pharmaceutical Compositions

[0334] The recombinant AAV vectors described herein can be used to prepare pharmaceutical compositions. In some embodiments, the pharmaceutical compositions disclosed herein comprise the recombinant AAV vectors disclosed herein and a pharmaceutically acceptable carrier, and optionally, other pharmaceutical agents, pharmaceutical preparations, stabilizers, buffers, carriers, adjuvants, diluents, etc. "Pharmaceutically acceptable" refers to a material that is not toxic or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects.

[0335] In some embodiments, the pharmaceutical composition comprises 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 may contain antioxidants, buffers, antibacterial agents, and solutes that make the composition isotonic with the blood of the intended subject to be administered. Aqueous and non-aqueous sterile suspensions, solutions, and emulsions may include suspending agents and thickening agents. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcohol / water solutions, emulsions, or suspensions, including saline and buffered media. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable vehicle and may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or a fixed oil. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases.

[0336] In some embodiments, the pharmaceutical compositions may be presented in unit / dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition, requiring only the addition of a sterile liquid carrier, such as saline or water for injection, immediately prior to use.

[0337] In some embodiments, the pharmaceutical compositions disclosed herein can be formulated for intravenous, intramuscular, intrathecal, or intracerebroventricular administration.

[0338] 4. Treatment Methods

[0339] The recombinant AAV (rAAV) vectors of the present disclosure or pharmaceutical compositions comprising the same can be administered to a subject in need thereof by any mode of delivery, including but not limited to intravenous, intraperitoneal, and intramuscular administration.

[0340] In some embodiments, the recombinant AAV vector of the present disclosure or a pharmaceutical composition comprising 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 a subject in need thereof simultaneously or at intervals.

[0341] The present application provides methods for reducing the risk of, preventing, and treating metastasis by administering to a patient a rAAV or a pharmaceutical formulation thereof as described herein.

[0342] In other embodiments, the recombinant AAV vectors of the present disclosure or pharmaceutical compositions comprising the same can be administered as a single intravenous dose or divided intravenous doses. In some embodiments, the dose for intravenous delivery can be 1×10 10 to 1×10 13 vg / kg, 2×10 10 to 1×10 13 vg / kg、3×10 10 to 1×10 13 vg / kg, 4×10 10 to 1×10 13 vg / kg, 5×10 10 to 1×10 13 vg / kg, 6×10 10 to 1×10 13 vg / kg、7×10 10 to 1×10 13 vg / kg、8×10 10 to 1×10 13 vg / kg、9×10 10 to 1×10 13 vg / kg, 1×10 11 to 1×10 13 vg / kg, 2×10 11 to 1×10 13 vg / kg、3×10 11 to 1×10 13 vg / kg, 4×10 11 to 1×10 13 vg / kg, 5×10 11 to 1×10 13 vg / kg, 6×10 11 to 1×10 13 vg / kg、7×10 11 to 1×10 13 vg / kg、8×10 11 to 1×10 13 vg / kg、9×10 11 to 1×10 13 vg / kg, 1×10 12 to 1×10 13 vg / kg, 2×10 12 to 1×10 13 vg / kg、3×10 12 to 1×10 13 vg / kg, 4×10 12 to 1×10 13vg / kg, 5×10 12 to 1×10 13 vg / kg, 6×10 12 to 1×10 13 vg / kg、7×10 12 to 1×10 1 3 vg / kg、8×10 12 to 1×10 13 vg / kg、9×10 12 to 1×10 13 vg / kg, 1×10 10 to 1×10 12 vg / kg, 2×10 10 to 1×10 12 vg / kg、3×10 10 to 1×10 12 vg / kg, 4×10 10 to 1×10 12 vg / kg, 5×10 10 to 1×10 12 vg / kg, 6×10 10 to 1×10 12 vg / kg、7×10 10 to 1×10 12 vg / kg、8×10 10 to 1×10 12 vg / kg、9×10 10 to 1×10 12 vg / kg, 1×10 11 to 1×10 12 vg / kg, 2×10 11 to 1×10 12 vg / kg、3×10 11 to 1×10 12 vg / kg, 4×10 11 to 1×10 12 vg / kg, 5×10 11 to 1×10 12 vg / kg, 6×10 11 to 1×10 12 vg / kg、7×10 11 to 1×10 12 vg / kg、8×10 11 to 1×10 12 vg / kg、9×10 11 to 1×10 12 vg / kg, 1×10 10 to 1×10 11 vg / kg, 2×10 10 to 1×10 11 vg / kg、3×10 10 to 1×10 11 vg / kg, 4×10 10 to 1×10 11 vg / kg, 5×10 10 to 1×10 11 vg / kg, 6×10 10 to 1×10 11 vg / kg、7×10 10 to 1×10 11 vg / kg、8×10 10 to 1×10 11 vg / kg or 9×10 10 to 1×10 11 (viral genome (vg) / kilogram (kg) (vg / kg)). In some embodiments, the recombinant AAV vector of the present disclosure or the pharmaceutical composition comprising the same is 1×10 11 In some embodiments, the recombinant AAV vector of the present disclosure or the pharmaceutical composition comprising the same is administered at a dose of 3×10 11 In some embodiments, the recombinant AAV vector of the present disclosure or the pharmaceutical composition comprising the same is administered at a dose of 1×10 12 In some embodiments, the recombinant AAV vector of the present disclosure or the pharmaceutical composition comprising the same is administered at a dose of 3×10 12 In some embodiments, the recombinant AAV vector of the present disclosure or the pharmaceutical composition comprising the same is administered in a single intravenous dose of 5×10 12 A single intravenous dose of vg / kg was administered.

[0343] In some embodiments, the present application provides a method of treating cancer in a patient by administering to the patient an effective amount of a rAAV vector as described herein or a pharmaceutical formulation thereof. In some embodiments, the cancer is neuroblastoma, melanoma, retinoblastoma, Ewing's sarcoma, small cell lung cancer, glioma, osteosarcoma, or soft tissue sarcoma. For example, in some embodiments, the cancer is neuroblastoma.

[0344] Ways to reduce the risk of metastasis

[0345] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VH CDR3 sequence selected from the group consisting of 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 has not been diagnosed with cancer. In some embodiments, the patient has not received cancer treatment.

[0346] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VH CDR3 sequence selected from the group consisting of 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. VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 sequences of the CDR3 sequences.

[0347] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VH CDR3 sequence selected from the group consisting of 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. VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 sequences of the CDR3 sequences.

[0348] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VH CDR3 sequence selected from the group consisting of 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. VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 sequences of the CDR3 sequences.

[0349] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:19 have 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.

[0350] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:15 have 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.

[0351] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:19 have 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.

[0352] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:15 have 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.

[0353] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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.

[0354] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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.

[0355] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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.

[0356] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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.

[0357] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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:9.

[0358] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 10.

[0359] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 11.

[0360] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 12.

[0361] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 13.

[0362] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of a rAAV vector or a pharmaceutical formulation thereof, wherein the rAAV vector comprises a transgene 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: 41.

[0363] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of a rAAV vector or a pharmaceutical formulation thereof, wherein the rAAV vector comprises a transgene 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: 42.

[0364] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of a rAAV vector comprising a transgene 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:43.

[0365] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of a rAAV vector comprising a transgene 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:44.

[0366] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of a rAAV vector comprising a transgene 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:45.

[0367] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of a rAAV comprising at least 85% sequence identity to SEQ ID NO: 53 (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%).

[0368] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of a rAAV comprising at least 85% sequence identity 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%).

[0369] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of a rAAV comprising 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%).

[0370] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of a rAAV comprising 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%).

[0371] In some embodiments, the present application provides a method of reducing the risk of metastasis by administering to a patient an effective amount of rAAV, wherein the rAAV vector comprises 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%).

[0372] Methods to prevent metastasis

[0373] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequence 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 has not been diagnosed with cancer. In some embodiments, the patient has not received cancer treatment.

[0374] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequence 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. CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 sequences.

[0375] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequence selected from the group consisting of 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. CDR2, VH CDR3, VL CDR1, VLCDR2 and VL CDR3 sequences.

[0376] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequence 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. CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 sequences.

[0377] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:19 have 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.

[0378] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 sequence corresponding to SEQ ID NO: 14 and SEQ ID NO: 15, respectively. The VH domain and VL domain sequences of NO:15 have 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.

[0379] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:19 have 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.

[0380] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:15 have 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.

[0381] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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.

[0382] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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.

[0383] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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.

[0384] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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.

[0385] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 9.

[0386] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 10.

[0387] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 11.

[0388] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 12.

[0389] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 13.

[0390] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of a rAAV vector comprising a transgene 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:41.

[0391] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of a rAAV vector comprising a transgene 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:42.

[0392] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of a rAAV vector comprising a transgene 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:43.

[0393] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of a rAAV vector comprising a transgene 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:44.

[0394] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of a rAAV vector comprising a transgene 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:45.

[0395] In some embodiments, the present application provides a method of preventing metastasis by administering to a patient an effective amount of a rAAV comprising at least 85% sequence identity to SEQ ID NO: 53 (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%).

[0396] In some embodiments, the present application provides a method of preventing metastasis by administering to a patient an effective amount of a rAAV comprising at least 85% sequence identity 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%).

[0397] In some embodiments, the present application provides a method of preventing metastasis by administering to a patient an effective amount of a rAAV comprising 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%).

[0398] In some embodiments, the present application provides a method of preventing metastasis by administering to a patient an effective amount of a rAAV comprising 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%).

[0399] In some embodiments, the present application provides a method of preventing metastasis by administering to a patient an effective amount of a rAAV comprising 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%).

[0400] Methods of treating metastasis

[0401] In some embodiments, the present application provides a method of treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequence 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. CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 sequences.

[0402] In some embodiments, the present application provides a method of treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequence 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. CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 sequences.

[0403] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of an rAAV vector or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequence selected from the group consisting of 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. CDR2, VH CDR3, VL CDR1, VLCDR2 and VL CDR3 sequences.

[0404] In some embodiments, the present application provides a method of treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequence 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. CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 sequences.

[0405] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 sequence corresponding to SEQ ID NO: 18 and SEQ ID NO: 21, respectively. The VH domain and VL domain sequences of NO:19 have 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.

[0406] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 sequence corresponding to SEQ ID NO: 14 and SEQ ID NO: 15, respectively. The VH domain and VL domain sequences of NO:15 have 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.

[0407] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 sequence corresponding to SEQ ID NO: 18 and SEQ ID NO: 21, respectively. The VH domain and VL domain sequences of NO:19 have 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.

[0408] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:15 have 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.

[0409] In some embodiments, the present application provides a method of treating metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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.

[0410] In some embodiments, the present application provides a method of treating metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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.

[0411] In some embodiments, the present application provides a method of treating metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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.

[0412] In some embodiments, the present application provides a method of treating metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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.

[0413] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 9.

[0414] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 10.

[0415] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 11.

[0416] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 12.

[0417] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 13.

[0418] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of a rAAV vector or a pharmaceutical formulation thereof, wherein the rAAV vector comprises a transgene 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: 41.

[0419] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of a rAAV vector comprising a transgene 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:42.

[0420] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of a rAAV vector comprising a transgene 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:43.

[0421] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of a rAAV vector comprising a transgene 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:44.

[0422] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of a rAAV vector comprising a transgene 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:45.

[0423] In some embodiments, the present application provides a method of treating metastasis by administering to a patient an effective amount of a rAAV comprising at least 85% sequence identity to SEQ ID NO: 53 (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%).

[0424] In some embodiments, the present application provides a method of treating metastasis by administering to a patient an effective amount of a rAAV comprising at least 85% sequence identity 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%).

[0425] In some embodiments, the present application provides a method of treating metastasis by administering to a patient an effective amount of a rAAV comprising 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%).

[0426] In some embodiments, the present application provides a method of treating metastasis by administering to a patient an effective amount of a rAAV comprising 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%).

[0427] In some embodiments, the present application provides a method of treating metastasis by administering to a patient an effective amount of rAA, wherein the rAAV comprises 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%).

[0428] Methods for promoting T cell-mediated killing of circulating tumor cells

[0429] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VHCDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VH CDR3 sequence selected from the group consisting of 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. CDR2 and VL CDR3 sequences.

[0430] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VHCDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VH CDR3 sequence selected from the group consisting of 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. CDR2 and VL CDR3 sequences.

[0431] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequence 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. CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 sequences.

[0432] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VHCDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VH CDR3 sequence selected from the group consisting of 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. CDR2 and VL CDR3 sequences.

[0433] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:19 have 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.

[0434] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:15 have 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.

[0435] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:19 have 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.

[0436] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:15 have 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.

[0437] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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: 5; NO:17 has an scFv 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%).

[0438] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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: 5. NO:16 has an scFv 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%).

[0439] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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: 7; NO:17 has an scFv 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%).

[0440] 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 or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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: 7; NO:16 has an scFv 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%).

[0441] 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 a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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:9.

[0442] 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 a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 10.

[0443] 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 a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 11.

[0444] 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 a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 12.

[0445] 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 a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 13.

[0446] 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 a rAAV vector comprising a transgene 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:41.

[0447] 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 a rAAV vector comprising a transgene 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:42.

[0448] 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 a rAAV vector comprising a transgene 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: 43.

[0449] 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 a rAAV vector comprising a transgene 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:44.

[0450] 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 a rAAV vector comprising a transgene 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: 45.

[0451] 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 rAAV comprising at least 85% sequence identity to SEQ ID NO: 53 (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%).

[0452] 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 rAAV comprising at least 85% sequence identity 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%).

[0453] 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 rAAV comprising 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%).

[0454] 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 rAAV comprising 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%).

[0455] 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 rAAV comprising 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%).

[0456] Methods for preventing cancer in tumor-prone patients

[0457] In some embodiments, the present application provides a method of preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VH CDR3 sequence selected from the group consisting of 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. CDR2 and VL CDR3 sequences.

[0458] In some embodiments, the present application provides a method of preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VH CDR3 sequence selected from the group consisting of 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. CDR2 and VL CDR3 sequences.

[0459] In some embodiments, the present application provides a method of preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VH CDR3 sequence selected from the group consisting of 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. CDR2 and VL CDR3 sequences.

[0460] In some embodiments, the present application provides a method of preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VH CDR3 sequence selected from the group consisting of 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. CDR2 and VL CDR3 sequences.

[0461] In some embodiments, the present application provides a method for preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:19 have 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.

[0462] In some embodiments, the present application provides a method of preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:15 have 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.

[0463] In some embodiments, the present application provides a method of preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:19 have 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.

[0464] In some embodiments, the present application provides a method of preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:15 have 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.

[0465] In some embodiments, the present application provides a method of preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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: 5. NO:17 has an scFv 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%).

[0466] In some embodiments, the present application provides a method of preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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: 5. NO:16 has an scFv 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%).

[0467] In some embodiments, the present application provides a method of preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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: 7; NO:17 has an scFv 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%).

[0468] In some embodiments, the present application provides a method of preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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: 7. NO:16 has an scFv 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%).

[0469] In some embodiments, the present application provides a method for preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 9.

[0470] In some embodiments, the present application provides a method for preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 10.

[0471] In some embodiments, the present application provides a method for preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 11.

[0472] In some embodiments, the present application provides a method for preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 12.

[0473] In some embodiments, the present application provides a method for preventing cancer in a patient susceptible to tumors (e.g., GD2+ tumors) by administering to the patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 13.

[0474] In some embodiments, the present application provides a method for preventing cancer in a patient susceptible to tumor development (e.g., GD2+ tumors) by administering to the patient an effective amount of a rAAV vector comprising a transgene 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: 41.

[0475] In some embodiments, the present application provides a method for preventing cancer in a patient susceptible to tumor development (e.g., GD2+ tumors) by administering to the patient an effective amount of a rAAV vector comprising a transgene 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:42.

[0476] In some embodiments, the present application provides a method for preventing cancer in a patient susceptible to tumor development (e.g., GD2+ tumors) by administering to the patient an effective amount of a rAAV vector comprising a transgene 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: 43.

[0477] In some embodiments, the present application provides a method for preventing cancer in a patient susceptible to tumor development (e.g., GD2+ tumors) by administering to the patient an effective amount of a rAAV vector comprising a transgene 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:44.

[0478] In some embodiments, the present application provides a method for preventing cancer in a patient susceptible to tumor development (e.g., GD2+ tumors) by administering to the patient an effective amount of a rAAV vector comprising a transgene 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:45.

[0479] In some embodiments, the present application provides a method of preventing cancer in a patient susceptible to developing a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of a rAAV comprising at least 85% sequence identity to SEQ ID NO: 53 (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%).

[0480] In some embodiments, the present application provides a method of preventing cancer in a patient susceptible to developing a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of a rAAV comprising at least 85% sequence identity 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%).

[0481] In some embodiments, the present application provides a method of preventing cancer in a patient susceptible to developing a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of a rAAV comprising 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%).

[0482] In some embodiments, the present application provides a method of preventing cancer in a patient susceptible to developing a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of a rAAV comprising 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%).

[0483] In some embodiments, the present application provides a method of preventing cancer in a patient susceptible to developing a tumor (e.g., a GD2+ tumor) by administering to the patient an effective amount of a rAAV comprising 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%).

[0484] Ways to prevent cancer recurrence

[0485] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VLCDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VH CDR3 sequence selected from the group consisting of 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. CDR2 and VL CDR3 sequences.

[0486] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VLCDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VH CDR3 sequence selected from the group consisting of 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. CDR2 and VL CDR3 sequences.

[0487] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VHCDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequence selected from 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. VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 sequences of the CDR3 sequences.

[0488] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (i) a GD2 binding site comprising a VL CDR1, VLCDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequence 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 a VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VH CDR3 sequence selected from the group consisting of 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. CDR2 and VL CDR3 sequences.

[0489] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:19 have 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.

[0490] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:15 have 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.

[0491] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., a 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 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 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; The VH domain and VL domain sequences of NO:19 have 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.

[0492] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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 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; The VH domain and VL domain sequences of NO:15 have 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.

[0493] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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: 5. NO:17 has an scFv 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%).

[0494] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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: 5. NO:16 has an scFv 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%).

[0495] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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: 7. NO:17 has an scFv 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%).

[0496] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein has: (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: 7. NO:16 has an scFv 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%).

[0497] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 9.

[0498] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 10.

[0499] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 11.

[0500] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 12.

[0501] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of a rAAV vector encoding a bispecific fusion protein or a pharmaceutical formulation thereof, wherein the bispecific fusion protein 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: 13.

[0502] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of a rAAV vector comprising a transgene 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: 41.

[0503] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of a rAAV vector comprising a transgene 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:42.

[0504] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of a rAAV vector comprising a transgene 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: 43.

[0505] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of a rAAV vector comprising a transgene 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:44.

[0506] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission of cancer (e.g., GD2+ cancer) by administering to the patient an effective amount of a rAAV vector comprising a transgene 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: 45.

[0507] In some embodiments, the present application provides a method of preventing cancer recurrence in a patient in remission of cancer (e.g., a GD2+ cancer) by administering to the patient an effective amount of a rAAV comprising at least 85% sequence identity to SEQ ID NO: 53 (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%).

[0508] In some embodiments, the present application provides a method of preventing cancer recurrence in a patient in remission of cancer (e.g., a GD2+ cancer) by administering to the patient an effective amount of a rAAV comprising at least 85% sequence identity 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%).

[0509] In some embodiments, the present application provides a method of preventing cancer recurrence in a patient in remission of cancer (e.g., a GD2+ cancer) by administering to the patient an effective amount of a rAAV comprising 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%).

[0510] In some embodiments, the present application provides a method of preventing cancer recurrence in a patient in remission of cancer (e.g., a GD2+ cancer) by administering to the patient an effective amount of a rAAV comprising 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%).

[0511] In some embodiments, the present application provides a method of preventing cancer recurrence in a patient in remission of cancer (e.g., a GD2+ cancer) by administering to the patient an effective amount of a rAAV comprising 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%).

[0512] Combination therapy

[0513] In another aspect of the present disclosure, the rAAV vector or pharmaceutical formulation thereof is administered concurrently with treatment of the primary tumor.

[0514] In some embodiments of the present disclosure, the rAAV vector or pharmaceutical formulation thereof is administered concurrently with surgical resection of the primary tumor, radiotherapy, chemotherapy, or immunotherapy.

[0515] In some embodiments, the rAAV vector as described herein, or a pharmaceutical formulation thereof, is administered in combination with a checkpoint inhibitor selected from a CTLA-4 inhibitor, a PD-1 inhibitor, and a PD-L1 inhibitor.

[0516] In some embodiments, the rAAV vector or 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 vector or pharmaceutical formulation thereof as described herein is administered in combination with ipilimumab.

[0517] In some embodiments, the rAAV or pharmaceutical formulation thereof as described herein is administered in combination with a PD-1 inhibitor selected from pembrolizumab, nivolumab, cemiprilimab, dostarlimab, JTZ-4014, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA 00012, AMP-224, and AMP-514. For example, in some embodiments, the rAAV vector or pharmaceutical formulation thereof as described herein is administered in combination with pembrolizumab or nivolumab.

[0518] In some embodiments, the rAAV vector or pharmaceutical formulation thereof as described herein 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, the rAAV vector or pharmaceutical formulation thereof as described herein is administered in combination with atezolizumab.

[0519] The present application provides methods for reducing the risk of, preventing, or treating metastasis, wherein the rAAV vectors described herein or pharmaceutical formulations thereof are administered concurrently with treatment of a primary tumor. In some embodiments, the primary tumor is a neuroblastoma, a melanoma, a retinoblastoma, an Ewing's sarcoma, a small cell lung cancer, a glioma, an osteosarcoma, or a soft tissue sarcoma. For example, in some embodiments, the primary tumor is a neuroblastoma.

[0520] In some embodiments, a rAAV vector as described herein, or a pharmaceutical formulation thereof, is administered in combination with one or more than one AAV encoding different bispecific fusion proteins targeting different tumor-associated antigens.

[0521] Example

[0522] The present disclosure, now generally described, will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the disclosure and are not intended to limit the disclosure.

[0523] Example 1: Molecular cloning of AAV transgene constructs

[0524] The bispecific fusion protein transgene was synthesized by operably joining the following sequences from 5' to 3': (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.

[0525] A transgenic cassette is synthesized by operably joining a CAG promoter sequence, a bispecific fusion protein transgene, and a bovine growth hormone (BGH) polyadenylation sequence. The transgenic cassette is cloned into an appropriate cloning vector (e.g., pUC) and confirmed by DNA sequencing. The confirmed construct is restriction digested and gel purified for subsequent cloning into an appropriate AAV8 backbone vector containing an AAV2 ITR site and a kanamycin resistance gene.

[0526] After ligation, the DNA was transformed into E. coli (e.g., VB UltraStable TM Builder, Chicago, IL), grown on kanamycin selective medium and purified. Gene constructs that had undergone successful ligation were identified by restriction digestion.

[0527] The clones were then amplified by bacterial transformation in E. coli. The correct plasmid sequence was reconfirmed by restriction digestion.

[0528] Transient transfection and viral packaging

[0529] Using a standard calcium phosphate transfection method (e.g., as described by Vandendriessche et al. (2007. J Thromb Haemost 5: 16-24), which is incorporated herein by reference), the validated AAV vectors were transiently transfected into HEK293 cells in combination with an adenoviral helper plasmid and a packaging construct that delivers the AAV rep gene and the AAV cap gene. Two days after transfection, AAV particles were harvested and purified and titrated using two consecutive rounds of cesium chloride density gradient ultracentrifugation.

[0530] Example 2: Binding of bispecific fusion proteins to GD2 and CD3

[0531] Purified AAV particles as described in Example 1 are used to transduce producer cells (eg, HEK293 cells) to express the bispecific fusion protein. The bispecific fusion protein is collected and purified from culture supernatants and / or cell lysates and assayed for GD2 and CD3 binding.

[0532] Human cancer cell lines expressing GD2 (e.g., cell lines with high levels of GD2 expression or cell lines engineered to express exogenous GD2) were used to determine the binding of bispecific fusion proteins to GD2. Various concentrations of bispecific fusion proteins 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 to cells incubated with a control antibody that binds to GD2.

[0533] Similarly, T cell lines or peripheral blood mononuclear cells (PBMCs) were used to determine the binding of bispecific fusion proteins to CD3. Various concentrations of bispecific fusion proteins 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 with the binding of cells incubated with an anti-CD3 control antibody.

[0534] Standard co-culture cytotoxicity assays were also performed using dilution titrations of the purified bispecific fusion protein in culture medium alone and in 100% mouse or confluent human serum. The concentration of bispecific fusion protein required to kill 10% (EC10), 50% (EC50), and 90% (EC90) of target cells under standard conditions after 48 hours was determined. The ED10 / 50 / 90 values ​​were determined for 10 different cell lines (e.g., T98G, U87MG, C3c GBM cells) that differed in their GD2 expression.

[0535] Example 3: Bispecific fusion protein induces T cell-mediated cytotoxicity in vitro

[0536] PBMCs were isolated from human peripheral blood buffy coats using density gradient centrifugation. PBMCs were then co-cultured with GD2-expressing human cancer cells in the presence of various concentrations of bispecific fusion protein or control α-GD2 antibody. Following co-culture, cells were lysed and assayed using a commercially available cytotoxicity assay (e.g., Non-radioactive cytotoxicity assay (Promega, Madison, WI) was used for analysis.

[0537] Example 4: Dosage determination

[0538] To determine the vector dose and serum levels in human patients, the vector was administered in half-log increments (1 × 10 11 , 3×10 11 , 1×10 12 and 3×10 12 ) Mice were administered various doses of AAV and serum levels were analyzed by retro-orbital bleeding at time points expected to be steady state (e.g., 28 days). The vector doses used in the animal studies were selected to achieve serum levels above the EC90 determined in Example 2 for most cell lines.

[0539] Example 5: AAV8 delivery of bispecific fusion proteins to prevent metastasis

[0540] To determine the efficacy of AAV8-delivered bispecific fusion proteins in preventing metastasis, mouse treatment groups consisted of: (1) no local control, (2) oncolytic HSV1, (3) surgical resection, and (4) radiotherapy. For comparison purposes, each treatment group included its own vehicle control (i.e., no bispecific fusion protein) group.

[0541] GD2+ human cancer cells (e.g., GFP+ or CD45-CD56+, CD81+, NB84+, GD2+ cells) are administered to the test group in the flank of mice at various concentrations, and then control or purified AAV8 is administered to the mice for in vivo expression of bispecific fusion proteins. The size of the primary tumor and the presence of metastatic lesions are monitored by measuring the bioluminescence of the transplanted tumors and satellite metastases (if any) at different time points. Metastasis is confirmed and / or counted at autopsy. T cell infiltration, activation, and / or depletion of metastatic tissues (e.g., lungs and liver) are analyzed by immunohistochemistry and / or flow cytometry. Peripheral T cells are also analyzed by flow cytometry for activation and checkpoint marker expression every 2 weeks. Flow cytometry is also used to monitor circulating tumor cells every 2 weeks.

[0542] Alternatively, any of a variety of neuroblastoma xenograft models is used (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, mice (e.g., Balb / c) are subcutaneously implanted with GD2+ human cancer cells expressing luciferase or green fluorescent protein (GFP). The tumors are allowed to grow to an appropriate size, and then control or purified AAV8 particles are administered intravenously to the mice for in vivo expression of the bispecific fusion protein. Primary tumor size and the presence of metastatic lesions are monitored by measuring the bioluminescence of the transplanted tumors and satellite metastases (if any) at various time points. Metastases are confirmed and / or counted at autopsy. By immunohistochemistry and / or flow cytometry analysis of T cell infiltration, activation and / or exhaustion of metastatic tissues (e.g., lung and liver). Peripheral T cells are also activated and expressed by flow cytometry analysis of checkpoint markers every 2 weeks. Flow cytometry is also used to monitor circulating tumor cells every 2 weeks.

[0543] Example 6: Determining the immunogenicity and loss of expression over time of AAV8-delivered bispecific fusion proteins

[0544] To determine whether the bispecific fusion protein delivered by AAV8 elicited an immune response, AAV was administered intravenously to immunocompetent C57B1 / 6 mice. The mice were monitored with 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. Body weight was monitored at each blood draw as a readout for safety / toxicity.

[0545] Example 7: Evaluation of the Effects of Anti-GD2 / Anti-CD3ε Fusion Protein on GD2-Expressing Cell Lines

[0546] AAV constructs were prepared that each encoded one of five anti-GD2 / anti-CD3ε bispecific fusion proteins containing one of two anti-GD2 scFvs (hu3F8V5 and 14G2a) and one of two anti-CD3 scFvs (OKT3 and L2K-07) connected by a linker. A hu3F8V5 / okt3 protein containing an additional spacer moiety was prepared (1169). AAV construct design is described in detail in Figures 2A-2B In vitro and in vivo testing Figure 2B In the flank tumor model, Figure 2BThe construct in failed to control flank tumors.

[0547] 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) exhibited optimal protein production in 293T cells.

[0548] To evaluate the effects of anti-GD2 / anti-CD3ε bispecific fusion proteins on GD2-expressing neuroblastoma cells, supernatants were collected from 293T cells engineered by AAV transduction to produce a control or one of the anti-GD2 / anti-CD3ε bispecific fusion proteins detailed above (1169, 1170, 1172, 1173, or 1175). SK-N-Be(2)-CD19, CHP-134-CD19, and SK-N-AS-CD19 cells were each incubated with human peripheral blood mononuclear cells (huPBMCs) and supernatants from 293T cells at an effector: target (E:T) ratio of 10:1. After 48 hours of incubation, target cell viability ( Figure 4 The results showed that co-treatment of GD2-expressing target cells with huPBMC and bispecific fusion proteins 1169, 1170, 1172, 1173, or 1175 resulted in increased target cell killing of SK-N-AS-CD19, but not SK-N-Be(2)-CD19 or CHP-134-CD19 cells. GD2 expression on target cells was analyzed ( Figures 5A-5B ), which showed that both SK-N-Be(2)-CD19 and CHP-134-CD19 cells tested lost surface GD2 expression. These results indicate that the anti-GD2 / anti-CD3ε bispecific fusion protein can induce target-specific cell killing.

[0549] 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 cells, as well as prototypes expressing CD19, were incubated with 293T supernatants and huPBMCs containing anti-GD2 / anti-CD3ε bispecific fusion proteins (1172, huOKT3 / 5F11-HDD, or CAG-193 / dCGMRE) at an E:T ratio of 10:1. After 48 h of incubation, target cell viability was measured ( Figure 6 These results indicate that protein 1172 induced superior cell killing compared to the other proteins tested, with reduced effects observed in CHP-134. Analysis of GD2 expression in target cells ( Figure 7 ), which showed that the tested CHP-134 parental and CD19 cells lost surface GD2 expression. These results indicate that anti-GD2 / anti-CD3ε bispecific fusion proteins can induce target-specific cell killing.

[0550] Before further experiments, SK-N-AS, SK-N-Be(2), and CHP-134 neuroblastoma cells were evaluated for GD2 expression. 5 × 10 5 GD2 staining was assessed by flow cytometry using anti-GD2 antibody. Figure 8 ). The results showed 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 retain 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 with 293T supernatant and huPBMC containing protein 1172 at concentrations of 0 pM, 10 pM, 100 pM, and 1000 pM at an E:T ratio of 10:1. After 48 hours of incubation, target cell viability (upper panel) and GD2 expression (lower panel) were measured ( Figure 9 ).

[0551] To further evaluate the target-specific cell killing ability of the anti-GD2 / anti-CD3ε bispecific fusion protein 1172, the GD2 surface expression of various neuroblastoma cells was assessed by flow cytometry ( Figures 10A-10B The cells were then incubated with 293T supernatant and huPBMC 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 is correlated with the surface expression of GD2 on target cells.

[0552] To evaluate the potential effect of human serum on the target-specific cell killing ability of the anti-GD2 / anti-CD3ε bispecific fusion protein 1172, various neuroblastoma cell lines were incubated with 293T supernatant containing 0 pM, 50 pM, 100 pM, 500 pM, 1000 pM, and 5000 pM of protein 1172; human serum at concentrations of 10%, 50%, and 100%; and huPBMC 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 has limited effect on the target-specific cell killing ability of the anti-GD2 / anti-CD3ε bispecific fusion protein 1172.

[0553] To evaluate the target-specific cell killing ability of the anti-GD2 / anti-CD3ε bispecific fusion protein 1172 against GD2-expressing lung cancer cells, H446, H446-Luc, and H2228 cells were incubated with 293T supernatant and huPBMC 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 the anti-GD2 / anti-CD3ε bispecific fusion protein 1172 successfully mediates the killing of GD2-expressing lung cancer cells.

[0554] Example 8: In vivo evaluation of the effects of anti-GD2 / anti-CD3ε fusion protein

[0555] To evaluate the antitumor effect of a prototype AAV8 anti-GD2 / anti-CD3ε bispecific fusion protein (GD2 scFv:5F11) with and without co-treatment with the oncolytic virus talimogene laherparepvec (TVEC), huPBMC-NSGS mice bearing SK-N-Be(2)-CD19 / Luc tumors were injected with 5×10 12 The mice were co-treated with AAV vectors (anti-GD2 / anti-CD3ε or GFP control) at gc / kg, with a TVEC co-treatment group. All mice were co-injected with a single dose of huPBMC. The experimental overview is provided in Figure 13A In and Figure 13B GD2 surface staining is shown. Images of mouse tumors on the indicated days are shown in Figure 13C Tumor growth measured by luminescence is shown in Figure 13D The survival rate of each group is shown in Figure 13E Total mouse body weight is shown in Figure 13F The results also showed toxicity in TVEC-treated mice, independent of other treatments. Subsequent testing revealed a loss of GD2 expression on SK-N-Be(2)-CD19 / Luc tumor cells.

[0556] To evaluate the antitumor effect of rAAV8-CAG-234-MRE anti-GD2 / anti-CD3ε bispecific fusion protein in two neuroblastoma xenograft models, huPBMC-NSGS mice bearing SK-N-Be(2)-Luc or CHP 134-Luc tumors were injected with 5×10 12 gc / kg of AAV vector or control. The experimental overview is provided in Figure 14A In, and Figure 14B GD2 surface staining of tumor cells is shown. 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 are shown in Figure 14D and 14E The survival rate of each group is shown in Figure 14F The concentration of anti-GD2 / anti-CD3ε bispecific fusion protein is shown in Figure 14G The standard curve of anti-GD2 / anti-CD3ε bispecific fusion protein concentration is shown in Figure 14H Total mouse body weight is shown in Figure 14I Images of CHP-Luc tumor-bearing mice on the indicated days are shown in Figure 14J Tumor growth measured by luminescence and tumor volume are shown in Figure 14K and 14L The survival rate of each group is shown in Figure 14M The concentration of anti-GD2 / anti-CD3ε bispecific fusion protein is shown in Figure 14N The standard curve of anti-GD2 / anti-CD3ε bispecific fusion protein concentration is shown in Figure 14O Total mouse body weight is shown in Figure 14P These results show a significant increase in survival observed for mice treated with the anti-GD2 / anti-CD3ε bispecific fusion protein in both models.

[0557] 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), huPBMC-NSGS mice bearing CHP134-Luc tumors were injected with 5×10 12 gc / kg of AAV vector or control. Three doses of huPBMC were administered on days 14, 21, and 28 after tumor implantation. A subset of mice were given five intraperitoneal doses of 100 μg anti-PDL1 (days 18, 21, 25, 28, 32, and 35) or three intratumoral doses of 1×10 5 pfu of HSV1716. The experimental overview is provided in Figure 15A Images of tumor-bearing mice treated with AAV vectors or control + anti-PDL1 on the indicated days are shown in Figure 15B Tumor growth measured by luminescence and tumor volume are shown in Figure 15C and 15D The survival rate of each group is shown in Figure 15E The concentration of anti-GD2 / anti-CD3ε bispecific fusion protein is shown in Figure 15F The standard curve of anti-GD2 / anti-CD3ε bispecific fusion protein concentration is shown in Figure 15GTotal mouse body weight is shown in Figure 15H Images of tumor-bearing mice treated with AAV vector, HSV1716, or AAV vector ...

Claims

1. A recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3': (a) 5' AAV inverted terminal repeat (ITR); (b) promoter; (c) a transgene encoding a bispecific fusion protein comprising: (i) a GD2 binding site comprising a light chain variable region (VL) and a heavy chain variable region (VH) of an anti-GD2 antibody, wherein the light chain variable region (VL) comprises the complementary determining region 1 (CDR1), complementary determining region 2 (CDR2) and complementary determining region 3 (CDR3) sequences of SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75, respectively, or SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81, respectively; and the heavy chain variable region comprises the CDR1, CDR2 and CDR3 of SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, respectively, 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 VH and VL of an anti-CD3 antibody; (d) a modified RNA stability regulatory element (MRE), and (e) 3'AAVITR.

2. The rAAV vector of claim 1, wherein the promoter is selected from a chicken β-actin promoter, an elongation factor 1α (EFlα) promoter, a simian virus 40 (SV40) promoter, or a CAG promoter.

3. The rAAV vector of any one of claims 1-2, wherein the promoter is a CAG promoter.

4. The rAAV vector of any one of claims 1-3, wherein the promoter comprises a sequence that is at least 95% identical to SEQ ID NO:

66.

5. The rAAV vector of any one of claims 1-4, wherein the anti-GD2 antibody VL and VH comprise sequences that are at least 95% identical to SEQ ID NO: 2 and SEQ ID NO: 1, respectively.

6. The rAAV vector of claim 5, wherein the GD2 binding site is a single-chain variable fragment (scFv).

7. The rAAV vector of claim 6, wherein the anti-GD2 antibody VL is fused to the anti-GD2 antibody VH using a scFv linker peptide comprising SEQ ID NO:

25.

8. The rAAV vector of any one of claims 5-7, wherein the anti-GD2 antibody VL is fused to the anti-GD2 antibody VH via a scFv linker peptide comprising the sequence of SEQ ID NO:

20.

9. The rAAV vector of any one of claims 1-4, wherein the anti-GD2 antibody VL and VH comprise sequences that are at least 95% identical to SEQ ID NO: 4 and SEQ ID NO: 3, respectively.

10. The rAAV vector of claim 9, wherein the GD2 binding site is a single-chain variable fragment (scFv).

11. The rAAV vector of claim 10, wherein the anti-GD2 antibody VL is fused to the anti-GD2 antibody VH via a scFv linker peptide comprising the sequence of SEQ ID NO:

20.

12. The rAAV vector of any one of claims 9-11, wherein the GD2 binding site comprises a sequence that is at least 95% identical to SEQ ID NO:

7.

13. The rAAV vector of any one of claims 1-12, wherein the anti-CD3 antibody VH comprises CDR1, CDR2 and CDR3 of SEQ ID NO:85, SEQ ID NO:86 and SEQ ID NO:87, respectively, and the anti-CD3 antibody VL comprises CDR1, CDR2 and CDR3 of SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:90, respectively.

14. The rAAV vector of claim 13, wherein the anti-CD3 antibody VH and VL comprise sequences that are at least 95% identical to SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

15. The rAAV vector of claim 13 or 14, wherein the CD3 binding site is a single chain variable fragment (scFv).

16. The rAAV vector of claim 15, wherein the anti-CD3 antibody VH is fused to the anti-CD3 antibody VL via a scFv linker peptide comprising a sequence identical to SEQ ID NO:

25.

17. The rAAV vector of any one of claims 13-16, wherein the anti-CD3 binding site comprises a sequence that is at least 95% identical to SEQ ID NO:

16.

18. The rAAV vector of any one of claims 1-12, wherein the anti-CD3 antibody VH comprises CDR1, CDR2, and CDR3 of SEQ ID NO:91, SEQ ID NO:92, and SEQ ID NO:93, respectively, and the anti-CD3 antibody VL comprises CDR1, CDR2, and CDR3 of SEQ ID NO:94, SEQ ID NO:95, and SEQ ID NO:96, respectively.

19. The rAAV vector of claim 18, wherein the anti-CD3 antibody VH and VL comprise sequences that are at least 95% identical to SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

20. The rAAV vector of claim 18 or 19, wherein the CD3 binding site is a single chain variable fragment (scFv).

21. The rAAV vector of claim 20, wherein the anti-CD3 antibody VH is fused to the anti-CD3 antibody VL via a scFv linker peptide comprising a sequence identical to SEQ ID NO:

25.

22. The rAAV vector of any one of claims 18-21, wherein the CD3 binding site comprises a sequence at least 95% identical to SEQ ID NO:

17.

23. The rAAV vector of any one of claims 1-22, wherein the bispecific fusion protein comprises an N-terminal signal peptide having a sequence at least 95% identical to SEQ ID NO:

26.

24. The rAAV vector of any one of claims 1-23, wherein 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.

25. The rAAV vector of any one of claims 1-24, wherein 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.

26. The rAAV vector of any one of claims 1-25, wherein the transgene further comprises a regulatory element on the 5' side or the 3' side of the sequence encoding the bispecific fusion protein.

27. The rAAV vector of claim 26, wherein the regulatory element is on the 3' side of the sequence encoding the bispecific fusion protein.

28. The rAAV vector of claim 27, wherein the regulatory element is derived from a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and comprises a sequence that is at least 95% identical to SEQ ID NO:

64.

29. The rAAV vector of any one of claims 1-28, wherein the transgene further comprises a Kozak sequence.

30. The rAAV vector of any one of claims 1-29, wherein the vector further comprises a polyadenylation sequence on the 3' side of the transgene sequence and on the 5' side of the 3'AAVITR.

31. The rAAV vector of claim 30, wherein the polyadenylation sequence is a bovine growth hormone (BGH) polyadenylation sequence that is at least 95% identical to SEQ ID NO:

65.

32. The rAAV vector of any one of claims 1-31, wherein the 3'AAVITR comprises a sequence that is at least 95% identical to SEQ ID NO:

59.

33. The rAAV vector of any one of claims 1-33, wherein the vector further comprises an antibiotic resistance gene sequence.

34. The rAAV vector of claim 33, wherein the antibiotic resistance gene is a kanamycin resistance gene.

35. The rAAV vector of any one of claims 1-34, wherein 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.

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

11.

37. 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 according to any one of claims 1-36 or a pharmaceutical formulation thereof.

38. 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 according to any one of claims 1-36 or a pharmaceutical formulation thereof.

39. A method for preventing metastatic disease in a patient, comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector according to any one of claims 1-36 or a pharmaceutical preparation thereof.

40. 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 preparation thereof according to any one of claims 1-36.

41. The method of any one of claims 37-40, wherein the rAAV or pharmaceutical preparation thereof is administered concurrently with treatment of a primary tumor.

42. The method of claim 41, wherein treatment of the primary tumor comprises surgical resection, radiation therapy, chemotherapy, or immunotherapy.

43. A method for preventing cancer in a patient prone to developing a GD2+ tumor, comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector according to any one of claims 1-36 or a pharmaceutical preparation thereof.

44. A method for preventing cancer recurrence in a GD2+ cancer remission patient, comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector or a pharmaceutical preparation thereof according to any one of claims 1-36.

45. The method of any one of claims 37-44, wherein the AAV or a pharmaceutical preparation thereof is administered with a checkpoint inhibitor selected from the group consisting of a CTLA-4 inhibitor, a PD-1 inhibitor, and a PD-L1 inhibitor.

46. ​​The method of claim 45, wherein the checkpoint inhibitor is selected from the group consisting of: pembrolizumab, ipilimumab, nivolumab, and atezolizumab.

47. A pharmaceutical preparation comprising the recombinant adeno-associated virus (rAAV) vector according to any one of claims 1-36 and a pharmaceutically acceptable carrier.

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