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

Abstract

Cancer remains a major global health problem and is the second leading cause of death in the United States. Breast cancer persists as the leading cause of death in women. Current treatment options for breast cancer are not effective for all patients and often can have significant adverse side effects. Provided herein are recombinant adeno-associated virus (rAAV) vectors that express bispecific fusion proteins that bind to HER2 and CD3, and methods of using them for reducing the risk of metastasis, prevention, or treatment. The present disclosure provides rAAV vectors for expressing bispecific fusion proteins. TIFF2025525027000066.tif77170

Description

Technical Field

[0001] Cross-reference This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 391,962, filed Jul. 25, 2022, which is hereby incorporated by reference in its entirety.

[0002] Field The present disclosure generally relates to adeno-associated virus (AAV) vectors for delivering transgene sequences encoding bispecific fusion proteins comprising HER2 binding sites and CD3 binding sites. The present disclosure further relates to methods of killing circulating tumor cells, thereby reducing the risk of cancer and metastatic disease, delaying its onset, and preventing it.

Background Art

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

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

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

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

[0007] HER2 (ErbB2) is a transmembrane glycoprotein belonging to the epidermal growth factor receptor family. It is a receptor tyrosine kinase that regulates cell survival, proliferation, and growth and thus plays a major role in many human malignancies. The ERBB2 gene is amplified or overexpressed in approximately 30% of human breast cancers. Patients with breast cancer overexpressing HER2 have significantly lower overall survival and shorter disease-free periods compared to patients whose cancer does not overexpress HER2. Furthermore, overexpression of HER2 leads to an increase in breast cancer metastasis. Overexpression of HER2 is also known to occur in many other cancer types, including aggressive uterine cancers such as ovarian cancer, esophageal cancer, bladder cancer, gastric cancer, salivary duct cancer, lung adenocarcinoma, and serous endometrial cancer of the uterus. SUMMARY OF THE INVENTION

[0008] Summary The present disclosure relates to methods of using and compositions of adeno-associated virus vectors for expressing bispecific fusion proteins for reducing the risk of, preventing, and treating cancer and metastasis.

[0009] In some aspects, the present disclosure is a recombinant adeno-associated virus (rAAV) vector that, from 5' to 3', a) a 5' AAV inverted terminal repeat (ITR); b) a promoter; c) the following: (i) a HER2 binding site comprising the variable light (VL) and variable heavy (VH) regions of an anti-HER2 antibody, (ii) a linker peptide, and (iii) A CD3 binding site comprising the VH and VL of an anti-CD3 antibody A transgene encoding a bispecific fusion protein comprising d) A modified RNA stability regulatory element (MRE), and e) A 3’ AAV ITR Provided is a recombinant adeno-associated virus (rAAV) vector comprising. In some embodiments, the promoter is selected from the group consisting of the chicken β-actin promoter, the elongation factor 1α (EF1α) promoter, the simian virus 40 (SV40) promoter, or the CAG promoter. In some embodiments, the promoter is the CAG promoter. In some embodiments, the promoter comprises a sequence that is at least 95% identical to SEQ ID NO: 87. In some embodiments, the anti-HER2 antibody VL comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 106, SEQ ID NO: 107, and SEQ ID NO: 108, respectively, and the anti-HER2 antibody VH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively. In some embodiments, the anti-HER2 antibody VL and VH each comprise a sequence that is at least 95% identical to SEQ ID NO: 5 and SEQ ID NO: 4, respectively. In some embodiments, the HER2 binding site is a single-chain variable region fragment (scFv). In some embodiments, the anti-HER2 antibody VL is fused to the anti-HER2 antibody VH using the scFv linker peptide comprising SEQ ID NO: 25. In some embodiments, the HER2 binding site comprises a sequence that is at least 95% identical to SEQ ID NO: 89. In some embodiments, the anti-HER2 antibody VL comprises the complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3) sequences of SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102, respectively, and the anti-HER2 antibody VH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively. In some embodiments, the anti-HER2 antibody VL and VH each comprise a sequence that is at least 95% identical to SEQ ID NO: 2 and SEQ ID NO: 1, respectively. In some embodiments, the HER2 binding site is a single-chain variable region fragment (scFv).In some embodiments, the anti-HER2 antibody VL is fused to the anti-HER2 antibody VH using an scFv linker peptide comprising the sequence of SEQ ID NO: 24. In some embodiments, the scFv comprises a sequence that is at least 95% identical to SEQ ID NO: 88. In some embodiments, the anti-HER2 antibody VL comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 112, SEQ ID NO: 113, and SEQ ID NO: 114, respectively, and the anti-HER2 antibody VH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, respectively. In some embodiments, the anti-HER2 antibody VL and VH each comprise a sequence that is at least 95% identical to SEQ ID NO: 8 and SEQ ID NO: 7, respectively. In some embodiments, the HER2 binding site is a single-chain variable region fragment (scFv). In some embodiments, the anti-HER2 antibody VL is fused to the anti-HER2 antibody VH using an scFv linker peptide comprising the sequence of SEQ ID NO: 26. In some embodiments, the HER2 binding site comprises a sequence that is at least 95% identical to SEQ ID NO: 90. In some embodiments, the anti-HER2 antibody VL comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID NO: 120, respectively, and the anti-HER2 antibody VH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, respectively. In some embodiments, the anti-HER2 antibody VL and VH each comprise a sequence that is at least 95% identical to SEQ ID NO: 10 and SEQ ID NO: 11, respectively. In some embodiments, the HER2 binding site is a single-chain variable region fragment (scFv). In some embodiments, the anti-HER2 antibody VL is fused to the anti-HER2 antibody VH by an scFv linker peptide comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the HER2 binding site comprises a sequence that is at least 95% identical to SEQ ID NO: 12.In some embodiments, the anti-HER2 antibody VL comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 126, respectively, and the anti-HER2 antibody VH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, respectively. In some embodiments, the anti-HER2 antibody VL and VH each comprise a sequence that is at least 95% identical to SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In some embodiments, the HER2 binding site is a single-chain variable region fragment (scFv). In some embodiments, the anti-HER2 antibody VL is fused to the anti-HER2 antibody VH by an scFv linker peptide comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the HER2 binding site comprises a sequence that is at least 95% identical to SEQ ID NO: 15. In some embodiments, the linker peptide comprises the same sequence as SEQ ID NO: 29. In some embodiments, the anti-CD3 antibody VH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, respectively, and the anti-CD3 antibody VL comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively. In some embodiments, the anti-CD3 antibody VH and VL each comprise a sequence that is at least 95% identical to SEQ ID NO: 16 and SEQ ID NO: 17, respectively. In some embodiments, the CD3 binding site is a single-chain variable region fragment (scFv). In some embodiments, the anti-CD3 antibody VH is fused to the anti-CD3 antibody VL using an scFv linker peptide comprising the same sequence as SEQ ID NO: 28. In some embodiments, the CD3 binding site comprises a sequence that is at least 95% identical to SEQ ID NO: 18. In some embodiments, the rAAV vector further comprises a Kozak sequence.In some embodiments, the rAAV vector further comprises a polyadenylation sequence 3' of the transgene sequence and 5' of the 3' AAV ITR. In some embodiments, the polyadenylation sequence is a bovine growth hormone (BGH) polyadenylation sequence that is at least 95% identical to SEQ ID NO: 81. 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 AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, or derivatives thereof. In some embodiments, the bispecific fusion protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In some embodiments, the transgene comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 58, or SEQ ID NO: 62.In some embodiments, the transgene comprises decreased CpG dinucleotides and / or increased methylation of CpG dinucleotides as compared to the parental equivalent. In some embodiments, the rAAV vector comprises a sequence that is at least 90% identical to SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 82, or SEQ ID NO: 86.

[0010] In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) vector that, from 5' to 3', a) a 5' AAV inverted terminal repeat (ITR) comprising a sequence that is at least 90% identical to SEQ ID NO: 75 or SEQ ID NO: 91; b) a promoter; c) the following: (i) a HER2 binding site comprising a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 106, SEQ ID NO: 107, and SEQ ID NO: 108, respectively, of an anti-HER2 antibody, and a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively; (ii) a linker peptide comprising a sequence according to SEQ ID NO: 29, and (iii) a CD3 binding site comprising a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, respectively, of an anti-CD3 antibody, and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively; wherein the transgene comprises a sequence encoding a bispecific fusion protein comprising the above; d) a modified RNA stability regulatory element (MRE), and e) A 3’ AAV ITR comprising a sequence that is at least 90% identical to SEQ ID NO: 75 or SEQ ID NO: 91 provided is a recombinant adeno-associated virus (rAAV) vector comprising the same.

[0011] In some aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector that, from 5’ to 3’, a) a 5’ AAV inverted terminal repeat (ITR) comprising a sequence that is at least 90% identical to SEQ ID NO: 75 or SEQ ID NO: 91; b) a promoter; c) a transgene encoding a bispecific fusion protein comprising a sequence that is at least 90% identical to SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23; d) a modified RNA stability regulatory element (MRE), and e) a 3’ AAV ITR comprising a sequence that is at least 90% identical to SEQ ID NO: 75 or SEQ ID NO: 91 provided is a recombinant adeno-associated virus (rAAV) vector comprising the same.

[0012] In some aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a sequence that is at least 90% identical to SEQ ID NO: 20.

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

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

[0015] In some aspects, the disclosure provides a method of preventing metastatic disease in a patient, the method comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector described herein or a pharmaceutical formulation thereof.

[0016] In some aspects, the disclosure provides a method of promoting T cell-mediated killing of circulating tumor cells in a patient, the method comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector described herein or a pharmaceutical formulation thereof. In some embodiments, the rAAV or its pharmaceutical formulation is administered concurrently with treatment of the primary tumor. In some embodiments, the primary tumor is a breast tumor. In some embodiments, treatment of the primary tumor comprises surgical resection, radiation therapy, chemotherapy, or immunotherapy.

[0017] In some aspects, the disclosure provides a method of preventing cancer in a patient having a predisposition to develop HER2+ tumors, the method comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector described herein or a pharmaceutical formulation thereof.

[0018] In some aspects, the disclosure provides a method of preventing cancer recurrence in a patient in remission from HER2+ cancer, the method comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector described herein or a pharmaceutical formulation thereof. In some embodiments, the rAAV or its pharmaceutical formulation is administered in combination with a checkpoint inhibitor selected from the group consisting of CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors. In some embodiments, the checkpoint inhibitor is selected from the group consisting of pembrolizumab, ipilimumab, nivolumab, and atezolizumab.

[0019] In some aspects, the disclosure provides a pharmaceutical formulation 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 of reducing the risk of metastatic disease in a patient, comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector or a pharmaceutical formulation thereof. In some aspects, the rAAV vector comprises, from 5' to 3', a 5' AAV inverted terminal repeat (ITR); a promoter; a transgene comprising a sequence encoding a bispecific fusion protein; and a 3' AAV ITR. In some aspects, the bispecific fusion protein comprises a HER2 binding site comprising a heavy chain variable region (VH) and a light chain variable region (VL) of an anti-HER2 antibody; a linker peptide; and a CD3 binding site comprising a VH and a VL of an anti-CD3 antibody.

[0021] 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 an adeno-associated virus (AAV) 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 HER2 binding site comprising a heavy chain variable region (VH) and a light chain variable region (VL) of an anti-HER2 antibody; a linker peptide; and a CD3 binding site comprising a VH and a VL of an anti-CD3 antibody.

[0022] In some aspects, the present disclosure provides a method of preventing metastatic disease in a patient, comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector or a pharmaceutical formulation thereof. In some aspects, the rAAV vector comprises, from 5' to 3', a 5' AAV inverted terminal repeat (ITR); a promoter; a transgene comprising a sequence encoding a bispecific fusion protein; and a 3' AAV ITR. In some aspects, the bispecific fusion protein comprises a HER2 binding site comprising a heavy chain variable region (VH) and a light chain variable region (VL) of an anti-HER2 antibody; a linker peptide; and a CD3 binding site comprising a VH and a VL of an anti-CD3 antibody.

[0023] In some aspects, the present disclosure provides a method of promoting T cell-mediated killing of circulating tumor cells in a patient, comprising administering to the patient an effective amount of an adeno-associated virus (AAV) 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 HER2 binding site comprising a heavy chain variable region (VH) and a light chain variable region (VL) of an anti-HER2 antibody; a linker peptide; and a CD3 binding site comprising a VH and a VL of an anti-CD3 antibody.

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

[0025] In some aspects, the present disclosure provides a method of preventing cancer in a patient having a predisposition to develop a HER2+ tumor, the method comprising administering to the patient an effective amount of an adeno-associated virus (AAV) 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 HER2 binding site comprising a heavy chain variable region (VH) and a light chain variable region (VL) of an anti-HER2 antibody; a linker peptide; and a CD3 binding site comprising a VH and a VL of an anti-CD3 antibody.

[0026] In some aspects, the present disclosure provides a method of preventing cancer recurrence in a patient in remission from HER2+ cancer, the method comprising administering to the patient an effective amount of an adeno-associated virus (AAV) 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 HER2 binding site comprising a heavy chain variable region (VH) and a light chain variable region (VL) of an anti-HER2 antibody; a linker peptide; and a CD3 binding site comprising a VH and a VL of an anti-CD3 antibody.

[0027] In some aspects, the present disclosure provides a recombinant adeno-associated virus vector comprising, from 5' to 3', a 5' AAV inverted terminal repeat (ITR); a promoter; a transgene comprising a sequence encoding a bispecific fusion protein having a sequence that is at least 95% identical to SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23; and a 3' AAV ITR.

[0028] In some aspects, the rAAV or its pharmaceutical formulation is administered with a checkpoint inhibitor selected from the group consisting of CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors. 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 that is at least 95% identical to SEQ ID NO: 75. In some aspects, the 3’ AAV ITR comprises a sequence that is at least 95% identical to SEQ ID NO: 91.

[0030] In some aspects, the promoter is selected from the chicken β-actin promoter, elongation factor 1α (EF1α) promoter, simian virus 40 (SV40) promoter, and CAG promoter. In some aspects, the promoter is the CAG promoter. In some aspects, the promoter comprises a sequence that is at least 95% identical to SEQ ID NO: 87.

[0031] In some aspects, the anti-HER2 antibody VL of the HER2 binding site has the complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3) sequences of SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102, respectively, and the anti-HER2 antibody VH of the HER2 binding site has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively. In some aspects, the anti-HER2 antibody VL and VH each have a sequence that is at least 95% identical to SEQ ID NO: 2 and SEQ ID NO: 1, respectively. In some aspects, the HER2 binding site is a single-chain variable region fragment (scFv). In some aspects, the scFv comprises a VL of the HER2 binding site fused to the anti-HER2 antibody VH of the HER2 binding site by an scFv linker peptide comprising the sequence of SEQ ID NO: 24. In some aspects, the scFv comprises a sequence that is at least 95% identical to SEQ ID NO: 88.

[0032] In some aspects, the anti-HER2 antibody VL of the HER2 binding site has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 106, SEQ ID NO: 107, and SEQ ID NO: 108, respectively, and the anti-HER2 antibody VH of the HER2 binding site has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively. In some aspects, the anti-HER2 antibody VL and VH of the HER2 binding site each have a sequence that is at least 95% identical to SEQ ID NO: 5 and SEQ ID NO: 4, respectively. In some aspects, the HER2 binding site is a single-chain variable region fragment (scFv). In some aspects, the anti-HER2 antibody VL of the HER2 binding site is fused to the anti-HER2 antibody VH of the HER2 binding site by an scFv linker peptide comprising SEQ ID NO: 25. In some aspects, the HER2 binding site comprises a sequence that is at least 95% identical to SEQ ID NO: 89.

[0033] In some aspects, the anti-HER2 antibody VL of the HER2 binding site has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 112, SEQ ID NO: 113, and SEQ ID NO: 114, respectively, and the anti-HER2 antibody VH of the HER2 binding site has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, respectively. In some aspects, the anti-HER2 antibody VL and VH of the HER2 binding site each have a sequence that is at least 95% identical to SEQ ID NO: 8 and SEQ ID NO: 7, respectively. In some aspects, the HER2 binding site is a single-chain variable region fragment (scFv). In some aspects, the anti-HER2 antibody VL of the HER2 binding site is fused to the anti-HER2 antibody VH of the HER2 binding site by an scFv linker peptide comprising SEQ ID NO: 26. In some aspects, the HER2 binding site comprises a sequence that is at least 95% identical to SEQ ID NO: 90.

[0034] In some aspects, the anti-HER2 antibody VL of the HER2 binding site has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID NO: 120, respectively, and the anti-HER2 antibody VH of the HER2 binding site has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, respectively. In some aspects, the anti-HER2 antibody VL and VH of the HER2 binding site each contain a sequence that is at least 95% identical to SEQ ID NO: 10 and SEQ ID NO: 11. In some aspects, the HER2 binding site is a single-chain variable region fragment (scFv). In some aspects, the anti-HER2 antibody VL of the HER2 binding site is fused to the anti-HER2 antibody VH of the HER2 binding site by an scFv linker peptide containing the amino acid sequence of SEQ ID NO: 27. In some aspects, the HER2 binding site contains a sequence that is at least 95% identical to SEQ ID NO: 12.

[0035] In some aspects, the anti-HER2 antibody VL of the HER2 binding site has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 126, respectively, and the anti-HER2 antibody VH of the HER2 binding site has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, respectively. In some aspects, the anti-HER2 antibody VL and VH of the HER2 binding site each comprise a sequence that is at least 95% identical to SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In some aspects, the HER2 binding site is a single-chain variable region fragment (scFv). In some aspects, the anti-HER2 antibody VL of the HER2 binding site is fused to the anti-HER2 antibody VH of the HER2 binding site by an scFv linker peptide comprising the amino acid sequence of SEQ ID NO: 27. In some aspects, the HER2 binding site comprises a sequence that is at least 95% identical to SEQ ID NO: 15.

[0036] In some aspects, the linker peptide comprises a sequence identical to SEQ ID NO: 29.

[0037] In some aspects, the anti-CD3 antibody VH of the CD3 binding site has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, respectively, and the anti-CD3 antibody VL has the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0038] In some aspects, the VL and VH of the anti-CD3 antibody of the CD3 binding site each comprise a sequence that is at least 95% identical to SEQ ID NO: 16 and SEQ ID NO: 17. In some aspects, the CD3 binding site is a single-chain variable region fragment (scFv). In some aspects, the VL of the anti-CD3 antibody of the CD3 binding site is fused to the VH of the anti-CD3 antibody of the CD3 binding site by an scFv linker peptide comprising a sequence identical to SEQ ID NO: 28. In some aspects, the CD3 binding site comprises a sequence that is at least 95% identical to SEQ ID NO: 18.

[0039] In some aspects, the bispecific fusion protein comprises a sequence that is at least 95% identical to SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.

[0040] In some aspects, the bispecific fusion protein has an N-terminal signal peptide comprising a sequence that is at least 95% identical to SEQ ID NO: 34.

[0041] In some aspects, the transgene comprises a sequence that is at least 95% identical to SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 58, or SEQ ID NO: 62.

[0042] In some aspects, the transgene further has a regulatory element 5' or 3' of the sequence encoding the bispecific fusion protein. In some aspects, the regulatory element is 3' of the sequence encoding the bispecific fusion protein. In some aspects, the regulatory element is derived from the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and comprises a sequence that is at least 95% identical to SEQ ID NO: 80.

[0043] In some aspects, the transgene further has a Kozak sequence.

[0044] In some embodiments, the vector further has a polyadenylation sequence 3' of the transgene sequence and 5' of the 3' AAV ITR. In some embodiments, the polyadenylation sequence is a bovine growth hormone (BGH) polyadenylation sequence that is at least 95% identical to SEQ ID NO: 81.

[0045] In some embodiments, the vector further has an antibiotic resistance gene sequence. In some embodiments, the antibiotic resistance gene is a kanamycin resistance gene.

[0046] In some embodiments, the vector comprises a sequence that is at least 95% identical to SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 82, or SEQ ID NO: 86.

[0047] In some embodiments, the present disclosure provides an adeno-associated virus (AAV) comprising a recombinant AAV vector according to any one of the above embodiments.

[0048] In some embodiments, the present disclosure provides a pharmaceutical formulation comprising an adeno-associated virus (AAV) according to any one of the above embodiments and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0050] Detailed Description The present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid encoding a bispecific fusion protein comprising the heavy chain variable region (VH) and light chain variable region (VL) of an anti-HER2 antibody and the VH and VL of an anti-CD3 antibody.

[0051] The present disclosure also provides methods for using the rAAVs described herein to reduce, prevent, or treat cancer and the risk of metastasis in a patient.

[0052] To facilitate understanding of the present disclosure, several terms and phrases are defined below.

[0053] As used herein, the terms "a" and "an" mean "one or more" and include the plural form, unless the context is inappropriate.

[0054] The terms "nucleic acid", "nucleotide", or "oligonucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly shown sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0055] The term "gene" can refer to a segment of DNA involved in the production or coding of a polypeptide chain. It can include regions before and after the coding region (leader and trailer), as well as intervening sequences (introns) between individual coding segments (exons).

[0056] A "promoter" is defined as one or more nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes a nucleic acid sequence near the transcription start site. A promoter can also optionally include distal enhancer or repressor elements that can be located thousands of base pairs away from the transcription start site.

[0057] An "regulatory element" as used herein refers to a nucleic acid sequence that can regulate the transcription of a gene (e.g., a transgene) and / or the stability or translation of the transcribed mRNA product. In some embodiments, the regulatory element can regulate the tissue-specific transcription of a gene. The regulatory element can include at least one transcription factor binding site, e.g., a transcription factor binding site for a muscle-specific transcription factor. A regulatory element as used herein increases or enhances promoter-driven gene expression when compared to the transcription of a gene from a promoter alone in the absence of the regulatory element. A regulatory element as used herein can occur at any distance (i.e., proximal or distal) relative to the transgene it regulates. A regulatory element as used herein can include a part of a larger sequence involved in transcriptional control, e.g., a part of a promoter sequence. However, a regulatory element alone is typically not sufficient to initiate transcription by itself and requires the presence of a promoter.

[0058] A nucleic acid is "functionally linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is functionally linked to a coding sequence if it affects the transcription of the coding sequence; or a ribosome binding site is functionally linked to a coding sequence if it is positioned to facilitate translation.

[0059] As used herein, the term "array of equivalent coding potentials" refers to a nucleic acid sequence that has functional equivalence to another reference nucleic acid. An array of equivalent coding potentials may or may not have the same primary nucleotide sequence. For example, with respect to a reference nucleic acid encoding an expressed polypeptide, an array of equivalent coding potentials can functionally encode the same expressed polypeptide and may contain the same primary nucleotide sequence as the reference nucleic acid, or may contain one or more alternative codons as compared to the reference nucleic acid. For example, an endogenous nucleic acid sequence encoding a polypeptide can be modified by codon optimization to yield a sequence that encodes the same polypeptide. A codon-optimized sequence can be one in which the codons in a polynucleotide encoding a polypeptide have been substituted to modify the activity, expression, and / or stability of the polynucleotide. For example, codon optimization can be used to vary the degree of sequence similarity of an array of equivalent coding potentials as compared to an endogenous gene sequence while retaining the potential to encode the protein product of the endogenous gene.

[0060] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, these terms encompass amino acid chains of any length, including full-length proteins and functional fragments thereof, wherein the amino acid residues are linked by covalent peptide bonds.

[0061] The terms "variable domain" (e.g., VH domain or VL domain) and "variable region" are used interchangeably and refer to the parts of an antibody or immunoglobulin domain that show variability in sequence and are involved in determining the specificity and binding affinity of a particular antibody. The variability is not evenly distributed throughout the entire variable domain of the antibody; rather, it is concentrated in specific subdomains of each of the heavy and light chain variable regions. These subdomains are called "hypervariable regions" or "complementarity-determining regions" (CDRs). The more conserved (i.e., non-hypervariable) parts of the variable domain are called "framework" regions (FRM or FR), which provide a scaffold for the six CDRs in three-dimensional space to form the antigen-binding surface.

[0062] As used herein, the terms "complementary" or "complementarity" refer to specific base pairing between nucleotides or nucleic acids. Complementary nucleotides are generally A and T (or A and U), and G and C.

[0063] As used herein, the term "transgene" refers to an exogenous gene artificially introduced into the genome of a cell, or an endogenous gene artificially introduced into a non-native locus in the genome of a cell. A transgene can refer to a segment of DNA involved in the production or coding of a polypeptide chain. A transgene can include regions before and after the coding region (leader and trailer), as well as intervening sequences (introns) between individual coding segments (exons).

[0064] As used herein, the terms "introduce" or "deliver" in the context of a nucleic acid, e.g., an AAV vector, refer to the transfer of a nucleic acid from outside the cell into the interior of the cell, e.g., a muscle cell. In some cases, introduction refers to the transfer of a nucleic acid from outside the cell into the interior of the cell nucleus. Various methods of such transfer are contemplated, including but not limited to electroporation, contact with nanowires or nanotubes, receptor-mediated internalization, transfer via cell-penetrating peptides, liposome-mediated transfer, etc.

[0065] As used herein, the terms "encapsidated" or "packaged" refer to the formation of AAV particles by including an AAV vector within a viral capsid.

[0066] The term "substantial identity" or "substantially identical", when used in the context of polynucleotide or polypeptide sequences, refers to a sequence having at least 60% sequence identity to a reference sequence. Alternatively, the percent identity can be any integer from 60% to 100%. Exemplary embodiments include, as described below, at least 60%, ^{-}65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% when compared to a reference sequence using the programs described herein, preferably BLAST, with standard parameters. One of ordinary skill in the art will recognize that these values can be appropriately adjusted to determine the corresponding identity of the proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[0067] For sequence comparison, typically one sequence functions as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, sub-sequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used or alternative parameters can be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0068] Algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating a search to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence as long as the cumulative alignment score increases. The cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the word hits in each direction stops when the cumulative alignment score decreases by an amount X from its reached maximum; when the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, a word size (W) of 28, an expectation value (E) of 10, M = 1, N = -2, and a comparison of both strands.In the case of amino acid sequences, the BLASTP program, by default, uses a word size (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0069] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that a match between two nucleotide sequences or amino acid sequences occurs by chance. For example, in a comparison of a test nucleic acid with a reference nucleic acid, if the minimum total probability is less than about 0.01, more preferably less than about 10 -5 less, and most preferably less than about 10 -20 less, the nucleic acid is considered to be similar to the reference sequence.

[0070] The terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably herein and, in some embodiments, refer to any mammalian subject, particularly a human, for whom diagnosis, treatment, or therapy is desired. As used for treatment purposes, “mammal” refers to any animal classified as a mammal, including humans, breeding animals, and livestock, as well as laboratory, zoo, sports, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. In some embodiments, the mammal is a human. None of these terms require medical practitioner monitoring and / or a cancer diagnosis or current cancer treatment.

[0071] As used herein, the term “efficient delivery” or “efficiently deliver” refers to the administration of a recombinant adeno-associated virus vector encoding a transgene that results in the expression of the transgene in a desired cell or tissue.

[0072] 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 produce a beneficial or desired result (e.g., expression of a protein, or a desired prophylactic or therapeutic effect). The effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treating" includes any effect, e.g., alleviation, mitigation, modulation, improvement or elimination, that results in the amelioration of a condition, disease, disorder, etc., or improvement of their symptoms.

[0073] Throughout this specification, when a composition is described as having, comprising, or including a particular component, or when a process and method are described as having, comprising, or including a particular step, it is further contemplated that there exist compositions of the present disclosure consisting essentially of, or consisting of, the recited components, and that there exist processes and methods according to the present disclosure consisting essentially of, or consisting of, the recited processing steps.

[0074] 1. Recombinant adeno-associated virus (AAV) vector As used herein, a "recombinant adeno-associated virus (rAAV) vector" refers to a vector (e.g., a nucleic acid vector) comprising a promoter and one or more transgenes, or polynucleotides of interest, flanked by AAV inverted terminal repeat (ITR) sequences. The rAAV vectors described herein can be replicated and packaged into viral particles when introduced into a host cell further comprising one or more vectors encoding the rep and cap gene products.

[0075] Inverted terminal repeat The inverted terminal repeat (ITR) is a 145-nucleotide palindromic sequence adjacent to the transgene. The 5’ and 3’ ITRs of the recombinant adeno-associated virus (rAAV) vector are necessary for both the integration of the transgene into the host cell genome (e.g., human chromosome 19) and encapsulation into AAV particles.

[0076] In some embodiments, the rAAV vectors of the disclosure include ITR sequences from any one AAV serotype, e.g., AAVrh.74, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13. In some embodiments, the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, or derivatives thereof. In some embodiments, the recombinant AAV vectors disclosed herein include AAV2 5’ and 3’ ITR sequences. In some embodiments, the recombinant AAV vectors disclosed herein include AAV8 5’ and 3’ ITR sequences.

[0077] In some embodiments, the recombinant AAV vectors described herein include a 5’ AAV2 ITR having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 75 (see Table 1A). In some embodiments, the 5’ AAV2 ITR includes a sequence having at least about 80% identity to SEQ ID NO: 75. In some embodiments, the 5’ AAV2 ITR includes a sequence having at least about 85% identity to SEQ ID NO: 75. In some embodiments, the 5’ AAV2 ITR includes a sequence having at least about 90% identity to SEQ ID NO: 75. In some embodiments, the 5’ AAV2 ITR includes a sequence having at least about 95% identity to SEQ ID NO: 75. In some embodiments, the 5’ AAV2 ITR includes a sequence having at least about 96% identity to SEQ ID NO: 75. In some embodiments, the 5’ AAV2 ITR includes a sequence having at least about 97% identity to SEQ ID NO: 75. In some embodiments, the 5’ AAV2 ITR includes a sequence having at least about 98% identity to SEQ ID NO: 75. In some embodiments, the 5’ AAV2 ITR includes a sequence having at least about 99% identity to SEQ ID NO: 75. In some embodiments, the 5’ AAV2 ITR includes a sequence having 100% identity to SEQ ID NO: 75. In some embodiments, the 5’ AAV2 ITR includes SEQ ID NO: 75. In some embodiments, the 5’ AAV2 ITR consists of SEQ ID NO: 75.

[0078] In some embodiments, the recombinant AAV vectors described herein comprise a 3’ AAV2 ITR having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 91 (see Table 1A). In some embodiments, the 3’ AAV2 ITR comprises a sequence having at least about 80% identity to SEQ ID NO: 91. In some embodiments, the 3’ AAV2 ITR comprises a sequence having at least about 85% identity to SEQ ID NO: 91. In some embodiments, the 3’ AAV2 ITR comprises a sequence having at least about 90% identity to SEQ ID NO: 91. In some embodiments, the 3’ AAV2 ITR comprises a sequence having at least about 95% identity to SEQ ID NO: 91. In some embodiments, the 3’ AAV2 ITR comprises a sequence having at least about 96% identity to SEQ ID NO: 91. In some embodiments, the 3’ AAV2 ITR comprises a sequence having at least about 97% identity to SEQ ID NO: 91. In some embodiments, the 3’ AAV2 ITR comprises a sequence having at least about 98% identity to SEQ ID NO: 91. In some embodiments, the 3’ AAV2 ITR comprises a sequence having at least about 99% identity to SEQ ID NO: 91. In some embodiments, the 3’ AAV2 ITR comprises a sequence having 100% identity to SEQ ID NO: 91. In some embodiments, the 3’ AAV2 ITR comprises SEQ ID NO: 91. In some embodiments, the 3’ AAV2 ITR consists of SEQ ID NO: 91.

[0079] (Table 1A) AAV ITR Sequences TIFF2025525027000002.tif45161

[0080] Promoter Promoters drive the expression of rAAV vector transgenes and are typically located upstream (or 5') of the transgenes they regulate expression of.

[0081] In some embodiments, the recombinant AAV vectors of the disclosure include mammalian promoters, such as human, non-human primate (e.g., cynomolgus monkey), mouse, horse, cow, pig, cat, and dog promoters. In some embodiments, the recombinant AAV vectors disclosed herein include strong and constitutively active promoters to drive high-level expression of the transgene. For example, in some embodiments, the promoter is a CAG promoter (cytomegalovirus immediate enhancer fused to the chicken β-actin promoter), a cytomegalovirus (CMV) promoter / enhancer, an elongation factor 1α (EF1α) promoter, a simian virus 40 (SV40) promoter, or a chicken β-actin promoter.

[0082] In some embodiments, the promoter described herein comprises a CAG promoter having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 87. In some embodiments, the CAG promoter comprises a sequence having at least about 80% identity to SEQ ID NO: 87. In some embodiments, the CAG promoter comprises a sequence having at least about 85% identity to SEQ ID NO: 87. In some embodiments, the CAG promoter comprises a sequence having at least about 90% identity to SEQ ID NO: 87. In some embodiments, the CAG promoter comprises a sequence having at least about 95% identity to SEQ ID NO: 87. In some embodiments, the CAG promoter comprises a sequence having at least about 96% identity to SEQ ID NO: 87. In some embodiments, the CAG promoter comprises a sequence having at least about 97% identity to SEQ ID NO: 87. In some embodiments, the CAG promoter comprises a sequence having at least about 98% identity to SEQ ID NO: 87. In some embodiments, the CAG promoter comprises a sequence having at least about 99% identity to SEQ ID NO: 87. In some embodiments, the CAG promoter comprises a sequence having 100% identity to SEQ ID NO: 87.

[0083] CAG promoter sequence: TIFF2025525027000003.tif189134

[0084] SV40 intron In some embodiments, the recombinant AAV vector of the disclosure comprises an SV40 intron. The SV40 intron is a regulatory element commonly used in gene therapy vectors and enhances the translation and stability of the expressed RNA transcript.

[0085] In some embodiments, the SV40 intron is downstream (i.e., 3') of the promoter and upstream (i.e., 5') of the transgene. In other embodiments, the SV40 intron can be downstream (i.e., 3') of the transgene.

[0086] In some embodiments, the SV40 intron comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 92. In some embodiments, the SV40 intron comprises SEQ ID NO: 92. In some embodiments, the SV40 intron consists of SEQ ID NO: 92. In some embodiments, the SV40 intron comprises a sequence having at least about 80% identity to SEQ ID NO: 92. In some embodiments, the SV40 intron comprises a sequence having at least about 85% identity to SEQ ID NO: 92. In some embodiments, the SV40 intron comprises a sequence having at least about 90% identity to SEQ ID NO: 92. In some embodiments, the SV40 intron comprises a sequence having at least about 95% identity to SEQ ID NO: 92. In some embodiments, the SV40 intron comprises a sequence having at least about 96% identity to SEQ ID NO: 92. In some embodiments, the SV40 intron comprises a sequence having at least about 97% identity to SEQ ID NO: 92. In some embodiments, the SV40 intron comprises a sequence having at least about 98% identity to SEQ ID NO: 92. In some embodiments, the SV40 intron comprises a sequence having at least about 99% identity to SEQ ID NO: 92. In some embodiments, the SV40 intron comprises a sequence having 100% identity to SEQ ID NO: 92.

[0087] SV40 intron sequence: TIFF2025525027000004.tif19132

[0088] Polyadenylation sequence In some embodiments, the recombinant AAV vectors of the present disclosure include a sequence encoding a polyadenylation sequence, such as the bovine growth hormone (BGH) polyadenylation sequence (SEQ ID NO: 81) or the SV40 polyadenylation sequence (SEQ ID NO: 139). Polyadenylation sequences are nucleic acid elements commonly used in gene therapy vectors that assist in the transport of RNA from the nucleus, the translation of RNA, and the stability of RNA.

[0089] In some embodiments, the recombinant AAV vector of the present disclosure comprises a sequence encoding a BGH poly(A) tail having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 81. In some embodiments, the BGH poly(A) tail comprises a sequence having at least about 80% identity to SEQ ID NO: 81. In some embodiments, the BGH poly(A) tail comprises a sequence having at least about 85% identity to SEQ ID NO: 81. In some embodiments, the BGH poly(A) tail comprises a sequence having at least about 90% identity to SEQ ID NO: 81. In some embodiments, the BGH poly(A) tail comprises a sequence having at least about 95% identity to SEQ ID NO: 81. In some embodiments, the BGH poly(A) tail comprises a sequence having at least about 96% identity to SEQ ID NO: 81. In some embodiments, the BGH poly(A) tail comprises a sequence having at least about 97% identity to SEQ ID NO: 81. In some embodiments, the BGH poly(A) tail comprises a sequence having at least about 98% identity to SEQ ID NO: 81. In some embodiments, the BGH poly(A) tail comprises a sequence having at least about 99% identity to SEQ ID NO: 81. In some embodiments, the BGH poly(A) tail comprises a sequence having 100% identity to SEQ ID NO: 81. In some embodiments, the BGH poly(A) tail comprises a sequence according to SEQ ID NO: 81. In some embodiments, the BGH poly(A) tail consists of a sequence according to SEQ ID NO: 81.

[0090] In some embodiments, the recombinant AAV vector of the present disclosure comprises a sequence encoding an SV40 poly(A) tail having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 139. In some embodiments, the SV40 poly(A) tail comprises a sequence having at least about 80% identity to SEQ ID NO: 139. In some embodiments, the SV40 poly(A) tail comprises a sequence having at least about 85% identity to SEQ ID NO: 139. In some embodiments, the SV40 poly(A) tail comprises a sequence having at least about 90% identity to SEQ ID NO: 139. In some embodiments, the SV40 poly(A) tail comprises a sequence having at least about 95% identity to SEQ ID NO: 139. In some embodiments, the SV40 poly(A) tail comprises a sequence having at least about 96% identity to SEQ ID NO: 139. In some embodiments, the SV40 poly(A) tail comprises a sequence having at least about 97% identity to SEQ ID NO: 139. In some embodiments, the SV40 poly(A) tail comprises a sequence having at least about 98% identity to SEQ ID NO: 139. In some embodiments, the SV40 poly(A) tail comprises a sequence having at least about 99% identity to SEQ ID NO: 139. In some embodiments, the SV40 poly(A) tail comprises a sequence having 100% identity to SEQ ID NO: 139. In some embodiments, the SV40 poly(A) tail comprises a sequence according to SEQ ID NO: 139. In some embodiments, the SV40 poly(A) tail consists of a sequence according to SEQ ID NO: 139.

[0091] BGH poly(A) tail sequence: TIFF2025525027000005.tif29128

[0092] SV40 poly(A) tail sequence: TIFF2025525027000006.tif14128

[0093] Enhancer In some embodiments, the recombinant AAV vectors of the present disclosure include one or more enhancer sequences. The enhancer sequences can increase the transcription level of the transgene, for example, by functioning as binding sites for transcription factors and co-regulatory factors that assist DNA looping and recruitment of the transcriptional machinery to the promoter.

[0094] In some embodiments, the enhancer is downstream (i.e., 3') of the 5' ITR and upstream (i.e., 5') of the promoter. In some embodiments, the enhancer is downstream (i.e., 3') of the promoter and upstream (i.e., 5') of the transgene. In some embodiments, the enhancer is downstream (i.e., 3') of the transgene and upstream (i.e., 5') of the 3' UTR.

[0095] Antibiotic resistance gene In some embodiments, the recombinant AAV vectors of the present disclosure include 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.

[0096] 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: 150. 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: 150. 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: 150. 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: 150. 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: 150. 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: 150. 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: 150. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having the nucleic acid sequence of SEQ ID NO: 150.

[0097] 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: 177. 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: 177. 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: 177. 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: 177. 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: 177. 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: 177. 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: 177. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having the nucleic acid sequence of SEQ ID NO: 177.

[0098] Kanamycin Variant 1 TIFF2025525027000007.tif97128

[0099] Kanamycin Variant 2 TIFF2025525027000008.tif97128

[0100] Kozak sequence In some embodiments, the recombinant AAV vector of the present disclosure includes a Kozak sequence. In some embodiments, the Kozak sequence is the AAV2 Kozak sequence. In some embodiments, the Kozak sequence is the AAV8 Kozak sequence. In some embodiments, the Kozak sequence is the AAV-rh74 Kozak sequence. Exemplary Kozak sequences are found in Table 1B below.

[0101] (Table 1B) Exemplary Kozak sequences TIFF2025525027000009.tif127128

[0102] In some embodiments, the Kozak sequence includes a sequence having at least 85% (e.g., 85%, 90%, 95%, 97%, 98%, or 99%) sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 151-174. In some embodiments, the Kozak sequence includes a sequence having at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 151-174. In some embodiments, the Kozak sequence includes a sequence having at least 90% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 151-174. In some embodiments, the Kozak sequence includes a sequence having at least 95% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 151-174. In some embodiments, the Kozak sequence includes a sequence having at least 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 151-174. In some embodiments, the Kozak sequence includes a sequence having at least 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 151-174. In some embodiments, the Kozak sequence includes a sequence having at least 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 151-174. In some embodiments, the Kozak sequence includes a sequence having any one of the nucleic acid sequences of SEQ ID NOs: 151-174.

[0103] αHER2-αCD3 transgene In some embodiments, the transgene of the present disclosure is a nucleic acid sequence encoding a bispecific fusion protein having a HER2 binding site and a CD3 binding site. In some embodiments, the HER2 binding site comprises the VH and VL of an anti-HER2 antibody, and the CD3 binding site comprises the VH and VL of an anti-CD3 antibody.

[0104] In some embodiments, the transgene is integrated into the genome of the cell or is episomally expressed.

[0105] HER2 binding site As used herein, the HER2 binding site includes a protein comprising the sequence of SEQ ID NO: 93 and, in some embodiments, a polypeptide or a complex of two or more polypeptides that specifically binds to the relevant isoforms and orthologs. In some embodiments, the HER2 binding site specifically binds to one or more epitopes on the extracellular domain of HER2.

[0106] TIFF2025525027000010.tif141130* Underlined text indicates the extracellular domain.

[0107] In some embodiments, the HER2 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-HER2 antibodies and their corresponding complementarity determining regions (CDRs) that can specifically bind to HER2 in combination. Table 2B lists the corresponding nucleotide sequences of the VH and VL domains of anti-HER2 antibodies.

[0108] (Table 2A) αHER2 VH / VL and CDR amino acid sequences TIFF2025525027000011.tif235161TIFF2025525027000012.tif196161

[0109] (Table 2B) αHER2 VH / VL nucleotide sequences TIFF2025525027000013.tif35161TIFF2025525027000014.tif245161TIFF2025525027000015.tif177161

[0110] In some embodiments, the HER2 binding site comprises VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences selected from the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences listed in Table 2A, determined under Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), the IMGT unique numbering scheme, Chothia (e.g., see Chothia C & Lesk A M, (1987), J. Mol. Biol. 196: 901-917), MacCallum (see MacCallum R M et al., (1996) J. Mol. Biol. 262: 732-745), or any other CDR determination method known in the art.

[0111] Unless otherwise indicated, the CDR sequences provided in Table 2A are determined under the Kabat numbering scheme.

[0112] In some embodiments, the HER2 binding site comprises: (i) VL CDR1 comprising the amino acid sequence of SEQ ID NO: 100; (ii) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 101; (iii) VL CDR3 comprising the amino acid sequence of SEQ ID NO: 102; (iv) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 97; (v) VH CDR2 comprising the amino acid sequence of SEQ ID NO: 98; and (vi) VH CDR3 comprising the amino acid sequence of SEQ ID NO: 99.

[0113] In some embodiments, the HER2 binding site comprises: (i) VL CDR1 comprising the amino acid sequence of SEQ ID NO: 106; (ii) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 107; (iii) VL CDR3 comprising the amino acid sequence of SEQ ID NO: 108; (iv) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 103; (v) VH CDR2 comprising the amino acid sequence of SEQ ID NO: 104; and (vi) VH CDR3 comprising the amino acid sequence of SEQ ID NO: 105.

[0114] In some embodiments, the HER2 binding site comprises: (i) VL CDR1 comprising the amino acid sequence of SEQ ID NO: 112; (ii) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 113; (iii) VL CDR3 comprising the amino acid sequence of SEQ ID NO: 114; (iv) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 109; (v) VH CDR2 comprising the amino acid sequence of SEQ ID NO: 110; and (vi) VH CDR3 comprising the amino acid sequence of SEQ ID NO: 111.

[0115] In some embodiments, the HER2 binding site comprises: (i) VL CDR1 comprising the amino acid sequence of SEQ ID NO: 118; (ii) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 119; (iii) VL CDR3 comprising the amino acid sequence of SEQ ID NO: 120; (iv) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 115; (v) VH CDR2 comprising the amino acid sequence of SEQ ID NO: 116; and (vi) VH CDR3 comprising the amino acid sequence of SEQ ID NO: 117.

[0116] In some embodiments, the HER2 binding site comprises: (i) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 124; (ii) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 125; (iii) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 126; (iv) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 121; (v) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 122; and (vi) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 123.

[0117] Table 2A further lists the amino acid sequences of exemplary VL and VH domains that can specifically bind to HER2 in combination. In some embodiments, the HER2 binding site of the present disclosure 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 VH domain and VL domain sequences listed in Table 2A, and includes a VL domain and a VH domain. In some embodiments, the HER2 binding site includes a VH having an amino acid sequence with at least 80% sequence identity to the amino acid sequence according to any one of SEQ ID NO: 1, 4, 7, 11, and 14; and a VL including an amino acid sequence with at least 80% sequence identity to the amino acid sequence according to any one of SEQ ID NO: 2, 5, 8, 10, and 13. In some embodiments, the HER2 binding site includes a VH having an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to any one of SEQ ID NO: 1, 4, 7, 11, and 14; and a VL including an amino acid sequence with at least 85% sequence identity to the amino acid sequence according to any one of SEQ ID NO: 2, 5, 8, 10, and 13. In some embodiments, the HER2 binding site includes a VH having an amino acid sequence with at least 90% sequence identity to the amino acid sequence according to any one of SEQ ID NO: 1, 4, 7, 11, and 14; and a VL including an amino acid sequence with at least 90% sequence identity to the amino acid sequence according to any one of SEQ ID NO: 2, 5, 8, 10, and 13. In some embodiments, the HER2 binding site includes a VH having an amino acid sequence with at least 95% sequence identity to the amino acid sequence according to any one of SEQ ID NO: 1, 4, 7, 11, and 14; and a VL including an amino acid sequence with at least 95% sequence identity to the amino acid sequence according to any one of SEQ ID NO: 2, 5, 8, 10, and 13.In some embodiments, the HER2 binding site comprises a VH comprising an amino acid sequence having at least 96% sequence identity to an amino acid sequence according to any one of SEQ ID NO: 1, 4, 7, 11, and 14; and a VL comprising an amino acid sequence having at least 96% sequence identity to an amino acid sequence according to any one of SEQ ID NO: 2, 5, 8, 10, and 13. In some embodiments, the HER2 binding site comprises a VH comprising an amino acid sequence having at least 97% sequence identity to an amino acid sequence according to any one of SEQ ID NO: 1, 4, 7, 11, and 14; and a VL comprising an amino acid sequence having at least 97% sequence identity to an amino acid sequence according to any one of SEQ ID NO: 2, 5, 8, 10, and 13. In some embodiments, the HER2 binding site comprises a VH comprising an amino acid sequence having at least 98% sequence identity to an amino acid sequence according to any one of SEQ ID NO: 1, 4, 7, 11, and 14; and a VL comprising an amino acid sequence having at least 98% sequence identity to an amino acid sequence according to any one of SEQ ID NO: 2, 5, 8, 10, and 13. In some embodiments, the HER2 binding site comprises a VH comprising an amino acid sequence having at least 99% sequence identity to an amino acid sequence according to any one of SEQ ID NO: 1, 4, 7, 11, and 14; and a VL comprising an amino acid sequence having at least 99% sequence identity to an amino acid sequence according to any one of SEQ ID NO: 2, 5, 8, 10, and 13. In some embodiments, the HER2 binding site comprises a VH comprising an amino acid sequence according to any one of SEQ ID NO: 1, 4, 7, 11, and 14; and a VL comprising an amino acid sequence according to any one of SEQ ID NO: 2, 5, 8, 10, and 13.

[0118] In some embodiments, the HER2 binding site of the present disclosure comprises (i) a VH having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 1; and (ii) a VL having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 2. In some embodiments, the HER2 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 HER2 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 HER2 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 HER2 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 HER2 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 HER2 binding site comprises a VH comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 1; and a VL comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 2. In some embodiments, the HER2 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 HER2 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 HER2 binding site comprises a VH comprising the amino acid sequence according to SEQ ID NO: 1; and a VL comprising the amino acid sequence according to SEQ ID NO: 2.

[0119] In some embodiments, the HER2 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: 4; 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: 5. In some embodiments, the HER2 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: 4; and a VL comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 5. In some embodiments, the HER2 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: 4; and a VL comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 5. In some embodiments, the HER2 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: 4; and a VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 5. In some embodiments, the HER2 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: 4; and a VL comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 5.In some embodiments, the HER2 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: 4; and a VL comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 5. In some embodiments, the HER2 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: 4; and a VL comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 5. In some embodiments, the HER2 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: 4; and a VL comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 5. In some embodiments, the HER2 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: 4; and a VL comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 5. In some embodiments, the HER2 binding site comprises a VH comprising the amino acid sequence according to SEQ ID NO: 4; and a VL comprising the amino acid sequence according to SEQ ID NO: 5.

[0120] In some embodiments, the HER2 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: 7; 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: 8. In some embodiments, the HER2 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: 7; and a VL comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the HER2 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: 7; and a VL comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the HER2 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: 7; and a VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the HER2 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: 7; and a VL comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 8.In some embodiments, the HER2 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: 7; and a VL comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the HER2 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: 7; and a VL comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the HER2 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: 7; and a VL comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the HER2 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: 7; and a VL comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the HER2 binding site comprises a VH comprising the amino acid sequence according to SEQ ID NO: 7; and a VL comprising the amino acid sequence according to SEQ ID NO: 8.

[0121] In some embodiments, the HER2 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: 11; 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: 10. In some embodiments, the HER2 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: 11; and a VL comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 10. In some embodiments, the HER2 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: 11; and a VL comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 10. In some embodiments, the HER2 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: 11; and a VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 10. In some embodiments, the HER2 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: 11; and a VL comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 10.In some embodiments, the HER2 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: 11; and a VL comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 10. In some embodiments, the HER2 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: 11; and a VL comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 10. In some embodiments, the HER2 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: 11; and a VL comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 10. In some embodiments, the HER2 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: 11; and a VL comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 10. In some embodiments, the HER2 binding site comprises a VH comprising the amino acid sequence according to SEQ ID NO: 11; and a VL comprising the amino acid sequence according to SEQ ID NO: 10.

[0122] In some embodiments, the HER2 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: 13. In some embodiments, the HER2 binding site comprises a VH comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 14; and a VL comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 13. In some embodiments, the HER2 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: 13. In some embodiments, the HER2 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: 13. In some embodiments, the HER2 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: 13.In some embodiments, the HER2 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: 13. In some embodiments, the HER2 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: 13. In some embodiments, the HER2 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: 13. In some embodiments, the HER2 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: 13. In some embodiments, the HER2 binding site comprises a VH comprising the amino acid sequence according to SEQ ID NO: 14; and a VL comprising the amino acid sequence according to SEQ ID NO: 13.

[0123] In some embodiments, the HER2 binding site comprises, but is not limited to, a single-chain variable region fragment (scFv), an antibody, a Fab, a Fab’, an F(ab’)2, a minibody, or a nanobody (VHH). For example, in some embodiments, the bispecific fusion protein of the present disclosure comprises an scFv polypeptide that specifically binds to HER2.

[0124] In some embodiments, the HER2 binding site of the present disclosure is in the scFv format. In some embodiments, the HER2-binding scFv of the present disclosure comprises an scFv linker polypeptide that functionally connects the VH domain and the VL domain. For example, in some embodiments, the HER2-binding scFv comprises, from the N-terminus to the C-terminus, the VL domain of an anti-HER2 antibody, the scFv linker polypeptide, and the VH domain of an anti-HER2 antibody. In other embodiments, the HER2-binding scFv comprises, from the N-terminus to the C-terminus, the VH domain of an anti-HER2 antibody, the scFv linker polypeptide, and the VL domain of an anti-HER2 antibody.

[0125] In some embodiments, the scFv linker polypeptide comprises a sequence selected from the linker sequences in Table 3A.

[0126] (Table 3A) scFv linker peptide sequences TIFF2025525027000016.tif57135

[0127] In some embodiments, the HER2-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 domain 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.

[0128] (Table 3B) Spacer peptide sequences TIFF2025525027000017.tif37135

[0129] Table 4A lists the amino acid sequences of exemplary HER2-binding scFvs. In some embodiments, the bispecific fusion proteins of the present disclosure comprise a HER2-binding scFv having a sequence that is at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the scFv sequences listed in Table 4A. Table 4B lists the corresponding nucleotide sequences of exemplary HER2-binding scFvs.

[0130] (Table 4A) HER2-binding scFv Amino Acid Sequences TIFF2025525027000018.tif153163TIFF2025525027000019.tif157163* Underlined and italicized The letters indicate the scFv linker sequence; Underlined The letters indicate the spacer sequence.

[0131] (Table 4B) HER2-binding scFv Nucleotide Sequences TIFF2025525027000020.tif60163TIFF2025525027000021.tif245163TIFF2025525027000022.tif162163* Underlined The letters indicate the scFv linker sequence, or the spacer sequence and the scFv linker sequence.

[0132] In some embodiments, the bispecific fusion protein of the present disclosure comprises an scFv that specifically binds to HER2 and that comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 3. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 85% identity to SEQ ID NO: 3. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 90% identity to SEQ ID NO: 3. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 95% identity to SEQ ID NO: 3. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 96% identity to SEQ ID NO: 3. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 97% identity to SEQ ID NO: 3. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 98% identity to SEQ ID NO: 3. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 99% identity to SEQ ID NO: 3. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having 100% identity to SEQ ID NO: 3.

[0133] In some embodiments, the bispecific fusion protein of the present disclosure comprises a scFv that specifically binds to HER2 and that comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 88. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 85% identity to SEQ ID NO: 88. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 90% identity to SEQ ID NO: 88. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 95% identity to SEQ ID NO: 88. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 96% identity to SEQ ID NO: 88. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 97% identity to SEQ ID NO: 88. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 98% identity to SEQ ID NO: 88. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 99% identity to SEQ ID NO: 88. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having 100% identity to SEQ ID NO: 88.

[0134] In some embodiments, the bispecific fusion protein of the present disclosure comprises a scFv that specifically binds to HER2 and comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 85% identity to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 90% identity to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 95% identity to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 96% identity to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 97% identity to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 98% identity to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 99% identity to SEQ ID NO: 6. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having 100% identity to SEQ ID NO: 6.

[0135] In some embodiments, the bispecific fusion protein of the present disclosure comprises a scFv that specifically binds to HER2 and comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 89. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 85% identity to SEQ ID NO: 89. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 90% identity to SEQ ID NO: 89. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 95% identity to SEQ ID NO: 89. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 96% identity to SEQ ID NO: 89. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 97% identity to SEQ ID NO: 89. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 98% identity to SEQ ID NO: 89. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 99% identity to SEQ ID NO: 89. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having 100% identity to SEQ ID NO: 89.

[0136] In some embodiments, the bispecific fusion protein of the present disclosure comprises a scFv that specifically binds to HER2 and that comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 9. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 85% identity to SEQ ID NO: 9. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 90% identity to SEQ ID NO: 9. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 95% identity to SEQ ID NO: 9. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 96% identity to SEQ ID NO: 9. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 97% identity to SEQ ID NO: 9. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 98% identity to SEQ ID NO: 9. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 99% identity to SEQ ID NO: 9. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having 100% identity to SEQ ID NO: 9.

[0137] In some embodiments, the bispecific fusion protein of the present disclosure comprises a scFv that specifically binds to HER2 and comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 90. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 85% identity to SEQ ID NO: 90. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 90% identity to SEQ ID NO: 90. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 95% identity to SEQ ID NO: 90. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 96% identity to SEQ ID NO: 90. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 97% identity to SEQ ID NO: 90. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 98% identity to SEQ ID NO: 90. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 99% identity to SEQ ID NO: 90. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having 100% identity to SEQ ID NO: 90.

[0138] In some embodiments, the bispecific fusion protein of the present disclosure comprises a scFv that specifically binds to HER2 and comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 140. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 85% identity to SEQ ID NO: 140. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 90% identity to SEQ ID NO: 140. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 95% identity to SEQ ID NO: 140. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 96% identity to SEQ ID NO: 140. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 97% identity to SEQ ID NO: 140. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 98% identity to SEQ ID NO: 140. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 99% identity to SEQ ID NO: 140. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having 100% identity to SEQ ID NO: 140.

[0139] In some embodiments, the bispecific fusion protein of the present disclosure comprises a scFv that specifically binds to HER2 and that comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 12. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 85% identity to SEQ ID NO: 12. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 90% identity to SEQ ID NO: 12. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 95% identity to SEQ ID NO: 12. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 96% identity to SEQ ID NO: 12. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 97% identity to SEQ ID NO: 12. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 98% identity to SEQ ID NO: 12. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 99% identity to SEQ ID NO: 12. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having 100% identity to SEQ ID NO: 12.

[0140] In some embodiments, the bispecific fusion protein of the present disclosure comprises a scFv that specifically binds to HER2 and that comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 141. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 85% identity to SEQ ID NO: 141. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 90% identity to SEQ ID NO: 141. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 95% identity to SEQ ID NO: 141. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 96% identity to SEQ ID NO: 141. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 97% identity to SEQ ID NO: 141. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 98% identity to SEQ ID NO: 141. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 99% identity to SEQ ID NO: 141. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having 100% identity to SEQ ID NO: 141.

[0141] In some embodiments, the bispecific fusion protein of the present disclosure comprises a scFv that specifically binds to HER2 and has an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 15. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 85% identity to SEQ ID NO: 15. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 90% identity to SEQ ID NO: 15. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 95% identity to SEQ ID NO: 15. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 96% identity to SEQ ID NO: 15. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 97% identity to SEQ ID NO: 15. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 98% identity to SEQ ID NO: 15. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having at least about 99% identity to SEQ ID NO: 15. In some embodiments, the scFv that specifically binds to HER2 comprises a sequence having 100% identity to SEQ ID NO: 15.

[0142] CD3 binding site The bispecific fusion proteins of the present disclosure can include a polypeptide or a complex of two or more polypeptides that specifically binds to CD3 on the surface of T cells. In some embodiments, the bispecific fusion proteins of the present disclosure bind to CD3 expressed on mature T lymphocytes, such as αβ T cells, γδ T cells, NK-T cells, mucosal-associated invariant T (MAIT) cells, and phenotypic subsets thereof. In some embodiments, binding of CD3 induces activation of T cells when crosslinked to HER2.

[0143] As used herein, the CD3 binding site is, in some embodiments, a polypeptide or a complex of two or more polypeptides that specifically binds to CD3 (SEQ ID NO: 142). For example, the CD3 binding site binds to the CD3ε chain.

[0144] CD3 sequence (ε chain): TIFF2025525027000023.tif24134

[0145] In some embodiments, the CD3 binding site includes a heavy chain variable region (VH) and a light chain variable region (VL). Table 5A lists the VH and VL domains of anti-CD3 antibodies, and their corresponding complementarity-determining regions (CDRs), that can specifically bind to CD3 in combination. Table 5B lists the corresponding nucleotide sequences of the VH and VL regions of anti-CD3 antibodies.

[0146] (Table 5A) αCD3 VH / VL sequences and CDRs TIFF2025525027000024.tif176161

[0147] (Table 5B) αCD3 VH / VL nucleotide sequences TIFF2025525027000025.tif187161TIFF2025525027000026.tif235161

[0148] In some embodiments, the CD3 binding site comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences listed in Table 5A, determined under the IMGT unique numbering scheme, Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, for example, Chothia C & Lesk A M, (1987), J. Mol. Biol. 196: 901-917), MacCallum (see MacCallum R M et al., (1996) J. Mol. Biol. 262: 732-745), or any other CDR determination method known in the art.

[0149] Unless otherwise indicated, the CDR sequences provided in Table 5A are determined under the Kabat numbering scheme.

[0150] In some embodiments, the CD3 binding site comprises: (i) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 127; (ii) VH CDR2 comprising the amino acid sequence of SEQ ID NO: 128; (iii) VH CDR3 comprising the amino acid sequence of SEQ ID NO: 129; (iv) VL CDR1 comprising the amino acid sequence of SEQ ID NO: 130; (v) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 131; and (vi) VL CDR3 comprising the amino acid sequence of SEQ ID NO: 132.

[0151] In some embodiments, the CD3 binding site comprises (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 133; (ii) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 134; (iii) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 135; (iv) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 136; (v) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 137; and (vi) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 138.

[0152] Table 5A further lists the amino acid sequences of exemplary VH and VL domains that can specifically bind to CD3 in combination. In some embodiments, the CD3 binding site of the present disclosure comprises VH and VL having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences listed in Table 5A.

[0153] In some embodiments, the CD3 binding site of the present disclosure comprises (i) a VH having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 16; 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: 17. In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 16; and a VL comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 17. 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: 16; and a VL comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 17. 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: 16; and a VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 17. 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: 16; and a VL comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 17.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: 16; and a VL comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 17. 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: 16; and a VL comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 17. 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: 16; and a VL comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 17. 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: 16; and a VL comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the CD3 binding site comprises a VH comprising the amino acid sequence according to SEQ ID NO: 16; and a VL comprising the amino acid sequence according to SEQ ID NO: 17.

[0154] In some embodiments, the CD3 binding site of the present disclosure comprises (i) a VH having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 95; 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: 96. In some embodiments, the CD3 binding site comprises a VH comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 95; and a VL comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 96. 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: 95; and a VL comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 96. 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: 95; and a VL comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 96. 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: 95; and a VL comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 96.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: 95; and a VL comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 96. 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: 95; and a VL comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 96. 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: 95; and a VL comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 96. 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: 95; and a VL comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 96. In some embodiments, the CD3 binding site comprises a VH comprising the amino acid sequence according to SEQ ID NO: 95; and a VL comprising the amino acid sequence according to SEQ ID NO: 96.

[0155] In some embodiments, the CD3 binding site comprises, but is not limited to, a single-chain variable region fragment (scFv), an antibody, a Fab, a Fab’, an F(ab’)2, a minibody, or a nanobody (VHH). For example, in some embodiments, the bispecific fusion protein of the disclosure comprises an scFv polypeptide that specifically binds to CD3, respectively.

[0156] In some embodiments, the CD3 binding site of the present disclosure is in scFv format. In some embodiments, the CD3-binding scFv of the present disclosure comprises an scFv linker polypeptide that functionally connects the VH domain and the VL domain. For example, in some embodiments, the CD3-binding scFv comprises, from 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.

[0157] In some embodiments, the scFv linker polypeptide comprises a sequence selected from the linker sequences in Table 3A.

[0158] Table 6A lists the amino acid sequences of exemplary CD3-binding scFvs. In some embodiments, the bispecific fusion protein of the present disclosure comprises a CD3-binding scFv 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.

[0159] (Table 6A) αCD3 scFv Amino Acid Sequences TIFF2025525027000027.tif65161* Underlined and italicized The letters indicate the scFv linker sequence.

[0160] (Table 6B) αCD3 scFv Nucleotide Sequences TIFF2025525027000028.tif152161TIFF2025525027000029.tif245161TIFF2025525027000030.tif45161* Underlined The letters indicate the scFv linker sequence.

[0161] In some embodiments, the bispecific fusion protein of the present disclosure comprises a scFv that specifically binds to CD3 and that comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 85% identity to SEQ ID NO: 18. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 90% identity to SEQ ID NO: 18. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 95% identity to SEQ ID NO: 18. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 96% identity to SEQ ID NO: 18. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 97% identity to SEQ ID NO: 18. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 98% identity to SEQ ID NO: 18. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 99% identity to SEQ ID NO: 18. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having 100% identity to SEQ ID NO: 18.

[0162] In some embodiments, the bispecific fusion protein of the present disclosure comprises a scFv that specifically binds to CD3 and comprises an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 94. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 85% identity to SEQ ID NO: 94. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 90% identity to SEQ ID NO: 94. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 95% identity to SEQ ID NO: 94. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 96% identity to SEQ ID NO: 94. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 97% identity to SEQ ID NO: 94. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 98% identity to SEQ ID NO: 94. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having at least about 99% identity to SEQ ID NO: 94. In some embodiments, the scFv that specifically binds to CD3 comprises a sequence having 100% identity to SEQ ID NO: 94.

[0163] Exemplary αHER2-αCD3 bispecific fusion protein Enumerated below are examples of the bispecific fusion proteins of the present disclosure that comprise a HER2 binding site fused to a CD3 binding site via a linker peptide.

[0164] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences selected from the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences listed in Table 2A; (ii) a linker peptide comprising a sequence selected from Table 7; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences listed in Table 5A. In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences listed in Table 5A; (ii) a linker peptide comprising a sequence selected from Table 7; and (iii) a HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences selected from the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences listed in Table 2A. The order of the HER2 binding site and the CD3 binding site is not limited. For example, the HER2 binding site is at the amino terminus of the bispecific fusion protein and the CD3 binding site is at the carboxy terminus of the bispecific fusion protein. In some embodiments, the CD3 binding site is at the amino terminus of the bispecific fusion protein and the HER2 binding site is at the carboxy terminus of the bispecific fusion protein.

[0165] (Table 7) Linker peptide TIFF2025525027000031.tif17135

[0166] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively; (ii) a linker peptide comprising the sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0167] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively; (ii) a linker peptide comprising the sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0168] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0169] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0170] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0171] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 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; (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, respectively.

[0172] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 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: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0173] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 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: 5 and SEQ ID NO: 4, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0174] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 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: 8 and SEQ ID NO: 7, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0175] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 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: 10 and SEQ ID NO: 11, respectively; (ii) a linker peptide comprising the sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0176] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 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 NOs: 13 and 14, respectively; (ii) a linker peptide comprising the sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NOs: 16 and 17, respectively.

[0177] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the scFv sequences listed in Table 4A; (ii) a linker peptide comprising a sequence selected from Table 7; and (iii) a CD3 binding site comprising an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the scFv sequences listed in Table 6A.

[0178] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 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: 88; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0179] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 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: 89; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0180] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 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: 90; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0181] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 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: 12; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0182] In some embodiments, the bispecific fusion protein of the present disclosure comprises: (i) a HER2 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: 15; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0183] In some embodiments, the bispecific fusion protein 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 domain of the HER2 scFv, at the C-terminus of the VL domain of the HER2 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.

[0184] In some embodiments, the bispecific fusion protein of the present disclosure comprises an amino acid sequence corresponding to the sequences listed in Table 8A. Table 8B lists the corresponding nucleotide sequences of the bispecific fusion protein.

[0185] (Table 8A) Bispecific fusion protein amino acid sequences TIFF2025525027000032.tif216161TIFF2025525027000033.tif89161* Underlined and italicized The letters indicate the scFv linker sequence; Underlined The letters indicate the spacer sequence; the bold letters indicate the linker sequence.

[0186] (Table 8B) Nucleotide sequence of the bispecific fusion protein TIFF2025525027000034.tif133161TIFF2025525027000035.tif245161TIFF2025525027000036.tif244161TIFF2025525027000037.tif230161

[0187] In addition to the sequences presented in Table 8A, the bispecific fusion proteins of the present disclosure may further include a signal peptide fused to the N-terminus. It is understood that the mature form of the protein with the signal peptide cleaved is expressed.

[0188] For example, the bispecific fusion proteins of the present disclosure may further include a signal peptide having an amino acid sequence having at least 85% identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 34.

[0189] Signal peptide sequence: TIFF2025525027000038.tif12128

[0190] 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 least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 19. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to SEQ ID NO: 19.

[0191] 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: 20. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 20. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 20. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 20. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 20. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 20. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 20. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 20. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to SEQ ID NO: 20.

[0192] 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: 21. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 21. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 21. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 21. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 21. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 21. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 21. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 21. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to SEQ ID NO: 21.

[0193] 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: 22. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 22. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 22. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 22. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 22. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 22. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 22. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 22. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to SEQ ID NO: 22.

[0194] 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: 23. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 23. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 23. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 23. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 23. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 23. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 23. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 23. In some embodiments, the bispecific fusion protein comprises an amino acid sequence having 100% identity to SEQ ID NO: 23.

[0195] Exemplary rAAV vectors In some embodiments, the recombinant adeno-associated virus (rAAV) vector of the present disclosure comprises 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: 58-62. In some embodiments, the transgene comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 58-62. In some embodiments, the transgene comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 58-62. In some embodiments, the transgene comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 58-62. In some embodiments, the transgene comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 58-62. In some embodiments, the transgene comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 58-62. In some embodiments, the transgene comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 58-62. In some embodiments, the transgene comprises a sequence having 100% identity to any one of SEQ ID NOs: 58-62.

[0196] In some embodiments, the rAAV vector of the present disclosure comprises a transgene sequence that is at least 85% sequence identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 59.

[0197] In some embodiments, the rAAV vectors of the present disclosure comprise a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 60 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0198] In some embodiments, the rAAV vectors of the present disclosure comprise a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 61 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0199] In some embodiments, the rAAV vectors of the present disclosure comprise a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 58 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0200] In some embodiments, the rAAV vectors of the present disclosure comprise a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 62 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0201] In some embodiments, the rAAV vectors of the present disclosure include one or more regulatory elements. In some embodiments, the one or more regulatory elements are 5' to the sequence encoding the bispecific fusion protein. In some embodiments, the one or more regulatory elements are 3' to the sequence encoding the bispecific fusion protein. For example, in some embodiments, the regulatory element is 3' to the sequence encoding the bispecific fusion protein and is derived from the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the regulatory element is at least 85% identical to SEQ ID NO: 80 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%). In some embodiments, the WPRE comprises a sequence having at least about 85% identity to SEQ ID NO: 80. In some embodiments, the WPRE comprises a sequence having at least about 90% identity to SEQ ID NO: 80. In some embodiments, the WPRE comprises a sequence having at least about 95% identity to SEQ ID NO: 80. In some embodiments, the WPRE comprises a sequence having at least about 96% identity to SEQ ID NO: 80. In some embodiments, the WPRE comprises a sequence having at least about 97% identity to SEQ ID NO: 80. In some embodiments, the WPRE comprises a sequence having at least about 98% identity to SEQ ID NO: 80. In some embodiments, the WPRE comprises a sequence having at least about 99% identity to SEQ ID NO: 80. In some embodiments, the WPRE comprises a sequence having 100% identity to SEQ ID NO: 80.

[0202] Regulatory element derived from WPRE: TIFF2025525027000039.tif68134

[0203] In some embodiments, the regulatory element is 3’ to the sequence encoding the bispecific fusion protein and is a modified RNA stability regulatory element (MRE). In some embodiments, the regulatory element is at least 85% identical to SEQ ID NO: 175 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%). In some embodiments, the MRE comprises a sequence having at least about 85% identity to SEQ ID NO: 175. In some embodiments, the MRE comprises a sequence having at least about 90% identity to SEQ ID NO: 175. In some embodiments, the MRE comprises a sequence having at least about 95% identity to SEQ ID NO: 175. In some embodiments, the MRE comprises a sequence having at least about 96% identity to SEQ ID NO: 175. In some embodiments, the MRE comprises a sequence having at least about 97% identity to SEQ ID NO: 175. In some embodiments, the MRE comprises a sequence having at least about 98% identity to SEQ ID NO: 175. In some embodiments, the MRE comprises a sequence having at least about 99% identity to SEQ ID NO: 175. In some embodiments, the MRE comprises a sequence having 100% identity to SEQ ID NO: 175.

[0204] In some embodiments, the MRE comprises a sequence having at least about 85% identity to SEQ ID NO: 176. In some embodiments, the MRE comprises a sequence having at least about 90% identity to SEQ ID NO: 176. In some embodiments, the MRE comprises a sequence having at least about 95% identity to SEQ ID NO: 176. In some embodiments, the MRE comprises a sequence having at least about 96% identity to SEQ ID NO: 176. In some embodiments, the MRE comprises a sequence having at least about 97% identity to SEQ ID NO: 176. In some embodiments, the MRE comprises a sequence having at least about 98% identity to SEQ ID NO: 176. In some embodiments, the MRE comprises a sequence having at least about 99% identity to SEQ ID NO: 176. In some embodiments, the MRE comprises a sequence having 100% identity to SEQ ID NO: 176.

[0205] Modified RNA stability regulatory element (MRE) variant 1 TIFF2025525027000040.tif68134

[0206] Modified RNA stability regulatory element (MRE) variant 2 TIFF2025525027000041.tif68134

[0207] In some embodiments, the rAAV vector of the present disclosure has a nucleotide sequence that is at least 85% sequence identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the nucleotide sequences listed in Table 9.

[0208] (Table 9) Exemplary AAV2 vector sequences TIFF2025525027000042.tif186161TIFF2025525027000043.tif244161TIFF2025525027000044.tif244161TIFF2025525027000045.tif244161TIFF2025525027000046.tif244161TIFF2025525027000047.tif244161TIFF2025525027000048.tif244161TIFF2025525027000049.tif244161TIFF2025525027000050.tif244161TIFF2025525027000051.tif244161TIFF2025525027000052.tif244161TIFF2025525027000053.tif244161TIFF2025525027000054.tif244161TIFF2025525027000055.tif244161TIFF2025525027000056.tif244161TIFF2025525027000057.tif244161TIFF2025525027000058.tif152161

[0209] In some embodiments, the rAAV vector comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 82-86. In some embodiments, the rAAV vector comprises a sequence having at least about 90% identity to any one of SEQ ID NOs: 82-86. In some embodiments, the rAAV vector comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 82-86. In some embodiments, the rAAV vector comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 82-86. In some embodiments, the rAAV vector comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 82-86. In some embodiments, the rAAV vector comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 82-86. In some embodiments, the rAAV vector comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 82-86. In some embodiments, the rAAV vector comprises a sequence having 100% identity to any one of SEQ ID NOs: 82-86.

[0210] In some embodiments, the rAAV vector of the present disclosure has a nucleotide sequence that is at least 85% sequence identical to SEQ ID NO: 83 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

[0211] 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: 84.

[0212] 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: 85.

[0213] 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: 82.

[0214] 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: 86.

[0215] In some embodiments, the rAAV vectors of the present disclosure include one or more components (e.g., regulatory elements, transgenes) that contain reduced CpG dinucleotides and / or increased methylation of CpG dinucleotides as compared to parental equivalents. In some embodiments, the CpG dinucleotides are reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% as compared to parental equivalents. In some embodiments, the CpG dinucleotides are reduced in the range of about 5% to about 90%, about 10% to about 80%, about 15% to about 75%, about 20% to about 70%, about 25% to about 65%, or about 30% to about 60%. In some embodiments, the methylation of CpG dinucleotides is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% as compared to parental equivalents. In some embodiments, the methylation of CpG dinucleotides is increased in the range of about 5% to about 90%, about 10% to about 80%, about 15% to about 75%, about 20% to about 70%, about 25% to about 65%, or about 30% to about 60%.

[0216] 2. Production of Recombinant Adeno-Associated Virus (AAV) Vectors Recombinant AAV particles can be produced by any standard method (e.g., WO 2001 / 083692, which is hereby incorporated by reference in its entirety; Masic et al. 2014. Molecular Therapy, 22(11):1900-1909; Carter, 1992, Current Opinions in Biotechnology, 1533-539; Muzyczka, 1992, Curr. Topics in Microbial, and Immunol., 158:97-129); Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al, J. Virol, 62: 1963 (1988); and Lebkowski et al, Mol. Cell. Biol, 7:349 (1988). Samulski et al, J. Virol., 63:3822-3828 (1989); U.S. Patent No. 5,173,414; WO 95 / 13365; U.S. Patent No. 5,658.776; WO95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US 96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. Vaccine 13: 1244-1250 (1995); Paul et al. Human Gene Therapy 4:609-615 (1993); Clark et al. Gene Therapy 3: 1124-1132 (1996); U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent No. 6,258,595).For example, in some embodiments, the AAV vectors described herein can be transformed into Escherichia coli to scale up DNA production, purified using any standard method (e.g., Maxi-Prep K), and verified by restriction digestion or sequencing. The purified AAV vector can then be combined with a plasmid containing the AAV rep and AAV cap genes, as well as an AAV helper plasmid, and transfected into a suitable packaging cell line (e.g., HEK293, HeLa, Sf9, PerC.6, MRC-5, WI-38, Vera, or FRhL-2 cells) using standard methods (e.g., calcium phosphate transfection, liposomes, polyethyleneimine, electroporation, etc.). The AAV rep and cap genes can be derived from any AAV serotype and may be the same as or different from those of the recombinant AAV vector ITR, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. In one embodiment, the recombinant AAV described herein contains AAV rep and cap genes derived from AAV2 and AAV9, respectively. The AAV helper plasmid can be derived from any AAV serotype and may be the same as or different from those of the recombinant AAV vector ITR, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. In one embodiment, the recombinant AAV described herein contains a plasmid having a helper gene derived from AAV2.

[0217] In some embodiments, the AAV rep and cap genes are derived from AAVrh.74. In some embodiments, the rep and cap genes comprise a nucleotide sequence having at least 85% (e.g., 85%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 178. In some embodiments, the rep and cap genes comprise a nucleotide sequence having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 178. In some embodiments, the rep and cap genes comprise a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 178. In some embodiments, the rep and cap genes comprise a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 178. In some embodiments, the rep and cap genes comprise a nucleotide sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 178. In some embodiments, the rep and cap genes comprise a nucleotide sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 178. 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: 178. In some embodiments, the rep and cap genes comprise a nucleotide sequence having the nucleic acid sequence of SEQ ID NO: 178.

[0218] (Table 10) Rep and Cap Sequences TIFF2025525027000059.tif142159TIFF2025525027000060.tif244159TIFF2025525027000061.tif244159TIFF2025525027000062.tif244159TIFF2025525027000063.tif244159TIFF2025525027000064.tif244159TIFF2025525027000065.tif59159

[0219] In some embodiments, the recombinant AAVs described herein are harvested from packaging cells and purified by standard methods in the art, such as by cesium chloride ultracentrifugation gradients or column chromatography (e.g., Clark et al, Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69: 427-443 (2002); U.S. Patent No. 6,566,118 and WO 98 / 09657, which are hereby incorporated by reference in their entirety).

[0220] In some embodiments, the rAAV of the disclosure comprises a nucleotide sequence that is at least 85% sequence identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the nucleotide sequences listed in Table 9.

[0221] In some embodiments, the rAAV of the present disclosure is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, or a derivative thereof. In some embodiments, the rAAV is AAV2, or a derivative thereof. In some embodiments, the rAAV is AAV8, or a derivative thereof. In some embodiments, the rAAV is AAV-rh74, or a derivative thereof.

[0222] 3. Pharmaceutical Composition The recombinant AAV vectors described herein can be used in the manufacture of pharmaceutical compositions. In some embodiments, the pharmaceutical compositions disclosed herein comprise the recombinant AAV vectors of the present disclosure and a pharmaceutically acceptable carrier, and optionally, other agents, pharmaceuticals, stabilizers, buffers, carriers, adjuvants, diluents, and the like. "Pharmaceutically acceptable" means a material that is not toxic or otherwise undesirable, i.e., the material can be administered to a subject without causing undesirable biological effects.

[0223] In some embodiments, the pharmaceutical composition can include sterile aqueous and non-aqueous injection solutions, which are optionally isotonic with the blood of the subject to which the pharmaceutical composition is to be delivered. The pharmaceutical composition can contain antioxidants, buffers, bacteriostatic agents, and solutes, which render the composition isotonic with the blood of the intended subject to which the composition is administered. Aqueous and non-aqueous sterile suspensions, solutions, and emulsions can include suspending and thickening agents. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters for injection such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions including physiological saline and buffered media. In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable vehicle and can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0224] In some embodiments, the pharmaceutical composition can be presented in unit / dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in a freeze-dried state that requires only the addition of a sterile liquid carrier, such as physiological saline or water for injection, immediately prior to use.

[0225] In some embodiments, the pharmaceutical compositions disclosed herein can be formulated for intravenous, intramuscular, intrathecal, or intraventricular administration.

[0226] 4. Method of Treatment The recombinant AAV of the disclosure or a pharmaceutical composition comprising the same can be administered to a subject in need thereof by any delivery mode including, but not limited to, intravenous, intraperitoneal, and intramuscular administration.

[0227] In some embodiments, the recombinant AAV of the disclosure or a pharmaceutical composition containing the same can be administered in one, two, three, four, five, or more doses. In some embodiments, when multiple doses are administered, the doses can be administered to the subject in need thereof simultaneously or at intervals.

[0228] This application provides a method for reducing, preventing, and treating cancer and the risk of metastasis by administering to a patient an rAAV vector as described herein, or a pharmaceutical formulation thereof.

[0229] In other embodiments, the recombinant AAV vector of the disclosure or a pharmaceutical composition containing the same can be administered as a single intravenous dose or a divided intravenous dose. In some embodiments, the dose for intravenous delivery is 1 X 10 10 ~1 X 10 13 vg / kg, 2 X 10 10 ~1 X 10 13 vg / kg, 3 X 10 10 ~1 X 10 13 vg / kg, 4 X 10 10 ~1 X 10 13 vg / kg, 5 X 10 10 ~1 X 10 13 vg / kg, 6 X 10 10 ~1 X 10 13 vg / kg, 7 X 10 10 ~1 X 10 13 vg / kg, 8 X 10 10 ~1 X 10 13 vg / kg, 9 X 10 10 ~1 X 10 13 vg / kg, 1 X 10 11 ~1 X 10 13 vg / kg, 2 X 10 11 ~1 X 10 13 vg / kg, 3 X 10 11 ~1 X 10 13 vg / kg, 4 X 10 11 ~1 X 10 13vg / kg, 5 X 10 11 ~1 X 10 13 vg / kg, 6 X 10 11 ~1 X 10 13 vg / kg, 7 X 10 11 ~1 X 10 13 vg / kg, 8 X 10 11 ~1 X 10 13 vg / kg, 9 X 10 11 ~1 X 10 13 vg / kg, 1 X 10 12 ~1 X 10 13 vg / kg, 2 X 10 12 ~1 X 10 13 vg / kg, 3 X 10 12 ~1 X 10 13 vg / kg, 4 X 10 12 ~1 X 10 13 vg / kg, 5 X 10 12 ~1 X 10 13 vg / kg, 6 X 10 12 ~1 X 10 13 vg / kg, 7 X 10 12 ~1 X 10 13 vg / kg, 8 X 10 12 ~1 X 10 13 vg / kg, 9 X 10 12 ~1 X 10 13 vg / kg, 1 X 10 10 ~1 X 10 12 vg / kg, 2 X 10 10 ~1 X 10 12 vg / kg, 3 X 10 10 ~1 X 10 12 vg / kg, 4 X 10 10 ~1 X 10 12 vg / kg, 5 X 10 10 ~1 X 10 12 vg / kg, 6 X 10 10 ~1 X 10 12 vg / kg, 7 X 10 10 ~1 X 10 12 vg / kg, 8 X 10 10 ~1 X 10 12vg / kg, 9 X 10 10 ~1 X 10 12 vg / kg, 1 X 10 11 ~1 X 10 12 vg / kg, 2 X 10 11 ~1 X 10 12 vg / kg, 3 X 10 11 ~1 X 10 12 vg / kg, 4 X 10 11 ~1 X 10 12 vg / kg, 5 X 10 11 ~1 X 10 12 vg / kg, 6 X 10 11 ~1 X 10 12 vg / kg, 7 X 10 11 ~1 X 10 12 vg / kg, 8 X 10 11 ~1 X 10 12 vg / kg, 9 X 10 11 ~1 X 10 12 vg / kg, 1 X 10 10 ~1 X 10 11 vg / kg, 2 X 10 10 ~1 X 10 11 vg / kg, 3 X 10 10 ~1 X 10 11 vg / kg, 4 X 10 10 ~1 X 10 11 vg / kg, 5 X 10 10 ~1 X 10 11 vg / kg, 6 X 10 10 ~1 X 10 11 vg / kg, 7 X 10 10 ~1 X 10 11 vg / kg, 8 X 10 10 ~1 X 10 11 vg / kg, or 9 X 10 10 ~1 X 10 11 (virus genome (vg) / kilogram (kg) (vg / kg)). For example, the recombinant AAV vector of the present disclosure or a pharmaceutical composition containing the same is 1 X 10 11It is administered as a single intravenous dose of vg / kg. In some embodiments, the recombinant AAV vector or pharmaceutical composition containing the same of the present disclosure is 3 X 10 11 It is administered as a single intravenous dose of vg / kg. In some embodiments, the recombinant AAV vector or pharmaceutical composition containing the same of the present disclosure is 1 X 10 12 It is administered as a single intravenous dose of vg / kg. In some embodiments, the recombinant AAV vector or pharmaceutical composition containing the same of the present disclosure is 3 X 10 12 It is administered as a single intravenous dose of vg / kg. In some embodiments, the recombinant AAV vector or pharmaceutical composition containing the same of the present disclosure is 5 X 10 12 It is administered as a single intravenous dose of vg / kg.

[0230] In some embodiments, the present application provides a method for treating cancer in a patient by administering to the patient an effective amount of the recombinant AAV vector or a pharmaceutical formulation thereof described herein. In some embodiments, the cancer is breast cancer, ovarian cancer, esophageal cancer, bladder cancer, gastric cancer, salivary duct cancer, adenocarcinoma, uterine cancer, lung cancer, glioma, head and neck cancer, urothelial cancer, cervical cancer, bronchial cancer, colon cancer, rectal cancer, melanoma, renal cell cancer, pancreatic cancer, prostate cancer, pharyngeal cancer, liver cancer, intrahepatic bile duct cancer, or thyroid cancer. For example, in some embodiments, the cancer is breast cancer.

[0231] Method for reducing the risk of metastasis In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of a recombinant adeno-associated virus (rAAV) vector, or a pharmaceutical formulation thereof, comprising an rAAV vector encoding a bispecific fusion protein having: (i) A HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively. In some embodiments, the patient is not diagnosed with cancer. In some embodiments, the patient has not received cancer treatment.

[0232] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) An HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0233] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of a recombinant adeno-associated virus (rAAV) vector, or a pharmaceutical formulation thereof, comprising an rAAV vector encoding a bispecific fusion protein having the following: (i) An HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0234] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) An HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0235] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0236] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0237] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL and VH domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL domain and VH domain sequences corresponding to SEQ ID NO: 5 and SEQ ID NO: 4, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising VH and VL domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0238] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 8 and SEQ ID NO: 7, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0239] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL and VH domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL and VH domain sequences corresponding to SEQ ID NO: 10 and SEQ ID NO: 11, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising VH and VL domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0240] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 13 and SEQ ID NO: 14, respectively; (ii) a linker peptide comprising the sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0241] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 88; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0242] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a HER2 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: 89; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0243] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) 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: 90, which comprises a HER2 binding site; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; and (iii) an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which comprises a CD3 binding site.

[0244] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) 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: 12, which comprises a HER2 binding site; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; and (iii) an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which comprises a CD3 binding site.

[0245] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a HER2 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: 15; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0246] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 19, or a pharmaceutical formulation thereof.

[0247] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 20, or a pharmaceutical formulation thereof.

[0248] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 21, or a pharmaceutical formulation thereof.

[0249] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 22, or a pharmaceutical formulation thereof.

[0250] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 23 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0251] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence having at least 85% sequence identity to SEQ ID NO: 59 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0252] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence having at least 85% sequence identity to SEQ ID NO: 60 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0253] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 61 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0254] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 58 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0255] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 62 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0256] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 83, or a pharmaceutical formulation thereof.

[0257] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 84, or a pharmaceutical formulation thereof.

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

[0259] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 82, or a pharmaceutical formulation thereof.

[0260] In some embodiments, the present application provides a method for reducing the risk of metastasis by administering to a patient an effective amount of an rAAV vector having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 86, or a pharmaceutical formulation thereof.

[0261] Method for preventing metastasis In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively. In some embodiments, the patient is not diagnosed with cancer (e.g., HER2 cancer). In some embodiments, the patient has not received cancer treatment.

[0262] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0263] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) An HER2-binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3-binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0264] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0265] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) An HER2-binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3-binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0266] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0267] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 5 and SEQ ID NO: 4, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0268] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 8 and SEQ ID NO: 7, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0269] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 10 and SEQ ID NO: 11, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0270] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 13 and SEQ ID NO: 14, respectively; (ii) a linker peptide comprising the sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0271] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 88; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0272] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a HER2 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: 89; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0273] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) 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: 90, which comprises a HER2 binding site; (ii) A linker peptide comprising the sequence of SEQ ID NO: 29; and (iii) An scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which comprises a CD3 binding site.

[0274] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) 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: 12, which comprises a HER2 binding site; (ii) A linker peptide comprising the sequence of SEQ ID NO: 29; and (iii) An scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which comprises a CD3 binding site.

[0275] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a HER2 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: 15; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0276] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 19, or a pharmaceutical formulation thereof.

[0277] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 20, or a pharmaceutical formulation thereof.

[0278] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 21, or a pharmaceutical formulation thereof.

[0279] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 22, or a pharmaceutical formulation thereof.

[0280] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 23, or a pharmaceutical formulation thereof.

[0281] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 59, or a pharmaceutical formulation thereof.

[0282] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 60, or a pharmaceutical formulation thereof.

[0283] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 61 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0284] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 58 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0285] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 62 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0286] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 83, or a pharmaceutical formulation thereof.

[0287] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 84, or a pharmaceutical formulation thereof.

[0288] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 85, or a pharmaceutical formulation thereof.

[0289] In some embodiments, the present application provides a method for preventing metastasis by administering to a patient an effective amount of an rAAV vector having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 82, or a pharmaceutical formulation thereof.

[0290] 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, that comprises at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 86.

[0291] Method for treating metastasis

[0290] 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, that encodes a bispecific fusion protein having: (i) a HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0292]

[0290] 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, that encodes a bispecific fusion protein having: (i) A HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0293] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) An HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0294] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0295] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0296] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0297] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 5 and SEQ ID NO: 4, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0298] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 8 and SEQ ID NO: 7, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0299] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 10 and SEQ ID NO: 11, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0300] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 13 and SEQ ID NO: 14, respectively; (ii) A linker peptide comprising the sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0301] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 88; (ii) A linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0302] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a HER2 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: 89; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0303] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) 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: 90, which is a HER2 binding site; (ii) A linker peptide containing the sequence of SEQ ID NO: 29; and (iii) An scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which is a CD3 binding site.

[0304] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) 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: 12, which is a HER2 binding site; (ii) A linker peptide containing the sequence of SEQ ID NO: 29; and (iii) An scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which is a CD3 binding site.

[0305] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a HER2 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: 15; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0306] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein 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: 19, or a pharmaceutical formulation thereof.

[0307] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 20, or a pharmaceutical formulation thereof.

[0308] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 21, or a pharmaceutical formulation thereof.

[0309] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 22, or a pharmaceutical formulation thereof.

[0310] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein 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: 23, or a pharmaceutical formulation thereof.

[0311] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 59, or a pharmaceutical formulation thereof.

[0312] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 60, or a pharmaceutical formulation thereof.

[0313] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 61, or a pharmaceutical formulation thereof.

[0314] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 58, or a pharmaceutical formulation thereof.

[0315] In some embodiments, the present application provides a method for treating metastases by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 62, or a pharmaceutical formulation thereof.

[0316] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of an rAAV vector having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 83, or a pharmaceutical formulation thereof.

[0317] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of an rAAV vector having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 84, or a pharmaceutical formulation thereof.

[0318] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of an rAAV vector having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 85, or a pharmaceutical formulation thereof.

[0319] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of an rAAV vector having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 82, or a pharmaceutical formulation thereof.

[0320] In some embodiments, the present application provides a method for treating metastasis by administering to a patient an effective amount of an rAAV vector having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 86, or a pharmaceutical formulation thereof.

[0321] A method for promoting T cell-mediated killing of circulating tumor cells In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having: (i) a HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0322] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0323] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0324] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0325] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0326] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0327] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 5 and SEQ ID NO: 4, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0328] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 8 and SEQ ID NO: 7, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0329] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 10 and SEQ ID NO: 11, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0330] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 13 and SEQ ID NO: 14, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0331] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) 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: 88, which is a HER2 binding site; (ii) a linker peptide containing the sequence of SEQ ID NO: 29; and (iii) an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which is a CD3 binding site.

[0332] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) 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: 89, which is a HER2 binding site; (ii) a linker peptide containing the sequence of SEQ ID NO: 29; and (iii) an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which is a CD3 binding site.

[0333] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a HER2 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: 90; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0334] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) 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: 12, which contains a HER2 binding site; (ii) A linker peptide containing the sequence of SEQ ID NO: 29; and (iii) An scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which contains a CD3 binding site.

[0335] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) 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: 15, which contains a HER2 binding site; (ii) A linker peptide containing the sequence of SEQ ID NO: 29; and (iii) An scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which contains a CD3 binding site.

[0336] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 19, or a pharmaceutical formulation thereof.

[0337] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 20, or a pharmaceutical formulation thereof.

[0338] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 21, or a pharmaceutical formulation thereof.

[0339] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 22 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0340] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 23 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0341] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence having at least 85% sequence identity to SEQ ID NO: 59 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0342] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 60 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0343] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 61 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0344] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 58 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0345] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 62 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0346] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 83, or a pharmaceutical formulation thereof.

[0347] In some embodiments, the present application provides a method for promoting T cell-mediated killing of circulating tumor cells by administering to a patient an effective amount of an rAAV vector comprising at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 84, or a pharmaceutical formulation thereof.

[0348] 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 having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 85, or a pharmaceutical formulation thereof.

[0349] 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 having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 82, or a pharmaceutical formulation thereof.

[0350] 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 having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 86, or a pharmaceutical formulation thereof.

[0351] A method for preventing cancer in a patient having a predisposition to developing a tumor In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to developing a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following: (i) An HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0352] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) An HER2-binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3-binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0353] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2-binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3-binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0354] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0355] In some embodiments, the present application provides a method of preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0356] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0357] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 5 and SEQ ID NO: 4, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0358] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 8 and SEQ ID NO: 7, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0359] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 10 and SEQ ID NO: 11, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0360] In some embodiments, the present application provides a method of preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 13 and SEQ ID NO: 14, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0361] In some embodiments, the present application provides a method of preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) 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: 88, which comprises a HER2 binding site; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; and (iii) an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which comprises a CD3 binding site.

[0362] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) 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: 89, which comprises a HER2 binding site; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; and (iii) an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which comprises a CD3 binding site.

[0363] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a HER2 binding site comprising a scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 90; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0364] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) 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: 12, which comprises a HER2 binding site; (ii) A linker peptide comprising the sequence of SEQ ID NO: 29; and (iii) An scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which comprises a CD3 binding site.

[0365] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) 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: 15, which comprises a HER2 binding site; (ii) A linker peptide comprising the sequence of SEQ ID NO: 29; and (iii) An scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which comprises a CD3 binding site.

[0366] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 19, or a pharmaceutical formulation thereof.

[0367] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 20, or a pharmaceutical formulation thereof.

[0368] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 21, or a pharmaceutical formulation thereof.

[0369] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 22 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0370] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 23 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0371] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 59 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0372] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 60 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0373] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 61 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0374] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 58 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0375] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector comprising a transgene sequence that is at least 85% sequence identical to SEQ ID NO: 62 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0376] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector comprising at least 85% sequence identity to SEQ ID NO: 83 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0377] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector comprising at least 85% sequence identity to SEQ ID NO: 84 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or a pharmaceutical formulation thereof.

[0378] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 85, or a pharmaceutical formulation thereof.

[0379] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 82, or a pharmaceutical formulation thereof.

[0380] In some embodiments, the present application provides a method for preventing cancer in a patient having a predisposition to develop a tumor (e.g., a HER2+ tumor) by administering to the patient an effective amount of an rAAV vector having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 86, or a pharmaceutical formulation thereof.

[0381] Method for preventing cancer recurrence In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., HER2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0382] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., HER2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0383] In some embodiments, the present application provides a method of preventing cancer recurrence in a patient in remission from cancer (e.g., HER2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0384] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., HER2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) An HER2-binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3-binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0385] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., HER2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2-binding site comprising VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 sequences corresponding to SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3-binding site comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences selected from the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 sequences corresponding to SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

[0386] In some embodiments, the present application provides a method of preventing cancer recurrence in a patient in remission from cancer (e.g., HER2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0387] In some embodiments, the present application provides a method of preventing cancer recurrence in a patient in remission from cancer (e.g., HER2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 5 and SEQ ID NO: 4, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0388] In some embodiments, the present application provides a method of preventing cancer recurrence in a patient in remission from cancer (e.g., HER2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL and VH domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL and VH domain sequences corresponding to SEQ ID NO: 8 and SEQ ID NO: 7, respectively; (ii) A linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) A CD3 binding site comprising VH and VL domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0389] In some embodiments, the present application provides a method of preventing cancer recurrence in a patient in remission from cancer (e.g., HER2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 binding site comprising VL and VH domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VL and VH domain sequences corresponding to SEQ ID NO: 10 and SEQ ID NO: 11, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising VH and VL domain sequences having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0390] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., HER2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) A HER2 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: 13 and SEQ ID NO: 14, respectively; (ii) a linker peptide comprising a sequence corresponding to SEQ ID NO: 29; and (iii) a CD3 binding site comprising a VH domain and a VL domain having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the VH domain and VL domain sequences corresponding to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0391] In some embodiments, the present application provides a method of preventing cancer recurrence in a patient in remission from cancer (e.g., HER2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) An scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 88, which comprises a HER2 binding site; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; and (iii) an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which comprises a CD3 binding site.

[0392] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., HER2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) An scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 89, which comprises a HER2 binding site; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; and (iii) an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which comprises a CD3 binding site.

[0393] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., HER2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) a HER2 binding site comprising a scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 90; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; 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: 18.

[0394] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., HER2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) An scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 12, which comprises a HER2 binding site; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; and (iii) an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which comprises a CD3 binding site.

[0395] In some embodiments, the present application provides a method for preventing cancer recurrence in a patient in remission from cancer (e.g., HER2+ cancer) by administering to the patient an effective amount of an rAAV vector encoding a bispecific fusion protein having the following, or a pharmaceutical formulation thereof: (i) An scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 15, which comprises a HER2 binding site; (ii) a linker peptide comprising the sequence of SEQ ID NO: 29; and (iii) an scFv having at least 85% sequence identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18, which comprises a CD3 binding site.

[0396] ...

Claims

**Claim 1** A recombinant adeno-associated virus (rAAV) vector, from 5' to 3', (a) a 5' AAV inverted terminal repeat (ITR); (b) a promoter; (c) the following: (i) a HER2 binding site comprising a variable light chain (VL) and a variable heavy chain (VH) of an anti-HER2 antibody, (ii) a linker peptide, and (iii) a CD3 binding site comprising a VH and a VL of an anti-CD3 antibody encoding a bispecific fusion protein; (d) a modified RNA stability regulatory element (MRE), and (e) a 3' AAV ITR comprising the recombinant adeno-associated virus (rAAV) vector. **Claim 2** The rAAV vector according to claim 1, wherein 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, or a CAG promoter. **Claim 3** The rAAV vector according to any one of claims 1 to 2, wherein the promoter is a CAG promoter. **Claim 4** The rAAV vector according to any one of claims 1 to 3, wherein the promoter comprises a sequence that is at least 95% identical to SEQ ID NO:

87. **Claim 5** The rAAV vector according to any one of claims 1 to 4, wherein the anti-HER2 antibody VL comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 106, SEQ ID NO: 107, and SEQ ID NO: 108, respectively, and the anti-HER2 antibody VH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively. **Claim 6** The rAAV vector according to claim 5, wherein the anti-HER2 antibody VL and VH each comprise a sequence that is at least 95% identical to SEQ ID NO: 5 and SEQ ID NO: 4, respectively. **Claim 7** The rAAV vector according to claim 5 or 6, wherein the HER2 binding site is a single-chain variable region fragment (scFv). **Claim 8** The rAAV vector according to claim 7, wherein the anti-HER2 antibody VL is fused to the anti-HER2 antibody VH using an scFv linker peptide comprising SEQ ID NO:

25. **Claim 9** The rAAV vector according to any one of claims 5 to 8, wherein the HER2 binding site comprises a sequence that is at least 95% identical to SEQ ID NO:

89.

10. The rAAV vector according to any one of claims 1 to 4, wherein the anti-HER2 antibody VL comprises the complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) sequences of SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102, respectively, and the anti-HER2 antibody VH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 97, SEQ ID NO: 98, and SEQ ID NO: 99, respectively.

11. The rAAV vector according to claim 10, wherein the anti-HER2 antibody VL and VH each comprise a sequence that is at least 95% identical to SEQ ID NO: 2 and SEQ ID NO: 1, respectively.

12. The rAAV vector according to claim 10 or 11, wherein the HER2 binding site is a single-chain variable region fragment (scFv).

13. The rAAV vector according to claim 12, wherein the anti-HER2 antibody VL is fused to the anti-HER2 antibody VH using an scFv linker peptide comprising the sequence of SEQ ID NO:

24.

14. The rAAV vector according to claim 13, wherein the scFv comprises a sequence that is at least 95% identical to SEQ ID NO:

88.

15. The rAAV vector according to any one of claims 1 to 4, wherein the anti-HER2 antibody VL comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 112, SEQ ID NO: 113, and SEQ ID NO: 114, respectively, and the anti-HER2 antibody VH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 109, SEQ ID NO: 110, and SEQ ID NO: 111, respectively.

16. The rAAV vector according to claim 15, wherein the anti-HER2 antibody VL and VH each comprise a sequence that is at least 95% identical to SEQ ID NO: 8 and SEQ ID NO: 7, respectively.

17. The rAAV vector according to claim 15 or 16, wherein the HER2 binding site is a single-chain variable region fragment (scFv).

18. The rAAV vector according to claim 17, wherein the anti-HER2 antibody VL is fused to the anti-HER2 antibody VH using an scFv linker peptide comprising SEQ ID NO:

26.

19. The rAAV vector according to any one of claims 15 to 18, wherein the HER2 binding site comprises a sequence that is at least 95% identical to SEQ ID NO:

90.

20. The rAAV vector according to any one of claims 1 to 4, wherein the anti-HER2 antibody VL comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 118, SEQ ID NO: 119, and SEQ ID NO: 120, respectively, and the anti-HER2 antibody VH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, respectively.

21. The rAAV vector according to claim 20, wherein the anti-HER2 antibody VL and VH each comprise a sequence that is at least 95% identical to SEQ ID NO: 10 and SEQ ID NO: 11, respectively.

22. The rAAV vector according to claim 20 or 21, wherein the HER2 binding site is a single-chain variable region fragment (scFv).

23. The rAAV vector according to claim 22, wherein the anti-HER2 antibody VL is fused to the anti-HER2 antibody VH by an scFv linker peptide comprising the amino acid sequence of SEQ ID NO:

27.

24. The rAAV vector according to any one of claims 20 to 23, wherein the HER2 binding site comprises a sequence that is at least 95% identical to SEQ ID NO:

12.

25. The rAAV vector according to any one of claims 1 to 4, wherein the anti-HER2 antibody VL comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 126, respectively, and the anti-HER2 antibody VH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, respectively.

26. The rAAV vector according to claim 25, wherein the anti-HER2 antibody VL and VH each comprise a sequence that is at least 95% identical to SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

27. The rAAV vector according to claim 25 or 26, wherein the HER2 binding site is a single-chain variable region fragment (scFv).

28. The rAAV vector according to claim 27, wherein the anti-HER2 antibody VL is fused to the anti-HER2 antibody VH by an scFv linker peptide comprising the amino acid sequence of SEQ ID NO:

27.

29. The rAAV vector according to any one of claims 25 to 28, wherein the HER2 binding site comprises a sequence that is at least 95% identical to SEQ ID NO:

15.

30. The rAAV vector according to any one of claims 1 to 29, wherein the linker peptide comprises the same sequence as SEQ ID NO:

29.

31. The rAAV vector according to any one of claims 1 to 30, wherein the anti-CD3 antibody VH comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, respectively, and the anti-CD3 antibody VL comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively.

32. The rAAV vector according to claim 31, wherein the anti-CD3 antibody VH and VL each comprise a sequence that is at least 95% identical to SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

33. The rAAV vector according to claim 31 or 32, wherein the CD3 binding site is a single-chain variable region fragment (scFv).

34. The rAAV vector according to claim 33, wherein the anti-CD3 antibody VH is fused to the anti-CD3 antibody VL using an scFv linker peptide comprising the same sequence as SEQ ID NO:

28.

35. The rAAV vector according to any one of claims 31 to 34, wherein the CD3 binding site comprises a sequence that is at least 95% identical to SEQ ID NO:

18.

36. The rAAV vector according to any one of claims 1 to 35, further comprising a Kozak sequence.

37. The rAAV vector according to any one of claims 1 to 36, further comprising a polyadenylation sequence 3' of the transgene sequence and 5' of the 3' AAV ITR.

38. The rAAV vector according to claim 37, wherein the polyadenylation sequence is a bovine growth hormone (BGH) polyadenylation sequence that is at least 95% identical to SEQ ID NO:

81.

39. The rAAV vector according to any one of claims 1 to 38, further comprising an antibiotic resistance gene sequence.

40. The rAAV vector according to claim 39, wherein the antibiotic resistance gene is a kanamycin resistance gene.

41. The rAAV vector according to any one of claims 1 to 40, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, or a derivative thereof.

42. The rAAV vector according to any one of claims 1 to 41, wherein the bispecific fusion protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO:

23.

43. The rAAV vector according to any one of claims 1 to 42, wherein the transgene comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 58, or SEQ ID NO:

62.

44. The rAAV vector according to any one of claims 1 to 43, wherein the transgene contains reduced CpG dinucleotides and / or increased methylation of CpG dinucleotides as compared to the parental equivalent.

45. The rAAV vector according to any one of claims 1 to 44, comprising a sequence that is at least 90% identical to SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 82, or SEQ ID NO:

86.

46. A recombinant adeno-associated virus (rAAV) vector, from 5' to 3', (a) a 5' AAV inverted terminal repeat (ITR) comprising a sequence that is at least 90% identical to SEQ ID NO: 75 or SEQ ID NO: 91; (b) a promoter; (c) the following: (i) a HER2 binding site comprising a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 106, SEQ ID NO: 107, and SEQ ID NO: 108, respectively, of an anti-HER2 antibody, and a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105, respectively; (ii) a linker peptide comprising a sequence according to SEQ ID NO: 29, and (iii) a CD3 binding site comprising a VH comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 127, SEQ ID NO: 128, and SEQ ID NO: 129, respectively, of an anti-CD3 antibody, and a VL comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 130, SEQ ID NO: 131, and SEQ ID NO: 132, respectively comprising a sequence encoding a bispecific fusion protein; (d) a modified RNA stability regulatory element (MRE), and (e) a 3' AAV ITR comprising a sequence that is at least 90% identical to SEQ ID NO: 75 or SEQ ID NO: 91 comprising the recombinant adeno-associated virus (rAAV) vector.

47. A recombinant adeno-associated virus (rAAV) vector, from 5' to 3', (a) A 5’ AAV inverted terminal repeat (ITR) comprising a sequence that is at least 90% identical to SEQ ID NO: 75 or SEQ ID NO: 91; (b) A promoter; (c) A transgene encoding a bispecific fusion protein comprising a sequence that is at least 90% identical to SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23; (d) A modified RNA stability regulatory element (MRE), and (e) A 3’ AAV ITR comprising a sequence that is at least 90% identical to SEQ ID NO: 75 or SEQ ID NO: 91 The recombinant adeno-associated virus (rAAV) vector comprising the above. **Claim 48** A recombinant adeno-associated virus (rAAV) vector comprising a sequence that is at least 90% identical to SEQ ID NO:

20. **Claim 49** A method for reducing the risk of metastatic disease in a patient, comprising administering to the patient an effective amount of the recombinant adeno-associated virus (rAAV) vector according to any one of claims 1 to 48 or a pharmaceutical formulation thereof. **Claim 50** A method for delaying the onset of metastatic disease in a patient, comprising administering to the patient an effective amount of the recombinant adeno-associated virus (rAAV) vector according to any one of claims 1 to 48 or a pharmaceutical formulation thereof. **Claim 51** A method for preventing metastatic disease in a patient, comprising administering to the patient an effective amount of the recombinant adeno-associated virus (rAAV) vector according to any one of claims 1 to 48 or a pharmaceutical formulation thereof. **Claim 52** A method for promoting T cell-mediated killing of circulating tumor cells in a patient, comprising administering to the patient an effective amount of the recombinant adeno-associated virus (rAAV) vector according to any one of claims 1 to 48 or a pharmaceutical formulation thereof. **Claim 53** The method according to any one of claims 49 to 52, wherein the rAAV or its pharmaceutical formulation is administered simultaneously with the treatment of the primary tumor. **Claim 54** The method according to claim 53, wherein the primary tumor is a breast tumor. **Claim 55** The method according to claim 53 or 54, wherein the treatment of the primary tumor comprises surgical resection, radiotherapy, chemotherapy, or immunotherapy. **Claim 56** A method for preventing cancer in a patient having a predisposition to develop HER2+ tumors, the method comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector according to any one of claims 1 to 48 or a pharmaceutical formulation thereof.

57. A method for preventing cancer recurrence in a patient in remission from HER2+ cancer, the method comprising administering to the patient an effective amount of a recombinant adeno-associated virus (rAAV) vector according to any one of claims 1 to 48 or a pharmaceutical formulation thereof.

58. The method according to any one of claims 49 to 57, wherein the rAAV or its pharmaceutical formulation is administered together with a checkpoint inhibitor selected from the group consisting of CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors.

59. The method according to claim 58, wherein the checkpoint inhibitor is selected from the group consisting of pembrolizumab, ipilimumab, nivolumab, and atezolizumab.

60. A pharmaceutical formulation comprising a recombinant adeno-associated virus (rAAV) vector according to any one of claims 1 to 48 and a pharmaceutically acceptable carrier.

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